Signal transducer and activator of transcription-3 inhibitor WP1066 affects human tongue squamous cell carcinoma proliferation and apoptosis in vitro and in vivo

Objective To investigate the antitumour molecular mechanisms of WP 1066 ( STAT-3 inhibitor ) to human tongue squamous cell carcinoma in vitro and in vivo.Methods WP1066 was used to inhibit the p-STAT-3 expression in Tscca human tongue squamous cell carcinoma cell line .Real-time PCR was used to detect the microRNA-21 expression after treatment with DMSO and WP 1066.Methyl thiazolyl tatrozolium ( MTT ) assay was employed to determine cell survival.Flow cytometry ( FCM ) was used to measure apoptosis .The expression level of STAT-3/p-STAT-3, programmed cell death protein 4(PDCD-4), antigen 67 ( Ki-67 ) , B cell lymphoma 2 ( Bcl-2 ) and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) was examined by Western blotting.Luciferase reporter gene assay was conducted to verify the regulation of STAT-3 to microRNA-21.Tscca xenograft tumor model was established in BALB /c nude mice and the tumors were divided into control , DMSO and WP1066 treated groups.The tumor tissues were measured by immunohistochemistry stain and terminal-deoxynucleoitidyl transferase mediated nick end labeling ( TUNEL) assay.Results STAT-3/p-STAT-3 protein was suppressed after treatment with WP 1066 (STAT-3:F=15.336,P =0.004,p-STAT-3:F=52.837,P=0.000).MicroRNA-21 relative expression level was down-regulated (F=8.197,P=0.019).Cell survival rate was significantly reduced after treatment with WP1066 than control groups ( F=94.388,P=0.000).Early apoptosis rate increased after treatment with WP1066 ( F =217.080, P =0.000 ).PDCD-4 and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3)protein expression was increased after treatment with WP 1066(PDCD-4:F=8.771,P=0.017;CCASP-3:F=26.611,P=0.001).Ki-67 and Bcl-2 protein was down regulated (Ki-67:F=5.854,P=0.039;Bcl-2:F=125.502,P=0.000).Luciferase reporter gene assay proved that STAT-3 combined with specific promoter region of microRNA-21.In vivo, the tumor volume after treatment with WP 1066 was significantly smaller than control groups by the end of observation (F=15.390,P=0.000) .Immunological histological chemistry indicated that PDCD-4 and CCASP-3 protein expression was up-regulated simultaneously while Ki-67 and Bcl-2 protein of tumor tissue was down-regulated after treatment with WP1066 than control groups.TUNEL assay suggested that apoptosis index rose after treatment with WP 1066 than control groups ( F =133.368, P =0.000 ).Conclusions WP1066 affected Tscca cancer cell proliferation and apoptosis via inhibiting STAT-3/microRNA-21.WP1066 provided new direction and possibility to human tongue squamous cell carcinoma therapy . Key words: Oral squamous cell carcinomac ;  Tongue neoplasms ;  Cell proliferation ;  Apoptosis ;