Use of proanthocyanidins in the preparation of anti-tumor stem cell drugs

Abstract

The application belongs to the technical field of biological medicine, and relates to application of procyanidine in preparation of an anti-tumor stem cell medicine, and is characterized in that the anti-tumor stem cell medicine is used for inhibiting tumor progression and / or inhibiting tumor stem cells. The anti-tumor stem cell medicine prepared by using procyanidine can inhibit tumor cell proliferation and tumorigenicity, inhibit tumor stem cells, and improve chemotherapeutic sensitivity of oxaliplatin.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of procyanidine in preparation of anti-tumor stem cell drugs. BACKGROUND

[0002] Colon cancer (CRC) is a common malignant tumor of the digestive tract. Its occurrence and development are affected by genetic, lifestyle, environment and other comprehensive factors. It is the third largest cancer in the world and ranks second in the world in terms of cancer-related deaths, which seriously threatens human life and health. Many studies have shown that the conventional CRC treatment including chemotherapy and radiotherapy has poor effect, which may be related to the failure to eradicate cancer stem cells (CSCs).

[0003] More and more studies have found that colon cancer stem cells (CCSCs) are a small subpopulation of cells with self-renewal, unlimited proliferation and differentiation capacity in colon cancer. CCSCs have strong radiotherapy and chemotherapy resistance, can maintain the growth of tumor cells, accelerate tumor progression, and cause metastasis and recurrence after cancer treatment. Although traditional cancer treatment methods can reduce the size of the tumor, they cannot target CSCs to eradicate cancer. Unfortunately, there are not many targeted drugs and treatment methods for CCSCs. Therefore, elucidating the regulatory mechanism of CCSCs evolution and developing treatment methods for CCSCs are important breakthroughs for effective treatment of CRC.

[0004] Procyanidine (PC) is a class of flavonoids. Studies have shown that plant anthocyanins have multiple pharmacological activities such as free radical scavenging, antioxidant, anti-aging, anti-inflammatory and inhibition of bacterial growth. In addition, more and more data also show that PC has an anticancer effect in various malignant tumors. PC can reverse epithelial-mesenchymal transition (EMT) by inhibiting the TGF-beta signaling pathway, and inhibit the migration and invasion of bladder cancer cells. Some studies have also found that PC can induce apoptosis and cell cycle arrest of non-small cell lung cancer cells through the JAK2 / STAT3 axis. However, the role and mechanism of PC in the development of CRC and CCSCs are not clear. SUMMARY

[0005] The application provides application of procyanidine in preparation of anti-tumor stem cell drugs to overcome the deficiencies of the prior art. The anti-tumor stem cell drug prepared from procyanidine can inhibit tumor cell proliferation and tumorigenicity, and inhibit tumor stem cells, and improve the chemotherapy sensitivity of oxaliplatin.

[0006] To this end, the present application provides an application of procyanidins in the preparation of an anti-tumor stem cell drug for inhibiting tumor progression and / or inhibiting tumor stem cells.

[0007] In some embodiments of the present application, the anti-tumor stem cell drug inhibits tumor progression and / or inhibits tumor stem cells by inhibiting the Wnt / β-catenin signaling pathway.

[0008] In some embodiments of the present application, the anti-tumor stem cell drug inhibits the Wnt / β-catenin signaling pathway by inhibiting the protein expression of β-catenin, p-GSK3β, and DVL1, DVL2, DVL3.

[0009] In some embodiments of the present application, the anti-tumor stem cell drug inhibits tumor stem cells by inhibiting the protein expression of tumor stem cell surface markers CD133 and CD44.

[0010] In some embodiments of the present application, the anti-tumor stem cell drug inhibits tumor stem cells by inhibiting the expression of key transcription factors c-Myc, Nanog, and OCT4 of tumor stem cells.

[0011] In some embodiments of the present application, the tumor progression includes the proliferation and tumorigenicity of tumor cells.

[0012] In some embodiments of the present application, the tumor includes colorectal cancer.

[0013] According to the present application, the anti-tumor stem cell drug is used to inhibit the progression of colorectal cancer and / or inhibit colorectal cancer stem cells.

[0014] According to the present application, the anti-tumor stem cell drug is used to inhibit the proliferation and tumorigenicity of colorectal cancer cells and / or inhibit colorectal cancer stem cells.

