Application of ansamitocin P-3 in preparation of medicine for treating gastric cancer

Abstract

The invention discloses an application of ansamitocin P-3 (Ansamitocin P-3) in preparation of a medicine for treating gastric cancer. The invention proves that the ansamitocin P-3 enables gastric cancer cells to die by influencing the apoptosis pathway of the cells through cytological research. Meanwhile, a stomach cancer abdominal cavity planting model of a nude mouse is constructed, and it is also observed that the ansamitocin P-3 has a killing effect on stomach cancer in the model. The invention provides a new application of ansamitocin P-3 in treatment of gastric cancer, and the ansamitocin P-3 has good clinical transformation value. The invention provides a new treatment means for gastric cancer patients with drug resistance.

Description

Technical Field

[0001] The present invention relates to a new gastric cancer treatment drug, and in particular to the use of ansamitocin P-3 in the preparation of a gastric cancer treatment drug. The present invention belongs to the field of medical technology. Background Art

[0002] Ansamitocin p-3 is a potent inhibitor of tubulin polymerization. 32 H 43 ClN2O9. The chemical structure is shown below:

[0003]

[0004] The study found that 5μM Ansamitocin p-3 completely inhibited the polymerization of microtubules isolated from bovine brain, but 80μM Ansamitocin p-3 did not cause microtubule polymerization. 16μM Ansamitocin p-3 also effectively depolymerized polymerized microtubules (IC50 = 3.8μM). Adding Ansamitocin p-3 to cultured cells prevented a certain concentration of dibutyryl cyclic adenosine 3':5'-phosphate from changing the morphology of AC cells from fibroepithelial cells to glioma-like cells. In addition, 16nM Ansamitocin p-3 treatment also caused the well-developed cytoplasmic network of A31 cells to rapidly disperse. Short-term treatment of A31 cells or KB cells with Ansamitocin p-3 also inhibited DNA synthesis. These results indicate that Ansamitocin p-3 acts by interacting with the microtubule assembly system, resulting in the inhibition of the formation of mitotic spindle fibers and ultimately cell death. [1] Ansamitocin p-3 has toxic effects on A-549, HT-29, and MCF-7 cells in a dose-dependent manner, with ED50 values ​​of 4×10 -7 ,4×10 -7 , and 2×10 -6 μg / mL [2] Ansamitocin p-3 was also toxic to HCT-116 cells, with an EC50 of 0.081 nM. [3] Ansamitocin p-3 enhances the effects of radiation in Drosophila cells and human cancer cells in a p53-dependent manner [4]. Ansamitocin p-3 (>1μg) treatment significantly inhibited the growth of leukemia SN36 and induced P388 leukemia cells to arrest in metaphase. Ansamitocin p-3 treatment at a dose of 25μg / kg per day significantly prolonged the lifespan of mice bearing intraperitoneal B16 malignant melanoma by 130%. Ansamitocin p-3 treatment also significantly prolonged the lifespan of mice bearing Ehrlich ascites carcinoma, Sarcoma 180, and P815 mastocytoma, but only slightly prolonged the lifespan of mice bearing ascites MOPC-104E myeloma, leukemia L1210, and leukemia C1498. [1] .

[0005] However, Ansamitocin P-3 has not been studied in upper gastrointestinal tumors, especially its role and mechanism in gastric cancer are still unclear. Drug treatment of gastric cancer is still a bottleneck in domestic tumor treatment. Compared with solid tumors such as breast cancer and colorectal cancer, there is a lack of specific drugs for the treatment of gastric cancer.

[0006] References:

[0007] [1]Ootsu K, et al. Effects of new antimitotic antibiotics, ansamitocins, on the growth ofmurine tumors in vivo and on the assembly of microtubules invitro. Cancer Res, 1980, 40(5), 1707-1717.

[0008] [2] Suwanborirux K, et al. Ansamitocin P-3, a maytansinoid, from Claopodium crispifolium and Anomodon attenuatus or associated actinomycetes. Experientia, 1990, 46 (1), 117-120.

[0009] [3]Young DH, et al. Aradioligandbinding assay for antitubulin activity in tumor cells. J Biomol Screen, 2006, 11(1), 82-89.

[0010] [4] Edwards A, et al. Combinatorial effect of maytansinol and radiation in Drosophila and human cancer cells. Dis Model Mech, 2011, 4(4), 496-503. Summary of the invention

[0011] The purpose of the present invention is to provide a new medicine for treating gastric cancer.

