Application of vitexin in preparation of antitumor drugs

Abstract

The invention discloses application of vitexin (the molecular formula of the vitexin is C19H18O8, the molecular weight of the vitexin is 374.34, and the CAS number of the vitexin is 479-91-4) in preparation of antitumor drugs. The method comprises the following steps: firstly, treating esophageal cancer cell lines KYSE70 and KYSE450 with vitexin, detecting cell proliferation conditions after 0, 24 hours, 48 hours and 72 hours, and determining that the vitexin has an inhibiting effect on the esophageal cancer cell lines; the vitexin can inhibit cell proliferation of skin cancer cell lines SK-MEL-5 and SK-MEL-28 and ovarian cancer cell lines SKOV3 and TOV112D under the same concentration, and compared with a control group, the growth speed of tumor cells is remarkably reduced along with increase of the concentration of the vitexin, so that the vitexin has a remarkable effect on treating esophageal cancer, skin cancer and ovarian cancer; secondly, through a Pull down experiment, it is found that vitexin targets PRDX1, and it is indicated that vitexin can inhibit proliferation of tumor cells in vivo and in vitro by targeting PRDX1.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the application of vitex luteolin in the preparation of anti-tumor drugs. Background Art

[0002] Cancer has become the leading cause of death in China and is a serious public health problem. According to statistics, esophageal cancer and liver cancer are also the most common types of cancer in China, and are also the most common causes of cancer death. Cancer is the result of the synergistic effects of environmental, dietary, lifestyle and genetic factors. The combination of carcinogens and cellular genetic factors leads to cellular genetic gene mutations and gradually develops into cancer. The early clinical symptoms of cancer are not obvious. When the clinical symptoms are more prominent, the patient's condition is already in the middle or late stages. Therefore, choosing safe and effective methods to prevent and treat cancer is of great significance to improving the patient's prognosis and quality of life.

[0003] Casticin (molecular formula: C 19 H 18 O8, molecular weight: 374.34, CAS number 479-91-4) is a traditional Chinese herbal medicine extracted from plants of the genus Vitex. It has high medicinal value and anti-inflammatory, antioxidant, antibacterial and anti-tyrosinase effects. It is mainly used to treat wind-heat colds. It has low toxicity and side effects and is safe to use, so it has high clinical value. Studies have found that vitex lutein can inhibit the growth of breast cancer, colorectal cancer, lung cancer and other cancers, and can regulate mitochondrial apoptosis and JNK signaling pathways to inhibit the proliferation of esophageal squamous cell carcinoma cells and promote cell apoptosis; in gastric cancer, vitex lutein can enhance TRAIL-induced apoptosis by upregulating DR5 and endoplasmic reticulum stress; in colorectal cancer, vitex lutein induces apoptosis by activating mitochondria-dependent signal cascades and apoptosis signal-regulating kinase 1 (c-Jun N-terminal Kinase, JNK). The above literature shows that vitex lutein is of great significance in inhibiting the occurrence and development of tumors, but the exact effect and mechanism of action of vitex lutein in esophageal cancer, skin cancer and ovarian cancer are still unclear. Therefore, clarifying the mechanism of action of vitex lutein in inhibiting the occurrence and development of tumor cells in esophageal cancer, skin cancer and ovarian cancer is of great significance for early prevention and clinical medication guidance for cancer patients. Summary of the invention

[0004] The purpose of the present invention is to provide the use of vitex luteolin in the preparation of anti-tumor drugs, which are drugs for preventing and treating esophageal squamous cell carcinoma, skin cancer or ovarian cancer.

[0005] Based on the above purpose, the present invention adopts the following technical solutions: The invention discloses an application of vitex luteolin in the preparation of an anti-tumor drug, wherein the anti-tumor drug is a drug for preventing and treating esophageal squamous cell carcinoma, skin cancer or ovarian cancer.

[0006] Furthermore, the drug for treating esophageal cancer, skin cancer or ovarian cancer is a drug for preparing a drug for inhibiting the proliferation of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells.

