CN109303919B - Application of Akt Inhibitor in Preparation of Drugs with Enhanced Anti-hepatocellular Carcinoma Activity of Lycorine - Google Patents

Abstract

The invention discloses the application of an Akt inhibitor in the preparation of a drug for enhancing the anti-hepatocellular carcinoma activity of lycorine. The invention provides the application of Akt inhibitor in the preparation of a product that enhances the anti-liver cancer activity of lycorine or the anti-liver cancer cell activity. Experiments have shown that the apoptosis rate of liver cancer cells treated with Akt inhibitor and lycorine combined is higher than the sum of the apoptosis rate of liver cancer cells treated with Akt inhibitor alone and the apoptosis rate of liver cancer cells treated with lycorine alone. Akt inhibitor enhanced the anti-hepatocellular carcinoma activity of lycorine.

Description

Application of Akt Inhibitor in Preparation of Drugs with Enhanced Anti-hepatocellular Carcinoma Activity of Lycorine

technical field

The invention relates to the use of Akt inhibitors, in particular to the application of Akt preparations in the preparation of lycorine-enhancing anti-hepatocellular carcinoma active drugs.

Background technique

Hepatocellular carcinoma (HCC) is one of the most common aggressive tumors worldwide. Liver cancer is a highly lethal tumor with median survival rates remaining below 1 year after diagnosis. Liver cancer is a heterogeneous disease arising from chronic liver diseases such as chronic inflammation and cirrhosis. To date, treatment strategies for liver cancer mainly include resection, transplantation, local ablation, and chemotherapy. Because the early symptoms of liver cancer are not obvious, most patients with liver cancer are diagnosed at an advanced stage and lose the opportunity for surgery. Most patients who receive surgery in time will still experience recurrence and metastasis. Among chemotherapeutic agents, sorafenib, a small-molecule multikinase inhibitor, is considered an aggressive treatment and the only approved systemic drug that prolongs survival in patients with advanced liver cancer. However, the life expectancy of patients with advanced liver cancer treated with sorafenib is only 8-11 months. In the past few decades, despite significant progress in conventional treatment options for patients with liver cancer, it remains one of the most lethal malignancies worldwide due to limited treatment, poor prognosis, and high recurrence rate. New effective and promising therapeutic strategies still require intensive exploration on a global scale.

Natural products with diverse biological activities and mechanisms often serve as excellent drugs for cancer prevention and anticancer drug discovery. Lycorine (structure shown in Fig. 1a), an active alkaloid of common folk medicines, has been explored with various biological activities including anticancer, antiviral, antimalarial, antibacterial and anti-inflammatory activities. Although the target or mechanism of lycorine has not been defined, the main biological activity and low cytotoxicity of lycorine make it a potential clinical drug or a potential potential drug. For example, it is of great interest in cervical cancer, leukemia, prostate cancer and multiple myeloma as a promising anticancer agent. However, its precise molecular mechanism remains unclear.

Akt, also known as protein kinase B (PKB), is a serine/threonine-specific protein kinase that plays an important role in various cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to enhance the anti-hepatoma activity or anti-hepatoma cell activity of lycorine.

In order to solve the above technical problems, the present invention provides the application of Akt inhibitors in the preparation of products that enhance the anti-cancer activity of lycorine or the activity against liver cancer cells.

The present invention also provides the application of Akt inhibitor and lycorine in the preparation of anti-liver cancer or anti-liver cancer cell products (such as medicines, vaccines, health products and/or food).

In the above application, the Akt inhibitor may specifically be LY294002, whose structural formula is as shown in formula 1.

In the above-mentioned application, those skilled in the art can determine the ratio of the Akt inhibitor and the lycorine according to the effect of anti-liver cancer or anti-liver cancer cells, such as the specific ratio of the Akt inhibitor and the lycorine. The Akt inhibitor can be 1 μmol: 2 μmol Lycorine.

The present invention also provides anti-liver cancer or anti-liver cancer cell products (such as medicines, vaccines, health products and/or food), which contain Akt inhibitors and lycorine.

In the above product against liver cancer or liver cancer cells, both the Akt inhibitor and the lycorine can be packaged separately.

In the above-mentioned anti-liver cancer or anti-liver cancer cell products, the ratio of the Akt inhibitor and the lycorine can be determined by those skilled in the art according to the effect of anti-liver cancer or anti-liver cancer cells. Specifically, the proportion of lycorine can be 1 μmol of the Akt inhibitor: 2 μmol of lycorine.

In the above-mentioned anti-liver cancer or anti-liver cancer cell products, the active ingredients of the product may be the Akt inhibitor and the lycorine, the active ingredients of the product may also contain other ingredients, and the other active ingredients of the product may be Those skilled in the art can determine according to the anti-liver cancer effect.

In the above-mentioned anti-liver cancer or anti-liver cancer cell product, the product may further contain a carrier or an excipient. The carrier materials include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), insoluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carriers Materials (such as cellulose acetate phthalate and carboxymethyl cellulose, etc.).

In the above anti-liver cancer or anti-liver cancer cell product, the Akt inhibitor may be LY294002.

In the above application or the above anti-liver cancer or anti-liver cancer cell product, the liver cancer may be hepatocellular carcinoma.

In the present application, the anti-hepatoma, anti-hepatoma cell, anti-hepatoma activity or anti-hepatoma cell activity may specifically be promoting liver cancer cell apoptosis and/or promoting liver cancer cell autophagy and/or reducing liver cancer cell viability.

Experiments have shown that the apoptosis rate of liver cancer cells treated with Akt inhibitor and lycorine combined is higher than the sum of the apoptosis rate of liver cancer cells treated with Akt inhibitor alone and the apoptosis rate of liver cancer cells treated with lycorine alone. Akt inhibitor enhanced the anti-hepatocellular carcinoma activity of lycorine.

Description of drawings

Figure 1 shows that lycorine inhibits the growth of hepatocellular carcinoma.

