Research In The Treatment Of PI3K Inhibitor BEZ235on MGMT-positive Glioma Cell Line

Malignant gliomas, highly invasive primary brain tumors, are heterogeneous.Glioblastoma multiforme (GBM) is the most common intracranial tumor and among themost lethal. The growth pattern of GB is very invasive, thus, it is nearly impossible tomake a complete surgical resection. The use of adjuvant therapies might be required toprolong survival.The current standard of care for GBM includes maximal safe resection of the tumorfollowed by the combination of radiation and chemotherapy with the oral DNA-alkylatingagent with good penetration into the blood-brain barrier, temozolomide (TMZ). It has beenwell established that patients responding to alkylating therapy with TMZ differs amongthese with the same histological diagnosis of GBM and depends on the methylation of themethylguanine-DNA methyltransferase (MGMT) promoter. NVP-BEZ235has beendemonstrated both in vivo and in vitro by inhibition of autophagy and increase theapoptosis enhance effect of TMZ on malignant glioma. PI3K inhibitors can inhibit thegrowth of tumor. Because the research at this stage the inhibition of PI3K, BEZ235as apotential anti-tumor drugs as the first PI3K inhibitors use in clinical trials. The mainpurpose of this study is to test the resistance to chemotherapy in MGMT positive gliomacell lines TMZ and BEZ235combination therapy is more effective than BEZ235and TMZcombine in treatment of tumor.BEZ235has also been demonstrated to enhance the therapeutic efficacy of TMZagainst malignant glioma by inhibiting autophagy and increasing apoptosis both in vitroand in vivo. Little is known about the mechanism of how it could enhance the therapeuticpotential of TMZ in GBM. Recently, one study has demonstrated that resveratrol reversesTMZ-resistance by downregulation of MGMT in T98G glioblastoma cells by theNF-κB-dependent pathway. NF-κB is the most important regulator. It is thus suggested thatdownregulation of NF-κB-MGMT pathway is a feasible strategy to overcomeTMZ-resistance. The involvement of PI3K in the regulation of NF-κB-MGMT pathway has been described previously.One study has demonstrated the inhibition of PI3K pathwayin GBM and implications for combination therapy with TMZ. Phosphoinositide3-kinaseinhibitors (PI3Ki) inhibit PI3K pathway to suppress tumor growth. For this reason, manyPI3Ki are being investigated to check their efficacy against cancers. BEZ235PI3Ki is onepotential drug that was the first PI3Ki to enter clinical trials. It has been shown thatBEZ235possesses antitumor activity, including against human gliomas. Thus, the mainpurpose of this pilot study is to test whether combination of TMZ and BEZ235is moreeffective than BEZ alone in antitumor activity.This study from the three aspects of the research of PI3K inhibitor BEZ235in gliomacells in the treatment of MGMT positive cell line:1.After BEZ235treatment and use MTTassay to detective the effect of cell proliferation.2.Using Methylation PCR detect thedegree of methylation and unmethylation effect.3.comparison the expression of MGMTprotein in every treatment group of A172and T98G, to further understand the combinationeffect.ObjectiveAccording to one of the PI3K/mTOR inhibitors of BEZ235combined withconventional glioma chemotherapy drug TMZ combination treatment, whether canimprove the MGMT-positive glioma cell lines resistant to TMZ treatment.MethodsThe selection of MGMT positive glioma cell line T98G and MGMT negative cell lineA172, seeded them to6-well plates and96-well plates, then divided into the control groupand the experimental group, the optimum BEZ235concentration in a cellline, separatelyfor the combined treatment of TMZ TMZ and BEZ235, respectively.1.Use MTT assay inthe24,48,72hours after treatment of experimental groups to detect proliferation inhibitionrate of tumor cell lines, to understand the inhibitory rate of cell proliferation in each groupafter treatment.2.Use Methylation PCR to detect methylation and unmethylation of groups.3.Use Western Blot to detect the levels of groups’ MGMT protein and quantification.Results1. To compare the cytotoxic effect of BEZ235alone and combination of BEZ235andTMZ, the%of cell viability was determined by comparing with that of TMZ-treated T98Gcell lines. The cell viability was significantly lower in combinations or BEZ235alonetreated cells of all concentration groups, except that at50nM BEZ235-treated one with or without TMZ as well as that at100nM BEZ235alone group.2. Compared with the twounmethylated groups, the relative MGMT unmethylation of treatment group with TMZ100μM+BEZ235800nM in T98G cells was significantly lower than that of TMZ100μMalone in T98G cells.3. This data was consistent with the gene methylation status: theunmethylation of MGMT gene increased MGMT protein expression in T98G cells and thisenhancement can be significantly reversed by BEZ235treatment.Conclusion1.Our results clearly demonstrated that TMZ induces cytotoxicity in A172cellssignificantly but only slightly in T98G cells, suggesting that A172is susceptible to TMZ;whereas T98G is resistant to TMZ. It has shown that BEZ235alone and the combination ofBEZ235and TMZ had lower cell viability during72h of treatment than that of24h oftreatment, suggesting that longer exposure of treatment may induce higher cytotoxicity tocells.2. It indicates that BEZ235might be used as an adjuvant to potentiate TMZ efficacyby modulating NF-κB-MGMT pathway and suppressing tumor activities.3.The result of MGMT promoter methylation status by methylation PCR clearlydemonstrated BEZ235which functioned as PI3K inhibitor, can significantly reverse theun-methylation to reduce MGMT protein expression.