[0015] In some embodiments of the present application, the anti-tumor stem cell drug comprises a therapeutically effective amount of procyanidins and a pharmaceutically acceptable excipient or adjuvant.

[0016] In some embodiments of the present application, the excipient or adjuvant comprises at least one of starch, dextrin, lactose, mannitol, sucrose, microcrystalline cellulose, sodium hydroxymethyl starch, talc.

[0017] In some embodiments of the present application, the concentration of procyanidins in the anti-tumor stem cell drug is 5-80 μM.

[0018] According to the present application, the concentration of the procyanidins is for example 5 μM, 8 μM, 10 μM, 12 μM, 15 μM, 18 μM, 20 μM, 22 μM, 25 μM, 28 μM, 30 μM, 32 μM, 35 μM, 38 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM.

[0019] In some embodiments of the present application, the anti-tumor stem cell drug is used for combined administration with one or more additional chemotherapeutic drugs or radiotherapy drugs.

[0020] According to the present application, the anti-tumor stem cell drug is used for combined administration with oxaliplatin.

[0021] According to the present application, the molar ratio of the procyanidins to oxaliplatin is 0.5-1.5:1, preferably 0.5-1:1.

[0022] According to the present application, the molar ratio of the procyanidins to oxaliplatin is for example 0.5:1, 0.6:1, 0.8:1, 1:1, 1.2:1, 1.5:1.

[0023] Advantages of the present application:

[0024] (1) The present application provides the use of procyanidins in the preparation of an anti-tumor stem cell drug. The anti-tumor stem cell drug prepared from procyanidins can be used to inhibit tumor cell proliferation and tumorigenicity, and inhibit tumor stem cells.

[0025] (2) The present application provides the use of procyanidins in the preparation of an anti-tumor stem cell drug. The anti-tumor stem cell drug prepared from procyanidins can be used to inhibit tumor cell proliferation and tumorigenicity by inhibiting the Wnt / β-catenin signaling pathway, and inhibit tumor stem cells.

[0026] (3) The present application provides the use of procyanidins in the preparation of an anti-tumor stem cell drug. The anti-tumor stem cell drug prepared from procyanidins can be used to inhibit colorectal cancer cell proliferation and tumorigenicity, and inhibit colorectal cancer stem cells; and can be used to inhibit colorectal cancer cell proliferation and tumorigenicity by inhibiting the Wnt / β-catenin signaling pathway, and inhibit colorectal cancer stem cells.

[0027] (4) The present application provides the use of procyanidins in the preparation of an anti-tumor stem cell drug. The anti-tumor stem cell drug prepared from procyanidins can be used in combination with oxaliplatin, which can improve the chemosensitivity of oxaliplatin. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1Figure showing the results of the effect of PC on tumor stem cells in Example 1; wherein, a- image of HT29 cells suspended to form spheres, b- image of HCT116 cells suspended to form spheres, c- number of tumor spheres in HT29 cells, d- number of tumor spheres in HCT116 cells.

[0029] Figure 2 Figure showing the results of the effect of PC on the expression of molecular markers of tumor stem cells in Example 1; wherein, a- Western blotting of CD133 and CD44 proteins in HT29 cells, b- Western blotting of CD133 and CD44 proteins in HCT116 cells, c- protein expression level of CD133 and CD44 in HT29 cells, d- protein expression level of CD133 and CD44 in HCT116 cells.

[0030] Figure 3 Figure showing the results of the effect of PC on the expression of molecular markers of tumor stem cells in Example 1; wherein, a- Western blotting of c-Myc, Nanog and OCT4 proteins in HT29 cells, b- Western blotting of c-Myc, Nanog and OCT4 proteins in HCT116 cells, c- protein expression level of c-Myc, Nanog and OCT4 in HT29 cells, d- protein expression level of c-Myc, Nanog and OCT4 in HCT116 cells.

[0031] Figure 4 Figure showing the results of the effect of PC on tumor cell proliferation in Example 2; wherein, a- survival rate of HT29 cells, b- survival rate of HCT116 cells.

[0032] Figure 5 Figure showing the results of the effect of PC on tumor cell proliferation in Example 2; wherein, a- image of HT29 cell clones, b- image of HCT116 cell clones, c- number of HT29 cell clones, d- number of HCT116 cell clones.

[0033] Figure 6 Figure showing the results of the effect of PC on tumorigenicity of tumor cells in Example 3; wherein, a- tumor growth curve in nude mice, b- image of ex vivo tumor, c- volume of ex vivo tumor.

