Thymoquinone Inhibits Proliferation In Gastric Cancer Via STAT3 Pathway In Vivo And In Vitro

Part 1 Mechanism of Thymoquinone regulating proliferation via STAT3 signaling pathways in three gastric cancer cellsObjective:The purpose of this study was to elucidate the mechanism of Thymoquinone regulating tumor cell proliferation apoptosis through the STAT3 pathway in three gastric cancer cellsMethods:HGC27?BGC823?SGC7901 cells were cultured in vitro and treated with Thymoquinone (0,10,25,50,75,100,125?mol/L) for 12h,24h and 36h, and then the proliferation inhibitory rates were detected by MTT assay. The protein expression of STAT3?p-STAT3?STAT5 was detected and analyzed by Western blot methodResults:Thymoquinone inhibited STAT3 activation in all of these cells. With the maximum inhibition in TQ concentration of 50 microns occurs and did not influence the expression of STAT3 protein. Western blot clearly showed that TQ hindered the STAT3 in HGC27 cell nuclear translocation. TQ did not inhibit STAT5 phosphorylation, or the expression of STAT3 protein. The results showed that the TQ inhibited of STAT3 tyrosine phosphorylation specially.Conclusion:in this three gastric cancer cell, TQ regulateed cell apoptosis through the inhibition of STAT3 complex amino acid phosphorylation, the expression of STAT3 protein has not changedPart 2 Related genes in HGC27 gastric cancer cell regulating tumor cell proliferation apoptosis via STAT3 signaling pathwaysObjective:to study which genes played a decisive role in HGC27 gastric cancer cells via STAT3 signaling pathwayMethods:in this part, set HGC27 gastric cancer cell as research object, detected by RT-PCR and STAT3-luciferase report gene analysis, determined by MTT, Hoechst 33258 V cell apoptosis, membrane protein staining assay. The protein expression of STAT3, p-STAT3, STAT5, p-STAT5, p-JAK2, JAK2, p-Src, Src, GAPDH and Lamin-A, survivin, Cyclin D, Bcl 2, Bax, PPAR, Caspase-3,7,9 were detected by Western blot method. Flow cytometry (FCM) was used to detect the cell cycle and cell apoptosis. In HGC27 cell, TQ induced apoptotic cell death in a dose-dependent related manner, and through the analysis and was detected by Hoechst 33258-FITC Annexin V/PI method. Compared with control group, difference was statistically significant due to P<0.05Results:TQ-induced down-regulation of STAT3 activation is associated with a reduction in Janus-activated kinase 2 (JAK2) and c-Src activity. TQ also down-regulates the expression of STAT3-regulated genes such as Bcl-2, cyclin D, surviving and VEGF, and activates caspase-3,7,9. Annexin V-FITC/PI analysis results showed that the TQ induced apoptosisin a concentration-depended related manner. Hoechst 33258 impregnation result contained the nuclear fragments of apoptotic body.Conclusion:TQ in HGC27 cells inhibited the expression of STAT3 genes, including cell cycle protein D1, the BCL-2, survivin, angiogenesis gene product (VEGF), the BCL-xL and survivin may be associated with the division of PARP, TQ activated caspase-3,7,9 in concentration-depended related manner.Part3 Effect of biology behavior research using Thymoquinone in gastric cancer cell in vivoObjective:the purpose in this part of the study was to detect the specific mechanism of Thymoquinone's effect on gastric cancer cell in the body subcutaneous transplantation tumor of nude mice.Method:in vivo, nude mice with gastric carcinoma subcutaneous transplantation tumor model was set up, divided into four groups, four mice each cage. After tumor model completed, nude mice were randomly assigned into four groups:(1) untreated, constant volume normal saline; (2) 10 mg/kg TQ; (3) 20 mg/kg TQ; (4) 30 mg/kg TQ. Intraperitoneal injected, three times a week. Weighed every 3 days, used caliper to measure tumor size. Mice were sacrificed, weighed after 30 days. TUNNEL method used to detect the number of gastric cancer cells. Compared with control group, statistical significance due to P< 0.05.Results:the experimental nude mice tumor weighed out after the death and stripping. Four groups of weight as:(762.7±35.2mg) (647.8±33.9mg) (595.3±30.3mg) (305.1±34.8mg), statistically significant due to P<0.05.Conclusion:compared with control group, after treatment tumor significantly reduced the weight and volume.