Targeted Blockage Of STAT3 Signaling Pathway With JSI-124 Inhibits The Proliferation Of Human Glioblastoma Multiforme Cells In Vitro

BackgroundGlioblastoma Multiforme(GBM)is the most common and lethal primary brain tumor in adults,and represents at least 80 percent of malignant gliomas.The current management of GBM generally includes cytoreductive surgery,followed by adjuvant radiotherapy with/without chemotherapy.Despite recent advances in both diagnostic modalities and therapeutic strategies,it has the worst prognosis among the malignant astrocytomas with median survival of less than one year after diagnosed.Recurrence remains a major therapeutic challenge in GBM.More effective therapeutic strategies are needed in clinic.Signal Transducer and Activator of Transcription(STAT)proteins are latent cytoplasmic transcription factors that play a critical role in mediating cytokine and growth factor signaling.Especially,signal transducer and activator of transcription 3 (STAT3)plays a key role in the regulation of cell growth,survival,apoptosis,and differentiation.Moreover,reports demonstrated that persistent activation and dysregulation of STAT3 have been frequently observed in a wide variety of primary cancers including GBM.Constitutively activated STAT3 plays a major role in pathogenesis of cancer by controlling the altered expression and regulation of STAT3 target genes,which can protect apoptosis,enhance cell proliferation,induce tumour angiogenesis and modulate immune functions in favour of evasion of immune surveillance.Thus,the critical role of STAT3 in the molecular pathogenesis provides an attractive molecular target for therapeutic intervention in GBM.Until now,there are many strategies to disturb activated STAT3 for cancer treatment,including antisense nucleotide,nucleic acid enzymes,goes live mutants expressed overt missing,RNA interference,suppression of upstream kinases, phosphoric acid cheese and Decoy oligonucleotide.JSI-124,a cell-permeable triterpenoid compound,acts as a selective inhibitor of JAK/STAT3,with no apparent effect on the Akt,extracellularsignal-regulated kinase,or c-Jun NH2-terminal kinase pathways.JSI-124 is isolated from natural plants,such as the Cucurbitaceae and Cruciferae.Recent reports have indicated that JSI-124 can inhibit cell proliferation and induce apoptosis via suppressing phosphorylation of STAT3 in a number of human cancer cell lines in vitro as well as to inhibit tumor growth of A549 lung adenocarcinoma and MDA-MB-468 breast cancer cells in nude mice.So the application of JSI-124 in GBM has an important therapeutic potential.In the current study,JSI-124 targeted inhibiting STAT3 pathway was employed to investigate the influence to human GBM cells.The study was divided into two parts.PartⅠBlocking Phosphorylated STAT3 Suppresses the Growth of Human Glioblastoma Multiforme CellsObjectiveTo analyze STAT3 and its phosphorylation protein levels and the significance of constitutively activation of STAT3 signaling pathway in the pathogenesis of human GBM,GBM resection specimens and cells were examined by Western blot.In order to identify the inhibition effects of JSI-124 on cell proliferation,GBM cell lines A172 and U251 were treated with JSI-124 in vitro and biological charaterizations were evaluated.This study tried to provide a basis for further evaluation of JSI-124 as a therapeutic agent against GBM.Methods1.We collected 9 primary GBM resection specimens(gradeⅣ,according to the WHO Grading System)and 3 cases of nomal human neural tissue.To analyze STAT3 and p-STAT3(Tyr705)levels,GBM resection specimens and cells were examined by Western blot,and phosphorylated-STAT3's frequence was evaluated in human GBM.2.Cell Counting Kit-8 assay was used to measure cell growth rate.We measured the cell growth of GBM cell lines A172 and U251 in the presence of different concentrations of JSI-124 after incubation for 1 to 3 days.Absorbance was measured with excitation at 450 nm and emission at 630 nm using a microplate