CN113350481A - Application of charantin MAP30 in preparation of medicines or chemotherapy supplements for preventing and treating ovarian cancer - Google Patents

Abstract

The present invention provides the use of bitter melon protein MAP30 in the preparation of medicines or chemotherapy supplements for preventing and treating ovarian cancer. In the present invention, it is found that the biologically active protein MAP30 isolated from the seeds of bitter gourd exhibits strong anti-cancer effect and anti-chemo-resistance effect on ovarian cancer cells, and MAP30 can inhibit cell migration, cell invasion and cell invasion through induction in various ovarian cancer cell lines. cell proliferation, whereas there was no such inhibitory effect in normal immortalized ovarian epithelial cells. When co-treated with cisplatin, MAP30 was able to elicit a synergistic effect of cytotoxicity in ovarian cancer cells. By intraperitoneal co-injection of low doses of cisplatin and MAP30, tumor spread and progression were significantly reduced in a tumor xenograft mouse model. Blood tests confirmed that MAP30 did not cause any side effects such as liver damage, kidney damage in mice. These findings of the present invention suggest that the bitter melon protein MAP30 may serve as a non-toxic additive that enhances the efficacy of chemotherapy and benefits current ovarian cancer patients.

Description

Application of charantin MAP30 in preparation of medicines or chemotherapy supplements for preventing and treating ovarian cancer

Technical Field

The invention belongs to the field of medicinal chemistry and pharmacology, and particularly relates to application of momordica charantia protein MAP30 in preparation of a medicament or a chemotherapy supplement for preventing and treating ovarian cancer.

Background

Ovarian cancer is one of the most fatal gynecological malignancies worldwide, including china. The prognosis for most cases of ovarian cancer is poor, and many patients die from advanced disease. Platinum-based drugs, such as cisplatin, are the most potent anticancer agents commonly used to treat advanced ovarian cancer. However, the major limitations of these drugs include initially responsive tumor acquisition resistance and severe side effects associated with high doses. Therefore, there is an urgent need to explore alternative anti-cancer approaches, such as the use of food and plant supplements in combination with existing chemotherapy approaches, to improve the efficacy of available drugs.

AMP-dependent protein kinase (AMPK) is a metabolic energy sensor that controls the energy balance in cells. It has recently been found that low AMPK activity contributes to cancer cell growth, whereas treatment with AMPK activators such as AICAR and metformin significantly inhibits cell growth.

There is increasing evidence that traditional Chinese medicine is a remarkable cancer treatment, but scientific research on the molecular mechanisms on which it depends is lacking. Evidence-based traditional Chinese medicine for cancer therapy shows that traditional Chinese medicine not only targets cancer cells, but also treats the whole body. Bioactive compounds from chinese medicine exert antitumor activity through multi-level and multi-signal transduction targeting. Therefore, the traditional Chinese medicine can be used as an auxiliary anticancer treatment.

Bitter melon (Momordica charrantia) is a common edible vegetable, but it has also been used in various ancient folk medical practices for the treatment of various diseases. Evidence now emerging has shown that extracts of momordica charantia (BME) can exert effects similar to natural AMPK in the inhibition of human cancer cell growth and induction of apoptosis, while having no toxic effect on normal cells. In particular, the momordica charantia anti-HIV protein (MAP30), one of the most biologically active components in BME, has been demonstrated to have immunomodulatory, anti-tumor, anti-viral and anti-Human Immunodeficiency Virus (HIV) activities, and to inhibit protein synthesis in vivo.

Disclosure of Invention

Since chemotherapy resistance is a major obstacle to clinical management of advanced ovarian cancer, the present study provides scientific data confirming that nontoxic MAP30 isolated from momordica charantia can be used as a supplement to enhance the effects of chemotherapy. This would not only benefit current ovarian cancer patients, but also have a large potential impact on public health policies in cancer treatment.

Therefore, the invention provides application of the momordica charantia protein MAP30 in preparing a medicament or a chemotherapy supplement for preventing and/or treating ovarian cancer.

The invention also provides application of the momordica charantia protein MAP30 and cisplatin in preparing a medicament or a chemotherapy supplement for preventing or treating ovarian cancer.

In particular, the ovarian cancer is advanced ovarian cancer or chemotherapy-resistant ovarian cancer.

Specifically, the prevention and/or treatment of ovarian cancer is inhibition of ovarian cancer cell growth, cell migration/invasion, ovarian cancer growth, and/or ovarian cancer colonization.

In another aspect, the invention provides the use of momordica charantia protein MAP30 in the preparation of an AMPK activator.

The invention also provides the use of the momordica charantia protein MAP30 in the preparation of a medicament for the prevention and/or treatment of diseases or conditions associated with the inhibition of AMPK activation.

In a further aspect, the present invention provides a pharmaceutical composition for use in the prevention and/or treatment of ovarian cancer comprising the momordica charantia protein MAP30 and cisplatin.

In one embodiment of the pharmaceutical composition, the weight ratio of the momordica charantia protein MAP30 to cisplatin is 1: (0.6 to 1.8), preferably 1: (1.0 to 1.4), more preferably 1: 1.2.

preferably, in the above use or pharmaceutical composition, the momordica charantia protein MAP30 is a natural momordica charantia protein MAP 30; further preferably, said native momordica protein MAP30 is isolated from momordica charantia, thailand or momordica charantia; more preferably, said native momordica charantia protein MAP30 is isolated from momordica charantia; further preferably, said native momordica charantia protein MAP30 is isolated from momordica charantia taiwan in china.

Preferably, the amino acid sequence of the momordica charantia protein MAP30 is shown in SEQ ID No.1 or 2.

Preferably, the natural momordica charantia protein MAP30 is a natural momordica charantia protein MAP30 modified by glycosylation; more preferably, the glycosylation modification site of the native momordica charantia protein MAP30 is located at asparagine 74 in the amino acid sequence shown in SEQ ID No. 1.

The preparation method of the natural momordica charantia protein MAP30 can comprise the following steps:

(1) soaking bitter gourd seeds, homogenizing, centrifuging, and collecting supernatant;

(2) eluting the supernatant loaded on the Affi-gel blue gel column by using 20mM Tris-HCl buffer solution containing 1M NaCl, and dialyzing the eluted part containing the natural bitter gourd protein MAP 30;

(3) with 20mM NH containing 1M NaCl₄The OAc buffer elutes the dialyzed fraction loaded onto the SP-Sepharose column and the eluted fraction comprising the native Momordica charantia protein MAP30 is dialyzed；

(4) FPLC-gel filtration of the dialyzed fraction loaded onto a Superdex 75HR 10/300 column with 50mM Tris-HCl buffer containing 0.1M NaCl, collecting the first peak at OD 280 nm;

preferably, the preparation method of the natural momordica charantia protein MAP30 comprises the following steps:

(1) soaking fructus Momordicae Charantiae seed, homogenizing, centrifuging, filtering to obtain supernatant;

(2) eluting the supernatant loaded on the Affi-gel blue gel column by using 20mM Tris-HCl buffer solution containing 1M NaCl, and dialyzing the eluted part containing the natural bitter gourd protein MAP30 in a dialysis tube with the molecular weight cut-off of 8 kDa;

(3) with 20mM NH containing 1M NaCl₄Eluting the dialyzed fraction loaded onto the SP-sepharose column with OAc buffer, dialyzing the eluted fraction comprising native charantin MAP30, and lyophilizing;

(4) the lyophilized powder was resuspended in 50mM Tris-HCl buffer containing 0.1M NaCl, the suspension loaded onto a Superdex 75HR 10/300 column was subjected to FPLC-gel filtration with the same buffer, the first peak at OD 280nm was collected, dialyzed against centricon with a molecular weight cut-off of 3kDa and lyophilized to a powder.

The anti-tumorigenic and anti-chemotherapeutic resistance effects of MAP30 in advanced ovarian cancer cells are still unknown. The invention discovers that the bioactive protein MAP30 separated from bitter gourd seeds shows strong anti-cancer effect and anti-chemotherapy drug resistance effect on ovarian cancer cells. The invention discovers that MAP30 exists in various bitter gourd in different quantities, and proves that different bitter gourd extracts have the function of resisting cancers of ovarian cancer cells. Functional studies of the present invention revealed that MAP30 can inhibit cell migration, cell invasion and cell proliferation by induction in various ovarian cancer cell lines, but not in normal immortalized ovarian epithelial cells. MAP30 was able to elicit a cytotoxic synergistic effect in ovarian cancer cells when co-treated with cisplatin. By co-injecting low doses of cisplatin and MAP30 intraperitoneally, there was a significant reduction in tumor spread and progression in the tumor xenograft mouse model. Importantly, bloodThe liquid test confirmed that MAP30 did not cause any side effects such as liver injury, kidney injury in mice. Mechanistically, MAP30 can induce AMPK activation and cellular S-phase arrest, whereas other AMPK activators, such as AICAR and metformin, cause G0/G1 arrest. In addition, MAP30 can regulate intracellular Ca²⁺Levels and in related pathways decrease calcineurin activity. Furthermore, MAP30 can regulate cellular metabolism of ovarian cells by inhibiting GLUT-1 and GLUT-3 mediated glucose uptake and inhibiting adipogenesis as well as lipid droplet formation. In summary, these findings of the present invention indicate that native MAP30 is a non-toxic additive that enhances the chemotherapeutic effect and benefits current ovarian cancer patients, and that its inhibition of cancer cell proliferation is significantly superior to recombinant MAP 30.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows that MAP30 has the effect of a CaMKK β/AMPK activator. (A) Western blot shows the effect of the recombinant MAP30 on the activity of AMPK and downstream targets FOXM1 and AKT thereof. (B) The time course shows the effect of native MAP 30. (C) Natural MAP30 in AMPK alpha 1 alpha 2^-/-Effect of ES2 on AKT/ERK/FOXM1 signaling in cells. (D) Transwell cell migration (upper panel) and Transwell cell invasion assay (lower panel) showed native MAP30 versus AMPK α 1 α 2, compared to negative control (Ctrl)^-/-The cell migration and invasion capacity of ES2 cells. (E) Isogenic WT and RG mutant HEK-293 cell lines showed that MAP30 induced phosphorylation of AMPK in a dose and time dependent manner in both cell lines. (F) Co-treatment with the CaMKK β inhibitor, STO-609(10 μ M) abolished AMPK phosphorylation induced by MAP30 in both cell lines. (G) The Fluo-4 NW calcium assay showed that after 24 hours of treatment with MAP30(5, 10, 20, and 40. mu.g/mL), MAP30 mediated an increase in the calcium-dependent concentration of cells in OVCA433 cells compared to control cells without MAP30 treatment (P.ltoreq.0.05). Treatment with calcium ionophore A23187 (2. mu.M) served as a positive control. (H) Knock-out of AMPK α 1 α 2 significantly reduced calcium uptake by OVCA433 cells, compared to OVCA433 control cells, 24 hours after MAP30(5, 10, 20, and 40 μ g/mL), leading to AMPK α 1 α 2^-/-OVCA433 the concentration-dependent increase of cellular calcium in cells was significantly hindered (. about.p.. ltoreq.0.01).

Figure 2 shows that MAP30 is a major component of BME and acts like a natural AMPK activator. (A) Western blot analysis verified the specificity of the mouse anti-MAP 30 antibody. A distal single band at 30kDa was observed in the sample containing pure MAP30 (0.5. mu.g). (B) Western blot analysis showed the components of MAP30 for Thailand (Thai) BME, Chinese BME and Chinese Taiwan (TW) BME. (C) XTT cell proliferation assays show dose-dependent inhibition of cell proliferation of ovarian cancer cells. (D) The lesion formation assay demonstrated that MAP30 inhibited the number of lesions formed in ovarian cancer cells. (E) Positive staining for annexin V and Propidium Iodide (PI) (apoptotic cells) was analyzed by flow cytometry in OVCA433 cells treated with MAP30(15 and 45 μ g/mL) and AICAR (1 mM). (F) Western blot analysis showed cleavage of caspase 3 and PARP (apoptosis) of MAP30 treated OVCA433 and ES2 cells. (G) the transwell migration assay showed that MAP30(10 and 40 μ g/mL) treated ES2 cells showed a significant decrease in the number of cells permeating through the membrane in a dose-dependent manner compared to untreated controls (upper panel). MAP30 treated ES2 cells (10 and 40 μ g/mL) showed less invasive cell passage through the basement membrane matrix compared to the control (lower panel).

Figure 3 shows that MAP30 alone inhibits the oncogenic properties of ovarian cancer cells. (A) Tumor xenograft nude mouse models showed treatment with MAP30 by intraperitoneal injection every two days (5 μ g/kg), for a total of five injections. (A) Mean tumor burden and (B) relative number of tumor nodules formed via MAP30(5 μ g/kg). (C) Fluorescence micrographs directed to the location of tumor nodules in the abdominal cavity of mice treated with or without MAP 30. (D) MAP30 (5. mu.g/kg) did not significantly reduce the body weight of both groups of mice throughout the experiment. (E) MAP30 (5. mu.g/kg) did not produce statistically significant changes in ascites in both groups of mice. (F) Metabolomics data plotted in the heatmap show the key difference in 37 fatty acid expression between control and ascites in MAP30(5 μ g/kg) treated animals. (G) MAP30(5 μ g/kg) treatment reduced total fatty acids in the ascites of the ES2 xenografted mouse tumor model compared to untreated controls. (H) MAP30(1.25, 2.5 and 10. mu.g/kg) did not cause an observable reduction in body weight in all groups of mice throughout the experiment. (I) Kaplan-Meier survival analysis showed no significant difference in survival curves between the low doses of MAP 30. (J) Biochemical tests showed that the low dose of MAP30 showed no significant side effects on liver and kidney function as the control. (K) Histopathology of liver, kidney and spleen in control and MAP30 treated groups.