[0007] Furthermore, the drug for treating esophageal cancer, skin cancer or ovarian cancer is a drug for promoting apoptosis of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells.

[0008] Furthermore, the esophageal squamous cell carcinoma cells are KYSE70 cells and / or KYSE450 cells.

[0009] Furthermore, the skin cancer cells are SK-MEL-5 cells and / or SK-MEL-28 cells.

[0010] Furthermore, the ovarian cancer cells are SKOV3 cells and / or TOV112D cells.

[0011] Furthermore, vitex luteolin at 0.25 μM to 1 μM can inhibit the proliferation of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells or promote apoptosis of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells.

[0012] Furthermore, vitex luteolin reduces the proliferation ability of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells or promotes apoptosis of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells by binding to PRDX1 protein.

[0013] Application of vitex luteolin in the preparation of PRDX1 protein inhibitors.

[0014] The natural small molecule compound vitex lutein in the present invention has high medicinal value, anti-inflammatory, antioxidant, antibacterial and anti-tyrosinase effects, and has low toxic and side effects, safe to use, and high clinical use value. The anti-cancer effect of the compound vitex lutein has been reported by scholars in breast cancer, colorectal cancer, lung cancer and other cancers, but the mechanism of the compound in esophageal cancer, skin cancer and ovarian cancer has not been reported; and the target protein in the present invention: PRDX1 protein is a new protein discovered in recent years as a treatment for cancer patients. The functional research of the protein has been reported by scholars, but the protein as a target of vitex lutein for the treatment of esophageal cancer, skin cancer and ovarian cancer patients has not been reported, and has not been applied to clinical treatment. Therefore, the present invention intends to prove through relevant experiments that the natural small molecule compound vitex lutein targets PRDX1 protein, induces tumor cell apoptosis and effectively inhibits the proliferation of tumor cells, thereby ultimately achieving clinical treatment of patients with esophageal cancer, skin cancer and ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The esophageal cancer cell lines KYSE70 and KYSE450 were seeded in 96-well plates, treated with vitexin (0, 0.25 μM, 0.5 μM, 1 μM), and the cell growth was detected by MTT assay at 0, 24 h, 48 h, and 72 h; Figure 2 The skin cancer cell lines SK-MEL-5 and SK-MEL-28 cells were seeded in 96-well plates, treated with vitexin (0, 0.25 μM, 0.5 μM, 1 μM), and the cell growth was detected by MTT assay at 0, 24 h, 48 h, and 72 h; Figure 3 The ovarian cancer cell lines SKOV3 and TOV112D cells were seeded in 96-well plates, and the cells were treated with vitexin (0, 0.25 μM, 0.5 μM, 1 μM). The MTT assay was used to detect the growth of cells at 0, 24 h, 48 h, and 72 h. Figure 4 After treating esophageal cancer cell lines KYSE70 and KYSE450 with vitexin (0, 0.25μM, 0.5μM, 1μM), PI and Annexin V staining flow cytometry was used to detect cell apoptosis; Figure 5 SK-MEL-5 and SK-MEL-28 cell lysates were incubated with vitexatin conjugate Sepharose 4B beads or with Sepharose 4B beads alone to pull down proteins, and PRDX1 expression was detected by western blotting.

[0016] Figures 1 to 4 middle,* p <0.05, ** p <0.01, *** p <0.001. DETAILED DESCRIPTION

[0017] Vitex lutein is a natural compound, and existing studies have shown the role of vitex lutein in the treatment of cancer. The technical solution of the present invention is further described in detail below in conjunction with the examples, but the protection scope of the present invention is not limited thereto.

[0018] The present invention mainly detects the effect of vitex lutein on the proliferation of esophageal cancer, skin cancer and ovarian cancer cells through cell proliferation experiment, cell apoptosis experiment and pull down experiment. The present invention first screens the effective drug concentration range of vitex lutein through literature search and MTT experiment, and on this basis, performs cell proliferation experiment and cell apoptosis experiment to further verify that vitex lutein inhibits the occurrence of tumor cells, thereby showing that vitex lutein has a significant effect in treating esophageal cancer, skin cancer and ovarian cancer.