In Figure 1, a is the structural formula of lycorine; b is the cell viability assay result; c is the single cell clone formation assay result; d-f are the tumorigenicity test results of xenograft tumors, and g is the immunohistochemical detection of tumors by Ki67 staining Relative changes of proliferation during growth, h is the result of HE staining immunohistochemical detection of heart, liver, spleen, lung and kidney.

Figure 2 shows that lycorine promotes autophagy in hepatocellular carcinoma.

In Figure 2, a is the Western blot analysis of the expression levels of p62 and LC-3B in HepG2 and SMMC-7721; b is the Western blot analysis of the expression levels of Atg5, Atg7 and Atg12 in HepG2 and SMMC-7721; c is the transmission electron microscope analysis, d and e are the autophagic flux analysis; f is the expression of p62 and LC-3B in hepatocellular carcinoma by western blot analysis; g is the expression of LC-3B in hepatocellular carcinoma detected by immunohistochemistry.

Figure 3 shows that inhibition of autophagy can further promote lycorine-induced apoptosis of liver cancer cells.

In Figure 3, a, c and f are the expression levels of p62, LC-3B, Cleaved Caspase-3, PARP1/2 and Actin in each treated cell by western blot analysis. b, d and e are the cell viability and apoptosis rate of each treated cell.

In Figure 3, in each picture of a and b, from left to right are control-treated cells, cells treated with lycorine alone, cells treated with 3-MA alone, and cells treated with lycorine and 3-MA in combination. In each picture of c and d, from left to right, control siRNA alone treated 1 cell, lycorine and control siRNA combined treatment 1 cell, LC-3B-1 treated cell alone, lycorine and LC-3B-1 Combined treatment of cells. In each picture of e and f, from left to right are control siRNA treatment 2 cells alone, lycorine and control siRNA combined treatment 2 cells, LC-3B-2 treatment cells alone, lycorine and LC-3B-2 Combined treatment of cells.

Figure 4 shows that lycorine promotes apoptosis and autophagy in liver cancer cells through the TCRP1/Akt/mTOR signaling pathway.

In Figure 4, a is the Western blot analysis of TCRP1 protein levels in HepG2 and SMMC-7721; b is real-time quantitative PCR analysis of TCRP1 mRNA levels in HepG2 and SMMC-7721 and Western blot analysis of TCRP1 protein levels in HepG2 and SMMC-7721 ;c is the result of western blot analysis showing that overexpression of TCRP1 significantly prevented the inhibitory effect of lycorine on the Akt/mTOR pathway; d is the result of western blotting analysis that overexpression of TCRP1 blocked lycorine-induced apoptosis and autophagy-related proteins Expression; e is the result of cell viability assay; f is autophagy flux analysis; g is TCRP1 overexpression strongly blocks the inhibitory effect of lycorine on colony formation; h is lycorine treatment reduces TCRP1 in HepG2 xenograft tumors , protein levels of Akt, 4-EBP1 and p70S6K.

Figure 5 shows the levels of phosphorylated Akt and TCRP1 detected by immunohistochemistry.

a and b are much stronger positive staining for phosphorylated Akt and TCRP1 in lycorine (10 mg/kg)-treated xenograft tumors; c and d are immunohistochemical detection of phosphorylated Akt in two liver cancer patients and TCRP1 levels.

Figure 6 shows that Akt inhibitors enhance the anti-cancer activity of lycorine. In both images, from left to right are control-treated cells, lycorine-treated cells alone, LY294002-treated cells alone, and cells treated with lycorine and LY294002 in combination.

Figure 7 shows that siRNA targeting TCRP1 enhances the anti-cancer activity of lycorine. In both pictures, from left to right, cells treated with control siRNA alone, cells treated with lycorine and control siRNA in combination, cells treated with siRNA _TCRP1 alone, and cells treated with lycorine and siRNA _TCRP1 in combination.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The materials and methods in the following examples are as follows:

Cell Culture and Cell Handling: Human HCC cell lines HepG2, SMMC-7721, HuH-7 and Chang hepatocytes were obtained from ATCC in April 2015. Spent cells were recovered within 1 month. Cell lines were identified by PCR-amplified short tandem repeat analysis. Cells were maintained at 37 °C in DMEM or RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere containing 5% _CO . Expression vectors for human TCRP1 and Akt were designed and purchased from Servicebio Technologies (Wuhan, China). Lycorine (purity >98%) was purchased from Shanghai Yuanye Biotechnology (Shanghai, China). Lycorine was prepared as a 20 mM stock solution by dissolving in dimethyl sulfoxide (DMSO) and stored in aliquots at -20°C. When treating cells, the medium was diluted and used immediately; when injecting into mice, PBS was used. Dilute to the corresponding concentration. 3-MA, MG-132 and LY294002 were purchased from Selleck (London, ON, Canada). MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA ). 3-MA was prepared as a 20 mM stock solution by dissolving in dimethyl sulfoxide (DMSO), and when treating cells, the medium was diluted and used immediately. LY294002 was prepared by dissolving in dimethyl sulfoxide (DMSO) as a 20 mM stock solution, and when treating cells, the medium was diluted and used immediately.

Tissue samples: 45 pairs of human liver cancer and their matched adjacent non-tumor tissue arrays were purchased from Shanghai Biochip Co., Ltd. (Shanghai, China). All of these samples were de-identified and the cohort had clinical outcome information.

Determination of cell viability by MTT method: Add MTT solution to the cells to be tested and incubate at 37°C for 4 hours. Then remove the medium. Add 100ul DMSO, and measure the absorbance at 570nm with a microplate reader.

Apoptosis Assay: Apoptosis was measured by staining cells using Muse™ Annexin V and Dead Cell Assay Kit (Millipore, Billerica, MA, USA) and in a benchtop Muse Cell Analyzer (Millipore, Billerica, MA, USA) Cell counts, cell cycle and apoptosis assays were performed according to the manufacturer's instructions.

Monoclonal formation assay (colony formation assay): Tumor cells were seeded into 6-well plates and cultured overnight. Cells were then treated with the indicated concentrations of agents for 48 hours. After rinsing with fresh medium, colonies were formed, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and then counted over the indicated time.