Figure 4 shows that MAP30 synergistically enhances cisplatin-mediated cytotoxicity against ovarian cancer cells. (A) The XTT cell proliferation assay demonstrated that the growth rate of OVCA433, HEY and HEYA8 cells was most significantly reduced by the combination of MAP30 (1-4. mu.g/mL) and cisplatin (0.5. mu.g/mL). (B) Flow cytometric analysis of annexin and Propidium Iodide (PI) staining of apoptotic cells revealed a dose-dependent increase in the apoptotic cell population following MAP30, and was more pronounced when cells were co-treated with MAP30(2.5 and 5. mu.g/mL) and cisplatin (0.5. mu.g/mL). Tumor xenograft nude mouse model describes the intraperitoneal inoculation of ES2 cells into 4-5 week old female nude mice. Approximately one week after tumor cell injection, treatment with MAP30 (2.5. mu.g/kg) and cisplatin (2mg/kg) was started and continued every two days for a total of five injections. (C) The average tumor burden and (D) the relative number of tumor nodules formed by co-treatment were much less than cisplatin alone and vehicle treated controls. (E) Co-treatment with MAP30 and cisplatin or cisplatin alone did not significantly reduce ascites in both groups of mice compared to vehicle-treated controls. (F) Co-treatment with MAP30 or cisplatin alone did not significantly reduce body weight in both groups of mice. (G) abdominal dissection with blue arrows indicating the location of tumor nodules in the abdominal cavity of PBS, cisplatin, and MAP30 and cisplatin treated mice.

FIG. 5 shows the oncogenic effect of MAP30 on ovarian cancer cells to disrupt OCM. (A) XTT cell proliferation assay revealed significant inhibition of cell proliferation in Omentum Conditioned Medium (OCM) -cultured OV2008 and SKOV3 cells treated with 1% (v/v) of chinese BME and MAP30(2.5 μ g/mL). (B) Colony formation experiments demonstrated that co-treatment of OCM cultured a2780cp cells with MAP30(2.5 μ g/mL) showed a significant reduction in colony numbers compared to OCM (0.02). (C) Wound healing experiments demonstrated that OCM (OCM control) treatment resulted in a significant increase in cell migration of SKOV3 and ES2 cells compared to untreated control medium (DMEM control). Whereas co-treatment of OCM treated SKOV3 and ES2 cells with MAP30(2.5 μ g/mL) or 10% (v/v) chinese BME positive control resulted in a significant delay in wound closure rate compared to their respective OCM controls. The width of the wound is indicated by the arrows in the photograph and the relative cell migration rates of the three independent experiments are expressed as relative gap width/time. (D) The Transwell migration assay revealed that co-treatment of MAP30(2.5 μ g/mL) or metformin (1.5mM) and a positive control of 10% (v/v) BME on OCM cultured SKOV3 cells showed a significant reduction in cell permeation through the cell membrane compared to the OCM control (. P < 0.001). (E) OCM cultured SKOV3 cells invaded less slowly through the basement membrane matrix of the Transwell invasion assay kit compared to the OCM control by treatment with MAP30 (2.5. mu.g/mL), 10% (v/v) BME or metformin (1.5 mM).

Figure 6 shows that MAP30 alters glycolysis and lipid metabolism in ovarian cancer cells. (A) Ovarian cancer cells exhibited higher aerobic glycolysis as evidenced by higher lactate production after 24 and 48 hours of culture compared to HOSE96 cells. (B) MAP30 (40. mu.g/mL) treatment significantly reduced lactate production in ovarian cancer cells. (C) Hexokinase activity and (D) Lactate Dehydrogenase (LDH) activity were reduced by MAP30(10 and 40. mu.g/mL) in a time and dose dependent manner. (E) glucose uptake assay showed reduced glucose uptake in ovarian cancer cells treated with MAP (10 and 40. mu.g/mL) with AICAR (1mM) as a positive control. (F) Western blot analysis showed that expression of GLUT1 and GLUT3 in ovarian cancer cells was inhibited by MAP30 (40. mu.g/mL) for 24 and 48 hours. (G) Nile red fluorescence of lipid-loaded ES2 in OCM culture. OCM treated with 1% DMEM and clean ascites was used as a negative control. (H) Ovarian cancer cells were starved overnight in FBS-free DMEM prior to OCM and MAP30 treatment, and subsequent cell lipolysis was determined by lipolysis assay. It was found that lipolysis of ovarian cancer cells was increased by treatment with MAP30(5 and 20. mu.g/mL) for 24 hours, with 1% FBS DMEM as a positive control. (I) MAP30 treatment reduced ATP production in ovarian cancer cells.

The non-denaturing gel in figure 7 shows the size of native MAP30 isolated from dried bitter gourd seeds (left panel). Western Blot analysis based on cDNA of MAP30 with 285 amino acids plus 6 histidine tags showed the size of recombinant MAP30 protein produced by E.coli (BL21(DE3)) (right panel) at a concentration of 0.47mg/ml, volume of 0.22ml, amount of 103. mu.g, buffer PBS, pH 7.4, lane A wild type MAP30 protein (signal peptide removed), lane B wild type MAP30 protein (signal peptide not removed), MK molecular weight marker.

XTT cell proliferation assay in figure 8 shows co-treatment of native and recombinant MAP30 proteins at various doses. At day 4, native MAP30 showed significantly higher inhibition of cell growth of a2780cp (-10%), OVCA433 (-30%) and ES2 (20%) (. P <0.05,. P <0.01,. P < 0.001).

Figure 9 shows that both native and recombinant MAP30 proteins are able to activate AMPK signaling, but the effect of native MAP30 protein is stronger.

FIG. 10 in combination with FIG. 7 shows that native MAP30 protein is 2-5kDa smaller in size than recombinant MAP30 protein.

Fig. 11 shows that MAP30 is the major component of BME and functions as a natural AMPK activator. (A) The XTT cell proliferation assay showed dose-dependent inhibition of cell proliferation of ovarian cancer cells, but had no effect on HOSE 11-12. (B) The lesion formation assay showed that MAP30 decreased the number of lesions formed in ovarian cancer cells (a2780cp, OVCA433 and ES2) at 5 and 10 μ g/mL, respectively, for one week. (C) Transwell migration assay showed that ES2 cells treated with MAP30(10 and 40 μ g/mL) showed a significant reduction in the number of cells penetrating the membrane in a dose-dependent manner compared to untreated controls (upper panel). ES2 cells treated with MAP30(10 and 40. mu.g/mL) showed fewer cells invaded by matrigel than the control (lower panel). (D) flow cytometry analyzed annexin V and Propidium Iodide (PI) positive staining (apoptotic cells) in MAP30(10 and 40. mu.g/mL) and AICAR (1mM) treated OVCA433 cells. (E) immunoblot analysis showed cleavage of caspase 3 and PARP (═ apoptosis) in OVCA433 and ES2 cells treated for 24-48h with MAP30(40 μ g/mL). All bars are expressed as mean + SEM. P <0.05, P <0.01, P <0.001 (compared to control).

FIG. 12 shows that MAP30 acts as a natural CaMKK β/AMPK activator. Immunoblot analysis showed the effect of (A) recombinant MAP30 (30. mu.g/mL) and (B) native MAP30 (40. mu.g/mL) on AMPK and its downstream targets FOXM1 and AKT activity in OVCA433 and ES2 cells. (C) Immunoblot analysis showed the inhibitory effect of recombinant MAP30 (30. mu.g/mL) and native MAP30 (40. mu.g/mL) on mTOR signaling activity in OVCA433 and ES2 cells. (D) Immunoblot analysis showed native MAP30 (40. mu.g/mL) versus OVCA433, ES2 and AMPK α 1 α 2^-/-Effect of AKT/ERK/FOXM1 Signaling in ES2 cells. (E) Transwell cell migration (upper panel) and invasion assay (lower panel) show different doses (0, 10 and 40. mu.g/mL) of native MAP30 versus AMPK α 1 α 2 compared to negative control (Ctrl)^-/-ES2 cell migration and invasion capacity lasting 16-24 hours. (F) Isogenic WT and RG mutant HEK293 γ 2 cell lines showed that MAP30 induced phosphorylation of AMPK in both cell lines in a dose and time dependent manner. (G) Immunoblot analysis showed that co-treatment with the CaMKK β inhibitor STO-609(10 μ M) abolished MAP30(40 μ g/mL) induced phosphorylation of AMPK in both cell lines. (H) The Fluo-4 NW calcium assay with calcium ionophore A23187 (2. mu.M) as a positive control was used to examine the concentration-dependent increase in cellular calcium in OVCA433 cells treated with MAP30(5, 10, 20 and 40. mu.g/mL) for 24 hours. (I) Knock-out of AMPK α 1 α 2 compared to control cells (Ctrl) treated with MAP30(5, 10, 20, and 40 μ g/mL) for 24 hours^-/-Then, OVCA433 AMPK α 1 α 2^-/-MAP 30-mediated concentration-dependent cellular calcium elevation in cells was significantly hindered. All bars are expressed as mean + SEM. P<0.05，**P<0.01， ***P<0.001 (compared to control).

Figure 13 shows that MAP30 induces iron apoptosis in ovarian cancer cells. (A) AMPK alpha 1 alpha 2 treated in microplate format with OVCA433 and the ES2 parent (Ctrl) and MAP30(20 and 40. mu.g/mL) and Erastin (5 and 2.5. mu.M) for 24 hours^-/-Cellular ROS detection was measured by DCFDA fluorescence intensity in cells. (B) Measurement of cellular GSH/GSSG ratio in OVCA433 and ES2 cells by fluorescent microplate format. OVCA433 and ES2 parental (Ctrl) and the use of MAP30(20 and 40. mu.g/mL) and Erastin (5. mu.M for OVCA433 cells, ES2 cells)2.5. mu.M) for 24 hours^-/-The cells showed a decrease in the GSH/GSSG ratio. (C) Immunoblot analysis revealed that MAP30 pairs wild-type and AMPK α 1 α 2^-/-GPX4 in OVCA433 and ES2 cells had similar inhibitory effects to Erastin. Treatment time for various doses of MAP30 and Erastin was 24 hours. GAPDH was used as an internal control. (D) Confocal imaging showed lipid peroxidation activity (green) measured in OVCA433 cells using C11 BODIPY 581/591(C11) (ThermoFisher) 24 hours after treatment with Erastin (10. mu.M), MAP30(20 and 40. mu.g/mL), and Fer-1 (10. mu.M) in combination with Erastin (10. mu.M) or MAP30 (40. mu.g/mL). PBS was used for negative control (Ctrl). C11^Non-oxUnoxidized C11 (red), C11^OxOxidized C11 (green). (scale bar 20 μm). (E) Flow cytometry analysis of OVCA433 cells treated with Erastin (10. mu.M) (red), MAP30 (40. mu.g/mL) (orange) and Fer-1 (10. mu.M) + MAP30 (40. mu.g/mL) (blue) for 24 hours. PBS was used as a negative control (Ctrl) (green) and stained with C-11BODIPY (ThermoFisher). (F) Flow cytometry analysis showed that the MAP30 (40. mu.g/mL, 24h) induced effect on lipid peroxidation activity of AMPK wild type versus AMPK α 1 α 2^-/-OVCA433 cells were more potent. Ferrostatin-1(Fer-1) (10. mu.M, 24 hours) was considered an inhibitor of iron apoptosis (left panel). The bar graphs show the relative median fluorescence intensity of the control and treated groups (MAP30 and/or Fer-1) analyzed by flow cytometry (right panel). All bars are expressed as mean + SEM. P<0.05， **P<0.01，***P<0.001 (compared to control).

Figure 14 shows that MAP30 alone inhibits the carcinogenicity of ovarian cancer cells. (A) The nude mouse tumor xenograft model showed MAP30 treatment by intraperitoneal injection once every two days for a total of five doses. (A) Average tumor burden and (B) relative number of tumor nodules formed after receiving MAP30 (250. mu.g/kg). (C) Fluorescence stereomicroscopy indicated the location of tumor nodules in the peritoneal cavity of mice treated with or without MAP30(250 μ g/kg). BF is bright field. (D) MAP30 (250. mu.g/kg) did not significantly reduce body weight in both groups of mice throughout the experiment. (E) Neither group of mice was statistically significantly affected by the use of MAP30 (250. mu.g/kg). (F) Metabolomics data plotted in the heatmap show the key difference in the expression of 37 fatty acids in ascites from control (Ctrl) and MAP30(250 μ g/kg) treated animals. (G) MAP30(250 μ g/kg) treatment reduced total fatty acids in the ascites of the ES2 xenografted mouse tumor model compared to the untreated control group. (H) MAP30(62.5, 125 and 500. mu.g/kg) did not significantly reduce the body weight of all groups of mice throughout the experiment. (I) Kaplan-Meier survival analysis showed no significant difference in survival curves between the low dose MAP 30. (J) Biochemical tests showed that the low dose of MAP30 had no significant adverse effect on liver and kidney function as controls. (K) Hematoxylin and eosin (H & E) staining showed histopathology of liver, kidney and spleen (Ctrl, 62.5. mu.g/kg and 500. mu.g/kg) in the control and MAP30 treated groups. (scale bar 100 μm). All bars are expressed as mean + SEM. P <0.05, P <0.01, P <0.001 (compared to control). ns is meaningless.

