Application of mettl3 inhibitor combined with cisplatin in cervical cancer

Abstract

The application name is the application of METTL3 inhibitor combined with chemotherapy drugs to cervical cancer. It belongs to the field of antitumor drugs. Experiments prove that METTL3 inhibitor STM2457 can be used to treat cervical cancer; at the same time, it is proved that STM2457 can increase the sensitivity of the chemotherapeutic drug cisplatin for treating cervical cancer. Through experiments, the effect of using METTL3 inhibitor STM2457 alone on the growth of cervical cancer cells is understood; the effect of METTL3 inhibitor STM2457 combined with the chemotherapeutic drug cisplatin on the survival, proliferation and apoptosis-inducing ability of cervical cancer cells is understood; in vivo experiments on animals verify the effect of METTL3 inhibitor STM2457 alone and combined with the chemotherapeutic drug cisplatin on cervical cancer. Through the above methods, new treatment strategies are used to solve the drug resistance of cervical cancer patients, so as to achieve the best effect of treating cervical cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of anti-tumor drugs, and uses METTL3 inhibitor STM2457 to enhance the sensitivity of the chemotherapeutic drug cisplatin and treat cervical cancer. BACKGROUND

[0002] Cervical cancer (CC) is one of the most common gynecological malignancies, and is the fourth most common cancer affecting women worldwide, and is also the sixth most common cancer among women in China. In the cancer data statistics in China, the new cases of cervical cancer have reached 110,000, and 5.9 million patients have died of cervical cancer, which is almost half of the data enough to show that cervical cancer seriously endangers the health of women. Previous studies have shown that the prophylactic vaccination against cervical cancer can effectively prevent the first level of cervical cancer, and the secondary prevention relying on screening and early diagnosis and treatment has also achieved obvious results. Among the cases of cervical cancer diagnosed in developing countries, more than 70% are locally invasive or metastatic, and compared with early cervical cancer, the prognosis of such recurrent or metastatic cervical cancer patients is poorer, which can lead to higher mortality. The treatment of early and part of advanced cervical cancer is mainly radical surgery, and surgery can bring certain control effect, but the local recurrence and metastasis rate still hovers around 50%, and the reason is that the patients are not sensitive to the chemotherapeutic drugs during the treatment. Therefore, finding new strategies for the treatment of cervical cancer is particularly urgent for patients with advanced or recurrent cervical cancer who are resistant to chemotherapeutic drugs.

[0003] Meth-yltransferase like 3 (METTL3) is an N 6 - methyladenosine (m 6 A) methyltransferase regulated by methylation modification. STM2457 is a pioneering, highly efficient, selective, and orally active METTL3 inhibitor. It has high specificity for METTL3 and has no inhibitory effect on other RNA methyltransferases. Currently, STM2457 is in the clinical research stage in humans, and the indication is advanced solid tumors. STM2457 has been reported to inhibit the progression of acute myeloid leukemia, prolong the survival of mice, and play an anticancer role. Whether STM2457 can treat cervical cancer still needs further research.

[0004] Cis-dichlorodiamine platinum (cisplatin, DDP) is a non-specific drug of cell cycle, which can cross-link with DNA strands and show cytotoxicity. Studies have shown that about 20% of cervical cancer patients fail to respond to chemotherapy, resulting in tumor recurrence and poor prognosis. The most serious disadvantage of cisplatin treatment is that tumor cells are prone to drug resistance. Therefore, it is urgent to improve the sensitivity of cisplatin for the treatment of cervical cancer patients. SUMMARY

[0005] To solve the drug resistance of cervical cancer patients to conventional chemotherapy drugs, the technical problem to be solved by the present application is to use METTL3 inhibitor STM2457 combined with chemotherapy drug cisplatin to improve the sensitivity of cervical cancer patients to chemotherapy drug cisplatin.

[0006] The technical scheme of the present application is as follows:

[0007] The present application selects human cervical cancer cells HELA, and constructs a subcutaneous tumor in nude mice using a murine cell line U14.

[0008] The research results of the present application show that different concentrations of METTL3 inhibitor STM2457 can inhibit the growth of cervical cancer cells.

[0009] Further, compared with single drug, METTL3 inhibitor STM2457 combined with chemotherapy drug cisplatin can inhibit the growth of cervical cancer cells and enhance the ability to induce apoptosis of cervical cancer cells.

[0010] Further, compared with single drug, STM2457 combined with chemotherapy drug cisplatin can inhibit the growth of subcutaneous tumor in nude mice.