Immunofluorescence assay: Cells were fixed with 4% paraformaldehyde for 30 min and incubated with 0.5% Triton X-100 in PBS and blocked with 5% BSA for 30 min. Slides were stained with anti-LC-3B antibody overnight at 4 °C followed by Alexa-Fluor 488-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature. Nuclei were stained with 2.5 μg/mL DAPI (Invitrogen, Carlsbad, CA, USA) and visualized by inverted fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).

Immunohistochemical detection: excised tissue sections, formalin-fixed, paraffin-embedded, then treated with anti-Ki67, anti-cleaved caspase 3, anti-PARP, anti-LC-3B and anti-TCRP1 antibodies overnight at 4°C , followed by a biotinylated secondary antibody. Immunoreactivity was visualized using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). Known positive controls were included in each experiment, and negative controls were obtained by omitting the primary antibody.

Western blot analysis: Antibodies used were as follows: SQSTM1/p62 (D5E2) antibody, Atg5 (D5F5U) antibody, Atg7 (D12B11) antibody, Atg12 (D88H11) antibody, anti-LC-3B (D11) antibody, phospho-p70S6 kinase (Thr -389) antibody, total p70S6K antibody, phospho-Akt (Ser-473) antibody, total Akt antibody, phospho-4-EBP1 (Thr-37/46) antibody, total 4-EBP1 antibody and Cleaved Caspase 3 (D175) antibody were A product of CST Corporation (Danvers, MA, USA). Anti-PARP1/2 (H250) and TCRP1 (E-13) antibodies were products of Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin (A5441) antibody was a product of Sigma-Aldrich (St. Louis, MO, USA).

Example 1. Autophagy inhibitor enhances the anti-cancer activity of lycorine (the combination of autophagy inhibitor and lycorine enhances the apoptosis of liver cancer cells)

1. Lycorine inhibits the growth of hepatocellular carcinoma

Cell viability was assayed by MTT assay to analyze the effect of lycorine on three typical human hepatocellular carcinoma cell lines

(HepG2, SMMC-7721, HuH-7), and a non-tumorigenic human hepatocyte cell line (Chang Liver). HepG2, SMMC-7721, HuH-7 and Chang Liver were cultured in medium containing 0, 10, 20, 30, 40, and 50 μM of lycorine for 48 hours, respectively, and the cell viability was measured by MTT method. The results showed that 10, 20 , 30, 40, and 50 μM of lycorine for 48 hours significantly decreased the viability of liver cancer cells. At the same time, lycorine did not affect the cell viability of normal hepatocyte Chang Liver at the same dose (b in Figure 1). . The HepG2, SMMC-7721, and HuH-7 treated with 0, 10, and 20 μM of lycorine for 48 hours were used for the detection of monoclonal formation. The results showed that 10 and 20 μM of lycorine treated HepG2, SMMC-7721, and HuH-7 At 48 hours, the cell proliferation of HepG2, SMMC-7721, and HuH-7 was significantly inhibited (C in Figure 1). The results of the tumorigenicity assay of the xenograft tumors showed that, as shown in d in Figure 1, the size of the tumors was significantly reduced after treatment with lycorine compared with the control group. Consistent with this, tumors in the normal group were much larger in weight than those treated with lycorine (Fig. 1, e). Compared with the control group, the growth rate of lycorine-treated xenograft tumors was significantly decreased (Fig. 1, f). To further evaluate the relative changes in proliferation in tumor growth, immunohistochemical detection was performed by Ki67 (an antigen that marks the state of cell proliferation) staining, and the results showed that compared with tumors in the normal group, Lycorine (10 mg/kg) and Lycorine (20 mg/kg) kg) group had a significant percentage decrease (g in Figure 1). However, there were no significant differences in cell morphology and body weight in the main target organ (h in Figure 1). Collectively, these results suggest that lycorine inhibits liver cancer proliferation in vitro and in vivo.

Among them, the tumorigenicity detection method of xenograft tumor is as follows:

HepG2 cells ( ⁵ x 106 in 0.2 mL PBS) were inoculated (by subcutaneous injection) into 7-week-old BALB/c female athymic nude mice (Taconic). When the tumor volume reached 100mm, the mice were randomly divided into ³ groups: normal group, Lycorine (10mg/kg) group (Lycorine 10mg/kg) and Lycorine (20mg/kg) group (Lycorine 20mg/kg), each group of 8 Only. And the solution was intraperitoneally injected every other day for 33 consecutive days. Lycorine (10mg/kg) group was injected with 120 μL of lycorine solution, so that the injection dose of lycorine was 10mg/kg body weight/day; Lycorine (20mg/kg) group Each animal was injected with 120 μL of lycorine solution, so that the injection dose of lycorine was 20 mg/kg body weight/day; the normal group was injected with an equal volume of PBS. Tumor volume and body weight were monitored every 3 days. After mice were sacrificed 33 days from the first dose, their tumors were weighed, photographed and fixed in 4% paraformaldehyde for immunohistochemical assays.

2. Lycorine promotes autophagy in hepatocellular carcinoma

In addition to apoptosis, autophagy is another mode of programmed cell death in response to cellular stress. The effect of lycorine on autophagosome formation was first investigated by evaluating LC-3B and p62, two canonical markers of autophagy, using media containing 0, 10, 20, 30 and 40 μM of lycorine, respectively. HepG2 and SMMC-7721 were cultured for 48 hours, and the cells were harvested with SQSTM1/p62 (D5E2) antibody, LC-3B (D11) antibody, Atg5 (D5F5U) antibody, Atg7 (D12B11) antibody, Atg12 (D88H11) antibody and anti- The expression of p62, LC-3B, Atg5, Atg7, Atg12 and Actin (as an internal reference) was analyzed by western blotting with β-actin (A5441) antibody, and the results showed that lycorine could up-regulate the expression of LC-3B at a certain dose dependent, while the expression of p62 was significantly reduced (a in Figure 2). Lycorine can significantly up-regulate the protein expressions of Atg5, Atg7 and Atg12 (Fig. 2b). To further validate lycorine-induced autophagy, intracellular morphological changes in liver cancer were investigated by transmission electron microscopy. The method was as follows: HepG2, SMMC-772 were incubated with media containing 0 and 40 μM lycorine, respectively, for 48 hours, and the cells were fixed with 4% glutaraldehyde. Ultrathin (50 nm) sections were cut in an ultramicrotome and stained with 1% uranyl acetate and lead citrate. Finally, sections were determined by electron microscope Hitachi H-7650 (Hitachi, Tokyo, Japan). The results showed that double-membrane vesicles (containing subcellular material) were significantly accumulated in 40 μM lycorine-treated cells (cells represented by Lycorine in c in Figure 2) compared with normal cells (0 μM lycorine-treated cells). (c in Figure 2).

