BNIP3 protects HepG2 cells against etoposide-induced cell death under hypoxia by an autophagy-independent pathway
Introduction
Tumor hypoxia is recognized as a poor prognosis factor associated with resistance to cancer therapy such as radiotherapy and chemotherapy [1], [2]. Inside the tumor, oxygen supply is compromised by the inadequate blood flow due to the abnormal vasculature network and by the high rate of cancer cell proliferation. Indeed, cancer cell proliferation also contributes to hypoxia by increasing the interstitial pressure and the diffusion distance from the blood vessels. Cancer cells localized in hypoxia areas are more resistant to chemotherapy than cells in well-oxygenated zones because drug delivery in hypoxia areas is limited and because oxygen is required for the cytotoxicity of some drugs [3]. Besides direct effects, hypoxia also promotes cancer cell resistance against chemotherapy by inducing adaptive mechanisms to hypoxia [4]. Regulation of gene expression, involving HIF-1, is an important component of the initiation of these adaptive responses to hypoxia. HIF-1 regulates the expression of over a hundred of genes and among them are genes encoding proteins involved in the regulation of angiogenesis, erythropoiesis, anaerobic metabolism, drug resistance and proliferation. In addition, cell survival and cell death are both regulated by hypoxia, in part through the regulation of programmed cell death I and II processes named apoptosis and autophagy, respectively. The regulation of apoptosis by hypoxia involves the regulation of the expression of genes such as Bid, Bax, Noxa, Survivin and Mcl-1 [5], [6], [7], [8]. In addition, hypoxia selects cancer cells with reduced apoptotic potential [9]. Autophagy is first a cellular response in which proteins, organelles and portions of cytoplasm are engulfed, digested and recycled to sustain cellular metabolism during stress such as nutrient starvation and hypoxia [10]. Although stimulation of autophagy might constitute a protective response, extensive and non-regulated autophagy may lead to cell death which may be either independent or linked to apoptosis [11].
Under hypoxia, the induction of autophagy is mainly due to the hypoxia-inducible protein BNIP3, encoded by a HIF-1 target gene. BNIP3 belongs to the BH3-only Bcl-2 family and contains a BH3 domain and a transmembrane domain that targets BNIP3 to the outer mitochondrial membrane. Unlike other BH3-only proteins, interactions with other proteins as well as its functions depend on the transmembrane domain but not on the BH3 domain [12]. Before its role in autophagy was discovered, BNIP3 was associated with cell death by multiple mechanisms. Currently, it seems that BNIP3 may participate in cell death by inducing necrosis, apoptosis or autophagy. In some cases, localization of BNIP3 into the outer mitochondrial membrane causes an increase in ROS production, cytochrome c release by PTP opening, loss of ΔΨm and leads to caspase-dependent cell death [13]. Bax and Bak seem to be essential for BNIP3-induced cell death characterized by apoptosis features [14]. Caspase-independent cell death through the release of endoG but not of cytochrome c was also observed following BNIP3 insertion into the outer mitochondrial membrane [15]. Finally, BNIP3 can also induce autophagic cell death. However, the precise mechanisms involved are still poorly understood. It seems that BNIP3 could be involved in autophagosome-lysosome fusion or in earlier stages such as Beclin-1 activation and mTOR inhibition [10], [16], [17]. Finally, BNIP3 has been linked to protective autophagy and cell survival [10], [18].
We have previously shown that human hepatoma HepG2 cells could be protected against etoposide-induced apoptosis under hypoxia. Given the potential role of both autophagy and BNIP3 in the regulation of cell death and cell survival, we investigated their putative role in etoposide-induced cell death and the protection provided by hypoxia.
Section snippets
Cell culture and hypoxia incubation
Human hepatoma HepG2 cells were maintained in culture in 75-cm2 polystyrene flasks (Costar, Lowell, USA) with 15 ml Dulbecco's modified Eagle's medium liquid (DMEM, Invitrogen, Carlsbad, USA) and 10% of foetal calf serum (Invitrogen, Carlsbad, USA) and incubated under an atmosphere containing 5% CO2. For hypoxia experiments (1% O2), cells were incubated in serum-free CO2-independent medium (Invitrogen, Carlsbad, USA) supplemented with 1 mM l-glutamine (Sigma, St Louis, USA) with or without
Hypoxia protects HepG2 cells against etoposide-induced apoptosis
We have previously shown that hypoxia protects HepG2 cells against etoposide-induced apoptosis [21], [22], [23]. To confirm these results, cells were incubated for 16 h in serum-free medium in the presence or in the absence of etoposide, under normoxia (21% O2) or under hypoxia (1% O2). At the end of the incubation, cells were fixed and nuclei were labelled with DAPI and observed with fluorescent microscopy (Fig. 1A). We observed nuclear fragmentation in cells exposed to etoposide under normoxia
Discussion
Our recent studies have demonstrated that hypoxia protects human hepatoma HepG2 cells against etoposide-induced apoptosis (Fig. 1A and B) [8], [21], [22], [23]. Hypoxia is an energy-limiting stress which leads to autophagy upregulation. Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of cytoplasm. The formation of autophagosome depends upon Atg proteins [34]. Autophagy can be stimulated by various stresses, including nutrient
Acknowledgements
J.-P. Cosse is recipient of a FRIA fellowship (Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture). This article presents results of the Belgian program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. The responsibility is assumed by its authors. We thank Prof. Bassam Janji (LHCE CRP-Santé, Luxembourg) for providing the pEGFP-LC3m plasmid and the department of electron microscopy (FUNDP).
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