[0011] The above research shows that METTL3 inhibitor STM2457 can inhibit the growth of cervical cancer cells and improve the sensitivity of cervical cancer cells to chemotherapy drug cisplatin.

[0012] Advantages:

[0013] The present application mainly uses METTL3 inhibitor STM2457 combined with chemotherapy drug cisplatin for the treatment of cervical cancer patients and cervical cancer patients with drug resistance to chemotherapy drugs. Through experiments, we found that STM2457 alone can inhibit cell growth. Further, combined with chemotherapy drug cisplatin, the ability to inhibit the growth of cervical cancer cells and induce apoptosis is increased. The experimental results suggest that METTL3 inhibitor STM2457 alone or combined with chemotherapy drug cisplatin has great application prospect, and provides a certain theoretical basis and new treatment idea for the clinical treatment of cervical cancer. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1The effect of STM2457 alone on the survival rate of human cervical cancer cells HELA.

[0015] Figures 2-6 The effect of STM2457 combined with cisplatin on the survival rate of human cervical cancer cells HELA, the ability to inhibit the proliferation of cervical cancer cells and induce apoptosis of cervical cancer cells.

[0016] Figures 7-8 The weight of subcutaneous tumors and the volume change of mice per day before and after the mice were sacrificed after subcutaneous modeling of mouse cervical cancer cells U14 in nude mice, and the use of STM2457 alone, cisplatin alone and the combination of the two. DETAILED DESCRIPTION

[0017] The present application will be described in detail below with reference to the accompanying drawings. The cervical cancer cells HELA required in the present application were purchased from Shanghai Kui Sai Biological QuiCell Cell Library. The culture medium, PBS and trypsin used were purchased from biosharp company. Fetal bovine serum was purchased from Shanghai Datexil Biological Technology Co., Ltd. 1% streptomycin / penicillin was purchased from American Gibco company. The drugs STM2457 and cisplatin used were purchased from American MCE company and diluted according to the drug instructions. The antibody Cleaved Caspase-3 used was purchased from Wuhan Yunclone Technology Co., Ltd. and diluted according to the instructions. The apoptosis kit was purchased from Beijing Solabio Technology Co., Ltd. The drugs, reagents and antibodies used in the experiment can be purchased from other commercial channels, and the experimental results are only used to illustrate the present application and not to limit the scope of the present application. The experimental method is operated according to the conventional operation steps or according to the experimental method provided by the reagent merchant.

[0018] Cell culture: human cervical cancer cells HELA were cultured in DMEM medium containing 10% fetal bovine serum, 1% streptomycin / penicillin. The cell culture conditions were: 37℃, 5% CO2 incubator.

[0019] Drug concentration: STM2457 concentration was 50mM, cisplatin concentration was 3.33mM. The concentration of STM2457 used in the experiment was 60μM. The fixed concentration of cisplatin used was 5μM.

[0020] 1) In vitro experiment

[0021] 1. The effect of different concentrations of STM2457 on the survival rate of human cervical cancer cells HELA

[0022] Select logarithmic growth, good state HELA to 96-well plate, about 1.5 x 10 3Cells were used, with three wells for each drug concentration. The drug concentration gradient was 0, 60, 80, 100, and 120 μM. The specific steps were as follows: Discard the original culture medium, wash the cells twice with 2 ml PBS, digest the cells with 1 ml trypsin for 1 min, add 2 ml culture medium to neutralize the trypsin, transfer the cell suspension to a centrifuge tube, and centrifuge at 1000 rpm for 3 min. Discard the supernatant, resuspend the cells in fresh culture medium, take 5 μL for cell counting, and calculate the required cell suspension. Seed 100 μL per well in a 96-well plate. After 24 h of cell adhesion, dilute 100 μL of STM2457 to contain drug concentrations of 0, 60, 80, 100, and 120 μM. After 24 h of drug treatment, add 10 μL of CCK8, incubate for 2 h, and then measure the absorbance at 450 nm using a microplate reader. (Appendix) Figure 1 The results showed that the survival rate of HELA cells decreased with increasing drug concentration, suggesting that STM2457 can inhibit the growth of HELA cells.

[0023] 2. Effect of STM2457 combined with the chemotherapy drug cisplatin on the survival rate of human cervical cancer cells HELA.

