CN111773301B - A kind of composition containing aloe vera extract and application thereof - Google Patents

Abstract

The invention belongs to the field of medicines, and discloses a composition containing an aloe extract and application thereof, wherein the composition comprises the aloe extract, camptothecin and derivatives thereof, the aloe extract, the camptothecin and the derivatives thereof are combined for use, so that DNA damage in cancer cells can be increased, the level of gamma H2AX protein can be up-regulated, the cell cycle can be retarded in the S phase, apoptosis can be induced, a WNT/beta-catenin signal channel can be inhibited, the two are synergistic, and the cytotoxic activity of the cancer cells can be enhanced, so that the anticancer effect can be achieved. The method can be applied to the preparation of anticancer drugs, WNT/beta-catenin pathway inhibitors and WNT/beta-catenin pathway signal inactivation agents.

Description

A kind of composition containing aloe vera extract and application thereof

technical field

The invention belongs to the field of medicine, in particular to a composition containing aloe vera extract and its application.

Background technique

Cancer is a common disease that seriously threatens the lives of people in my country and the world, ranking first in the cause of death. In recent years, despite great progress in early diagnosis and early detection of tumors, and standard chemotherapy and radiotherapy have been used clinically for decades, the overall survival rate of most clinically advanced patients has not improved significantly. When normal cells evolve into cancer cells, they will have six basic characteristics of cancer cells: signals to maintain proliferation, avoid proliferation inhibition, resist cell death, induce angiogenesis, unrestricted replication, and activate invasion and metastasis. The entire carcinogenesis process can be divided into cancer occurrence stage, cancer progression stage and cancer metastasis stage. Most of the diagnosed cancer patients are in the middle or late stage. The large number of cancer cells exist, replicate and metastasize, threatening the life of the patients. However, there is still no good drug that can kill cancer cells in a targeted manner. Normal cells are less damaged.

The WNT/β-catenin signaling pathway is involved in various developmental processes and the occurrence and development of many cancers, especially colorectal cancer and liver cancer. Key regulatory steps in the canonical WNT signaling pathway include phosphorylation, ubiquitination and subsequent degradation of β-catenin. This process involves a specialized cytoplasmic destruction complex composed of the scaffold protein Axin and neoplastic polyposis (APC), glycogen synthase kinase-3α/β (GSK-3), casein kinase 1 (CKI) It consists of three core components. Once the WNT ligand binds to the receptor complex to form a seven-transmembrane domain fryzzle protein or a one-pass transmembrane protein of the LDL receptor-related protein family (especially LRP5 or LRP6), information transduction inhibits Axin-mediated Phosphorylation of β-catenin causes unphosphorylated β-catenin to accumulate and transport to the nucleus to form complexes with DNA-binding proteins of the TCF/LEF (T cell factor/lymphoid enhancer factor) family and activate WNT target gene expression . At present, there are few drugs targeting the WNT/β-catenin signaling pathway, and they have not shown excellent effects.

Therefore, there is an urgent need to provide a composition for inhibiting the WNT/β-catenin pathway in cancer cells.

SUMMARY OF THE INVENTION

The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. Therefore, the present invention provides a composition containing aloe vera extract, which can inhibit the WNT/β-catenin pathway in cancer cells.

An aloe vera extract-containing composition includes aloe vera extract and camptothecin and derivatives thereof.

Preferably, the composition includes aloe vera extract and a camptothecin derivative; further preferably, the camptothecin derivative is 10-hydroxycamptothecin (HPT).

Preferably, in the composition, the content of the aloe vera extract is 50-400 μg/ml, and the content of the camptothecin and its derivatives is 2.5-50 μM; further preferably, the aloe vera extract The content of the compound is 50-150 μg/ml, and the content of the camptothecin and its derivatives is 5-30 μM.

Preferably, the aloe vera extract-containing composition is obtained by leaching the aloe vera extract with water or/and alcohol; further preferably, the aloe vera extract is obtained by leaching with water.

A medicine, comprising the aloe extract-containing composition and auxiliary materials.

The medicament is in any dosage form to be pharmaceutically acceptable, such as a tablet, powder, or capsule.

The application of the aloe extract-containing composition in the preparation of anti-cancer drugs; preferably, the application of the aloe extract-containing composition in the preparation of anti-cancer drugs.

Application of the composition in the preparation of WNT/β-catenin pathway inhibitor.

