| With the growth and aging of the population, as well as an increasing prevalence of established risk factors such as smoking, overweight, physical inactivity, and changing reproductive patterns associated with urbanization and economic development. Cancer constitutes an enormous burden on society. Based on GLOBOCAN estimates, about 14.1 million new cancer cases and 8.2 million deaths occurred in 2012 worldwide. Among them, worldwide, the incidence and mortality rates of gastric cancer account for the fourth and third place in male patients, respectively, and account for both five in female patients. Chemotherapy is commonly used in the treatment of gastric cancer, and the commonly used chemotherapy drugs are 5-fluorouracil, cisplatin and doxorubicin, which have been toxic side effects, easy to resistance and other defects. Therefore, it is important to search for low toxicity and efficient anti-cancer drugs inhibit gastric cancer development.Thymoquinone (TQ), as the active constituents of black cumin (Nigella sativa) seed oil, is also called 2-Isopropyl-5-methyl-1,4-benzo-quinone, C10H12O2. TQ has been shown to suppress the proliferation, induced apoptosis and drug resistance, and so on in various tumor cells, including colorectal carcinoma, pancreatic carcinoma, breast adenocarcinoma, prostate cancer, et al. A phase I study had reported no significant systemic toxicities in adult patients with solid tumors or hematological malignancies who were treated with TQ. It was also found that the human body could tolerate a dose of TQ 75 to 2600 mg/day. Because of its high efficiency, low toxicity and natural features in cancer prevention and treatment, TQ gradually attracted the attention of scholars in the world, but the research in tumors of digestive system especially in gastric cancer is rare. Our previous study found that TQ could inhibit proliferation and induce apoptosis in gastric cancer cells, presumably with the STAT3 signaling pathway.Signal transducer and activator of transcription (STAT) have been shown to play a fundamental role in tumor cell survival and proliferation. STAT3 is a key molecule of the JAK/STAT signaling pathway and can be activated by a variety of ligands that respond to massive signals such as IL-6, TNF-a, and VEGF. Aberrant expression and constitutive activation of STAT3 are involved in a variety of human malignancies such as gastric, colon, breast and lung. Once activated, STAT3 undergoes phosphorylation-induced homodimerization, leading to nuclear translocation, DNA binding, and subsequent gene transcription (e.g. Bcl-xL?Bcl-2?survivin?cyclin D? Mcl-1). In addition, STAT3 can control vascular endothelial growth factor (VEGF) expression, which is necessary for angiogenesis and the maintenance of tumor vasculature. STAT3 has been implicated in the inhibition of immune responses to tumor growth by blocking expression of pro-inflammatory factors. The STAT3 gene is located on chromosome 17q21. Several studies have found STAT3 gene polymorphism may modulate the expression of STAT3, and thereby affecting its activity.Based on preliminary studies, we further explore the specific mechanisms of TQ regulating proliferation and apoptosis in gastric cancer cells, as well as a comprehensive analysis of STAT3 gene polymorphisms and cancer susceptibility.Part 1 Thymoquinone inhibits growth in gastric cancer cells in vitro via STAT3 pathwayPurpose Previous studies have found that TQ could inhibit proliferation and induce apoptosis in gastric cancer cells. However, the specific mechanism is still unknown. Here, the aim of this study was to elucidate the mechanism of TQ-induced apoptosis in human gastric cancer cells in vitro.Methods First, gastric cancer cells were treated with 25,50 and 75 ?M TQ for 12,24 or 36 h, or 50 ?M TQ, the STAT3 gene level was detected by RT-PCR; the expression of STAT3 pathway related proteins were measured by Western-blot, STAT3 nuclear translocation were also analyzed by Western-blot, Cell cycle analysis was measured by Flow Cytomtry; Then, gastric cancer cells were treated with 50 ?M TQ, TQ plus IL-6 10 ng/ml or TQ plus WP10665 ?M for 12,24 or 36 h, the rate of proliferation was measured by MTT assay, the colony-forming ability was assayed by soft agar colony formation assay, conditions of apoptosis were measured by Hoechst33258 and Annexin V-FITC/PI of Flow Cytomtry, the expression of apoptosis related proteins were measured by Western-blot.Results The results show that TQ inhibited STAT3 phosphorylation and the level of STAT3 gene in a dose-dependent and time-dependent manner, which corrected with the inhibition of c-Src and JAK2 activation. Howver, TQ did not inhibit STAT5 phosphorylation. In addition, TQ could inhibit STAT3 nuclear translocation and STAT3 related proteins. TQ induced the accumulation of cells in sub-Gl phase. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after TQ and combination treatment, and the effect of TQ combined with WP1066 was the most strongest. The expression of Bax increased, whereas that of Bcl-2, survivin and cyclin-D, VEGF decreased. In addition, stimulation of PARP, caspase-3 caspase-7 and caspase-9 activity was observed.Conclusions TQ may suppress gastric cancer cells survival and proliferation through STAT3 signaling pathway.Part 2 Thymoquinone inhibits growth in gastric cancer cells in vivo via STAT3 pathwayPurpose Previous studies have found that TQ could inhibit proliferation and induce apoptosis in gastric cancer cells. However, the specific mechanism is still unknown. Here, the aim of this study was to elucidate the mechanism of TQ-induced apoptosis in human gastric cancer cells in vivo.Method Gastric cancer cells were subcutaneously inoculated into the lower right flank of the nude mice. After establishing mouse xenograft model, TQ, TQ plus IL-6 or TQ plus WP1066 was administrated via intraperitoneally injection every 3 days. Tumor growth was monitored using calipers. The nude mice were killed at 30 days. Status of nude mice and inhibitory effect of tumor growth was observed, the tumor volume was calculated, and growth curve was plotted. The morphological changes were observed and the STAT3 protein expression in tissue was detected by immunohistochemistry and Western blot. Representative histological sections of TUNEL were taken from mice bearing transplantation tumor were detected.Results The results show that marked changes in apoptotic morphology and apoptosis rates were clearly observed after TQ and combination treatment. The expression of STAT3 protein decreased and TQ markedly inhibited the growth of tumors via apoptosis induction, as demonstrated by a TUNEL assay.Conclusion TQ combined with STAT3 intervention agents could inhibit tumor growth and induce apoptosis in mouse xenograft model. Taken together, the results suggest STAT3 signaling pathway may be involved in the process of TQ inhibit the growth of gastric cancer cells.Part 3 Association between polymorphisms of STAT3 and susceptibility of cancer:A Meta-analysisPurpose Persistent activation of STAT3 signaling has been demonstrated to influence important processes in tumors such as survival, proliferation, apoptosis, angiogenesis, and invasion, and modulates the expression of target genes that are involved in various physiological functions. Recently, the STAT3 gene polymorphisms have been implicated in cancer risk, but the reported results are inconsistent. Here we performed a meta-analysis to explore the correlation between STAT3 polymorphism and cancer risk.Methods All eligible case-control studies published up to March 2015 were identified by searching PubMed, EMABSE, Web of Science, CNKI, VIP, WanFang, CJFD and CBM databases. Two reviewers independently identified the literature according to inclusion and exclusion criteria. Meta-analysis was performed using RevMan 5.2 and Stata 12.0 software.Results A total of 21 studies comprising 22 case-control studies were included. Overall, a significantly decreased risk was found for rs 12949918 polymorphism (recessive model:OR=0.77,95%CI:0.68-0.87, TC vs. TT:OR=0.87,95%CI:0.79-0.96, CC vs. TT:OR=0.71,95%CI:0.62-0.81); while there was no significant association for rs12949918 polymorphism under dominant model, and for other four polymorphisms under all genetic models. In subgroup analysis by ethnicity, for rs 12949918 polymorphism, similar results were detected among Caucasians, similarly, a significant decreased risk was observed in Asians under dominant and CC vs. TT model; for rs2293152 polymorphism, significant association was detected among Caucasians under dominant model.Conclusion STAT3 rs12949918 polymorphism is associated with the susceptibility of cancer, and genotype TC/CC may decrease the risk of suffering from cancer. |
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