Abstract
Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing’s sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1). In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1α stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1α in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1α. Inhibition of the phosphoinositide 3-kinase (PI3K) pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target to reverse drug resistance in paediatric osteosarcoma. The novel finding of HIF-1α independent drug resistance suggests however other hypoxia related targets may be more relevant in paediatric osteosarcoma.
Highlights
Cytotoxic drug resistance in hypoxia can vary between tumour type and with drug used. [13,50,51] Evidence exists of the importance of hypoxia in osteosarcoma, but the effect of hypoxia on the response of osteosarcoma cells to clinically relevant cytotoxic drugs has not been reported
Significant resistance to etoposide, cisplatin and doxorubicin in hypoxia was seen in all 3 osteosarcoma cell lines, consistent with previous data showing hypoxia-induced resistance to cisplatin, doxorubicin and etoposide in a range of different tumour types. [6,7,8,10,11,13,14,23,25,27,29,31,35,36,38,48,52] Drug-induced apoptosis was reduced in HOS and U2OS cells exposed to cisplatin, doxorubicin and etoposide and in 791T cells exposed to cisplatin and doxorubicin, suggesting reduced apoptosis as the underlying mechanism for hypoxia-induced drug resistance
Hypoxia-induced resistance to cisplatin-induced apoptosis has been previously reported in a number of tumour cell types [25,35,39,48,52,53] as has hypoxia-induced resistance to doxorubicin [14,27,35] and etoposide. [7,10,13,27,36,53] 791T cells showed highly significant (p = 0.0012) resistance to etoposide in hypoxia (Figure 1) they consistently showed an equivalent degree of etoposide-induced apoptosis in normoxia and hypoxia, suggesting that other resistance mechanisms may be active in these cells
Summary
Osteosarcoma is the most common primary malignancy of bone and occurs most frequently in late childhood and early adulthood. [1] The introduction of dose intensive combination chemotherapy has increased the overall survival for osteosarcoma patients to over 70%. [2,3] in those with metastasis and in those who relapse, prognosis remains poor with survival rates of only 20– 30%. [4,5] There has been no improvement in the survival of osteosarcoma patients in the last 20 years and new therapeutic options are urgently needed.In vitro evidence of hypoxia-induced drug resistance exists for a wide variety of cytotoxic agents in a wide variety of adult tumour types. [6,7,8,9,10,11,12] Hypoxia is able to induce resistance to etoposide and vincristine in neuroblastoma cells and doxorubicin, vincristine and actinomycin-D in rhabdomyosarcoma and Ewing’s sarcoma cells. [13,14] Markers of hypoxia including hypoxia-inducible factor-1 (HIF-1), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CA IX) can be detected in osteosarcomas [15,16,17] and the presence of these markers correlates with poor patient outcome, suggesting that hypoxia has an important role in osteosarcoma. [15,17,18] The effect of hypoxia on drug response in osteosarcoma has not been shown.The main transcription factor responsible for the cellular adaptation to hypoxia is HIF-1. Hydroxylation does not occur and the alpha subunits stabilise, dimerise with HIF-1b and translocate to the nucleus where they regulate the transcription of over 100 target genes, many of which are directly or indirectly involved in drug resistance. [6,8,9,10,11,13,22,23,24,25,26,27,28,29] in many tumour types HIF-1 is a valid target to reverse hypoxia-induced drug resistance. [12,31,34,35,36,37,38,39] Importantly, inhibiting this activation sensitises cells to cytotoxic agents in hypoxia, and they are possible targets to reverse hypoxia-induced drug resistance Wild-type p53 is inactivated in some tumour cells in hypoxia, inducing resistance to p53-mediated apoptosis [30,31,32,33], and in some tumour types hypoxia-induced drug resistance occurs only in cell lines with wild-type p53. [25] Activation of the phosphoinositol-3-kinase (PI3K) pathway, nuclear factor kappa-B (NFkB), cycloxygenase-2 (COX-2), activator protein-1 (AP-1), cjun, Pim-1 and STAT-3 in hypoxia have all been found to induce drug resistance, mainly by a reduction in drug-induced apoptosis. [12,31,34,35,36,37,38,39] Importantly, inhibiting this activation sensitises cells to cytotoxic agents in hypoxia, and they are possible targets to reverse hypoxia-induced drug resistance
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