PX-478, an inhibitor of hypoxia-inducible factor-1alpha, enhances radiosensitivity of prostate carcinoma cells.
ABSTRACT: Overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF-1alpha is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF-1alpha inhibitor PX-478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX-478 for 20 hr, and HIF-1alpha protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX-478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX-478 decreased HIF-1alpha protein in PC3 and DU 145 cells. PX-478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU-145. PX-478 (20 mumol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF-1alpha and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 mumol/L concentration. PX-478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of gammaH2AX expression in the irradiated cells. PX-478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX-478 as a clinical radiation enhancer.
Project description:The aim of this study is to investigate the clinical significance of hypoxia inducible factor-1? (HIF-1?) expression in esophageal squamous cell cancer (ESCC) and clarify the effects of PX-478, a selective HIF-1? inhibitor, on ESCC both in vitro and in vivo. HIF-1?, cyclooxygenase-2 (COX-2) and programmed death ligand-1 (PD-L1) were markedly overexpressed in ESCC tissue and associated with poorer survival. In vitro, both COX-2 and PD-L1 expression of ESCC cells were significantly induced by CoCl2 treatment, but inhibited by HIF-1? knock-down or PX-478 treatment. Furthermore, PX-478 significantly inhibited tumor cell proliferation by inhibiting the G2/M transition and promoting apoptosis of ESCC cells. In addition, inhibited epithelial-mesenchymal transition was observed after PX-478 treatment. In vivo, PX-478 significantly decreased tumor volume following subcutaneous implantation. Together, our results indicated that PX-478 had significant antitumor activity against HIF-1? over-expressing ESCC tumors in vitro and in vivo. These results opened up the possibility of inhibiting HIF-1? for targeted therapy of ESCC.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is the worst prognoses among all the malignancies. Now, gemcitabine (Gem) is the first line chemotherapeutic drug for advanced pancreatic cancer. However, Gem is usually ineffective to the PDAC because of high degree of drug resistance. Hypoxia and immune suppressive milieu are the best-described hallmarks of PDAC; therefore, we investigated the impact of hypoxia inducible factor-1 (HIF-1) inhibitor, PX-478, in combination with Gem on the induction of immunogenic cell death (ICD). We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice. Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects. Pancreatic cell lines treated with Gem and PX-478 can induce an increase in eIF2? phosphorylation was correlated with down-regulation of HIF-1? and elicited exposure of CRT and release of HMGB1 and ATP. Only co-treated cells induced DC maturation/phagocytosis and IFN-? secretion by cytotoxic T lymphocytes. Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization. We propose that inhibition HIF-1? elicits Gem-induced immune response and eliminates PDAC cells by inducing ICD.
Project description:Disseminated prostate cancer cells colonize the skeleton to progress into macroscopic lesions only if they successfully adapt to the bone microenvironment. We previously reported that the ability of prostate cancer cells to generate skeletal tumors in animal models correlated with the expression of the alpha-receptor for Platelet-Derived Growth Factor (PDGFRa). In this study we aimed to identify PDGFRa-regulated genes responsible for the acquisition of a bone-metastatic prostate phenotype. We performed genome-wide expression comparative analyses of human prostate cancer cell lines that differ for PDGFRa expression and propensity to establish tumors in the skeleton of animal models. We investigated the genes that were differentially regulated in the highly bone-metastatic PC3-ML cells and their low-metastatic counterpart PC3-N cells, and the genes differentially regulated between PC3-N and PC3-N with overexpression of PDGFRa (PC3NRa). We have previously shown that DU-145 cells lack PDGFRa and fail to survive longer than three days as disseminated tumor cells after homing to the mouse bone marrow. Interestingly, and in contrast to PC3-N cells, the exogenous expression of PDGFRa did not promote metastatic bone-tropism of DU-145 cells in our model. Thus, we examined the genes that were differentially regulated between DU-145 and DU-145(Ra) and excluded them from our candidate genes. Finally, to refine our findings and compensate for PC3 and DU-145 genetic disparity, we performed a comparative analysis of the genes differentially regulated between two bone metastatic single-cell progenies that were derived from PC3-ML cells. Seven human prostate cancer cell lines were analyzed in total for this study. Each cell line was analyzed in duplicate from two different passages in culture.
