Cancer treatment medicine composition and application thereof in preparation of esophageal cancer treatment medicine

Abstract

The invention relates to the field of biological medicine, in particular to a cancer treatment medicine composition and application thereof in preparation of esophageal cancer treatment medicine. The purpose of the present invention is to provide a cancer treatment pharmaceutical composition, which comprises a therapeutically effective amount of an inhibition component, and the inhibition component is used for inhibiting the expression of ALKBH3. The pharmaceutical composition provided by the invention can effectively inhibit the expression level of the ALKBH3 gene, has an inhibiting effect on growth, proliferation and migration of esophageal cancer cells, and can also induce esophageal cancer cell DNA damage and increase the sensitivity of tumor cells to radiotherapy and chemotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to a cancer therapeutic drug composition and its application in the preparation of a therapeutic drug for esophageal cancer. Background Art

[0002] Esophageal cancer is one of the most common malignant tumors in humans. Globally, the incidence and mortality rates of esophageal cancer rank among the top. Most patients are already in the advanced stage when significant symptoms appear, resulting in a five-year survival rate of less than 20%. Esophageal cancer is mainly divided into two histological types: squamous cell carcinoma and adenocarcinoma, with squamous cell carcinoma being particularly common in the Asian population. Esophageal cancer patients usually undergo treatments such as surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, but tumor recurrence and metastasis are the main reasons for treatment failure.

[0003] Radiotherapy and chemotherapy are important treatment modalities for esophageal cancer, applicable to radical treatment of locally advanced patients, neoadjuvant treatment before surgery, and adjuvant treatment after surgery. For patients with locally advanced esophageal cancer who are not suitable for surgery, concurrent chemoradiotherapy is the only radical treatment strategy. However, the resistance of esophageal cancer to radiotherapy and chemotherapy leads to local recurrence or distant metastasis in some patients after treatment, significantly reducing the survival period of patients. Therefore, exploring new therapeutic and chemoradiotherapy sensitization targets for esophageal cancer helps improve the therapeutic efficacy of esophageal cancer and is of great significance for improving the prognosis of esophageal cancer patients.

[0004] In the prior art, reports on the ALKBH3 gene mainly focus on its role in promoting tumor progression, for example:

[0005] 1. Gu et al. found through various experimental methods such as dot blot assay, immunofluorescence, and Western blot that the expression level of ALKBH3 is upregulated in uveal melanoma and is associated with poor prognosis in patients. Knockdown of ALKBH3 can increase the level of m¹A, thereby inhibiting the proliferation, migration, and invasion abilities of uveal melanoma cells and playing an inhibitory role in tumor growth both in vitro and in vivo. Mechanistically, histone lactylation promotes the malignant progression of cancer by enhancing the expression of ALKBH3. (See: Gu X, et al. Histone lactylation-boosted ALKBH3 potentiates tumor progression and diminished promyelocytic leukemia protein nuclear condensates by m¹A demethylation of SP100A. Nucleic Acids Research, 2024, 52, 2273–2289.)

[0006] 2. Wang et al. found through various experimental methods, such as immunohistochemistry, qRT-PCR, cell proliferation and colony formation assays, that ALKBH3 is overexpressed in hepatocellular carcinoma and is associated with tumor differentiation degree, TNM stage, and distant metastasis. Patients with high ALKBH3 expression showed worse disease-free survival and overall survival compared to those with low ALKBH3 expression. Additionally, knocking down ALKBH3 could inhibit the proliferation ability of liver cancer cells in vitro and the tumorigenic ability in vivo. (See: Wang Q, Wang G, Wang Y, et al. ALKBH3 expression is associated with tumor recurrence and unfavorable prognosis in hepatocellular carcinoma. J Gastroenterol Hepatol. 2018. doi:10.1111 / jgh.14117)

[0007] The above research indicates that ALKBH3, as a key tumor promoter and epigenetic regulator, has the potential to be a therapeutic target for esophageal cancer. However, currently, there are no reports at home and abroad on the research of inhibiting ALKBH3 in the preparation of anti-esophageal cancer drugs and radiosensitizers and chemosensitizers for esophageal cancer radiotherapy and chemotherapy. Summary of the Invention

[0008] The object of the present invention is to provide a cancer treatment drug composition, which can effectively inhibit the expression level of the ALKBH3 gene, has an inhibitory effect on the growth and proliferation ability and migration of esophageal cancer cells, and can also induce DNA damage in esophageal cancer cells.

