Adenovirus-mediated Delivery Of BFGF Small Interfering RNA Reduces STAT3Phosphorylation And Induces The Apoptosis In Glioma Cells U251

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. Despite technological advances in surgical resection followed by the application of combined radiotherapy and chemotherapy, GBM carries a dismal prognosis primarily due to its aggressive proliferation in the brain regulated by complex molecular mechanisms. Basic fibroblast growth factor (bFGF), over-expressed in GBM, has been correlated with growth, progression, and vascularity of human malignant gliomas. Thus, bFGF may be a promising target for novel therapeutic approaches in glioma. Previously, we reported that adenovirus-mediated delivery of bFGF small interfering RNA (Ad-bFGF-siRNA) showed antitumor effects and enhanced the sensitivity of glioblastoma cells to chemotherapy in glioma cell U251. However, the major mechanisms involved remain unknown. Signal transducer and activator of transcription3(STAT3) is constitutively active in GBM and correlates positively with the glioma grades. In addition, as a specific transcription factor, STAT3serves as the convergent point of various signaling pathways activated by multiple growth factors and/or cytokines. Therefore, we hypothesized that the proliferation inhibition and apoptosis induction by Ad-bFGF-siRNA may result from the interruption of STAT3phosphorylation.This study will be divided to three aspects:First, the changes of STAT3signaling pathway in the cell by the treatment with Ad-bFGF-siRNA were explored. The human glioblastoma cell line U251was infected with the recombinant adenoviruses Ad-bFGF-siRNA constructed and identified before, to incubated for24,48, or72h respectively. Cells were then lysed and total protein was extracted. Western blot was performed to detect the expression of STAT3, pSTAT3(Ser727) and pSTAT3(Tyr705), and then we selected the three classic signaling pathways related to the STAT3pathway:JAK-STAT3, Ras-Raf-MEK-MAPK (ERK) and Src, and dectect the key upstream kinases in the three classic pathway and downstream targets of STAT3to explore how the recombinant adenoviruses with bFGF-siRNA affect the STAT3signaling pathway. Cells treated with DMEM and an adenovirus vector expressing green fluorescent protein (Ad-GFP) or null (Ad-null) were used as the controls. The results revealed that Ad-bFGF-siRNA interferes with the activation of STAT3in a time-dependent manner, The expression of total STAT3, ERK1/2and JAK2remained similar among three groups across different time points, the level of pSTAT3Tyr705with Ad-bFGF-siRNA treatment was markedly decreased at all three time points, and the expression of pSTAT3Ser727moderately decreased at24and48h and then restored to the control level at72h. In agreement with the down-regulation of pSTAT3Ser727, the activation of ERK1/2was also decreased in a similar manner, the decrease in phosphorylated JAK2was also observed in a similar manner with pSTAT3Tyr705. the decrease in total and phosphorylated Src were both observed only at48h in the Ad-bFGF-siRNA treatment group, and as the downstream targets of STAT3, CyclinDl and Bcl-xl were also significantly decreased compared with the levels in the Ad-GFP and control groups. In summary, Ad-bFGF-siRNA could reduce the phosphorylation of STAT3by down regulating the activation of ERK1/2, JAK2, but not Src signaling transduction, which subsequently down regulated downstream targeted substrates of STAT3, CyclinD1and Bcl-xl.Second, the extracellular levels of cytokines closely related to STAT3were explored. Due to its ability to activate STAT3, the IL-6secretion is responsible for the persistent activation of STAT3, we performed the ELISA to test the IL-6level in the supernatant of U251cells infected by Ad-bFGF-siRNA for0-24h,24-48h,48-72h respectively. The result showed that IL-6secretion in the Ad-bFGF-siRNA group was lower than that in the control and Ad-GFP groups during both24-48h and48-72h periods. In turn, U251cells infected with Ad-bFGF-siRNA for48h were treated with serum-free DMEM in the presence or absence of recombinant IL-6for24h. we performed the Western blot to detect the changes of phosphorylation of STAT3, the result showed the phosphorylation of STAT3at both Tyr705and Ser727was elevated after stimulated with IL-6for24h. Therefore, Ad-bFGF-siRNA could reduce the phosphorylation of STAT3by down regulating the IL-6secretion, and the reduction of the phosphorylation of STAT3may be responsible for down regulating the IL-6secretion.Finally, the possible apoptotic pathway triggered by Ad-bFGF-siRNA were explored. Mitochondrial transmembrane potential (△Ψm) was measured with Flow cytometry and fluorescence microscopy, when cells were infected with Ad-bFGF-siRNA for72h. Western blot was performed to examine the levels of three important players in apoptosis:Cytochrome C, Caspase3, and Bax. The result revealed that Ad-bFGF-siRNA reduces the mitochondrial transmembrane potential (△Ψm) and elevate the expression of Cytochrome C, Caspase3, and Bax and ultimately induces apoptosis in U251cells.Generally speaking, from this study we can conclude:In this paper, we found a potential mechanism that in glioma cells U251, Ad-bFGF-siRNA could reduce the phosphorylation of STAT3by down regulating the IL-6secretion and the activation of ERK1/2and JAK2signaling transduction, which subsequently down regulated downstream targeted substrates of STAT3and ultimately resulted in the collapse of mitochondrial membrane potentials as well as the induction of mitochondrial-related apoptosis.