| ObjectAccording to the investigation report of WHO, gastric carcinoma takes the second leading position of cancer-associated death worldwide nowadays. Besides of traditional methods, such as surgery, chemotherapy or radiotherapy, immunotherapy was considered to be a promising therapy against cancer. Although biotherapy for gastric carcinoma has been developed at bench, clinical practice at bedside is still limited by technical difficulties, higher cost or elaborate procedures. Therefore, there is urgent need for novel effective and systematical therapeutical strategies for gastriccarcinoma.As investigations demonstrated, PRRs (pattern recognition receptors) were not only expressed on immune cells, but also were expressed on nonparenchymal cells, including tumor cells, which indicate that PRRs may hand down other molecular events different with endogenous immune. Many researchers indicated that PRRs in tumor cells may play a "double blade sword" role in tumor progression. In different tumor cells and tissues, different PRRs may lead to different effect. Many PRRs could promote tumor progression, while others may suppress tumor development.TLR3, which expressed both in membrance and endosome, can recognize double strand RNA (dsRNA) and mimics polyinosinic-polycytidylic acid (poly(I:C)), and initiate anti-viral immunal response. Meanwhile, RLRs can activated by dsRNA and poly(I:C) either, along with the production of type I IFN. The effect of poly(I:C) in tumor immune therapy have been well demonstrated. Poly(I:C) was not only used as an immune adjuvant in clinical trials, but also triggering apoptosis of a kinds of cancers. However, the effect of poly(I:C) on gastric carcinoma have not been well explored. Our research aims to demonstrate the effect of poly(I:C) on human gastric adenocarcinoma cell lines and investigate related molecular mechanisms, which suggests a new approach based on TLRs for anti-tumor therapeutic application. MethodsFirst, the basic mRNA levels of TLR1-TLR10in AGS and BGC-823cells were determined by semi-quantitative RT-PCR. After treated with poly(I:C) for24h, the proliferation rates of AGS and BGC-823cells were measured by MTT assay, CCK-8assay and EdU incorporation assay, and NK cell cytolysis activity against AGS and BGC-823cells were analyzed by MTT assay, CFSE-7-AAD assay. Cellular total RNA was extracted, and cytokine mRNAs were detected by real-time PCR. FACS was used for analysis of the expression of TLR3, Cyclin D1and many cytolysis associated ligands on gastric cancer cells and the level of CD107a, perforin and TNF-a of NK cells.Second, we transfected poly(I:C) into AGS and BGC-823cells with LipofectamineTM2000, and determined cell apoptosis by Annexin V/PI staining. mRNA level of RLRs, type I IFN pathway associated key moleculars, proinflammatory cytokines were measured by real-time PCR. Western Blotting was used to analyze the activation of NF-κB. Cells were treated as anti-IFNR or added IFN-(3to determine the effect of IFN-β in pro-apoptotic effect. Subsequently, real-time PCR and FACS were used to analyze the expression of Bcl-2family. Finally, we established gastric tumor xenograft nude mice model to determine the supression growth effect on gastric adenocarcinoma of poly(I:C).At last, after transfection with poly(I:C), the cytolysis sensibility against NK cells and the expression of cytolysis-associated ligands were analyzed by MTT and FACS, respectively. Furthermore, after NK cells were incubated with the supernatant of BGC-823cells transfected with poly(I:C), proliferation and cytolysis activity against BGC-823were determined by MTT assay, migration were measured by transwell, as well as cytolysis associated recepotors and cytokines were analyzed by FACS.Results(1) When treated with poly(I:C), the proliferation rate of AGS and BGC-823cells were inhibited, accompanied with the decreased level of DNA replication, Cyclin Dl and the mRNA level of several cytokines; Meanwhile, the cytolysis activity of NK cell was increased, concomitant with increased secretion of CD107a, perforin and TNF-a.(2) We observed robust pro-apoptotic effect of poly(I:C) on gastric adenocarcinoma cells in vitro and inhibition of the growth of gastric adenocarcinoma significantly in gastric tumor xenograft nude mice model. This pro-apoptotic signaling was initiated not only from RIG-I and MDA5, but also from LGP2, whereas independent of type Ⅰ IFN. Further studies showed that Bcl-2family may be involved in the direct apoptosis pathway.(3) The cytolytic activity of NK cells against poly(I:C) treated gastric adenocarcinoma cells was augmented, concomitant with the increased expression of NKG2D ligand MICA/B and Fas. Furthermore, the supernatant of poly(I:C) treated gastric adenocarcinoma cells promoted the proliferation and migration, as well as the cytolytic activity of NK cells accompanied by up-regulation of activatory receptors and cytolysis associated molecules. The increased cytolytic activity of NK cells was mediated by type Ⅰ IFN secretion from poly(I:C) treated gastric adenocarcinoma cells.ConclusionDirectly added poly(I:C) into culture medium can suppress the proliferation of gastric adenocarcinoma cells and enhance the sensitivity of gastric adenocarcinoma cell to NK cell cytolysis. Meanwhile, transfected poly(I:C) into cytoplasm could both elicit direct pro-apoptotic effects on gastric adenocarcinoma, at the same time, enhance innate immune responses via improve tumor microenvironment and alter the tumor immunosuppression status. The multi-pronged effects poly(I:C) by combine the direct cytotoxic effect and effective immune actication effect suggests a new approach based on PRRs for anti-tumor therapeutic application. |
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