Figure 15 shows that MAP30 synergistically enhances cisplatin-mediated cytotoxicity in ovarian cancer cells. (A) XTT cell proliferation assays demonstrated that MAP30 in combination with cisplatin significantly reduced the growth rate of OVCA433, HEY and HEYA8 cells to the greatest extent. MAP30 (5-20. mu.g/mL) was used in combination with cisplatin (0.3. mu.g/mL) in OVC433 cells, and MAP30 (1-4. mu.g/mL) was used in combination with cisplatin (0.5. mu.g/mL) in HEY and HEYA8 cells. (B) Flow cytometric analysis of annexin-V and Propidium Iodide (PI) staining of apoptotic cells showed a dose-dependent increase in the apoptotic cell population following MAP30 treatment, which was more pronounced when cells were co-treated with MAP30(2.5 and 5. mu.g/mL) and cisplatin (0.5. mu.g/mL) for 48 h. (C) Morphological examination of OVCA433 cells treated with increasing concentrations of MAP30 and/or cisplatin for 3 days (upper panel). (scale bar 100 μm). Cell viability of OVCA433 cells treated with increasing concentrations of MAP30 and/or cisplatin for 3 days and determination of IC50 (lower panel). (D) The matrix plot shows the percentage of viable cells after 3 days of treatment with increasing concentrations of MAP30 and/or cisplatin (left panel). The synergy matrix (middle panel) and surface plot (right panel) show the synergy between cisplatin and MAP30 for OVCA433 cells (N ═ 3). All bar or line plots are presented as mean + SEM. P <0.05, P <0.01, P <0.001 (compared to control).

Figure 16 shows that MAP30 enhances cisplatin-mediated tumor growth in vivo. To establish a nude mouse tumor xenograft model, ES2 cells were inoculated intraperitoneally into 4-5 week-old female nude mice. Approximately one week after tumor cell injection, treatment with MAP30 (125. mu.g/kg) and cisplatin (2mg/kg) was started, once every two days, for a total of five injections. (A) Representative photographs of peritoneal spreads with arrows (blue) indicate the location of tumor nodules in the peritoneal cavity of mice treated with vehicle PBS (Ctrl), cisplatin (2mg/kg), and MAP30 (125. mu.g/kg) and cisplatin (2 mg/kg). (B) The mean tumor burden and (C) the relative number of tumor nodules formed after co-treatment were much lower than cisplatin and vehicle treated controls alone. (D) Co-treatment of MAP30 and cisplatin or cisplatin alone significantly reduced ascites in both groups of mice compared to vehicle-treated controls. (E) Co-treatment with MAP30 and cisplatin alone did not significantly reduce body weight in both groups of mice. All bars are expressed as mean + SEM. P <0.05, P <0.01, P <0.001 (compared to control).

Figure 17 shows that MAP30 alters glycolysis and lipid metabolism in ovarian cancer cells. (A) Ovarian cancer cells showed enhanced aerobic glycolysis as shown by higher lactic acid production after 24 and 48 hours in culture compared to HOSE 96-9-18 cells. (B) MAP30(40 μ g/mL) treatment for 24 hours (left panel) and 48 hours (right panel) significantly reduced lactate production in ovarian cancer cells. AICAR (1mM) and A23187 (2. mu.M) were used as positive controls for AMPK activation. (C) MAP30(10 to 40. mu.g/mL) reduced hexokinase activity and (D) Lactate Dehydrogenase (LDH) activity in a time and dose dependent manner. (E) Glucose uptake assays showed a decrease in glucose uptake in ovarian cancer cells following treatment with AICAR (1mM) as a positive control, MAP30(10 to 40. mu.g/mL). (F) Immunoblot analysis showed that after 24 to 48 hours of treatment with MAP30(40 μ g/mL), expression of GLUT1 and GLUT3 was reduced in ovarian cancer cells, while GLUT4 remained unchanged. (G) Nile red fluorescence of lipid-loaded ES2 in OCM cultures for 48 hours. Cleanasite-treated 1% DMEM and OCM were used as negative controls. (H) Ovarian cancer cells were starved overnight in DMEM without FBS prior to treatment with OCM and MAP30, and subsequent cell lipolysis was measured by lipolysis assay. This finding shows that lipolysis of ovarian cancer cells is increased 24 to 48 hours after treatment with MAP30(10 and 40 μ g/mL) with 1% FBS DMEM as a positive control. (I) MAP30(10 and 40. mu.g/mL) treatment for 48 hours reduced ATP production in ovarian cancer cells.

Figure 18 shows a graphical summary.

Figure 19 shows that MAP30 is the major component of BME and functions as a natural AMPK activator. (A) Immunoblot analysis confirmed the specificity of laboratory-bred mice against the primary MAP30 antibody. A distant single band at 30kDa was observed in the sample containing pure MAP30(0.5 mg). (B) Immunoblot analysis showed the content of MAP30 in thailand (Thai) BME, chinese BME and chinese Taiwan (TW) BME.

Figure 20 shows that MAP30 induces S-phase cell cycle arrest in ovarian cancer cells. (left panel) flow cytometry analysis showed cell cycle distribution of A2780cp, OVCA433 and ES2 cells treated with MAP30(15 and 40mg/mL) or AICAR (1mM) for 24 hours. The left peak indicates non-replicating cells (2n) at G0/G1. The right peak at about double PI intensity represents the DNA content (4n) cells that are fully replicated at G2/M. Cells between the two peaks undergo S-phase DNA replication. (right panel) representative quantitative plots depict the proportion of cells at each stage of the cell cycle in each treatment. All ovarian cancer cell lines a2780cp, OVCA433 and ES2 showed an increase in S phase cell cycle population, especially in MAP30(40 μ G/mL) treatment, while in AICAR (1mM) treatment these cell lines arrested at G0/G1 phase cell cycle. Data are presented as mean ± SEM. P <0.05, P <0.01(N ═ 3) compared to control group.

Figure 21 shows that MAP30 induces apoptosis. Immunofluorescence analysis of ovarian cancer cells ES2 showed that 48h incubation of activated caspase 3 or 7 (green) induced by MAP30(40mg/mL) was assayed using CellEventTM caspase 3/7 green detection reagent. Scale bar 100 mm.

Figure 22 shows that MAP 30-induced AMPK activation synergized with cisplatin in both platinum-sensitive and drug-resistant ovarian cancer cells. Cell viability of HEY cells treated with increasing concentrations of MAP30 and/or cisplatin for 3 days and IC50 determined (upper left panel). Morphological examination of HEY cells treated with increasing concentrations of MAP30 and/or cisplatin for 3 days (upper right panel). The matrix plot shows the percentage of viable cells after 3 days of treatment with increasing concentrations of MAP30 and/or cisplatin (lower left panel). Synergy matrix (middle lower panel) and surface mapping (lower right panel) show the synergy of cisplatin and MAP30 in HEY cells (N ═ 3).

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.

Unless defined otherwise, terms related to technology and science in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Cell lines and human clinical samples. Human ovarian cancer cell lines A2780cp (provided by professor Benjamin Tsang of University of Ottawa), OVCA433 (provided by professor George SW Tsao of School of Biological Sciences, HKU), HEY, HEYA8 (provided by professor AWS Wong of School of Biological Sciences, HKU), ES2 cells and HEK293 cells of the human embryonic kidney line were purchased from the American Type Culture Collection (American Type Culture Collection, ATCC, Rockville, Md.). Immortalized Human Ovarian Surface Epithelial (HOSE) cell line HOSE 96-9-18 (provided by professor G.S.W. Tsao of School of biological Sciences, HKU). Human retinal tissue collected from surgical procedures performed at Mary Hospital is approved by the institute of University of Hong Kong Board of the University of Hong Kong/Hospital island network of medicine (HKU/HA HKW IRB) (IRB reference: UW 11-298).

Example 1 purification of native MAP30 and preparation of recombinant MAP30

Chinese dried balsam pear seeds were purchased from local Chinese herbal medicine shops. To purify the dayHowever, MAP30, 200g of dried seeds of Momordica charantia were soaked in double distilled water (ddH)₂O) overnight. Seeds were placed in 1.5L ddH using Waring blender₂Homogenization in O. The slurry was centrifuged at 30000g twice at 4 ℃ for 20 minutes each. The supernatant was filtered using a double-layer filter paper, and the volume of the filtrate was adjusted to 2L with 20mM Tris-HCl (pH7.6) buffer.

The supernatant was loaded onto an 18cm x 5cm Affi-gel blue gel column (1537301, Bio-Rad) (bed volume 350mL) equilibrated with the same buffer. After loading the sample, the column was washed with 3L of buffer to remove unadsorbed material. The adsorbed material was eluted with 1L of 20mM Tris-HCl (pH7.6) buffer containing 1M NaCl. The adsorbed fraction comprising native MAP30 was purified by ddH in a dialysis tube with a molecular weight cut-off of 8kDa (132665, Spectra/Por) at 4 deg.C₂O dialyzed thoroughly overnight.

The dialyzed fraction was adjusted to 20mM ammonium acetate (NH)₄OAc) (ph4.5) and loaded onto an 18cm x 5cm SP-agarose column (17-0729-01, GE Healthcare) equilibrated with the same buffer (bed volume: 350 mL). After removal of unadsorbed material with the same buffer, the column was loaded with 1.5L of 20mM NH containing 0.2M NaCl₄OAc (pH4.5) buffer to remove loosely bound material. Thereafter, 1L of 20mM NH containing 1M NaCl was used₄The OAc (pH4.5) buffer eluted the column, yielding a fraction containing native MAP 30. The fractions were dialyzed (132665, Spectra/Por) in a dialysis tube at 4 ℃ with ddH₂O was dialyzed thoroughly overnight, frozen in liquid nitrogen, and lyophilized to powder form with a lyophilizer (Thermo Fisher Scientific).

The dried powder was resuspended to 15mg/mL with 50mM Tris-HCl (pH7.6) buffer containing 0.1M NaCl. FPLC-gel filtration was performed using an AKTA purifier (GE Healthcare) on a Superdex 75HR 10/300 column (17-5174-01, GE Healthcare) pre-equilibrated with the same buffer. By size exclusion, the elution profile exhibited two major absorption peaks at OD 280nm, the first peak being purified native MAP 30. The first peak fraction was collected, dialyzed against centricon (VS15T92, Sartorious) with a molecular weight cut-off of 3kDa and lyophilized to powder form ready for further experiments. SDS-PAGE results with samples from different elution volumes of Superdex 75 showed that the fraction of peak 1 gave a single 30kDa protein band belonging to MAP 30. 40mg of MAP30 could be isolated from 100g of Momordica charantia seeds with this purification method.

Recombinant MAP30 was provided by professor JB Zhan, university of zhejiang. Briefly, the MAP30 cDNA was cloned into the pET-28a expression plasmid (Novagen) and introduced into E.coli BL21(DE3) for expression. Following IPTG (1mM) induction, the bacteria were harvested by centrifugation, followed by sonication, washing and affinity purification using His-tag, recombinant MAP30 could be isolated from the soluble fraction according to the manufacturer's (Novagen) instructions.

Example 2 pharmacological Activity test of MAP30 against ovarian cancer

CRISPR/Cas 9-mediated gene knockout

AMPK α 1 and α 2 gene knockouts were performed by transfection of pL-crispr. efs. gfp plasmid (gifted by professor DG Hardie of Dundee University, UK) carrying sgRNA oligonucleotides for AMPK α 1 and α 2. After transfection, puromycin was used for 24 hours, followed by selection with replacement of fresh medium. Western blot analysis was used to detect positive knockout clones for AMPK α 1 and α 2.

Western blot analysis

Protein lysates were isolated from cells using Cell lysis buffer (Cell Signaling Technology) containing protease inhibitor cocktail (Roche) and phenylmethylsulfonyl fluoride (Sigma Chemical Co.). After separation by 10% SDS-PAGE, the resulting mixture was transferred to a polyvinylidene fluoride (PVDF) membrane. Using enhanced chemiluminescent reagent solution (Amersham)^TMECL) were run and visualized by X-ray film.

3. Cell cycle and apoptosis assays

Ovarian cancer cells were stained with 250 μ L of 50 μ g/mL propidium iodide (PI (P1304MP, Invitrogen) in PBS in the dark for 15 min. flow cytometry was performed using BD FACSCAntoTM II System (BD Biosciences.) the proportion of cells per phase of the cell cycle was analyzed using Modfit LT Software (Verity Software House.) to detect apoptosis, treated cells were stained according to the manufacturer's protocol (BD Biosciences) as described in annexin V FITC apoptosis detection kit TDSAfter analysis, the mixture passes through BD FACSVersese^TMSystem (BD biosciences) the percentage of apoptotic cells was analyzed.

Fluo-4 NW calcium and calcineurin cell Activity assay

To assess the amount of intracellular calcium, a Fluo-4 NW calcium assay kit (F36206, Thermo) was used according to the manufacturer's protocol. To determine the phosphatase activity of cellular calcineurin (PP2B), a complete colorimetric assay was performed using the calcineurin cellular activity assay kit (BML-AK816, Enzo).

5. Determination of ATP levels

The amount of ATP in ovarian cancer cells was determined using a luminescent ATP detection assay kit (ab113849, Abcam).

6. Glucose uptake and lactate production

The glucose uptake levels of ovarian cancer cells were determined using a glucose uptake assay kit (colorimetric method) (ab136955, Abcam). To determine lactate production and secretion in ovarian cancer cells, an L-lactate test kit (colorimetric method) (ab65331, Abcam) was used according to the manufacturer's protocol.

