Application of trichosanthin in preparation of cervical cancer cis-platinum drug resistance reversal agent

Abstract

The invention discloses application of trichosanthin in preparation of a cervical cancer cis-platinum drug resistance reversal agent, belongs to the technical field of new application of drugs, and particularly relates to new application of trichosanthin in reversal of cervical cancer cis-platinum drug resistance. The radix trichosanthis protein can inhibit proliferation of a HeLa cell strain (HeLa / DDP cell strain) resistant to cisplatin when being singly used, can increase the sensitivity of the cisplatin resistant cell strain to drug resistance when being combined with cisplatin, and can increase HeLa / DDP cell apoptosis by up-regulating expression of pro-apoptotic proteins Bax and c-PARP and down-regulating expression of apoptosis inhibition protein Bcl-2, so that the purpose of reversing the platinum drug resistance of cervical cancer is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of new drug applications. The trichosanthin of the present invention can significantly inhibit the proliferation of HeLa cell lines (HeLa / DDP cell lines) resistant to cisplatin and reverse the sensitivity of HeLa / DDP cells to cisplatin, thereby improving the prognosis of patients. Background Art

[0002] The statistics of Cancer Statistics 2024 show that despite the gradual promotion of early screening, the application of HPV vaccines and the continuous improvement of diagnosis and treatment technologies, cervical cancer is still a common malignant tumor that threatens the life and health of women worldwide. Cisplatin (DDP)-based concurrent chemoradiotherapy (CCRT) is currently the standard method for treating FIGO stage IIb-IVa cervical cancer, but relevant clinical studies have shown that about 30%-40% of patients cannot achieve complete remission, and cervical cancer patients' resistance to cisplatin is an important reason for poor prognosis. Trichosanthin (TCS) is a type I ribosome inactivating protein extracted from the tuber of the medicinal plant Trichosanthes kirilowii. It can inactivate the ribosomes of eukaryotic organisms by cutting the N-glycosidic bond on adenine 4324 of 28S rRNA, thereby preventing the synthesis of proteins. It is clinically used for the treatment of diseases such as ectopic pregnancy, placenta accreta, and hydatidiform mole, with good efficacy and few side effects. There are no reports on the research of trichosanthin reversing cisplatin resistance in cervical cancer at home and abroad. Based on the above research background, the present invention studies the new use of trichosanthin, clarifies the inhibitory effect of trichosanthin on the proliferation of cisplatin-resistant HeLa cell line (HeLa / DDP cell line), and combines trichosanthin with cisplatin to act on HeLa / DDP cells, determines the drug concentration with the best synergistic effect, and preliminarily explores the mechanism of action of trichosanthin reversing cisplatin resistance in HeLa / DDP cells, providing a new treatment approach for patients with cisplatin-resistant cervical cancer. Summary of the invention

[0003] Cisplatin is the drug of choice for concurrent chemoradiotherapy of cervical cancer. However, cisplatin resistance is one of the main reasons that limit clinical efficacy and the progress of tumor treatment in some patients. There have been no reports on the study of reversing cisplatin resistance in cervical cancer by trichosanthin at home and abroad. The present invention studies the new use of trichosanthin, clarifies the reversal effect of trichosanthin on cisplatin resistance, and combines trichosanthin with cisplatin to determine the best anti-cancer concentration of the two drugs according to the combined drug index (CI value), and clarifies the effects of trichosanthin alone and trichosanthin combined with cisplatin on the proliferation, migration and apoptosis of cervical cancer HeLa cells.

[0004] In one aspect, the present invention provides a use of trichosanthin in the preparation of a drug for treating cervical cancer.

[0005] Application of trichosanthin in the preparation of drugs for inhibiting the proliferation of HeLa / DDP cells to treat cervical cancer.

[0006] In some embodiments, the present invention provides a novel use of trichosanthin in the preparation of a cisplatin resistance reversal agent for treating cervical cancer. Trichosanthin can significantly inhibit the proliferation of HeLa / DDP cells at a mass concentration of 0.01-0.2 μg / ml, and the highest IC 50 It is 0.06±0.01 μg / ml.

[0007] On the other hand, the present invention provides a use of trichosanthin as a reversal agent in the preparation of a drug-resistant drug for treating cervical cancer.