Project description:We aimed to investigate gene expression associated with radiosensitisation of normoxic and hypoxic prostate cancer cells by the class I/II histone deacetylase inhibitor (HDACi) vorinostat. A pronounced deregulation of DNA repair and chromatin organization genes by vorinostat in DU 145 than in PC-3 or 22Rv1 was found and was a likly mechanism underlying radiosensitisation of DU 145. Expression of these genes was generally not affected by hypoxia and was altered by vorinostat in DU 145 towards the baseline levels of PC-3 and 22Rv1. A 56-gene expression signature associated with radiosensitisation under normoxia and hypoxia, including 8 genes with baseline expression characteristic of the radiosensitising effect was generated. These findings propose a hypoxia independent expression signature to predict the radiosensitising effect of vorinostat. Overall design: DU 145, PC-3 and 22Rv1 cell lines, for which differences in intrinsic radiosensitivity have been demonstrated in previous work, were exposed to vorinostat (1µM, 24h) and hypoxia (0.2% O2, 24h), subjected to gene expression profiling and irradiated at 2 and 5 Gy. Samples collected for gene expression analysis were taken prior to irradiation. Vorinostat mediated radiosensitisation occurred under normoxia and hypoxia in the intrinsically radioresistant DU 145, but not in the radiosensitive PC-3 and 22Rv1. To identify a gene expression signature most likely playing a major role in the vorinostat mediated radiosensitisation, a supervised analysis of global gene expression data were performed.
Project description:Mammalian cells increase transcription of genes for adaptation to hypoxia through the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) protein. How cells transduce hypoxic signals to stabilize the HIF-1alpha protein remains unresolved. We demonstrate that cells deficient in the complex III subunit cytochrome b, which are respiratory incompetent, increase ROS levels and stabilize the HIF-1alpha protein during hypoxia. RNA interference of the complex III subunit Rieske iron sulfur protein in the cytochrome b-null cells and treatment of wild-type cells with stigmatellin abolished reactive oxygen species (ROS) generation at the Qo site of complex III. These interventions maintained hydroxylation of HIF-1alpha protein and prevented stabilization of HIF-1alpha protein during hypoxia. Antioxidants maintained hydroxylation of HIF-1alpha protein and prevented stabilization of HIF-1alpha protein during hypoxia. Exogenous hydrogen peroxide under normoxia prevented hydroxylation of HIF-1alpha protein and stabilized HIF-1alpha protein. These results provide genetic and pharmacologic evidence that the Qo site of complex III is required for the transduction of hypoxic signal by releasing ROS to stabilize the HIF-1alpha protein.
Project description:Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1alpha (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor).Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1alpha, and HIF-1alpha siRNA to test if HIF-1alpha activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1alpha and VEGF in transcriptional and translational levels.During hypoxia (0.5% O2), 5 muM cadmium chloride completely inhibited HIF-1alpha expression and reversed the protection by hypoxia preconditioning. HIF-1alpha siRNA (15 nM) reduced HIF-1alpha expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1alpha dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1alpha, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1alpha.These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1alpha, but that VEGF is not a component of the protection.
Project description:The hypoxia-inducible factor 1alpha (HIF-1alpha) is the master regulator of the cellular response to hypoxia. A key regulator of HIF-1alpha is von Hippel-Lindau protein (pVHL), which mediates the oxygen-dependent, proteasomal degradation of HIF-1alpha in normoxia. Here, we describe a new regulator of HIF-1alpha, the hypoxia-associated factor (HAF), a novel E3-ubiquitin ligase that binds HIF-1alpha leading to its proteasome-dependent degradation irrespective of cellular oxygen tension. HAF, a protein expressed in proliferating cells, binds and ubiquitinates HIF-1alpha in vitro, and both binding and E3 ligase activity are mediated by HAF amino acids 654 to 800. Furthermore, HAF overexpression decreases HIF-1alpha levels in normoxia and hypoxia in both pVHL-competent and -deficient cells, whereas HAF knockdown increases HIF-1alpha levels in normoxia, hypoxia, and under epidermal growth factor stimulation. In contrast, HIF-2alpha is not regulated by HAF. In vivo, tumor xenografts from cells overexpressing HAF show decreased levels of HIF-1alpha accompanied by decreased tumor growth and angiogenesis. Therefore, HAF is the key mediator of a new HIF-1alpha-specific degradation pathway that degrades HIF-1alpha through a new, oxygen-independent mechanism.