7. Determination of hexokinase and Lactate Dehydrogenase (LDH) Activity

The level of hexokinase activity was determined using the hexokinase activity test kit (colorimetry) (ab136957, Abcam) and the level of LDH activity was determined using the LDH test kit (colorimetry) (ab102526, Abcam) according to the manufacturer's instructions.

8. Cell proliferation and lesion formation assay

Cell proliferation was assessed by XTT cell proliferation kit (Roche, Basel, Switzerland). For lesion formation assays, approximately 1000 cells were cultured in each well of a six-well plate and incubated with different treatments. Colonies were stained with crystal violet and counted.

9. Basal Membrane matrix cell migration and invasion assay

The migration and invasion capacity of the cells was examined by transwell cell migration and invasion kit (Corning, NY, USA) according to the manufacturer's instructions. Migrated/invaded cells were stained and counted by microscope.

10. In vivo tumorigenicity test

To investigate the effect of MAP30 on tumor growth in vivo, 2X 10 was used⁶Each ovarian cancer cell was transplanted intraperitoneally into five female BALB/cAnN-nu nude mice per group, which were four weeks old. When a significant tumor developed after about one week, the experimental group received intraperitoneal injections of MAP30(62.6, 125, 250, and 500. mu.g/kg) once every 2 days. For the control group, PBS was injected intraperitoneally only. Whole animal studies were conducted according to guidelines approved by The Committee of Use of Live Animals (The Committee on The Use of Live Animals in Teaching and Research of The University of Hong Kong) (CULATR No.: 2560-11).

11. Data analysis

All experiments were independently repeated at least three times unless otherwise indicated. Values are expressed as mean ± standard error of mean, and compared using the two-tailed Student's t test. Fisher's exact test (for parametric data) and Mann-Whitney test (for nonparametric data) were used, and P.ltoreq.0.05 was considered statistically significant.

12. Results

(1) MAP30 acts as a natural AMPK activator

Studies of the present invention have shown that natural AMPK activators such as BME and pharmacological AMPK activators a23187, AICAR and metformin inhibit the growth of human ovarian and cervical cancer cells caused by AKT/ERK/FOXM1 and AKT/FOXO3a/FOXM1 signaling, respectively. It has been widely reported that recombinant MAP30 can mediate anticancer effects against human cancers. To test whether there was any difference between recombinant MAP30 and native MAP30 in AMPK activation, Western blot analysis was first performed in the present invention, and the results showed that both types of MAP30(40 μ g/mL) could mediate increased phosphorylation at Thr172 of AMPK and at Ser79 of ACC while decreasing the expression level of FOXM1 and phosphorylation of AKT (Ser473) and ERK in a time-dependent manner (fig. 1A and 1B), indicating that native MAP30 could mediate anticancer effects by inhibiting AKT/ERK/FOXM1 and AKT/FOXO3a/FOXM1 signaling instead of recombinant MAP 30. Furthermore, CRISPR/Cas 9-mediated knockout of AMPK α 1 and α 2 genes in ovarian cancer cell lines such as ES2 cells is robustReduced MAP 30-mediated inhibition of AKT/ERK/FOXM1 signaling activity (FIG. 1C). In addition, knock-out AMPK α 1 α 2 in ES2 cells compared to ES2 control cells treated with MAP30(10 and 40 μ g/mL)^-/-Resulting in a 5-fold decrease in cell migration and invasion capacity (fig. 1D).

Unlike common AMPK activators, the present inventors found that MAP30 is similar to BME, amplifying AMPK phosphorylation in WT and R531G cells in a dose and time dependent manner (fig. 1E), suggesting that MAP30 mediated AMPK activation may constitute an AMP independent mechanism. In addition, phosphorylation of AMPK was significantly impaired in response to MAP30 in WT and R531G cells treated with the CaMKK β inhibitor STO-609(10 μ M) (fig. 1F). To further confirm the calcium-dependent pattern of MAP 30-induced AMPK activation in ovarian cancer cell lines, a Fluo-4 NW calcium assay was performed to monitor the cellular calcium concentration. Cellular calcium concentrations in OVCA433 cells were increased in a dose-dependent manner by treatment with MAP30(5, 10, 20, and 40 μ G/mL) compared to controls without MAP30 treatment (fig. 1G). In contrast, knock-out of AMPK α 1 and α 2 significantly reduced the sharp rise in calcium (fig. 1H), confirming a key role for CaMKK β in MAP 30-mediated AMPK activation.

These data support the notion that MAP30, like BME, activates AMPK in inhibiting cell growth, migration/invasion of ovarian cancer cells via CaMKK β signaling, while inhibiting AKT/ERK/FOXM1 transduction.

(2) MAP30 is one of the important components of fructus Momordicae Charantiae extract (BME)

The present invention evaluated the amount of MAP30 in each extract of momordica charantia. Western blot analysis demonstrated the highest level of MAP30 (. about.12 ng/. mu.L) in Thailand (Thai) BME, followed by Chinese BME (. about.9.6 ng/. mu.L) and Taiwan (TW) BME (6 ng/. mu.L) (FIGS. 2A and 2B).

To determine the role of native MAP30 in cell growth, three human ovarian cancer cell lines were treated with different dilutions of native MAP30(5, 10, and 20 μ g/mL). XTT cell proliferation assay functionally demonstrated that the effect of native MAP30 is similar to recombinant MAP30, which can reduce cell viability of ovarian cancer cells by 40% to 80% in a dose-dependent manner compared to its control (fig. 2C). Notably, there was no significant effect on the proliferation of immortalized human ovarian surface epithelial (HOSE 11-12) cells (FIG. 2C). Consistently, the lesion formation assay confirmed that the number of colonies was significantly reduced by about 50% to 90% in a2780cp, OVCA433 and ES2 cells treated with native MAP30(5 and 10 μ g/mL) (fig. 2D). These results indicate that native MAP30 is one of the major bioactive components of BME that exerts a growth-delaying effect on ovarian cancer cells.

(3) MAP30 induces apoptosis and inhibits cancer cell migration and invasion

Similar to AICAR, ovarian cancer cells were treated with MAP30(15 and 45 μ g/mL, 24h), using annexin V-propidium iodide/flow cytometry analysis, which showed a 65% and 150% increase in apoptosis in a dose-dependent manner (fig. 2E). Western blot analysis demonstrated that treatment with MAP30 (45. mu.g/mL) increased cleavage of PARP and caspase 3 in OVCA433 and ES2 cells (FIG. 2F), suggesting that MAP30 induces apoptosis in human ovarian cancer cells through an intrinsic apoptotic mechanism.

Furthermore, the Transwell cell migration assay showed that the cell migration ability of ES2 cells was greatly hindered by MAP30 in a time and concentration dependent manner. After treatment with MAP30(10 and 40. mu.g/mL), there was an approximately 40% to 60% reduction in cell migration (FIG. 2G). Furthermore, the Transwell invasion assay showed that in ES2 cells treated with MAP30(10 and 40 μ G/mL), the number of cells invading through the basement membrane matrix was also reduced by 40% to 60% (fig. 2G).

(4) MAP30 inhibits tumor growth in vivo without causing side effects

In this study, a highly metastatic human ovarian cancer cell line ES2 with a GFP marker was inoculated intraperitoneally into female nude mice. When a significant tumor developed about one week, MAP30(250 μ g/kg) was administered intraperitoneally every 2 days into tumor-bearing mice for a total of 5 injections, and then all mice were sacrificed. The results revealed that tumor-bearing mice treated with MAP30(250 μ g/kg) exhibited about 1.5-fold significant tumor growth and tumor nodule number inhibition compared to the control group (fig. 3A, 3B and 3C). These findings show that MAP30 has a strong in vivo efficacy against human ovarian cancer growth without any effect on mouse body weight (fig. 3D). Interestingly, MAP30 (250. mu.g/kg) treated and control groups both had similar amounts of ascites volume (FIG. 3E), suggesting that there may be a change in the biological composition of ascites between the two groups. Indeed, GC-MS analysis of the target metabolites in ascites was therefore carried out and it was found that the MAP30 treated group showed about a 50% reduction in total fatty acids compared to the control group. Most of them are unsaturated fatty acids, such as C16: 1. c18: 1. c18: 2. c22: 1. c22: 2. c24:1, etc., are important for the biosynthesis and bioenergy requirements of tumor cells (FIGS. 3F and 3G).

Although the studies of the present invention indicate that the MAP30 protein is non-toxic, there is interest in examining the toxicity of the protein in mouse models. Mice treated with an excessively high dose of MAP30(1000 μ g/kg) were found to cause significant weight loss and died even after 1 month (fig. 3H and 3I). However, for mice treated with low dose MAP30(62.5 and 125 μ g/kg) or treated for more than 1 month, no significant changes in body weight were observed, even no death (fig. 3H and 3I). However, biochemical analysis revealed that no changes in the levels of serum creatinine, alanine, transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase were observed in mice treated with multiple doses (62.5, 125 and 500 μ g/kg) of MAP30 compared to the vehicle control group (fig. 3J), indicating no adverse effects on kidney and liver function, except for a small deviation of AST in the MAP30(500 μ g/kg) treated group, which may have some small liver damage (fig. 3J). However, histopathological studies showed that liver, kidney and spleen structures of mice treated with multiple doses of MAP30(62.5 and 500 μ g/kg) appeared normal compared to the control group (fig. 3K), representing that there was no serious toxic effect of in vivo administration of MAP30, but some unforeseen side effects may occur at higher doses of MAP 30. Therefore, it is necessary to observe the health of mice in the use of MAP30 for a long time.

(5) MAP30 sensitizes ovarian cancer cells to cisplatin-induced cytotoxicity

Given that MAP30 is the major bioactive component of BME, it should sensitize ovarian cancer cells to cisplatin/paclitaxel-induced cytotoxicity. Cell proliferation of ovarian cancer cells was only inhibited by about 50% and 20% by treatment with MAP30 or cisplatin alone (FIG. 4A). However, co-treatment with MAP30 and cisplatin (0.5. mu.g/mL) resulted in at least 75% increase in cytostatic in ovarian cancer cells (FIG. 4A). Furthermore, flow cytometry analysis showed that HEYA8 cells not only caused a significant, dramatic increase in the population of apoptotic cell death in a dose-dependent manner, but also increased the sensitivity of HEYA8 cells to cisplatin-mediated cytotoxicity by 160% and 445% in a dose-dependent manner (fig. 4B).

To further investigate whether MAP30 enhanced cisplatin-induced cytotoxicity in vivo, ES2 tumor-bearing nude mice were injected intraperitoneally with MAP30(125 μ g/kg) and cisplatin (2mg/kg) once every three days for a total of five injections, and then all mice were sacrificed. Consistent with the in vitro data, co-treatment of MAP30 and cisplatin resulted in a significant tumor growth delay in the abdominal cavity of mice, as was a 4-fold reduction in tumor weight and a 2-fold reduction in tumor nodules compared to cisplatin alone (fig. 4C, 4D, and 4G). In addition, mean ascites volume was significantly reduced by cisplatin alone or by co-treatment with MAP30 and cisplatin (fig. 4E), while the body weight of the mice was not severely affected at the end of the experiment (fig. 4F). These findings indicate that MAP30 is not only harmless in vivo, but also helps to inhibit tumor growth by enhancing cisplatin-induced cytotoxicity.

(6) MAP30 blocks the effects of the tumor microenvironment on ovarian cancer cells

Previous studies have shown that the carcinogenic properties of ovarian cancer are enhanced when co-cultured in ascites or Omentum Conditioned Medium (OCM). As expected, the cell growth rate of human ovarian cancer cell lines such as OV2008 and SKOV3 was significantly enhanced by OCM treatment (fig. 5A), suggesting that OCM can stimulate cell growth of ovarian cancer cells and also support oncogenic effects of OCM in the metastatic cascade. However, the XTT cell proliferation assay showed significant reduction in cell proliferation rates of human ovarian cancer cell lines OV2008 (P.ltoreq.0.001) and SKOV3 (P.ltoreq.0.05) cells compared to their OCM control by treatment with MAP30 (10. mu.g/mL) and 1% (v/v) BME (positive control) (FIG. 5A). Moreover, the lesion formation assay showed a significant reduction in the number of colonies in MAP30(10 μ g/mL) treated a2780cp by about 60% compared to OCM control (fig. 5B). Wound healing trials revealed that wound closure rates were significantly accelerated for ovarian cancer cell lines such as SKOV3 and ES2 upon treatment with OCM, resulting in about 30% and 60% increase in cell migration compared to controls, respectively (fig. 5C). Furthermore, Transwell cell migration assays were also performed to better quantify the migration effects of OCM on human ovarian carcinoma cells. Consistent with previous wound healing assays, the data showed an approximately 2.5-fold increase in cell migration rate of SKOV3 cells treated with OCM (OCM control) compared to control medium (DMEM control) (fig. 5D). The Transwell cell invasion assay showed a significant increase in the number of cells invaded by the basement membrane matrix by OCM-cultured ovarian cancer cell line SKOV3 compared to untreated control medium (DMEM control) (fig. 5E). These findings support the notion that OCM has the ability to promote cell migration and cell invasion in human ovarian cancer cells, the stimulatory effects of which are counteracted by the natural AMPK activator MAP 30.