To determine autophagic flux, cells HepG2, SMMC-7721, were transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3) according to the manufacturer's instructions (Shanghai Jikai Gene Chemical Technology). The transfected cells were then incubated with media containing 0 and 40 μM of lycorine for 48 hours, respectively. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 minutes and washed with PBS. Finally, GFP/mRFP images were visualized with a laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan). Among them, mRFP is used to label and track LC3, and the weakening of GFP can indicate the fusion of lysosomes and autophagosomes to form autolysosomes. Indicating autophagic lysosomes, the intensity of autophagic flux can be clearly seen through the counting of different colored spots. In addition, HepG2 and SMMC-772 were incubated with media containing 0 and 40 μM lycorine, respectively, for 48 hours, the cells were stained with DAPI for nuclei, and 10 minutes later, the cells were immunized with anti-LC-3B (D11) antibody. chemical detection. The results showed that the use of tandem mRFP-GFP-tagged LC-3, after lycorine treatment, resulted in increased LC-3-II conversion, promotion of LC-3 lipidation and puncta, and accumulation of yellow punctate autophagosomes (Fig. 2). d and e). In Figure 2 d and e, the normal group and Control are cells treated with 0 μM lycorine, and Lycorine is cells treated with 40 μM lycorine.

Furthermore, the effect of lycorine on autophagy in liver cancer in vivo was assessed by western blot assay and immunohistochemical staining. HepG2 cells ( ⁵ x 106 in 0.2 mL PBS) were inoculated (by subcutaneous injection) into 7-week-old BALB/c female athymic nude mice (Taconic). When the tumor volume reached 100 mm ³ , the mice were randomly divided into 3 groups: normal group, Lycorine (10 mg/kg) group and Lycorine (20 mg/kg) group, with 8 mice in each group. And the solution was intraperitoneally injected every other day for 33 consecutive days. Lycorine (10mg/kg) group was injected with 120 μL of lycorine solution, so that the injection dose of lycorine was 10mg/kg body weight/day; Lycorine (20mg/kg) group Each animal was injected with 120 μL of lycorine solution, so that the injection dose of lycorine was 20 mg/kg body weight/day; the normal group was injected with an equal volume of PBS. Tumor volume and body weight were monitored every 3 days. After mice were sacrificed 33 days from the first administration, tumors were fixed in 4% paraformaldehyde, and tumor cells were detected by immunohistochemistry with anti-LC-3B (D11) antibody, and tumor cells were treated with SQSTM1/ p62 (D5E2) antibody, LC-3B (D11) antibody and anti-β-actin (A5441) antibody were subjected to western blotting to analyze the expression levels of p62, LC-3B and Actin (as internal control). The results showed that compared with the normal group, lycorine treatment significantly increased the level of LC-3B-II in HepG2 tumors, and at the same time, the level of p62 was significantly decreased (f in Figure 2). In f in Figure 2, the 0 column in the Lycorine (mg/kg) row is in the normal group, the 10 column in the Lycorine (mg/kg) row is the Lycorine (10 mg/kg) group, and the Lycorine (mg/kg) row in the 20 columns were in the Lycorine (20 mg/kg) group.

Consistent with this, a similar trend was observed by immunohistochemical staining for LC-3B. In the lycorine-treated tumors, the number of LC-3B-positively stained autophagic cells was significantly higher than that in the normal group (Fig. 2, g). Taken together, these results suggest that lycorine promotes autophagy in hepatocellular carcinoma.

3. Inhibition of autophagy can further promote lycorine-induced apoptosis of liver cancer cells

3.1 The autophagy inhibitor 3-MA enhances the anti-cancer activity of lycorine, and 3-MA and lycorine have a synergistic effect in anti-cancer

Although apoptosis and autophagy are two distinct modes of programmed cell death, they have complex interconnections to maintain intracellular homeostasis. To study the effect of lycorine on autophagy and apoptosis in hepatoma cells, 3-methyladenine (3-MA), an autophagy inhibitor (blocks autophagy), was selected with and without 3- MA-treated lycorine-induced hepatoma cells.

Four treatments were set up in the experiment, which were control treatment, lycorine alone, 3-MA alone, and lycorine and 3-MA combined. The specific experimental methods are as follows:

Control treatment: HepG2, SMMC-7721 were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37°C for 50 hours respectively, and the cells were collected to obtain control treated cells.

Lycorine alone treatment: HepG2, SMMC-7721 were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin for 2 hours at 37°C, and then lycorine was added to the medium to lycolic acid. The content of alkali was 40 μM and the cells were further cultured at 37° C. for 48 hours respectively, and the cells were collected to obtain cells treated with lycorine alone.

3-MA alone treatment: HepG2, SMMC-7721 were treated with 3-MA medium (3-MA was added to DMEM medium containing 10% FBS and 1% penicillin/streptomycin to 3 mM of 3-MA) The obtained medium) were cultured at 37° C. for 50 hours respectively, and the cells were collected to obtain cells treated with 3-MA alone.

Co-treatment with lycorine and 3-MA: HepG2, SMMC-7721 were treated with 3-MA medium (3-MA to 3-MA was added to DMEM medium containing 10% FBS and 1% penicillin/streptomycin The content is the medium obtained by 3mM) after culturing respectively for 2 hours at 37°C, then adding lycorine to the medium to the content of lycorine is 40 μM and continuing to culture for 48 hours at 37°C, collecting cells to obtain Lycoris Cells were treated in combination with base and 3-MA.