The application of the composition in the preparation of a WNT/β-catenin pathway semaphore inactivating agent.

With respect to the prior art, the beneficial effects of the present invention are as follows:

The composition containing aloe vera extract in the present invention includes aloe vera extract and camptothecin and its derivatives, and the combined use of aloe vera extract and camptothecin and its derivatives can increase DNA damage in cancer cells, up-regulate γH2AX At the protein level, it blocks the cell cycle in S phase, induces apoptosis and inhibits the WNT/β-catenin signaling pathway. The two synergistically enhance the cytotoxic activity of cancer cells to achieve anti-cancer effects.

Description of drawings

1 is a graph showing the results of the cytotoxic MTT test in Example 1 and Example 2;

Fig. 2 is the result of comet analysis of HepG2 cells in Example 1 and Example 2;

Fig. 3 is a graph of HepG2 cell cycle analysis in Example 1 and Example 2;

Figure 4 is an analysis diagram of HepG2 cell apoptosis in Example 1 and Example 2;

Figure 5 is a protein level map of the WNT/β-catenin pathway in HepG2 cells in Example 1 and Example 2;

Among them, **P<0.05, **P<0.01, compared with the control group; P<0.05, ##P<0.01, compared with the HPT group.

Detailed ways

In order to make those skilled in the art understand the technical solutions of the present invention more clearly, the following examples are now given for illustration. It should be noted that the following examples do not limit the protection scope of the present invention.

The aloe vera extract used in the following examples was purchased from Yifang Pharmaceutical Co., Ltd. (Guangzhou, China). Unless otherwise specified, other raw materials, reagents or devices can be obtained from conventional commercial channels, or can be obtained by existing known methods.

Example 1

The aloe vera extract was weighed, and the aloe vera extract was ultrasonically extracted in water at 80°C for 30 minutes before filtering the extract. Aloe vera extract was stored at -20°C until use. The HPT was taken and mixed with aloe vera extract to prepare composition 1, wherein the concentration of HPT was 10 μM, and the content of aloe vera extract was 50 μg/ml.

Example 2

The aloe vera extract was weighed, and the aloe vera extract was ultrasonically extracted in water at 80°C for 30 minutes before filtering the extract. Aloe vera extract was stored at -20°C until use. The HPT was taken and mixed with aloe vera extract to prepare composition 2, wherein the concentration of HPT was 10 μM, and the content of aloe vera extract was 100 μg/ml.

Product effect test

The compositions prepared in Examples 1 and 2 were tested as follows.

Materials and methods

1. Drugs and reagents

Human hepatocellular carcinoma HepG2 cells were provided by the American Culture Collection (ATCC) (Rockville, MD, USA);

DMEM medium, penicillin-streptomycin solution, fetal bovine serum (FBS) and 0.25% trypsin EDTA solution were purchased from GIBCO Invitrogen (Carlsbad, CA, USA);

10-Hydroxycamptothecin (HPT) was purchased from Chengdu Mansite Biotechnology Co., Ltd. (Chengdu, China);

Ultrapure water was obtained from Milli-Q pure water system (Bedford, MA, USA, Millipore Company);

Mass spectrometry grade methanol, acetonitrile and acetic acid were purchased from Anaqua Chemical Supply (Houston, TX, USA);

Stable isotope labeled 13C1015N5-adenosine triphosphate (ATP13C15N), dimethyl sulfoxide (DMSO), trypsin-EDTA solution and 3-[(4,5)-dimethyl-2thiazole]-2,5-diphenyl Tetrazolium bromide (MTT) was purchased from SigmaAldrich Chemical Company (St. Louis, Michigan, USA/St.Louis, MO, USA);

Aqueous 30% ammonium hydroxide ( _NH4OH ), ammonium acetate ( _NH4OAc ) were purchased from Sigma-Aldrich (St. Louis, MI, MO, USA).

HPLC quality methanol, acetonitrile and acetic acid (AcOH) were purchased from JT Baker Chemical Company (Phillipsburg, NJ, NJ, USA);

Formic acid was purchased from Fisher Scientific (Fair Lawn, NJ, USA); ether was purchased from Tedia (Fairfield, OH, USA);

Tetrafluoroboric acid (HBF4) and trimethylsilyldiazomethane (TMSD; 2M in hexane) were supplied by AlfaAesar (Ward Hill, MA, USA);

The column Sepax GP-C18 (2.1 x 150 mm, 1.8 μm) was obtained from Sepax Technologies (Newark, DE, USA).