[0009] Another object of the present invention is to provide an application of a cancer treatment drug composition in the preparation of drugs for treating esophageal cancer.

[0010] The embodiments of the present invention are achieved through the following technical solutions:

[0011] A cancer treatment drug composition, comprising: a therapeutically effective amount of an inhibitory component, and the inhibitory component is used to inhibit the expression of ALKBH3.

[0012] Preferably, the inhibitory component comprises: HUHS105.

[0013] Preferably, the cancer treatment drug composition further comprises: a therapeutically effective amount of cisplatin.

[0014] An application of the described cancer treatment drug composition in the preparation of drugs for treating esophageal cancer.

[0015] Preferably, the esophageal cancer treatment drug is used to increase the sensitivity of tumor cells to radiotherapy and chemotherapy.

[0016] The present invention has at least the following beneficial effects:

[0017] After the esophageal cancer cell lines KYSE520 and ECA109 were treated with HUHS105 in the present invention, the expression of the ALKBH3 gene was successfully inhibited.

[0018] In the present invention, the inhibitory effect of HUHS105 combined with radiotherapy and chemotherapy on the growth of esophageal cancer cells is higher than that of the group treated with HUHS105 alone, the radiotherapy group alone, and the chemotherapy group alone, showing an obvious radiosensitization and chemosensitization effect.

[0019] The pharmaceutical composition provided by the present invention can effectively inhibit the expression level of the ALKBH3 gene, has an inhibitory effect on the growth, proliferation and migration of esophageal cancer cells, can also induce DNA damage in esophageal cancer cells and increase the sensitivity of tumor cells to radiotherapy and chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 : IC50 curves and ALKBH3 protein expression levels of KYSE520 and ECA109 cells in the HUHS105 treatment group and the control group;

[0022] Figure 2 : Colony formation of KYSE520 and ECA109 cells in the HUHS105 treatment group and the control group; Figure 3 : Scratch healing of KYSE520 and ECA109 cells in the HUHS105 treatment group and the control group; Figure 4 : Number of γ-H2AX foci formed by radiotherapy in KYSE520 and ECA109 cells in the HUHS105 treatment group and the control group; Figure 5 : Cell survival of KYSE520 and ECA109 cells in the HUHS105 treatment group and the control group at different radiotherapy doses; Figure 6 : Cisplatin sensitivity of KYSE520 and ECA109 cells in the HUHS105 treatment group and the control group. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention.

[0024] Example 1: A cancer treatment pharmaceutical composition, comprising: a therapeutically effective amount of an inhibitory component for inhibiting ALKBH3 expression.

[0025] Example 2: The inhibitory component comprises: HUHS105.

[0026] Example 3: The cancer treatment pharmaceutical composition further comprises: a therapeutically effective amount of cisplatin.

[0027] Example 4: Use of a cancer treatment pharmaceutical composition as described above in the preparation of a drug for treating esophageal cancer.

[0028] Example 5: The drug for treating esophageal cancer is used to increase the sensitivity of tumor cells to radiotherapy and chemotherapy.

[0029] A therapeutically effective amount means the amount of a compound that is effective in preventing, alleviating or improving the symptoms of a disease in a subject being treated or prolonging the survival period.