Western blot analysis control treatment with SQSTM1/p62 (D5E2) antibody, LC-3B (D11) antibody, Cleaved Caspase 3 (D175) antibody, anti-PARP1/2 (H250) antibody and anti-β-actin (A5441) antibody The expression levels of p62, LC-3B, Cleaved Caspase-3, PARP and Actin (as an internal reference) in cells, cells treated with lycorine alone, cells treated with 3-MA alone, and cells treated with lycorine and 3-MA combined, the results showed that 3-MA significantly attenuated lycorine-induced autophagy in hepatoma cells (HepG2 and SMMC-7721) (Fig. 3a). Interestingly, 3-MA further increased lycorine-induced apoptosis by activating CleavedCaspase 3 and PARP (Fig. 3a).

PARP (poly ADP-ribose polymerase) is the cleavage substrate of caspase, a core member of apoptosis.

Cell viability was measured by MTT method using the control-treated cells, lycorine alone, 3-MA alone, and lycorine and 3-MA combined treatment cells by using Muse™ Annexin V and a dead cell assay kit (Millipore , Billerica, MA, USA) stained cells to measure apoptosis, the results are shown in b in Figure 3, the apoptosis rate of HepG2 cells treated with lycorine and 3-MA combined (60.18% ± 1.02%) was higher than that of stone The sum of the apoptosis rate of HepG2 cells treated with alliline alone (40.85%±1.13%) and the apoptosis rate of HepG2 cells treated with 3-MA alone (7.53%±0.13%); the combined treatment of lycorine and 3-MA The apoptosis rate of SMMC-7721 cells (63.52% ± 2.22%) was higher than that of SMMC-7721 cells treated with lycorine alone (46.33% ± 2.35%) and SMMC-7721 cells treated with 3-MA alone The sum of the apoptosis rate (7.21%±0.11%). It indicated that 3-MA enhanced the anti-cancer activity of lycorine, and 3-MA and lycorine had a synergistic effect in anti-cancer.

Example 2. siRNA targeting LC-3B enhances the anti-liver cancer activity of lycorine, and siRNA targeting LC-3B and lycorine have a synergistic effect in anti-liver cancer

NCBI Reference Sequence: NM_022818.4 (Update Date: 10-JUL-2017) of the human microtubule associated protein 1 light chain 3beta (Homo sapiens microtubule associated protein1 light chain 3beta, MAP1LC3B, LC-3B for short) gene.

In this experiment, random double-stranded RNA was selected as the negative control siRNA. The name of the random double-stranded RNA was siNC. The nucleotide sequence from the 5' end to the 3' end of one chain of siNC was (5'-uucuccgaacgugucacguTT-3') The nucleotide sequence from the 3' end to the 5' end of the other chain of siNC is (3'-TTaagaggcuugcacagugca-5').

In this experiment, two siRNAs targeting human LC-3B were selected, named LC-3B-1 and LC-3B-2, respectively. The nucleotide sequence of one strand of LC-3B-1 from the 5' end to the 3' end is (5'-gcucuucuagaauuguuuaTT-3' (Sequence 1 in the Sequence Listing)), and the nucleotide sequence from the 3' end to the 5' end of the other chain of LC-3B-1 is (3'-TTcgagaagaucuuaacaaau-5'( Sequence 2)) in the sequence listing. The nucleotide sequence from the 5' end to the 3' end of one chain of LC-3B-2 is (5'-guagaagauguccgacuuaTT-3' (sequence 3 in the sequence listing)), and the nucleotide sequence of the other chain of LC-3B-2 is The nucleotide sequence from the 3' end to the 5' end is (3'-TTcaucuucuacaggcugaau-5' (SEQ ID NO: 4 in the sequence listing)).

In the sequences of the above three siRNAs, u, c, g, a and T are uracil ribonucleotides, cytosine ribonucleotides, guanine ribonucleotides, adenine ribonucleotides and thymine deoxynucleotides, respectively. ribonucleotides.

Eight treatments were set up in the experiment, namely control siRNA alone treatment 1, control siRNA alone treatment 2, lycorine and control siRNA combined treatment 1, lycorine and control siRNA combined treatment 2, LC-3B-1 alone treatment, lycorine Alkali and LC-3B-1 combined treatment, LC-3B-2 single treatment, lycorine and LC-3B-2 combined treatment. The specific experimental methods are as follows:

The experimental methods of control siRNA alone treatment 1 and control siRNA alone treatment 2 are the same, the experimental method of lycorine and control siRNA combined treatment 1 is the same as that of lycorine and control siRNA combined treatment 2, the experimental method of control siRNA alone treatment 1 is the same as that of lycorine and lycorine. Control siRNA combined treatment 1 was used as a control for LC-3B-1, and control siRNA treatment 2 alone with lycorine and control siRNA combined treatment 2 was used as a control for LC-3B-2.

Control siRNA alone treatment 1, control siRNA alone treatment 2: HepG2 and SMMC-7721 cells were seeded in 6-well plates, and 4 × 10 ⁵ cells were seeded in each well, with 10% FBS and 1% penicillin/streptomycin siNCs were transfected with Lipofectamine RNAi Max after culturing at 37°C for 12h, and siNCs were transfected 12h later. After 24h of secondary transfection, siNCs were replaced with fresh DMEM medium containing 10% FBS and 1% penicillin/streptomycin The culture was continued at 37°C for 48 hours, and the cells were collected to obtain control siRNA-treated 1 cell and control siRNA-treated 2 cells alone. The concentration of siNC in each transfection was 30 nM.

Combined treatment of lycorine and control siRNA 1, combined treatment of lycorine and control siRNA 2: HepG2 and SMMC-7721 cells were seeded in 6-well plates, and 4 × 10 ⁵ cells were seeded in each well. The cells were cultured with 1% penicillin/streptomycin DMEM medium at 37°C for 12 hours, and then transfected with Lipofectamine RNAi Max. After 12 hours, the secondary transfection was performed. After the secondary transfection of siNCs for 24 hours, the medium containing 40 μM lycorine was changed. (Add lycorine to the DMEM medium containing 10% FBS and 1% penicillin/streptomycin until the content of lycorine is 40 μM) and continue to culture at 37° C. for 48 hours, respectively, and collect the cells to obtain lycorine and control. 1 cell was co-treated with siRNA, and 2 cells were co-treated with lycorine and control siRNA. The concentration of siNC in each transfection was 30 nM.

