Original Articles2-Methoxyestradiol synergizes with sorafenib to suppress hepatocellular carcinoma by simultaneously dysregulating hypoxia-inducible factor-1 and -2
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.