Characterization of a new monoclonal anti-glypican-3 antibody specific to the hepatocellular carcinoma cell line, HepG2

Abstract

AIMTo characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan (HSPG), clone 1E4-1D9.METHODSThe antigen recognized by mAb 1E4-1D9 was immunoprecipitated and its amino acid sequence was analyzed LC/MS. The transmembrane domain, number of cysteine residues, and glycosylation sites were predicted from these entire sequences. Data from amino acid analysis was aligned with glypican-3 (). The competitive reaction of mAb 1E4-1D9 and anti-glypican-3 on HepG2 cells was demonstrated by indirect immunofluorescence and analyzed by flow cytometry. Moreover, co-immunoprecipitation of mAb 1E4-1D9 and anti-glypican-3 was performed in HepG2 cells by Western immunoblotting. The recognition by mAb 1E4-1D9 of a specific epitope on solid tumor and hematopoietic cell lines was studied using indirect immunofluorescence and analyzed by flow cytometry.RESULTSMonoclonal antibody 1E4-1D9 reacted with an HSPG isolated from human liver and a band of 67 kD was detected under both reducing and non-reducing conditions. The specific antigen pulled down by mAb 1E4-1D9, having a MW of 135 kD, was analyzed. The results showed two sequences of interest, gi30722350 (1478 amino acid) and gi60219551 (1378 amino acid). In both sequences no transmembrane regions were observed. Sequence number gi30722350 was 99.7% showed a match to FYCO1, a molecule involved in induction of autophagy. Sequence number gi60219551 contained 15 cysteines and 11 putative glycosylation sites with 6 predicted N-glycosylation sites. It was also matched with all PDZ domain proteins. Moreover, it showed an 85.7% match to glypican-3. Glypican-3 on HepG2 cells competitively reacted with both phycoerythrin-conjugated anti-glypican-3 and mAb 1E4-1C2 and resulted in an increase of double-stained cell population when higher concentration of mAb 1E4-1D9 was used. Moreover, antigens precipitated from HepG2 cell by anti-glypican-3 could be detected by mAb 1E4-1D9 and vice versa. The recognition of antigens, on other solid tumor cell lines, by mAb 1E4-1D9 was studied. The results demonstrated that mAb 1E4-1D9 reacted with Huh7, HepG2, HT29, MCF7, SW620, Caco2, B16F1, U937, K562 and Molt4 cells. It was also found to be weakly positive to SW1353 and HL60 and negative to H460 and Hela cell lines.CONCLUSIONAll findings show that mAb 1E4-1D9 specifically recognizes glypican-3. Moreover, a new partner molecule of glypican-3, FYCO1 is proposed based on the results from co-precipitation studies.

Association between microRNA‑527 and glypican‑3 in hepatocellular carcinoma

“…Similar to the HCC tissues, several HCC cell lines are reported to express GPC-3 ( 43 ). The expression of GPC-3 was confirmed in several HCC cell lines, including the Huh-7 cell line ( Fig.…”

“…Hence, the pathophysiology of HCCs is not well understood. Previous studies have reported the molecular mechanisms of miRNA in the carcinogenic process ( 42 , 43 ). miRNAs regulate gene expression, and one miRNA is estimated to regulate >200 target genes ( 5 , 6 ).…”

A Novel Strategy for Screening Tumor-Specific Variable Domain of Heavy-Chain Antibodies

“…In addition to phage binding towards tumor-specific cells, HepG2 cells showed partial positive binding. These partial bindings could be considered insignificant because many studies have reported that partial binding between HepG2 and heterologous monoclonal antibodies often occurs, as the HepG2 cell line does not have specific cancer markers and shares some biological surface proteins expressed in most normal epithelial tissues, lymphatic tissues, and cancer stem cells, including cells of HNSCC, breast, colon, liver, ovarian, pancreatic, and gastric cancer [ 33 , 34 , 35 , 36 ].…”

The RUFYs, a Family of Effector Proteins Involved in Intracellular Trafficking and Cytoskeleton Dynamics

“…So far, no correlation has been found between RUFY4 and any type of cancer. FYCO1 has also been implicated in colorectal cancer progression ( Sillars-Hardebol et al, 2010 ) and recent studies have concluded that FYCO1 may serve as a biomarker in bladder cancer ( Eissa et al, 2017 ) or hepatocellular carcinoma ( Vongchan and Linhardt, 2017 ; Figure 4C ). Plus, FYCO1 can indirectly associated with cell invasion ( Pedersen et al, 2020 ).…”