Sodium orthovanadate overcomes sorafenib resistance of hepatocellular carcinoma cells by inhibiting Na+/K+-ATPase activity and hypoxia-inducible pathways

Abstract

The resistance to sorafenib highly affects its clinical benefits for treating hepatocellular carcinoma (HCC). Sodium orthovanadate (SOV) is a phosphate analog that displays anti-cancer activities against various types of malignancies including HCC. The present study has demonstrated that SOV is able to overcome sorafenib resistance and strengthens sorafenib in suppressing sorafenib-resistant HCC cells in vitro and in animal models. Similar to its action on parental HCC cells, SOV induced cell cycle arrest at G2/M phases by regulating cyclin B1 and cyclin-dependent kinase 1, and apoptosis by reducing mitochondrial membrane potential, in sorafenib-resistant HCC cells. More importantly, SOV inhibited ATPase activity, which was significantly elevated in sorafenib-resistant HCC cells. SOV also reduced the expression of HIF-1α and HIF-2α and their nuclear translocation, resulting in downregulation of their downstream factors including vascular endothelial growth factor, lactate dehydrogenase-A and glucose transporter 1. Its ability to inhibit ATPase activity and hypoxia-inducible pathways enabled SOV to efficiently suppress both normoxic and hypoxic cells, which compose cancer cell populations inside sorafenib-resistant HCC tumors. The present results indicate that SOV may be a potent candidate drug for overcoming the resistance to sorafenib in treating HCC.

Figure 1

Increased ATPase activity contributes to sorafenib resistance of HCC cells. (a) HepG2, HepG2-SR, Huh7 and Huh7-SR cells were lysed for measuring ATPase activity, which was represented by the amount of phosphate release from cells by using malachite green reagent. (b,c) The expression of Na⁺/K⁺-ATPase α3 subunit protein in the above cells was detected by immunoblotting (b) and immunocytochemistry (c). (d,e) HepG2-SR and Huh7-SR cells were transfected with control siRNA or siRNA targeting the α3 subunit. Cells were harvested 48 h later, and then subjected to immunoblotting (d) and ATPase activity assays (e). (f) Cells transfected with control siRNA or siRNA targeting the α3 subunit were incubated for 48 h in the presence or absence of sorafenib (5 μM). Cell viability (%) was measured. The density of each immunoblotting band was normalized to β-actin. “*” Indicates P < 0.05, and “**” P < 0.001.

Figure 2

Sodium orthovanadate (SOV) reduces ATPase activity in sorafenib-resistant HCC cells. (a) HepG2-SR and Huh7-SR cells were incubated for 30 min with SOV at various concentrations. Cells were harvested and lysed, and the amounts of phosphate release were measured. (b,c) Cells were incubated with vehicle or SOV at 5 μM for 6, 12 or 24 h, and then stained with K⁺ fluorescent dye PBFI-AM. (b) Fluorescence intensity was measured. (c) Representative images were taken from PBFI-AM-stained cells incubated with vehicle or SOV (5 μM) for 24 h. (d,e) Cells were incubated with 5 μM of SOV for 24 h, and then subjected to qRT-PCR for measuring the expression of Na⁺/K⁺-ATPase α3 subunit mRNA (d) and immunoblotting for its protein expression (e). The density of each immunoblotting band was normalized to β-actin. “N.S.” indicates no significance. “*” (P < 0.05) and “**” (P < 0.001) vs. vehicle-treated cells.

Figure 3

SOV inhibits the proliferation of HCC cells. HepG2, HepG2-SR, Huh7 and Huh7-SR cells were incubated for 48 h with various concentrations of SOV. (a) Cell viability (%) was compared with respective untreated cells. With a logarithmic regression analysis, the values of IC₅₀ for each cell type were calculated. (b) HepG2-SR and Huh7-SR cells treated with SOV at concentrations of 0, 5 and 10 μM were cytometrically analyzed for determining cell cycle distribution, and representative histograms are shown. (c) The percentages of cells arrested at G2/M phases were plotted. (d) Cells were subjected to immunoblotting. The density of each immunoblotting band was normalized to β-actin. “*” (P < 0.05) and “**” P < 0.001 vs. vehicle-treated cells.

