Application of combination of Apicidin and Bevacizumab in preparation of medicine for treating cervical cancer

Abstract

The invention provides application of combination of Apicidin and Bevacizumab in preparation of a medicine for treating cervical cancer, and belongs to the technical field of anti-tumor medicines. Researches show that the Apicidin improves the sensitivity of cervical cancer cells to the targeted drug Bevacizumab, the combined use of the Apicidin and the targeted drug Bevacizumab can more effectively inhibit the growth of the cervical cancer cells, the ability of inducing the apoptosis of the cervical cancer cells is also enhanced, and the Apicidin and the targeted drug Bevacizumab have a good synergistic effect. Therefore, the application prospect of the combination of the Apicidin and the targeted drug Bevacizumab is huge, a more effective feasible scheme is provided for clinical treatment of cervical cancer, and a new theoretical basis is provided for clinical combined treatment of recurrence and drug resistance of cervical cancer.

Description

Technical Field

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

[0002] Cervical cancer (CC) is one of the most common malignancies among human cancers and a huge challenge facing women worldwide. It is usually caused by human papillomavirus (HPV) infection. Although cervical cancer screening and HPV vaccination have been widely used, a large number of cases still exist. Surgery, radiotherapy, and chemotherapy are currently the main treatments for cervical cancer. However, the high incidence of tumor recurrence and disease progression after these conventional therapies is very worrying, and new therapies need to be developed. In addition, these traditional monotherapies non-selectively target actively proliferating cells, ultimately leading to the simultaneous destruction of healthy and cancerous cells. In contrast, combination therapy combining two therapeutic drugs has become the cornerstone of cancer treatment. By targeting different pathways, combination therapy can reduce the incidence of cancer cell 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 antitumor activity in a variety of human cancer types. Acetylation and deacetylation of nucleosomal core histones play an important role in chromatin structure regulation and gene transcription regulation. Histone acetylation is controlled by the activities of two enzyme families, histone acetyltransferase (HAT) and histone deacetylase (HDAC). Imbalance in histone acetylation status leads to abnormal cell behavior in morphology, cycle, differentiation, and carcinogenesis. In recent years, an increasing number of structurally diverse HDAC inhibitors have been discovered, including trichostatin A, trichostatin, sodium butyrate, etc., which inhibit cell proliferation by arresting the cell cycle, induce differentiation and morphological changes in oncogene-transformed cells, and exhibit antitumor activity in vivo. In addition, apicidin has been reported to have anti-angiogenic potential both in vitro and in vivo, indicating that it indirectly inhibits the hypoxia-induced angiogenesis 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 vascular endothelial growth factor (VEGF) to activate cell surface tyrosine kinase receptors (VEGFR-1, -2), generating downstream signals to reduce endothelial cell proliferation, migration, immature endothelial cell growth, increased vascular permeability, and ultimately inhibit the formation of new blood vessels. However, angiogenesis is also associated with immunosuppression. Therefore, angiogenesis and immunosuppression may occur simultaneously during tumor formation and progression. In fact, a variety of pro-angiogenic factors, especially vascular endothelial growth factor (VEGF-A), the main stimulant of angiogenesis, have immunosuppressive functions. Therefore, targeting angiogenic pathways has been used to restore anti-tumor immune responses and plays a very important role in inhibiting tumor growth.

[0005] Research shows that bevacizumab may not work as well as expected in some patients with cervical cancer, possibly because of the development of drug resistance. Summary of the invention

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

[0007] The present invention provides the use of Apicidin combined with Bevacizumab in preparing a medicine 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 cervical cancer cells are Hela, Siha or U14.

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

[0011] The above studies show that Apicidin increases the sensitivity of cervical cancer cells to the targeted drug Bevacizumab. The combination of the two can more effectively inhibit the growth of cervical cancer cells and enhance the ability to induce apoptosis of cervical cancer cells, which has a good synergistic effect. Therefore, the application prospects of Apicidin combined with the targeted drug Bevacizumab are huge, providing a more effective and feasible solution for the clinical treatment of cervical cancer, and providing a new theoretical basis for the combined treatment of clinical cervical cancer recurrence and drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1This study studies the effects of different concentrations of Apicidin alone on the survival rate of human cervical cancer cells Hela, Siha and mouse cervical cancer cells U14.

[0013] Figure 2 This study shows the effect of different concentrations of Apicidin alone on the proliferation rate of human cervical cancer cells Hela, Siha and mouse cervical cancer cells U14.

[0014] Figure 3 The proliferation of human cervical cancer cells Hela, Siha and mouse cervical cancer cells U14 was photographed using different concentrations of Apicidin alone.