(7) MAP30 inhibits glycolysis and de novo lipogenesis in ovarian cancer cells

In this study, the L-lactate assay consistently demonstrated that lactate production was higher in various ovarian cancer cell lines than in immortalized human ovarian surface epithelial (HOSE 96) cells during two days of culture (fig. 6A). Such enhanced lactate production in ovarian cancer cells is a significant feature that is attached to the treatment of the native AMPK activator MAP30(40 μ g/mL) and the positive controls AICAR (1mM) and a23187(2 μ M) (fig. 6B), suggesting that a reduction in glycolysis occurs in ovarian cancer cells via MAP30 mediated AMPK activation. Such a decrease in glycolysis is limited by the activity of glycolytic enzymes such as hexokinase and LDH, since the hexokinase and LDH activity of OVCA433 cells was significantly inhibited by more than 50% in a time and dose dependent manner upon MAP30 treatment for 24 and 48 hours (fig. 6C and 6D). Importantly, the net reduction in AMPK-activated glycolysis mediated by MAP30 contributes to the reduction in carbon flux. For this purpose, glucose uptake assays were performed, with MAP30(10 and 40 μ g/mL) behaving similarly to the drug AMPK activator AICAR (1mM), inhibiting glucose uptake of approximately 30% to 60% in OVCA433 cells in a dose-dependent manner. Western blot analysis further confirmed that expression of GLUT1 and GLUT3 was significantly decreased in a time-dependent manner in OVCA433 and ES2 cells treated with MAP30(40 μ g/mL) (fig. 6F). This data indicates that MAP30 is able to inhibit glucose uptake by reducing the GLUT-1 and-3 transporters. To further investigate the role of MAP30 in the glucose metabolism kinetics of tumor growth in vivo, 18F-FDG Micro-PET/CT was performed.

Since the omentum is considered to be the preferred site for ovarian cancer metastasis, it is postulated that lipids specifically regulate ovarian cancer cells in the omentum microenvironment to promote malignant cell survival and metastatic cancer cell spread. In the present invention, OCM-cultured ovarian cancer cell line OVCA433 showed increased lipid droplet formation using Nile Red fluorescent staining, while MAP30 co-treatment (5 and 10. mu.g/mL) significantly reduced lipid droplet numbers by 50-70% (FIG. 6G). To further determine whether MAP30 accelerates catabolic lipolysis in human ovarian cancer cells, lipolysis assays were performed showing that MAP30(10 and 40 μ g/mL) induced an approximately 2-4 fold increase in cellular lipolysis in ovarian cancer cells (fig. 6H). Moreover, ATP detection assays revealed a significant reduction in ATP production in ovarian cancer cells due to MAP30(10 and 40 μ g/mL) treatment (fig. 6I). This is understood as the activation of AMPK promotes catabolism and inhibits anabolism to conserve cellular energy. The findings of the present invention together indicate that MAP30 mediated AMPK activation triggers a series of catabolic processes in the metabolic profile of ovarian cancer cells, including decreased lipid synthesis from surrounding sources and enhanced fatty acid oxidation even in the fatty acid-enriched ascites microenvironment.

(8) The natural MAP30 protein is superior to the recombinant MAP30 protein

Although we found that recombinant and native MAP30 were similar in size (-30 kDa) (see fig. 7) and both had a role in AMPK activation and anti-cancer, the post-translational modification of native MAP30 had other functional roles not possessed by recombinant MAP30 protein. Preliminary data have shown that native MAP30 shows about 10-30% greater inhibition of cell proliferation of ovarian cancer cells compared to recombinant MAP30 (see figure 8). Furthermore, the toxicity of the recombinant protein may reduce the safety of applying MAP30 in cancer therapy.

The natural MAP30 protein consists of 286 amino acids, has a molecular weight of 30kDa, and has an amino acid sequence shown in SEQ ID NO. 1. Wherein the N-terminal MVKCLLLSFLIIAIFIGVPTAKG is a signal peptide which is removed upon secretion of the mature MAP30 protein from plant cells (Lee-Huang S.et al. FEBS Lett.272:12-18 (1990)). The recombinant MAP30 protein consists of 284 amino acids, the amino acid sequence of the recombinant MAP30 protein is shown in SEQ ID NO.2 (ACCESSION AAB35194), the calculated molecular weight is 31.9kDa, and the estimated pI is 9.0. In the recombinant MAP30 protein, the above-mentioned signal peptide sequence was replaced by a histidine-tag (His-tag), which did not affect the functional effects of MAP 30. However, native MAP30 showed a stronger AMPK activation effect (see fig. 9), and a stronger cell growth delaying effect. Western Blot analysis showed the difference (. about.2-5 kDa) between native MAP30 protein and recombinant MAP30 protein at the N-terminus (see FIGS. 7 and 10). In addition to the differences in protein length, a glycosylation site was present at amino acid 74 of MAP30 protein. In native MAP30 protein, this site was glycosylated, but in the recombinant MAP30 protein produced by e.coli, it was not glycosylated, as e.coli does not have a protein glycosylation system. Translational modification of the MAP30 protein resulted in a functional difference between the native MAP30 protein and the recombinant MAP30 protein. In conclusion, the differences in protein structure and glycosylation between the native MAP30 protein and the recombinant MAP30 protein resulted in differences in cytotoxicity and AMPK activation in ovarian cancer cells. The differences in protein structure are the main reason why the native MAP30 protein has better AMPK activation and other cytotoxic effects on ovarian cancer cells.

In addition, the rapid production of recombinant MAP30 protein also caused some toxicity due to the accumulation of protein aggregates.

(9) Discussion of the related Art

Acquired chemotherapy resistance is currently a major obstacle in the clinical management of ovarian cancer. This has led to an urgent need to find alternative treatment regimens or new pharmaceutical agents. Chinese medicine has been widely used in ancient China and India for the treatment of human diseases including cancer. It not only targets cancer cells, but also treats the entire body, such as the gas, blood and immune system. Today, traditional Chinese medicine is used for the purpose of supplementing alternative medicines. Bioactive compounds from chinese medicine have interesting functions in anticancer by modulating cancer cells at multiple levels and multiple targets. This makes the traditional Chinese medicine and its bioactive compounds potentially useful applications as ideal adjuvants in modern cancer therapy. Bitter gourd extract (BME) has previously been reported to exert inhibitory effects on ovarian Cancer cells by activating AMPK/mTOR signaling (Yung MM et al. integer Cancer ther. 2016Sep; 15(3): 376-89.). When co-administered with cisplatin, BME may exhibit a synergistic effect that enhances cisplatin-mediated cytotoxicity in ovarian cancer cells. In this study, MAP30 protein was isolated from dry seeds of momordica charantia. Functional and biochemical analysis showed MAP30 to be one of the major bioactive components in BME. The present invention was able to demonstrate that different functional anticancer effects are positively correlated with the amount of MAP30 in each BME.

Indeed, BME comprises a variety of bioactive compounds and has been shown to have anti-cancer effects against a variety of human cancers. For example, Kuguacin J (KJ) isolated from an extract of momordica charantia has been reported to sensitize ovarian cancer cells to paclitaxel resistance. In addition, the ribonuclease RNase MC2 can induce apoptotic mediators in apoptosis that cause human breast cancer. Even for biologically inactive compounds, a novel peptide BG-4 from Momordica charantia has been shown to promote apoptosis in human colon cancer. However, these components did not show any relationship to or function of AMPK. It is generally known that momordica charantia has been used in ancient china and india for the treatment of diabetes, similar to metformin isolated from goat beans for use as a first line treatment for diabetes. This suggests that the main function of momordica charantia is AMPK activator. Coincidently, this MAP30 protein has been shown to act as a natural AMPK activator, inducing AMPK activation in an AMP-dependent manner through the CaMKK β pathway and inhibiting mTOR signaling, inhibiting the newly discovered signaling AKT/ERK/FOXM1, showing inhibitory effects on ovarian cancer cells.

AMPK is not only a key energy sensor in normal cells, but is also a crucial factor in tumor development. AMPK is known to be a key sensor for maintaining cellular energy balance regulating multiple cellular metabolic pathways. Emerging evidence has shown that malignant cells require high AMPK activity to initially overcome metabolic stress from the tumor microenvironment, while advanced cancers prefer low AMPK activity to enhance cell growth and progression. This suggests that late stage and more aggressive cancer cells prefer low AMPK activity to reduce metabolic limitations, such as mTOR, for exerting high carcinogenicity. On the other hand, this confers an advantage to AMPK activators in selectively disrupting ovarian cancer cell function. The ability of native MAP30 isolated from non-toxic momordica charantia to increase AMPK activity through the CaMKK β pathway and in an AMP-independent manner suggests that it targets cancer cells from another perspective whose survival and other cellular activities are not affected by AMP stress conditions, such as a nutrient-rich microenvironment. Indeed, the present study shows that the native MAP30 can significantly inhibit tumor growth, particularly tumor colonization in tumor xenograft mouse models, as well as cell growth, cell migration/invasion. mTOR signaling is generally known to be an important pathway in cellular metabolism, growth, proliferation, survival and resistance to drugs. However, mTOR inhibitors are not sufficient to achieve a broad and potent anticancer and anti-chemotherapeutic drug resistance effect. MAP30, however, appears to offset this deficiency. The findings of the present invention show that MAP30 may not only inhibit oncogenic AKT/ERK/FOXM1 signaling, but also regulate cellular metabolism, such as reducing glucose uptake by inhibiting GLUT-1 and GLUT-3 expression, as well as lipid droplet formation, possibly even affecting the resistant lipolysis of ovarian cancer cells in a nutrient-free microenvironment. To further demonstrate these in vivo effects, treatment of tumor-bearing mice with MAP30 will be performed by the 18F-FDG Micro-PET/CT assay used to study the glucose uptake capacity of tumor cells in the mice, as is being done. The discovery of the present invention can provide a scientific basis for its potential use in combating the chemotherapeutic resistance of ovarian cancer cells to existing chemotherapeutic drugs.

And (4) conclusion: the findings of the present invention indicate that MAP30 protein is one of the major bioactive components of momordica charantia, exerts a potent inhibitory effect on the carcinogenic properties of ovarian cancer cells by activating CaMKK β/AMPK signaling, and inhibits AKT/ERK/FOXM1 signaling activity as well as regulates glycolysis and adipogenesis. The bioactive protein has the potential to develop into a chemotherapeutic supplement for ovarian cancer.

Example 3 MAP30 protein from Momordica charantia has therapeutic effects on ovarian cancer in vivo by altering metabolism and inducing apoptosis of iron and has synergistic activity with cisplatin

A comprehensive study was conducted to characterize the effect of native MAP30 isolated from momordica charantia seeds on ovarian cancer cells. The use of Omentum Conditioned Medium (OCM) and mouse tumor xenograft model demonstrated that non-toxic MAP30 in combination with cisplatin exhibited potent anticancer and chemoresistance effects on ovarian cancer cells. These findings indicate that MAP30 can be used as a supplement to augment the therapeutic outcome of existing platinum-based chemotherapy regimens.

The material and the method are as follows:

1. cell lines and human clinical samples

The human ovarian cancer cell line a2780cp was provided by professor Benjamin Tsang at ottawa university, canada. HEY and HEYA8 human ovarian carcinoma cells were provided by professor AWS Wong of the institute of bioscience, hong kong university. ES2 human epithelial ovarian cancer cells and the human embryonic kidney cell line HEK293 were purchased from American type culture Collection (ATCC, Manassas, Va., USA). Additional human ovarian cancer cell lines OVCA433 and SKOV3, as well as two immortalized Human Ovarian Surface Epithelial (HOSE) cell lines HOSE11-12 and HOSE 96-9-18 were provided by professor G.S.W.Tsao of the biomedical college of hong Kong university. Professor DG Hardie, university of england dundi, provided a pair of WT and R531G AMPK γ 2 isogenic HEK293 cells. All cell lines were mycoplasma free. Human omentum tissue was collected from procedures performed in Mary Hospital (IRS reference: UW 11-298) with prior approval by the agency review Committee of the hong Kong university/Hospital administration, hong Kong Western networking (HKU/HA HKW IRS).

2. Purification of native MAP30 from dried seeds of bitter gourd

Natural MAP30 was isolated from dried seeds of Momordica charantia (Momordica charantia L.) Kunth.

CRISPR/Cas 9-mediated gene knockout

Knock-out of AMPK α 1 and α 2 genes was performed by transfection of the pL-crispr. efs. gfp plasmid (provided by professor DG Hardie, university of dungdi, england) carrying sgRNA oligonucleotides for AMPK α 1 and α 2. Following transfection, puromycin was used for selection. Immunoblot analysis was used to detect positive knockout clones for AMPK α 1 and α 2.

4. Immunoblot analysis

Using a mixture containing protease inhibitors (Roche, Basel, Switzerland) and benzylCell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) of sulfonyl fluoride (Sigma-Aldrich corp., st. louis, MO, USA) protein lysates were isolated from cells. Proteins were separated using 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. Immunoassays using enhanced chemiluminescent reagent solutions and visualisation on X-ray film, or by

CLx Imaging was performed using Image studio software (Li-Cor Biosciences) for analysis (LI-COR Biosciences, Lincoln, Nebraska, USA).

5. Cell cycle and apoptosis assays

Ovarian cancer cells were stained with 250 μ L of 50 μ g/mL PBS solution of Propidium Iodide (PI) (P1304MP, Invitrogen) in the dark for 15 minutes. Flow cytometry was performed using the BD FACSCAntoTM II system (BD Biosciences, San Jose, Calif., USA). The proportion of cells at each stage of the cell cycle was analyzed using Modfit LT Software (Verity Software House, Topsham, ME, USA). To detect apoptosis, the treated cells were stained using Annexin V FITC apoptosis detection kit TDS according to the manufacturer's protocol (BD Biosciences). After flow cytometry, BD FACSVerse was used^TMThe system (BD Biosciences) analyzed the percentage of apoptotic cells. To examine the level of activated caspase-3 and-7 in ovarian cancer cells after MAP30 treatment, treated cells in 48-well microplates (CLS3548, Corning, NY, USA) were analyzed using CellEventTM caspase 3/7 green detection reagent (C10423, Invitrogen) according to the manufacturer's protocol.

