Cancer Letters

Cancer Letters

Volume 355, Issue 1, 1 December 2014, Pages 96-105
Cancer Letters

Original Articles
2-Methoxyestradiol synergizes with sorafenib to suppress hepatocellular carcinoma by simultaneously dysregulating hypoxia-inducible factor-1 and -2

https://doi.org/10.1016/j.canlet.2014.09.011Get rights and content

Abstract

Sorafenib is the approved systemic drug of choice for advanced hepatocellular carcinoma (HCC), but has demonstrated limited benefits because of drug resistance. 2-Methoxyestradiol (2ME2) has been shown to be a promising anticancer drug against various types of cancers and acts by dysregulating hypoxia-inducible factor (HIF)-1. Hypoxic cancer cells are extremely resistant to therapies since they elicit strong survival ability due to the cellular adaptive response to hypoxia, which is controlled by HIF-1 and HIF-2. The present study has demonstrated that sorafenib downregulated the expression of HIF-1α, making the hypoxic response switch from HIF-1α- to HIF-2α-dependent pathways, resulting in upregulation of HIF-2α, which contributes to the insensitivity of hypoxic HCC cells to sorafenib. HIF-2α played a dominant role in regulating VEGF, thus sorafenib in turn increased the expression of VEGF (a downstream molecule of both HIF-1 and HIF-2) and cyclin D1 (a downstream molecule of HIF-2), but reduced the expression of LDHA (a downstream molecule of HIF-1), in hypoxic HCC cells. 2ME2 significantly reduced the expression of both HIF-1α and HIF-2α, and their downstream molecules, VEGF, LDHA and cyclin D1, rendering hypoxic HCC cells to increased sensitivity to 2ME2. 2ME2 also inhibited the nuclear translocation of HIF-1α and HIF-2α proteins, but had no effect on their mRNA expression. 2M2 synergized with sorafenib to suppress the proliferation and induction of apoptosis of HCC cells in vitro and in vivo, and inhibited tumoral angiogenesis. These results indicate that 2ME2 given in combination with sorafenib acts synergistically for treating HCC.

Introduction

Hepatocellular carcinoma (HCC), the second most frequent cause of cancer death in men worldwide [1], is notoriously resistant to chemotherapeutic drugs [2]. Sorafenib is the only approved first-line systemic drug for advanced HCC [2]. It targets multiple cellular signalling pathways and tyrosine kinase receptors including vascular endothelial growth factor (VEGF) receptor (VEGFR), thus inducing apoptosis, and inhibiting cell proliferation and tumor angiogenesis [3]. Unfortunately, sorafenib has demonstrated limited survival benefits with very low response rates due to drug resistance [3]. Although the exact mechanisms for the resistance to sorafenib have not yet been fully elucidated, some approaches have been launched to circumvent resistance by combining it with other anticancer drugs [4], [5].

Hypoxic microenvironments inside solid tumors are one of the major causes of drug resistance [6], [7]. Sorafenib executes its anticancer effects largely through its anti-angiogenic activity against HCC [6], but the correlation of sorafenib resistance and hypoxic microenvironment is attractive because anti-angiogenesis is speculated to lead to tumor starvation and subsequent hypoxia [8]. The discovery of hypoxia-inducible factors (HIFs) as master driving forces of the cellular adaption to hypoxia has provided a fundamental molecular link to the clinical dilemma [7]. HIFs regulate a vast array of genes encoding proteins involved in tumoral angiogenesis, and the glycolysis, proliferation, metastasis and apoptosis of cancer cells [9]. Each HIF is a heterodimer composed of an α-subunit and a β-subunit, and binds hypoxia-response elements (HREs) in the promoters of the targeted genes [9]. HIF-1α and HIF-2α complex with HIF-1β (also known as ARNT, aryl hydrocarbon receptor nuclear translocator) to form a heterodimer, and are degradable in an oxygen-dependent manner [10]. Both HIF-1α and HIF-2α are upregulated in HCC tissues, and contribute to tumor progression and resistance to pharmacological therapy [11], [12].

