Hypoxia-induced Protection Research Articles

Receptor-Mediated Mitophagy Rescues Cancer Cells under Hypoxic Conditions.

Simple SummaryStimulation of cell death is a promising strategy for tumor cell elimination. Many tumors develop under hypoxic conditions, which alter tumor cell biology and makes them resistant to cell death stimuli. One of the mechanisms responsible for tumor cell resistance to treatment could be the stimulation of mitophagy (mitochondrial quality control), which eliminates mitochondria that can trigger the mitochondrial pathway in apoptosis. We found that hypoxia stimulates mitophagy via enhanced expression of BNIP3 and BNIP3L proteins, which are involved in receptor-mediated mitophagy.Targeting mitochondria with thenoyltrifluoroacetone (TTFA), an inhibitor of Complex II in the respiratory chain, stimulated cisplatin-induced apoptosis in various cell lines in normoxia but not in hypoxia. This can be explained by the elimination of mitochondria involved in triggering apoptotic cell death by mitophagy, either Parkin-dependent or receptor-mediated. Treatment with TTFA alone or in combination with cisplatin did not cause accumulation of PINK1, meaning that under hypoxic conditions cells survive through activation of a receptor-mediated pathway. Hypoxia triggers the accumulation of BNIP3 and BNIP3L (also known as NIX), key participants in receptor-mediated mitophagy. Under hypoxic conditions, stimulation of autophagy, as assessed by the accumulation of lipidated form of LC3 (LC3II), was observed. To exclude the contribution of canonical macroautophagy in LC3II accumulation, experiments were performed using U1810 cells lacking ATG13, a key enzyme of macroautophagy. Despite the absence of ATG13, hypoxia-mediated accumulation of LC3II was not affected, underlying the importance of the receptor-mediated pathway. In order to prove the protective role of BNIP3 against cisplatin-induced apoptosis, BNIP3-deficient A549 cells were used. Surprisingly, a BNIP3 knockout did not abolish hypoxia-induced protection; however, in cells lacking BNIP3, a compensatory upregulation of BNIP3L was detected. Thus, in the absence of BNIP3, mitophagy could be maintained by BNIP3L and lead to cell death suppression due to the elimination of proapoptotic mitochondria. When both BNIP3 and BNIP3L were knocked out, the inhibitory effect of hypoxia on apoptosis was diminished, although not abolished completely. Undoubtedly, receptor-mediated mitophagy is likely to be one of the mechanisms responsible for cell death suppression under hypoxic conditions.

Normobaric intermittent hypoxic training regulates microglia phenotype and enhances phagocytic activity.

The effects of intermittent hypoxic training or conditioning on many pathological conditions have been widely investigated. One of the pathological conditions dealt with intermittent hypoxic training is ischemic stroke. Well-known mechanisms of intermittent hypoxia-induced protection are related to increased energy metabolism and the enhanced antioxidant effects. In the last decades, the role of microglia in the progress of ischemic stroke-related brain damage has been focused. The dual-edge function of microglia indicates that the microglia-mediated inflammatory response is definitely beneficial in the early stage of ischemic stroke, but long-term activation of microglia is rather detrimental during the recovery process. The effect of IHT on microglia polarization is not investigated. This study focused on whether IHT regulates the polarization of microglia without dampening its classic phagocytic function. This study will provide pivotal information regarding the effects of IHT on the long-term effects on the recovery process from ischemic stroke.

Abstract 1534: Hes1 regulates hypoxia induced chemoresistance of myeloma cells

Abstract Bone marrow (BM) microenvironment plays a critical role in response of tumor cells to chemotherapeutic drugs. Hypoxia has been shown to contribute to pathogenesis not only solid but also hematological tumors. While normal BM environment is hypoxic, tumor growth is associated with increased hypoxia in BM niche. We have previously demonstrated that activation of Notch signaling could be one of the mechanisms responsible for survival of multiple myeloma (MM) cells from chemotherapeutic drugs. Here we investigated whether hypoxia could modulate Notch signaling and induce chemoresistance of MM cells. Three MM cell lines were treated with doxorubicin or melphalan in hypoxic (1% O2) or normoxic conditions. Using Annexin V binding assay we demonstrated that hypoxia induced chemoresistance in only one (H929) cell line studied. This effect was associated with significant up-regulation of expression of Notch target gene Hes1 and its protein level while Hes1 level was unchanged in other two MM cell lines. Inhibition of Hes1 with siRNA canceled the protective effect of hypoxia on MM cells. To further confirm the involvement of Notch pathway in regulation of drug sensitivity, Hes-1 was overexpressed in MM cells in normoxic condition using vector pIRES-AcGFP-Hes-1. MM cells with increased Hes1 level became less sensitive to chemotherapeutics as compared to cells transfected with control vector. Hes-1 is considered to be a Notch target gene. However, hypoxia did not affect Notch receptors expression level in MM cells. The effect of hypoxia on Hes1 was Notch-independent as was evident from Notch/CBF1 reporter assay. In addition, inhibition of Notch receptor cleavage with gamma-secretase inhibitor GSI-XII (Calbiochem) did not affect hypoxia induced MM cell chemoresistance. Up-regulation of Hes-1 was induced by HIF-1a as treatment of MM cells with hypoxia mimetic deferoxamine resulted in up-regulation of Hes1 and inhibition of HIF-1a with siRNA abrogated hypoxia-induced protection of MM cells. Thus, our study, for the first time, demonstrates that hypoxia could induce chemoresistance of MM cells through Notch-independent up-regulation of Hes-1. These results could be important not only for understanding the cancer biology but for designing therapeutic intervention in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1534. doi:1538-7445.AM2012-1534