[0019] Materials and Methods 1 Experimental Materials 1.1 Cell lines The human esophageal squamous cell carcinoma cell lines, including KYSE70 and KYSE450, human skin cancer cell lines SK-MEL-5 and SK-MEL-28, and human ovarian cancer cell lines SKOV3 and TOV112D, were purchased from the Shanghai Cell Bank. The cell lines required for the experiment were cultured in a mixed medium of 10% FBS and 1% penicillin-streptomycin. All cells were cultured in a constant temperature cell culture incubator at 37°C, 5% CO2, and appropriate humidity. All cells were stored in Henan Hormel Cancer Research Institute.

[0020] 1.2 Reagent preparation Prepare vitex luteolin solution, item number: 479-91-4, its molecular weight is 374.34, weigh 374.34 mg of powder into a sterile EP tube and fully dissolve it in 1 mL DMSO to make the prepared drug concentration 100 mmoL / L, and store it at -80℃ in the dark after packaging; vitex luteolin is a natural compound extracted from Vitex quinata, and its chemical structure is as follows.

[0021] 1.3 Instruments and equipment: 1.3.1 Constant temperature CO2 incubator (Thermo Scientific, USA) 1.3.2 Clean bench (Thermo Scientific, USA) 1.3.3 Constant temperature centrifuge (Beckman Coulter, USA) 1.3.4 High temperature and high pressure steam sterilizer (Beijing Taiya Saifu Company) 1.3.5 Fume hood (Beijing Zhongbaoyuan Office Equipment Co., Ltd.) 1.3.6 Metal Bath (Thermo Fisher, USA) 1.3.7 Transfer membrane instrument (Hoefer, USA) 2 Experimental methods 2.1 Cell proliferation assay Human esophageal cancer cell lines KYSE70 and KYSE450, skin cancer cell lines SK-MEL-5 and SK-MEL-28, and ovarian cancer cell lines SKOV3 and TOV112D were seeded in 96-well plates at 1000 cells / well. After 48 hours of attachment, the cells were incubated with different final concentrations of vitexin (0, 0.25 μM, 0.5 μM, 1 μM) for 0, 24 h, 48 h, and 72 h. The proliferation of tumor cells was detected by an ELISA instrument at a wavelength of 570 nm / 620 nm.

[0022] 2.2 Apoptosis Esophageal cancer cell lines KYSE70 and KYSE450 were cultured in 6 cm culture dishes and incubated overnight in a 37°C, 5% CO2 incubator. The cells were incubated with different concentrations of vitexin for 24 or 48 h, and a certain volume of Binding Buffer was added according to the number of samples to resuspend the cells and transfer them to flow tubes. Cell apoptosis was detected and analyzed by flow cytometry, and the data were processed using Graphpad prism software.

[0023] 2.3 Protein pull-down assay SK-MEL-5 and SK-MEL-28 cell lysates were incubated with vitexin conjugate Sepharose 4B beads or with Sepharose 4B beads alone in reaction buffer (50 mM Tris-HCI, pH=7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP-40 and 0.2 mM PMSF, 20x protease inhibitors) and incubated overnight at 4°C with gentle shaking. The beads were washed three times with wash buffer (50 mM Tris-HCI, pH=7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP-40 and 0.2 mM PMSF) and the binding was detected by western blotting.

[0024] Experimental Results 1. If Figure 1 As shown in the figure, the effect of vitex lutein on the growth of esophageal cancer cell lines KYSE70 and KYSE450 was observed. The cell growth trend was observed and it was found that when the concentration of vitex lutein was 0.25 μM, 0.5 μM, and 1 μM, the proliferation of KYSE70 cells was significantly inhibited after 24 h of culture. When the concentration of vitex lutein was 0.25 μM, the proliferation of KYSE450 cells was significantly inhibited after 48 h and 72 h of culture, indicating that vitex lutein showed a significant inhibitory effect on the growth of esophageal cancer cells.