6. Lipid peroxidation assay and determination of GSH/GSSG Activity

To evaluate lipid peroxidation or iron apoptosis (ferroposis) of ovarian cancer cells co-treated for 24 hours with MAP30 (40. mu.g/mL), and either Erastin (5. mu.M) alone (B1524, APExBIO Technology LLC., Boston, MA, USA), or the iron apoptosis inhibitor Ferrostatin-1(Fer-1) (10. mu.M) (SML0583, Sigma-Aldrich, St. Louis, MI, USA) in combination with MAP30 (40. mu.g/mL), C11 BODIPY 581/591C11 dye (lipid DIP 581/591C11 dye (lipid apoptosis) was usedMass peroxidation sensor) (2.5. mu.M) (D3861, ThermoFisher Scientific, Waltham, MA, USA). Using BD FACSLric^TMThe flow cytometry system (BD Biosciences, San Jose, CA, USA) performed flow cytometry analysis, analyzing at least 10000 cells per cell sample. Oxidation of C11 BODIPY 581/591 was indicated by an increase in green fluorescence intensity in the FITC channel (which shows oxidized probe). Data were analyzed using FlowJo version 7.6 software. For confocal imaging experiments, each experimental group was examined for treated ovarian cancer cells and imaged using a confocal imaging system (Carl Zeiss LSM 800). Three independent biological replicates were performed for each condition according to the manufacturer's protocol.

Changes in the GSH/GSSG ratio of ovarian cancer cells following co-treatment with MAP30 and/or Erastin were detected using GSH/GSSG ratio detection assay kit II (fluorescent-green) (ab205811, Abcam, Cambridge, MA, USA) according to the manufacturer's instructions.

Fluo-4 NW calcium assay

To assess the amount of intracellular calcium, a Fluo-4 NW calcium assay kit (F36206, Thermo) was used according to the manufacturer's protocol.

Determination of ATP levels and Reactive Oxygen Species (ROS) production

The amount of ATP in ovarian cancer cells was evaluated using a luminescent ATP detection assay kit (ab113849, Abcam). Cellular ROS production was assessed using the DCFDA-Cellular ROS assay (ab113851, Abcam) according to the manufacturer's protocol.

9. Glucose uptake and lactate production

The level of glucose uptake in ovarian cancer cells was measured using a glucose uptake assay kit (colorimetric) (ab136955, Abcam). To measure lactate production in and lactate secretion from ovarian cancer cells, an L-lactate test kit (colorimetric method) (ab65331, Abcam) was used according to the manufacturer's protocol.

10. Determination of hexokinase and Lactate Dehydrogenase (LDH) Activity

Hexokinase activity was measured using the hexokinase activity test kit (colorimetry) (ab136957, Abcam) and LDH activity was measured using the LDH test kit (colorimetry) (ab102526, Abcam) according to the manufacturer's instructions.

11. Relative quantification of target metabolites of fatty acids

Target metabolites in ascites were analyzed using gas chromatography-mass spectrometry (GC-MS) at the LKS medical institute proteomics and metabonomics Core laboratory (CPOS-PM Core) at Hong Kong University (HKU). GC/MS chromatograms were acquired in SCAN and SIM mode using an Agilent 7890B GC-Agilent 7010 triple quadrupole mass spectrometer system. Data analysis was performed using Agilent MassHunter workstation quantitative analysis software (CPOS-PM Core, HKU).

12. Cell proliferation and lesion formation assay

Cell proliferation was assessed using the XTT cell proliferation kit (Roche, Basel, Switzerland). For lesion formation assays, approximately 1000 cells were cultured in each well of a six-well plate and incubated with different treatments. Colonies were stained with crystal violet and counted.

13.CellTiter-

3D cell viability assay

Suspending ovarian cancer cells in a suspension containing 2% Geltrex^TMMedia of matrix (Gibco BRL, Gaithersburg, Md., USA) and seeded in triplicate at a density of about 1000 cells per well in Nunclon with ultra low cell attachment surface^TMSphera^TM96U-microwell plates (Thermo Fisher Scientific). The cells were subsequently grown for one week and then treated for three days with serially diluted concentrations of each drug alone (MAP30 and cisplatin) or a combination at a fixed molar ratio. After drug treatment, CellTiter-

The 3D cell viability assay detects viable cells. IC50 values were determined for each drug using non-linear regression, and drug synergy was determined using the Combenefit algorithm.

14. Matrigel cell migration and invasion assay

The migration and invasion capacity of cells was examined using the Transwell cell migration and invasion kit (Corning, NY, USA) according to the manufacturer's instructions. Migrated/invaded cells were stained and counted using a microscope.

15. In vivo tumorigenicity test

To investigate the effect of MAP30 on tumor growth in vivo, 2X 10 was used⁶Each ES2 ovarian carcinoma cell was injected intraperitoneally (i.p.) into a group of five four-week-old BALB/cAnN-nu female nude mice. When a significant tumor was formed within about one week, the experimental group was treated every 2 days by injection of MAP30(5 μ g). Control group was intraperitoneally injected with PBS only. Fluorescence stereomicroscopy (Nikon, japan) was used to capture fluorescence and bright field images of tumor nodules scattered within the peritoneal cavity of mice. The entire animal study was conducted according to guidelines approved by the Committee for use of live animals in the university of hong Kong (CULATR No. 3817-15).

16. Data analysis

All experiments were repeated at least three times independently unless otherwise stated. Values are expressed as mean ± SEM and compared using two-tailed student's t-test. Fisher's exact test (for parametric data) and Mann-Whitney test (for nonparametric data) were used, and P.ltoreq.0.05 was considered statistically significant.

As a result:

MAP30 is the main component of Momordica charantia extract (BME)

BME is a natural AMPK activator that inhibits growth, cell migration, and invasion of ovarian cancer cells. It was observed that BME from different sources can activate AMPK to different extents, suggesting a dose-dependent effect of MAP 30. Monoclonal MAP30 antibody was used to demonstrate the highest MAP30 levels (-12 ng/. mu.l) in thailand (Thai) BME, followed by china BME (-9.6 ng/. mu.l) and chinese Taiwan (TW) BME (6 ng/. mu.l) (fig. S1A and S1B), indicating that the different levels of MAP30 from three varieties of momordica charantia are completely consistent with their ability to activate AMPK in ovarian cancer cells.

Functional studies showed that MAP30 inhibited cell viability of ovarian cancer cells (a2780cp, OVCA433 and ES2) by 40% to 80% in a dose-dependent manner compared to controls using XTT cell proliferation assays (fig. 1A). Notably, cell proliferation of immortalized human ovarian surface epithelial (HOSE 11-12) cells was not significantly inhibited (FIG. 11A). Lesion formation analysis showed that the number of colonies in ovarian cancer cells (a2780cp, OVCA433 and ES2) was significantly reduced from 50% to 90% after treatment with MAP30(5 and 10 μ g/mL) compared to controls (fig. 11B). To test whether MAP30 inhibited migration and invasion of ovarian cancer cells, ES2 ovarian cancer cells were treated with MAP30(15 and 40 μ g/mL) for 15 and 24 hours and subjected to a Transwell cell migration/invasion assay. Quantitative analysis showed that MAP30(15 and 40 μ g/mL) treatment for 15 and 24h reduced the cell migration and cell invasion capacity of ES2 cells by 40-70% in a dose and time dependent manner (fig. 11C). These tumor suppressive effects were also found in ovarian cancer cells treated with BME. Based on the functional similarity of BME in ovarian cancer cells and MAP30 content response, MAP30 is the main bioactive component of BME. MAP30 exhibited selective cytotoxicity and inhibition of ovarian cancer cells, but no cytotoxicity to normal ovarian epithelial cells.

MAP30 triggering cell cycle arrest and apoptosis in ovarian cancer cells

To describe the basic molecular mechanism by which MAP30 inhibits tumor growth, the effect of MAP30 on cell cycle progression of ovarian cancer cells was investigated using flow cytometry analysis of DNA content. Cell cycle analysis showed that MAP30 caused a clear arrest in S phase in various ovarian cancer cells with highly variable levels, unlike AICAR-induced cell cycle arrest in G1 phase (fig. S2). MAP30 is the major component of BME and it may have a similar functional role as BME in AMPK-mediated cell growth and apoptosis. Thus, it was examined whether MAP30 functions like other AMPK activators (such as AICAR) in causing apoptosis in ovarian cancer cells. AICAR (1mM, 24h) was used as a positive control. OVCA433 ovarian cancer cells treated with MAP30(10 and 40 μ g/mL, 24h) showed a dose-dependent increase in the rate of apoptosis of 2 to 3 fold compared to the negative control (fig. 11D). The CellEventTM caspase 3/7 green assay revealed that MAP30 induced mitochondria-mediated caspase activation in ES2 following co-treatment with MAP30 and cisplatin (figure 21). Immunoblot analysis further demonstrated that treatment with MAP30(40 μ g/mL) increased cleavage and caspase 3 activity of PARP in OVCA433 and ES2 cells (fig. 11E). These results indicate that MAP30 induces cell cycle S-phase arrest and apoptosis in ovarian cancer cells.

MAP30 acting as a natural AMPK activator

The study shows that BME is a natural AMPK activator, and remarkably inhibits the carcinogenic capacity of ovarian cancer and cervical cancer by inhibiting AKT/ERK/FOXM1 and AKT/FOXO3a/FOXM1 signaling pathways respectively. This study compared the functional differences between native MAP30 (isolated from dried bitter gourd seeds) and recombinant MAP30 protein (produced by e. Immunoblot analysis showed that native MAP30 protein (40 μ g/mL) and recombinant MAP30 protein (30 μ g/mL) significantly increased phosphorylation at Thr172 of AMPK and Ser79 of ACC, but decreased levels of FOXM1 and phosphorylation of AKT and ERK (Ser473) in a time-dependent manner (fig. 12A and 12B). Considering that both recombinant MAP30(30 μ g/mL) and native MAP30(40mg/mL) can activate AMPK, they were also shown to inhibit mTOR signaling activity in another AMPK downstream target, i.e. ovarian cancer cells (fig. 12C). This finding confirms that native and recombinant MAP30 exhibit anti-cancer effects on ovarian cancer cells through negative regulation of the AKT/ERK/FOXM1 and mTOR signaling cascades. Notably, the role of these proteins is consistent with the functional effects of BME on ovarian and cervical cancer cells. To further demonstrate the down-regulation of the AKT/ERK/FOXM1 signaling cascade and its associated oncogenic properties, a CRISPR/Cas9 gene knockout system was used to delete AMPK- α 1 and- α 2(AMPK α 1 α 2) in ovarian cancer cells^-/-). As a result, it was confirmed that AMPK-. alpha.1 and-. alpha.2 (AMPK. alpha.1. alpha.2)^-/-) The complete depletion significantly attenuated MAP 30-mediated inhibition of the AKT/ERK/FOXM1 signaling cascade (fig. 12D) and resulted in an approximately 2-fold decrease in the cell migration and invasion capacity of ovarian cancer cells (fig. 12E). These results indicate that inhibition of MAP30 occurs in an AMPK-dependent manner.

Unlike most drug AMPK activators, MAP30 functions similarly to BME, in that both factors are dose-sumTime-dependent increases phosphorylation of AMPK in WT and R531G AMPK γ 2 isogenic HEK293 cells (fig. 12F). These results indicate that MAP 30-mediated AMPK activation may be involved in AMP-independent mechanisms. Treatment with the CaMKK β inhibitor STO-609(10 μ M) significantly attenuated phosphorylation of AMPK in WT and R531G HEK293 cells in response to MAP30 (fig. 12G). To further confirm the calcium-dependent pattern of MAP 30-induced AMPK activation in ovarian cancer cells, a Fluo-4 NW calcium assay was used. The results show calcium ion (Ca) in OVCA433 cells compared to controls co-treated with MAP30²) Was increased dose-dependently (fig. 12H). In contrast, CRISPR/Cas 9-mediated gene knock-out of AMPK α 1 and α 2(AMPK α 1 α 2)^-/-) Reduce intracellular Ca²⁺Increase (fig. 12I), which confirms a key role of CaMKK β in MAP 30-mediated AMPK activation.

Taken together, these data support that MAP30 is the major component of BME, activating AMPK and inhibiting the AKT/ERK/FOXM1 and mTOR pathways via the CaMKK β pathway, as well as the associated cell growth, cell migration/invasion of ovarian cancer cells.