[0008] Application of trichosanthin as a reversal agent in the preparation of a drug that inhibits HeLa / DDP cell proliferation and achieves drug resistance in the treatment of cervical cancer.

[0009] The cervical cancer drug resistance includes cervical cancer cisplatin resistance.

[0010] The concentration of the trichosanthes protein is 0.01-0.2 μg / ml.

[0011] In another aspect, the present invention provides a use of trichosanthin in combination with cisplatin in the preparation of a drug for treating cervical cancer.

[0012] The application of the trichosanthin in combination with cisplatin in the preparation of a drug for inhibiting the proliferation of HeLa / DDP cells to achieve the treatment of cervical cancer.

[0013] The trichosanthin is used in combination with cisplatin in the preparation of a drug for increasing the sensitivity of HeLa / DDP cells to cisplatin to achieve the treatment of cervical cancer.

[0014] The mass concentration of the trichosanthes protein is 0.00625-0.2 μg / ml; the mass concentration of cisplatin is 1-32 μg / ml.

[0015] The combination of trichosanthin and cisplatin can increase the sensitivity of HeLa / DDP cells to cisplatin and reduce the dose of cisplatin.

[0016] Using the technical solution of the present invention, it was found that the half inhibitory drug concentration (IC 50 ) was 8.04±0.15 μg / ml, and the half inhibitory drug concentration (IC 50was 2.35 ± 0.18 μg / ml, and the sensitization index was 3.4 or above.

[0017] According to the CCK8 experiment, calculate the cell proliferation inhibition rates of trichosanthin, cisplatin, and the combination of trichosanthin and cisplatin. Use CompuSyn software to calculate the CI value and draw the combination index (CI value) curve graph. According to the drug combination selection principle (CI > 1 antagonism; CI = 1 addition; 0.6 < CI < 1 synergy; CI < 0.6 strong synergy), determine the best combined drug effect of trichosanthin and cisplatin. The combination of trichosanthin and cisplatin can significantly inhibit the migration and invasion of HeLa / DDP cells, increase the proportion of apoptosis of HeLa / DDP cells, up-regulate the expression of pro-apoptotic proteins Bax and c-PARP, and down-regulate the expression of anti-apoptotic protein Bcl-2.

[0018] In summary, the present invention discloses a new use of trichosanthin as a cisplatin resistance reversing agent for cervical cancer. Trichosanthin can significantly inhibit the proliferation of HeLa / DDP cells at a mass concentration of 0.01 - 0.2 μg / ml. When trichosanthin is combined with cisplatin, it can increase the sensitivity of HeLa / DDP cells to cisplatin, and the sensitization index is above 3.4.

[0019] Trichosanthin in the present invention has a new use as a cisplatin resistance reversing agent for cervical cancer, providing a new treatment approach for patients with cisplatin-resistant cervical cancer. Brief Description of the Drawings

[0020] Figure 1 It is the effect diagram of the inhibitory effect of trichosanthin, cisplatin, and the combination of trichosanthin and cisplatin on the proliferation of HeLa / DDP cells.

[0021] Figure A: Inhibitory effect of TCS on the proliferation of HeLa / DDP cells; Figure B: Inhibitory effects of DDP alone and in combination on the proliferation of HeLa / DDP cells. Note: For each concentration group of TCS, DDP alone and in combination compared with the blank control group, **P < 0.01, ***P < 0.001. For the combination drug group compared with the same concentration group of DDP alone: P < 0.001, ±s, n = 3; independent sample t-test was used.

[0022] Figure 2 It is the combination index (CI value) curve graph. Figure A: CI curve graph of HeLa / DDP cells at 48 h; Figure B: Drug concentration combinations of TCS and DDP and the corresponding inhibition rates and CI values of HeLa / DDP cells at 48 h.

[0023] Figure 3The figure shows the effect of trichosanthin, cisplatin alone, and the combination of trichosanthin and cisplatin on the migration ability of HeLa / DDP cells. Figure A is a scratch test; Figure B is a bar chart of statistical analysis of the scratch test.

[0024] Note: Comparison between TCS, DDP single-drug group and combined-drug group and control group (0μg / ml):*** P <0.001; The combined drug group was compared with the TCS group and the DDP group:## P <0.01,### P <0.001, all using independent samples t test, t-test, ± s , n=3.