[0025] 2. If Figure 2 As shown in the figure, the effect of vitex lutein on the growth of skin cancer cell lines SK-MEL-5 and SK-MEL-28. By observing the cell growth trend, it was found that when the concentration of vitex lutein was 0.5 μM and 1 μM, the proliferation of SK-MEL-5 cells was significantly inhibited after 24 h of culture. When the concentration of vitex lutein was 0.25 μM, the proliferation of SK-MEL-28 cells was significantly inhibited after 72 h of culture, indicating that vitex lutein showed a significant inhibitory effect on the growth of skin cancer cells.

[0026] 3. If Figure 3 As shown in the figure, the effect of vitex lutein on the growth of ovarian cancer cell lines SKOV3 and TOV112D cells. By observing the cell growth trend, it was found that when the concentration of vitex lutein was 0.5μM and 1 μM, the proliferation of SKOV3 cells was significantly inhibited after 48 h and 72 h of culture. When the concentration of vitex lutein was 0.25μM, 0.5μM, and 1μM, the proliferation of TOV112D cells was significantly inhibited after 24 h of culture, indicating that vitex lutein showed a significant inhibitory effect on the growth of ovarian cancer cells.

[0027] 4. If Figure 4 As shown in the figure, esophageal cancer cell lines KYSE70 and KYSE450 were cultured in 6 cm culture dishes. When the cell density reached 60-70%, the cells were treated with 0, 0.25μM, 0.5μM, and 1μM concentrations of vitex lutein. After 24 hours of culture in a cell culture incubator, samples were collected and cell apoptosis was detected by flow cytometry. The experimental results showed that with the increase of drug concentration, the number of apoptotic cells was significantly increased. When the concentration of vitex lutein was 1μM, the effect of promoting apoptosis of KYSE70 cells was very significant after 24 hours of culture. When the concentration of vitex lutein was 0.5μM, the effect of promoting apoptosis of KYSE450 cells was extremely significant after 24 hours of culture.

[0028] like Figure 5 As shown, SK-MEL-5 and SK-MEL-28 cell lysates were incubated with vitexatin conjugate agarose 4B beads or agarose 4B beads alone to pull down proteins, and PRDX1 expression was detected by western blotting. The results of the protein pull-down experiment showed that vitexatin can directly target PRDX1.

Claims

1. The use of vitex luteolin in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is a drug for preventing and treating esophageal squamous cell carcinoma, skin cancer or ovarian cancer.

2. The use according to claim 1, characterized in that: The drug for treating esophageal cancer, skin cancer or ovarian cancer is a drug for preparing a drug for inhibiting the proliferation of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells.

3. The use according to claim 1, characterized in that: The drug for treating esophageal cancer, skin cancer or ovarian cancer is a drug for promoting apoptosis of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells.

4. The use according to claim 2 or 3, characterized in that: The esophageal squamous cell carcinoma cells are KYSE70 cells and / or KYSE450 cells.

5. The use according to claim 2 or 3, characterized in that: The skin cancer cells are SK-MEL-5 cells and / or SK-MEL-28 cells.

6. The use according to claim 2 or 3, characterized in that: The ovarian cancer cells are SKOV3 cells and / or TOV112D cells.

7. The use according to claim 2 or 3, characterized in that: Vitex luteolin at 0.25µM ~1 µM can inhibit the proliferation or promote the apoptosis of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells.

8. The use according to claim 2 or 3, characterized in that: Vitex luteolin reduces the proliferation ability of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells or promotes apoptosis of esophageal squamous cell carcinoma cells, skin cancer cells or ovarian cancer cells by binding to PRDX1 protein.

9. Application of vitex luteolin in the preparation of PRDX1 protein inhibitors.

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