Impact Of O~6-methylguanine-DNA Methyltransferase Expression On The Therapy Of Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) accounts for 75-80% of all RCC and is the most common histological type. RCC is notoriously refrac-tory to radiation therapy and standard chemotherapy. Until recently,the outcome of medical treatment with cytotoxic agents for RCC was disappointing. To date, many mechanisms have been proposed toinduce drug resistance of cancer cells. These include overexpression of P-glycoprotein, decreased expression of DNA topoisomerase II, over-expression of an antiapoptotic gene bcl-2, overexpression of DNA re-pair proteins and so on.A large number of environmental alkylating carcinogens, endogenous alkylating species and various tumor chemotherapeutic agents attack DNA at the 06 position of guanine, forming O6-alkylguanine. This lesion is considered to be a major cause of mutations and malignant transformation induced by 06-alkylating agents. O6-alkylguanine is repaired by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). MGMT alkylating agent play a dual role in tumor chemotherapy. On the one hand, MGMT can protect the normal tissues from alkylating agent of injury and the second cancer On the other hand MGMT can cause tumor tissue of alkylating agent chemotherapy drug resistance.The expression pattern of MGMT is different in different types of tumors. The expression level ofMGMT is found to be lower than their tissues of origin in some tumors. However, the MGMT expression profile in ccRCC remains unclear. Methylation inhibitors 5-Aza-CdR can be able to reverse the abnormal methylation of DNA, activate the inactivation of tumor suppressor gene expression, so they are a potential antitumor drugs.In this study, we sought to investigate the expression profile of MGMT inccRCC and analyzed the role of MGMT in the drug resistance of ccRCC. At the same time, We will apply 5-Aza-1 CdR to kidney cancer treatment,to explore the application of 5-Aza-CdR can improve kidney cancer cell sensitivity of mitomycin.Part I The effects of MGMT gene expression on drug resistance in transparent kidney cell carcinoma一、MGMT expression is up-regulated in ccRCC tissues and cell lines.1、We examined MGMT protein expression in 60 formalin-fixed specimens by immunohistochemistry. MGMT protein expression in tumors was in general up-regulated compared with that in normal kidney tissues. The patients were divided into the MGMT low or none-expression group(staining score is "" or’+’) andMGMT high-expression group (staining score is’++’or’+++’) based on immunohistochemistry scores. The high-expression rate of the MGMT was 62% (37/60) in ccRCC samples。 we confirmed the up-regulation of MGMT by qRT-PCR. The results revealed that the level ofMGMT mRNAwas up-regulated by 2-fold in 17/20 (85%) ccRCC tissues compared with their adjacent normal tissues.!2、We also detectedMGMT expression level in two ccRCC cell lines and one normal kidney cell lines HK-2. We found that MGMT mRNA expression level was higher in all two ccRCC cell lines than that in HK-2 cells。.Western blot analysis confirmed high expression of MGMT protein in two ccRCC cell lines。二、MGMT expression is associated higher grades/stages of ccRCCImmunohistochemistry analysis was performed to detect MGMT expression level in 60 ccRCC samples.MGMT expression was positively correlated with the tumor pathologic grade and clinical stage. The percentage of high expression of MGMT was 40.0,67.7 and 77.3% in ccRCC at G1, G2 and G3/G4, respectively (P= 0.04). The percentage of high expression of MGMT was 39.1,70.1 and 80.0%in ccRCC at T1, T2 and T3/T4,respectively (P=0.02).三、Knockdown of MGMT sensitizes ACHN and 786-0 cells to BCNU or TMZ1、After treatment with siRNA for 48 h, MGMT protein expression in ACHN cells was significantly decreased compared with that in control siRNA-treated cells。2% The MTT method was used to assess cell viability. Knockdown of MGMT decreased cell survival at all doses of BCNU or TMZ, as compared with the random control.3、The protective role of MGMT against alkylation-induced cell killing was further studied in ccRCC line 786-0. Knockdown of MGMT also significantly increased the sensitivity of 786-0 cells to BCNU or TMZ.Part Ⅱ The effects of 5-Aza-CdR on chemotherapy sensitivity in kidney cancer cell lines CAKI-1 and 786-0一、Determined by MTT method to detect mitomycin on CAKI-1 cell inhibitory actionWe use determined by MTT method to detect different concentration mitomycin on CAKI 24 h-1 cell growth inhibition rate, found that mitomycin can inhibit cell increment, and render a dose dependent.二、Preliminary results show methylation inhibitors 5-Aza-CdR can enhance kidney cell 786-0 sensitivity of mitomycinMTT assay show that 5-Aza-CdR can inhibit growth of the kidney cancer cell lines 786-0 obviously. Different concentration of 5-Aza-CdR combined mitomycin inhibition of tumor cell present dose dependent, with the increase of concentration of 5-Aza-CdR, the inhibition rate of tumor cells increased significantlyPart Ⅲ Conclusions and Innovation一、Conclusions1、MGMT expression is up-regulated in ccRCC tissues and cell lines.2、 MGMT expression is associated higher grades/stages of ccRCC3、MGMTparticipate in alkylating agent class chemotherapy drug resistance process in renal clear cell carcinoma4、Methylation inhibitors 5-Aza-CdR can enhance kidney cell 786-0 sensitivity of mitomycin.二、Innovation and defects1、This study confirmed for the first time that MGMT expression is up-regulated in ccRCC tissues and cell lines., and the expression of MGMT is associated higher grades/stages of ccRCC. We found that Knockdown of MGMT sensitizes ACHN and 786-0 cells to BCNU or TMZ.However, how MGMT participate in the progress of renal clear cell carcinoma and the mechanism of the resistance of alkylating agent is unclear.2、Preliminary results show methylation inhibitors 5-Aza-CdR can enhance kidney cell 786-0 sensitivity of mitomycino Wherher the effects of 5-Aza-CdR on kidney cell 786-0 sensitivity of mitomycin though influencing the expression of MGMT or activity needs Further research.