[0024] Select logarithmically growing, well-formed HELA (Helicobacter pylori) and lay it in a 96-well plate, with each well approximately 1.5 x 10⁻⁶ cm². 3 Cells were used in 3 wells for each drug concentration. The final concentration of STM2457 was 60 μM, and the final concentration of cisplatin was 5 μM. 100 μL of drug-containing culture medium was added to each well. The order of drug addition was: blank, STM2457, cisplatin, and STM2457 + cisplatin. The specific steps were as follows: Discard the original culture medium, wash the cells twice with 2 ml PBS, digest the cells with 1 ml trypsin for 1 min, add 2 ml culture medium to neutralize the trypsin, transfer the cell suspension to a centrifuge tube, and centrifuge at 1000 rpm for 3 min. Discard the supernatant, resuspend the cells in fresh culture medium, take 5 μL for cell counting, and calculate the required cell suspension. Plate the cells in a 96-well plate with 100 μL of culture medium per well, creating 4 drug concentration gradients. After 24 h of cell adhesion, dilute the drug. After 24 h of drug treatment, add 10 μL of CCK8, incubate for 2 h, and then measure the absorbance at 450 nm using a microplate reader. (See attached image) Figure 2 The results showed that, compared with the single-drug group, treatment with cisplatin in combination with STM2457 significantly inhibited the growth of HELA cells. These results suggest that STM2457 can enhance the ability of cisplatin to inhibit HELA cell growth.

[0025] 3. Effect of STM2457 combined with the chemotherapy drug cisplatin on the proliferation of human cervical cancer cells HELA.

[0026] Select logarithmically growing, well-formed HELA (Helicobacter pylori) and lay it in a 96-well plate, with each well approximately 1.5 x 10⁻⁶ cm². 3Cells were used, with three wells for each drug concentration. The final concentration of STM2457 was 60 μM, and the final concentration of cisplatin was 5 μM. 100 μL of drug-containing culture medium was added to each well. The order of drug addition was: blank, STM2457, cisplatin, and STM2457 + cisplatin. The specific steps were as follows: Discard the original culture medium, wash the cells twice with 2 ml PBS, digest the cells with 1 ml trypsin for 1 min, add 2 ml culture medium to neutralize the trypsin, transfer the cell suspension to a centrifuge tube, and centrifuge at 1000 rpm for 3 min. Discard the supernatant, resuspend the cells in fresh culture medium, take 5 μL for cell counting, and calculate the required cell suspension. Plate 96-well plates with 100 μL of culture medium per well, for a total of four drug concentration gradients. After 24 hours of cell adhesion, on the first day, 10 μL of CCK8 was added to 12 wells of a 96-well plate and incubated for 2 hours. The absorbance was measured at 450 nm using a microplate reader. On subsequent days, at the same time, the drug was diluted and added to 12 wells of a 96-well plate. After 24 hours of drug treatment, 10 μL of CCK8 was added and incubated for 2 hours. The absorbance was measured at 450 nm using a microplate reader. The same results were obtained for 4 consecutive days.

[0027] Select logarithmically growing, well-formed HELA to form 6-well plates, with approximately 1 x 10⁻⁶ cells per well. 3 Cells were used, with three wells for each drug concentration. The final concentration of STM2457 was 60 μM, and the final concentration of cisplatin was 5 μM. 3 ml of drug-containing culture medium was added to each well. The order of drug addition was: blank, STM2457, cisplatin, and STM2457 + cisplatin. The specific steps were as follows: Discard the original culture medium, wash the cells twice with 2 ml PBS, digest the cells with 1 ml 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 1000 rpm for 3 min. Discard the supernatant, resuspend the cells in fresh culture medium, take 5 μL for cell counting, and calculate the required cell suspension. Plate the cells in 6-well plates with 3 ml of culture medium per well, for a total of 4 drug concentration gradients. After 96 hours, the drug was diluted and allowed to act for 7 days. The culture medium was then discarded. The cells were washed three times with 2 ml of PBS. 1 ml of 4% paraformaldehyde general tissue fixative was added to each well for 30 min. The cells were then washed three times with 2 ml of PBS. 1 ml of crystal violet staining solution was added to each well for 30 min. The cells were washed three times with 2 ml of PBS. The cells were then air-dried in a fume hood and photographed.

[0028] Appendix Figure 3 , 4 The results showed that, compared with the single-drug group, treatment with cisplatin in combination with STM2457 significantly inhibited the proliferation of HELA cells. These results suggest that STM2457 can enhance the ability of cisplatin to inhibit HELA cell proliferation.

[0029] 4. Effect of STM2457 combined with the chemotherapy drug cisplatin on HELA cell apoptosis.

