The Mechanism Study Of N-3 Polyunsatured Fatty Acid On Gastric Cancer Treatment

[Background] Docosahexaenoic acid (DHA) and Eicosapentaenoic Acid (EPA) areω-3 polyunsaturated fatty acids (co-3PUFAs).ω-3PUFAs have many beneficial effects and have been widely used in prevention and treatment of various diseases. The main beneficial effects can be summarized as following:(1)Provide energy and nutrition support in fat emulsion; (2)Reduce inflammatory cytokines, such as IL-1,IL-6,TNFa, and regulate the immune effect; (3) Reduce inflammation in multiple organ trauma stress; (4)Treatment of cardiovascular disease by lipid-lowering, anticoagulant and other beneficial effects. Recently, more and more empirical evidences show thatω-3 polyunsaturated fatty acids have a significant beneficial effect in cancer treatment. Meanwhile, our preliminary studies in vitro and in vivo have further confirmed that co-3 polyunsaturated fatty acids can also sensitize effect of chemotherapy when combined with chemotherapy drugs.[Research Objective] In vitro cell culture level and in vivo tumor-bearing mice level, we investigate the anticancer mechanism of co-3PUFA and 5-Fuorouracil (5-FU) alone or in combination in treatment of gastric cancer, thus may provide new ideas for comprehensive treatment of gastric cancer.Part I:In vitro cell culture level, investigate the effect ofω-3 polyunsaturated fatty acid alone or combined with 5-FU chemotherapy to treat gastric cancer cell lines[Cell Culture Methods]In vitro method:①In vitro culturing cells in 96-well plates, MTT assay was used to investigate the inhibition effect of DHA and 5-FU alone or in combination to treat gastric cancer cells SGC7901;②Use DCFH-DA probe to detect ROS level of gastric cancer cell SGC7901 treated by DHA and 5-FU alone or in combination;③Use dual luciferase reporter gene assay to detect NF-κB signaling pathway changes of gastric cancer cell SGC7901 treated by DHA and 5-FU alone or in combination;④After gastric cancer cells treated by DHA and 5-FU alone or in combination, use RT-PCR method to detect mRNA expression of tumor proliferation and invasion-related gene MMP2, COX2, CyclinDl,which is the downstream gene of NF-κB signaling pathway;⑤After gastric cancer cells treated by DHA and 5-FU alone or in combination, use Westernblot method to detect protein expression of tumor proliferation and invasion-related gene MMP2, COX2, CyclinDl;⑥Use MDA assay kit to investigate the lipid peroxidation status of gastric cancer cells treated by DHA and 5-FU alone or in combination;⑦Use PI flow cytometry to detect gastric cancer cell cycle after treated by DHA and 5-FU alone or in combination.[In vitro Results]①MTT test:Both DHA and 5-FU can inhibit tumor cell proliferation, DHA combined 5-FU chemotherapy can further inhibit tumor cell proliferation and reach the enchancing chemotherapy sensitivity effect.②DCFH-DA Determination of Active Oxygen:Both DHA and 5-FU can increase reactive oxygen species, DHA combined 5-FU chemotherapy, can further increase the ROS level of tumor cells, and reach the enchancing chemotherapy sensitivity effect.③NF-κB signaling pathway by dual-luciferase reporter gene assay:Tumor cells'NF-κB signaling pathway can be significantly activated in itself, DHA inhibited NF-κB signaling pathway, and 5-FU chemotherapy can activate NF-κB signaling pathway. DHA combined 5-FU chemotherapy can inhibit the elevated levels of chemotherapy-induced NF-κB signaling pathway.④RT-PCR results:5-FU chemotherapy can activate NF-κB signaling pathway, and accordingly makes the downstream gene, which is associated with tumor proliferation and invasion, MMP2, COX2, CyclinDl elevated in mRNA expression level. Combining with DHA can inhibit the increased mRNA expression level of chemotherapy-induced NF-κB pathway downstream gene MMP2, COX2, CyclinDl.⑤Westernblot results:Similar with RT-PCR results,5-FU chemotherapy can activate NF-κB signaling pathway, and accordingly makes the downstream gene MMP2, COX2, CyclinDl elevated in protein expression level. Combining with DHA can inhibit the increased protein expression level of chemotherapy-induced NF-κB pathway downstream gene MMP2, COX2, CyclinDl.⑥MDA determination:DHA can increase lipid peroxidation of MDA production in gastric cancer cells SGC7901. However,5-FU chemotherapy has no significant effect in lipid peroxidation.