LC-3B-1 treatment alone: HepG2 and SMMC-7721 cells were seeded in 6-well plates at ⁴ × 10 cells per well in DMEM medium containing 10% FBS and 1% penicillin/streptomycin LC-3B-1 was transfected with Lipofectamine RNAi Max after 12 hours of incubation at 37°C, and LC-3B-1 was transfected after 12 hours. After 124 hours of secondary transfection, LC-3B-1 was replaced with fresh water containing 10% FBS and 1% penicillin/streptomycin The DMEM medium was further cultured at 37 °C for 48 hours, and the cells were collected to obtain LC-3B-1-treated cells. The concentration of LC-3B-1 in each transfection was 30 nM.

Combined treatment with lycorine and LC-3B-1: HepG2 and SMMC-7721 cells were seeded in 6-well plates, ⁴ × 105 cells per well, with 10% FBS and 1% penicillin/streptomycin LC-3B-1 was transfected with Lipofectamine RNAi Max after culturing in DMEM medium at 37°C for 12h, followed by secondary transfection after 12h, and the medium containing 40 μM lycorine was changed after secondary transfection of LC-3B-1 for 24h. (Add lycorine to the DMEM medium containing 10% FBS and 1% penicillin/streptomycin until the content of lycorine is 40 μM) at 37° C. for 48 hours, collect the cells to obtain lycorine and LC -3B-1 co-treated cells. The concentration of LC-3B-1 in each transfection was 30 nM.

LC-3B-2 treatment alone: HepG2 and SMMC-7721 cells were seeded in 6-well plates at ⁴ x 10 cells per well in DMEM medium containing 10% FBS and 1% penicillin/streptomycin LC-3B-2 was transfected with Lipofectamine RNAi Max after 12 hours of incubation at 37°C, and the secondary transfection was performed after 12 hours. After 24 hours of secondary transfection, LC-3B-2 was replaced with fresh water containing 10% FBS and 1% penicillin/streptomycin The DMEM medium was further cultured at 37°C for 48 hours, and the cells were collected to obtain LC-3B-2 treated cells alone. The concentration of LC-3B-2 in each transfection was 30 nM.

Combined treatment with lycorine and LC-3B-2: HepG2 and SMMC-7721 cells were seeded in 6-well plates at ⁴ × 10 cells per well, and treated with 10% FBS and 1% penicillin/streptomycin. LC-3B-2 was transfected with Lipofectamine RNAi Max after culturing in DMEM medium at 37°C for 12h, followed by secondary transfection after 12h, and 24h after secondary transfection of LC-3B-2, the medium containing 40 μM lycorine was changed (Add lycorine to the DMEM medium containing 10% FBS and 1% penicillin/streptomycin until the content of lycorine is 40 μM) at 37° C. for 48 hours, collect the cells to obtain lycorine and LC -3B-2 co-treated cells. The concentration of LC-3B-2 in each transfection was 30 nM.

Western blot analysis with SQSTM1/p62 (D5E2) antibody, LC-3B (D11) antibody, Cleaved Caspase 3 (D175), anti-PARP1/2 (H250) antibody and anti-β-actin (A5441) antibody Control siRNA alone 1 cell treated, 2 cells treated with control siRNA alone, 1 cell treated with lycorine and control siRNA, 2 cells treated with lycorine and control siRNA, LC-3B-1 treated cells alone, lycorine and LC-3B- 1 The expression levels of p62, LC-3B, Cleaved Caspase-3, PARP and Actin (as an internal reference) in cells treated with combined treatment, cells treated with LC-3B-2 alone, and cells treated with lycorine and LC-3B-2 combined, the results showed that two LC-3B-targeting siRNAs, LC-3B-1 and LC-3B-2, significantly attenuated lycorine-induced autophagy in hepatoma cells (HepG2 and SMMC-7721) (c and f in Figure 3). ). Both LC-3B-1 and LC-3B-2, two siRNA targeting LC-3B, reduced lycorine-induced LC-3B content in HepG2 and SMMC-7721 cells. This indicated that siRNA knockdown of endogenous LC-3B significantly reduced lycorine-induced autophagy.

1 cell was treated with control siRNA alone, 2 cells were treated with control siRNA alone, 1 cell was treated with lycorine and control siRNA in combination, 2 cells were treated with lycorine and control siRNA in combination, cells were treated with LC-3B-1 alone, lycorine and LC-3B-1 combined treatment of cells, LC-3B-2 alone treatment of cells, lycorine and LC-3B-2 combined treatment of cells were determined by MTT assay cell viability, by using Muse™ Annexin V and dead cell assay kit ( Millipore, Billerica, MA, USA) stained cells to measure apoptosis, the results are shown in Figure 3 d and e, the apoptosis rate of HepG2 cells treated with lycorine and LC-3B-1 combined (68.32%±1.53 %) was higher than the apoptosis rate of HepG2 cells treated with lycorine and control siRNA combined 1 (47.73%±1.81%) and the apoptotic rate of HepG2 cells treated with LC-3B-1 alone (3.12%±0.38%). and; the apoptosis rate of HepG2 cells treated with lycorine and LC-3B-2 (57.62%±4.28%) was higher than that of HepG2 cells treated with lycorine and control siRNA (34.25%±2.92%). %) and the sum of the apoptosis rates (6.23%±2.35%) of HepG2 cells treated with LC-3B-2 alone. The apoptosis rate of SMMC-7721 cells treated with lycorine and LC-3B-1 (64.39%±1.62%) was higher than that of SMMC-7721 cells treated with lycorine and control siRNA (42.33%). ±3.85%) and the sum of the apoptosis rates of SMMC-7721 cells treated with LC-3B-1 alone (4.04% ± 0.29%); the apoptosis of SMMC-7721 cells treated with lycorine and LC-3B-2 combined The rate of apoptosis (58.98%±0.33%) was higher than the apoptosis rate of SMMC-7721 cells treated with lycorine and control siRNA combined 2 (38.66%±4.30%) and the apoptosis rate of SMMC-7721 cells treated with LC-3B-2 alone. The sum of mortality (3.14%±0.32%). This indicates that LC-3B-1 and LC-3B-2, two siRNAs targeting LC-3B, enhance the anti-cancer activity of lycorine, and the siRNA targeting LC-3B and lycorine have a synergistic effect in anti-liver cancer. . These results strongly suggest that inhibition of autophagy can further promote lycorine-induced apoptosis in HCC cells.

