Application of Apicidin combined with Bevacizumab in the preparation of a medicament for treating cervical cancer

Abstract

The present invention provides the use of Apicidin in combination with Bevacizumab in the preparation of a medicament for treating cervical cancer, and the present invention belongs to the technical field of anti-tumor drugs. Research shows that Apicidin enhances the sensitivity of cervical cancer cells to the targeted drug Bevacizumab. The combined use of the two can more effectively inhibit the growth of cervical cancer cells, and the ability to induce apoptosis of cervical cancer cells is also enhanced, showing good synergistic effects. Therefore, the application prospect of Apicidin in combination with the targeted drug Bevacizumab is huge, providing a more effective feasible solution for the clinical treatment of cervical cancer and a new theoretical basis for solving the combined treatment of cervical cancer recurrence and drug resistance in clinical practice.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and particularly to the application of Apicidin combined with Bevacizumab in the preparation of drugs for treating cervical cancer. Background Art

[0002] Cervical cancer (CC) is one of the most common malignant tumors in human cancers and a huge challenge faced by women globally, usually caused by human papillomavirus (HPV) infection. Although cervical cancer screening and HPV vaccination have been widely used, there are still a large number of cases. Surgery, radiotherapy, and chemotherapy are the main current methods for treating cervical cancer. However, the high incidence of tumor recurrence and disease progression after these conventional therapies is very concerning, and new therapies need to be developed. In addition, these traditional single therapies non-selectively target actively proliferating cells, ultimately resulting in the simultaneous destruction of healthy and cancer cells. Instead, combination therapies that combine two therapeutic drugs have become the cornerstone of cancer treatment. By targeting different pathways, combination therapies can reduce the incidence of cancer cell drug resistance and can reduce toxicity by reducing the required dose of a single drug.

[0003] Apicidin is a cyclic peptide histone deacetylase (HDAC) inhibitor that has been shown to exhibit anti-tumor activity in multiple human cancer types. The acetylation and deacetylation of nucleosome core histones play important roles in chromatin structure regulation and gene transcription regulation. The acetylation of histones is controlled by the activities of two enzyme families, histone acetyltransferase (HAT) and histone deacetylase (HDAC). Imbalance in the histone acetylation state leads to abnormal behaviors of cells in terms of morphology, cycle, differentiation, and carcinogenesis. In recent years, more and more structurally diverse HDAC inhibitors have been discovered, including trichostatin A, trapoxin, sodium butyrate, etc. They inhibit cell proliferation by arresting the cell cycle, induce the differentiation of oncogene-transformed cells and morphological changes, and exhibit anti-tumor activity in vivo. In addition, it has been reported that Apicidin has anti-angiogenic potential both in vitro and in vivo, indicating that it indirectly inhibits the hypoxia-induced angiogenic mediator vascular endothelial growth factor (VEGF) and inhibits the migration and proliferation of endothelial cells.

[0004] Bevacizumab is an anti-angiogenic drug that targets vascular endothelial growth factor (VEGF-A). It mainly inhibits the activation of tyrosine kinase receptors (VEGFR-1, -2) on the cell surface by vascular endothelial growth factor (VEGF), generating downstream signals to reduce the proliferation and migration of vascular endothelial cells, the growth of immature endothelial cells, and the increase in vascular permeability, and ultimately inhibits the formation of new blood vessels. However, angiogenesis is also associated with immunosuppression. Therefore, during tumor formation and progression, angiogenesis and immunosuppression may occur simultaneously. In fact, multiple pro-angiogenic factors, especially vascular endothelial growth factor (VEGF-A), the main stimulant of angiogenesis, have immunosuppressive functions. Therefore, targeting the angiogenic pathway has been used to restore the anti-tumor immune response, which plays a very important role in inhibiting tumor growth.

[0005] Studies have shown that Bevacizumab may not achieve the expected efficacy in some cervical cancer patients, which may be due to the development of drug resistance. Summary of the Invention

[0006] To solve the problems of recurrence and drug resistance in cervical cancer patients, the present invention provides the following technical solutions:

[0007] The present invention provides the use of Apicidin in combination with Bevacizumab in the preparation of a drug for treating cervical cancer.

