Poly(I:C) And Poly(dA:dT) Induce IFN-β Production In SNB19Glioma Cells

Glioma is the most common neoplasm of the central nervous system, and one of the mostmalignant tumors. Despite comprehensive treatment with the combination of surgery,chemotherapy and radiotherapy, its prognosis remains extremely poor. In the field of novelcancer therapies, immunotherapy has accumulated vast amount of laboratory endeavours anddata of preclinical investigations. Of the latest advances in the research of cancerimmunothrapy, activation of pattern recognition receptors, which are the outpost of theimmune system sensing pathogen-associated molecular patterns (PAMPs) activating the entireimmune system, has exhibited its potential of translating anti-infection immune response intoanti-tumor immune response using artifical mimicks of PAMPs. Poly (I:C) is a type ofsynthetic double stranded RNA intensively studied in cancer immunotherapies through theabove-mentioned strategy. Poly (I:C) was previously believed to activate the patternrecognition receptor TLR3/TRIF signalling pathway in the immune cells and the cancer cellsto stimulate the production of a variety of kinds of cytokines and chemokines and thusactivate the immune system directed against the cancer cells. Recent studies havedemonstrated evidences suggesting the role of pattern recognition receptor RIG-I and MDA-5in the poly (I:C) based immunotherapy. These facts have added complexity to these effectsamong different types of cancers, and in the study of glioma therapies, the function andmechanisms of Poly (I:C) has not been clearly addressed. Therefore, we investigated thisissue beginning with the identification of TLR3expression in the glioma cell lines. Withdetection by flowcytometry, we found that in glioma cell line SNB19, U251, U87, SHG44,LN-118and U118MG, the expression of TLR3exhibited an intracellar distrbution. For thisreason, we transfected Poly (I:C) with lipofectamine2000into the cancer cells. Forcomparison,2additional dsRNA (5’ppp-dsRNA，Poly(A:U)) and6dsDNA (ISD，HSV-60Naked，VACV-70Naked，Poly (dG:dC)-Poly (dG:dC)，Poly (dA:dT)-Poly (dA:dT)，Poly(dA:dT)) were selected to stimulate a variety of cancer cells lines, and detected the levelof expression of IFN-β mRNA. As a result, Poly (I:C) widely stimulated the expression of IFN-βmRNA in all tested cancer cell lines, and its efficacy was significantly higher than theother two dsRNA. Notably, in the series of dsDNAs, we found Poly (dA:dT) to possess thesimilar ability compared with Poly (I:C), and both of them exhibited dose-dependentstimulation of IFN-β mRNA expression in SNB19glioma cells, which was confirmed on theprotein level by ELISA. Furthermore, in a dose dependent manner, Poly (I:C) and Poly(dA:dT) stimulated the mRNA expression of IL-6, TNF-a, CXCL10and CCL5, which alsosuggested their comprehensive role in the glioma immunotherapy. In order to illustrate thepossible underlying mechanism, we selected the possible molecular target including TRIF(TLR3pathway), RIG-I and IPS-1(RIG-I/IPS-1pathway) and STING (STING pathway).Downregulatioin of RIG-I and IPS-1mRNA expression significantly interfered with Poly (I:C)and Poly (dA:dT) stimulation of IFN-β mRNA expressioin in SNB19cells, whiledownregulation of TRIF and STING did not result in a signifant effect. These resultssuggested that the possible role of RIG-IPS-1pathway, instead of TLR3-TRIF pathway, inPoly (I:C) and Poly (dA:dT) stimulated immunogenic effect in glioma cells. Furthermore, wealso detected the inhibitory effect of Poly (I:C) and Poly (dA:dT) on SNB19cells. Whentransfected with Poly (I:C) or Poly (dA:dT), cell growth of SNB19cells were significantlyinhibited after24h under microscope observation, which was quantified and confirmed byMTT. With flowcytometry, we detected obvious effect of apoptosis induction with Poly (I:C)and Poly (dA:dT) in SNB19cells，which positively correlated with dose and duration of agentadministration. Cell cycle analysis determined cell cycle block at S phase. Importantly,growth inhibition and apoptosis induction were achieved with intracellar stimulation, whilesitmulation without the facilitation of lipofectamine transfection failed to produce anysignificant effect. In conclusion, sitmulation of pattern recognition receptors with double-stranded nucleotide PAMPs such as Poly(I:C) and Poly(dA:dT) is a promising strategy inglioma immunotherapy.