[0012] In order to achieve the above object, the present invention adopts the following technical means:

[0013] The present invention proves through cytological research that ansamitocin P-3 causes gastric cancer cell death by affecting the cell apoptosis pathway. At the same time, the present invention constructs a nude mouse abdominal cavity implantation model of gastric cancer, in which it is also observed that ansamitocin P-3 has a killing effect on gastric cancer.

[0014] Based on the above research, the present invention proposes the use of ansamitocin P-3 in the preparation of a drug for treating gastric cancer. The chemical formula of the ansamitocin P-3 is C 32 H 43 ClN2O9, the chemical structure is as follows:

[0015]

[0016] Preferably, the drug is an injectable drug.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention proposes a new use of ansamitocin P-3 in treating gastric cancer. The present invention finds through research that ansamitocin P-3 can kill gastric cancer cells by affecting cell apoptosis, can play a good tumor killing effect in gastric cancer, and has a good clinical transformation value. The present invention provides a new treatment direction for gastric cancer patients with drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the killing effect of ansamitocin P-3 on gastric cancer cell lines;

[0020] Among them, A: Ansamitocin P-3 concentration curve of HGC27 cell line; B: Ansamitocin P-3 concentration curve of MKN45 cell line;

[0021] Figure 2 The effect of ansamitocin P-3 on the cell cycle of gastric cancer cells;

[0022] Among them, A: flow cytometry HGC27 cell cycle ratio (NC is the control group, P-3 is the ansamitocin treatment group); B: flow cytometry MKN45 cell cycle ratio (NC is the control group, P-3 is the ansamitocin treatment group); C: flow cytometry HGC27 apoptotic cell ratio (NC is the control group, P-3 is the treatment group); D: flow cytometry MKN45 apoptotic cell ratio (NC is the control group, P-3 is the treatment group);

[0023] Figure 3 The animal experiment results show that Ansamitocin P-3 has a killing effect on gastric cancer;

[0024] Among them, A: luminescence of gastric cancer cells in the peritoneal cavity of the DMSO control group; B: luminescence of gastric cancer cells in the peritoneal cavity of the ansamitocin P-3 treatment group. DETAILED DESCRIPTION

[0025] The following examples are used to further describe the present invention, and the advantages and disadvantages of the present invention will become clearer as the description proceeds. However, the following examples are exemplary only and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0026] Example 1 The killing effect of ansamitocin P-3 on gastric cancer cell lines

[0027] Using HGC27 and MKN45 gastric cancer cell lines, we evaluated the proliferation of different cell lines by cell counting kit-8 (CCK-8) assay. Gastric cancer cell lines with good growth status were cultured in advance. 5000 cells were placed in each well of a 96-well plate, and 5 replicate wells were set for each cell line. The three wells with the closest results were taken to calculate the average absorbance. After culturing and waiting for the cells to adhere to the wall, the value was recorded as 0 hours, and then the absorbance was recorded for 24 hours. Ansamitocin P-3 complete medium with concentrations of 0.001μM, 0.01μM, 0.1μM, 1μM, 10μM, and 100μM were added to the two gastric cancer cell lines, respectively, and cultured for 24 hours. The culture medium containing 10μl CCK-8 reagent was replaced 1 hour in advance, incubated in the dark for 1 hour, and the absorbance after 1 hour was measured at 450nm using a 96-well plate microplate reader. Finally, the average value and statistical value were calculated, and a curve graph was drawn. The results are shown in Figure 1 As shown, the results showed that in the MKN45 gastric cancer cell line, the IC50 concentration of ansamitocin P-3 was 0.95 μM, and in the HGC27 gastric cancer cell line, the IC50 concentration was 16.23 μM.

[0028] Example 2 Ansamitocin P-3 kills gastric cancer cells via apoptosis pathway

