Sodium orthovanadate inhibits growth of human hepatocellular carcinoma cells in vitro and in an orthotopic model in vivo

doi:10.1016/j.canlet.2014.05.018

Cancer Letters

Volume 351, Issue 1, 28 August 2014, Pages 108-116

https://doi.org/10.1016/j.canlet.2014.05.018 Get rights and content

Highlights

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Sodium orthovanadate (SOV) inhibited growth of the human HCC cells in vitro.
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SOV inhibited growth of the human HCC cells in an orthotopic model in vivo.
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SOV could regulate the proliferation, cell cycle and apoptosis of HCC cells.
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SOV could inhibit autophagy in HCC cells, which plays a prodeath role.

Abstract

The transition metal vanadium is widely distributed in the environment and exhibits various biological and physiological effects in the human body. As a well known vanadium compound, sodium orthovanadate (SOV) has shown promising antineoplastic activity in several human cancers. However, the effects of SOV on liver cancer are still unknown. In this study, for the first time, we showed that SOV could effectively suppress proliferation, induce G2/M cell cycle arrest and apoptosis, and diminish the mitochondrial membrane potential (MMP) of HCC cells in vitro. In addition, our in vitro results were recapitulated in vivo, showing that SOV exhibited a dose-dependent inhibition of growth of human HCC in an orthotopic model, evidenced by the reduction in tumor size, proliferation index and microvessel density, and increase in cell apoptosis. Most important, we found that SOV could inhibit autophagy in HCC cells in vitro and in vivo, which plays a prodeath role. Thus, our findings suggest that SOV could effectively suppress the growth of human HCC through the regulations of proliferation, cell cycle, apoptosis and autophagy, and thus may act as a potential therapeutic agent in HCC treatment.

Introduction

Hepatocellular carcinoma (HCC) is the third most common cause of death from cancer worldwide and has a very poor prognosis [1]. Despite extensive exploration of novel therapies, there has been little success in improving the treatment of HCC. Surgical therapy can offer the only chance for long-term cure, but tumor resection is feasible for <15% of patients, and recurrence and metastasis remained the major obstacles to more prolonged survival after surgery [2], [3], [4], [5]. Therefore, more effective therapeutic strategies for treatment of HCC are urgently needed.

The transition metal vanadium is widely distributed in the environment and exhibits various biological and physiological effects in the human body. It has also become more and more important for the development and growth of some organisms as one of the dietary microelements. As a well known vanadium compound, sodium orthovanadate (SOV) has shown numerous biological activities, including the inhibition of nonselective protein tyrosine phosphatases, activation of tyrosine kinases, mitogenic, neuroprotective and antidiabetic effects [6]. Recent studies have also shown that it could exhibit antineoplastic activity in several human cancers cells, including lung, kidney and prostate cancers [7], but the effects of SOV in liver cancer have not yet been reported.

Autophagy is an evolutionarily conserved process involving lysosomal degradation of cytoplasmic and cellular organelles, which occurs in all eukaryotic cells from yeast to mammals. This process is now emerging as an important issue as apoptosis in response to drug therapy in cancer cells [8], [9], [10], [11], [12], [13], [14]. Several anticancer drugs have been shown to regulate autophagy as well as apoptosis. Recently, some studies have shown that autophagy constitutes a potential target for cancer therapy and the induction of autophagy in response to therapeutics can be viewed as having a prodeath or a prosurvival role, which contributes to the anticancer efficacy of these drugs as well as drug resistance.

In this study, for the first time, we showed that SOV exhibited a dose-dependent inhibition of growth of the human HCC cells in vitro and in an orthotopic model. The mechanism may be due to the regulations of proliferation, cell cycle, apoptosis and autophagy. Most important, we found that SOV could induce cell apoptosis and inhibit autophagy in human HCC cells, in vitro and in vivo, simultaneously. Additionally, further reducing autophagy by 3-methyladenine (3MA) significantly enhanced SOV-induced apoptosis in HCC cells, while rapamycin could reverse such autophagy inhibition and reduced the apoptosis-inducing effect of SOV in HCC cells, both in vitro and in vivo, consequently, these data indicates that such autophagy inhibition effect plays a prodeath role.

Section snippets

Cell culture, reagents and antibodies

The human HCC cell lines HepG2, Hep3B and SK-Hep-1 were obtained from the American Type Culture Collection (Rockville, USA). Cell lines were routinely cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) in a 5% CO₂ atmosphere at 37 °C (all reagents were from HyClone China Ltd., China). SOV, rapamycin and 3-MA were purchased from Sigma–Aldrich, the antibodies against BECN1, LC3, cyclin B1, cdc2, PARP,

Inhibitory effect of SOV on proliferation in HCC cells

HepG2, SK-Hep-1 and Hep3B cells were incubated with increasing concentrations of SOV (7.5 μM, 15 μM and 30 μM) for 72 h and cell viability was determined with a CCK-8 kit. Here we showed that SOV significantly suppressed the proliferation of HCC cells in a dose-dependent manner (Fig. 1). After 72 h treatment with SOV, there was significant difference in the cell viability index between control and 15 μM or 30 μM SOV treated cells (p < 0.05), whereas lower dose of SOV (7.5 μM) also caused a slight but

Discussion

The present study has demonstrated the anti-cancer effects of SOV in the treatment of human HCC cells.SOV suppressed growth of HCC cells in a dose-dependent manner both in vitro and in vivo. The underling mechanisms may be involved of regulations of proliferation, cell cycle and apoptosis. Upon further exploration, we found that for the first time SOV might serve as a novel autophagy inhibitor in cancer therapy.

Recently, remarkable advances have been made in anti-hepatoma mechanisms of drugs.

Conflict of Interest

The authors have declared no conflict of interest.

Acknowledgements

This study was jointly supported by grants from Heilongjiang Postdoctoral Foundation (LBH-Z11066 and LBH-Z12201), Science and Technology Research Project of Heilongjiang Province Education Department (1154z1005), China Postdoctoral Science Foundation (2012M510990, 2012M520769 and 2013T60387), Natural Science Foundation of Heilongjiang Province of China (QC2013C094) and the National Natural Scientific Foundation of China (81100305 and 81270527).

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¹: These authors contributed equally.

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