Role of hypoxia in the hallmarks of human cancer

Hypoxia has been recognized as one of the fundamentally important features of solid tumors and plays a critical role in various cellular and physiologic events, including cell proliferation, survival, angiogenesis, immunosurveillance, metabolism, as well as tumor invasion and metastasis. These responses to hypoxia are at least partially orchestrated by activation of the hypoxia‐inducible factors (HIFs). HIF‐1 is a key regulator of the response of mammalian cells to oxygen deprivation and plays critical roles in the adaptation of tumor cells to a hypoxic microenvironment. Hypoxia and overexpression of HIF‐1 have been associated with radiation therapy and chemotherapy resistance, an increased risk of invasion and metastasis, and a poor clinical prognosis of solid tumors. The discovery of HIF‐1 signaling has led to a rapidly increasing understanding of the complex mechanisms involved in tumor hypoxia and has helped greatly in screening novel anticancer agents. In this review, we will first introduce the cellular responses to hypoxia and HIF‐1 signaling pathway in hypoxia, and then summarize the multifaceted role of hypoxia in the hallmarks of human cancers. J. Cell. Biochem. 107: 1053–1062, 2009. © 2009 Wiley‐Liss, Inc.     

A redox-silent analogue of tocotrienol inhibits hypoxic adaptation of lung cancer cells

We have previously reported that a redox-silent analogue of α-tocotrienol (T3), 6-O-carboxypropyl-α-tocotrienol (T3E) shows more potential anti-carcinogenic property than T3 in a lung cancer cell (A549 cell). However, the mechanisms by which T3E exerts its potential anti-carcinogenic effect is still unclear. As tumor malignancy is associated with hypoxia adaptation, in this study, we examined whether T3E could suppress survival and invasion in A549 cells under hypoxia. Hypoxia treatment drastically-induced activation of the protein tyrosine kinase, Src, and its regulated signaling required for hypoxia adaptation of A549 tumor cells. The survival and invasion capacity of the tumor cells under hypoxia was suppressed by T3E via the inactivation of Src. More specifically, T3E-dependent inhibition of Src-induced Akt activation contributed to suppression of cell survival under hypoxia, and the reduction of fibrinolytic factors such as plasminogen activator-1(PAI-1) via the decrease of hypoxia-inducible factor-2α by T3E led to inhibition of hypoxic invasion. Overall these results suggest that T3E suppresses hypoxia adaptation of A549 cells by the inhibition in hypoxia-induced activation of Src signaling.     

ROS generation in endothelial hypoxia and reoxygenation stimulates MAP kinase signaling and kinase-dependent neutrophil recruitment

Reactive oxygen species (ROS)-induced injury has been shown to occur during the reperfusion phase of ischemia-reperfusion and ROS are known to induce signaling events. We hypothesized that oxygen sensing in endothelial cells is also dependent on internal redox changes during hypoxia and that endothelial cells respond to changing oxygen environments via signaling, switching to an inflammatory phenotype. Endothelial cells exposed to relative hypoxia or the mitochondrial inhibitors rotenone, antimycin A, or FCCP show loss of mitochondrial membrane potential. During hypoxia, an increase in cytoplasmic ROS and glutathione S-transferase activity occurred, suggesting changes in intracellular redox state, mimicked with rotenone or FCCP but inhibited by antimycin A. Phosphorylation of stress-responsive mitogen-activated protein kinases occurred in hypoxia and was rapid and prolonged. Phosphorylation was inhibited by vitamin C, N-acetyl cysteine, or antimycin A. Chelation of intracellular calcium inhibits phosphorylation but the mitochondrial transition pore inhibitor cyclosporin A had no effect. Reoxygenation caused a further round of signaling, which was rapid but transient. Functionally, adhesion of neutrophils after hypoxia-reoxygenation under flow is ROS, P-selectin, and MAPK dependent. Therefore, changes in cellular signaling and phenotype are abrogated by ROS scavengers and suggest their use as therapeutic agents in ischemia-reperfusion.     

p66Shc is involved in promoting HIF-1alpha accumulation and cell death in hypoxic T cells

Hypoxia results in adaptationally appropriate alterations of gene expression through the activation of hypoxia-inducible factor (HIF)-1 to overcome any shortage of oxygen. Peripheral blood mononuclear cells may be exposed to low oxygen tensions for different times as they migrate between blood and various tissues. We and others have previously shown that T-cell adaptation to hypoxia is characterized by a modulation of cytokine expression and an inhibition of T-cell activation. We have recently demonstrated that the adaptor protein p66Shc negatively regulates T-cell activation and survival. We here show that hypoxia enhances HIF-1alpha accumulation and vascular endothelial growth factor production in T cells. Hypoxic T cells expressed high levels of p21(WAF1/CIP1), of the pro-apoptotic molecules BNIP3, a classic HIF target gene, and BAX, as well as low levels of the anti-apoptotic molecule BCLxl, associated with an induction of cell death. We found out that hypoxic T cells expressed p66Shc. Furthermore, using T-cell transfectants expressing p66Shc, as well as T cells derived from mice p66Shc-/-, we defined a role of p66Shc in T-cell responses to hypoxia. Of interest, hypoxic p66Shc-positive transfectants expressed higher level of HIF-1alpha than negative controls. Thus, p66Shc may play an important role in downstream hypoxic signaling, involving HIF-1alpha protein accumulation and cell death in T lymphocytes.     