[0008] Preferably, the molar concentration ratio of the combined use of Apicidin and Bevacizumab is 10-40:10-320.

[0009] Preferably, the cancer cells of the cervical cancer are Hela, Siha or U14.

[0010] Preferably, the drug induces apoptosis of Hela, Siha or U14.

[0011] Based on the above studies, Apicidin enhances the sensitivity of cervical cancer cells to the targeted drug Bevacizumab. The combined use of the two can more effectively inhibit the growth of cervical cancer cells, and the ability to induce apoptosis of cervical cancer cells is also enhanced, showing good synergistic effects. Therefore, the application of Apicidin in combination with the targeted drug Bevacizumab has great prospects, providing a more effective feasible solution for the clinical treatment of cervical cancer and a new theoretical basis for the combined treatment of cervical cancer recurrence and drug resistance in clinical practice. Brief Description of the Drawings

[0012] Figure 1To investigate the effects of Apicidin at different concentrations on the survival rates of human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14 when used alone.

[0013] Figure 2 To investigate the effects of Apicidin at different concentrations on the proliferation rates of human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14 when used alone.

[0014] Figure 3 To take pictures of the proliferation of human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14 when treated with Apicidin at different concentrations alone.

[0015] Figure 4 To take pictures of the migration of human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14 when treated with Apicidin at different concentrations alone.

[0016] Figure 5 To detect the expression of related apoptotic proteins in human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14 when induced by Apicidin at different concentrations alone.

[0017] Figure 6 To induce apoptosis in human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14 with Apicidin at different concentrations alone. For the small graphs in the first and third rows, the abscissas from left to right are 10 3 、10 4 、10 5 、10 6 、10 7 ; for the small graphs in the second row, the abscissas from left to right are 10 1 、10 3 、10 5 、10 7 ; for the small graphs in the first and third rows, the ordinates from bottom to top are 10 2 、10 3 、10 4 、10 5 、10 6 ; for the small graphs in the second row, the ordinates from bottom to top are 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 .

[0018] Figure 7To investigate the effects of Apicidin combined with the targeted drug Bevacizumab on the survival rates of human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14.

[0019] Figure 8 To investigate the effects of Apicidin combined with the targeted drug Bevacizumab on the proliferation rates of human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14.

[0020] Figure 9 To take pictures of the proliferation of human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14, after treatment with Apicidin combined with the targeted drug Bevacizumab.

[0021] Figure 10 To take pictures of the migration of human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14, after treatment with Apicidin combined with the targeted drug Bevacizumab.

[0022] Figure 11 To investigate the enhanced induction of the expression of related apoptotic proteins in human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14, by Apicidin combined with the targeted drug Bevacizumab.

[0023] Figure 12 To investigate the enhanced induction of apoptosis in human cervical cancer cells Hela and Siha, as well as mouse cervical cancer cells U14, by Apicidin combined with the targeted drug Bevacizumab. The abscissas of the small graphs in the first row are 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 from left to right, and the ordinates are 10 2 、10 3 、10 4 、10 5 、10 6 from bottom to top; the abscissas of the small graphs in the second row are 10 1 、10 3 、10 5 、10 7 from left to right, and the ordinates are 10 1 、10 2 、10 3 、10 4 、10 5 from bottom to top; the abscissas of the small graphs in the third row are 10 3 、10 4 、105 , 10 6 , with the vertical axis from bottom to top being 10 2 , 10 3 , 10 4 , 10 5 .

[0024] Figure 13 shows the changes in the size of subcutaneous tumors in nude mice after being modeled with mouse cervical cancer cell line U14 and treated with Apicidin combined with the targeted drug Bevacizumab.

[0025] Figure 14 shows the statistical results of the changes in the size of subcutaneous tumors in nude mice after being modeled with mouse cervical cancer cell line U14 and treated with Apicidin combined with the targeted drug Bevacizumab.