[0015] Figure 4 Photographs were taken of the migration of human cervical cancer cells Hela and Siha and mouse cervical cancer cells U14 using different concentrations of Apicidin alone.

[0016] Figure 5 The expression of related apoptosis proteins in human cervical cancer cells Hela, Siha and mouse cervical cancer cells U14 was induced by using different concentrations of Apicidin alone.

[0017] Figure 6 The apoptosis of human cervical cancer cells Hela, Siha and mouse cervical cancer cells U14 induced by Apicidin at different concentrations. The horizontal coordinates of the first and third rows of small figures are 10 from left to right. 3 , 10 4 , 10 5 , 10 6 , 10 7 , the horizontal coordinates of the second row of small pictures are 10 from left to right 1 , 10 3 , 10 5 , 10 7 , the vertical coordinates of the first and third rows of small pictures are 10 from bottom to top 2 , 10 3 , 10 4 , 10 5 , 10 6 , the vertical coordinates of the second row of small pictures are 10 from bottom to top 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 .

[0018] Figure 7This study investigates the effects of Apicidin combined with the targeted drug Bevacizumab on the survival rate of human cervical cancer cells Hela and Siha and mouse cervical cancer cells U14.

[0019] Figure 8 This study investigates the effects of Apicidin combined with the targeted drug Bevacizumab on the proliferation rate of human cervical cancer cells Hela and Siha and mouse cervical cancer cells U14.

[0020] Fig. 9 Photographs were taken of the effects of Apicidin combined with the targeted drug Bevacizumab on the proliferation of human cervical cancer cells Hela, Siha and mouse cervical cancer cells U14.

[0021] Fig.10 Photographs were taken of the migration effects of Apicidin combined with the targeted drug Bevacizumab on human cervical cancer cells Hela, Siha and mouse cervical cancer cells U14.

[0022] Fig.11 This is the expression of related apoptosis proteins induced by Apicidin combined with the targeted drug Bevacizumab in cervical cancer cells Hela, Siha and mouse cervical cancer cells U14.

[0023] Fig.12 Apicidin combined with the targeted drug Bevacizumab can enhance the apoptosis of cervical cancer cells Hela, Siha and mouse cervical cancer cells U14. The horizontal axis of the first row of small figures is 10 from left to right. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , the vertical coordinates are 10 from bottom to top 2 , 10 3 , 10 4 , 10 5 , 10 6 ; The horizontal coordinates of the second row of small pictures are 10 from left to right 1 , 10 3 , 10 5 , 10 7 , the vertical coordinates are 10 from bottom to top 1 , 10 2 , 10 3 , 10 4 , 10 5 ; The horizontal coordinates of the third row of small pictures are 10 from left to right 3 , 10 4 , 105 , 10 6 , the vertical coordinates are 10 from bottom to top 2 , 10 3 , 10 4 , 10 5 .

[0024] Fig.13 This is the change in subcutaneous tumor size in nude mice after treatment of mouse cervical cancer cell U14 model with Apicidin combined with targeted drug Bevacizumab.

[0025] Fig.14 These are the statistical results of the changes in subcutaneous tumor size in nude mice after treatment with Apicidin combined with the targeted drug Bevacizumab for the treatment of mouse cervical cancer cell U14 model.

[0026] Fig.15 The changes in nude mouse body weight after the treatment of mouse cervical cancer cell U14 model with Apicidin combined with targeted drug Bevacizumab.

[0027] Fig.16 HE staining results of tumor tissues in nude mice after treatment of mouse cervical cancer cell U14 with Apicidin combined with targeted drug Bevacizumab.

[0028] Fig.17 The Ki67 staining results of tumor tissues in nude mice after the treatment of mouse cervical cancer cell U14 model with Apicidin combined with the targeted drug Bevacizumab. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the 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 in an incubator at 37°C and 5% CO2.

[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 3ml PBS, digest the cells with 1ml trypsin for 1min, add 2ml culture medium to neutralize the trypsin, transfer the cell suspension to a centrifuge tube, and centrifuge at 2000rmp for 1.5min. Discard the supernatant, add new culture medium to resuspend the cells, take 10μl to count, and calculate the required cell suspension. Spread 96-well plates with 100μl per well. After 24h of cell adhesion, dilute Apicidin to 100μl containing drug concentrations of 0, 5, 10, 20, 30, and 40μM. After 24h of drug action, add 10μl of CCK8, incubate in a 37°C incubator for 2h, and measure the absorbance value at 450nm with an enzyme reader. The cell survival rate is as follows. Figure 1 Similarly, 0.2×10 4 10 cells / well were plated in a 96-well plate. After 24 hours of cell attachment, Apicidin was diluted to 100 μl with drug concentrations of 0, 10, 20, and 30 μM, and 10 μL of CCK-8 solution was added to each well on days 1 to 5. After incubation at 37°C for 2 hours, the absorbance value was measured at 450 nm by an ELISA reader. The cell proliferation rate was calculated as follows: Figure 2 shown. Figure 1 , 2 The results showed that with the increase of drug concentration, the survival rate and proliferation rate of Hela, Siha and U14 cells decreased, suggesting that Apicidin can inhibit the growth of Hela, Siha and U14 cells.