[0029] HGC27 and MKN45 gastric cancer cell lines were treated with complete medium containing ansamitocin P-3 at a concentration of 0.2 nM for 24 hours as the drug group (P-3). At the same time, gastric cancer cells cultured in complete medium without ansamitocin P-3 were set as the control group (NC). They were incubated with anhydrous ethanol at -20°C overnight before the experiment. First, fully digest with 1 ml of 0.25% trypsin (without EDTA) in a culture flask and then add 1 ml of complete culture medium to terminate the digestion. Centrifuge at 1000 rpm / min in a centrifuge for 5 minutes, aspirate the supernatant, wash the cells with PBS, and repeat twice. After the last centrifugation, resuspend the cells with PBS and use a cell counting plate to calculate the cell concentration. Mix the counted PBS and cell clusters thoroughly, take 5×10 5 Resuspend the cells in the liquid of 100 μl of PBS, centrifuge at 300×g for 5 minutes, and discard the supernatant. Repeat 3 samples for each cell type and store them on ice. Dissolve RNase AReagent, mix well, and put on ice for later use. Add 1 ml of PBS to wash the cells again, centrifuge at 300×g for 5 minutes, and discard the supernatant. Add 300μl of PBS to resuspend the cells. Add 1.2 ml of pre-cooled alcohol, mix well, and place in a -20℃ refrigerator for 1 hour. Centrifuge at 300×g for 5 minutes, leaving only the cells, and then add 1 ml of PBS to resuspend the cells, and let stand at room temperature for 15 minutes. Centrifuge at 300×g for 5 minutes again, add 100μl of RNase AReagent to the remaining cells and blow the cells well, and bathe at 37℃ for 30 minutes. Add 400μl of PI Reagent (50μg / ml) and mix well, and incubate at 2-8℃ away from light for 30 minutes. Immediately detect on the machine after endowment, and record the red fluorescence at the excitation wavelength of 488nm. The data are collected and analyzed. Figure 2 As shown in A and B, the results show that after being treated with ansamitocin P-3, both gastric cancer cells were arrested in the G2 / M phase, and the number of arrested cells in the drug group was about twice that of the control group.

[0030] In addition, the HGC27 and MKN45 cells in the logarithmic growth phase treated with ansamitocin P-3 and the cells in the control group were respectively cultured at 1×10 6 10 cells were inoculated into a 6-well plate. When the cells were completely attached to the wall, the cell supernatant was collected. The cells were rinsed twice with PBS. The PBS was recovered and the residual liquid was sucked out. 1 ml of EDTA-free trypsin was added and incubated in a cell culture incubator for 2 minutes. The trypsin was neutralized with the collected mixed solution. The cell suspension was collected by gently blowing with the pipette tip and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded and the cell pellet was washed twice with PBS. An appropriate amount of 1×Annexin V Binding Solution was added to the cell pellet to make the cell concentration reach 1×106 pc / ml, 5μl Annexin V, FITC conjugate and 5μl PI Solution were added to each tube of the experimental group, the flow tube was wrapped with tin foil and incubated for 15 minutes, and finally 400μl 1×Annexin VBinding Solution was added to each tube, and the test was completed within 1 hour. The results are shown in Figure 2 As shown in C and D, the results show that the number of apoptotic cells in the drug group increased greatly, which was about 3 times the number of apoptotic cells in the control group.

[0031] Example 3 Animal Experiment on the Killing Effect of Ansamitocin P-3 on Gastric Cancer

[0032] The gastric cancer cells with luciferase label were digested and washed. The cell concentration was adjusted to 1×10 7 / ml, turn each mouse abdomen upward, take 100μl of cell fluid and inject it into the abdominal cavity of Balb / c nude mice through the groin. After observing that the cell fluid will not flow out, put the mouse back into the cage. The mice were randomly divided into two groups. The drug group used a dose of 0.03mg / kg (DMSO was used to prepare the stock solution of Ansamitocin P-3), which was diluted with a solvent of polyethylene glycol 300 (40%) + Tween 80 (5%) + saline (53%) and injected intraperitoneally. The control group was injected with the same dose of solvent and DMSO. After three consecutive injections, rest for four days. Ensure that the living environment of the mice is relatively sterile, change the bedding twice a week, and replenish water every two days. After 7 days, the two groups of mice were injected with potassium salt of fluorescein, with a concentration of 15mg / ml and an injection volume of 10μl / g. Fluorescence imaging was performed using an IVIS machine 10 minutes after injection. After 5 days, the results of peritoneal implantation in the control group and the drug group were observed, and photos were taken to compare the luminescence between the two groups. Figure 3 As shown, the results showed that after continuous intraperitoneal injection of Ansamitocin P-3 at a dose of 0.3 mg / kg for 3 days, the tumor cells in mice were significantly reduced.

Claims

1. Use of Ansamitocin P-3 in the preparation of a drug for treating gastric cancer, wherein the chemical formula of Ansamitocin P-3 is C 32 H 43 ClN2O9, the chemical structure is as follows:

2. The use according to claim 1, characterized in that The medicine is an injectable medicine.

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