The Role And Molecular Mechanism Of FOXM1 And Thiostrepton In Growth And Metastasis Of Laryngeal Squmous Cell Carcinoma

Objective:To investigate the effect of down-regulation of FoxM1 by inhibitor thiostrepton on proliferation, apoptosis, invasion, metastasis and clone informing ability of laryngeal carcinoma cells in vitro and its mechanism. To investigate the effect of FoxM1 inhibition by thiostrepton on growth of laryngeal carcinoma cells in vivo and its mechanism. To investigate the effect of down-regulation of FoxMl by FoxMl shRNA on clone informing ability of laryngeal carcinoma cells in vitro and growth in nude mice in vivo.Methods:The mRNA and protein expression of FoxMl in Hep-2 cells after treating with thiostrepton were detected by RT-PCR and western blot. Cell viability of Hep-2 cells by treating with thiostrepton was studied by CCK-8 assay.Hep-2 cells was stained with CFSE, and CFSE fluorescence intensity of the stained tumor cells which were treated with thiostrepton at 24,48 and 72 h was detected. Cell cycle of Hep-2 cells after treating with thiostrepton was detected by flow cytometry. The mRNA and protein expression of cyclinD1 and cyclinE1 in Hep-2 cells after treating with thiostrepton were detected by RT-PCR and western blot.We used annexin V/PI dual staining for confirmation of thiostrepton-induced apoptosis in Hep-2 cells. Hep-2 cells were treated with various doses of thiostrepton for 48 h and apoptosis was measured by Tunel analysis. The mRNA and protein expression of Bcl-2, Bax, and p53 in Hep-2 cells after treating with thiostrepton were detected by RT-PCR and western blot. We then tested the effect of thiostrepton on the mitochondrial membrane potential. Hep-2 cells were treated with thiostrepton for 24 and 48 h and labeled with JC1 dye, and mitochondrial membrane potential was measured by flow cytometry. The protein expression of cyto chrome c, cleaved caspase-9, cleaved caspase-3 and cleaved PARP in Hep-2 cells after treating with thiostrepton were detected by western blot. We used annexin V/PI dual staining for confirmation of apoptosis of Hep-2 cells, pretreatment of with 60 μM z-VAD-fmk, followed by thiostrepton treatment and western blot to detect the expression of cleaved caspase-3 in Hep-2 cells. The protein expression of FADD,cleaved caspase-8, cIAP1, XIAP and survivin in Hep-2 cells after treating with thiostrepton were detected by western blot.Migration and invasion tests were used to examine the effect of FoxMl inhibition by thiostrepton on metastasis and invasion abilities of Hep-2 cells. The mRNA and protein expression of MMP-2 and MMP-9 in Hep-2 cells after treating with thiostrepton were detected by RT-PCR and western blot.Anchorage-independent growth assay was used detect the effect of FoxM1 inhibition by thiostrepton on clone informing ability of Hep-2 cells. Xenograft model of tumor formation was used to investigate how thiostrepton influences tumorigenesis in vivo. The protein expression of FoxM1, Ki67 and cleaved caspase-3 in laryngeal carcinoma tissue in nude mice after treating with thiostrepton were detected by immunohistochemistry and western blot.FoxM1shRNA Hep-2 cell line was established by affected with lentivirus. Anchorage-independent growth assay was used detect the effect of FoxMl inhibition by shRNA on clone informing ability of Hep-2 cells. Xenograft model of tumor formation was used to investigate how FoxMl inhibition by shRNA influences tumorigenesis in vivo.Results:FoxMl mRNA and protein expressions were significantly decreased in a dose-dependent manner after thiostrepton treatment. Down-regulation of FoxM1 expression by thiostrepton caused inhibition of Hep-2 cell viability. FoxMl inhibition by thiostrepton suppressed the proliferation of Hep-2 cells. Thiostrepton induced a gradual time-dependent S phase arrest. Cyclin D1 and cyclin El were also down-regulated after treating with thiostrepton. FoxM1 inhibition by thiostrepton induced apoptosis of Hep-2 cells. Thiostrepton caused decreased Bcl-2 expression and increased Bax and p53 expression. Thiostrepton induced loss of mitochondrial membrane potential in Hep-2 cells with a dose-and time-dependent manner and induced cytochrome c released from mitochondria into cytosol. Cytochrome c activated the downstream caspases cleaved caspase 9, cleaved caspase-3 and cleaved PARP. FoxMl inhibition by thiostrepton resulted in the activation of FADD and cleaved caspase-8 in Hep-2 cells. Thiostrepton treatment caused a dose-dependent down-regulation of cIAP1 and survivin. However, XIAP was also down-regulated but not at a dose-dependent manner. FoxMl inhibition by thiostrepton inhibited metastasis and invasion abilities of Hep-2 cells by down-regulation of MMP-2 and MMP-9. In addition, FoxM1 inhibition by thiostrepton inhibited clone informing ability of Hep-2 cells. FoxMl inhibition by thiostrepton inhibited tumorigenesis of Hep-2 cells in vivo. The expression level of Ki67 was down-regulated and cleaved caspase-3 up-regulated detected by immunohistochemical staining. FoxM1 shRNA Hep-2 cell line was established by affecting with lentivirus. Down-regulation of FoxMl by shRNA inhibited clone informing ability of Hep-2 cells in vitro and tumor growth in vivo.Conclusion:Down-regulation of FoxMl by thiostrepton inhibit the proliferation of Hep-2 cells by arresting cell cycle at S phase and down-regulation of cyclinD1 and cyclinE1. Down-regulation of FoxMl by thiostrepton induce apoptosis of Hep-2 cells through mitochondrial-and caspase-dependent intrinsic pathway and Fas-dependent extrinsic pathway as well as IAP family. Inhibition of FoxMl by thiostrepton inhibit the metastasis and invasion abilities of Hep-2 cells by down-regulation of MMP-2 and MMP-9. FoxMl inhibition by thiostrepton or FoxMl shRNA inhibit tumorigenesis of Hep-2 cells in vivo.