reader (Bio-Rad).The relative growth rate was calculated as follows:experimental absorbance/untreated control absorbance at each time point.3.A172 and U251 cells were exposed to indicated concentrations of JSI-124 for 24 h and cell morphology was evaluated by phase-contrast microscopy.4.Western blot was used to analyze the effects of JSI-124 on STAT3 and p-STAT3(Tyr705)protein expression of A172 and U251 cells with the different dose and time-dependent manner.Results1.Overexpressed and constitutively activated STAT3 in human GBM cell lines and samples.The result of western blot analysis revealed STAT3 were constitutively activated in the human GBM samples and cells,while normal human astrocytes(NHA) contained very low levels of or no activated STAT3.Furthermore,there were 88.89% (8 of 9 cases)present of p-STAT3(TyrT05)in human GBM samples.2 of 9 GBM tumors contained low levels of activated STAT3.Additionally,overexpressed STAT3 and p-STAT3(TyrT05)were exhibited in both U251 and A172 cells.It supported that the two cell lines could be used as ideal object in our study in vitro.Both p-STAT3(Tyr705)and STAT3 were also present at the similar levels in U251 and A172 cells.2.JSI-124 suppressed the proliferation of A172 and U251 cells.The results CCK-8 assay showed that JSI-124 suppressed cell proliferation in a time-and dose-dependent manner in both GBM cells.Compared to control cells, U251 and A172 cells treated with 100 nM of JSI-124 for 48 h resulted in＞50% suppression of cell proliferation.Significant differences was found in experimental group and control group(P＜0.05).3.JSI-124 induced morphological transformation of A172 and U251 CellsThe results demonstrated that the cell morphology and density of A172 and U251 cells were affected significantly after treatment with JSI-124 for 24 h and exhibited a dose dependent manner,while dimethyl sulfoxide(DMSO)had no influence on the shape and numbers of cells.Cell count decreased significantly in U251 and A172 cells at the dose of 100 nM or 200 nM,respectively,while JSI-124 had no influence on the morphology of cells.However,the shape and numbers of U251 and A172 cells were changed significantly when treated with JSI-124 at 200 nM and 400 nM,respectively. Microscopic observation of A172 and U251 cells treated with 1000 nM JSI-124 which revealed major alterations in their morphology and cell count decreased significantly. We noted one of the morphologic changes from a flat polygonal appearance to a smaller round shape with shorter tapering processes.4.JSI-124 decreased the levels of phosphorylated STAT3 in A172 and U251 cells.Dimethyl sulfoxide(DMSO)had no effect on the protein expression of phosphorylated STAT3 and total STAT3 in A172 cells.Total STAT3 protein levels in A172 and U251ccells treated with JSI-124 for 24 h didn't show significant change; however,phosphorylation of tyrosine 705 of STAT3 was inhibited by JSI-124 in dose and time-dependent fashion.Phosphorylated STAT3 levels decreased significantly in JSI-124 treated U251 and A172 cells at the dose of 100 nM or 200 nM,respectively. The rate of inhibition by JSI-124 is the highest in dose of 400 nM.Furthermore,the inhibition of the levels of phosphorylated STAT3 by JSI-124 was time-dependent that the relative expressions were reduced after treated for more than 24 h.Conclusion1.High frequency of STAT3 activation is presented in both GBM tumors and cell lines,and the critical role of STAT3 in the molecular pathogenesis of human GBM which play a major role in GBM carcinogenesis provides an attractive molecular target for therapeutic intervention in GBM.2.Persistent activation and dysregulation of STAT3 are presented in two GBM cell lines U251 and A172,so U251 and A172 cells could be used as ideal models in our current study.3.JSI-124 which acts as a cell-permeable inhibitor of STAT3 can specifically block STAT3 signaling pathway by decreasing the levels of phosphorylated STAT3. But,dephosphorylation of STAT3 by JSI-124 is a relatively slow event.4.JSI-124 