| Glioblastoma (WHO IV) is the most common intracranial tumor with the worst prognosis. Although its incidence (3 / 100, 000) is less than most tumors originated from other systems, because of its rapid growth and invasion of central nervous system and resistance to the conventional therapies, it's prognosis is far worse than the other tumors. Glioblastoma is characterized by short course of disease and high mortality. Imaging-guided neuro-surgery, stereotactic radiation therapy and cytotoxic chemotherapy could not fundamentally cure glioblastoma. Gliolastoma patients soon die from tumor recurrence after initial surgery within six months. The median survival of glioblastoma patients who received subtotal surgery alone is only 7.0 months, and that of those who received combination treatment with subtotal surgery and radiotherapy is only 12.1 months. Previous studies show that, although temozolomide-based chemotherapy can effectively prolong the overall survival and enhance the quality of life of patients, because of the active GSTs system and abundant MGMT content in glioblastoma cells, about 48% -58% glioblastoma patients are resistant to temozolomide-based chemotherapy. And then these patients have to accept other drugs such as cisplatin whose therapeutic potency is not sound good (median survival: 15 months vs. 25.5 months). Therefore, development of adjuvant agents for temozolomide-based chemotherapy which could reverse drug-resistance, expand adaptable people, and improve the therapeutic efficiency is of clinical significance.Resveratrol is a natural polyphenols with broad anti-tumor activity. It can also inhibit atherosclerosis and protect neurons from ischemia. Our previous studies found that resveratrol significantly inhibited the proliferation of and induced caspase-dependent apoptosis in glioblastoma cells. In addition, resveratrol was documented to induce apoptosis primarily through inhibition of YKL-40expression of and de-phosphorylation of ERK-1/2. Moreover, accumulating studies also indicated that resveratrol participated in the multiple biochemical activities, such as down-regulating expression of HIF-1α, VEGF and NF-kappaB, up-regulating expression of tumor suppressor p53 and inactivating STAT3, and so on. Given that NF-kappaB is an important positive positive transcriptional regulator of MGMT while p53 is the most important negative transcriptional regulator of MGMT, we therefore investigated whether resveratrol can decrease MGMT expression and subsequently reverse temozolomide- resistance through repressed NF-kappaB expression and increased p53 expression in the following experiments.At first we confirmed the anti-glioblastoma bioactivity of resveratrol in A172 and T98G cells which have less active Notch-1 and mutant p53. Results showed that resveratrol significantly inhibited cell viability of and induced apoptosis in glioblastoma A172 and T98G cells in a time and dose-dependent manner. Also we observed that resveratrol significantly promoted the activation of Notch-1, restored the expression and nuclear translocation of wild-type p53. In addition, resveratrol treatment led to significant dephosphorylation of Akt-1, increased expression of pro-apoptotic Bax protein, decreased expression of the pro-survival Bcl-2 protein, and ultimately activation of caspase-3-mediated enzymatic cascade. Notch-1 is an important regulating factor involved in neural development and tumor progression, in order to investigate whether Notch-1 activation played critical role in resveratrol-induced apoptosis in glioblastoma cells, we then treated A172 and T98G cells in combination with resveratrol and Notch-1 inhibitor MRK-003. Results indicated that simultaneous MRK-003 treatment could completely blocke resveratrol-induced activation of Notch-1, and partially rescued apoptosis. We also found that simultaneous MRK-003 treatment significantly attenuated resveratrol-induced up-regulated expression and nuclear translocation of p53. Moreover, the expression alterations of several downstream apoptosis-regulating proteins such as Bax and Bcl-2 which were induced by resveratrol were also partially attenuated. Results suggested that Notch-1 activation-dependent p53 restoreation contributed to resveratrol-induced apoptosis in glioblastoma A172 and T98G cells.In a preliminary study, we examined the expression level of MGMT in all the glioblastoma cell lines maintained in our laboratory. It was evidenced that only T98G cells were MGMT positive and temozolomide-resistant. We then tested the therapeutic potency of combination treatment with resveratrol and temozolomide in glioblastoma T98G cells. It was found that treatment with temozolomide alone (100μM, a clinical relevant concentration for 24 hours could only induced apoptosis in a small number of T98G cells. In contrast, combination treatment with resveratrol and temozolomide had strengthened growth-inhibition effect (98.2±1.1% vs. 61.4±4.5%, p = 0.001) and apoptosis-indusive potency (11.5±2.3% vs. 42.5±1.8%, p = 0.001). Results showed that resveratrol was capable to sensitize glioblastoma T98G cells to temozolomide treatment. Combination treatment with resveratrol and temozolomide achieved a significant synergistic effect. Because T98G cells were resistant to temozolomide principlly due to the abundant intracellular MGMT, we then carried out western blot analysis to detect the expression level of MGMT in glioblastoma T98G cells treated with or without resveratrol and temozolomide. Data suggested that temozolomide alone significantly induced MGMT expression (149.6±8.3%, p = 0.001), and simultaneous resveratrol treatment almost completely abolished temozolomide-up-regulated MGMT expression (p = 0.001). Thus, resveratrol was able to completely deplete MGMT in glioblastoma T98G cells, which may account for the sensitized temozolomide-response beside the enhanced capability of resveratrol to induce apoptosis in glioblastoma cells. Since NF-kappaB is the most important positive MGMT transcriptional regulator, we then tested the endogenous expression of proteins in NF-kappaB signaling pathway. It was known that kappaB-ras1 and I-kappa-B-αare two most important inhibitors of NF-kappaB. Western blot anslyses indicated that neither resveratrol nor temozolomide applied alone or in combination, restored kappaB-ras1 expression in glioblastoma T98G cells. In addition, these treatments did not affect the expression level of I-kappa-B-α. Temozolomide alone could significantly induce tumor cell NF-kappaB p65 subunit expression (109.2±2.8%, p = 0.03), while combination treatment with resveratrol and temozolomide could significantly decrease the expression of NF-kappaB p65 (74.6±3.5%, p = 0.001). Taken together, we conclude that temozolomide-resistance in glioblastoma T98G cells on the one hand resulted from the endogenous abundant MGMT expression, on the other hand resulted from continuous temozolomide treatment-induced MGMT expression, partially through up-regulating NF-kappaB expression. Resveratrol is capable to activate Notch-1, which subsequently (1) promote the expression of p53 protein and nuclear translocation; (2) inhibit the expression of NF-kappaB p65 subunit; (3) block the intracellular up-regulated expression of MGMT in response to continuous temozolomide treatment. Current study indicated that combination treatment with resveratrol and temozolomide had a synergistic effect on inducing apoptosis in glioblastoma cells and reversed the temozolomide-resistance. Taken together, resveratrol was a reliable adjuvant agent for temozolomide-based chemotherapy and warranted further study.
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