<p>Hypoxia-Induced Aquaporin-3 Changes Hepatocellular Carcinoma Cell Sensitivity to Sorafenib by Activating the PI3K/Akt Signaling Pathway</p>

Abstract

Purpose: Hypoxia-induced changes are primarily activated in patients with hepatocellular carcinoma (HCC) and long-term sorafenib exposure, thereby reducing the sensitivity to the drug. Aquaporin-3 (AQP3), a member of the aquaporin family, is a hypoxia-induced substance that affects the chemosensitivity of non-hepatocellular tumors. However, its expression and role in the sensitivity of hypoxic HCC cells to sorafenib-induced apoptosis remain unclear. The purpose of this study was to detect changes in AQP3 expression in hypoxic HCC cells and to determine whether these changes alter the sensitivity of these cells to sorafenib. Materials and Methods: Huh7 and HepG2 hypoxic cell models were established and AQP3 expression was detected using quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Furthermore, the role of AQP3 in cell sensitivity to sorafenib was evaluated via flow cytometry, Western blotting, and a CCK-8 assay. Results: The results of qPCR and Western blotting showed that AQP3 was overexpressed in the Huh7 and HepG2 hypoxic cell models. Furthermore, AQP3 protein levels were positively correlated with hypoxia-inducible factor-1α (HIF-1α) levels. Compared with cells transfected with lentivirus-GFP (Lv-GFP), hypoxic cells transfected with lentivirus-AQP3 (Lv-AQP3) were less sensitive to sorafenib-induced apoptosis. However, the sensitivity to the drug increased in cells transfected with lentivirus-AQP3RNAi (Lv-AQP3RNAi). Akt and Erk phosphorylation was enhanced in Lv-AQP3-transfected cells. Compared with UO126 (a Mek1/2 inhibitor), LY294002 (a PI3K inhibitor) attenuated the AQP3-induced insensitivity to sorafenib observed in hypoxic cells transfected with Lv-AQP3. Combined with LY294002-treated cells, hypoxic cells transfected with Lv-AQP3RNAi were more sensitive to sorafenib. Conclusion: The study results show that AQP3 is a potential therapeutic target for improving the sensitivity of hypoxic HCC cells to sorafenib.

Sorafenib-induced Aquaporin-3 Downregulation is Coupled With Proliferation Inhibition and Increased Apoptosis in Hepatocellular Carcinoma Cells

“…Sorafenib and UO126 (Sigma-Aldrich, St. Louis, Missouri, USA) were purchased and dissolved in 100% DMSO to prepare a 10mM stock solution, which was then diluted with DMEM to the desired concentration, with a nal concentration of 0.1% DMSO, recommended for in vitro studies. Unless otherwise stated, all antibodies were obtained from Life Span BioSciences (LSBIO, Seattle, Washington, USA) 32 . The enhanced chemiluminescence detection kit was purchased from Amersham Pharmacia Biotech-UK.…”

“…Protein concentration was determined via the bicinchoninic acid protein assay as per the Bio-Protocol (bioprotocol, Philadelphia, Pennsylvania, USA). An equivalent of 30μg of the protein extract was resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro transferred (wet) to polyvinylidene di uoride membranes (EMD Millipore Corporation, Billerica, Massachusetts, USA) 32 . The membranes were initially blocked with 5% BSA in TBST (137mM NaCl, 20mM Tris HCl [pH 7.6], and 0.1% [v/v] Tween 20) for 1 h, followed by incubation overnight at 4°C with primary antibodies (1:1000) against AQP3, Erk, p-Erk, Akt, p-Akt, p53, p-p53, p21, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and GAPDH/beta-tubulin (as loading controls).…”

“…The membranes were washed with PBS 3x each 5 min and then incubated with Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) at room temperature for 1 h. Immunodetection was performed using an ECL Western Blotting Detection Kit (Beyotime, Shanghai, China). The relative protein expression levels were quanti ed by densitometric measurement of ECL reaction bands and normalized to GAPDH/beta-tubulin levels 32 .…”

“…GAPDH F, 5 -GATCATCAGCAATGCCTCCT-3 and R, 5 -GAGTCCTTCCACGATACCAA-3 . The data have been deposited in a publicly accessible database (GenBank) with accession number NM_004925.5 and NM_002046.7 for AQP3 and GAPDH, respectively 32 .…”

Effect of the Hypoxia Inducible Factor on Sorafenib Resistance of Hepatocellular Carcinoma

“…In addition, Yeh et al showed that galectin-1 is elevated in sorafenib-resistant HCC cells, both in vitro and in vivo, promoting tumor metastasis and increasing tumor invasion, suggesting that galectin-1 plays a role in downstream regulation of the AKT/mTOR/HIF-1 signaling pathway (113). Hypoxia induces overexpression of AQP3 in hypoxic HCC cells, thereby altering sensitivity of these cells to sorafenib by activating the PI3K/Akt signaling pathway (114).…”