The breast cancer cells response to chronic hypoxia involves the opposite regulation of NF-kB and estrogen receptor signaling

The progression of cancer is associated with tumor's ability to outgrow the existing vasculature resulting in chronic hypoxic pressure, however the molecular mechanism of cancer cell response to chronic hypoxia is poorly understood. In this study we have analyzed the reorganization of estrogen receptor (ER) signaling in breast cancer cells under chronic hypoxia and examined the role of interrelations between ER and NF-kB signaling in cell adaptation to hypoxia. Using long-term culturing of MCF-7 breast cancer cells in hypoxia-mimetic conditions (cobalt chloride) we have established a hypoxia-tolerant subline characterized by HIF-1 hyperexpression that retained the tolerance to hypoxia even when the cells were returned to normoxic conditions.The hypoxia-tolerant cells were characterized by non-affected ER signaling, irreversible suppression of NF-kB activity, and increased sensitivity to cytokine-induced apoptosis. Estradiol treatment suppressed the NF-kB activity in both parent and hypoxia-tolerant MCF-7 cells. In contrast to MCF-7 cells, the exposure of estrogen-independent MCF-7/T2 subline to chronic hypoxia was not accompanied by noticeable changes in NF-kB activity or cell sensitivity to cytokines. Taken together, the results presented demonstrate the importance of interrelations between ER and NF-kB signaling in the response of estrogen-dependent breast cancer cells to chronic hypoxia.     

Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.     

Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.     

急性低氧与间断低氧适应对家兔局部血流分布的影响

本实验目的在于探讨急性低氧和间断低氧适应对局部血流分布的影响。我们将26只家兔分为急性低氧,低氧适应和常氧对照三组。在麻醉状态下用放射性标记的蟾蜍红细胞分别测定左心室、双侧肾、双侧肾上腺的血流量;并分区测定了大脑皮质、海马、丘脑下部、脑干的局部脑血流。吸入10％低氧混合气1小时后,急性低氧组脑局部、左心室、肾上腺的血流显著高于对照。经2周间断低氧适应后,低氧适应组脑局部(脑干除外)、左心室、肾上腺的血流下降。两组动物低氧时的肾血流变化不明显。结果提示,2周间断低氧适应能改变局部血流分布,血流的再分布有利于改善机体的抗低氧能力。     

Effect of chronic intermittent hypoxia on biological behavior and hypoxia‐associated gene expression in lung cancer cells

Hypoxia plays an important role in the development of solid tumors and is associated with their therapeutic resistance. There exist three major forms of hypoxia: acute, chronic, and intermittent hypoxia. Previous studies have shown that cancer cells could behave in the form of adaptation to hypoxia in tumor growth, which could result in their biological changes and determine their responses to the therapies. To investigate the tumor cells' adaptation to hypoxia, we recreated two models using two lung cancer cell lines in the presence of intermittent hypoxia, which is characterized by changes in oxygen pressure within the disorganized vascular network. We investigated biological behaviors such as cell cycle, proliferation, radiation sensitivity, apoptosis and migration, hypoxia signal pathway in the lung cancer cells treated with chronic intermittent hypoxia, as well as the role of hypoxia inducible factor 1 there, hypoxia‐inducible genes analyzed by real‐time RT‐PCR chip in H446 cells treated with the model. The results indicated the changes of some hypoxia target gene expressions of those induced by hypoxia, some of which were confirmed by real‐time RT‐PCR. The cells mediated by irradiation induced resistance to radiation and apoptosis and increased metastasis in lung cancer cells. It was found that such changes were related to hypoxia inducible factor 1, alpha subunit (HIF‐1α). J. Cell. Biochem. 111: 554–563, 2010. © 2010 Wiley‐Liss, Inc.     