[0034] Figure 7Figure of the results of the influence of PC on the Wnt / β-catenin signaling pathway in Example 4; wherein, a, Western blotting diagram of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 proteins in HT29 cells, b, Protein expression levels of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 in HT29 cells, c, Western blotting diagram of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 proteins in HCT116 cells, d, Protein expression levels of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 in HCT116 cells.

[0035] Figure 8 Figure of the results of the influence of PC on the Wnt / β-catenin signaling pathway in Example 4; wherein, a, Western blotting diagram of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 proteins in HT29 cells, b, Protein expression levels of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 in HT29 cells, c, Western blotting diagram of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 proteins in HCT116 cells, d, Protein expression levels of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 in HCT116 cells.

[0036] Figure 9 Figure of the results of the influence of PC on tumor stem cells through the Wnt / β-catenin signaling pathway in Example 5; wherein, a, Image of HCT116 and HT29 cells suspended into spheres, b, Number of tumor spheres in HCT116 and HT29 cells.

[0037] Figure 10 Figure of the results of the influence of PC on tumor stem cell molecular marker expression through the Wnt / β-catenin signaling pathway in Example 5; wherein, a, Western blotting diagram of c-Myc, Nanog and OCT-4 proteins in HT29 cells, b, Protein expression levels of c-Myc, Nanog and OCT-4 in HT29 cells, c, Western blotting diagram of c-Myc, Nanog and OCT-4 proteins in HCT116 cells, d, Protein expression levels of c-Myc, Nanog and OCT-4 in HT29 cells.

[0038] Figure 11Figure of the result of the influence of PC on the proliferation of tumor cells through the Wnt / β-catenin signaling pathway in Example 6; wherein a, HCT116 cell survival rate, b, HT29 cell survival rate.

[0039] Figure 12 Figure of the result of the influence of PC on the proliferation of tumor cells through the Wnt / β-catenin signaling pathway in Example 6; wherein a, HCT116 and HT29 cell clone image, b, HCT116 and HT29 cell clone number.

[0040] Figure 13 Figure of the result of the inhibition of tumor cells by combined administration of PC and L-OHP in Example 7; wherein a, HT29 cell inhibition rate, b, HT29 cell IC50, c, HCT116 cell inhibition rate, d, HCT116 cell IC50.

[0041] Figure 14 Figure of the result of the inhibition of tumor cells by combined administration of PC and L-OHP through the Wnt / β-catenin signaling pathway in Example 7; wherein a, HT29 cell inhibition rate, b, HT29 cell IC50, c, HCT116 cell inhibition rate, d, HCT116 cell IC50. DETAILED DESCRIPTION

[0042] In order to make the present application more easily understood, the present application will be described in detail below with reference to examples, which are only illustrative and do not limit the scope of application of the present application.

[0043] The present application provides the use of procyanidin in the preparation of an anti-tumor stem cell drug. The anti-tumor stem cell drug prepared from procyanidin can inhibit tumor cell proliferation and tumorigenicity, and inhibit tumor stem cells, and improve the chemosensitivity of oxaliplatin.

[0044] 1. Cell culture:

[0045] Colorectal cancer cell lines HCT116 and HT29 cells were obtained from the Chinese Academy of Sciences Cell Bank. HT29 cells were cultured in McCoy's 5A medium (VivaCell) containing 10% FBS (Gibco) and 1% penicillin-streptomycin (Hyclone); HCT116 cells were cultured in PMI-1640 medium (Hyclone) containing 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO2. Cells were digested with trypsin (Hyclone) and subcultured every 3-4 days.

[0046] 2. Reagents and antibodies:

[0047] PC, L-OHP, PEG300, and Tween-80 were purchased from MedChem Express. LiCl was purchased from Adamas-beta. Primary antibodies for CD133, CD44, OCT-4, c-Myc, Nanog, and p-GSK3β (Ser9) were purchased from Cell Signaling Technology. Primary antibodies for β-catenin, DVL1, DVL2, DVL3, and GAPDH were from Santa Cruz. GSK3β antibody was purchased from ABclone. α-tublin antibody was purchased from Beyotime. Primary antibody diluent, skim milk powder, protease inhibitors, and phosphatase inhibitors were all purchased from Beyotime. Secondary antibodies were purchased from Zhongshan Jinqiao. DMSO was purchased from Solarbio.