2. Plant raw materials

Aloe vera/Aloe barbadensis miller in this application is dried aloe vera exudate, purchased from Yifang Pharmaceutical Co., Ltd. (Guangzhou, China).

3. LC-MS/MS analysis of aloe vera extract

The standards of aloin A (≥98%), aloin B (≥98%), aloe verain (≥98%) and aloe verain (≥98%) were purchased from Baoji Chenguang Biological Co., Ltd. (Baoji, China); Ultrapure water was obtained from Milli-Q water purification system (Bedford, MA, USA, Millipore Corporation); mass spectrometry grade acetonitrile was purchased from Anaqua Chemical Supply (Houston, Texas, USA/Houston, TX, USA) .

Aloe vera extracts were performed on a Thermo Fisher TSQ LC-MS/MS system consisting of an Accela autosampler, an Accele pump, and a Quantum Access triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose/San Jose, CA, USA , CA, USA). Chromatographic separation was performed using a Sepax GP-C18 column (2.1 mm x 150 mm, 1.8 μm, Thermo Fisher Scientific, San Jose, CA, USA). The eluent was 30% acetonitrile water, the liquid flow rate was set to 0.3 mL/min, the column temperature was kept at 35 °C, and the ion spray voltage in negative ion mode was set to 3000 V at a temperature of 350 °C and an auxiliary air pressure of 15 psi. . As shown in Table 1, quantitative analysis was performed using mass spectrometry multiple reaction monitoring (MRM) technology.

Table 1. Settings for Mass Spectrometry Multiple Reaction Monitoring (MRM)

Name Parent Product Polarity Aloin A (Aloin A) 417.000 297.000 negative Aloin B (Aloin B) 417.000 297.000 negative Aloenin 409.300 247.000 negative Aloesin (Aloe Vera) 393.300 273.230 negative

4. Cell Culture

HepG2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin and placed in an incubator at 37°C, 5% _CO Incubation. HepG2 cells were seeded in 100 mm x 20 mm dishes (Corning Inc, Corning, NY, USA). After overnight culture, cells were divided into the following groups: control group (C), cells were incubated with medium alone for 48 hours; 10-hydroxycamptothecin group (HPT), cells were first incubated with medium for 24 hours hours, and then exposed to 10.0 μM HPT for 24 hours; Aloe vera extract group (Aloe), cells were exposed to 50.0 μg/mL or 100.0 μg/mL of aloe vera extract for 48 hours; composition 1 and composition 2), cells were treated with composition 1 and composition 2 for 24 hours, respectively. On the day of cell harvest, additional cell culture dishes were used for cell counts to normalize the nucleotide pool and to determine viability by trypan blue exclusion assay.

5. Cytotoxicity test

Austria)，参考波长为 650nm。使用血细胞计数器测定细胞数。通过GraphPad Prism软件(GraphPad Software 公司，美国加利福尼/CA,USA)计算IC 50值[最大抑制浓度的一半(50％)]。细胞存活率(％)＝[OD(处理)-OD(空白)]/[OD(对照，未处理)-OD(空白)]×100。药物相互作用通过组合指数(CI)方法进行评估，而CI<1，CI＝1或CI>1分别表示协同作用，累加作用或拮抗作用。CI值通过CompuSyn软件计算(ComboSyn公司，美国纽约/NY, USA)。The inhibitory effect of different groups on HepG2 cells was determined by cytotoxic MTT assay. HepG2 cells were seeded at a density of 1×10 ⁴ cells/well in 96-well plates (LabServ, Thermo Fisher Scientific Co, Beijing, China). After incubation, they were treated for 24, 48 and 72 hours with a concentration of HPT, aloe vera or a composition (including composition 1 and composition 2). MTT solution (0.5 mg/mL final concentration in medium) was added to each well and incubated for a further 4 hours. The medium was removed and 100 μL of DMSO was added to each well to dissolve the purple crystalline formazan. Absorbance was measured at 570 nm using a microplate UV/VIS spectrophotometer (Infinite M200 PRO, Tecan Austria GmbH 5082, Gredich, Austria/

Austria), the reference wavelength is 650 nm. Cell numbers were determined using a hemocytometer. IC50 values [half (50%) of the maximal inhibitory concentration] were calculated by GraphPad Prism software (GraphPad Software, Inc., California/CA, USA). Cell viability (%)=[OD(treated)-OD(blank)]/[OD(control, untreated)-OD(blank)]×100. Drug interactions were assessed by the Combination Index (CI) method, with CI < 1, CI = 1 or CI > 1 indicating synergy, additive or antagonism, respectively. CI values were calculated by CompuSyn software (ComboSyn Corporation, New York/NY, USA).

