Effects Of New Alkylating Agent Temozolomide Easter On The Glioma Cells And Its Possible Mechanisms

Objective: 1. To investigate the effects of temozolomide easter on glioma cells. 2. To test whether TMZ-HE could overcome TMZ resistance. 3. To explore the possible mechanisms of apoptosis induced by temozolomide easterMethods: 1. Methyl thiazolyl tetrazolium(MTT) assay was used to measure the effects on the proliferation of LN229 and T98 G cells cultured with graded concentrations of TMZ and TMZ-HE for 24, 48, 72 and 96 h. The long term effect of TMZ and TMZ-HE was measured by colony formation assay. The Annexin V and propidium iodide(PI) assay by flow cytometer were applied to detect the apoptosis and necrosis induced by TMZ and TMZ-HE. 2. The changes of cell cycle were detected by FCM. Western blot detected the expressing of resistant protein MGMT in T98 G after treatment with TMZ and TMZ-HE.Western blot detected the expressing of γH2AX, Bax and Bcl-2 in LN229 and T98 G cells after treatment with TMZ and TMZ-HE. 3. Constructed resistant cell lines LN229/MGMT which MGMT in LN229/MGMT was over-expressed, and resistant cell lines SHG-44/TMZ which MGMT in SHG-44/TMZ was negative, then the effect of TMZ-HE on resistant cells was detected by MTT assay.Results: 1. Our data showed that TMZ-HE effectively inhibited the proliferation of GBM cells simultaneously induced apoptosis. These effects were much stronger than TMZ. 1) MGMT expression was detected in T98 G and LN18, and MGMT expression in T98 G was higher than LN18. MGMT in LN229 was negative. 2) TMZ-HE obviously suppressed the proliferation of LN229, LN-18 and T98 G glioma cells in a concentration-and time-dependent manner compared with TMZ, and the IC50 value of TMZ was much higher than TMZ-HE. 3) TMZ-HE induced more cellular apoptosis of the LN229 and T98 G cells in a concentration-dependent manner.2. Novel alkylating agent TMZ-HE could reverse TMZ resistance. 1) The results of Western Blot showed that MGMT expression was detected in LN229/MGMT, and MGMT expression in SHG-44/TMZ was negative. 2) The results of MTT showed that TMZ-HE exhibits its activity on TMZ resistant cells and the effect on resistant cell lines with MGMT positive was more than that with MGMT negative.3. TMZ-HE induced a rapid and profound S-phase accumulation, and blocked DNA repair protein MGMT and was associated with early activation of DNA damage signaling. The mechanisms of apoptosis induced by TMZ-HE may be that they acted on the targets Bcl-2 and Bax, down-regulated the gene of Bcl-2 and up-regulated the gene of Bax. 1) The results of Western Blot showed that TMZ or TMZ-HE can significantly increase DNA lesion singalling γH2AX 2) The results of Western Blot showed that TMZ or TMZ-HE can reduce the expression of anti-apoptotic protein Bcl-2 and increase the expression of protein Bax. 3) High concentration of TMZ-HE induced a distinct cell cycle arrest with accumulation of cells in S phase that is not observed for TMZ. 4) The results of Western Blot showed that TMZ or TMZ-HE can significantly reduce the expression of MGMT in T98 G cells, TMZ-HE could block the expressing of MGMT in T98 G earlier than TMZConclusions: 1) Our data showed that TMZ-HE effectively inhibited the proliferation of GBM cells simultaneously induced apoptosis. These effects were much stronger than TMZ. 2) Novel alkylating agent TMZ-HE could reverse TMZ resistance. 3) TMZ-HE induced a rapid and profound S-phase accumulation, and blocked DNA repair protein MGMT and was associated with early activation of DNA damage signaling. The mechanisms of apoptosis induced by TMZ-HE may be that they acted on the targets Bcl-2 and Bax, down-regulated the gene of Bcl-2 and up-regulated the gene of Bax.