Hypoxia stimulates proliferation of human hepatoma cells through the induction of hexokinase II expression
Introduction
Hepatocellular carcinoma (HCC) is a neoplasm most commonly originating from the diseased liver. The common risk factor for the development of HCC is chronic liver diseases caused by hepatitis B or C viral infection [1], [2]. The presence of risk factor may offer the possibility for detecting small or early HCCs with regular hepatic imaging and serum alpha-fetoprotein determination [3]. HCCs at this stage are readily treated by local ablation therapy employing either ethanol injection or radiofrequency application [4], [5]. On the other hand, multiple or large HCCs, which are not amenable to such local therapies, can alternatively be treated with transarterial chemoembolization (TAE) [6].
The therapeutic efficacy of TAE is based on the anoxic or hypoxic insult on cells. However, HCC cells likely have a compensatory mechanism rendering cells in a hypoxic microenvironment to survive or even proliferate more efficiently than cells in a normoxic condition. This assumption is based on the following two clinical observations. First, cell death is rarely observed in the center of HCC nodules, where cells are relatively exposed to hypoxia, and this is true even in large ones. Second, the surviving cells in HCC nodules having been treated with TAE, which confers a strong hypoxic insult, are sometimes growing faster than those in neighboring nodules.
Oxygen-dependent mitochondrial adenosine triphosphate (ATP) generating system is primarily responsible for generating energy required for maintaining cell survival. In a hypoxic state, a glycolytic system is operating as a salvage pathway of generating ATP. Hexokinase (HK), the first enzyme in this pathway, is essential for maintaining the high glycolytic phenotype [7], [8]. Among the four mammalian HK types (HK I-IV), HK II is the predominantly overexpressed form in HCCs [9], [10]. Among several events involved in this enzyme expression, gene amplification [11] and promoter activation [10], [12], [13], [14] appear to be major contributors.
Hypoxia-inducible factor-1 (HIF-1) is a heterodimer protein composed of HIF-1α and β subunits and plays a role in O2 homeostasis [15], [16], [17]. Whereas HIF-1β is a constitutively expressed protein, HIF-1α is degraded in the ubiquitin-proteasome pathway under normoxic conditions [18]. However, it is stabilized under hypoxic conditions followed by its heterodimerization with HIF-1β to form a functional HIF-1 complex. This heterodimer complex transactivates many kinds of hypoxia-inducible genes such as erythropoietin, angiogenic factors including VEGF, and glycolytic enzymes, by binding to the hypoxia-responsive element (HRE) in promoters of these genes [19]. HRE has also been identified in the HK II promoter, and correspondingly HK II is reported to be upregulated by HIF-1 in cancer cells other than HCCs [14], [20].
Therefore, we hypothesized that HK II is actively participating in cell survival in HCCs in a hypoxic state. To test this hypothesis, we formulated the following questions: (i) Does hypoxia stimulate HCC cell growth? (ii) Is this enhanced cell growth induced by HK II expression via a HIF-1α dependent mechanism? (iii) If so, does HK II inhibition suppress cell growth? and finally, (iv) What is the mechanism of cell death? Collectively, the results of the current study demonstrate that human HCC cells proliferate more efficiently in a hypoxic condition through HK II induction, and the inhibition of this enzyme effectively induces apoptotic cell death. These results implicate that HK II induction is participating in HCC progression and the blockage of HK II may therapeutically be efficacious in human HCCs.
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Cell line and culture
Several human hepatoma cell lines were chosen for this study: Huh-BAT (Huh-7 cells stably transfected with a bile acid transporter [21], which are derived from a well differentiated HCC [22]), HepG2 and SNU-475 cells which are derived from a poorly differentiated HCC [23]. Cells were either grown in DMEM (Huh-BAT and HepG2 cells) or in RPMI 1640 (SNU-475 cells) supplemented with 10% fetal bovine serum, penicillin 100,000 U/l and streptomycin 100 mg/l. In all the experiments performed in this
Does hypoxia stimulate HCC cell growth?
HCC cells were serum starved and either cultured in a normoxic or hypoxic condition. In contrary to cells in a normoxic state, cells grown in a hypoxic chamber proliferated more efficiently (Fig. 1). This finding implicates that hypoxia stimulates HCC cellular growth.
Is HK II expression induced by hypoxia?
To explore the mechanism of enhanced proliferation of HCC cells in a hypoxic state, we next examined HIF-1α, HK I, HK II, HSP70 and mcl-1 expression levels in these cells. As shown in Fig. 2, HIF-1α and HK II expression levels were
Discussion
The principal findings of this study relate to the hypoxia-induced activation of a salvage pathway of energy generation in human HCC cells. The results demonstrate that hypoxia stimulates human HCC cell growth through the induction of HK II expression, and the inhibition of this enzyme effectively induces apoptotic cell death. These results implicate that HK II induction is participating in HCC progression and therefore, the blockage of HK II may have a therapeutic implication. Each of these
Acknowledgements
The authors thank Ms. Sung-Hee Lee and Ms. Soo-Mi Lee for their excellent technical assistance. This study was supported by grants from the Korean Foundation of Liver Research (2003) and the National Cancer Control R&D Program 2003, Ministry of Health and Welfare, Republic of Korea.
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