Factor, Jesper B. Andersen, Elizabeth A. Conner, Snorri S. Thorgeirsson Background/Aims: Cyclooxygenase-2 (COX-2) is a rate-limiting key enzyme catalyzing the conversion of arachidonic acid (AA) into prostaglandins (PGs). Compelling evidence has documented increased expression of COX-2 in various

human cancers including hepatocellular carcinoma (HCC). Therefore, selective blockage of COX-2 activity may represent an effective strategy for anti-cancer therapy. This study was designed to construct an intrabody against COX-2 and MK-1775 datasheet to determine its effect on HCC cell growth (intrabodies can be directed to intra-cellular compartments to neutralize and block the function of target proteins). Methods: A single-chain fragment of antibody variable region (scFv) against COX-2 (OX-1) was isolated see more by antibody phage display. And then an expression plasmid pIn-tra-OX1 harboring an endoplasmic reticulum (ER)-retained scFv gene against human COX-2 (Intra-OX1) was constructed. The activity of scFv was characterized by ELISA and immunopre-cipitation. The expression and subcellular distribution of Intra-OX1 in HepG2 cells were detected by RT-PCR, Western blot and fluorescence staining. Cell cycle and apoptosis were detected by flow cytometry. The antitumor efficacy of Intra-OX1 on HCC in vivo was assessed by intratumoral

injection of pIn-tra-OX1 to subcutaneous tumors in nude mice. Results: ELISA and immunoprecipitation showed that OX1 specifically bound to human recombinant COX-2 and endogenous COX-2 in HepG2 cells. OX1 inhibited the oxygenation of AA catalyzed by COX-2 enzymes. In HepG2 cells transfected with pIntra-OX1, Intra-OX1 was expressed efficiently and localized in the endoplasmic reticulum. Co-immunoprecipitation assay showed that Intra-OX1 in HepG2/pIntra-OX1 cells could recognize and bind to COX-2. Expression of Intra-OX1 inhibited PGE2 release from HepG2 cells. Compared with the control, the expression of Intra-OX1 significantly inhibited the growth of HepG2 cells, resulted

in cell cycle arrest in the 上海皓元 G0/G1 phase (P<0.01), and induced more cell apoptosis (P<0.01). Intra-OX1 also significantly suppressed the growth of subcutaneous tumors in nude mice in vivo after the intratumoral injection of pIntra-OX1. Expression of Intra-OX1 decreased the levels of EGFR, STAT3 in HepG2 cells. Conclusion: Anti-COX-2 intrabody inhibited HCC growth both in vitro and in vivo through blockage of COX-2 activity. Further studies are warranted to determine whether this approach can be utilized therapeutically for hepatocellular carcinoma. Disclosures: The following people have nothing to disclose: Yan Wu, An Cui, Xun Zhou, Wenhan Wang, Nannan Yao, Hanwei Li, Chang Han, Tong Wu, Guiying Li Background: A number of studies have reported crucial roles of cancer-associated fibroblasts (CAFs) in providing cancer cells with proliferative, survival, invasive and metastatic propensities favouring tumourigenesis.