Hypoxia-induced cisplatin resistance is reversible and growth rate independent in lung cancer cells

doi:10.1016/j.canlet.2011.03.014

Cancer Letters

Volume 308, Issue 2, 28 September 2011, Pages 134-143

https://doi.org/10.1016/j.canlet.2011.03.014 Get rights and content

Abstract

Hypoxia frequently occurs in solid tumors and is known to contribute to chemotherapy resistance. However, the mechanisms leading to chemotherapy resistance are not entirely known. We investigated hypoxia-induced resistance to cisplatin in NSCLC cell lines. We show that chronic moderate hypoxia induced resistance to cisplatin in NSCLC cells without involvement of selection pressure. Our data suggest that stabilization of the hypoxia-inducible factor 1 alpha and down-regulation of the pro-apoptotic protein BAX play a role in this process. Furthermore, we provide evidence that hypoxia-induced resistance to cisplatin is not due to the reduced growth rate of cancer cells under hypoxic conditions.

Introduction

Lung cancer is the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. Platinum-based chemotherapy represents the gold standard for treating NSCLC [1]. However, the efficacy is limited partially due to primary chemotherapy resistance. Resistance to cisplatin is highly complex and may occur due to various reasons i.e. decreased accumulation of cisplatin or increased repair of DNA damage [2], [3]. In addition, hypoxia-induced chemotherapy-resistance may play a role.

It is well acknowledged that in solid tumors hypoxic conditions prevail and that this contributes to treatment failure and bad prognosis [4]. There are two possibilities how hypoxia might induce resistance in cancer cells. On the one hand hypoxia was found to cause cell death via apoptosis and thereby select for apoptosis resistant cells [5], [6], [7]. On the other hand it was shown that adaptation to hypoxia leads to chemotherapy resistance [8], [9], [10], [11], [12], [13]. The level of oxygen seems to be the critical factor deciding which mechanism comes to play [14]. At O₂ concentrations below 0.1% selection may be most important while at oxygen concentrations around 1% the literature is inconsistent [15], [16].

Proliferation of cancer cells is inhibited by low oxygen tensions [17], [18]. It is commonly believed that the potency of DNA-damaging agents, like cisplatin, to cause apoptosis is strongly dependent on the growth rate [19], [20], [21]. Thus, a reduction of growth rate by hypoxia could be an important mechanism leading to resistance to cisplatin.

We investigated hypoxia-induced resistance to cisplatin in NSCLC cell lines with different growth rates and confirmed that NSCLC cells develop a strong growth rate-independent resistance to cisplatin in hypoxia but do not undergo apoptosis in hypoxia per se. We found that this hypoxia-induced chemoresistance was reversible after re-oxygenation. This clearly indicates a reversible hypoxia-specific cellular adaptation process leading to primary resistance against cisplatin.

Section snippets

Cell lines

The human NSCLC cell line NCI-H358 (CRL-5807) was purchased from American Type Culture Collection (ATCC®, Manassas, VA). The human NSCLC cell line A549 was purchased from Cell Lines Service (Eppelheim, Germany). Cells were cultured in DMEM-F12 (Gibco, Paisley, UK) or RPMI 1640 (ATCC®) culture medium supplemented with 10% fetal calf serum (FCS, Biowest, Nuaillé, France), 2 mM l-glutamine (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in a humidified incubator with 21% oxygen and

Effects of hypoxia on proliferation and viability in human NSCLC cells

To analyze the influence of hypoxia (1% O₂) on proliferation and viability of NSCLC cells, A549 and NCI-H358 cells were exposed to hypoxia for up to 96 h. Cells were investigated every 24 h with electronic pulse area analysis. A remarkable reduction of growth rate was found in hypoxic A549 cells (Fig. 1A). NCI-H358 cells exhibited a strongly reduced growth rate compared to A549 cells in normoxia and their growth rate was only insignificantly reduced in hypoxia (Fig. 1A). Hypoxia-induced reduction

Discussion

In this study we show that chronic moderate hypoxia induces resistance to cisplatin in A549 and NCI-H358 NSCLC cells without involvement of selection pressure. The cells display a delayed regain of sensitivity to cisplatin after re-oxygenation. Hypoxia reduced the growth rate of A549 cells and, insignificantly, NCI-H358 cells. Despite this different response to hypoxia, hypoxia-induced cisplatin resistance was similar in both cell lines. Serum starvation mimicked the effect of hypoxia on cell

Conflicts of interest

None declared.

Acknowledgements

We are grateful to Elisabeth Wirnsperger, Maria Schloffer, Alexandra Hof, and Mag. Peter Blümel for excellent technical assistance. We thank the core facilities at the Center for Medical Research, Medical University of Graz, for their support.

C. Wohlkoenig is supported by the Medical University of Graz (PhD Program Molecular Medicine).

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Hypoxia-induced cisplatin resistance is reversible and growth rate independent in lung cancer cells

Abstract

Introduction

Section snippets

Cell lines

Effects of hypoxia on proliferation and viability in human NSCLC cells

Discussion

Conflicts of interest

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Cancer Lett.

Semin. Radiat. Oncol.

Cancer Treat. Rev.

J. Biol. Chem.

Curr. Opin. Genet. Dev.

Adjuvant chemotherapy, with or without postoperative radiotherapy, in operable non-small-cell lung cancer: two meta-analyses of individual patient data

Lancet

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Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours

Nature

Cyclic exposure to hypoxia and reoxygenation selects for tumor cells with defects in mitochondrial apoptotic pathways

FASEB J.

Effects of acute versus chronic hypoxia on DNA damage responses and genomic instability

Cancer Res.

Role of hypoxia inducible factor-1 alpha in modulation of apoptosis resistance

Oncogene

Mechanism of HIF-1alpha-dependent suppression of hypoxia-induced apoptosis in squamous cell carcinoma cells

Cancer Sci.

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