MAP30 Induction of iron apoptosis in ovarian cancer cells

MAP30 causes intracellular Ca in ovarian cancer cells²⁺Increase in (FIG. 12G), and Ca²⁺May increase cytoplasmic oxidative stress and mitochondrial dysfunction. Excessive cytosolic Reactive Oxygen Species (ROS) may lead to lipid peroxidation and iron apoptosis, which are alternatives to programmed cell death. To investigate whether MAP30 caused iron apoptosis, it was investigated whether MAP30 enhanced ROS production in ovarian cancer cells. MAP30 was compared to the iron apoptosis activator Erastin and it was observed that MAP30 increased intracellular ROS production in ovarian cancer cells in a dose-dependent manner (fig. 13A). Notably, MAP30 is at AMPK α 1 α 2^-/-Less ROS production was induced in ovarian cancer cells (fig. 13A). The reduced ratio of glutathione to oxidized glutathione (GSH/GSSG) is the central cellular antioxidant system that prevents oxidative damage. To investigate whether MAP30 reduced the GSH/GSSG ratio during ROS elevation like Erastin, G was measured in ovarian cancer cells co-treated with MAP30 and ErastinSH/GSSG ratio. MAP30 significantly reduced the GSH/GSSG ratio in a dose-dependent manner and was shown to be negatively correlated with ROS levels (fig. 13B). In AMPK alpha 1 alpha 2^-/-In ovarian cancer cells, the effect of MAP30 on GSH/GSSG ratio was reduced, but there was no difference in the effect of Erastin (fig. 13B). Studies have shown that Erastin prevents cellular cysteine uptake by inhibiting the cystine/glutamate reverse transporter (xCT) and glutathione peroxidase 4(GPX4), while GSH depletion induces lipid peroxidation and iron apoptosis. Thus, the effect of MAP30 on GPX4 and Erastin was compared. Immunoblot analysis showed that MAP30 was present in wild type and AMPK α 1 α 2^-/-GPX4 protein was reduced in a dose-dependent manner in ovarian cancer cells similar to Erastin. By using BODIPY^TM581/591C11 as a lipid peroxidation probe demonstrated that MAP30 induces lipid peroxidation in ovarian cancer cells and that a potent inhibitor of apoptosis, Ferrostatin-1(Fer-1), inhibited this effect (FIGS. 13D and 13E). Because of the MAP30 vs. AMPK α 1 α 2^-/-The effect of ROS production and GSH/GSSG ratio depletion in ovarian cancer cells was minor, so MAP 30-mediated lipid peroxidation in these cells was studied. Flow cytometry analysis revealed that MAP30 significantly induced lipid peroxidation, which was counteracted by Fer-1 in ovarian cancer cells (fig. 13F). However, MAP30 is only present in AMPK α 1 α 2^-/-Lipid peroxidation was slightly induced in ovarian cancer cells (fig. 13F). These findings confirm that MAP30 is an inducer of iron apoptosis in ovarian cancer cells. However, the mechanism by which loss of AMPK activity reduces the efficiency of MAP30 in inducing iron apoptosis is not clear. Further studies were necessary to elucidate the functional role of AMPK in MAP 30-induced iron apoptosis.

MAP30 inhibits tumor growth in vivo without adverse effects

A highly metastatic human ovarian cancer cell line ES2, labeled with Green Fluorescent Protein (GFP), was inoculated intraperitoneally into female nude mice. After a significant tumor occurred within about one week, MAP30 (250. mu.g/kg) was injected intraperitoneally into tumor-bearing mice every other day for a total of 5 injections, and then sacrificed. Tumor-bearing mice treated with MAP30 showed significant tumor growth inhibition with an approximately 1.5-fold reduction in tumor nodule numbers compared to the control group (fig. 14A, 14B and 14C). These findings indicate that MAP30 has a potent in vivo efficacy against human ovarian cancer growth without any adverse effect on mouse body weight (fig. 14D). Notably, similar ascites volumes were found in the MAP30(250 μ g/kg) treated group and the control group (fig. 14E), indicating a change in the biological composition of ascites between the two groups. Thus, GC-MS analysis of the target metabolites in ascites was performed, showing about 50% reduction in total fatty acids for the MAP30 treated group compared to the control group. Most fatty acids were unsaturated fatty acids, such as C16:1, C18:1, C18:2, C22:1, C22:2, and C24:1, which meet the basic biosynthetic and bioenergy requirements of tumor cells (fig. 14F and 14G).

Although the study showed that the MAP30 protein is non-toxic, the toxicity of the protein was examined in a mouse model. Mice treated with high dose of MAP30 (500. mu.g/kg) showed approximately 10% weight loss (FIGS. 14H and 14I). However, mice treated with lower doses of MAP30(62.5 and 125 μ g/kg) for more than 1 month showed no significant change in body weight, and no death was observed (fig. 14H and 14I). Biochemical analysis revealed no observable changes in serum creatinine, alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) or alkaline phosphatase levels in mice treated with 62.5 and 125 μ g/kg MAP30 compared to vehicle control (fig. 14J), indicating no adverse effects on kidney and liver function. A small deviation in AST was observed with high dose MAP30(500 μ g/kg) treatment, indicating some mild liver injury (fig. 14J). However, histopathology showed that liver, kidney and spleen structures of mice treated with low and high doses of MAP30(62.5 and 500 μ g/kg, respectively) were similar to the control group (fig. 14K), indicating that there was no serious toxic effect of MAP30 administration in vivo. However, at high doses MAP30 may have some unexpected side effects. Therefore, it is necessary to study the long-term toxicity of MAP30 in mice.

Synergistic enhancement of cisplatin-mediated cytotoxicity of MAP30 in vitro

Since MAP30 induced apoptosis and iron apoptosis in ovarian cancer cells, it was examined whether MAP30 sensitizes ovarian cancer cells to cisplatin/paclitaxel-induced cytotoxicity. Three advanced ovarian cancer cell lines were co-treated with different concentrations of cisplatin and/or MAP30 using a 2D culture system. Treatment with MAP30 alone or cisplatin alone inhibited cell proliferation in ovarian cancer cells by 50% and about 20%, respectively (fig. 15A). However, co-treatment with MAP30 and cisplatin (0.5. mu.g/mL) increased the cytostatic rate of ovarian cancer cells to at least 75% (FIG. 15A). Flow cytometry analysis showed a significant and dose-dependent increase in HEYA8 cell population undergoing apoptotic death, with MAP30 increasing cisplatin-mediated cytotoxicity by about 1.6-4.5 fold in a dose-dependent manner compared to controls (fig. 15B).

Increasing evidence notes various limitations of the 2D system in testing cancer cells for chemoresistance, including its poor mimic in vivo on the tumor microenvironment. Recently, in vitro three-dimensional culture systems (3D systems) have been developed that replicate the in vivo tumor microenvironment and more accurately represent drug responses. Therefore, it was further investigated whether MAP30 synergistically enhanced cisplatin cytotoxicity using a 3D culture system. CellTiter-

3D cell viability analysis for assessment at 2% Geltrex^TMSensitivity of 3D spherical ovarian cancer cells cultured in stroma (Gibco BRL) to native MAP30 and/or cisplatin treatment. MAP30 or cisplatin treatment alone produced a dose-dependent inhibitory effect on cell viability of OVCA433 (fig. 15C) and HEY spheroids (fig. 22). Next, it was determined whether co-treatment with MAP30 and cisplatin enhanced the therapeutic effect compared to each drug alone. Ovarian cancer cell lines, such as OVCA433 and HEY cells, were treated with MAP30 and cisplatin in a fixed molar ratio at serially diluted concentrations. The combination of the two drugs induced significantly more significant slowing of cell viability compared to the individual treatments in each of OVCA433 (fig. 15C) and HEY cells (fig. 22). The effect of the drug combination was determined using the Combenefit algorithm and the synergy matrix showed that the dual inhibition produced excellent synergistic anti-cancer effects on OVCA433 (fig. 15D) and HEY cells (fig. 22). These data indicate that MAP30 synergistically enhances cisplatin cytotoxicity in ovarian cancer cells.

MAP30 enhances cisplatin-induced cytotoxicity in vivo

To further investigate whether MAP30 enhances cisplatin-induced cytotoxicity in vivo, ES2 tumor-bearing nude mice were injected intraperitoneally with MAP30 (125. mu.g/kg) and cisplatin (2mg/kg) once every three days for five total injections, and then sacrificed. Consistent with the results of the in vitro study, co-treatment with MAP30 and cisplatin resulted in a significant reduction in tumor growth, as indicated by a 4-fold reduction in tumor weight (fig. 16A and 16B) and a 2-fold reduction in tumor nodules (fig. 16A and 16B) compared to cisplatin alone treatment. At the end of the experiment, treatment with cisplatin alone or with MAP30 in combination with cisplatin significantly reduced the mean ascites volume (fig. 16D) without significantly affecting the body weight of the mice (fig. 16E). These findings confirm that MAP30 is harmless in vivo and helps to inhibit tumor growth by enhancing cisplatin-induced cytotoxicity.

MAP30 inhibits glycolysis and de novo fat synthesis in ovarian cancer cells

There is increasing evidence that AMPK is a metabolic tumor suppressor that controls tumor development and progression. This study investigated whether MAP30 alters tumor metabolism to achieve its inhibitory effect on the oncogenic properties of ovarian cancer. L-lactic acid assay analysis showed that ovarian cancer cells typically produced relatively higher levels of lactic acid than immortalized human ovarian surface epithelial control cells throughout two days of culture (FIG. 17A). This increased lactate production in ovarian cancer cells was clearly offset by positive control treatment with the native AMPK activator MAP30(40 μ g/mL) and with drug AMPK activators such as AICAR (1mM) and a23187(2 μ M) (fig. 17B), indicating that the reduction in glycolysis in ovarian cancer cells occurs through MAP30 mediated AMPK activation. Notably, the decrease in glycolysis was positively correlated with a decrease in glycolytic enzyme activity such as hexokinase and LDH, which was significantly inhibited by 30-80% and 50-90% after MAP30(10-40 μ g/mL) treatment for 24 to 48 hours, respectively (fig. 17C and 17D). Notably, it is hypothesized that the net reduction in glycolysis through MAP 30-mediated AMPK activation is due to a drop in carbon flux. Thus, glucose uptake assays were performed. MAP30(10-40 μ g/mL) behaved similarly to the drug AMPK activator AICAR (1mM) and inhibited glucose uptake in OVCA433 cells by about 30% to 60% in a dose-dependent manner (fig. 17E). Immunoblot analysis further confirmed that expression of GLUT1 and GLUT3 glucose transporters significantly decreased in a time-dependent manner after treatment with MAP30(40 μ g/mL), but that another glucose transporter GLUT4 remained unchanged (fig. 17F). These data indicate that MAP30 inhibits glucose uptake by reducing the GLUT-1 and-3 transporters.

Omentum is the preferential site for metastasis of ovarian cancer. Thus, it is hypothesized that free fatty acids enriched in the omentum microenvironment specifically modulate ovarian tumor cells, thereby promoting malignant cell survival and metastatic cancer cell spread. Lipid droplet formation was increased using nile red fluorescent staining for OCM-cultured ovarian cancer cell line OVCA433, while co-treatment with MAP30(5 and 10 μ G/mL) reduced the lipid droplet number by 50-70% (fig. 17G). To further determine whether MAP30 accelerates catabolic lipolysis in human ovarian cancer cells, lipolysis assays were performed. The results showed that MAP30(10 and 40. mu.g/mL) induced an approximately 2-4 fold increase in cellular lipolysis in ovarian cancer cells (FIG. 17H). ATP detection assay showed a significant reduction in ATP production in ovarian cancer cells due to MAP30(10 and 40 μ g/mL) treatment (fig. 17I). Overall, the findings of the present invention indicate that MAP30 interrupts glucose uptake and lipid droplet formation in ovarian cancer cells, indicating that MAP30 will inhibit the invasiveness and metastasis of ovarian cancer in the ascites microenvironment.

Discussion:

traditional Chinese medicines target cancer cells and treat the entire body, including the qi, blood, organs and immune system. Traditional Chinese medicine is used as a supplementary substitute drug. Bioactive compounds from chinese medicine have significant anti-cancer activity by modulating multiple levels and targets in cancer cells. Therefore, the traditional Chinese medicine and the bioactive compound thereof can be used as ideal auxiliary agents for modern cancer treatment. In china and india, bitter melon has been used medically to improve digestion and treat diabetes and obesity and is known for its "plant insulin" activity. Momordica charantia extract (BME) exerts an inhibitory effect on ovarian cancer cells by activating AMPK/mTOR signaling. The present study isolated MAP30 protein from dried bitter gourd seeds. Functional and biochemical analysis showed that MAP30 is the major bioactive component in BME.

BME comprises a variety of bioactive compounds, and it exhibits anti-cancer effects in a variety of human cancers. For example, Kukukocin J (KJ) isolated from extract of momordica charantia reduces resistance of ovarian cancer cells to paclitaxel. Rnase MC2 induces apoptotic mediators and apoptosis in human breast cancer. A novel peptide BG-4 from Momordica charantia can promote apoptosis of human colon cancer. However, these studies did not show any relationship or function of AMPK. Bitter gourd has been used in china and india for the treatment of diabetes, as it is used for the same purpose as the first line treatment for diabetes, metformin. This activity indicates that bitter gourd has a functional effect on AMPK activation. The balsam pear seeds are used for treating diabetes and obesity in traditional Chinese medicine. This use may be due to the abundance of bioactive metabolites in bitter melon seeds, which regulate cellular metabolism, as recently discovered using metabolomic profiling studies. MAP30 protein isolated from dried bitter gourd seeds was reported to promote apoptosis in prostate cancer cells. This result prompted us to isolate MAP30 protein from dried bitter gourd seeds. The yield of MAP30 protein was 40mg per 100g of balsam pear seeds. Notably, MAP30 proved to be the major component of BME. MAP30 activated AMPK via CaMKK β pathway in an AMP-independent manner and inhibited mTOR and the novel AKT/ERK/FOXM1 signaling pathway, thereby producing an inhibitory effect on ovarian cancer.