can suppress the proliferation of human GBM cells.PartⅡEffects of Blocking STAT3 Signaling Pathway by JSI-124 on Cell Cycle and Apoptosis and Its Mechanisms in Human GBM CellsObjectiveTo investigate the effects of blocking STAT3 signaling pathway by phosphotyrosine STAT3 level inhibitor JSI-124 on cell cycle and apoptosis and the relevant mechanisms on proliferative inhibition in human GBM cell lines A172 and U251 in vitro.Methods1.U251 and A172 cells were treated with various concentrations of JSI-124 for 36 h,followed by Annexin V and PI binding assay.After incubation for 24 h,we further investigated the effects of JSI-124 on the transcription levels of Bcl-xl and survivin genes in GBM cells by Real-time PCR.And,the mRNA expression level ofβ-actin was determined by conventional RT-PCR.2.NIH3T3 cells were used as negative control.FACS was applied to detect U251,A172 and NIH3T3 cells cell cycle after cultured with JSI-124 for 36 h. Real-time PCR was used to analyze cdc2 and cyclin B1 mRNA levels in U251 and A172 cells exposed to JSI-124.Meanwhile,the protein expression levels of cdc2 and cyclin B1 were examined by Western blot.Results1.JSI-124 effectively promoted apoptosis by downregulating antiapoptotic genes in GBM cells.The increased apoptosis was dose dependent.Results showed that the percentage of apoptotic A172 cells increased from 12.75±1.95%(DMSO,vehicle control)to 19.76±5.00 and 31.46±6.83%with 100 and 200 nM concentrations of JSI-124 respectively by 36h.Like A172 cells,JSI-124 could significantly induce apoptosis of U251 cells at concentrations of 0 and 100 nM from12.77±3.48%to 27.56±4.84%.We examined the transcription levels of Bcl-xl and survivin in GBM cells by Real-time PCR.The transcription levels of Bcl-xl and survivin in A172 cells were decreased by 65.66%(P＜0.01)and 85.22%(P＜0.01),respectively.Similarly,43.79%(P＜0.05)and 73.13%(P＜0.01)had changed in the transcription levels of Bcl-xl and survivin in U251 cells,respectively.2.JSI-124 induced accumulation of GBM cells in G2/M phase via downregulating cdc2 and cyclin B1.After treated with 200 nM JSI-124 for 36 h,the percentage of A172 cells in G2/M phase elevated from 10.78±1.48%to 42.88±5.14%and accompanied by a significant decrease in S phase(from 24.54±2.33%to 14.28±1.53%).Similarly, JSI-124 induced U251 cell accumulated in G2/M phase.While the cell cycle of NIH3T3 cells treated with 200 nM JSI-124 showed no obvious alteration(G2/M phase from 5.68±0.74%to 7.17±0.99%;S phase from 12.83±0.15%to 15.67±0.06%).We analyzed their mRNA levels in U251 and A172 cells exposed to JSI-124 by using Real-time PCR.The transcription levels of cdc2 and cyclin B1 in A172 cells were decreased to 0.22±0.02(P＜0.01)and 0.11±0.03(P＜0.01),respectively.In U251 cells, the mRNA levels of cdc2 and cyclin B1 were decreased to 0.69±0.07 and 0.37±0.08 (P＜0.05),respectively.Meanwhile,the protein levels of cdc2 and cyclin B1 also showed significant decrease in A172 cells treated with JSI-124 and a small but significant decrease in the expression levels of cdc2 was observed in U251 cells, which was consistent with transcription levels. Conclusion1.JSI-124 can effectively promot apoptosis by downregulating antiapoptotic genes in GBM cells.2.JSI-124 can induce accumulation of GBM cells in G2/M phase via downregulating cdc2 and cyclin B1.In summary,high frequency of STAT3 activation is presented in both GBM tumors and cell lines.JSI-124 contributes to significant inhibition of GBM cells proliferation by inducing apoptosis and cell cycle arrest at G2/M phase,which is help to throw lights on future studies that should extend these findings to in vivo tumor models.JSI-124 could reduce the expressions of Bcl-xl and Survivin,promoting the apoptosis in GBM cells.And this inhibitory effect is accompanied by abrogation of STAT3-mediated cell cycle though blocking the expression levels of cdc2 and cyclin B1.Thus,targeted STAT3 signaling pathway with JSI-124 may serve as a novel therapeutic strategy for GBM and other tumors.