Figure 4

SOV induces apoptosis of sorafenib-resistant HCC cells. HepG2-SR and Huh7-SR cells were incubated for 48 h with various concentrations of SOV. (a) Apoptosis rates (%) were measured, and representative histograms of cytometrically analyzed cells are shown. (b) Representative images were taken from cells stained with Annexin V/propidium iodide. (c) Cells were subjected to immunoblotting. The density of each immunoblotting band was normalized to β-actin. (d) Cells were analyzed for measuring the activity of caspase-3. “*” (P < 0.05) and “**” (P < 0.001) vs. vehicle-treated cells.

Figure 5

SOV inhibits the expression of HIF-1α and HIF-2α proteins and their nuclear translocation in sorafenib-resistant HCC cells. (a) HepG2-SR and Huh7-SR cells were incubated with SOV (0, 5 or 10 μM) under hypoxia (1% O₂) for 24 h. Cells were lysed and immunoblotted. Band density was normalized to β-actin. (b,c) Huh7-SR cells were incubated with SOV (0, 5 or 10 μM) under normoxia or hypoxia (1% O₂) for 24 h, and then subjected to immunoblotting (b). (c) Hypoxic Huh7-SR cells were subjected to qRT-PCR for detecting the expression of mRNAs. The level of mRNA from untreated cells was defined as 1. (d) Huh7-SR cells incubated with vehicle or SOV (10 μM) under hypoxia (1% O₂) for 24 h, and then immunostained with Abs against HIF-1α (red), HIF-2α (green) and DAPI (cellular nuclei, blue). (e) The nuclear and cytoplasmic fractions of vehicle- or SOV (10 μM)-treated hypoxic Huh7-SR cells were immunoblotted. The band density of HIF-1α or HIF-2α protein in nuclear fractions was normalized to ARNT, and that in cytoplasmic fractions, β-actin. (f) The ratio of HIF-1α or HIF-2α protein in nuclear/cytoplasmic fractions was calculated. “*” (P < 0.05) and “**” (P < 0.001) indicate a significant difference from respective vehicle-treated cells.

Figure 6

SOV synergizes with sorafenib to inhibit the proliferation and induce the apoptosis of hypoxic sorafenib-resistant HCC cells. (a) Huh7-SR cells were incubated for 24 h with serial concentrations of SOV (0, 1, 2, 4 or 8 μM) in the absence or presence of sorafenib (2.5 μM) under normoxia or hypoxia (1% O₂) for 24 h. Cell viability was assessed and the inhibitory rate (%) was calculated. (b,c) Huh7-SR cells were incubated for 24 h with vehicle, or sorafenib (2.5 μM), or SOV (2 μM), or the combination of sorafenib and SOV under normoxia or hypoxia (1% O₂). Cells were subjected to flow cytometry for analyzing apoptosis (b) and immunoblotting (c). Band densities were normalized to β-actin. “*” Indicates P < 0.05, and “**” P < 0.001. ^“#” (P < 0.05) and ^“##” (P < 0.001) indicate a significant reduction, ^“ϕ” (P < 0.05) and ^“ϕϕ” (P < 0.001), a significant increase, from respective vehicle-treated cells.

Figure 7

SOV strengthens sorafenib in suppressing sorafenib-resistant tumors in vivo. (a) Animal experimental schedule was described in Materials and Methods. (b) The bodyweights of mice were monitored. (c) The size (mm³) of tumors was recorded. (d) Tumors harvested at the end of experiments were weighed and representative tumors photographed. “*” Indicates P < 0.05, and “**” P < 0.001.

Figure 8

A schematic diagram of proposed mechanisms by which sodium orthovanadate displays its ability to enhance the effects of sorafenib against sorafenib-resistant HCC cells. Abbreviations: ARNT, aryl hydrocarbon receptor nuclear translocator; CDK1, cyclin-dependent kinase 1; HIF-1α, hypoxia-inducible factor-1α; HIF-2α, hypoxia-inducible factor-2α; GLUT1, glucose transporter 1; HREs, hypoxia-response elements; LDHA, lactate dehydrogenase-A; PARP, poly (ADP-ribose) polymerase; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.