[0048] 3. Statistical Analysis:

[0049] All experiments were independently repeated three times or as specified in the legend. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using t-tests (two-tailed) or one-way ANOVA. GraphPad Prism 6.0 software was used for statistical analysis. *p < 0.05, **p < 0.01 were considered statistically significant.

[0050] Example 1: Tumor Stem Cell Experiment

[0051] (1) The effect of PC on tumor stem cells was analyzed by suspension spheroidization experiment. The specific procedure is as follows: 1×10 4 HT29 and HCT116 cells were seeded into low-adhesion 6-well plates and cultured in DMEM / F12 serum-free medium (Gibco) containing 4 μg / mL insulin (Beyotime), 2% B27 (Thermo Scientific), 40 ng / mL EGF (Beyotime) and bFGF (Beyotime), and 1% penicillin-streptomycin. A series of PC concentrations (0, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM) were added to the HCT116 and HT29 cells, respectively, and cultured for 7 days. Representative images of cell spheroids were taken under a microscope, and spheroids with a diameter of up to 50 μm were counted. Statistical analysis results are as follows: Figure 1 As shown.

[0052] like Figure 1 The results (a, b, c, d) show that PC significantly reduced the size and number of tumor spheres in HT29 and HCT116 cells in a concentration-dependent manner. These data indicate that PC inhibits the phenotypic transformation of colorectal cancer stem cells.

[0053] (2) Further, the effect of PC on the expression of molecular markers of tumor stem cells was analyzed by Western blotting, and the specific process was as follows:

[0054] After HCT116 and HT29 cells were treated with a series of concentrations of PC (0, 5 μΜ, 10 μΜ, 20 μΜ, 40 μΜ, 80 μΜ) for 72 h, the cells were washed with PBS three times, 100 μL of RIPA strong lysis solution containing 2% protease inhibitor and 2% phosphatase inhibitor was added to each well, and the cells were lysed on ice for 15 min. After the cells at the bottom of the well were scraped, the lysis was continued on ice for 15 min. Then the whole lysis solution was centrifuged, and the supernatant was used as the protein sample. The protein concentration was determined by BCA kit (Bi Yun Tian), and then 60 μg of the sample was loaded for SDS-PAGE electrophoresis, followed by transfer to a PVDF membrane (Millipore). The membrane was blocked with 5% skim milk, and the corresponding molecular weight proteins on the membrane were cut horizontally. The corresponding primary antibody was incubated at 4°C overnight. The PVDF membrane was removed from the primary antibody, washed with 1x TBST three times, and the secondary antibody was incubated for 2 h. The PVDF membrane was washed with 1x TBST three times, and α-tubulin or GAPDH was used as an internal control. The protein signal was detected by ECL luminescent liquid and statistical analysis of the protein expression of CD133, CD44 and c-Myc, Nanog and OCT-4 in HT29 and HCT116 cells, and the statistical analysis results are shown in Figure 2 and Figure 3

[0055] As shown in the results of Figure 2 (a, b, c, d), after PC treatment of HT29 and HCT116 cells, the protein expression levels of stem cell surface markers CD133 and CD44 in the cells were significantly reduced. As shown in the results of Figure 3 (a, b, c, d), after PC treatment, the protein expression of stem cell key transcription factors c-Myc, Nanog and OCT-4 in HT29 and HCT116 cells was significantly down-regulated. These data further indicated that PC inhibited the protein expression of colorectal cancer stem cell surface markers CD133 and CD44 and the protein expression of stem cell key transcription factors Nanog, c-Myc and OCT4, and PC could effectively inhibit colorectal cancer stem cells.

[0056] Example 2 Proliferation test of tumor cells

[0057] (1) The effect of PC on tumor cell proliferation was analyzed by CCK-8 experiment, and the specific process was as follows: 1x10 4 ​HCT116 and HT29 cells were seeded into 96-well plates and cultured for 18-24 h, and then PC at a series of concentrations (0, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM) was added to the HCT116 and HT29 cells, respectively, and cultured for 48 h. Then, CCK-8 working solution was prepared by mixing culture medium at a ratio of 10:1, and added to the 96-well plates. After incubation in the dark for 2 h in an incubator, the absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated. The statistical analysis results are shown in Figure 4

[0058] As shown in Figure 4 (a and b), PC inhibited the survival of HT29 and HCT116 cells in a concentration-dependent manner. These data suggest that PC inhibits the proliferation of colorectal cancer cells.

