The Study On The Evolution Of The Resistant Characteristic Of The TMZ-resistant Human Glioma Cell Line

Glioblastoma (GBM WHOⅣ) is the most common and malignant intracranial tumor with diffusely infiltrative growth characteristics and dismal prognosis. The median survival of patients ranges from 12 to 15 months despite of aggressive surgery combined with radiation and chemotherapy. The highly lethal nature of this tumor partly accounts for its chemoresistance of GBM cells. The resistance of GBM cells to alkylating agents is mediated by many factors. Among these factors, the association between O6-methylguanine-DNA methyltransferase (MGMT) and chemo-resistance is more concerned. MGMT is a key enzyme in the DNA repair network, which catalyzes the transfer of the methyl group from O6-methylguanine to a cysteine residue of its active site. In this single step reaction, DNA-lesions caused by alkylating substances are repaired. MGMT subsequently is irreversibly inactivated and degraded.Although advances in chemotherapeutic drugs, alkylating agents remain the most common and effective administration in GBM, based on their lipophilic nature, which can appreciably permeate blood-brain barrier (BBB), and their intrinsic cytotoxic activities. Temozolomide (TMZ), an orally administered cytotoxic alkylating agent with broad-spectrum antitumor activity, is a bioavailable imidazotetrazine derivative of the alkylating agent dacarbazine. It penetrates BBB into the brain easily and undergoes rapid chemical conversion in the systemic circulation at physiological pH to its active compound, MTIC (5-(3-methyltriazen-l-yl)imidazole-4-carboximide), which degrades to a DNA-alkylating species, subsequently, is cytotoxic via the formation of O6-and N7-alkylguanine lesions in DNA, this procedure does not require hepatic metabolism for activation. TMZ becomes the most perspective chemotherapeutic drug against gliomas, on base that it is more effective, easier to administer and has fewer side-effects than its competitors. However, it has also failed to improve prognosis in all patients with GBM. The unsatisfactory outcome with chemotherapy has chiefly been originated from GBM tumor cells intrinsic or acquired chemoresistance.Thalidomide, a glutamic acid derivative, is considered as a potential drug to treat hyper-vascularized glioma due to its anti-angiogenesis effect. In a number of phase II clinical trials, thalidomide combined with temozolomide has been proved to prolong the median survival of patients with malignant glioma significantly, meanwhile, the side effect of this combination is not obvious.The mechanism of combination of thalidomide combined with temozolomide to treat gliomas is cleared gradually. In our previous study, we found thalidomide may inhibit PI3K/AKT/mTOR signaling pathway through activation of PTEN, a tumor suppress gene, to reinfore the induced autophagy. Meanwhile, the combination of both may induce apoptosis through activation of caspase-3. Furthermore, there is no study to examine whether the resistant characteristic of the TMZ-resistant human glioma cell line could be changed by temozolomide combined with thalidomide.The present study was divided into two parts.In the first part of this study was aimed to investigate how and why the chemoresistance to TMZ evolved in U251/TR cells, a TMZ-resistant cell line under TMZ administrator, and provide experimental evidence for optimal TMZ therapy strategy. U251/TR, was established by stepwise exposure of human U251 parental cells to TMZ induced culture for 8 months. MTT assay was used to calculate the resistance index and cell viability percentage. Western blotting(WB),RT-PCR,immunohistochemistry (IHC) and immunofluorescence(IF) were used to detect MGMT expression for the analysis of chemoresistant evolution and its mechanism in U251 cells during the TMZ induction procedure. U251/TR showed approximately 7-fold resistance to TMZ (RF=6.67±0.53). The MGMT protein expression was significantly increased in U251/TR cells compared with the parental U251 cells (p<0.01). The growth of U251/TR cells was inhibited significantly by TMZ with double the final induced strength from day 3 to 5 compared with DMSO control group (p<0.01, for all) and MGMT protein level in those treated cells was decreased accordingly after TMZ administration (p<0.05).The second part focused on whether the resistant characteristic of the TMZ-resistant human glioma cell line could be changed by thalidomide combined with temozolomide. MTT assay was used to calculate the resistance index and cell viability percentage. Western blotting(WB),RT-PCR,and immunofluorescence (IF) were used to detect MGMT expression for the analysis of U251/TR cells treated with temozolomide combined with thalidomide. MGMT promoter profile was analyzed by Methylation-specific polymerase-Chain-reaction analysis (MSP). Compared to negative control group, significant inhibition of proliferation was shown in TMZ+THD group after 24 h and TMZ group after 48 h. The MGMT protein levels in TMZ+THD group and TMZ group decreased significantly (p<0.01), moreover, there is no difference between both groups. We also found no change in promoter methylation in all groups.Conclusions1. Our results demonstrated that the primary mechanism of U251/TR cells resistant to TMZ is owing to increased activity of MGMT and the chemoresistance was evolving during the TMZ induction procedure. TMZ could overcome the TMZ-resistance itself by consuming MGMT steadily, which suggests that metronomic TMZ regimen could improve the chemo-sensitivity in TMZ resistant glioma clinically.2. Thalidomide combined with temozolomide could improve the cytotoxicity in U251/TR cells. The mechanism needs further evaluation in future prospective trials.