[0026] Figure 15 shows the changes in the body weight of nude mice after being modeled with mouse cervical cancer cell line U14 and treated with Apicidin combined with the targeted drug Bevacizumab.

[0027] Figure 16 shows the HE staining results of the tumor tissue in nude mice after being modeled with mouse cervical cancer cell line U14 and treated with Apicidin combined with the targeted drug Bevacizumab.

[0028] Figure 17 shows the Ki67 staining results of the tumor tissue in nude mice after being modeled with mouse cervical cancer cell line U14 and treated with Apicidin combined with the targeted drug Bevacizumab. Specific implementation manners

[0029] The technical solutions provided by the present invention will be described in detail below in combination with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] The mouse cervical cancer cell line U14 required for the following examples was purchased from the Hefei Wanwu Biotechnology Delph Cell Bank, and the human cervical cancer cells Hela and Siha were purchased from Procell (Wuhan, China). The culture medium, PBS, and trypsin used were purchased from Wuhan Sevier Company. Fetal bovine serum trypsin was purchased from Gibco, USA. 1% streptomycin / penicillin was purchased from China Biyuntian Company. The drugs Apicidin and Bevacizumab used were purchased from MCE, USA, and diluted according to the drug instructions. The antibodies Cleaved-caspase-3 (GB11767C-100); Bax (GB12690-100); Bcl-2 (GB124830-100) used were purchased from Wuhan Sevier Company and diluted according to the instructions. The apoptosis kit was purchased from Tongren Company, Japan. The drugs, reagents, antibodies, etc. used in the experiment can be purchased from other commercial channels, and the experimental results are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental method is operated according to the conventional operating steps or according to the experimental method provided by the reagent manufacturer.

[0031] Cell culture: Human cervical cancer cells Hela and Siha and mouse cervical cancer cells U14 were cultured in DMEM medium containing 10% fetal bovine serum and 1% streptomycin / penicillin. The cell culture conditions were: 37°C, 5% CO 2 in the incubator.

[0032] Example 1

[0033] In vitro experiments

[0034] 1. Effects of different concentrations of Apicidin on the survival rate and migration of cervical cancer cells Hela, Siha, and U14

[0035] Hela, Siha, and U14 cells with good logarithmic growth were selected to plate 96-well plates, with 1×10 4 cells for survival assay, 0.2 × 10 4 Cells / well were used for proliferation assay, 5 duplicate wells were set for each drug concentration, and the concentration gradient of Apicidin drug diluted with DMEM medium was: 0, 5, 10, 20, 30, 40 μM.

[0036] The specific steps are as follows: Discard the original culture medium, wash the cells twice with 3 ml of PBS, digest the cells with 1 ml of trypsin for 1 min, add 2 ml of culture medium to neutralize the trypsin, transfer the cell suspension to a centrifuge tube, and centrifuge at 2000 rmp for 1.5 min. Discard the supernatant, resuspend the cells with fresh culture medium, take 10 μl for counting, and calculate the required cell suspension. Seed the 96-well plate at 100 μl per well. After the cells adhere for 24 h, dilute Apicidin to 100 μl with drug concentrations of 0, 5, 10, 20, 30, and 40 μM respectively. After the drug acts for 24 h, add 10 μl of CCK8, incubate in an incubator at 37 °C for 2 h, and then measure the absorbance value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The cell survival rate is as Figure 1 shown. Similarly, seed the 96-well plate at 100 μl of culture medium containing 0.2×10 4 cells / well. After the cells adhere for 24 h, dilute Apicidin to 100 μl with drug concentrations of 0, 10, 20, and 30 μM respectively. Add 10 μL of CCK-8 solution to each well on days 1-5. After incubating at 37 °C for 2 h, measure the absorbance value at 450 nm with an ELISA reader. The cell proliferation rate is as Figure 2 shown. Figure 1 , 2 The results of

[0037] show that with the increase in drug concentration, the survival rate and proliferation rate of Hela, Siha, and U14 cells decrease. It is suggested that Apicidin can inhibit the growth of Hela, Siha, and U14 cells.