Although HIF-1α and HIF-2α share a high degree of sequence identity, a similar protein structure and several common targets such as VEGF, they mediate unique patterns of downstream gene induction [9]. HIF-1α is ubiquitously expressed, while HIF-2α is only expressed by certain cell-types including hepatocytes [9]. HIF-1α plays a dominant role in the response to acute hypoxia, whereas HIF-2α drives the response to chronic hypoxia, and the regulatory feedback of HIF-1α may be responsible for the selectivity [13], [14]. Importantly, depletion of HIF-1α increases the expression of HIF-2α in HCC cells via the reciprocal compensatory mechanism [12], and this switch provides a mechanism for more aggressive growth of tumors under hypoxia [15], [16]. It has been reported that sorafenib inhibits the synthesis of HIF-1α in HCC cells [17]. We have recently reported that upregulation of HIF-2α induced by sorafenib contributed to the resistance by activating the transforming growth factor (TGF)-α/epidermal growth factor receptor (EGFR) pathway in HCC cells [18]. These results indicate that simultaneously blocking HIF-1α and HIF-2α may improve the response of resistant hypoxic HCC cells to sorafenib.

2-Methoxyestradiol (2ME2) is produced in vivo by catechol-O-methyltransferase-mediated O-methylation of 2-hydroxyestradiol [19]. 2ME2 was initially considered to be an inactive end product of estrogen metabolite, but now has emerged as a promising anticancer agent and is currently under investigation for treating cancers in clinical trials. 2ME2 has exhibited anti-proliferative, anti-angiogenic and pro-apoptotic activities against several types of cancers including HCC [20], [21]. The major mechanisms accounting for its anticancer activities involve HIF-1 dysregulation and microtubule disruption [19]. Given the high degree of identity between HIF-1 and HIF-2, the possible HIF-2 dominance in regulating angiogenesis in HCC, and the switch from HIF-1- to HIF-2-dependent ways when HIF-1 is suppressed by sorafenib, we hypothesized that 2ME2 could enhance the efficacy of sorafenib in treating HCC by its ability to dyregulate HIF-1 and HIF-2.

Section snippets

Cell culture

Human HCC HepG2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and Huh7 cells from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum. Hypoxic cells were induced by incubating the cells in a hypoxia chamber containing 1% O2, 5% CO2, and 95% N2 at 37 °C for 24 h.

Antibodies and reagents

The antibodies (Abs) used in this

Upregulation of HIF-2α contributes to the insensitivity of hypoxic HCC cells to sorafenib

HCC cells were incubated with serial concentrations of sorafenib for different periods, and cell viability was assessed. Sorafenib inhibited the proliferation of both HepG2 and Huh7 cells in a concentration- and time-dependent manner (Fig. 1A). With a logarithmic regression analysis, the values of IC50 for HepG2 cells were calculated to be 11.2 µM (R2 = 0.893), 7.23 µM (R2 = 0.928), and 4.68 µM (R2 = 0.963), when cells were incubated with sorafenib for 24, 48 and 72 h, respectively. The values

Discussion

Sorafenib retains a unique pharmacotherapeutic option for the treatment of HCC. However, there is no effective systemic therapy so far after failure of sorafenib therapy, and the response rate of HCC to sorafenib is very low [3], [27], [28]. Therefore, there is an urgent need to seek alternative potential drugs to enhance the efficacy of sorafenib to combat HCC. The present study has demonstrated that 2ME2, a promising anticancer drug in clinical trials, synergizes with sorafenib to suppress

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was funded by grants from the National Natural Scientific Foundation (81272467), Shandong Provincial Science & Technology Development Planning (2010GSF10230), Health and Family Planning Commission of Shandong Province (2013WS0133), The Youth Scientific Fund of Heilongjiang Province (QC2013C098), and The First Affiliated Hospital of Harbin Medical University (2014B22), China. We thank Dr. Shiva Reddy (the University of Auckland, New Zealand) for revising the manuscript. L. Ma and G. Li

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    Li Ma and Guangxin Li contributed equally to this work.

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