Example 3. Akt inhibitor enhances the anti-hepatoma activity of lycorine (Akt inhibitor and lycorine in combination enhance the apoptosis of liver cancer cells)

1. Lycorine promotes apoptosis and autophagy of liver cancer cells through TCRP1/Akt/mTOR signaling pathway

Experiments were carried out with reference to the corresponding experimental methods in Example 1.

Tongue cancer resistance-associated protein 1 (TCRP1) has been reported to promote tumorigenesis in radiation-resistant oral squamous cell carcinoma by selectively activating PI3K/Akt and NF-KB pathways. As shown in a in Figure 4, lycorine treatment significantly reduced the expression of TCRP1 protein level in hepatoma cells. However, lycorine treatment did not significantly alter the mRNA level of TCRP1 (Fig. 4, b), suggesting that TCRP1 protein was largely transferred through proteasomal degradation. Subsequently, treatment with MG-132, a proteasome inhibitor, significantly abolished the inhibitory effect of lycorine on TCRP1 protein levels in hepatoma cells (Fig. 4, b). Taken together, these results suggest that lycorine may reduce TCRP1 protein levels by promoting the TCRP1 protein degradation pathway.

Next, it was found that overexpression of TCRP1 in hepatoma cells significantly prevented the inhibitory effect of lycorine on the Akt/mTOR pathway (Fig. 4c). In addition, TCRP1 overexpression also blocked lycorine-induced apoptosis and autophagy-related protein expression, and cell death (d-f in Figure 4). In addition, TCRP1 overexpression strongly blocked the inhibitory effect of lycorine on colony formation (Fig. 4g). Consistent with this, lycorine treatment reduced the protein levels of TCRP1, Akt, 4-EBP1 and p70S6K in HepG2 xenograft tumors (h in Figure 4). Thus, immunohistochemical staining demonstrated much stronger positive staining for phosphorylated Akt and TCRP1 in lycorine-treated xenograft tumors compared to control xenograft tumors (Figure 5, a and b). Next, the clinical correlation between Akt and TCRP1 in liver cancer patient tissues was evaluated. As expected, TCRP1 expression positively correlated with p-Akt (c and d in Figure 5), supporting the results in cultured cells and mouse tumor models. In conclusion, the results of this study suggest that lycorine plays a key role in promoting apoptosis and autophagy in liver cancer cells through the TCRP1/Akt/mTOR signaling pathway.

2. Akt inhibitor enhances the anti-cancer activity of lycorine (the combination of Akt inhibitor and lycorine enhances the apoptosis of liver cancer cells)

The Akt inhibitor used in this experiment is LY294002, and its structural formula is shown in formula 1.

Four treatments were set up in the experiment, namely control treatment, lycorine alone, LY294002 alone, and lycorine and LY294002 combined. The specific experimental methods are as follows:

Control treatment: HepG2, SMMC-7721 were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37°C for 50 hours respectively, and the cells were collected to obtain control treated cells.

Lycorine alone treatment: HepG2, SMMC-7721 were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin for 2 hours at 37°C, and then lycorine was added to the medium to lycolic acid. The content of alkali was 40 μM and the cells were further cultured at 37° C. for 48 hours respectively, and the cells were collected to obtain cells treated with lycorine alone.

LY294002 treatment alone: HepG2, SMMC-7721 were treated with LY294002 medium (a medium obtained by adding LY294002 to 20 μM LY294002 to DMEM medium containing 10% FBS and 1% penicillin/streptomycin) at 37°C After culturing for 50 hours, the cells were collected to obtain cells treated with LY294002 alone.

Combined treatment of lycorine and LY294002: HepG2, SMMC-7721 were treated with LY294002 medium (LY294002 was added to DMEM medium containing 10% FBS and 1% penicillin/streptomycin to a concentration of 20 μM of LY294002) ) After culturing for 2 hours at 37° C., add lycorine to the culture medium until the content of lycorine is 40 μM, and continue to culture at 37° C. for 48 hours, respectively, to collect cells to obtain cells treated with lycorine and LY294002.

Control-treated cells, lycorine alone, LY294002 alone and combined lycorine and LY294002 were measured by staining cells using Muse™ Annexin V and Dead Cell Assay Kit (Millipore, Billerica, MA, USA). The results showed that the apoptosis rate of HepG2 cells treated with lycorine and LY294002 (62.82%±3.82%) was higher than that of HepG2 cells treated with lycorine alone (37.66%±3.35%) and The sum of the apoptosis rates of HepG2 cells treated with LY294002 alone (4.85%±0.34%); the apoptotic rates of SMMC-7721 cells treated with lycorine and LY294002 combined (60.59%±4.68%) were higher than those treated with lycorine alone The sum of the apoptosis rate of SMMC-7721 cells (39.19% ± 4.16%) and that of LY294002-treated SMMC-7721 cells alone (3.99% ± 0.28%) (Figure 6). It indicated that LY294002 enhanced the anti-cancer activity of lycorine, and LY294002 and lycorine had a synergistic effect in anti-cancer.

Example 4. siRNA targeting TCRP1 enhances the anti-hepatocellular carcinoma activity of lycorine (the combination of a reagent that inhibits the expression of TCRP1 and lycorine enhances the apoptosis of hepatoma cells)

In this experiment, random double-stranded RNA was selected as the negative control siRNA. The name of the random double-stranded RNA was siNC. The nucleotide sequence from the 3' end to the 5' end of the other chain of siNC is (3'-TTaucuggaaccaugagcugcuagaaa-5').