[0059] (2) Further, the effect of PC on tumor cell proliferation was analyzed by a colony formation experiment, and the specific process was as follows: 1×10 3 HCT116 and HT29 cells were seeded into 6-well plates and cultured for 24 h, and then PC at a series of concentrations (0, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM) was added to the HCT116 and HT29 cells, respectively, and cultured for 10 days. Then, the original culture medium was discarded, the cells were washed twice with PBS, and the cells were fixed with 4% paraformaldehyde. Finally, the cells were stained with crystal violet (Biyun Tian) and photographed, and the number of colonies was counted. The statistical analysis results are shown in Figure 5

[0060] As shown in Figure 5 (a, b, c, d), PC significantly reduced the number of colonies of HT29 and HCT116 cells. These data suggest that PC inhibits the proliferation of colorectal cancer cells.

[0061] Example 3 Tumorigenicity test of tumor cells

[0062] The effect of PC on the tumorigenicity of tumor cells was analyzed by a xenotransplant tumor model experiment, and the specific process was as follows:

[0063] A total of 12 4-week-old male BALB / c nude mice were purchased from Zhejiang Vantoll Life Animal Technology Co., Ltd., and all the animals were raised in a specific pathogen-free environment for research. Each nude mouse was subcutaneously injected with 1×10 7 ​​ / 200 μL of HT29 cell suspension. After 7 days of inoculation, the mice were randomly divided into two groups, 6 in each group; the administration group was treated with PC at a concentration of 100 mg / kg by gavage, and the control group was treated with solvent (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% normal saline) by gavage, once a day. The maximum length (a) and the short diameter (b) of the tumor of the nude mice were measured and recorded every other day, and the tumor volume of the nude mice was calculated (formula: V = 0.52 x a x b 2 ); the nude mice were euthanized after 21 days of experiment, and the tumor was dissected out and the final volume was measured. The statistical analysis results are shown in Figure 6 .

[0064] As shown in the results of Figure 6 (a), the tumor growth rate of the PC administration group of nude mice was significantly lower than that of the control group of nude mice; as shown in the results of Figure 6 (b and c), the final tumor volume of the PC administration group was significantly reduced compared with the control group. These data indicate that PC inhibits the growth and tumorigenicity of colorectal cancer cells in vivo.

[0065] Example 4 Wnt / β-catenin signaling pathway test

[0066] (1) Effect of PC on Wnt / β-catenin signaling pathway The Western blotting method was used for analysis, and the specific process was as follows: HCT116 and HT29 cells were treated with a series of concentrations of PC (0, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM) for 72 h, and the same Western blotting method steps as in Example 1 were used, with GAPDH as an internal control, to detect protein signals and statistically analyze the protein expression of β-catenin, p-GSK3β, GSK3β and DVL1, DVL2, DVL3 in HCT116 and HT29 cells, and the statistical analysis results are shown in Figure 7 .

[0067] As shown in the results of Figure 7 (a, b, c, d), after PC treatment, the protein expression of β-catenin, p-GSK3β and dishevelled proteins DVL1, DVL2, DVL3 in HCT116 and HT29 cells was significantly reduced. These results indicate that PC inhibits the protein expression of β-catenin, p-GSK3β and DVL1, DVL2, DVL3 in colorectal cancer cells, and PC can inhibit the Wnt / β-catenin signaling pathway in colorectal cancer cells.

[0068] (2) Further, the influence of PC on Wnt / β-catenin signaling pathway was analyzed by Western blotting method, the specific process as follows: the experimental group was treated with PC (10 μM) + LiCl (10 mM) for 72 h, and the control group was treated with 10 μM PC for 72 h. The same Western blotting method as in Example 1 was used, and α-tubulin was used as an internal control. The protein signals were detected, and the protein expression of β-catenin, p-GSK3β, GSK3β, DVL1, DVL2 and DVL3 in HCT116 and HT29 cells was statistically analyzed. The statistical analysis results are shown in Figure 8 .

[0069] As shown in Figure 8 (a, b, c, d), LiCl, as an agonist of Wnt / β-catenin pathway, activates the Wnt / β-catenin pathway by phosphorylating the Ser9 site of GSK3β. In HT29 and HCT116 cells, LiCl effectively reversed the down-regulation of β-catenin and p-GSK3β protein induced by PC. These results show that PC inhibits the Wnt / β-catenin signaling pathway in colorectal cancer cells.