[0025] Figure 4 Figure 1: The effect of trichosanthin, cisplatin and combined drug use on apoptosis of HeLa / DDP cells detected by flow cytometry. Figure A: Flow cytometry apoptosis of HeLa / DDP cells; Figure B: Quantitative statistical analysis of the proportion of early apoptosis and late apoptosis in each group. Comparison between the combined drug group and the blank control group and the DDP single drug group: *** P <0.001, all using independent samples t test, t-test, ± s , n=3.

[0026] Figure 5 Western Blot detection of the effects of trichosanthin, cisplatin and combined use on HeLa / DDP cell-related apoptosis proteins. Figure A: Changes in the expression levels of HeLa / DDP cell apoptosis-related proteins Bax, Bcl-2, and PARP in each experimental group; Figure B: Protein expression gray value bar chart Note: TCS and DDP single-drug group and TCS+DDP combined-drug group were compared with blank control group, * P <0.05,** P <0.01,*** P <0.001; Comparison between combined medication group and DDP group: # P <0.05 (c-PARP / β-actin), ### P <0.001 (Bax / Bcl-2), ± s , n=3, all independent samples t test. DETAILED DESCRIPTION

[0027] Cell culture and grouping Cervical cancer HeLa / DDP cell line was purchased from Shanghai Fuyuan Biotechnology Co., Ltd. and cultured and preserved by the Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy of Three Gorges University. Culture conditions: 5% CO2, 37℃ humidified incubator, 1640 medium containing 10% fetal bovine serum, 1×105 U / L penicillin, and 100 mg / L streptomycin. The experiment was divided into 4 groups: no drug addition group (blank control group); cisplatin (DDP) single drug group; trichosanthin (TCS) single drug group; trichosanthin and cisplatin combined drug group.

[0028] Example 1 Trichosanthin (TCS), cisplatin (DDP) alone and TCS combined with DDP inhibit the proliferation of HeLa / DDP cells CCK8 assay to detect cell proliferation HeLa / DDP cells in the logarithmic growth phase were collected, the cell density was adjusted to 2×105 cells / ml, 200 μL of cell suspension was added to each well of a 96-well plate, and the plate was placed in a cell culture incubator overnight. On the second day, fresh 1640 culture medium was used according to the experimental groups. The drug concentrations were as follows: DDP monotherapy group (0 μg / ml, 2 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml), TCS monotherapy group (0 μg / ml, 0.00625 μg / ml, 0.0125 μg / ml, 0.025 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml), and the concentrations of TCS and DDP combined drugs were (0.00625 μg / ml, 1 μg / ml), (0.01 μg / ml, 2 μg / ml), (0.025 μg / ml, 4 μg / ml), (0.05 μg / ml, 8μg / ml), (0.1μg / ml, 16μg / ml), (0.2μg / ml, 32μg / ml).

[0029] Three replicate wells were set up for each concentration, and the experiment was repeated three times, with the detection time being 48 hours. After 48 hours of culture, the old culture medium was aspirated, and the CCK8 stock solution was diluted with basal culture medium to a CCK8 working solution with a concentration of 10%. 100μL of CCK8 working solution was added to each experimental well. The plate was then placed in a cell culture incubator and cultured in the dark for 30 minutes. After 30 minutes of culture, the 96-well plate was taken out and placed in a full-wavelength enzyme-linked immunosorbent assay to measure the OD value of each well at a wavelength of 460 nm. The formula for calculating the cell growth inhibition rate is: drug inhibition rate = 1- OD value of the experimental group / OD value of the control group. From Figure 1It can be seen that both TCS and DDP have inhibitory effects on the proliferation of HeLa / DDP cells in a concentration-dependent manner. The half-maximal inhibitory concentration (IC 50 50) of TCS on HeLa / DDP cells for 48 h is 0.06 ± 0.01 μg / ml, and the half-maximal inhibitory concentration (IC 50 50) of DDP on HeLa / DDP cells for 48 h is 8.04 ± 0.15 μg / ml. After the combined action of DDP and TCS, its half-maximal inhibitory concentration (IC 50 50) decreases to 2.35 ± 0.18 μg / ml. The sensitization index of TCS to DDP is 3.4 (8.04 / 2.35), and the inhibition rate of each concentration group of the combined drug is also significantly increased compared with the single drug group of DDP at the same concentration.