⑦DHA and 5-FU on the cell cycle:PI flow cytometry results suggest that DHA can block tumor cells to enter S phase,5-Fu significantly reduce the number of S phase cells. In combination, two-drug produced the effect of G0/G1 phase arrest more significant. The DHA or 5Fu alone could reduce the proliferation index of SGC7901 gastric cancer cell line, while in combined group the PI was reduced more significantly than any other groups (P<0.05). DHA producing arrest in G0/G1 phase may be related with reducing the expression of CyclinDl.Part II:in vivo tumor-bearing mice level, investigate the effect of co-3 polyunsaturated fatty acid alone or combined with chemotherapy to treat the mice pre-gastric carcinoma[Tumor Bearing Mice Methods]In vivo Methods:Mice pre-gastric cancer cell line (MFC) subcutaneous injected into 615 mice to constitute a tumor-bearing mice model, the mice were divided into four groups: Group 1 tumor-bearing mice without intervention; Group 2 tumor-bearing mice were intervened with oral fish oil; Group 3 mice were intervened with 5FU chemotherapy; Group 4 tumor-bearing mice were intervened with oral fish oil and chemotherapy. Oral fish oil were pharmaceutical drugs made in Beijing BeiHui, which is high-purity (DHA, EPA> 80%, ethyl ester type) soft capsules, and the mice fed with 2g/kg per day. Chemotherapy is using clinical 5-FU intravenous injection, intraperitoneal injected into tumor bearing mouse every two days (20mg/kg), from the day of mice being subcutaneously injected with tumor cells to the day of treated mice being killed after 14 days'intervention. Carefully recorded the following index:①tumor-bearing mice'general status, food intake and body weight changes in time;②mice tumor volume and weight changes;③At the end of experiment, extract tumor tissue RNA of each group mice, using RT-PCR method, compare the mRNA expression of tumor invasion and proliferation related gene MMP2, COX2, cyclinDl;④Extract the tumor tissue protein of each group mice, using Westernblot method to detect CyclinDl expression in protein leverl;⑤sing RT-PCR method, compare the mRNA expression of tumor inflammatory factor related gene IL-1β,1L-6;⑥Extract tumor tissue protein of each group mice, use the MDA assay kit to detect the lipid peroxidation status;⑦make paraffin section of mouse lung and tumor tissue, observation the microscopic tissue with HE staining.[In vivo Results]①General status, food intake and body weight changes of tumor-bearing mice:the mice intervened with oral fish oil showed generally good, no obvious symptoms; Intraperitoneal injection of 5-Fu chemotherapy can significantly affect the food intake of the tumor bearing mice. With the growth of tumors, mice in each groups decreased food intake gradually, the mice's general status deteriorated noticeably, body weight decreased, and turn out tumor cachexia symptoms. Mice intervened with oral fish oil (group 2,4), compared with the corresponding non-fish oil intervention groups (group 1,3), the body weight loss were reduced significantly. Oral fish oil can improve cancer cachexia.②Tumor volume and tumor weight changes of the tumor bearing mice:The tumor bearing mice intervened with chemotherapy (group3,4), compared with mice intervened without chemotherapy (group 1,2), tumor volumes were significantly smaller (P<0.05). The tumor bearing mice intervened with oral fish oil (group2,4), compared with mice intervened without oral fish oil (groupl,3), tumor volumes weren't significantly different(P<0.05). Similar with body weight, the carcass weight decreased sharply with tumor volume increased. Mice intervened with oral fish oil, compared to group mice intervened without oral fish oil, carcass weight were significantly higher (P<0.05).③mRNA expression of downstream gene MMP2, COX2, cyclinDl:oral fish oil or 5FU chemotherapy inhibited tumor tissue COX2, CyclinDl, MMP2 in mRNA expression (three genes in 2,3,4 group were significantly lower than those in group 1, p< 0.05).④Tumor tissue gene CyclinDl protein expression by Westernblot test:oral fish oil or 5FU chemotherapy inhibited tumor tissue CyclinDl expression (Cyclin D1 in 2,3,4 group was significantly lower than that in group 1, p<0.05).⑤Tumor tissue inflammatory factor gene IL-1β, IL-6 mRNA expression:compared to the control group (group 1), intervened with chemotherapy or fish oil(2,3,4 group), tumor tissue gene IL-1β, IL-6 mRNA expression were strongly inhibited (p<0.01).⑥Lipid peroxidation by MDA test:oral fish oil significantly increased the MDA level in tumor tissue (group 2 and 4 were significantly higher than group 1 and 3, p<0.05). Chemotherapy did not increase the tumor tissue MDA level (no significant difference between group 1 vs.3, p>0.05).⑦Mice lung tumor tissue HE staining of paraffin sections:tumor and lung tissue in each group mice showed no significant difference. [Conclusion of this study]1. In vitro cell culture level:n-3PUFA can inhibit NF-KB/IkB cell signal pathway and the downstream proliferation and invasion-related gene MMP2, COX2, CyclinDl expression, n-3PUFA can also increase cellular oxidative stress response to enhance chemotherapy sensitizing effect, and reduce the proliferation index of SGC7901 gastric cancer cell line.2. In vivo tumor-bearing mice level:co-3PUFA can reduce the expression of tumor tissue inflammatory factors IL-1β, IL-6 and improve cancer cachexia;ω-3PUFA combined with chemotherapy can suppress tumor proliferation and invasion-related gene MMP2, COX2, CyclinDl expression. Combining withω-3PUFA has some beneficial effects in cancer chemotherapy.3. Compared to the significant side effects of chemotherapy drugs,ω-3PUFA has anti-inflammation, regulation of blood fat, all without side effects. This experimental result provides the lab basis for the further clinical application ofω-3PUFA to treatment of cancer patients.