[0070] Example 5 Influence of Wnt / β-catenin signaling pathway on tumor stem cells

[0071] (1) The influence of PC on tumor stem cells through Wnt / β-catenin signaling pathway was analyzed by suspension sphere formation experiment, the specific process as follows: HT29 and HCT116 cells were cultured by the same method as in Example 1; the control group was treated with 10 μM PC alone, and the experimental group was treated with PC (10 μM) + LiCl (10 mM) for 7 days. Representative images of tumor spheres were taken, and spheres with a diameter of 50 μm in one sample were counted under a microscope. The statistical analysis results are shown in Figure 9 .

[0072] As shown in Figure 9 (a and b), compared with HCT116 and HT29 cells treated with PC alone, the size and number of tumor spheres in HCT116 and HT29 cells treated with LiCl and PC increased, and the activation of Wnt / β-catenin signaling pathway could effectively reverse the suspension sphere formation ability of PC-inhibited HT29 and HCT116 cells, further proving that PC inhibits the phenotypic transformation of colorectal cancer stem cells through Wnt / β-catenin signaling pathway.

[0073] (2) Further, the effect of PC on tumor stem cells through Wnt / p-catenin signaling pathway was analyzed by Western blotting method, and the specific process was as follows: 10 μM of PC was used in the control group, and PC (10 μM) + LiCl (10 mM) was used in the experimental group, and HCT116 and HT29 cells were treated. The same Western blotting method steps as in Example 1 were used, α-tubulin was used as an internal control, the protein signal was detected, and the protein expression of c-Myc, Nanog and OCT-4 in HCT116 and HT29 cells was statistically analyzed, and the statistical analysis results are shown in Figure 10 .

[0074] As shown in Figure 10 (a, b, c, d), compared with the control group, the protein expression levels of c-Myc, Nanog and OCT-4 in HT29 and HCT116 cells treated with PC were significantly down-regulated, but the down-regulation could be significantly improved after using LiCl. These results show that enhancing the activity of Wnt / p-catenin pathway can block the inhibitory effect of PC on CRC stem cells, and PC inhibits the phenotypic transformation of colorectal cancer stem cells through Wnt / p-catenin signaling pathway.

[0075] Example 6 Effect of Wnt / p-catenin signaling pathway on tumor proliferation test

[0076] (1) The effect of PC on tumor cell proliferation through Wnt / p-catenin signaling pathway was analyzed by CCK-8 experiment, and the specific process was as follows: the experimental group was pretreated with LiCl (10 mM) 24 h in advance, and then a series of concentrations of PC (0, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM) were added to HCT116 and HT29 cells for 48 h; the control group directly used a series of concentrations of PC (0, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM) to add to HCT116 and HT29 cells for 48 h; the same CCK-8 experiment steps as in Example 2 were used to calculate the cell viability. The statistical analysis results are shown in Figure 11 .

[0077] As shown in Figure 11 (a and b), LiCl as an agonist of Wnt / p-catenin signaling pathway rescued the inhibitory effect of PC on cell proliferation, further proving that PC inhibits the proliferation of colorectal cancer cells through Wnt / p-catenin signaling pathway.

[0078] (2) Further, the effect of PC on tumor cell proliferation through Wnt / β-catenin signaling pathway was analyzed by colony formation experiment, the specific process as follows: the control group used 10 μM of PC, the experimental group used PC (10 μM) + LiCl (10 mM), respectively, HCT116 and HT29 cells were treated for 10 days, the same colony formation experiment as in Example 2 was used for detection. The statistical analysis results are shown in Figure 12 .

[0079] As shown in Figure 12 (a and b), compared with HCT116 and HT29 cells treated with PC alone, the clonogenicity of HCT116 and HT29 cells treated with LiCl and PC was enhanced, the activation of Wnt / β-catenin signaling pathway could effectively reverse the inhibition of PC on the clonogenicity of HT29 and HCT116 cells, further proving that PC inhibited the proliferation of colorectal cancer cells through Wnt / β-catenin signaling pathway.