The mitochondrial thioredoxin system regulates nitric oxide-induced HIF-1alpha protein

Hypoxia-inducible factor-1 (HIF-1), consisting of two subunits, HIF-1alpha and HIF-1beta, is a key regulator for adaptation to low oxygen availability, i.e., hypoxia. Compared to the constitutively expressed HIF-1beta, HIF-1alpha is regulated by hypoxia but also under normoxia (21% O(2)) by several stimuli, including nitric oxide (NO). In this study, we present evidence that overexpression of mitochondrial-located thioredoxin 2 (Trx2) or thioredoxin reductase 2 (TrxR2) attenuated NO-evoked HIF-1alpha accumulation and transactivation of HIF-1 in HEK293 cells. In contrast, cytosolic-located thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount and activity under NO treatments. Taking into consideration that thioredoxins affect the synthesis of HIF-1alpha by altering Akt/mTOR signaling, we herein show that p42/44 mitogen-activated protein kinase and p70S6 kinase are involved. Moreover, intracellular ATP was increased in Trx1-overexpressing cells but reduced in cells overexpressing Trx2 or TrxR2, providing thus an understanding of how protein synthesis is regulated by thioredoxins.     

Oxygen-mediated regulation of tumor cell invasiveness. Involvement of a nitric oxide signaling pathway

Tumor hypoxia is associated with a poor prognosis for patients with various cancers, often resulting in an increase in metastasis. Moreover, exposure to hypoxia increases the ability of breast carcinoma cells to invade the extracellular matrix, an important aspect of metastasis. Here, we demonstrate that the hypoxic up-regulation of invasiveness is linked to reduced nitric oxide signaling. Incubation of human breast carcinoma cells in 0.5% versus 20% oxygen increased their in vitro invasiveness and their expression of the urokinase receptor, an invasion-associated molecule. These effects of hypoxia were inhibited by nitric oxide-mimetic drugs; and in a manner similar to hypoxia, pharmacological inhibition of nitric oxide synthesis increased urokinase receptor expression. The nitric oxide signaling pathway involves activation of soluble guanylyl cyclase (sGC) and the subsequent activation of protein kinase G (PKG). Culture of tumor cells under hypoxic conditions (0.5% versus 20% oxygen) resulted in lower cGMP levels, an effect that could be prevented by incubation with glyceryl trinitrate. Inhibition of sGC activity with a selective blocker or with the heme biosynthesis inhibitor desferrioxamine increased urokinase receptor expression. These compounds also prevented the glyceryl trinitrate-mediated suppression of urokinase receptor expression in cells incubated under hypoxic conditions. In contrast, direct activation of PKG using 8-bromo-cGMP prevented the hypoxia- and desferrioxamine-induced increases in urokinase receptor expression as well as the hypoxia-mediated enhanced invasiveness. Further involvement of PKG in the regulation of invasion-associated phenotypes was established using a selective PKG inhibitor, which alone increased urokinase receptor expression. These findings reveal that an important mechanism by which hypoxia increases tumor cell invasiveness (and possibly metastasis) requires inhibition of the nitric oxide signaling pathway involving sGC and PKG activation.     

G proteins as regulators in ethylene-mediated hypoxia signaling

Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O₂ concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead.Key words: submergence, hypoxia, ethylene, G protein, reactive oxygen species, H₂O₂     

Oxygen-dependent PAF receptor binding and intracellular signaling in ovine fetal pulmonary vascular smooth muscle

Circulating levels of platelet-activating factor (PAF) are high in the fetus, and PAF is active in maintaining high PVR in fetal hypoxia (Ibe BO, Hibler S, Raj J. J Appl Physiol 85: 1079-1085, 1998). PAF synthesis by fetal pulmonary vascular smooth muscle cells (PVSMC) is high in hypoxia, but how oxygen tension affects PAF receptor (PAF-r) binding in PVSMC is not known. We studied the effect of oxygen tension on PAF-r binding and signaling in fetal PVSMC. PAF binding was saturable. PAF-r density (B(max): fmol/10(6) cells; means +/- SE, n = 6), 25.2 +/- 0.77 during hypoxia (Po(2) <40 Torr), was higher than 13.9 +/- 0.44 during normoxia (Po(2) approximately 100 Torr). K(d) was twofold lower in hypoxia than normoxia. PAF-r protein expression, 35-40% greater in hypoxia, was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting translational regulation. IP(3) release, an index of PAF-r-mediated cell signaling, was greater in hypoxia (EC(50): hypoxia, 2.94 +/- 0.61; normoxia, 5.85 +/- 0.51 nM). Exogenous PAF induced 50-90% greater intracellular calcium flux in cells during hypoxia, indicating hypoxia augments PAF-r-mediated cell signaling. PAF-r phosphorylation, with or without 5 nM PAF, was 40% greater in hypoxia. These data show 1) hypoxia upregulates PAF-r binding, PAF-r phosphorylation, and PAF-r-mediated intracellular signaling, evidenced by augmented IP(3) production and intracellular Ca(2+) flux; and 2) hypoxia-induced PAF-r phosphorylation results in activation of PAF-r-mediated signal transduction. The data suggest the fetal hypoxic environment facilitates PAF-r binding and signaling, thereby promoting PAF-mediated pulmonary vasoconstriction and maintenance of high PVR in utero.