[0030] Example 2 Determination of the combined drug concentration of TCS and DDP According to the CCK8 experiment, calculate the cell proliferation inhibition rates of TCS, DDP, and the combination of TCS and DDP. Use CompuSyn software to calculate the CI value and draw the combined drug index (CI value) curve graph (as Figure 2 shown). The CI values of the six combined drugs are all below 1, showing a synergistic effect. The CI values of some high-concentration groups are also below 0.6, showing a strong synergistic effect (CI > 1 antagonism; CI = 1 addition; 0.6 < CI < 1 synergism; CI < 0.6 strong synergism). According to the CI curve graph of the two drugs TCS and DDP, select the concentration ratio of the two drugs corresponding to the CI value < 0.6 of the combined drug: TCS (0.03 μg / ml) and DDP (4 μg / ml) as the drug concentrations for the subsequent experiment. The subsequent experimental groups of HeLa / DDP cells are: control group (control), TCS group (0.03 μg / ml), DDP group (4 μg / ml), TCS + DDP (0.03 μg / ml + 4 μg / ml). The experimental time is 48 h for all.

[0031] Example 3 Inhibition of migration of HeLa / DDP cells by Trichosanthin (TCS), cisplatin (DDP) alone and the combination of TCS and DDP For the cell scratch experiment, collect HeLa / DDP cells in good condition in the logarithmic growth phase and seed them at a density of 2×10 5The cells were inoculated at a density of 10 cells / well in a six-well plate and cultured overnight in complete medium. On the second day, the cells were observed under an inverted microscope. When the cell confluence reached 70%, scratches were prepared. Use a 200 μl pipette tip to draw vertical lines along the ruler. Gently blow the cells twice with PBS to remove the scratched cells. According to the experimental groups, 6 ml of low-concentration culture medium containing drugs prepared in advance (culture medium containing 2% FBS) was added, and a control group (only adherent cells and low-concentration culture medium containing 2% FBS) was set up. Immediately take pictures under an optical microscope and store them, and then place them in a cell culture incubator for cultivation. Take pictures at 24 h and 48 h of cultivation. Figure 3 As shown in the figure, the spacing between the scratches of HeLa / DDP cells in the blank control group became smaller and smaller as time went on, and the cells basically healed after 48 hours. Compared with the blank control group, the migration ability of the TCS and DDP single-drug treatment groups weakened, and the rate of increase in the healing area slowed down. The cell migration ability of the combination drug group was significantly stagnant, with almost no migration ability. Compared with the single-drug group, the combination drug group had a significant difference in the ability to inhibit the migration of tumor cells.

[0032] Example 4 Trichosanthin (TCS), cisplatin (DDP) alone and TCS combined with DDP inhibit apoptosis of HeLa / DDP cells Flow cytometry was used to detect cell apoptosis. HeLa / DDP cells in logarithmic growth phase were plated at 2×10 5 The number of cells was inoculated into a six-well plate and cultured in a cell culture incubator overnight. On the second day, the old culture medium was discarded, and 6 ml of culture medium containing different drugs was added according to the experimental groups, and the culture was continued. After 48 hours, the cells were washed with cold 1×PBS and the cell pellet was collected. The collected cells were stained according to the instructions of the apoptosis kit. 300 μL of 1×Binding buffer working solution was added to the EP tube of each experimental treatment group to suspend the cells, and 1.2 ml of 1×Binding buffer working solution was added to the blank control group to suspend the cells, and then evenly divided into four parts and loaded into 4 new EP tubes, and no dye was added, FITC-AnnexinV single staining, PI single staining, FITC-Annexin V+PI double staining were respectively used as 4 control groups. First, 5 μL of Annexin V-FITC staining solution was added to the cell suspension in the treatment group in the dark, and it was gently mixed and incubated in the dark for 5 min. After 5 min, add 2 μL of PI staining solution to each tube of sample in the dark and mix gently. Continue to incubate in the dark. Detect apoptosis indicators by flow cytometry within 30 min and analyze the image results. Figure 4As shown, the apoptosis rate (the sum of early apoptosis and late apoptosis) of the control group was 6.7%±0.7%, the apoptosis rate of the TCS group was 19.3%±0.6%, the DDP group was 11.9%±1.0%, and the TCS+DDP combination group was 41.7%±3.8%. Compared with the blank control group, the TCS and DDP single-drug groups and the combination group increased the apoptosis rate of HeLa / DDP cells, and the apoptosis rate of the combination group was significantly higher than that of the DDP single-drug group. After statistical analysis, the difference was statistically significant, indicating that the combination group can promote the apoptosis of HeLa / DDP cells more than the DDP single-drug group.