[0037] Seed Hela, Siha, and U14 cells in the logarithmic growth phase at 0.05×10 4 cells / well in a 6-well plate. After one week, co-incubate different concentrations of Apicidin (0, 10, 20, 30 μM) with the cells for 7 days. Observe the formation of dot-like clones at the bottom of the 6-well plate. Fix the cells with 4% tissue fixative, stain with 1% crystal violet for 30 min, and take pictures for analysis. Figure 3 The results show that with the increase in drug concentration, Apicidin can inhibit the proliferation of cervical cancer cells Hela, Siha, and U14.

[0038] Suspend the cells with serum-free medium, adjust the cell concentration to 5×10 4 cells / well, seed them in the upper chamber of a Transwell chamber, and add serum-containing medium to the lower chamber. Add different concentrations of Apicidin (0, 10, 20, 30 μM) and incubate with the cells for 24 h. Fix the upper chamber with 4% tissue fixative, stain with crystal violet for 30 min, wipe the inside of the upper chamber with a cotton swab, observe and take pictures under a microscope, and count and analyze. Figure 4 The results show that with the increase in drug concentration, Apicidin can inhibit the migration of cervical cancer cells Hela, Siha, and U14.

[0039] 2. Effects of Apicidin on the apoptosis of cervical cancer cells Hela, Siha, and U14

[0040] Take Hela, Siha, and U14 cells in logarithmic growth phase, digest and centrifuge the cells. After resuspending the cells, inoculate them into 6-cm culture dishes. After the cells are completely adherent for 24 hours, treat the cells with drugs. The concentrations of Apicidin drug are: 0, 10, 20, 30 μM. After acting for 18 h, perform the next operation.

[0041] Protein extraction: For the cells treated with drugs, discard the supernatant, wash twice with PBS, add an appropriate amount of cell lysate, and lyse on ice for 5 min. Then scrape the lysate into an EP tube with a cell scraper and lyse on ice for 20 min. Centrifuge at 4°C, 12,000 rpm for 20 min, and collect the supernatant. After measuring the protein concentration by the BCA working method, dilute the supernatant with 5× protein loading buffer to 1×, boil in a metal bath at 100°C for 10 min, and then load the sample. According to the operation steps of the Western blot experiment, detect the expression of apoptosis-related Cleaved-caspase-3, Bax, and Bcl-2. Apoptosis detection: Dilute 10× AnnexinV binding Buffer to 1× AnnexinV binding Buffer according to the operation manual of the apoptosis kit. For the cells treated with drugs, discard the supernatant, wash twice with PBS, digest the cells with trypsin, and centrifuge. Discard the supernatant, add 1 ml of 1× AnnexinV binding Buffer to resuspend the cells, and centrifuge. Discard the supernatant and repeat the above steps once. Add 400 μl of 1× AnnexinV binding Buffer to resuspend the cells. Then add 5 μl of FITC / PI reagent respectively, keep in the dark for 15 min, and then detect on the machine. The results are as Figure 5 、 6 shown. As the drug concentration increases, the expression levels of apoptosis-related proteins Cleaved-caspase-3 and Bax in Hela, Siha, and U14 cells increase, the expression level of Bcl-2 decreases, and the number of apoptotic cells increases. It is suggested that Apicidin can induce apoptosis in Hela, Siha, and U14 cells.

[0042] 3. Effects of Apicidin combined with the targeted drug Bevacizumab on the survival rate and migration of cervical cancer cells Hela, Siha, and U14

[0043] Select Hela, Siha, and U14 in logarithmic growth phase and in good condition and seed them in 96-well plates, about 1×10 per well 4Cells per well were used for the survival assay, and 5 replicate wells were set for each drug concentration. Different concentrations of Bevacizumab alone (0, 10, 20, 40, 80, 160, and 320 μM) could reduce the survival rates of cervical cancer cells Hela, Siha, and U14 (as Figure 7 shown).

