Thymoquinone Prevents And Ameliorates DSS-induced Colitis In Mice And Induces Apoptosis In Gastric Cancer In Vivo And In Vitro

Thymoquinone (TQ), as the active constituents of black cumin (Nigella sativa) seed oil, is also called2-Isopropy1-5-methy1-1,4-benzo-quinone, C10H12O2. It is reported to exhibit antioxidant, anti-inflammatory, and anti-cancer effects, and it also has been used in the treatment of various diseases, while been minimally toxic to normal cells.TQ has protective effect on several animal models of inflammatory response. For instance, it has been shown to suppress acetic acid-induced colitis in rats, experimental autoimmune encephalomyelitis, experimental rheumatoid arthritis, murine septic peritonitis, reduce carrageenan-induced paw edema in rats, inhibit COX-2expression and prostaglandin production in a mouse model of allergic airway inflammation, protect against doxorubicin-induced cardiotoxicity in mice, prevent cisplatin-induced nephrotoxicity in mice, et al.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 TQ75to2600mg/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.Gastric cancer is one of the most common malignant tumors in China, which mortality accounted for second in the malignant tumor. The incidence of gastric cancer is increasing year by year, seriously endangering the health of Chinese. Chemotherapy is commonly used in the treatment of gastric cancer, and the commonly used chemotherapy drugs are5-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. However, there is little research on the role of TQ in gastric cancer.13years ago, Badary found that TQ was a powerful chemopreventive agent against BP-induced forestomach tumors in mice, and the possible modes of action of TQ may be through its antioxidant and anti-inflammatory activities, coupled with enhancement of detoxification processes.Numerous evidences have reported different modes of anti-inflammatory and anti-cancer action, including ROS generation, anti-proliferation, apoptosis induction and synergism with conventional medicine. Moreover, TQ could attenuate toxicity associated with the use of conventional medicine without compromising therapeutic efficacy. Despite the therapeutic efficacy in cancer cells and animal models has been proved, there have been little studies with the theary effect of colitis and gastric cancer with TQ. More systematic studies have to be performed systematically before TQ can be used into treatment of various carcinomas and inflammatory disorders.Part1Thymoquinone prevents and ameliorates Dextran Sulfate Sodium-induced Colitis in MicePurpose Thymoquinone (TQ), an active ingredient of the seed oil extract of Nigella sativa Linn, previously has been shown to possess antitumor, antioxidant and anti-inflammatory bioactivity. Whether TQ has any effect on colitis remains controversial. The aim of this study was to determine whether treatment with TQ prevents and ameliorates colonic inflammation in a mouse model of colitis.Methods C57BL/6murine colitis was induced by the administration of dextran sodium sulfate (DSS)(3%W/V) in the drinking water supplied to the mice for7consecutive days. The mice with colitis were treated with5mg/kg,10mg/kg or25mg/kg TQ in different time, and changes in mortality and body weight, macroscopic and microscopic colitis scores were examined. In addition, biochemical analyses were conducted. Results The treatment of DSS-induced colitis in mice with TQ prevented and significantly reduced the mortality and body weight loss. These results were associated with amelioration of colitis-related damage, as measured by macroscopic and microscopic colitis scores. In addition, there was a significant reduction in colonic myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels and an increase in glutathione (GSH) levels. And the treatment of mice in a dose-dependent manner. Conclusions These results indicate that TQ administration can prevent and improve murine DSS-induced colitis. These findings suggest that TQ could serve as a potential therapeutic agent for the treatment of patients with colitis.Part2Thymoquinone inhibits growth in gastric cancer cells both in vitro and in vivoPurpose Thymoquinone (TQ) has been shown to inhibit the growth of many types of human cancers in vitro and in vivo. However, the mechanism of inducing apoptosis in gastric cancer cells remains to be clarified. The aim of this study was to demonstrate the role of TQ, and elucidate the mechanism of TQ-induced apoptosis in human gastric cancer cells in vitro and vivo.Methods Gastric cancer cells were treated with25,50and100μM TQ for12,24or36h, or50μM TQ, the rate of proliferation was measured by CCK-8assay; conditions of apoptosis were measured by Hoechst33258and Annexin V-FITC/PI of Flow Cytomtry; the protein of Bax, Bcl-2, Bcl-xl, surviving, cyclin-D, Cyt-c, procaspase-3and procaspase-9, were measured by Western-blot. Gastric cancer cells were subcutaneously inoculated into the lower right flank of the nude mice. TQ (10,20and40mg/kg) was administrated via intraperitoneally injection every3days. Tumor growth was monitored using calipers. The nude mice were killed at30days. Status of nude mice and inhibitory effect of tumor growth was observed, the tumor volume was calculated, and growth curve was plotted. Representative histological sections of TUNEL were taken from mice bearing transplantation tumor were detected.Results Our results indicate that TQ inhibited the growth of gastric cancer cells in a dose-dependent and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after TQ treatment. The expression of Bax and Cytochrome c (Cyt-c) increased, whereas that of Bcl-2, Bcl-xl, surviving and cyclin-D decreased. In addition, stimulation of caspase-3and caspase-9activity was observed. We investigated the effect of combination of TQ as a mechanism to potentiate the inhibitory effect of gastric cancer cells xenograft model in nude mice subcutaneously, and TQ markedly inhibited the growth of tumors via apoptosis induction, as demonstrated by a TUNEL assay.Conclusions TQ may induce apoptosis in human gastric cancer cells primarily through a mitochondria-related pathway, and TQ has significant anti-cancer potential in vivo, which markedly inhibited the growth of tumor via apoptosis induction.Part3Thymoquinone augments5-fluorouracil-induced apoptosis in gastric cancer cellsPurpose In this part, in gastric cancer cells and xenograft model in nude mice subcutaneously, the mechanism of TQ augments5-Fu to inducte apoptosis and inhibit proliferation was elucidate.Methods,50μM TQ combined with75μg/ml5-Fu in gastric cancer cells. The rate of proliferation was measured by CCK-8assay; conditions of apoptosis were measured by Hoechst33258and Annexin V-FITC/PI of Flow Cytomtry; the protein of Bax, Bcl-2, Cyt-c, procaspase-3and procaspase-9, were measured by Western-blot.20mg/kg TQ combined with19mg/kg5-FU were administrated via intraperitoneally injection every3days in xenograft model. Tumor growth was monitored using calipers. The nude mice were killed at30days. Status of nude mice and inhibitory effect of tumor growth was observed, the tumor volume was calculated, and growth curve was plotted. Representative histological sections of TUNEL were taken from mice bearing transplantation tumor were detected.Results Our results indicate that TQ increased sensitization of gastric cancer cells to5-FU-induced cell apoptosis. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after TQ treatment. The expression of Bax and Cytochrome c (Cyt-c) increased, whereas that of Bcl-2decreased. In addition, stimulation of caspase-3and caspase-9activity was observed. We investigated the effect of combination of TQ and5-FU as a mechanism to potentiate the inhibitory effect of gastric cancer cells xenograft model in nude mice subcutaneously, and TQ markedly inhibited the growth of tumors and augments the effect of5-FU via apoptosis induction, as demonstrated by a TUNEL assay.Conclusions TQ may increased sensitization of gastric cancer cells to5-FU-induced cell apoptosis. And our data also suggest that combination of TQ and5-FU markedly inhibited the growth of tumor via apoptosis induction. Our results indicate that TQ could potentially be developed as a novel therapeutic agent against gastric cancer.