In this experiment, an siRNA targeting TCRP1 was selected and named as siRNA. The nucleotide sequence from the 5' end to the 3' end of one strand of siRNA _TCRP1 is (5'-uagucccaguuaugcuccagaguuuuTT-3'), and the nucleotide sequence of the other strand of siRNA _TCRP1 from 3' The nucleotide sequence from 'end to 5' end is (3'-TTaucagggucaauacgaggucucaaa-5'.

In the sequences of the above two siRNAs, u, c, g, a and T are uracil ribonucleotides, cytosine ribonucleotides, guanine ribonucleotides, adenine ribonucleotides and thymine deoxynucleotides, respectively. ribonucleotides.

Four treatments were set up in the experiment, namely control siRNA alone, lycorine and control siRNA combined, siRNA _TCRP1 alone, and lycorine and siRNA _TCRP1 combined. The specific experimental methods are as follows:

Control siRNA treatment alone: HepG2 and SMMC-7721 cells were seeded in 6-well plates, ⁴ × 10 cells per well, and cultured at 37°C in DMEM medium containing 10% FBS and 1% penicillin/streptomycin After 12 h, Lipofectamine RNAi Max was used to transfect siNC, and 12 h later, secondary transfection was performed. After 24 h of secondary transfection, siNC was replaced with fresh DMEM medium containing 10% FBS and 1% penicillin/streptomycin, and the culture was continued at 37 °C. At 48 hours, cells were harvested and cells treated with control siRNA alone were obtained. The concentration of siNC in each transfection was 30 nM.

Combined treatment with lycorine and control siRNA: HepG2 and SMMC-7721 cells were seeded in 6-well plates at ⁴ × 10 cells per well, and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin After culturing at 37 °C for 12 h, siNCs were transfected with Lipofectamine RNAi Max, and 12 h later, secondary transfections were performed. Add lycorine to the DMEM medium of streptomycin until the content of lycorine is 40 μM) and continue to culture at 37° C. for 48 hours respectively, collect the cells, and obtain the cells treated with lycorine and control siRNA. The concentration of siNC in each transfection was 30 nM.

siRNA _TCRP1 treatment alone: HepG2 and SMMC-7721 cells were seeded in 6-well plates, ⁴ × 10 cells per well, and cultured at 37°C in DMEM medium containing 10% FBS and 1% penicillin/streptomycin After 12h, Lipofectamine RNAi Max was used to transfect siRNA _TCRP1 . After 12h, secondary transfection was performed. After 24h of secondary transfection, siRNA _TCRP1 was replaced with fresh DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37°C, respectively. The culture was continued for 48 hours, and the cells were harvested to obtain cells treated with siRNA _TCRP1 alone. Among them, the siRNA _TCRP1 concentration of each transfection was 30 nM.

Combined treatment with lycorine and siRNA _TCRP1 : HepG2 and SMMC-7721 cells were seeded in 6-well plates at ⁴ × 10 cells per well, and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin After culturing at 37°C for 12 h, siRNA _TCRP1 was transfected with Lipofectamine RNAi Max. After 12 h, secondary transfection was performed. After 24 h of secondary transfection of siRNA _TCRP1 , the medium containing 40 μM lycorine was changed (to 10% FBS and 1% lycorine). Add lycorine to the DMEM medium of penicillin/streptomycin until the content of lycorine is 40 μM) and continue to culture at 37°C for 48 hours respectively, collect the cells, and obtain the cells treated with lycorine and siRNA _TCRP1 . Among them, the siRNA _TCRP1 concentration of each transfection was 30 nM.

Cells were treated with control siRNA alone, cells treated with lycorine and control siRNA in combination, cells treated with siRNA _TCRP1 alone, and cells treated with lycorine and siRNA _TCRP1 in combination by using Muse™ Annexin V and a dead cell assay kit (Millipore, Billerica, MA. , USA) stained cells to measure apoptosis, the results showed that the apoptosis rate (61.22%±1.49%) of HepG2 cells treated with lycorine and siRNA _TCRP1 was higher than that of HepG2 cells treated with lycorine and control siRNA. The sum of the apoptosis rate (36.90%±3.88%) and the apoptosis rate of HepG2 cells treated with siRNA _TCRP1 alone (5.19%±0.31%); the apoptosis rate of SMMC-7721 cells treated with lycorine and siRNA _TCRP1 combined (58.24 %±4.37%) was higher than the apoptosis rate of SMMC-7721 cells treated with lycorine and control siRNA combined (35.55%±2.96%) and the apoptotic rate of SMMC-7721 cells treated with siRNA _TCRP1 alone (4.04%±0.35 %) (Fig. 7). It indicated that siRNA targeting TCRP1 enhanced the anti-hepatocellular carcinoma activity of lycorine, and siRNA targeting TCRP1 and lycorine had a synergistic effect in anti-liver cancer.

<110> Tianjin University of Traditional Chinese Medicine

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Claims (9)

1. The application of the Akt inhibitor in preparing the product for enhancing the anti-liver cancer activity of lycorine is LY 294002.
2. 2. Use according to claim 1, characterized in that: the liver cancer is hepatocellular carcinoma.
3. The application of the Akt inhibitor in preparing the product for enhancing the anti-hepatoma cell activity of lycorine is LY 294002.
4. 4. Use according to claim 3, characterized in that: the liver cancer is hepatocellular carcinoma.
5. The application of the Akt inhibitor and lycorine in preparing the anti-liver cancer product is LY 294002.
6. 6. Use according to claim 5, characterized in that: the anti-liver cancer product is an anti-liver cancer cell product.
7. 7. An anti-hepatoma product, characterized in that: the product contains an Akt inhibitor and lycorine, wherein the Akt inhibitor is LY 294002.
8. 8. The product of claim 7, wherein: the product for resisting liver cancer is a product for resisting liver cancer cells.
9. 9. The product according to claim 7 or 8, characterized in that: the liver cancer is hepatocellular carcinoma.

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