[0044] The final concentration of Apicidin was 20 μM, and the final concentration of Bevacizumab was 80 μM. 100 μl of medium containing the drug was added to each well. The order of adding drugs was: blank (NC), Apicidin (Ap), Bevacizumab (Bev), Apicidin + Bevacizumab (Ap + Bev). The specific steps were as follows: discard the original medium, wash the cells twice with 3 ml of PBS, digest the cells with 1 ml of trypsin for 1 min, add 2 ml of medium to neutralize the trypsin, transfer the cell suspension to a centrifuge tube, and centrifuge at 2000 rmp for 1.5 min. Discard the supernatant, resuspend the cells with fresh medium, take 10 μl for counting, and calculate the required cell suspension. According to 100 μl of medium per well and 0.2×10 4 cells per well, seed the 96-well plate, with a total of 4 drug concentrations, and 5 replicate wells for each drug concentration. After the cells adhered for 24 h, add the diluted drug concentration. On days 1 - 5, add 10 μL of CCK-8 solution to each well, incubate in an incubator at 37 °C for 2 h, and then measure the absorbance value at 450 nm with an enzyme-linked immunosorbent assay reader, as Figure 8 shown.

[0045] As can be seen from Figure 7 , 8 compared with the single drug treatment group, after treatment with Apicidin combined with Bevacizumab, the growth of Hela, Siha, and U14 cells was significantly inhibited. The results suggest that Apicidin can enhance the ability of the targeted drug Bevacizumab to inhibit the growth of Hela, Siha, and U14 cells.

[0046] Seed Hela, Siha, and U14 cells in the logarithmic growth phase at 0.05×10 4 cells per well in a 6-well plate. After one week, incubate Apicidin and Bevacizumab alone or in combination with the cells for 7 days, observe the formation of dot-like clones at the bottom of the 6-well plate, fix the cells with 4% tissue fixative, stain with 1% crystal violet for 30 min, and take pictures for analysis. As Figure 9 shown, the combined administration of Apicidin and Bevacizumab can significantly inhibit the proliferation of cervical cancer cells.

[0047] Suspend the cells with serum-free medium and adjust the cell concentration to 5×10 4Cells were seeded at a density of Figure 10 per well into the upper chamber of a Transwell insert, and the lower chamber was filled with serum-containing medium. Apicidin and Bevacizumab were added alone or in combination and incubated with the cells for 24 h. The upper chamber was fixed with 4% tissue fixative, stained with crystal violet for 30 min, the interior of the upper cavity was wiped with a cotton swab, observed and photographed under a microscope, and counted and analyzed. As

[0048] shown in the results, the combined administration of Apicidin and Bevacizumab more significantly inhibited the migration of cervical cancer cells.

[0049] Logarithmically growing Hela, Siha, and U14 cells were digested and centrifuged. After resuspending the cells, they were seeded into 6-cm culture dishes, with 6 dishes plated. After the cells were completely adherent for 24 h, the cells were treated with drugs. Drug concentrations: the final concentration of Apicidin was 20 μM, and the final concentration of Bevacizumab was 80 μM. The order of drug addition was: blank (NC), Apicidin (Ap), Bevacizumab (Bev), Apicidin + Bevacizumab (Ap + Bev). After the combined drugs acted for a period of time and the cell shrinkage and lysis reached 40%, the next step was carried out. Protein extraction: For the cells treated with drugs, the supernatant was discarded, the cells were washed twice with PBS, an appropriate amount of cell lysis buffer was added, and after lysing on ice for 5 min, the lysate was scraped into an EP tube with a cell scraper. After lysing on ice for 20 min, centrifugation was carried out at 4°C, 12,000 rpm for 20 min, and the supernatant was collected. After measuring the protein concentration by the BCA working method, the supernatant was diluted to 1× with 5× protein loading buffer, boiled in a metal bath at 100°C for 10 min, and then loaded. According to the operation steps of the Western blot experiment, the expressions of apoptosis-related Cleaved-caspase-3, Bax, and Bcl-2 were detected. Apoptosis detection: 10× Annexin V binding Buffer was diluted to 1× Annexin V binding Buffer according to the operation manual of the apoptosis kit. For the cells treated with drugs, the supernatant was discarded, the cells were washed twice with PBS, then digested with trypsin and centrifuged. The supernatant was discarded, the cells were resuspended with 1 ml of 1× Annexin V binding Buffer and centrifuged. The supernatant was discarded, and the above steps were repeated once. The cells were resuspended with 400 μl of 1× Annexin V binding Buffer. Then, 5 μl of FITC / PI reagent was added respectively, and after incubating in the dark for 15 min, the cells were detected by flow cytometry. The results are shown in Figure 11 、 12As shown, compared with the use of either drug alone, the combined use of Apicidin with the targeted drug Bevacizumab significantly increased the expression of apoptosis-related proteins Cleaved-caspase-3 and Bax, significantly decreased the expression of Bcl-2, and significantly increased the number of apoptotic cells. The results suggest that Apicidin can enhance the ability of the targeted drug Bevacizumab to induce apoptosis in cervical cancer cells Hela, Siha, and U14.

