Study On The Anticancer Mechanism Of Temozolomide Analogs

Temozolomide(TMZ)is an oral safe and effective imidazotetrazine alkylating agent drug,which is easy to pass through the blood-brain barrie and has weak bone marrow suppression.It has been used as a line of anti-glioma chemotherapy drugs in clinical use and it has also a good effect on other tumors,such as melanoma.However,it has been found that 06-methyl guanine-DNA methyltransferase(MGMT)can repair TMZ-induced DNA damage while TMZ plays an anti-tumor effect requiring a functional normal mismatch repair(MMR)system.Thus,MGMT-overexpressing and MMR-deficient tumor cells are not sensitive to TMZ.In addition,like other chemotherapy drugs,TMZ can also produce acquired resistance after a period of time and the main mechanism of resistance is related to MGMT overexpression and MMR deficiency,which limits the clinical application of TMZ.Therefore,it is of great significance to develop TMZ analogues which can overcome the TMZ resistance problem.Previously,MTT assay screened TMZ derivatives 377 and 465 that could inhibit the growth of TMZ-resistant tumor cells.And analogs 377 and 465 display good anticancer activity against the MGMT-overexpressing glioma T98G and MMR deficient colorectal carcinoma HCT116 cell lines with IC50 value of 62.50 ?M,44.23?M and 33.09 ?M,25.37 ?M respectively.Studies have reported that TMZ can cause G2/M arrest in MGMT low expression and normal MMR function,lead to DNA double strand breaks and induce apoptosis,and ultimately play an anti-tumor effect.Therefore,we examined the effect of analogues on HCT116 and T98G cells,and found that 20?M 465 could cause G2/M phase arrest in HCT116 cells;100?M 377 and 50?M 465 could cause G2/M cycle arrest of T98G cells;100 ?M 377 and 465 can lead to DNA double-strand breaks in T98G cells and cause apoptosis.Previous studies have also found that 50?M 465 can lead to DNA double strand breaks and apoptosis in HCT116 cells.More interestingly,we found that 465 could downregulate the expression of MGMT in the protein and mRNA level.Therefore,we further studied the mechanism of 465 downregulating MGMT.Studies have reported that many factors could affect the expression of MGMT gene,such as MGMT promoter hypermethylation which can lead to chromosome compression,isolated transcription factor and inhibition of transcription.Therefore,we investigated the effect of MGMT promoter methylation on HCT116 cells were treated with 50?M 465 for 48h by Bisulfite Sequencing PCR.The promoter methylation level of MGMT was higher in HCT116 cells,whereas 465 did not affect the methylation level of MGMT promoter.In addition,studies have reported that transcription factor p53 can bind directly to the MGMT promoter sequence,thereby inhibiting its transcription.When HCT116 cells were treated with 50?M 465 for 48h,we found that the expression of wild-type p53 increased.We then demonstrated that 465 could increase the binding of wild-type p53 to the MGMT promoter in HCT116 cells by Chromatin Immunoprecipitation Assay.To further confirm that increased wild-type p53 could inhibit MGMT expression after HCT116 cells treated with 465,we used 50 ?M 465 to treat with HCT116 cells which were transfected with p53 shRNA interference plasmids.We found that the expression level of MGMT in cells was increased after cells treated with 465 when wild-type p53 was knocked down.These data indicate that 465 can increase the expression of wild-type p53,which reduce the expression of MGMT.In addition,in order to investigate the effect of TMZ derivatives 377 and 465 on the expression of MGMT in different p53 background cells,we treated T98G(mutant p53)and H1299(p53-/-)cells with different concentrations of 377 and 465 for 48 h.We found that 50 ?M 377 and 465 did not affect the expression of MGMT in both cells,whereas 100?M 377 and 465 down-regulated MGMT expression in T98G cells and 100?M 465 could decrease the expression of MGMT in H1299 cells.These results indicate that MGMT has different sensitivities to TMZ analogues in different p53 background cells.And it was reported that the MGMT promoter sequence had multiple binding sites for Sp1,and Sp1 could bind directly to the MGMT promoter sequence and promote transcription.We found that the expression of Spl decreased in HCT116 cells after them treated with 50?M 465 for 48h.Then,the combination of Spl and Spl binding site probes was detected by Electrophoretic Mobility Shift Assay.It was found that HCT116 cells treated with 465 cells could reduce the binding between Spl protein in the nucleus and probe.These data indicate that 465 could down-regulate Sp1 expression in HCT116 cells and reduce Sp1 binding to MGMT promoter,which affects MGMT expression.In summary,TMZ derivatives 377 and 465 can cause MGMT-overexpressing T98G glioma cells and MMR-deficient HCT116 colon cancer cells G2/M phase arrest,then lead to DNA double strand breaks and cell apoptosis.465 could downregulate the expression of MGMT in HCT116 cells at the transcriptional level.And it affected the expression of MGMT,not by affecting the methylation level of MGMT promoter,but by transcription factors wild-type p53 and Sp1.In addition,the sensitivity of MGMT to TMZ analogs 377 and 465 in different p53 background cells was different.