Hypoxia induced paclitaxel resistance in human ovarian cancers via hypoxia-inducible factor 1alpha.
ABSTRACT Chemoresistance severely restricts the anti-cancer medicines from effectively treating human ovarian cancer, which has been shown to develop and survive in the specific hypoxic environments. To understand the relationship between hypoxia and chemoresistance, we investigated the potential role of hypoxia in the pathophysiology of chemoresistance, especially focusing on hypoxia-inducible factor 1alpha (HIF-1alpha).
The A2780 ovarian cancer cells are cultured in gradient hypoxic conditions (5% O(2), 3% O(2), and 1% O(2)), the sensitivity of the cells to paclitaxel and the cell inhibitory rate were determined by MTT assay. The expression and the transcriptional activity of HIF-1alpha were examined by western blot, Immunocytochemical staining, reverse transcription-polymerase chain reaction (RT-PCR), and the dual luciferase reporter system, respectively. The cell cycle distribution was analyzed by flow cytometry. In addition, we silence HIF-1alpha expression by performing RNA interference.
MTT assay demonstrates that hypoxic challenge substantially reduces the susceptibility of cells to paclitaxel at all the tested concentrations. Coincident with this is the activation of HIF-1alpha in nuclear, which displays the increased transcriptional activity and high protein expression. Hypoxic manipulation (5% O(2), approximately 1% O(2)) significantly increased the cell population at G0/G1. Interestingly, knockdown of endogenous HIF-1alpha significantly alleviates the chemoresistance and promotes G1/S transition with the increased sensitivity of A2780 cells to paclitaxel under each hypoxic condition.
It suggests that HIF-1alpha, stimulated by hypoxia, exerts a pivotal role in chemoresistance by G0/G1 arrest. Eliminating hypoxic conditions or silencing HIF-1alpha by siRNA might provide a potent tool to enhance paclitaxel effectiveness in treatment of human ovarian cancer.
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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.PLoS ONE 01/2013; 8(6):e65304. · 3.73 Impact Factor
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ABSTRACT: High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.PLoS ONE 01/2013; 8(2):e57056. · 3.73 Impact Factor
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ABSTRACT: Introduction: Developing in vitro models for studying cell biology and cell physiology is of great importance to the fields of biotechnology, cancer research, drug discovery, toxicity testing, as well as the emerging fields of tissue engineering and regenerative medicine. Traditional two-dimensional (2D) methods of mammalian cell culture have several limitations and it is increasingly recognized that cells grown in a three-dimensional (3D) environment more closely represent normal cellular function due to the increased cell-to-cell interactions, and by mimicking the in vivo architecture of natural organs and tissues. Areas covered: In this review, we discuss the methods to form 3D multi-cellular spheroids, the advantages and limitations of these methods, and assays used to characterize the function of spheroids. The use of spheroids has led to many advances in basic cell sciences, including understanding cancer cell interactions, creating models for drug discovery and cancer metastasis, and they are being investigated as basic units for engineering tissue constructs. As so, this review will focus on contributions made to each of these fields using spheroid models. Expert opinion: Multi-cellular spheroids are rich in biological content and mimic better the in vivo environment than 2D cell culture. New technologies to form and analyze spheroids are rapidly increasing their adoption and expanding their applications.Expert opinion on biological therapy 07/2012; 12(10):1347-60. · 3.22 Impact Factor