Glypican-3 (GPC3) is a member of the family of heparan sulfate proteoglycans [25, 26]. Heparan sulfate proteoglycans (HSPGs) have profound effects on both tumor cell growth kinetics and metastasis formation . HSPGs have the functional capacity to regulate a myriad of molecular interactions that induce tumor cell proliferation and metastasis . Glypican-3 expression is higher in several tissues, such as the gastrointestinal tract, in human embryos .
In different types of tumors, GPC3 expression and its carcinogenic role are variable. In some tissues, such as ovary [19, 30], breast [20, 31] and lung (adenocarcinoma) , this glypican is downregulated, acting as a tumor suppressor, whereas in other tumors, it is overexpressed and functions as an oncoprotein, as observed in liver [33–35], lung (squamous cell carcinoma) [32, 36], melanoma  and embryonal tumors . In a recent study, Gailey and Bellizzi  analyzed GPC3 protein expression in squamous cell carcinoma (SCCs) of diverse anatomic sites and in invasive urothelial carcinomas from the urinary bladder. They observed GPC3 staining was present in 17.3% of 156 tumors, including those of the anus (10.0%), cervix (27.3%), esophagus (28.6%), larynx (30.0%), lung (50.0%), tongue base/tonsil (12.5%), urinary bladder (12.2%), ventral tongue/floor of mouth (12.5%) and vagina (40.0%).
In the present study, GPC3 expression was downregulated in primary clear cell renal cell carcinoma samples and cell lines. To the best of our knowledge, this report is the first that has detected downregulation of the GPC3 gene in clear cell renal cell carcinoma cell lines. Okon et al.  have shown a point of distinction between GPC3 expression in chromophobe carcinoma and clear cell renal cell carcinoma; GPC3 expression was upregulated in chromophobe carcinoma and downregulated in clear cell carcinoma, agreeing with our expression results in tissue samples and cell lines.
Gailey and Bellizzi  also observed an absence of GPC3 expression in squamous cell carcinomas (SCCs) from the penis, skin and vulva. As opposed to the results of this study in clear cell renal cell carcinoma, GPC3 overexpression has emerged as a positive marker in liver cancer because it is highly expressed in 70-100% of hepatocellular carcinomas (HCCs) but not in normal adult liver tissue [33–35]. In addition to being proposed as a marker for liver tumor diagnosis, GPC3 has also been evaluated as a target for antibody- and cell-based therapies of HCC .
Cell growth was reduced in cells overexpressing GPC3 protein, as measured by colony formation and proliferation rates 48 h post-transfection in both the 786-O and ACHN cell lines used in this study. Lin et al.  and Murthy et al.  found that GPC3 re-expression in ovarian cancer cell lines resulted in inhibition of the efficiency of colony formation. Some studies have shown that GPC3 plays an important role in cell growth and differentiation [41, 42], such as in the case of hepatocellular carcinoma . The data obtained in the present study suggest that GPC3 inhibits cell proliferation in clear cell renal cell carcinoma.
Therefore, we studied whether the reduced proliferation rate in cells overexpressing GPC3 occurs through the induction of apoptosis or through cell cycle arrest. Apoptosis, or programmed cell death, is a crucial point in the carcinogenic process. Cancer cells can overcome the apoptosis mechanism, and tumor progression continues [26, 44]. We observed that most cells, whether overexpressing or lacking GPC3, were viable at all time points in ACHN and 786-O cell lines. Our results suggest that cells overexpressing GPC3 in renal cell carcinoma do not induce apoptosis; therefore, we hypothesized that the inhibition of cell proliferation in renal carcinoma cells might occur due to cell cycle arrest.
To verify whether GPC3 overexpression arrests the cell cycle in renal cell carcinoma, we used flow cytometry to perform cell cycle analysis. The observed growth-repressive effect of GPC3 overexpressing cells was reflected by their arrest in the G1 cell cycle phase, which caused these cells to proliferate less in both cell lines. In this study, we observed changes in cell proliferation through the use of MTT assays in the ACHN cell line 48 h post-transfection. Using flow cytometry, we were able to identify changes in the cell cycle and, consequently, in cell proliferation 24 h after transfection for the same cell line. This difference is likely due to the increased sensitivity of the flow cytometry methodology to detecting changes in cell cycle and proliferation compared with the MTT assay. One previous study demonstrated that inhibiting GPC3 expression released hepatocyte carcinoma cells from G1 arrest and thus modulated cell cycle progression in this type of cancer .
Moreover, cell surface HSPGs inhibit invasion by promoting tight cell–cell and cell–extracellular matrix (ECM) adhesion. Previous studies documented diminished quantity and quality of heparan sulfate isolated from transformed cells compared with normal cells . This alteration in heparan sulfate accompanies a reduction in the adhesive capacity of transformed cells. Low levels of cell surface heparan sulfates also correlate with high metastatic activity in melanomas [47, 48]. Furthermore, GPC3 downregulation was more significant in invasive areas, a result which further supports an inhibitory role for GPC3 in tumor progression in hepatocellular carcinomas . Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders. Conversely, glypican-3 expression is decreased in human breast cancers, and ectopic expression of GPC3 inhibits growth of breast cancer cell lines . Additionally, glypican-3 was associated with the inhibition of invasion and metastasis of a mammary carcinoma cell line in vivo
. Thus, cells with normal epithelial morphology exhibit retention of cell surface HSPGs and tight attachment to the extracellular matrix. Cells that are beginning to invade exhibit reduced adhesion to the ECM, loss of epithelial morphology, and diminished levels of HSPG expression, whereas deeply invading cells completely lose HSPG expression .