Chinese Medicine The Source Of The Cytotoxic Activity Of Two Anti-cancer Drugs With Tumor Suppressor Gene Pdcd4 Expression Correlation

Traditional Chinese medicine is playing a more and more important role in the treatment of tumors. Some medicinal plants and insect drugs of traditional Chinese medicine has great potential in the anti-tumor domain. People are paying close attention to them.Mylabris has been used as a medicine for the treatment of tumors for more than2,000 years in China. The effective ingredient in mylabris is cantharidin, which can inhibits the synthesis of protein and affects cell growth and differentiation.Norcantharidin (NCTD) is a new anticancer drug developed and producted by China. NCTD is the demethyl derivative of cantharidin,which has significant anti-tumor activity and small side effects. What's more,NCTD is the only anticancer drug that can increase the number of leucocyte.Camptothecin(CPT) is an alkaloid extracted from Camptotheca acuninata, the only selectiveinhibitor of DNA topoisomeraseⅠ(TopoⅠ). Later people abstracted 10-HCPT(Hydroxycamptothecin, HCPT) from CPT. The chemical structure of 10-HCPT is similar to CPT's, only a hydrogen beening replaced by hydroxyl. Compared with CPT, HCPT has stronger anti-tumor effect and lower toxicity, that make camptothecin drugs play a significant role in theprevention and treatment of tumors.Retinoic acid (RA) is a derivative of vitamin A, also known as vitamin A acid. retinoic acid has a variety of isomeride. All-trans retinoic acid (ATRA) is an important one of them. ATRA is an important bioactive substances in vivo, It has been found play an important role in embryonic development, inhibiting cell proliferation and promoting cell differentiation and apoptosis, as well as in visual cycle.Pdcd4 (Programmed cell death 4) is an apoptosis gene.The up-regulated expression of Pdcd4 was seen in the apoptosis process of mice cells induced by many factors. Recent studies show that Pdcd4 is also a tumor suppressor:The expression of Pdcd4 in human lung cancer, breast cancer, colorectal cancer, prostate cancer and was down-regulated. Up-regulating Pdcd4 expression in mouse skin could inhibit epithelial cancer induced by carcinogenic substances. As a new tumor suppressor , Pdcd4 may become a new therapeutic target for anti-cancer therapy in the future.The main content of this study and the results:To obtain Pdcd4 high expression BGC-823 cell lines and the control cell lines, lipid-mediated transfection was used to transfect Pdcd4cDNA, empty carrier and mutant Pdcd4cDNA respectively into human gastric adenocarcinoma BGC-823 cells. Choosing human ovarian carcinoma SK-OV-3 cells as the host ,we use the same method to obtain Pdcd4 down-regulated expression cancer cell line and thecontrol cell line.Western blot was used to analysis the transfection results and the expression of Pdcd4 protein in the BGC-823 transfected cells treated by NCTD and HCPT. The results confirmed that we had established stable Pdcd4 up-regulated expression and Pdcd4 down-regulated expression cell lines. The results showed that after treated by 60 umol / L NCTD 72 hours, the expression of Pdcd4 protein was significantly down-regulated in the three transfected cells; after treated by 80 umol / L HCPT 72 hours, the expression of Pdcd4 protein was significantly up-regulated in the three transfected cells. The mechanism is still unclear.In the interest of whether Pdcd4 can affect the sensitivity of BGC-823 transfected cells to anti-cancer drugs, MTT method was used to detect the cytotoxic activity of NCTD and HCPT to the transfected cell lines. The results showed that low concentrations of NCTD effecting long–term and high concentrations of NCTD effecting short–term both increased the sensitivity of Pdcd4 up-regulated cells to NCTD. The results of HCPT is similar to NCTD's.To find out the cytotoxicity mechanism of NCTD and HCPT and its relation with Pdcd4,Flow cytometry was used to analyze the effects of NCTD and HCPT to the cell cycle and apoptosis of the BGC-823 transfected cell lines. The transfected human gastric adenocarcinoma BGC-823 cell lines was treated Respectively with NCTD and HCPT in a concentration of 60 umol / L and 80 umol / L . Cells were Collected respectively after 0,24,48 and 72 hours, flow cytometry was used to analysis the cell cycle and apoptosis of the treated cells. Flow cytometry results showed that the three transfected cell lines were arrested at G2-M stage by NCTD and arrested at S stage by HCPT.But there was no significant difference between the transfected cells.