[0030] HELA cells were taken for logarithmic growth, and the cells were digested and centrifuged. After resuspending the cells, they were inoculated into 6 cm culture dishes, 6 dishes were prepared. After 24 hours when the cells were completely adherent, the cells were treated with drugs. The drug concentrations were as follows: the final concentration of STM2457 was 60 μM, and the final concentration of cisplatin was 5 μM. The order of adding drugs was as follows: blank, STM2457, cisplatin, STM2457 + cisplatin. After the combined drugs were used for a period of time, the cells shrunk and lysed to 40%. The next step was performed. Protein extraction: after the drug treatment, the supernatant was discarded, the cells were washed twice with 2 ml of PBS, the cells were digested with 1 ml of trypsin for 1 min, 2 ml of medium was added to neutralize the trypsin, and the cell suspension was transferred to a centrifuge tube, which was centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the protein in the tube was collected, an appropriate amount of cell lysis buffer was added, and the lysis was performed on ice for 30 min, then the mixture was centrifuged at 4°C and 14000 rpm for 15 min, and the supernatant was collected. After measuring the protein concentration by BCA method, the supernatant was diluted to 1 x with 5 x protein loading buffer, and then boiled in a metal bath at 100°C for 10 min, and then loaded. According to the Western blotting experimental procedure, the expression of cleaved caspase-3 apoptosis protein was detected. Apoptosis detection: according to the instructions of the apoptosis kit, 10 x Annexin V binding Buffer was diluted to 1 x Annexin V binding Buffer. After the drug treatment, the supernatant was discarded, the cells were washed twice with PBS, the cells were trypsinized and centrifuged. The supernatant was discarded, and the cells were resuspended with 1 ml of 1 x 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 x Annexin V binding Buffer. Then 5 μL of FITC / PI reagent was added, and the mixture was incubated in the dark for 10 min, and then loaded onto the machine for detection. Appendix Figure 5 、 Figure 6 The results showed that compared with the single drug, the expression of apoptosis-related proteins increased significantly after STM2457 was combined with the chemotherapeutic drug cisplatin, and the amount of apoptotic cells increased. The results suggest that STM2457 can enhance the ability of the chemotherapeutic drug cisplatin to induce apoptosis of HELA cells.

[0031] 2) In vivo experiment

[0032] Effect of STM2457 combined with the chemotherapeutic drug cisplatin on the subcutaneous tumor of nude mice.

[0033] On the basis of the results of in vitro experiments, in vivo functional experiments were performed. 5 x 10 6 HELA cells were injected subcutaneously into the right groin of nude mice to establish a xenotransplantation model. The tumorigenicity of nude mice was observed, and when the tumor grew to about 100 mm 3Mice were randomly divided into 6 groups of 3 mice each, based on their weight and tumor size. The groups were as follows: control group (PBS), cisplatin group, STM2457 group, and 5-FU + cisplatin group. The drug concentrations used were: cisplatin (4 mg / kg / mouse, once every four days, intraperitoneal injection) and STM2457 (50 mg / kg / mouse, once every four days, intraperitoneal injection). Each mouse in the control group received an intraperitoneal injection of 100 μL of PBS, while each mouse in the treatment groups received an intraperitoneal injection of 100 μL of PBS containing the drug. Tumor volume was measured every four days. After 12 days of treatment, mice exhibiting emaciation and cachexia were euthanized, and tumors were dissected and weighed. Figure 7 The results showed that, during drug treatment of nude mice, the drug-treated group inhibited the growth of subcutaneous tumors compared to the control group. The combined drug treatment group showed an even stronger ability to inhibit subcutaneous tumor growth in nude mice. (Appendix) Figure 8 The results showed that, compared with the monotherapy group, subcutaneous tumors treated with STM2457 in combination with the chemotherapy drug cisplatin were smaller in both weight and volume. (See attached image) Figure 7 Appendix Figure 8 The results suggest that STM2457 or cisplatin alone inhibited the growth of subcutaneous tumors in nude mice, and that the ability of STM2457 to inhibit tumor growth was enhanced when combined with the chemotherapy drug cisplatin.

Claims

1. Use of STM2457 in combination with cisplatin in the preparation of a drug for treating cervical cancer.

2. Use according to claim 1, characterized in that, The dosage ratio of STM2457 to cisplatin is 60 μM: 5 μM.

3. Use according to claim 1, characterized in that, The cervical cancer is a cancer formed by human cervical cancer cells HeLa.

4. Use according to claim 3, characterized in that, The drug is used for inhibiting the proliferation of cervical cancer cells HeLa and inducing the apoptosis of cervical cancer cells HeLa.

5. The use according to claim 1, characterized in that, The drug is used for inhibiting the growth of subcutaneous transplanted tumors of nude mice.

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