2005;5:1980C1986

2005;5:1980C1986. reactivity to the CD151 antigen but also prevented tumor progression in HCC. Keywords: hepatocellular carcinoma, tetraspanin CD151, integrin 61, monoclonal antibody, therapeutical agent INTRODUCTION Hepatocellular carcinoma (HCC) is the fifth most common malignant neoplasm worldwide [1]. Disease outcomes have been attributed to (1) early distant metastasis and high rates of recurrence after intervention [2] and (2) a lack of effective and MP470 (MP-470, Amuvatinib) curative interventions [3, 4]. An increased understanding of the molecular mechanisms underlying this disease may give us insight into potential targets for developing new preventive and therapeutic options for HCC [5]. Tetraspanin CD151, one of the most important members of the tetraspanins, has been identified as an important player in physiological processes [6] and the progression of malignant tumors [7], including skin [8], pancreas [9], lung [10], colon [11] and breast tumors [12, 13]. Consistent with relevant reports, MP470 (MP-470, Amuvatinib) our serial studies implicated MP470 (MP-470, Amuvatinib) CD151 in several pathological processes, including tumor cell mobility [14], tumor neo-angiogenesis [15] and epithelial-mesenchymal transition (EMT) [4] in HCCs. A distinct feature of CD151 is its ability to self-assemble or associate with other transmembrane molecules to form tetraspanin-enriched microdomains (TEM) [6]. Among the associated partners, integrins are the principal group of proteins that interact with tetraspanin CD151 [6]. Previously, we also identified a group of partners that associated with CD151 in the HCC cell line HCCLM3 [16]. Additionally, we validated the role of the CD151/integrin 61 complex in the progression of HCC [16]. Therefore, both CD151 and CD151-enriched microdomains appears to be promising targets in the treatment of HCC [17]. However, simple inhibition of CD151 in HCC is evidently inappropriate because CD151 plays essential roles in normal physiological processes, including cell adhesion, motility, activation and proliferation [6, 18C20]. Based on the above evidence, the dissociation of CD151-depedent TEM could be an effective strategy for inhibiting CD151’s tumor-promoting abilities without disrupting its physiological functions [17]. Molecular targeted therapies have provided researchers with a broader perspective on the management of cancer, including colorectal and non-small cell lung cancers [21, 22]. Clinical trials have demonstrated that sorafenib, a multikinase inhibitor against Raf-1, B-Raf, VEGFR2, PDGFR and c-Kit receptors, improves progression-free survival in advanced HCC [23]. The benefit derived from sorafenib for advanced HCC patients is significant, but the drug prolongs mean survival by only 3 months compared with placebo [23]. Moreover, adverse events such as diarrhea, fatigue, weight loss, and hand-food skin reactions are frequent and limit the use of sorafenib for HCC patients. Therefore, it is important to develop alternative monoclonal antibodies for HCC [24]. In this study, we generated a CD151 mAb 9B (IgG1, ) against the CD151/integrin 61-binding domain and determined its bioactivity in HCC cells. RESULTS Characterization of CD151 mAb 9B A mouse anti-human CD151 mAb against the CD151/integrin 61 binding site [25] (Figure ?(Figure1A,1A, QRD194-196 site) was successfully produced and designated as CD151 mAb 9B. SDS-PAGE and Coomassie brilliant blue revealed two bands, including a light chain at 28 kDa and a heavy chain at 52 kDa (Figure ?(Figure1B).1B). The immunoisotype of CD151 mAb 9B was identified as IgG1 (Figure ?(Figure1C).1C). Western blotting using CD151 mAb 9B as the first antibody showed a band at 28 kDa in both HCCLM3 cell lysates and recombinant CD151 proteins (Figure ?(Figure1D).1D). A co-immunoprecipitation (IP) assay also showed that CD151 mAb 9B effectively immunoprecipitated CD151 protein as efficiently as the anti-CD151 antibody purchased from Abcam, which recognizes a different epitope of CD151. Interestingly, the integrin 6 protein cannot be detected Rabbit Polyclonal to DHPS by Western blotting from immuno-complex mixtures immunoprecipitated by CD151 mAb 9B, whereas integrin 6 can be detected from immuno-complex mixtures immunoprecipitated by the other anti-CD151 antibody. These results may indicate that CD151 mAb 9B competitively binds to the epitope through which integrin 6 binds to CD151 (Figure ?(Figure1E).1E). It was confirmed in our previous study that CD151 forms a complex with integrin 6, inducing Akt signaling. The results of MP470 (MP-470, Amuvatinib) the current study showed that treatment with 0.2 mg/ml of CD151 mAb 9B significantly decreased p-Akt expression in HCCLM3 cells (Figure ?(Figure1F1F). Open in a separate window Figure 1 Characterization of CD151 mAb 9BA. Schematic representation of the anti-human CD151 mAb 9B against the CD151/integrin 61-binding site. B. SDS-PAGE and Coomassie brilliant blue for the anti-human CD151 mAb 9B. C. The immunotype of CD151 mAb 9B. D. Western blot analysis for CD151 in HCCLM3.