Physiologic Hypoxia Protects Chronic Myelogenous Leukemia Progenitors From Elimination by Imatinib Mesylate.

Abstract 2181Poster Board II-158The use of Abl tyrosine kinase inhibitors (TKI), such as imatinib mesylate (IM), has seen a major advance in the control of chronic myelogenous leukemia (CML). However, since TKIs do not seem to eliminate CML stem cells, the use of TKIs may not represent a curative approach. Recent reports have shown that the bone marrow (BM) microenvironment, which facilitates the proliferation and renewal of resident hematopoietic stem cells (HSC), is hypoxic. The average BM O2 tension has been measured at 6-7% O2 in humans (physiologic hypoxia), including in patients with leukemia (Fiegl et al. Blood, 2009), while levels in the HSC niche are estimated to be even lower (<1% O2). In light of the above, it is likely that CML stem cells also reside in the HSC niche, and are similarly adapted for survival and self-renewal in this environment. Furthermore, although hypoxia is known to be associated with both radio- and chemo-resistance in solid tumors, its role in leukemia has has not been thoroughly investigated, particularly in the context of resistance to targeted therapies. Accordingly, we hypothesized that hypoxia protects CML progenitor cells from elimination by IM, a phenomenon which may contribute to disease persistence. To test the above, we examined if hypoxia modified the response of CML progenitor cells toward IM. Primary chronic phase (CP) CML samples “from either BM or peripheral blood (PB)” were obtained at the time of diagnosis from seven individuals with clinically-defined IM-sensitive (IM-S) or IM-resistant (IM-R) disease. Cells were then incubated with IM (0, 0.25, 1 or 5μM) under hypoxic (0.5% O2) or normoxic (21% O2) conditions for 96 hours. The treated cells were then harvested and plated in methylcellulose for 14 days (under 21% O2), after which the number of colony-forming cells (CFCs) were counted by two independent observers. In the absence of IM, four of the seven samples had a significant increase in CFCs (1.5-2.4 fold) when cultured in 0.5% O2 vs 21% O2, suggesting that primary CML CFCs may be better maintained in hypoxia. Furthermore, when treated with IM, six out of seven samples cultured under 0.5% O2 demonstrated dramatic increases in CFCs compared to those treated under 21% O2: a 2.3 to 9.0-fold increase at 1μM, and a 4.4 to 35.0-fold increase at 5μM IM. We also found that the protective effect of hypoxia was independent of the original source of the CFCs (BM or PB). In addition, we found that progenitors from both IM-S and IM-R patients were protected from IM under 0.5% O2, suggesting that hypoxia-induced protection is a general feature of CML progenitors. In order to establish a model for further study, we also tested if hypoxia elicits similar responses in four human CML cell lines (K562, AR230, LAMA84, and BV173). In contrast to primary CML cells, we found that hypoxia actually impaired the ability of all four cell lines to form colonies, and also did not confer protection from IM. These results show that CML cell lines have adapted to conditions of normoxia, and are thus inappropriate models to study the hypoxic response in CML. Additional data will be presented describing the mechanisms that may underlie the protective effect of hypoxia, as well as compounds that can counteract such effects. Ongoing experiments using the LT-CIC assay will also determine the response of primitive CML progenitors to growth under conditions of physiologic hypoxia vs normoxia. In conclusion, our results show that physiologic hypoxia protects CML progenitors from IM, and suggest that blocking of hypoxia-induced survival pathway(s) in CML progenitor cells may facilitate the elimination of residual CML progenitors. Disclosures:No relevant conflicts of interest to declare.

Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity

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.