[0080] Example 7 PC combined with L-OHP administration experiment

[0081] (1) The inhibitory effect of PC combined with L-OHP on tumor cells was analyzed by CCK-8 experiment, the specific process as follows: the experimental group used a series of concentrations of L-OHP (0, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM) and a series of concentrations of PC (0, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM) were added to HCT116 and HT29 cells for 48 h; the control group directly used a series of concentrations of L-OHP (0, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM) were added to HCT116 and HT29 cells for 48 h; the same CCK-8 experiment method as in Example 2 was used for detection and calculation of cell viability. The statistical analysis results are shown in Figure 13 .

[0082] As shown in Figure 13 (a and c), compared with L-OHP alone, PC combined with L-OHP had a greater inhibitory effect on HCT116 and HT29 cells; as shown in Figure 13 (b and d), compared with L-OHP alone, PC combined with L-OHP significantly reduced the IC50 of HCT116 and HT29 cells. These results showed that PC enhanced the inhibitory effect of L-OHP on HT29 and HCT116 cells, and PC combined with L-OHP improved the sensitivity of colorectal cancer cells to L-OHP.

[0083] (2) PC combined with L-OHP inhibited the tumor cells through Wnt / β-catenin signaling pathway The CCK-8 experiment was used to analyze the inhibition of HCT116 and HT29 cells after LiCl treatment, and the specific process was as follows: the experimental group was pretreated with LiCl (10 mM) 24 h in advance, and then a series of concentrations of PC (0, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM) and a series of concentrations of L-OHP (0, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM) were added to HCT116 and HT29 cells for 48 h; the control group 1 directly used a series of concentrations of L-OHP (0, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM), and the control group 2 directly used a series of concentrations of L-OHP (0, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM) and a series of concentrations of PC (0, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM) were added to HCT116 and HT29 cells for 48 h; the same CCK-8 experiment procedure as in Example 2 was used to calculate the cell viability. The statistical analysis results are shown in Figure 14

[0084] As shown in the results of Figure 14 (a and c), the inhibition rate of HCT116 and HT29 cells was reduced after LiCl treatment compared with the combined administration of PC and L-OHP alone; as shown in the results of Figure 14 (b and d), the IC50 of HCT116 and HT29 cells was up-regulated after LiCl treatment compared with the combined administration of PC and L-OHP alone. These results show that LiCl eliminates the effect of PC on the improvement of the sensitivity of cells to L-OHP chemotherapy, and PC combined with L-OHP improves the sensitivity of colorectal cancer cells to L-OHP through the Wnt / β-catenin signaling pathway.

[0085] It should be noted that the above-described examples are only used to explain the present application and do not constitute any limitation on the present application. The present application is described by referring to typical examples, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present application can be modified as specified within the scope of the claims of the present application, and the present application can be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials, and examples, it does not mean that the present application is limited to the specific examples disclosed therein, but rather, the present application can be extended to all other methods and applications having the same function.​

Claims

1. The use of proanthocyanidins in combination with oxaliplatin in the preparation of a drug against tumor stem cells, characterized in that, The anti-tumor stem cell drug is used for inhibiting tumor progression and / or inhibiting tumor stem cells; the tumor is colorectal cancer; The concentration of procyanidins in the anti-tumor stem cell drug is 5-80 µM; The molar ratio of procyanidins to oxaliplatin is 0.5-1.5:

1.

2. Use according to claim 1, characterized in that, The combination of procyanidins and oxaliplatin improves the sensitivity of colorectal cancer cells to L-OHP through the Wnt / β-catenin signaling pathway.

3. Use according to claim 1, characterized in that, The anti-tumor stem cell drug comprises a therapeutically effective amount of procyanidins and a pharmaceutically acceptable excipient.

4. Use according to claim 1, characterized in that, The molar ratio of procyanidins to oxaliplatin is 0.5-1:

1.

5. The use according to claim 1, characterized in that, The molar ratio of procyanidins to oxaliplatin is 0.5:

1.

6. Use according to claim 1, characterized in that, The molar ratio of procyanidins to oxaliplatin is 0.6:

1.

7. The use according to claim 1, characterized in that, The molar ratio of procyanidins to oxaliplatin is 0.8:

1.

8. The use according to claim 1, characterized in that, The molar ratio of procyanidins to oxaliplatin is 1:

1.

9. The use according to claim 1, characterized in that, The molar ratio of procyanidins to oxaliplatin is 1.2:

1.

10. The use according to claim 1, characterized in that, The molar ratio of procyanidins to oxaliplatin is 1.5:1.

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