AMPK is a key energy sensor in healthy cells and a key factor in tumor development. AMPK is a key sensor for maintaining cellular energy balance that regulates multiple cellular metabolic pathways. Emerging evidence suggests that malignant cells require higher AMPK activity to initially overcome metabolic stresses from the tumor microenvironment, but relatively lower AMPK activity is beneficial in promoting cell growth and development in advanced cancers. These data indicate that advanced and more aggressive cancer cells prefer low AMPK activity to reduce metabolic limitations, such as mTOR, to exert high carcinogenicity. These data also provide an advantage for AMPK activators in selectively impairing ovarian cancer cell function without affecting normal cells. Native MAP30 isolated from non-toxic momordica charantia increases AMPK activity through the CaMKK β pathway and in an AMP-independent manner, which provides another perspective for targeting cancer cells whose survival and other cellular activities are not affected by AMP stress conditions, such as a nutrient-rich microenvironment. The present study shows that native MAP30 significantly inhibited cell growth by S-phase cell cycle arrest, cell migration/invasion, and tumor growth or colonization in a mouse tumor xenograft model. mTOR signaling is an important pathway for cellular metabolism, growth, proliferation, survival, and resistance. However, mTOR inhibitors do not exhibit a broad and potent anti-cancer and anti-chemo-resistant effect. MAP30 does not exhibit this drawback.

Emerging evidence suggests that the ascites microenvironment provides growth factors and reprograms the metabolism of cancer cells to promote the progression of ovarian cancer through peritoneal metastasis. Thus, targeting cancer cell metabolism is a promising approach to compromise ovarian cancer aggressiveness in the tumor microenvironment. The findings of the present invention indicate that MAP30 inhibits the oncogenic AKT/ERK/FOXM1 signaling cascade and regulates cellular metabolism, e.g. by inhibiting the expression of the major glucose transporters GLUT1 and GLUT3, lipid droplet formation, and even lipolysis to reduce glucose uptake. In the murine ovarian cancer model of the invention, glycolysis and inhibition of lipid metabolism result in a significant reduction in tumor spread. The findings of the present invention demonstrate that MAP30 activates the AMPK pathway via CaMKK beta, resulting in Ca in ovarian cancer cells²⁺Is increased. Unconstrained Ca²⁺The signal may produce mitochondrial damage and increase ROS stress. Excess ROS induce caspase-dependent apoptotic cell death pathway activation in ovarian cancer cells.

In addition to mediating caspase activation and apoptosis, excess ROS may induce lipid peroxidation of cell membranes and lead to iron-dependent cell death, i.e., iron apoptosis. Studies have shown that ovarian cancer cells take up Free Fatty Acids (FFA) from the ascites microenvironment to support their metastatic progression in peritoneal metastasis. These metastatic ovarian cancer cells typically have a higher ratio of unsaturated FFA to saturated FFA in the ascites microenvironment. Due to multiple unsaturationFatty Acids (PUFAs) are excellent substrates for lipid peroxidation and are increased in metastatic ovarian cancer cells, and therefore whether MAP30 induces iron apoptosis to inhibit metastatic colonization in ovarian cancer was investigated. Erastin is a synthetic lethal compound that triggers iron apoptosis in cancer cells through xCT/GPX4 and GSH depletion. Using Erastin as a positive control, MAP30 was found to inhibit GPX4 as well, decreasing the GSH/GSSG ratio, and increasing the amount of ROS, enhancing lipid peroxidation. However, Ferrostatin-1(Fer-1) prevented the induction of lipid peroxidation, confirming that MAP30 is an inducer of iron apoptosis in ovarian cancer cells. Erastin is a class 1 iron apoptosis inducer (FIN) that induces iron apoptosis through the inactivation of GPX and depletion of intracellular GSH. Similar to Erastin, MAP30 depletes the intracellular antioxidant system, leading to accumulation of lipid peroxides, enhancing the susceptibility of ovarian cancer cells to free radical oxidative damage and cell death. On the other hand, since MAP30 is a natural AMPK activator, it is necessary to investigate whether AMPK activity is associated with iron apoptosis. Indeed, several recent studies have shown that AMPK activity mediates iron apoptosis, but the results in different cell types remain controversial. For example, Xong x. et al demonstrate that AMPK phosphorylates BECN1, which blocks system xCT and enhances iron apoptosis through Erastin-type induction in colon adenocarcinoma cells. In contrast, Lee H et al showed that stress-activated AMPK inhibited iron apoptosis in ischemic tissues through RSL 3-driven induction. In this study, it was found that depletion of AMPK reduced the MAP 30-induced Ca²⁺Influx and ROS, but result in a lesser reduction in GSH/GSSG ratio and lipid peroxidation in ovarian cancer cells. These results indicate that the presence or absence of AMPK has some effect on MAP 30-induced iron apoptosis in ovarian cancer cells, but its intrinsic mechanism remains to be elucidated.

And (4) conclusion:

MAP30 is a natural AMPK activator and modulates multiple levels and targets simultaneously. Notably, these activities of MAP30 enhanced its anti-cancer response and anti-chemoresistance in ovarian cancer, supporting its use as a supplement in advanced ovarian cancer to improve the efficacy of current chemotherapy regimens in a synergistic manner.

Although the present invention has been described to a certain degree, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Sequence listing

<110> university of hong Kong

<120> application of momordica charantia protein MAP30 in preparation of drugs or chemotherapy supplements for preventing and treating ovarian cancer

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Met Val Lys Cys Leu Leu Leu Ser Phe Leu Ile Ile Ala Ile Phe Ile

1 5 10 15

Gly Val Pro Thr Ala Lys Gly Asp Val Asn Phe Asp Leu Ser Thr Ala

20 25 30

Thr Ala Lys Thr Tyr Thr Lys Phe Ile Glu Asp Phe Arg Ala Thr Leu

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Pro Phe Ser His Lys Val Tyr Asp Ile Pro Leu Leu Tyr Ser Thr Ile

50 55 60

Ser Asp Ser Arg Arg Phe Ile Leu Leu Asn Leu Thr Ser Tyr Ala Tyr

65 70 75 80

Glu Thr Ile Ser Val Ala Ile Asp Val Thr Asn Val Tyr Val Val Ala

85 90 95

Tyr Arg Thr Arg Asp Val Ser Tyr Phe Phe Lys Glu Ser Pro Pro Glu

100 105 110

Ala Tyr Asn Ile Leu Phe Lys Gly Thr Arg Lys Ile Thr Leu Pro Tyr

115 120 125

Thr Gly Asn Tyr Glu Asn Leu Gln Thr Ala Ala His Lys Ile Arg Glu

130 135 140

Asn Ile Asp Leu Gly Leu Pro Ala Leu Ser Ser Ala Ile Thr Thr Leu

145 150 155 160

Phe Tyr Tyr Asn Ala Gln Ser Ala Pro Ser Ala Leu Leu Val Leu Ile

165 170 175

Gln Thr Thr Ala Glu Ala Ala Arg Phe Lys Tyr Thr Glu Arg His Val

180 185 190

Ala Lys Tyr Val Ala Thr Asn Phe Lys Pro Asn Leu Ala Ile Ile Ser

195 200 205

Leu Glu Asn Gln Trp Ser Ala Leu Ser Lys Gln Ile Phe Leu Ala Gln

210 215 220

Asn Gln Gly Gly Lys Phe Arg Asn Pro Val Asp Leu Ile Lys Pro Thr

225 230 235 240

Gly Glu Arg Phe Gln Val Thr Asn Val Asp Ser Asp Val Val Lys Gly

245 250 255

Asn Ile Lys Leu Leu Leu Asn Ser Arg Ala Ser Thr Ala Asp Glu Asn

260 265 270

Phe Ile Thr Thr Met Thr Leu Leu Gly Glu Ser Val Val Asn

275 280 285

<210> 2

<211> 284

<212> PRT

<213> Momordica charantia

<220>

<221> MUTAGEN

<222> (1)..(21)

<223> histidine tag

<400> 2

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Asp Val Asn Phe Asp Leu Ser Thr Ala Thr Ala

20 25 30

Lys Thr Tyr Thr Lys Phe Ile Glu Asp Phe Arg Ala Thr Leu Pro Phe

35 40 45

Ser His Lys Val Tyr Asp Ile Pro Leu Leu Tyr Ser Thr Ile Ser Asp

50 55 60

Ser Arg Arg Phe Ile Leu Leu Asn Leu Thr Ser Tyr Ala Tyr Glu Thr

65 70 75 80

Ile Ser Val Ala Ile Asp Val Thr Asn Val Tyr Val Val Ala Tyr Arg

85 90 95

Thr Arg Asp Val Ser Tyr Phe Phe Lys Glu Ser Pro Pro Glu Ala Tyr

100 105 110

Asn Ile Leu Phe Lys Gly Thr Arg Lys Ile Thr Leu Pro Tyr Thr Gly

115 120 125

Asn Tyr Glu Asn Leu Gln Thr Ala Ala His Lys Ile Arg Glu Asn Ile

130 135 140

Asp Leu Gly Leu Pro Ala Leu Ser Ser Ala Ile Thr Thr Leu Phe Tyr

145 150 155 160

Tyr Asn Ala Gln Ser Ala Pro Ser Ala Leu Leu Val Leu Ile Gln Thr

165 170 175

Thr Ala Glu Ala Ala Arg Phe Lys Tyr Thr Glu Arg His Val Ala Lys

180 185 190

Tyr Val Ala Thr Asn Phe Lys Pro Asn Leu Ala Ile Ile Ser Leu Glu

195 200 205

Asn Gln Trp Ser Ala Leu Ser Lys Gln Ile Phe Leu Ala Gln Asn Gln

210 215 220

Gly Gly Lys Phe Arg Asn Pro Val Asp Leu Ile Lys Pro Thr Gly Glu

225 230 235 240

Arg Phe Gln Val Thr Asn Val Asp Ser Asp Val Val Lys Gly Asn Ile

245 250 255

Lys Leu Leu Leu Asn Ser Arg Ala Ser Thr Ala Asp Glu Asn Phe Ile

260 265 270

Thr Thr Met Thr Leu Leu Gly Glu Ser Val Val Asn

275 280

Claims (10)

1. Use of bitter melon protein MAP30 in the preparation of a medicament for preventing and/or treating ovarian cancer or a chemotherapy supplement. 2. The use of balsam pear protein MAP30 and cisplatin in the preparation of a medicament for preventing or treating ovarian cancer or a chemotherapy supplement. 3. The use according to claim 1 or 2, wherein the ovarian cancer is advanced ovarian cancer or chemotherapy-resistant ovarian cancer. 4. The use according to any one of claims 1 to 3, wherein the prevention and/or treatment of ovarian cancer is inhibition of ovarian cancer cell growth, cell migration/invasion, ovarian cancer growth and/or ovarian cancer colonization. 5. The use of bitter melon protein MAP30 in the preparation of AMPK activator. 6. Use of bitter melon protein MAP30 in the preparation of a medicament for preventing and/or treating diseases or symptoms related to the inhibition of AMPK activation. 7. A pharmaceutical composition for preventing and/or treating ovarian cancer, comprising balsam pear protein MAP30 and cisplatin. 8. The pharmaceutical composition according to claim 7, wherein the mass ratio of the bitter melon MAP30 to cisplatin is 1:(0.6-1.8), preferably 1:(1.0-1.4), more preferably 1:1.2 . 9. The use according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 or 8, wherein the balsam pear protein MAP30 is natural balsam pear protein MAP30; Preferably, the natural bitter melon protein MAP30 is isolated from Thai bitter melon or Chinese bitter melon; more preferably, the natural bitter melon protein MAP30 is isolated from Chinese bitter melon; further preferably, the natural bitter melon protein MAP30 is from Taiwan, China Bitter gourd isolated; Preferably, the amino acid sequence of the bitter melon protein MAP30 is shown in SEQ ID NO.1 or 2; Preferably, the natural balsam pear protein MAP30 is a glycosylation-modified natural balsam pear protein MAP30; more preferably, the glycosylation modification site of the natural balsam pear protein MAP30 is located at No. 1 in the amino acid sequence shown in SEQ ID NO.1 74 asparagine. 10. purposes according to claim 9 or pharmaceutical composition, wherein the preparation method of described natural bitter melon protein MAP30 comprises the following steps: (1) after soaking the bitter gourd seeds, carry out homogenization, centrifuge, and get the supernatant; (2) adopt the 20mM Tris-HCl buffer solution containing 1M NaCl to elute the supernatant loaded on the Affi-gel blue gel column, and dialyze the eluted part containing the natural balsam pear protein MAP30; (3) eluting the dialyzed fraction loaded on the SP-Sepharose column with 20 mM NH ₄ OAc buffer containing 1 M NaCl, and dialyzing the eluted fraction containing native Momordica charantia MAP30; (4) FPLC-gel filtration was performed on the dialyzed portion loaded onto the Superdex 75HR 10/300 column using 50 mM Tris-HCl buffer containing 0.1 M NaCl, and the first peak at OD 280 nm was collected; Preferably, the preparation method of the natural bitter melon protein MAP30 comprises the following steps: (1) after soaking the balsam pear seeds, carry out homogenization, centrifuge, and filter to get the supernatant; (2) The supernatant loaded on the Affi-gel blue gel column was eluted with 20 mM Tris-HCl buffer containing 1 M NaCl, and the eluted part containing natural Momordica charantia MAP30 was placed in a dialysis tube with a molecular weight cut-off of 8 kDa dialysis; (3) using 20mM NH ₄ OAc buffer containing 1M NaCl to elute the dialyzed fraction loaded onto the SP-Sepharose column, dialyze the eluted fraction containing native Momordica charantia MAP30, and lyophilize; (4) The lyophilized powder was resuspended in 50 mM Tris-HCl buffer containing 0.1 M NaCl, and the suspension loaded on a Superdex 75HR 10/300 column was subjected to FPLC-gel filtration using the same buffer, and collected at OD 280 nm. The first peak at 3 kDa was dialyzed against centricon with a molecular weight cut-off of 3 kDa and lyophilized to a powder.

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