6. Crystal violet staining test

To visualize the efficacy of the drug on cells, cell viability was assessed by crystal violet staining. Briefly, HepG2 cells in 6-well plates were treated with 10.0 μM HPT medium, 50.0 μg/mL aloe medium, 100.0 μg/mL aloe medium or composition medium (including composition 1 and composition 2). (2.0×10 ⁵ cells/well). Subsequently, cells were stained with 0.5% crystal violet. After washing with PBS, colonies were counted using the Quantity One software clone counting function (LifeScience Research & Clinical Diagnostics, Bio-Rad Ltd, California, USA).

7. Immunofluorescence Assay

英国剑桥 /Cambridge,UK)。然后，在用PBS洗涤3次后，将细胞与合适的二抗山羊抗小鼠IgG H&

594(

稀释比例＝1:500，英国剑桥/Cambridge,UK)在5％BSA中于室温下黑暗中孵育1小时。最后，使用1μg/mL的蓝色荧光DAPI(4',6-二脒基-2- 苯基吲哚；Sigma-Aldrich)对核DNA进行染色。使用荧光显微镜进行成像分析(Leica Microsystems公司，德国韦茨拉尔/Wetzlar,Germany)。HepG2 cells were grown on glass circular culture sheets in 6-well plates, and HPT, aloe vera and combination groups (including composition 1 and composition 2) were added and incubated for 48 hours respectively. At the end of treatment, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, cells were washed three times with PBS and incubated in 5% (w/v) BSA for 30 min at room temperature to block cells, followed by incubation with the corresponding primary antibody gamma H2A.X for 12 h at 4°C (phospho S139 )[9F3](ab26350), dilution ratio=1:100;

Cambridge, UK/Cambridge, UK). Then, after washing 3 times with PBS, cells were incubated with the appropriate secondary antibody, goat anti-mouse IgG H&

594(

Dilution = 1:500, Cambridge, UK/Cambridge, UK) in 5% BSA for 1 hour at room temperature in the dark. Finally, nuclear DNA was stained with 1 μg/mL of blue fluorescent DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich). Imaging analysis was performed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

8. HPT-induced DNA damage and comet assay

HepG2 cells were seeded in 6-well plates at a density of 4×10 ⁵ /well and incubated for 24 hours. Cells were divided into the following groups: cells in the control group incubated with medium only, and cells exposed to 40.0 μM HPT for 1 h after pretreatment in 50.0 μg/mL or 100.0 μg/mL aloe for 24 h. Control group, cells incubated with medium only; injury group, cells exposed to 40.0 μM HPT for 1 h after pretreatment in 50.0 μg/mL or 100.0 μg/mL aloe for 24 h. After 1 h of HPT treatment, cells were washed with cold PBS and harvested, and cells were tested for DNA damage by alkaline comet electrophoresis according to previously published methods. Comets were detected with a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Its images were analyzed by the Comet Image Analysis System (Globalebio Corporation, Beijing, China). The total intensity (DNA content), tail length, percentage of DNA in the tail, and tail moment were used to assess the extent of DNA damage.

9. Cell Cycle Analysis

Cells were seeded in 6-well culture plates in duplicate at 4.0 x ¹⁰⁵ /well and incubated with the indicated concentrations of HPT, aloe or compositions (including composition 1 and composition 2) for 48 hours. They were then harvested and fixed with 70% (v/v) cold ethanol overnight at 4°C. Fixed cells were collected by centrifugation, resuspended in PBS, and incubated with 5 mg/mL propidium iodide (Sigma-Aldrich) and 10 mg/mL RNase A (Sigma-Aldrich) for 30 at room temperature in the dark minute. Cells were then analysed with a flow cytometer (Muse™ cell analyzer, Merck Millipore, Darmstadt, Germany). Finally, the percentage of cells at different stages (G0/G1, S and G2/M) was calculated using Modfit software (Verity Software House, USA).