Novel anti-oxidative role of calreticulin in protecting A549 human type II alveolar epithelial cells against hypoxic injury

Short-term hypoxic pretreatment is an effective approach to protect the lung from subsequent prolonged hypoxic injury under conditions such as lung transplantation, shock, and trauma. However, the signaling pathways are not well understood. By use of high-throughput, two-dimensional electrophoresis combined with mass spectrometry, we found that short-term hypoxic treatment upregulated calreticulin (CRT), an endoplasmic-reticulum stress protein, in A549 human type II alveolar epithelial cells. Genetic manipulation of CRT expression in A549 cells through small interferring RNA inhibition or overexpression demonstrated a positive correlation between CRT expression level and cell viability in subsequent prolonged hypoxia, which indicates that CRT is a key mediator of short-term hypoxia-induced cell protection. Importantly, CRT overexpression prevented reactive oxygen species (ROS) accumulation during prolonged hypoxia by inducing the expression of thioredoxin (TRX), an antioxidant, in A549 cells. Furthermore, CRT promoted the nuclear translocation of nuclear factor-E2-related factor 2, the transcription factor of TRX. Finally, overexpressing an inactive TRX mutant reversed the effects of CRT on ROS accumulation and cell protection. Our results demonstrate that CRT stimulates the anti-oxidant pathway and contributes to short-term hypoxia-induced protection in A549 type II alveolar epithelial cells, which may have potential therapeutic ramifications for hypoxic pulmonary diseases.

Hypoxia Inhibits Paclitaxel-Induced Apoptosis through Adenosine-Mediated Phosphorylation of Bad in Glioblastoma Cells

Solid tumors contain hypoxic cells that are resistant to radiotherapy and chemotherapy. The resistance in glioblastoma has been linked to the expression of antiapoptotic Bcl-2 family members. In this study, we found that in human glioblastoma cells hypoxia induces the phosphorylation of the Bcl-2 family protein Bad, thus protecting hypoxic cells from paclitaxel-induced apoptosis. Akt activation is required for the hypoxia-induced protection. In contrast, the extracellular signal-regulated kinase 1/2 activities have only a partial effect, being able to modulate Bad phosphorylation but not paclitaxel-induced apoptosis in hypoxia. We also demonstrated that the degradation of adenosine with adenosine deaminase, the knockdown of A(3) adenosine receptor expression by gene silencing, and the blockade of this receptor through A(3) receptor antagonists blocked the hypoxia-induced phosphorylation of Bad and the prolonged cell survival after treatment with paclitaxel in hypoxia. Thus, the adenosinergic signaling may be an essential component in the hypoxia survival pathway. These results suggest that hypoxia-induced chemoresistance of human glioblastoma cells may occur in a novel mechanism involving activation of adenosine-A(3) receptor-Akt pathway, which mediates Bad inactivation and favors cell survival.

Hypoxia protects HepG2 cells against etoposide-induced apoptosis VIA a HIF-1-independent pathway

Tumor hypoxia has been described to increase the resistance of cancer cells to radiation therapy and chemotherapy. It also supports the invasiveness and metastatic potential of the tumor. However, few data are available on the transduction pathway set up under hypoxia and leading to this resistance against anti-cancer therapies. HIF-1, the main transcription factor activated by hypoxia, has been recently shown to participate to this process although its role as an anti- or a pro-apoptotic protein is still controversy. In this study, we showed that hypoxia protected HepG2 cells against etoposide-induced apoptosis. The effect of hypoxia on cell death was assayed by measuring different parameters of the apoptotic pathway, like DNA fragmentation, caspase activity and PARP-1 cleavage. The possible implication of HIF-1 in the anti-apoptotic role of hypoxia was investigated using HIF-1α siRNA. Our results indicated that HIF-1 is not involved in the hypoxia-induced anti-apoptotic pathway. Another transcription factor, AP-1, was studied for its potential role in the hypoxia-induced protection against apoptosis. Specific inhibition of AP-1 decreased the protection effect of hypoxia against etoposide-induced apoptosis. Together, all these data underline that hypoxia could mediate its anti-apoptotic role via different transcription factors depending on the cellular context and pro-apoptotic stimuli.

Hypoxia confers protection against apoptosis via PI3K/Akt and ERK pathways in lung cancer cells

Hypoxia confers protection against apoptosis in cancer cells, and this hypoxia-induced resistance to apoptosis has been suggested to be associated with genetic and adaptive changes. However, it is not clear whether survival signals, such as the PI3K/Akt and ERK pathways are involved. We investigated the roles of these pathways in hypoxia-induced protection against apoptosis in lung cancer cells. Treatment of cells with either ultraviolet (UV) or etoposide induced apoptosis time-dependently in A549 and NCI-H157 cells. However, though hypoxia alone neither induced apoptosis nor reduced cell survival, it suppressed the apoptosis induced by UV or etoposide. Moreover, hypoxia activated the PI3K/Akt and ERK pathways, and blocking the activation of either pathway reversed resistance to UV- and etoposide-induced apoptosis in response to hypoxia. These results suggest that hypoxia confers resistance to UV- or etoposide-mediated apoptosis in lung cancer cells via the activations of the PI3K/Akt and the ERK pathways.