[0030] Compounds suitable for the compositions and methods of the present invention can be used in various formulations. Some formulations affect the rate at which the compound enters the bloodstream of a patient. Thus, some formulations are immediate release formulations, while other formulations are delayed release, sustained release or long-acting release formulations.

[0031] The term "pharmaceutical composition" means that the compounds of the present invention can be formulated with other chemical components (such as diluents, lubricants, swelling agents, disintegrants or carriers). Pharmaceutical compositions facilitate the administration of compounds that produce a primary therapeutic effect to an organism. There are various techniques for administering compounds in the art, including but not limited to oral, injection, inhalation, spraying, parenteral and topical administration. Pharmaceutical compositions can also be obtained by reacting the compound with an inorganic or organic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0032] The term "carrier" defines a compound that facilitates the incorporation of a compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier because it helps many organic compounds to be taken up into the cells or tissues of an organism.

[0033] The term "diluent" defines a compound diluted in water that will dissolve the target compound and stabilize the biologically active form of the compound. In the art, salts dissolved in buffer solutions are used as diluents. A commonly used buffer solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of the solution at low concentrations, buffer diluents rarely alter the biological activity of the compound.

[0034] In certain embodiments, the same substance can be used as a carrier, diluent, or excipient, or have any two of these roles or all three roles. Thus, a single additive in a pharmaceutical composition can have multiple functions.

[0035] The pharmaceutical compositions of the present invention can be manufactured using techniques known in the art, such as by means of conventional mixing, dissolving, granulating, sugar coating, milling, emulsifying, encapsulating, entrapping, or tableting methods.

[0036] Experiment:

[0037] 1. Materials

[0038] The esophageal cancer cell lines KYSE520 and ECA109 were purchased from the American Type Culture Collection (ATCC) and cultured in our laboratory;

[0039] RPMI 1640 medium was purchased from Zhejiang Senrui Biotechnology Co., Ltd.;

[0040] Fetal bovine serum was purchased from Novoprotein Scientific Inc.;

[0041] Trypsin, phosphate buffered saline (PBS), and RIPA protein lysate were purchased from Zhejiang Senrui Biotechnology Co., Ltd.;

[0042] Electrophoresis buffer and electrotransfer buffer were purchased from Hangzhou Aoqian Biotechnology Co., Ltd.;

[0043] ALKBH3 antibody and Actin antibody were purchased from Abmart.

[0044] 2. Application process

[0045] Cell culture and subculture: KYSE520 and ECA109 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. The cells were digested with trypsin, the cell suspension was collected and centrifuged, the supernatant was discarded, the cell pellet was resuspended with RPMI 1640, and then the cell suspension was transferred to a culture dish. The culture dish was gently shaken and then placed in an incubator containing 5% CO2 at 37°C for culture. The cell status was observed in a timely manner, and treatments such as medium replacement, subculture, cryopreservation, and plating were selected according to the cell status.

[0046] Drug treatment of cells: Inoculate an appropriate amount of cells into a six-well plate. Note that when plating, mix the cells evenly so that the cell density reaches 50%-70% the next day. Prepare solutions of different concentrations of HUHS105 (such as 0 μM, 20 μM, 40 μM, 60 μM, etc.) with the culture medium. Add the HUHS105 solutions of different concentrations to the six-well cell plate and gently shake the cell plate to mix evenly. Subsequently, place the cell culture plate in an incubator at 37 °C and 5% CO2 for culture. After 24 h of treatment, perform subsequent detections.

[0047] Collect the cells treated with HUHS105 and detect the protein expression level of ALKBH3 using Western blot assay. The specific steps are as follows: Aspirate the culture medium in the culture dish and wash the cells twice with pre-cooled PBS. After pouring out the PBS, add the pre-prepared lysis buffer mixed with RIPA and PMSF (100:1). After lysing on ice for half an hour, collect and place it in a high-speed centrifuge, and centrifuge at 14400 rpm for 15 min at 4 °C. Operate according to the instructions of the Thermo Fisher BCA protein quantification kit, and use an enzyme-linked immunosorbent assay reader to measure the absorbance of the sample at 562 nm to obtain the protein concentration of the experimental sample.