10. Apoptosis detection

Apoptosis was quantified using the Muse™ Annexin V&Dead Cell Kit (Darmstadt, Germany). Cells from HPT group, aloe vera group or combination group (including composition 1 and composition 2) were obtained by trypsinization, washed twice with cold PBS and resuspended in 1% FBS. The cells were stained by addition of Muse™ Annexin V&Dead Cell and then analyzed by flow cytometer (Muse™ cell analyzer, Merck Millipore, Darmstadt, Germany).

11. Western blot analysis

英国剑桥)、兔Caspase-3抗体(稀释比＝1:1000；Cell Signaling Technology公司，美国马萨诸塞州丹弗斯)、兔caspase-8抗体(稀释比＝1:1000；Cell Signaling Technology)、兔caspase-9抗体(稀释比＝1：1000；Cell Signaling Technology)、小鼠PARP抗体 (稀释比＝1:1000；

英国剑桥)、兔Bak抗体(稀释比＝1：1000；

英国剑桥)、小鼠Bax抗体(稀释比＝1:1000，

英国剑桥)及β-Actin抗体 (稀释比＝1:2000，Santa Cruz Biotechnology，美国加利福尼亚州)在4℃下过夜。在上述步骤之后，将其与HRP偶联抗体(稀释比＝1：10000；Cell Signaling Technology) 共同孵育1小时。通过增强的化学发光试剂(Invitrogen，英国苏格兰佩斯利)显示蛋白质谱带，使用Image J 1.46r软件(National Institutes ofHealth，美国马里兰州贝塞斯达)分析谱带。Cells from each group were collected, extracted and lysed in RIPA buffer (Cell Signaling Technologies, Beverly, MA, USA). Protein concentrations were determined using Bradford reagent (Bio-Rad Laboratory, Hercules, CA, USA). Briefly, cell lysates were mixed with 5x SDS loading buffer (4:1, v/v) and heated at 100°C for 5 minutes with the lid tightly closed. The thus treated lysate (20 μg) was subjected to 10% sodium dodecyl sulfonate tract-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, cell extracts from SDS-PAGE were transferred to nitrocellulose membranes (Merck Millipore, USA), combined with Wnt/β-Catenin Activated Target Antibody Sample Kit #8655 (dilution ratio = 1:1000; Cell Signaling Technology) , rabbit β-Catenin (dilution ratio = 1:1000; Cell Signaling Technology), mouse gammaH2A.X (phospho S139) [9F3] (dilution ratio = 1:1000;

Cambridge, UK), rabbit caspase-3 antibody (dilution ratio = 1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit caspase-8 antibody (dilution ratio = 1:1000; Cell Signaling Technology), rabbit caspase -9 antibody (dilution ratio=1:1000; Cell Signaling Technology), mouse PARP antibody (dilution ratio=1:1000;

Cambridge, UK), rabbit Bak antibody (dilution ratio = 1:1000;

Cambridge, UK), mouse Bax antibody (dilution = 1:1000,

Cambridge, UK) and β-Actin antibody (dilution = 1:2000, Santa Cruz Biotechnology, CA, USA) overnight at 4°C. Following the above steps, it was incubated with HRP-conjugated antibody (dilution ratio = 1:10000; Cell Signaling Technology) for 1 hour. Protein bands were visualized by enhanced chemiluminescence reagents (Invitrogen, Paisley, Scotland, UK) and analyzed using Image J 1.46r software (National Institutes of Health, Bethesda, MD, USA).

Result analysis

1. LC-MS/MS analysis of aloe vera extract

The constituents of aloe vera water extract were analyzed by LC-MS/MS. By comparing with the reference compounds, they were identified according to their retention time and cleavage mode of aloin A, aloin B, aloe verain and aloe verain. Aloe A, Aloe B, Aloe Ning and Aloe Vera in the aloe extract in this application were further quantified as markers, and their contents were 1249.0, 1080.0, 23.8 and 4.3 μg/g, respectively.

2. Aloe vera extract combined with HPT exerts a synergistic effect in HepG2 cells

The intrinsic cytotoxicity of aloe vera was determined by the cytotoxic MTT (3-[4,5-dimethyl-2-thiazole]-2,5 diphenyltetrazolium bromide) assay. HepG2 cells were treated with different concentrations of aloe vera (0-200.0 μg/mL) for different times (24, 48, 72 h).

In Figure 1, A represents the cell viability of HepG2 cells treated with different concentrations of aloe vera extract (0-200.0 μg/mL) for 24, 48 and 72 hours, the abscissa represents the concentration of aloe vera extract, and the ordinate represents the cell viability , it can be seen from the figure that when the concentration of aloe vera extract is less than or equal to 100.0 μg/mL, there is no obvious toxic effect on HepG2 cells.

