Abstract
Backgroundit is now well established that hypoxia renders tumor cells resistant to radio- but also chemotherapy. However, few elements are currently available as for the mechanisms underlying this protection.Resultsin this study, physiological hypoxia was shown to inhibit apoptosis induced in HepG2 cells by etoposide. Indeed, hypoxia reduced DNA fragmentation, caspase activation and PARP cleavage. The DNA binding activity of 10 transcription factors was followed while the actual transcriptional activity was measured using specific reporter plasmids. Of note is the inhibition of the etoposide-induced activation of p53 under hypoxia. In parallel, data from low density DNA microarrays indicate that the expression of several pro- and anti-apoptotic genes was modified, among which are Bax and Bak whose expression profile paralleled p53 activity. Cluster analysis of data unravels several possible pathways involved in the hypoxia-induced protection against etoposide-induced apoptosis: one of them could be the inhibition of p53 activity under hypoxia since caspase 3 activity parallels Bax and Bak expression profile. Moreover, specific downregulation of HIF-1α by RNA interference significantly enhanced apoptosis under hypoxia possibly by preventing the hypoxia mediated decrease in Bak expression without altering Bax expression.Conclusionthese results are a clear demonstration that hypoxia has a direct protective effect on apoptotic cell death. Moreover, molecular profiling points to putative pathways responsible for tumor growth in challenging environmental conditions and cancer cell resistance to chemotherapeutic agents.
Highlights
The adverse effect of tumor hypoxia on the outcome of clinical radiotherapy as well as chemotherapy is well established
HepG2 cells incubated in the presence of 50 μM etoposide during 16 hours did undergo apoptosis as shown by an increase in caspase activity, in active caspase 3 abundance, in PARP cleavage and in DNA fragmentation (Fig. 1)
Hypoxia inhibited the etoposideinduced apoptosis: a marked decrease in caspase activity and DNA fragmentation was observed in addition to a nearly complete inhibition of PARP cleavage and caspase 3 activation (Fig. 1)
Summary
The adverse effect of tumor hypoxia on the outcome of clinical radiotherapy as well as chemotherapy is well established. Hypoxic conditions elicit cellular responses designed to improve cell survival through an adaptive process. Regulation of gene expression through HIF-1 (hypoxia-inducible factor-1) and via other transcription factors plays an important role in this process. These changes in gene expression enable tumors to take advantage of the physiological response mechanisms to hypoxia to improve their own survival as well as their metastatic properties [1]. The two hydroxylated prolines are recognized by the protein pVHL, which is part of an ubiquitin ligase complex, targeting the HIF-1α subunit for degradation by the proteasome. The products of HIF-1 target genes allow the cell to adapt to the hypoxic conditions [2,3]
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