[0048] Mix the 5× Loading Buffer and the protein sample at a ratio of 1:4, and place it in a pre-activated metal bath heater. Boil at 100 °C for 5 min to denature the protein. Then add an appropriate amount of 1× Loading Buffer to each group of samples to make the protein concentrations of each group consistent. Use a Biorad electrophoresis apparatus to perform electrophoresis at a constant voltage of 100 V for 60 min. Cut out a PVDF membrane of appropriate size, soak it in methanol for 30 s, and install the clips in the transfer buffer in the order of black side (-), sponge, filter paper, gel, membrane, filter paper, filter paper, sponge, white side (+) from bottom to top. Place it in a transfer tank and transfer at a constant current in the pre-cooled transfer buffer for 30 - 120 minutes (the transfer time depends on the specific molecular weight of the protein). Prepare 5% milk with TBST, place the transferred membrane in the milk, and incubate it at room temperature on a shaker for 2 h. Place the protein band in the corresponding primary antibody and put it in a 4 °C refrigerator overnight. The next day, place the protein band in an antibody incubation box and wash it 3 times with TBST, 10 min each time. Prepare an appropriate amount of secondary antibody diluent with TBST at a concentration of 1:3000, incubate the protein band at room temperature on a shaker for 2 h, and then wash it 3 times with TBST, 10 min each time. Prepare the developing solution at a ratio of 1:1 and store it in a dark box. Soak the washed band in the developing solution, and then use ImageLab software to develop and analyze the band to obtain the protein expression trend. The results are shown in Figure 1. As the concentration of HUHS105 increases, the expression levels of the ALKBH3 gene in KYSE520 and ECA109 cells gradually decrease.

[0049] Cell colony formation assay: Digest and centrifuge the cells in the logarithmic growth phase to prepare a cell suspension. Count the cells using a cell counting chamber and adjust the cell density to an appropriate concentration of 500 cells / mL. Add 2 mL of medium to each well, gently shake the culture plate to evenly distribute the cells. Place the culture plate in a cell culture incubator at 37 °C and 5% CO2, and change the fresh medium every 2 - 3 days for continuous culture for 10 - 14 days. Discard the medium, gently rinse the cells 2 - 3 times with PBS, add 1 mL of methanol to fix for 15 minutes, then discard the methanol. Subsequently, add 1 mL of crystal violet staining solution and stain for 15 minutes. Then, slowly rinse with water to remove the excess staining solution and air dry. Observe under a microscope and count the number of cell colonies containing more than 50 cells. The results are as Figure 2 shown. As the concentration of the HUHS015 drug increases, the number of colony formations of both KYSE520 and ECA109 esophageal cancer cells shows a downward trend. This proves that HUHS015 has an inhibitory effect on the growth and proliferation ability of esophageal cancer cells.

[0050] Cell scratch healing assay: Cells in the logarithmic growth phase were digested and centrifuged to prepare a cell suspension, which was then inoculated into a 6-well plate. The seeding density was determined according to the cell type, and 2 mL of medium was added to each well. The 6-well plate was placed in an incubator for culture. When the cells reached confluence, a 20-μL pipette tip was used to scratch the cells in the center of the well. The cells were rinsed 3 times with PBS to wash away the scratched cells, and the cells were treated with HUHS015 at concentrations of 0 μM, 5 μM, 10 μM, and 15 μM, respectively. The initial state of the cell scratch was recorded at 0 h after treatment, and then the healing of the cell scratch was recorded at 24 h (for KYSE520 cells) and 12 h (for ECA109 cells) after treatment, respectively. The results are as Figure 3 shown. As the concentration of the HUHS015 drug increased, the scratch healing percentage of both KYSE520 and ECA109 esophageal cancer cells showed a downward trend. This indicates that HUHS015 can inhibit the migration ability of esophageal cancer cells, and this inhibitory effect is enhanced within a certain range as the drug concentration increases, that is, it proves that HUHS015 has an inhibitory effect on the migration ability of esophageal cancer cells.

[0051] Number of γ-H2AX foci formed in KYSE520 and ECA109 cells in the HUHS105 treatment group and the control group after radiotherapy induction;

[0052] KYSE520 and ECA109 cells were treated with different concentrations of HUHS105 (0 μM, 5 μM, 10 μM, 15 μM), and then induced with 8 Gy of radiation. The cells were fixed and immunofluorescently stained at 0.5 h, 4 h, and 24 h after treatment to detect the number of γ-H2AX foci formed in the cells. As Figure 4 shown, compared with the control group, the number of γ-H2AX foci formed in the HUHS015 treatment group cells was significantly increased at 0.5 h, 4 h, and 24 h, and there were statistical differences. This indicates that HUHS015 can induce DNA damage in esophageal cancer cells, and as the drug concentration increases, the degree of DNA damage intensifies. That is, it proves that HUHS015 has the effect of inducing DNA damage in esophageal cancer cells.

[0053] Radiosensitivity experiment of KYSE520 and ECA109 cells after HUHS015 treatment

[0054] In two esophageal cancer cell lines, KYSE520 and ECA109, the cells were treated with DMSO (as a control) and HUHS015, respectively, and then irradiated with different radiotherapy doses (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy). The survival fraction of the cells at different radiotherapy doses was calculated by the colony formation assay to evaluate the survival of the cells. FromFigure 5 It can be seen that at the same radiotherapy dose, the cell survival fraction of the HUHS015 treatment group is lower than that of the control group cells, and the difference is statistically significant. This indicates that HUHS015 can increase the sensitivity of esophageal cancer cells to radiotherapy, that is, after treatment with HUHS015, esophageal cancer cells are more likely to die after radiotherapy, and HUHS015 has a radiosensitizing effect.

[0055] The above irradiation conditions are as follows: The cells are irradiated using an Elekta medical linear accelerator, and the irradiation parameters are set as 6MV-X rays, a source-skin distance of 100 cm, and an irradiation field of 40 cm × 40 cm. The monitor units (MU) are set according to the actual required irradiation dose D.

[0056] The chemosensitivity of the HUHS105 treatment group and the control group of KYSE520 and ECA109 cells;

[0057] KYSE520 and ECA109 cells were treated with DMSO and HUHS015 respectively, and then the cells were exposed to different concentrations of cisplatin (the concentration was 0 - 20 μM in the KYSE520 cell experiment and 0 - 80 μM in the ECA109 cell experiment). The changes in cell viability were detected by the CCK-8 assay. As Figure 6 can be seen, at the same cisplatin concentration, the viability of KYSE520 and ECA109 cells treated with HUHS015 is lower than that of the control group cells. This indicates that HUHS015 can increase the chemosensitivity of esophageal cancer cells, that is, after treatment with HUHS015, the chemosensitivity of esophageal cancer cells increases.

[0058] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cancer treatment pharmaceutical composition, characterized in that, Comprising: A therapeutically effective amount of an inhibitory component for inhibiting ALKBH3 expression.

2. The cancer treatment pharmaceutical composition according to claim 1, wherein The inhibitory component comprises: HUHS105.

3. The cancer treatment pharmaceutical composition according to claim 1 or 2, characterized in that, Further comprising: A therapeutically effective amount of cisplatin.

4. Use of the cancer therapeutic pharmaceutical composition according to any one of claims 1 - 3 in the preparation of a therapeutic drug for esophageal cancer.

5. The application according to claim 4, characterized in that, The therapeutic drug for esophageal cancer is used to increase the sensitivity of tumor cells to radiotherapy and chemotherapy.

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