Hypoxic Preconditioning Protects against Ischemic Brain Injury

Animals exposed to brief periods of moderate hypoxia (8% to 10% oxygen for 3 hours) are protected against cerebral and cardiac ischemia between 1 and 2 days later. This hypoxia preconditioning requires new RNA and protein synthesis. The mechanism of this hypoxia-induced tolerance correlates with the induction of the hypoxia-inducible factor (HIF), a transcription factor heterodimeric complex composed of inducible HIF-1alpha and constitutive HIF-1beta proteins that bind to the hypoxia response elements in a number of HIF target genes. Our recent studies show that HIF-1alpha correlates with hypoxia induced tolerance in neonatal rat brain. HIF target genes, also induced following hypoxia-induced tolerance, include vascular endothelial growth factor, erythropoietin, glucose transporters, glycolytic enzymes, and many other genes. Some or all of these genes may contribute to hypoxia-induced protection against ischemia. HIF induction of the glycolytic enzymes accounts in part for the Pasteur effect in brain and other tissues. Hypoxia-induced tolerance is not likely to be equivalent to treatment with a single HIF target gene protein since other transcription factors including Egr-1 (NGFI-A) have been implicated in hypoxia regulation of gene expression. Understanding the mechanisms and genes involved in hypoxic tolerance may provide new therapeutic targets to treat ischemic injury and enhance recovery.

Hypoxia induces an autocrine-paracrine survival pathway via platelet-derived growth factor (PDGF)-B/PDGF-beta receptor/phosphatidylinositol 3-kinase/Akt signaling in RN46A neuronal cells.

In neurons, hypoxia activates intracellular death-related pathways, yet the antiapoptotic mechanisms triggered by hypoxia remain unclear. In RN46A neuronal cells, minimum media growth conditions induced cell death as early as 12 h after the cells were placed in these conditions (i.e., after removal of B-27 supplement). However, apoptosis occurred in hypoxia (1% O2) only after 48 h, and in fact hypoxia reduced the apoptosis associated with trophic factor withdrawal. Furthermore, hypoxia induced time-dependent increases in expression of platelet-derived growth factor (PDGF) B mRNA and protein, as well as PDGF-beta receptor phosphorylation. Although exogenous PDGF-BB induced only transient Akt activation, hypoxia triggered persistent activation of Akt for up to 24 h. Inhibition of phosphatidylinositol 3-kinase (PI3K) or of PDGF-beta receptor phosphorylation abrogated both hypoxia-induced and exogenous PDGF-BB-induced Akt phosphorylation, and it completely abolished hypoxia-induced protection from media supplement deprivation, which suggests that the long-lasting activation of Akt during hypoxia and the prosurvival induction were due to endogenously generated PDGF-BB. Furthermore, these inhibitors decreased hypoxia-inducible factor 1alpha (HIF-1alpha) DNA binding, which suggests that the PDGF/PDGF-beta receptor/Akt pathway induces downstream HIF-1alpha gene transcription. We conclude that in RN46A neuronal cells, hypoxia activates an autocrine-paracrine antiapoptotic mechanism that involves up-regulation of PDGF-B and PDGF-beta receptor-dependent activation of the PI3K/Akt signaling pathway to induce downstream transcription of survival genes.

Time-Dependent Changes in Catecholamine Turnover in Spontaneously Hypertensive Rats Exposed to Hypoxia

In three separate experiments, 4 to 5-week-old spontaneously hypertensive rats (SHR) and normotensive controls (Wistar-Kyoto [WKy]) were exposed to hypobaric hypoxia (simulated altitude = 3658 m) for 3 hr, 3 days, or 3 weeks. Comparable groups were maintained in ambient laboratory conditions (normoxia). Hypoxia prevented the increase in blood pressure noted in 8-week-old normoxic SHR. Right ventricular hypertrophy first occurred after 3 days of hypoxia, and was found in both SHR and WKy. Catecholamine turnover was measured using the tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine. In myocardium, both strains evidenced hypoxia-induced changes in norepinephrine (NE) turnover, which was increased at 3 hr, normalized at 3 days, and increased again at 3 weeks. Reduced basal NE concentration at 3 days indicated a temporary deficit in synthetic capacity, which would allow maintenance of a heightened neuronal output. Catecholamine turnover in right and left ventricles differed little in response to hypoxia, in spite of differential hemodynamic demands on SHR versus WKy or on right versus left ventricle. In contrast to findings in myocardium, significant interactive effects between strain and altitude exposure were noted for adrenal catecholamine turnover. Specifically, hypoxia exerted a suppressive influence in SHR that was not evident in WKy, and this may represent an important component of hypoxia-induced protection against the development of spontaneous hypertension.