[0050] Example 2

[0051] In vivo experiments

[0052] Based on the results of in vitro experiments, in vivo functional experiments were conducted in nude mice. U14 cells in the logarithmic growth phase were taken and resuspended in pre-cooled PBS. 1×10 6 / 100μlU14 cells. Establish a mouse cervical cancer subcutaneous tumor model. Observe the tumor formation in nude mice. When the tumor grows to about 100mm 3 At the time of the experiment, the mice were randomly divided into 4 groups according to the weight and tumor size of nude mice, with 5 mice in each group, and given drug treatment. The groups were as follows: control group (PBS), Apicidin group (AP), Bevacizumab group (BEV), Apicidin + Bevacizumab group (AP + BEV). The drug concentrations used were: PBS group (100 μl / day intraperitoneal injection), Apicidin group (5 mg / kg / 2 days intraperitoneal injection), Bevacizumab group (5 mg / kg / 2 days intraperitoneal injection); Apicidin + Bevacizumab group (5 mg / kg + 5 mg / kg / 2 days intraperitoneal injection). The behavior period was 14 days of treatment. After the start of treatment, the length (a) and width (b) of the tumor were measured with a vernier caliper every three days, and the volume of the tumor was calculated. The specific calculation formula was: Volume V = 0.5ab 2 After 15 days of drug treatment, the nude mice showed cachexia such as weight loss. The mice were killed by spinal dislocation, and the tumor tissues were removed by dissection and photographed. The corresponding EP tubes were marked and fixed in 4% tissue fixative. Figures 13 to 15 The results showed that during the drug treatment of nude mice, the drug group inhibited the weight and growth of subcutaneous tumors of nude mice compared with the control group. The combined drug group had a stronger ability to inhibit the weight and growth of subcutaneous tumors of nude mice. Figures 16 to 17 The results showed that HE staining showed that compared with the single drug group, the Apicidin combined with Bevacizumab group had the most severe tumor tissue disintegration or necrotic damage. Immunohistochemistry (IHC) test showed that compared with the single drug group, Apicidin combined with Bevacizumab more effectively reduced the expression of Ki67 in tumor tissue. Figure 17The blue dots are cell nuclei. Cells with yellowish-brown or brown precipitates in the cell nuclei are Ki67-positive cells. The experimental results show that in the combination treatment group, the nuclear staining is light and the positive index is low. Figures 13 to 17 The results suggest that Apicidin inhibits the growth of subcutaneous tumors in nude mice, and at the same time, the ability of Apicidin combined with Bevacizumab to inhibit tumor growth is enhanced.

[0053] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of Apicidin combined with Bevacizumab in the preparation of drugs for the treatment of cervical cancer.

2. The use according to claim 1, characterized in that The molar concentration ratio of the combined use of Apicidin and Bevacizumab is 10-40:10-320.

Images