| Cisplatin is a neutral, inorganic square planar complexes. It can reacted with DNA, inducing to produce the unique biological effects, then repair the damage of DNA to cell survival or activation of the apoptotic program irreversible, leading to cell apoptotic. However, the interaction of cisplatin with the DNA, mainly through a series of spontaneous hydration reaction, which relates with the water molecules to replace the process of the cis-chloro ligand. This single hydration form is recognized as a form of the highly reactive, but such form formation rate is often limited by many endogenous nucleophilic substances, such as:glutathione (GSH), methionine metallothionein, and protein. Therefore, when the cisplatin enters cells, it has the potential vulnerability or more of these substances, as well as other cellular components result cytoplasm inactivated.Cytotoxic chemotherapeutic drugs such as cisplatin, which mainly reacts with the purine bases in the DNA of the nucleophilic N7locus, thereby forming the composite form of DNA-protein and DNA-DNA chain and inter-chain cross-linking. However, there is evidence that the chain formed adducts can induced cytotoxic effects occurred. In addition, there are reports that found a similar chain crosslinking DACH-sulfate platinum complexes in cultured cells, and this complex is mainly for the difference between a different quantity and quality of DNA bases. Cisplatin resistant tumor cells cells provide new clinical ideas for the possible destruction of such cause. Since the DNA adduct formed chain can cause a lot of cisplatin-induced nuclear damage, therefore, the decision of the level and degree of the total platinum and DNA binding cytotoxicity. Although cisplatin affect DNA replication, but its influence there is no correlation between the generation of DNA synthesis and cell toxicity. Therefore, each recognition protein may start one or more specific biological signal source of stress, so that the DNA damage to exhibit seemingly unrelated biological effect, this replication and transcription of the with adduct cisplatin destruction process is consistent, but such biological effects do not necessarily lead to cell death directly. In terms of the for cisplatin role of cell survival and pro-apoptotic signal simultaneously activated cisplatin treatment, the relative intensity and/or duration of activation and integration of downstream signaling molecules, and ultimately produce different biological effects and resulting in generation of cisplatin resistance. Colorectal cancer is the more common malignancies in the clinical, its incidence ranks third, caused by a higher mortality rate. Today, the treatment of tumors constant development and updating, an endless stream of new anti-cancer drug cisplatin is still more commonly used chemotherapy drugs in clinical cancer cell-killing effect, especially when in its application dose more significant but because of its serious side effects and drug resistance, limit its wider application. The experiments show that the the AKT abnormal expression of increase may be associated with cisplatin resistance, cisplatin by what signaling pathways to regulate AKT expression is less involved.Part1cisplatin induce the ROS production and combination with NAC to research the inhibition and apoptosis ratio in human colon cancer cellsPurpose cisplatin enter into the cells resulted in the ROS increased at an abnormal level. ROS play an important role in the reaction in the regulation of oxidative stress, cell proliferation and apoptosis, regulate the activity of signaling pathways. However, the mechanism of inhibiting ROS how to increase the chemosensitivity of cisplatin remains unclear. The purpose of this paper is to verify cisplatin generation of ROS and inhibit ROS whether increased sensitivity of cisplatin.Methods the colon cancer cells(HCT-116) served as the experimental subjects. Set the ROS specific inhibitor of NAC concentration at20μmol/L, the cisplatin concentration at100μmol/L, combined them treatment of colon cancer HCT-116cell lines. ROS generated HCT-116detected by flow cytometry and immunofluorescence; MTT method and flow cytometry cisplatin (I group) and NAC combined with cisplatin (I group) to detected the growth and apoptosis of colon cancer cell line,and to verify the NAC to increase the chemosensitivity of cisplatin.Results Cisplatin can cause abnormal accumulation of intracellular ROS, cisplatin combined with NAC can enhance cisplatin inhibition and apoptosis in colon cancer cells HCT-116Conclusions NAC can enhance cisplatin chemotherapy sensitivity, the clinical results of this study provide a theoretical basis for increased sensitivity to cisplatin. Part2cisplatin combined with NAC regulated the role of AKT and activated the expression of AKT in colon cancersPurpose The level of AKT in a number of tumor cells, its mainly served as anti-apoptotic effects. However, with application cisplatin treatment in colon cancer cells AKT was also overexpressed, but cisplatin how to activated the AKT was still unknown. In this paper, observed at the cellular level, the role of cisplatin in colon cancer cells, and to study whether AKT expression, cisplatin and combined with NAC inhibited the expression of AKT; application STAT3SiRNA and STAT3plasmid transfected cells to detect the expression of AKT; application chromatin immunoprecipitation to detected the STAT3regulated the promoter of AKT at the transcriptional level.Method Setting the cisplatin concentration at0.25μM,50μM,100μM and200μM, and interfere with the colon cancer cells at6h; Using STAT3SiRNA and STAT3plasmid transfected colon cancer cells. RT-PCR and Western-blot assay detected the level of AKT. Application chromatin immunoprecipitation detect STAT3combined with AKT promoter at the transcriptional level.Results The expression levels of AKT cisplatin concentration is proportional to within a certain range, cisplatin and NAC combination of lowering of STAT3and AKT expression levels. STAT3SiRNA transfection of colon cancer cells can be reduced and increased expression levels of AKT and using the plasmids of STAT3produced the opposite effect. Application chromatin immunoprecipitation method detected STAT3binding with AKT thereby affecting at a transcriptional level.Conclusion Cisplatin activate STAT3/AKT signaling pathway involved in the development of resistance, The STAT3can regulate AKT expression at the protein level and transcriptional level, application of NAC can significantly improve the sensitivity to cisplatin,100μM cisplatin in combination with20μM NAC can achieve greatest effect and produce a low probability of resistancePart3Cisplatin combined with NAC in vivo study of colon cancer cells in vivo tumorigenicity Purpose To study the impact of cisplatin combined with NAC on human colon cancer cell line HCT-116cells formed tumor growth.Method BALB/C mice were randomly levels were divided into three groups, namely subcutaneous injection of HCT-116cell lines, cultured for30days, the mice were sacrificed to remove subcutaneous tumors were calculated injection of cisplatin and cisplatin combined with NAC tumor xenografts size and weight, as well as by immunohistochemical staining to detect the expression of AKT in transplanted tumor tissue.Results three groups of nude mice were tumorigenic success. CDDP+NAC cell group xenografts average size and of CDDP significantly lower than the control group cells group, the difference was statistically significant (P<0.05); Immunohistochemistry showed that AKT the CDDP group of transplanted tumor expression levels were significantly higher than in the CDDP+the xenografts are formed in the NAC cell group expression levels.Conclusion NAC lowered the expression of AKT, suggesting that NAC may be involved in the regulation of AKT expression. |
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