Antitumor Effects Of The Intrabody Against COX-2

Hepatocellular carcinoma,(HCC)is a serious threat to human health.There is no effective treatment for HCC now because it's hard to diagnosis at early stage and easy to relapse after surgery.Cyclooxygenase-2(COX-2)is a key enzyme in the synthesis of prostaglandins.As an inflammatory factor,COX-2 plays a very important role in inflammation and cancer.Studies have shown that COX-2 is overexpressed in HCC,and COX-2 inhibitors exert chemopreventive and therapeutic effects in HCC.Therefore COX-2 is a potential target for HCC therapy.However,traditional COX-2 inhibitors,such as NSAIDs,have side effects of hurting gastrointestinal function,and selective COX-2 inhibitors present high cardiovascular risk.So it is important to explore a specific and safe inhibitory strategy to COX-2 in anti-COX-2 cancer therapy.Intracellular antibodies(intrabodies)have shown a great promise and potential in anti-cancer therapy due to their high binding specificity and stability.Therefore,our previous studies have constructed an eukaryotic expression plasmid pBg-Intra-OX-1 coding an endoplasmic reticulum-retained anti-COX-2 intrabody.In this study,pBg-Intra-OX-1 was transfected into HepG2 cells(HCC cell line),to detect its expression and anti-tumor effects in HCC cells.The results are as follows:pBg-Intra-OX-1 was transfected into HepG2 cells,and a stable transfected HepG2 cell line expressing Intra-OX-1 was obtained.Then expression and subcellular distribution of Intra-OX-1 was detected by RT-PCR,Western-blot,and immunofluorescence staining analysis.Co-immunoprecipitation assay was conducted to verify the intracellular activity of Intra-OX-1 and the results showed that the Intra-OX-1 could bind effectively to COX-2 in HepG2 cells.the colony formation ability was measured by soft agar colony formation assay for cell growth and proliferation analysis.The cell apoptosis changes of the stable transfected cells were detected by Flow Cytometry.The results demonstrated that Intra-OX-1 inhibited HepG2 cells growth and proliferation,but promoted cell apoptosis.Through wound healing assay and transwell assay,we also found that Intra-OX-1 inhibited the migration and invasion of HepG2 cells.In order to investigate the molecular mechanism of Intra-OX-1,real-time Q-PCR was used to detect mRNA expression of cancer-related genes in HepG2 cells with or without Intra-OX-1.The results showed that Intra-OX-1 down-regulated CDK1,CDK2,CDK4 mRNA expression levels,and up-regulated p21 mRNA expression level in HepG2 cells,these changes may be associated with Intra-OX-1-inducing cell proliferation inhibition.At the same time,Intra-OX-1 can down-regulated mRNA levels of several cancer-related genes,such as EGFR,VEGF,C-Met,gp130,IL-6,stat3.These results suggest that the expression of anti-COX-2 introbody,Intra-OX-1,inhibits HepG2 cells growth,proliferation,invasion and migration,promotes cell apoptosis.Intra-OX-1 shows a good anti-tumor effect.This study provides experimental evidence and a sound basis for anti-COX-2 HCC gene therapy.