Resveratrol Sensitivities Of Gastric Cancer Cells And Their Relevance With STAT3 Signaling: A Preliminary Study

BackgroundGastric cancer was the fourth largest common cancer in the world and had the second highest death rate in the tumor. Now 5-FU, Cisplatin, Paclitaxel, Oxaliplatin and Oxaliplatin are commonly used in clinical treatment as chemotherapy drugs, but these drugs are more or less toxicity to the patient. So look for non-toxic side effect of the drug also has a very important clinical significance. Resveratrol is widely present in Polygonum Cuspidatum, grapes and other 70 kinds of natural plants. Resveratrol has many biological functions, including anti-inflammatory, anti-oxidant, anti-tumor and nerve protection, but also non-toxic side effects in humans and animals. So resveratrol has a good prospect on the prevention and treatment of gastric cancer. Numerous studies show that Resveratrol may be by intracellular signaling pathways(Wnt, Notch or JAK / STAT signaling pathway, etc.) to control the activation or inhibition of proliferation or apoptosis. There are also reported in the literature, STAT3 is not only in bladder cancer, breast cancer, pancreatic cancer and cervical cancer, but also is still abnormal activation and increased expression in gastric cancer. But the study of the impact of Resveratrol to the gastric cancer and its relationship with STAT3 signaling pathway is still little. Objective(1) Study the sensitivity of Resveratrol to the Gastric cancer cell line MGC-803, BGC-823 and AGS and its Characterized.(2) Analysis the expression of STAT3, p-STAT3 and STAT3 signaling pathway negative regulator of SOCS3, p-SHP2 and PIAS3 after 100μM Resveratrol was used in gastric cancer cell line MGC-803, BGC-823 and AGS around 48 h and analyze the possible mechanism.(3) Analysis of the role of JAK inhibitor AG490 on MGC-803, BGC-823 and AGS cells before and after 48 h, gastric cancer cell growth and apoptosis, so as to explore the role in the development of gastric cancer cells in the process of STAT signaling pathway plays and the intrinsic link about the anti-tumor activity of the signaling pathway may exist. Methods(1) Gastric cancer cell lines MGC-803 and BGC-823 were cultured in L-DMEM containing 10% domestic excellent FBS and then collected after 100μM Resveratrol treated for 48 hours. AGS cell was cultured in L-DMEM containing 10% imported FBS and then collected after 100μM Resveratrol treated for 48 hours.(2) Morphological observation and quantitative analyses of Gastric cancer cell growth, that were used to detect the sensitivity of cells to Resveratrol, were conducted before and after 100μM Resveratrol and 80μM AG490 treated through H&E staining.(3) MTT assay was used to detect the growth inhibition of gastric cancer cells in Resveratrol.(4)Using FCM to detect cell cycle and apoptosis after AG490 and Resveratrol-treated.(5)ICC, RT-PCR and Western-blotting were used to detect the expression change of STAT3, p-STAT3 and STAT3 negative regulation factors SOCS3, SHP2 and PIAS3 of JAK/STAT signal pathway in three cell lines before and after 80μM JAK inhibitor AG490 treated for 48 h.(6) Using ICC to detect the expression of p-STAT3 after AG490 and Resveratrol-treated for 48 h. Results(1) H&E staining found that significant changes have taken place in gastric cancer cell line MGC-803, BGC-823 and AGS cells treated with Resveratrol at 100μΜ after 48h; the growth of MGC-803 was inhibited and A small amount of apoptosis; the growth of BGC-823 and AGS was inhibited and induced apoptosis; The cytoplasm in AGS was narrowing and confluent to some big cells. Before and after 48 h, the morphological in gastric cancer cell line MGC-803, BGC-823 and AGS have changed in 80μΜ AG490, reducing the number.(2) MTT assay showed that Resveratrol concentration gradient treatment of gastric cancer cell line MGC-803, BGC-823 and AGS 48 h, the proliferation and activity of the cells was negatively correlated with the concentration of Resveratrol, which is more sensitive to BGC-823 and AGS.(3) FCM test results found that: In gastric cancer cell line MGC-803 and BGC-823 and AGS after 100μM Resveratrol treated for 48 hour, the cell cycle blocked in G1 phase, and all had different degrees of apoptosis. In MGC-803 and BGC-823 after 80μM AG490 treated for 48 hour, cell cycle blocked in the G1 phase, and had different degrees of apoptosis. In AGS after 80μM AG490 treated for 48 hour, cell cycle blocked in S phase, and there was a certain growth inhibition. In general, gastric cancer cell line MGC-803, BGC-823 and AGS for Resveratrol and AG490 had certain sensitivity: MGC-803 for Resveratrol had less sensitivity, BGC-823 and AGS for Resveratrol showed a trend of more sensitive, MGC-803 and BGC-823 and AGS for AG490 had more sensitivity.(4) ICC showed that Resveratrol treatment after 100μΜ 48 h, in gastric cancer cell line MGC-803 downregulation of STAT3, and p-STAT3 expression did not change significantly, SOCS3 upregulation, PIAS3 downregulated, p-SHP2 no significant change; In BGC-823, STAT3 and p-STAT3 were downregulated, negative regulator of p-SHP2 and PIAS3 were upregulated, whereas no significant change SOCS3. AGS cell lines, in addition to reduced SOCS3 expression were no other significant changes.(5) Western blotting showed that Resveratrol treatment after 100μΜ 48 h, the expression of STAT3 in gastric cancer cell line MGC-803 in slightly lower, p-STAT3 and p-SHP2 no significant change. In AGS, STAT3 and p-STAT3 were no significant changes, and p-SHP2 downregulated, but no significant change; In BGC-823, STAT3 and p-STAT3 were downregulated, negative regulator of p-SHP2 no significant change.(6) RT-PCR showed that Resveratrol was treated 100μΜ gastric cancer cell line MGC-803, BGC-823 and AGS after 48 h, MGC-803 and BGC-823 downregulation of STAT3, AGS in STAT3 slightly down; negative in MGC-803 in no significant changes in regulatory factors SHP2, SOCS3 has increased, while PIAS3 downregulation; significantly increased negative regulator of SHP2 and PIAS3 expressed in BGC-823, SOCS3 no significant change; in the negative regulation of AGS and PIAS3 no obvious factor SHP2 change, and downregulation of SOCS3. Conclusions(1) Resveratrol can inhibit the growth of gastric cancer cell line MGC-803, BGC-823 and AGS cells and promote apoptosis of BGC-823 and AGS.Cells were arrested in G1 phase.(2) BGC- 823 and MGC- 803 cell line of RES sensitivity difference may be related to degree of inhibition of STAT3 signaling pathways.(3) Resveratrol, AG490 processing degree of BGC- 823 cell apoptosis, resveratrol may exist many targets.(4) AG490 inhibits the growth of gastric cancer cells by inhibiting the activation of STAT3, downregulate p-STAT3.(5) Different gastric cancer cell line sensitivity to Resveratrol is not entirely consistent with the state of STAT3 activation pathway, suggesting that there may be more cell signaling pathway synergistically in Resveratrol on gastric cancer cell proliferation and apoptosis, and gastric cancer individualized treatment with a necessity.