[0033] Example 5 Western blotting was used to detect the effects of each group of drugs on the apoptotic protein levels of HeLa / DDP cells Western Blot detection of related protein expression levels. According to the experimental groups, the cells treated with drugs were collected, and the cells were fully lysed with protein lysis buffer on ice for 30 min. The cells were transferred to a pre-cooled 4℃ high-speed centrifuge and centrifuged at 12000 rpm for 15 min. The supernatant was placed in a pre-cooled EP tube. The protein sample was prepared according to the instructions of the BCA kit and placed in a microplate reader. The absorbance value (OD value) of the protein sample was measured at a wavelength of 562 nm, and the protein concentration was calculated according to the standard curve (standard curve: y=937.7x-20.855). The required protein loading volume was calculated according to the protein quantification and the measured protein concentration. The loading buffer was added according to the volume ratio of 4:1 between the protein sample volume and the loading buffer volume, and SDS-polyacrylamide gel electrophoresis was performed after boiling in a boiling water bath for 10 min. The voltage of the stacking gel was set to 60 V and the voltage of the separation gel was set to 80 V. The protein was wet-transferred to the PVDF membrane. After blocking the membrane with 8% skim milk prepared in TBST for 1 hour, replace the primary antibody and incubate the membrane on a shaker at 4°C overnight (generally no more than 16 hours). The next day, wash the membrane with TBST 3 times for 10 min at room temperature. Incubate the secondary antibody on a shaker at room temperature for 1 hour, and wash the membrane again 3 times for 10 min. ECL chemiluminescence color development, X-ray film exposure, development, and fixation, using β-Actin as an internal reference. Use Image J image analysis software to analyze the grayscale values ​​of the target protein and internal reference protein bands, and perform statistical analysis and comparison. The target proteins to be checked are: PARP, Bax, Bcl-2, etc. The experiment uses β-actin as the internal reference protein. Figure 5As shown in the figure, the protein levels of pro-apoptotic proteins Bax and c-PARP in the TCS group, DDP group and TCS+DDP group were increased compared with the blank control group, while the protein level of apoptosis inhibitor protein Bcl-2 was decreased. Compared with the DDP monotherapy group, the protein levels of pro-apoptotic proteins Bax and c-PARP in the combined drug group were increased, while the protein level of apoptosis inhibitor protein Bcl-2 was decreased, indicating that the combined drug can promote HeLa / DDP cell apoptosis better than DDP monotherapy.

Claims

1. Application of trichosanthin in the preparation of drugs for the treatment of cervical cancer.

2. The use according to claim 1, characterized in that: Application of trichosanthin in the preparation of drugs for inhibiting the proliferation of HeLa / DDP cells to treat cervical cancer.

3. Application of trichosanthin as a reversal agent in the preparation of drugs for the treatment of cervical cancer resistance.

4. The use according to claim 3, characterized in that: Application of trichosanthin as a reversal agent in the preparation of a drug that inhibits HeLa / DDP cell proliferation and achieves drug resistance in the treatment of cervical cancer.

5. The use according to claim 4, characterized in that: The cervical cancer drug resistance includes cervical cancer cisplatin resistance.

6. The use according to any one of claims 1 to 5, wherein the concentration of the trichosanthes protein is 0.01-0.2 μg / ml.

7. The application of trichosanthin in combination with cisplatin in the preparation of drugs for the treatment of cervical cancer.

8. The use according to claim 7, characterized in that: The application of trichosanthin combined with cisplatin in the preparation of drugs for inhibiting the proliferation of HeLa / DDP cells and treating cervical cancer.

9. The use according to claim 8, characterized in that: The application of trichosanthin in combination with cisplatin in the preparation of drugs for increasing the sensitivity of HeLa / DDP cells to cisplatin to treat cervical cancer.

10. The use according to claims 7-9, characterized in that: The mass concentration of the trichosanthes protein is 0.00625-0.2 μg / ml; the mass concentration of cisplatin is 1-32 μg / ml.

Images