B in Figure 1 represents cell survival for 48 hours treated with HPT alone or with composition 1 (10 μM HPT and 50 μg/ml aloe vera extract), composition 2 (10 μM HPT and 100 μg/ml aloe vera extract) As can be seen from the figure, HPT treatment alone or treatment with composition 1 and composition 2 will lead to a decrease in the survival rate of HepG2 cells, and the survival rate of HepG2 cells after treatment with the composition is even lower. effect.

In Figure 1, C represents the colonies formed by the cells of each treatment group, Petri dish 1 represents the control group, without adding aloe vera extract and HPT, Petri dish 2 represents the treatment with 50 μg/ml aloe vera extract, Petri dish 3 represents the treatment with 100 μg/ml aloe vera extract /ml of aloe vera extract, Petri dish 4 represents treatment with 10 μM HPT, Petri dish 5 represents co-treatment with 10 μM HPT and 50 μg/ml aloe vera extract, Petri dish 6 represents treatment with 10 μM HPT and 100 μg/ml of aloe vera extract was co-treated; as can be seen from the figure, the control group and the aloe vera extract alone treatment group, the cells grew well, and the treatment with 10 μ M of HPT would affect the growth of the cells, while the composition treatment would significantly affect the cell growth. grow.

D in the figure represents the number of cells in each group in the colony formation test. The count of cells forming colonies, the abscissa is each treatment group, and the ordinate is the number of cells. It can be seen from the figure that the combination of aloe-HPT significantly reduced the growth of HepG2 cells.

The interaction between aloe vera extract and HPT was assessed by the combination index (CI) method, where CI<1, CI=1 or CI>1 indicated synergy, additive effect or antagonism, respectively. Table 2 shows the IC50 values (half the maximal inhibitory concentration) and CI values for HPT treatment alone and for the combination treatment. From the IC ₅₀ and combination index (CI) values in the table, it can be seen that aloe vera extract and HPT have obvious synergistic effects on HepG2 cells.

Table 2. Cytotoxicity assessment of aloe vera extracts and compositions on HepG2 cells

Drug IC₅₀ CI value HPT 41.5±8.0 _ 50.0 μg/mL Aloe+HPT (composition 1) 10.4±2.9** 0.447±0.101 100.0 μg/mL Aloe+HPT (composition 2) 2.6±1.4** 0.311±0.001

Note: *P<0.05, **P<0.01, indicating a significant difference with the HPT group.

3. Aloe-HPT combination increases DNA damage in HepG2 cells

HPT is a DNA synthesis inhibitor that inhibits DNA topoisomerase I and causes DNA damage during DNA replication. Therefore, the degree of DNA damage caused by DNA strand breaks may reflect the cytotoxicity of HPT in cancer cells. To facilitate DNA damage analysis, comet assays were performed on HepG2 cells that were treated differently.

In Figure 2, A is the comet analysis of each group, the comet test image obtained by fluorescence microscope (original magnification: 200×), the figure is the control group, without adding aloe vera extract and HPT; with 50 μ g/ml of aloe vera Extract treatment; treated with 100 μg/ml aloe vera extract; treated with 40 μM HPT; co-treated with 10 μM HPT and 50 μg/ml aloe vera extract; with 10 μM HPT and 100 μg/ml Aloe vera extract co-processing. As can be seen from the figure, compared with the control group, there is no difference in the tail value of the group treated with aloe vera extract alone; after incubation for 1 h in the 40.0 μM HPT group alone, the tail in HepG2 cells can be observed; in the combination of aloe vera and HPT, it can be found longer tail.

In Fig. 2, B is the analysis diagram of the tail value, the abscissa is each treatment group, and the ordinate is the tail length; in Fig. 2, C is the analysis diagram of the tail moment value, the abscissa is each treatment group, and the ordinate is the tail moment value; It can be seen that compared with the control group, there is no difference in the tail value of the group treated with aloe vera extract alone; after incubation for 1 h in the 40.0 μM HPT group alone, the tail in HepG2 cells can be observed; in the combination of aloe vera + HPT, a longer tail can be found tail length and higher tail moment values.