Project description:BACKGROUND: Neuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1alpha and HIF-1beta subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1beta in astrocytes is mediated by HIF-1alpha. In this study, we demonstrate the role of HIF-1alpha in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively. METHODS: Primary fetal human astrocytes or mouse astrocytes generated from HIF-1alpha+/+ and HIF-1alpha+/- mice were subjected to hypoxia (<2% oxygen) or 125 muM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1alpha, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1alpha with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1alpha was analyzed by reporter gene assay. RESULTS: Sequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1alpha inducer, CoCl2, strongly up-regulated HIF-1alpha expression in astrocytes. Mouse HIF-1alpha+/- astrocytes had lower basal levels of HIF-1alpha and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1alpha+/+ and HIF-1alpha+/- astrocytes was correlated with the levels of HIF-1alpha in cells. Both hypoxia and CoCl2 also up-regulated HIF-1alpha and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes. CONCLUSION: These findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1alpha is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.
Project description:Gut microbes positively affect the physiology of many animals, but the molecular mechanisms underlying these benefits remain poorly understood. We recently reported that bacteria-induced gut hypoxia functions as a signal for growth and molting of the mosquito Aedes aegypti In this study, we tested the hypothesis that transduction of a gut hypoxia signal requires hypoxia-induced transcription factors (HIFs). Expression studies showed that HIF-? was stabilized in larvae containing bacteria that induce gut hypoxia but was destabilized in larvae that exhibit normoxia. However, we could rescue growth of larvae exhibiting gut normoxia by treating them with a prolyl hydroxylase inhibitor, FG-4592, that stabilized HIF-?, and inhibit growth of larvae exhibiting gut hypoxia by treating them with an inhibitor, PX-478, that destabilized HIF-?. Using these tools, we determined that HIF signaling activated the insulin/insulin growth factor pathway plus select mitogen-activated kinases and inhibited the adenosine monophosphate-activated protein kinase pathway. HIF signaling was also required for growth of the larval midgut and storage of neutral lipids by the fat body. Altogether, our results indicate that gut hypoxia and HIF signaling activate multiple processes in A. aegypti larvae, with conserved functions in growth and metabolism.
Project description:Hypoxia-inducible transcription factor-1alpha (HIF-1alpha), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a "master" gene in the tissue hypoxia response during tumor development. However, its role in the apoptosis of non-small cell lung cancer remains unknown. Here, we have studied the effects of HIF-1alpha on apoptosis by modulating HIF-1alpha gene expression in A549 cells through both siRNA knock-down and over-expression.A549 cells were transfected with a HIF-1alpha siRNA plasmid or a HIF-1alpha expression vector. Transfected cells were exposed to a normoxic or hypoxic environment in the presence or absence of 25 mM HEPES and 2-deoxyglucose (2-DG) (5 mM). The expression of three key genes of the glycolysis pathway, glucose transporter type 1(GLUT1), phosphoglycerate kinase 1(PGK1), and hexokinase 1(HK1), were measured using real-time RT-PCR. Glycolysis was monitored by measuring changes of pH and lactate concentration in the culture medium. Apoptosis was detected by TUNEL assay and flow cytometry.Knocking down expression of HIF-1alpha inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, over-expression of HIF-1alpha accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1alpha on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1alpha over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES.During hypoxia in A549 cells, HIF-1alpha promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis.