Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest in the G(0)/G(1) phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G(0)/G(1) phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells.     

The in vitro sensitivity of chronically hypoxic EMT6/SF cells to X-radiation and hypoxic cell radiosensitizers

We have investigated the effect of extreme, prolonged hypoxia on the radiosensitivity of EMT6/SF cells in vitro. As cells were kept hypoxic for 1-24 h, their radiosensitivity increased, but no further change was noted for hypoxic incubation beyond 24 h. Chronically hypoxic (45 h) cells were more radiosensitive than acutely hypoxic (1 h) cells by a factor of 1.43. When chronically hypoxic cells were re-aerated, the increased radiosensitivity persisted, although it was reduced. Misonidazole (MISO) radiosensitization was equally effective under conditions of acute and chronic hypoxia. In contrast, MISO, SR2555 and SR2508 were more cytotoxic in chronically hypoxic cultures than in acutely hypoxic cells. Measurements suggested that intracellular thiols may play an important role in the effects observed.     

NADPH oxidase subunit 4 mediates cycling hypoxia-promoted radiation resistance in glioblastoma multiforme

Cycling hypoxia is a well-recognized phenomenon within animal and human solid tumors. It mediates tumor progression and radiotherapy resistance through mechanisms that involve reactive oxygen species (ROS) production. However, details of the mechanism underlying cycling hypoxia-mediated radioresistance remain obscure. We have previously shown that in glioblastoma, NADPH oxidase subunit 4 (Nox4) is a critical mediator involved in cycling hypoxia-mediated ROS production and tumor progression. Here, we examined the impact of an in vivo tumor microenvironment on Nox4 expression pattern and its impact on radiosensitivity in GBM8401 and U251, two glioblastoma cell lines stably transfected with a dual hypoxia-inducible factor-1 (HIF-1) signaling reporter construct. Furthermore, in order to isolate hypoxic tumor cell subpopulations from human glioblastoma xenografts based on the physiological and molecular characteristics of tumor hypoxia, several techniques were utilized. In this study, the perfusion marker Hoechst 33342 staining and HIF-1 activation labeling were used together with immunofluorescence imaging and fluorescence-activated cell sorting (FACS). Our results revealed that Nox4 was predominantly highly expressed in the endogenous cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor microenvironment. Moreover, when compared to the normoxic or chronic hypoxic cells, the cycling hypoxic tumor cells derived from glioblastoma xenografts have much higher Nox4 expression, ROS levels, and radioresistance. Nox4 suppression in intracerebral glioblastoma-bearing mice suppressed tumor microenvironment-mediated radioresistance and enhanced the efficiency of radiotherapy. In summary, our findings indicated that cycling hypoxia-induced Nox4 plays an important role in tumor microenvironment-promoted radioresistance in glioblastoma; hence, targeting Nox4 may be an attractive therapeutic strategy for blocking cycling hypoxia-mediated radioresistance.     

The radiation response of cells recovering after chronic hypoxia

Experiments were performed to study the influence of hypoxic pretreatment on the radiation response of A431 human squamous carcinoma cells. Reaeration for 10 min after chronic hypoxia (greater than 2 h) was found to enhance the radiosensitivity of A431 cells, and the maximal effect was seen for those cells reaerated after 12 h of hypoxia. The radiosensitivity enhancement for reaerated cells after 12 h of hypoxia was maximized by 5 min after the return to aerobic conditions and reached the control level by 12 h of reaeration. This enhanced radiosensitive state was characterized by a reduced shoulder region and increased slope of the radiation dose-response curve for cells in both the exponential and plateau phases of growth. There was a slight increase in the number of G1 and decrease in the number of S and G2 + M cells for both exponential- and plateau-phase cultures following 12 h hypoxic treatment. Although growth inhibition induced by 12 h of hypoxia was seen for cells in the exponential phase, there was no cell number change in the plateau-phase culture after hypoxia. Plating efficiency (PE) of cells in both growth phases was reduced by 30% after hypoxia. Furthermore, in the exponential-phase culture, the extent of reduction in PE after hypoxia was similar among cells in different phases of the cell cycle. Although S-phase cells in exponentially growing cultures were relatively more resistant to radiation than G1 and G2 + M cells, the cell age-response pattern was the same whether the cells had been aerobic or hypoxic before reaeration and irradiation. Furthermore, the enhancement ratio associated with reaeration after 12 h of hypoxia for these three subpopulations of cells was 1.3. Our results indicate that the increase in radiosensitivity due to reaeration after chronic hypoxia is unlikely to be related to the changes of cell cycle stage and growth phase during hypoxic treatment.     

The in vitro sensitivities to radiation and misonidazole of mouse bone marrow cells derived from different microenvironments

Previous studies had indicated that haematopoietic cells (CFU-GM) which reside within compact bone are resistant to ionizing radiation and sensitive to the cytotoxic action of misonidazole (MISO) relative to cells which reside within the core of mouse femurs. It was postulated that the microenvironment within compact bone might be relatively hypoxic. CFU-GM from femur cores (Fraction 1) and from compact bone (Fraction 3) have been exposed to ionizing radiation and to the hypoxic cell radiosensitizer, MISO, under controlled conditions of oxygenation in vitro. The inherent radiosensitivity of aerated Fraction 1 CFU-GM is similar to their in vivo radiosensitivity. An oxygen enhancement ratio of 2.2 is observed for these cells in vitro. On the other hand, the in vitro radiosensitivity of hypoxic Fraction 3 CFU-GM was similar to their in vivo radiosensitivity. The oxygen enhancement ratio for Fraction 3 cells was 1.5, significantly lower than that observed for Fraction 1 cells. When CFU-GM cells were exposed to MISO under hypoxic conditions in vitro it was found that Fraction 3 CFU-GM were more sensitive to its cytotoxic action than were cells from Fraction 1. These data are consistent with the interpretation that some CFU-GM reside in an environment of relative hypoxia within the compact bone of the mouse femur.     

Role of Twist in vasculogenic mimicry formation in hypoxic hepatocellular carcinoma cells in vitro

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive human tumor cells to form matrix-rich networks de novo when cultured on a three-dimensional matrix, thus mimicking embryonic vasculogenesis. Some studies have shown that tumor hypoxia can promote tumor cells to form vessel-like tubes in vitro and express genes associated with VM. Although, the mechanisms involved in hypoxia-induced VM remain elusive, we hypothesized that the epithelial–mesenchymal transition (EMT) regulator Twist may play a major role in hypoxia-induced VM. We investigated this hypothesis in vitro by pretreating hepatocellular carcinoma cells under hypoxic conditions. Following the hypoxia treatment, the cells formed typical pipe-like VM networks. Moreover, the expression of VM markers was increased. Hypoxia-induced VM was accompanied by the increased expression of Twist. Twist siRNA reversed the effects of hypoxia on VM. These results suggest that the overexpression of Twist correlates to hypoxia-induced VM in hepatocellular carcinoma cells.     

Hypoxia inhibits TRAIL-induced tumor cell apoptosis: Involvement of lysosomal cathepsins

Tumor hypoxia interferes with the efficacy of chemotherapy, radiotherapy, and tumor necrosis factor-α. TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a potent apoptosis inducer that limits tumor growth without damaging normal cells and tissues in vivo. We present evidence for a central role of lysosomal cathepsins in hypoxia and/or TRAIL-induced cell death in oral squamous cell carcinoma (OSCC) cells. Hypoxia or TRAIL-induced activation of cathepsins (B, D and L), caspases (-3 and -9), Bid cleavage, release of Bax and cytochrome c, and DNA fragmentation were blocked independently by zVAD-fmk, CA074Me or pepstatin A, consistent with the involvement of lysosomal cathepsin B and D in cell death. Lysosome stability and mitochondrial membrane potential were reduced in hypoxia and TRAIL-induced apoptosis. However, TRAIL treatment under hypoxic condition resulted in diminished apoptosis rates compared to treatment under normoxia. This inhibitory effect of hypoxia on TRAIL-induced apoptosis may be based on preventing Bax activation and thus protecting mitochondria stability. Our data show that TRAIL or hypoxia independently triggered activation of cathepsin B and D leading to apoptosis through Bid and Bax, and suggest that hypoxic tissue regions provide a selective environment for highly apoptosis-resistant clonal cells. Molecular therapy approaches based on cathepsin inhibitors need to address this novel tumor-preventing function of cathepsins in OSCC.     

Inhibitory effect of a naphthazarin derivative, S64, on heat shock factor (Hsf) activation and glutathione status following hypoxia

The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment. Resistance of hypoxic cells to ionizing radiation and anticancer drugs has in part been attributed to changes in altered gene expression by hypoxia. We previously reported an activation of heat shock factor (Hsf) in murine tumor RIF cells following hypoxia and suggested that a subsequent accumulation of heat shock protein(s) (Hsp) is likely to contribute to the malignant progression of hypoxic tumor cells (Baek et al., 2001). In this study, we showed that hypoxia induced a DNA-binding activity of Hsf and activation of hsp70 gene expression in colon cancer Clone A cells, and that a naphthazarin derivative, S64, significantly inhibited the hypoxia-inducible hsp70 gene expression in Clone A cells. We also showed that S64 significantly reduced the cellular glutathione levels in this cell line. Considering the proposed effects of Hsp and glutathione on radiation and chemotherapy sensitivity, we suggest that the inhibitory effects of S64 on Hsf activation and cellular glutathione levels have potentially important clinical implications. We believe that the previously reported in vitro and in vivo anti-tumor effect of S64 (Song et al., 2000a, 2001) might be attributed, at least in part, to its effect on Hsf activation and/or glutathione depletion. We also believe that the detailed molecular mechanisms underlying the effects of S64 on Hsf and glutathione level following hypoxia deserve a more rigorous future study, the results of which could offer novel strategy to manipulate the resistance mechanisms of solid tumors.     

The influence of hypoxia on the relative sensitivity of human tumor cells to 62.5 MeV (p-->Be) fast neutrons and 4 MeV photons

Fast neutrons have been used in the clinical radiation therapy of tumors largely because of experimental evidence that their cytotoxic effects are much less dependent on oxygen levels than those of low-LET photons. The potential therapeutic advantage of fast neutrons based on hypoxia alone can be calculated as the "hypoxic gain factor", which is the ratio of the OERs for the fast-neutron compared to the photon beams. The hypoxic gain factor that is generally anticipated based on studies with established mammalian cell lines is about 1.6. However, surprisingly few studies have examined the influence of hypoxia on the fast-neutron radiosensitivity of human tumor cells of different histological types. For this reason, we have determined the OERs of five human tumor cell lines exposed to 62.5 MeV (p-->Be) cyclotron-generated fast neutrons or 4 MeV photons from a clinical linear accelerator. The OERs for four chemotherapy-naive cell lines, HT29/5, Hep2, HeLa and RT112, were invariably greater for photons than for neutrons, but all of these values were lower than expected on the basis of the previous literature. Despite their low OERs, these cell lines showed hypoxic gain factors that were within the range of 1.31-1.63, indicating that such effects cannot entirely explain the disappointing clinical results obtained with fast neutrons. In contrast, comparison of the surviving fractions at clinically relevant doses (1.6 Gy of neutrons and 2.0 Gy of photons) for these four tumor cell lines suggested that little benefit should result from neutron treatment. Only the cisplatin-resistant OAW42-CP line showed a significant hypoxic gain factor by this method of analysis. We conclude that, at the dose fractions used in clinical radiation therapy, there may not be a radiobiological precedent for higher local control rates after fast-neutron irradiation of hypoxic tumor cells.     

Hypoxia/reoxygenation: a dynamic regulator of lysyl oxidase-facilitated breast cancer migration

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.     

Paracrine effects of hypoxic fibroblast-derived factors on the MPT-ROS threshold and viability of adult rat cardiac myocytes

Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. With the use of an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts and examined two distinct effects of hypoxic fibroblast-conditioned medium (HFCM) on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of tumor necrosis factor (TNF)-alpha by cardiac fibroblasts. HFCM significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high-precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Furthermore, exposure of cardiac myocytes to HFCM for 5 h led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium spiked with TNF-alpha at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, because both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes.     

Gliotoxin enhances radiotherapy via inhibition of radiation-induced GADD45a, p38, and NFkappaB activation

The purpose of the study was to elucidate the mechanism underlying the enhancement of radiosensitivity to 60Co gamma-irradiation in human hepatoma cell line HepG2 pretreated with gliotoxin. Enhancement of radiotherapy by gliotoxin was investigated in vitro with human hepatoma HepG2 cell line. Apoptosis related proteins were evaluated by Western blotting. Annexin V/PI and reactive oxygen species (ROS) were quantified by Flow Cytometric (FACS) analysis. Gliotoxin (200 ng/ml) combined with radiation (4 Gy) treated cells induced apoptosis. Cells treated with gliotoxin (200 ng/ml) prior to irradiation at 4 Gy induced the expression of bax and nitric oxide (NO). The gliotoxin-irradiated cells also increased caspase-3 activation and ROS. Gadd45a, p38, and nuclear factor kappa B (NFkappaB) activated in irradiated cells was inhibited by Gliotoxin. Specific inhibitors of p38 kinase, SB203580, significantly inhibited NFkappaB activation and increased the cytotoxicity effect in cells exposed to gliotoxin combined with irradiation. However, SB203580 did not suppress the activation of Gadd45a in irradiated cells. Gliotoxin inhibited anti-apoptotic signal pathway involving the activation of Gadd45a-p38-NFkappaB mediated survival pathway that prevent radiation-induced cell death. Therefore, gliotoxin, blocking inflammation pathway and enhancing irradiation-induced apoptosis, is a promising agent to increase the radiotherapy of tumor cells.     

Similar radiation sensitivities of acutely and chronically hypoxic cells in HT 1080 fibrosarcoma xenografts

It has been suggested that chronically hypoxic tumor cells may be more radiosensitive than acutely hypoxic or even aerobic cells. In the present study we have used the fact that chronically, but not acutely, hypoxic cells that are transformed with a vector containing an enhanced green fluorescent protein (EGFP) driven by a hypoxia-responsive promoter become green (high EGFP) at low oxygen concentrations and can be viably sorted from transplanted tumors in vitro. We showed that the fluorescence of HT 1080 human fibrosarcoma cells stably transfected with this vector increases constantly with decreasing O2 concentrations (<2%, longer than 1 h, half maximum approximately 0.2% for longer than 8 h), and that cells subjected to repeated cycles of hypoxia/reoxygenation (simulating acutely hypoxic cells) showed only background fluorescence. To test the radiosensitivity of acutely and chronically hypoxic cells in tumors, we isolated high-EGFP ("chronically hypoxic") and low-EGFP cells (containing both acutely hypoxic and aerobic cells) from HT 1080 xenograft tumors by fluorescence-activated cell sorting (FACS), immediately after in situ treatment with 20 Gy (ambient or clamped), and plated the cells to determine clonogenic survival in vitro. We found that the survival of high-EGFP cells after irradiation was not affected by clamping, suggesting that all, or almost all, of these cells were fully (chronically) hypoxic. Also, the survival of the low-EGFP cells irradiated under clamped conditions (acutely hypoxic cells) was not significantly different from that of the high-EGFR cells (chronically hypoxic) cells irradiated under nonclamped (or clamped) conditions. We therefore conclude that, at least in this tumor model, the radiation sensitivity of chronically hypoxic cells is similar to that of the acutely hypoxic cells.     

Tumour necrosis factor-α suppresses the hypoxic response by NF-κB-dependent induction of inhibitory PAS domain protein in PC12 cells

Inflammation is often accompanied by hypoxia. However, crosstalk between signalling pathways activated by inflammation and signalling events that control adaptive response to hypoxia is not fully understood. Here we show that exposure to tumour necrosis factor-α (TNF-α) activates expression of the inhibitory PAS domain protein (IPAS) to suppress the hypoxic response caused by hypoxia-inducible factor (HIF)-1 and HIF-2 in rat pheochromocytoma PC12 cells but not in human hepatoma Hep3B cells. This induction of IPAS was dependent on the nuclear factor-κB (NF-κB) pathway and attenuated hypoxic induction of HIF-1 target genes such as tyrosine hydroxylase (TH) and vascular endothelial growth factor (VEGF). HIF-dependent reporter activity in hypoxia was also decreased following TNF-α treatment. Knockdown of IPAS mRNA by small interfering RNA (siRNA) restored the TNF-α-suppressed hypoxic response. These results indicate that TNF-α is a cell-type specific suppressor of HIFs and suggest a novel crosstalk between stimulation by inflammatory mediators and HIF-dependent hypoxic response.     

Hypoxia-resistant profile implies vulnerability of cancer stem cells to physiological agents,which suggests new therapeutic targets

We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state. Experiments were performed in vivo and in vitro using CSCs obtained from various anaplastic tumors: human melanoma, leukemia, and rat hepatoma cells. Pretreatment of melanoma CSCs with pyruvate significantly reduces their self-renewal in vitro and tumorigenicity in vivo. The metabolic network underlying the cytotoxic effect of the physiological factors was thoroughly defined, principally using AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem stage. This network, based on a tight integration of aerobic glycolysis, cellular redox state, and folate metabolism, is centered on the cellular NADP/NADPH ratio that controls the redox pathway of folate utilization in purine synthesis. On the whole, this study indicates that pyruvate, FH₄, and glutamine display anticancer activity, because CSCs are committed to survive and maintain their stemness in hypoxia. When CSC need to differentiate and proliferate, they shift from anaerobic to aerobic status, and the few mitochondria available makes them susceptible to the injury of the above physiological factors. This vulnerability might be exploited for novel therapeutic treatments.