In Figure 2, D is the immunofluorescence staining of DAPI and γH2AX in HepG2 cells (original magnification: 400×), from left to right, DAPI, γH2AX and Merge imaging (Merge is the imaging overlay of DAPI and γH2AX); immunofluorescence The staining results showed that aloe vera extract combined with HPT increased the level of γH2AX in a dose-dependent manner in HepG2 cells.

E in Figure 2 is the expression of γH2AX in Western blotting, in which the horizontal order from left to right is the control group, without the addition of aloe vera extract and HPT; treated with 100 μg/ml of aloe vera extract; treated with 15 μM HPT ; Co-treatment with 15 μM HPT and 50 μg/ml aloe vera extract; co-treatment with 15 μM HPT and 100 μg/ml aloe vera extract. Compared with the HPT alone group, the co-treatment group with HPT and aloe vera extract (ie the composition group) had significantly higher levels of γH2AX protein.

It can be seen from Figure 2 that the synergistic effect of aloe vera extract and HPT on DNA damage is related to the degree of accumulation of γH2AX.

10-Hydroxycamptothecin (HPT) is a camptothecin derivative, a specific inhibitor of topoisomerase I, which can form a cleavable complex between DNA and nuclear topoisomerase I enzymes after DNA cleavage The compound binds and stabilizes, preventing DNA resealing and allowing cleavable complexes to accumulate. Thus, the cleavable complex hinders the repair of temporary DNA single-strand breaks and prevents DNA replication, resulting in fatal DNA double-strand breaks, which in turn activate endonucleases, which initiate further DNA breaks and ultimately lead to cellular die. The comet assay (also known as the single cell gel assay (SCG)) is a sensitive and rapid method for detecting DNA strand breaks in single cells. The length of the comet tail was positively correlated with the degree of damage, and the data from the comet assay demonstrated that although aloe vera alone had no effect on DNA damage, aloe vera was able to stimulate HPT-induced DNA damage in a dose-dependent manner.

γH2AX is a basic histone with a molecular weight of 14 kD and a member of the H2 histone family. This nuclear protein is synthesized during the G1 and S phases of the cell cycle and plays an important role in reorganization between immunoglobulin transition regions. After double-strand DNA breaks, γH2AX is phosphorylated on serine 139. After treatment with the composition, the formation of γH2AX increased, thus further illustrating that aloe vera extract significantly promoted HPT-induced DNA double-strand breaks in HepG2 cells. The aloe extract and the HPT composition of the present invention have a synergistic effect on inducing DNA double-strand breaks in HepG2 cells.

4. Effects of aloe+HPT on cell cycle arrest

To investigate whether the antiproliferative effect of HPT on HepG2 cells is triggered by cell cycle arrest, cells treated with aloe vera extract, HPT alone, and the combination for 48 hours were analyzed using propidium iodide (PI)-stained flow cytometry Periodic distribution.

Figure 3 is the percentage of HepG2 cells at different stages, the abscissa is each group (respectively, control group, without adding aloe vera extract and HPT; treated with 50 μg/ml aloe vera extract; treated with 100 μg/ml aloe vera extract ; treated with 40 μM of HPT; co-treated with 10 μM of HPT and 50 μg/ml of aloe vera extract; co-treated with 10 μM of HPT and 100 μg/ml of aloe vera extract) at different periods, the ordinate is cells percentage.

Dysregulation of cell cycle progression and apoptosis are common features of cancer, and as shown in Figure 3, exposure of HepG2 cells to HPT resulted in a decrease in G0/G1 phase and an increase in G2/M phase compared to controls. In the cells treated with aloe extract and HPT, the proportion of cells in S phase increased, while the proportion of cells in G2/M phase decreased. Furthermore, treatment with the composition resulted in significant accumulation in the G0/G1 phase and significant depletion in the G2/M phase compared to the HPT alone-treated group. The results showed that the combined treatment of aloe vera extract and HPT significantly induced S-phase arrest in HepG2 cells compared with the control group.

5. Aloe-HPT induces caspase-dependent apoptosis

To identify whether the growth inhibitory effect of HPT-Aloe was caused by apoptosis, the occurrence of apoptosis was confirmed by flow cytometry stained with Annexin V & DeadCell dual reagents.

