Hypoxia and retinoic acid-inducible NDRG1 expression is responsible for doxorubicin and retinoic acid resistance in hepatocellular carcinoma cells
Introduction
Hepatocellular carcinoma (HCC) development is a major cause of mortality in patients with chronic liver disease. HCC is generally considered to be a hypervascular tumor [1] and therefore, transarterial chemoembolization (TACE) is employed as a standard treatment in unresectable HCCs on the basis of hypervascularity. However, advanced infiltrative HCCs seldom evidence hypervascularity and are usually refractory to TACE [2], [3], although they grow more aggressively than mass-forming hypervascular HCCs. Moreover, the surviving cancer cells in HCC nodules treated with TACE, which confers a profound hypoxic insult, occasionally grow more rapidly than those in neighboring nodules [4]. Therefore, hypoxia is a strong stimulus for the generation of signals that enable HCC cells to survive and proliferate under hypoxic conditions [5].
Doxorubicin, a potent activator of p53, is commonly utilized for HCC chemotherapy, delivered via the intra-hepatic artery [6]. However, its efficacy is rather low, and its systemic injection has not thus far provided any significant and substantial benefits in terms of tumor regression and overall survival [7]. Moreover, hypoxic tumor cells generally show chemoresistance [8] and therefore, the surviving HCC cells following TACE or HCC cells of infiltrative-type are expected to be highly resistant to doxorubicin.
Retinoic acid (RA), a metabolite of vitamin A, exhibited anti-tumor activity against a variety of cancers, and is currently utilized as a therapeutic or chemo-preventative agent in the treatment of several human cancers, including acute promyelocytic leukemia [9]. With regard to HCCs, RA significantly reduced the incidence of second primary HCCs and exhibited survival benefits in patients who underwent resection or local ablation of HCCs [10], [11]. Moreover, a phase II trial of a synthetic RA combined with TACE versus TACE alone in advanced HCC patients is currently underway. However, HCC cells occasionally show resistance to RA cytotoxicity [12].
N-myc downstream-regulated gene-1 (NDRG1) is a hypoxia- and RA-inducible protein, and has previously been implicated in carcinogenesis [13]. Doxorubicin is a potent p53 activator, and this NDRG1 protein is also a downstream target of p53. As hypoxic tumor cells are generally chemoresistant [8] and HCC cells frequently evidence RA resistance [12], we hypothesized that NDRG1 expression is responsible for doxorubicin and RA resistance in HCC cells, particularly under hypoxic conditions. In order to evaluate this hypothesis, we formulated the following questions: (i) Does hypoxia or RA increase NDRG1 expression in HCC cells? (ii) Are hypoxic HCC cells resistant to doxorubicin cytotoxicity? (iii) Does the suppression of NDRG1 expression sensitize HCC cells to doxorubicin and/or RA cytotoxicity, and finally (iv) What is the underlying mechanism of cell death? Collectively, these results show that hypoxia- and RA-inducible NDRG1 expression is responsible for doxorubicin and RA resistance in HCC cells. Thus, the selective interruption of NDRG1 signaling may prove therapeutically useful in HCCs, particularly in advanced infiltrative-type tumors that are exposed to a hypoxic environment.
Section snippets
Cell line and culture
Human HCC cell lines were used in this study: Huh-7 cells (derived from a well-differentiated HCC [14]) and RA-resistant SNU-761 cells (derived from a poorly-differentiated HCC [15]). Cells were grown in DMEM supplemented with 10% fetal bovine serum, 100,000 U/l of penicillin and 100 mg/l of streptomycin. In all of the experiments conducted in this study, the cells were serum-starved overnight in order to avoid the confounding variable of serum-induced signaling. According to the experimental
Hypoxia and RA induction of NDRG1 expression in HCC cells
We first attempted to determine whether hypoxia and RA induce NDRG1 expression in HCC cells. For this purpose, we used Huh-7 cells for the hypoxia experiments and RA-resistant SNU-761 cells for RA treatment. As shown in Fig. 1, NDRG1 expression level was increased by both hypoxia and RA, thereby indicating the hypoxia- and RA-inducibility of NDRG1 expression in human HCC cells.
Doxorubicin resistance in hypoxic HCC cells
We next attempted to determine whether hypoxic HCC cells are less sensitive to doxorubicin cytotoxicity. As shown in
Discussion
NDRG1 was originally regarded as a differentiation-related gene, which mainly participates in cell differentiation, organ formation, and embryonic development [17], [18], [19]. Recently, many studies have also reported its association with carcinogenesis in a broad variety of tumors [13], [20]. However, there remain controversies regarding its functional role in carcinogenesis and cancer progression. For example, a metastatic suppressor function was implied in prostate and colon cancers [21],
Conflicts of interest
None declared.
Acknowledgements
Financial support: This study was supported by the Seoul National University Hospital Research Fund (#06-2007-0109) and the Korea Health 21 R&D Project (#0412-CR01-0704-0001).
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