[0037] Hela, Siha and U14 cells in the logarithmic growth phase were cultured at 0.05×10 4 Cells / well were seeded in 6-well plates. One week later, different concentrations of Apicidin (0, 10, 20, 30 μM) were incubated with the cells for 7 days. The dot-like colony formation at the bottom of the 6-well plates was observed. The cells were fixed with 4% tissue fixative, stained with 1% crystal violet for 30 min, and photographed for analysis. Figure 3 The results showed that as the drug concentration increased, Apicidin could inhibit the proliferation of cervical cancer cells Hela, Siha, and U14.

[0038] Resuspend the cells in serum-free medium and adjust the cell concentration to 5 × 10 4 Cells were inoculated into the upper chamber of the Transwell chamber, and serum-containing culture medium was added to the lower chamber. Different concentrations of Apicidin (0, 10, 20, 30 μM) were added and incubated with the cells for 24 h. The upper chamber was fixed with 4% tissue fixative, stained with crystal violet for 30 min, and the interior of the upper chamber was wiped with a cotton swab, observed and photographed under a microscope, and counted and analyzed. Figure 4 The results showed that with the increase of drug concentration, Apicidin could inhibit the migration of cervical cancer cells Hela, Siha, and U14.

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

[0040] Take logarithmically growing Hela, Siha, and U14 cells, digest and centrifuge the cells. Resuspend the cells and inoculate them into a 6 cm culture dish. After 24 hours of complete cell adhesion, treat the cells with drugs. The concentrations of Apicidin are 0, 10, 20, and 30 μM. After 18 hours of action, proceed to the next step.

[0041] Protein extraction: After drug-treated cells, discard the supernatant, wash twice with PBS, add an appropriate amount of cell lysis buffer, lyse on ice for 5 minutes, scrape the cells to get the lysis buffer into an EP tube, lyse on ice for 20 minutes, centrifuge at 4°C, 12000rpm, 20 minutes, and collect the supernatant. After measuring the protein concentration by BCA working method, add 5× protein loading buffer to dilute the supernatant to 1×, boil in a metal bath at 100°C for 10 minutes, and then load the sample. According to the Western blotting experimental steps, detect the expression of apoptosis-related Cleaved-caspase-3, Bax, and Bcl-2. Apoptosis detection: dilute 10×AnnexinVbinding Buffer to 1×AnnexinVbinding Buffer according to the instructions of the apoptosis kit. After drug-treated cells, discard the supernatant, wash twice with PBS, trypsinize the cells, and centrifuge. Discard the supernatant, add 1ml 1×AnnexinVbinding Buffer to resuspend the cells, and centrifuge. Discard the supernatant and repeat the above steps once. Add 400 μl of 1×Annexin V binding buffer to resuspend the cells. Then add 5 μl of FITC / PI reagent respectively, protect from light for 15 minutes, and then detect on the instrument. The results are as follows Figure 5 , 6 As shown, with the increase of drug concentration, the expression of apoptosis-related proteins Cleaved-caspase-3 and Bax in Hela, Siha, and U14 cells increased, the expression of Bcl-2 decreased, and the number of apoptotic cells increased, indicating that Apicidin can induce apoptosis in Hela, Siha, and U14 cells.

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

[0043] Hela, Siha, and U14 cells with good logarithmic growth were selected to plate 96-well plates, with about 1×10 4Cells / well were used for survival assay, and 5 replicate wells were set for each drug concentration. Different concentrations of Bevacizumab (0, 10, 20, 40, 80, 160, and 320 μM) alone could reduce the survival rate of cervical cancer cells Hela, Siha, and U14 (e.g. Figure 7 shown).