In Figure 4, A is the distribution map of apoptotic cells before and after treatment, and the abscissa in the figure is each group (respectively, control group, without adding aloe vera extract and HPT; with 50 μg/ml of aloe vera extract treatment group; with 100 μg/ml of aloe vera extract treatment group; g/ml of aloe vera extract; treated with 40 μM of HPT; co-treated with 10 μM of HPT and 50 μg/ml of aloe vera extract; with 10 μM of HPT and 100 μg/ml of aloe vera extract Co-treatment group) in early, middle and late stages, the ordinate is the percentage of cells. As can be seen from A in Figure 4, the percentages of early and late apoptotic cells were significantly increased after exposure to HPT (with or without aloe vera extract). Because caspase activation is considered to be a common apoptotic pathway in tumor cells. Western blot was used to assess the functional role of caspases in apoptosis.

In Figure 4, B is the evaluation of the levels of PARP, caspase-3, caspase-9, Cleaved-caspase-9 and Bax by Western blot analysis, β-actin (β-actin) was used as a loading control, in the figure, horizontal In turn, the control group was used without adding aloe vera extract and HPT; treated with 100 μg/ml aloe vera extract; treated with 15 μM HPT; co-treated with 15 μM HPT and 50 μg/ml aloe vera extract; M of HPT and 100 μg/ml of aloe vera extract were co-treated. In Figure 4, C represents the representative graph of the relative expression levels of PARP, Cleaved-PARP, caspase-3, caspase-9, Cleaved-caspase-9 and Bax, and the abscissa is PARP, Cleaved-PARP, caspase-3, caspase in each group -9, the relative expression of Cleaved-caspase-9 and Bax, the ordinate is the relative expression level value.

As can be seen from B and C in Figure 4, PARP, caspase-9 and Bax in cells treated with aloe extract + HPT were significantly reduced, while Cleaved-PARP, Cleaved-caspase-9 and caspase-3 were strongly activated. These results confirmed that aloe vera extract could promote HPT-induced apoptosis through a cysteine protease-dependent mechanism.

6. Aloe-HPT composition inhibits WNT/β-catenin pathway in HepG2 cells

In order to further understand the mechanism of the synergistic effect of aloe vera and HPT, the WNT/β-catenin signaling pathway proteins were studied by western blotting experiments.

In Figure 5, A is the protein level map in the WNT/β-catenin pathway assessed by Western blotting, with β-actin as the loading control, and the horizontal order in the figure is the control group, without adding aloe vera extract and HPT; with 100 μg/ ml of aloe vera extract; treated with 15 μM HPT; co-treated with 15 μM HPT and 50 μg/ml aloe vera extract; co-treated with 15 μM HPT and 100 μg/ml aloe vera extract, longitudinal Each protein; in Figure 5, B is the relative expression level of WNT/β-catenin pathway protein, and the abscissa is each group (control group, without adding aloe vera extract and HPT; with 100 μg/ml of aloe vera extract treatment; with 15 μM of HPT; co-treated with 15 μM of HPT and 50 μg/ml of aloe vera extract; with 15 μM of HPT and 100 μg/ml of aloe vera extract) of Met, β-catenin, c-Jun , c-Myc oncogene, MMP7 protein, the ordinate is the relative expression level value.

Inhibition of the canonical WNT pathway results in the arrest of β-catenin translocation to the nucleus and suppresses the activity of nuclear TCF/LEF transcription factors, thereby disrupting target genes in Wnt-dependent signaling, including CD44, cyclin D1, c-Jun, LEF1 , Met, MMP-7, c-Myc and TCF1/TCF7. As can be seen from A and B in Figure 5, the aloe extract and HPT composition effectively induced the down-regulation of the protein levels of Met, β-catenin and c-Myc; up-regulated in a dose-dependent manner. The synergistic effect of the aloe extract and the HPT composition is related to the regulation of the cellular WNT/β-catenin signaling pathway, and the composition can inhibit the WNT/β-catenin pathway in HepG2 cells.

Claims (4)

1. An anti-liver cancer composition containing aloe extract is characterized by comprising aloe extract and 10-hydroxycamptothecin;

the content of the aloe extract is 50-400 μ g/ml, and the content of the 10-hydroxycamptothecin is 2.5-50 μ M;

the aloe extract is obtained by water extraction.

2. The composition of claim 1, wherein the aloe vera extract is present in an amount of 50-150 μ g/ml and the 10-hydroxycamptothecin is present in an amount of 5-30 μ M.

3. An anti-hepatoma drug comprising the composition of claim 1 or 2 and an adjuvant.

4. Use of the composition of claim 1 or 2 for the preparation of a medicament against liver cancer.

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