Research On The Effect Of Rapamycin On Glucose Metabolism In Human Glioma Cells And Its Anti-tumor Mechanism

Objective Glioma is the most common primary brain tumor, and may recur after the surgical removal of glioma. At present, the study of tumor cell proliferation and apoptosis is very common. In clinical, many widely used anticancer drugs are effective in treating tumors through inhibiting proliferation and inducing apoptosis of malignant tumor cells. PI3K/Akt/mTOR signaling pathway is frequently activated in a variety of tumors, and involves in tumor growth and development. MTOR, as a downstream molecule of the PI3K/Akt pathway, plays a central role in regulating the survival and proliferation of many malignant tumors cells including glioma. Rapamycin is an inhibitor of mTOR, and involves in the regulation of PI3K/Akt/mTOR signaling pathway in the malignant cells, via inhibiting mTOR activity. However, the anti-cancer effect of rapamycin on glioma as well as the involved molecular mechanism has not been thoroughly studied.In various malignant tumor cells, glycolysis is commonly at a high level, mainly due to the abnormally elevated expression and activity of glycolytic enzymes, suggesting that the glycolytic pathway may provide sourceful targets for anti-cancer treatment. MicroRNA-143has been recently reported to be regulated by mTOR signal pathway, and its expression is abnormally reduced in a variety of malignant tumors, and its targets include glycolytic rate-limiting enzyme hexokinase2(HK2). However, the roles of mTOR pathway, miRNA-143, and glycolysis, as well as their relationship in the regulation of glioma remain unclear.In this study, human glioma cell lines U251and CHG-5were used to explore the regulatory effects of rapamycin on proliferation, apoptosis, and glycolysis of glioma cells, and we also invesitgated the involved molecular mechanism.MethodRapamycin (100mM) was used to treat human glioma cell lines U251and CHG-5. MTT was performed to analyze the proliferation capacity of U251and CHG-5cells treated by100mM rapamycin with time. FCM was used to analyze the apoptosis levels of U251and CHG-5cells treated by100mM rapamycin with time. Western Blotting was used to examine the protein expression of p-S6K, on behalf of the activity of mTOR, in U251and CHG-5cells treated by100mM rapamycin with time. Glucose uptake and lactic acid secretion were assayed in U251and CHG-5cells treated by100mM rapamycin with time. The activity and protein expression levels of hexokinase2(HK2), pyruvate kinase (PK) and lactate dehydrogenase (LDH), all of which were key enzymes in glycolysis, were determined in U251and CHG-5cells treated by100mM rapamycin with time. The expression levels of miR-143, which directly negatively regulates HK2, were assayed in U251and CHG-5cells treated by100mM rapamycin with time.ResultIn U251and CHG-5cells treated by100mM rapamycin at0,6,12,24h, respectively, with the treatment time, the cell proliferation rate was gradually decreased, while the apoptosis levels showed an increasing trend. Western Blotting result showed that with the treatment time, the protein expression of p-S6k was gradually downregulated, indicating that the activity of mTOR was inhibited. Moreover, with the treatment time, glucose uptake and lactic acid secretion were decreased in U251and CHG-5cells, and the activity and protein expression levels of HK2, PK and LDH were all downregulated, suggesting that the glycolysis level was repressed. Furthermore, the miR-143expression level was gradually decreased with the treatment time of rapamycin in U251and CHG-5cells.ConclusionRapamycin can inhibite cell proliferation and induce cell apoptosis in human glioma U251and CHG-5cells via downregulating the activity of PI3K/Akt/mTOR signaling pathway. Moreover, Rapamycin can also inhibiting the glycolysis in U251and CHG-5cells, which may contribute to the inhibition of cell proliferation. More importantly, rapamycin can upregulate the expression of miR-143through downregulate the acivity of PI3K/Akt/mTOR signaling, which can further inhibit the protein expression of the glycolysis rate-limiting enzyme HK2, the target of miR-143, and hence decrease the glycolysis level in U251and CHG-5cells.