[0044] The final concentration of Apicidin is 20μM, and the final concentration of Bevacizumab is 80μM. Add 100μl of drug-containing culture medium to each well. The order of drug addition is: blank (NC), Apicidin (Ap), Bevacizumab (Bev), Apicidin + Bevacizumab (Ap + Bev). The specific steps are as follows: discard the original culture medium, wash the cells twice with 3ml PBS, digest the cells with 1ml trypsin for 1min, add 2ml culture medium to neutralize the trypsin, transfer the cell suspension to a centrifuge tube, and centrifuge at 2000rmp for 1.5min. Discard the supernatant, add new culture medium to resuspend the cells, take 10μl to count, and calculate the required cell suspension. According to 0.2×10 4 10 cells / well were plated in a 96-well plate, with a total of 4 drug concentrations, and 5 replicate wells were set for each drug concentration. After 24 hours of cell attachment, the diluted drug concentration was added. On days 1-5, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in a 37°C incubator for 2 hours. The absorbance value was measured at 450 nm using an ELISA reader. Figure 8 shown.

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

[0046] Hela, Siha and U14 cells in the logarithmic growth phase were cultured at 0.05×10 4 Cells / well were seeded in a 6-well plate. One week later, Apicidin and Bevacizumab were incubated with the cells alone or in combination for 7 days. The formation of dot-like colonies at the bottom of the 6-well plate was observed. The cells were fixed with 4% tissue fixative, stained with 1% crystal violet for 30 minutes, and photographed for analysis. Fig. 9 As shown, the combined administration of Apicidin and Bevacizumab can significantly inhibit the proliferation of cervical cancer cells.

[0047] Resuspend the cells in serum-free medium and adjust the cell concentration to 5 × 10 4Cells were inoculated into the upper chamber of the Transwell chamber, and serum-containing culture medium was added to the lower chamber. Apicidin and Bevacizumab were added alone or in combination and incubated with the cells for 24 hours. The upper chamber was fixed with 4% tissue fixative and stained with crystal violet for 30 minutes. The interior of the upper chamber was wiped with a cotton swab, observed and photographed under a microscope, and counted and analyzed. Fig.10 The results showed that the combined administration of Apicidin and Bevacizumab more significantly inhibited the migration of cervical cancer cells.

[0048] 4. Effect of Apicidin combined with targeted drug Bevacizumab on apoptosis of cervical cancer cells Hela, Siha, and U14

[0049] Take logarithmically growing Hela, Siha, and U14 cells, digest and centrifuge the cells. After resuspending the cells, inoculate them into 6 cm culture dishes and spread 6 dishes. After 24 hours of complete cell adhesion, treat the cells with drugs. Drug concentration: The final concentration of Apicidin is 20μM, and the final concentration of Bevacizumab is 80μM. The order of drug addition is: blank (NC), Apicidin (Ap), Bevacizumab (Bev), Apicidin + Bevacizumab (Ap + Bev). After a period of combined drug treatment, the cells shrink and the lysis reaches 40%. Proceed to the next step. Protein extraction: After drug-treated cells, discard the supernatant, wash twice with PBS, add an appropriate amount of cell lysis buffer, lyse on ice for 5 minutes, scrape the cells and take the lysis buffer into the EP tube, lyse on ice for 20 minutes, centrifuge at 4°C, 12000rpm, 20 minutes, and collect the supernatant. After measuring the protein concentration by the BCA working method, add 5ⅹ protein loading buffer to dilute the supernatant to 1ⅹ, boil it in a metal bath at 100℃ for 10 minutes, and then load the sample. According to the protein blotting experimental procedures, detect the expression of apoptosis-related Cleaved-caspase-3, Bax, and Bcl-2. Apoptosis detection: dilute 10ⅹAnnexinVbinding Buffer into 1ⅹAnnexinV binding Buffer according to the operating instructions of the apoptosis kit. After drug-treated cells, discard the supernatant, wash twice with PBS, digest the cells with trypsin, and centrifuge. Discard the supernatant, add 1ml 1ⅹAnnexin Vbinding Buffer to resuspend the cells, and centrifuge. Discard the supernatant and repeat the above steps once. Add 400μl of 1ⅹAnnexin V binding Buffer to resuspend the cells. Then add 5μl of FITC / PI reagent respectively, protect from light for 15 minutes, and then detect on the machine. The results are as follows. Fig.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 developed 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 13-15 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 16-17 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. Fig.17The blue dots are cell nuclei, and cells with brown or brown precipitates in the nuclei are Ki67-positive cells. The experimental results showed that the nuclear staining in the combined treatment group was light and the positive index was low. Figures 13 to 17 The results suggest that Apicidin inhibited the growth of subcutaneous tumors in nude mice, and the ability of Apicidin combined with Bevacizumab to inhibit tumor growth was enhanced.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection 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.

3. The use according to claim 2, characterized in that The cervical cancer cells are Hela, Siha or U14.

4. The use according to claim 3, characterized in that The drug induces apoptosis of Hela, Siha or U14.

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