Sex-Specific Differences in Autophagic Responses to Experimental Ischemic Stroke.

Ischemic stroke triggers a series of complex pathophysiological processes including autophagy. Differential activation of autophagy occurs in neurons derived from males versus females after stressors such as nutrient deprivation. Whether autophagy displays sexual dimorphism after ischemic stroke is unknown. We used a cerebral ischemia mouse model (middle cerebral artery occlusion, MCAO) to evaluate the effects of inhibiting autophagy in ischemic brain pathology. We observed that inhibiting autophagy reduced infarct volume in males and ovariectomized females. However, autophagy inhibition enhanced infarct size in females and in ovariectomized females supplemented with estrogen compared to control mice. We also observed that males had increased levels of Beclin1 and LC3 and decreased levels of pULK1 and p62 at 24 h, while females had decreased levels of Beclin1 and increased levels of ATG7. Furthermore, the levels of autophagy markers were increased under basal conditions and after oxygen and glucose deprivation in male neurons compared with female neurons in vitro. E2 supplementation significantly inhibited autophagy only in male neurons, and was beneficial for cell survival only in female neurons. This study shows that autophagy in the ischemic brain differs between the sexes, and that autophagy regulators have different effects in a sex-dependent manner in neurons.

Effect of melatonin supplementation in combination with neoadjuvant chemotherapy to miR-210 and CD44 expression and clinical response improvement in locally advanced oral squamous cell carcinoma: a randomized controlled trial.

BACKGROUND: Squamous cell carcinoma of the oral cavity (OSCC) is the sixth most common malignancy. Surgery is mainstay treatment for oral cancers. Surgery in locally advanced OSCC presents many challenges primarily because the head and neck have critical structures that can be damaged by tumor or treatment. It is thought that neoadjuvant chemotherapy (NC) in locally advanced OSCC is able to shrink tumor size. Chemoresistancy is a problem due to hypoxic microenvironment characterized by increased expression of HIF-1α. It is also regulated by miR-210 as well as increased expression of CD44 and CD133. Melatonin has a powerful antioxidant and oncostatic effects that are expected to improve tumor hypoxia and clinical response. Fifty patients with OSCC were included and randomized. miR-210 and CD44 expression were measured before and after intervention using qRT-PCR absolute quantification, and clinical response was evaluated according to RECIST 1.1 criteria. This study aims to determine the effect of melatonin in improving the clinical response of patients with locally advanced oral squamous cell carcinoma (OSCC) after neoadjuvant chemotherapy to miR-210 and CD44 expression. RESULTS: Melatonin administration reduced miR-210 levels but not significant (p = 0.767). CD44 expression also decreased in the melatonin group compared with placebo yet was not significant (p = 0.103). There was a decrease in the expression of miR-210 and CD44 followed by a decrease in the percentage of residual tumor but not significant (p = 0.114). CONCLUSION: In OSCC, the addition of 20-mg melatonin to neoadjuvant chemotherapy (NC) reduced the expression of miR-210 and CD44 and decreased the percentage of tumor residue; however, no statistically significant result was observed. TRIAL REGISTRATION: This study is registered to ClinicalTrials.gov under trial registration number: NCT04137627 with date of registration on October 22, 2019-retrospectively registered, accessible from: https://clinicaltrials.gov/ct2/show/NCT04137627.

In vitro design of mesenchymal to epithelial transition of prostate cancer metastasis using 3D nanoclay bone-mimetic scaffolds.

Nanocomposite scaffolds show extensive applications in regenerative medicine and have shown promise as in vitro analogues of human tissue that can be used for the study of diseases. The complex nature of cancer metastasis is recently investigated using several 3D scaffold models. Herein, we report a polymer-nanoclay-based in vitro tumour model that recapitulates early stage of prostate cancer (PCa) colonization during skeletal metastasis on bone mimetic scaffolds. A unique cell culture system termed as "sequential culture (SC)" has been applied to create a bone-mimetic niche for colonization of PCa cells. Human mesenchymal stem cells (MSCs) were seeded on the bone-mimetic scaffolds, where they differentiated into bone cells and then formed mineralized bone matrix without osteogenic supplements. Further, PCa was seeded on MSCs-seeded scaffolds. Sequentially cultured PCa cells with MSCs formed self-organized multicellular tumoroids with distinct tight cellular junctions and hypoxic core regions. Extensive quantitative reverse transcription-polymerase chain reaction experiments were performed to evaluate the expressions of genes related to osteotropic bone metastasis of PCa. On the nanoclay scaffolds, the MSCs differentiated to mature osteoblasts and epithelial to mesenchymal transition was inhibited whereas mesenchymal to epithelial transition was enhanced, as also the hypoxia increased angiogenesis, and finally, PCa cells initiated osteoblastic lesion. Further, the SC technique has significant effects on expression of key metastasis-related genes. Therefore, the SC-based tumour model can be applied to recapitulate more consistent osteotropic cancer cell behavior in understanding tumour biology. This model also can be implemented for drug screening to target colonization stage of PCa cells in the bone microenvironment.

Effects of erythropoietin and methylprednisolone on AQP4 expression in astrocytes.

Methylprednisolone sodium succinate (MPSS) has been suggested as a treatment for spinal cord injury (SCI), but its use has been limited due to its adverse effects. Erythropoietin (EPO) has been suggested as a promising candidate for limiting SCI in mammals. The aim of the present study was to investigate the effects of EPO in combination with MPSS on astrocytes following ischemic injury in vitro. Astrocytes were isolated from the cerebral cortex of postnatal day 3 Sprague­Dawley rats and cultured in vitro. Astrocyte ischemic injury was induced by oxygen and glucose deprivation for 4 h, and reperfusion was simulated by subsequent culture under normoxic conditions. The effects of EPO and MPSS on the expression of aquaporin­4 (AQP4) were investigated. Ischemic astrocytes were treated with EPO (10 U/ml), MPSS (10 µg/ml), or EPO (10 U/ml) in combination with MPSS (10 µg/ml) during reperfusion. The cell viability of astrocytes was assessed using an MTT assay. The mRNA and protein expression levels of AQP4 were determined using reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. The role of the protein kinase C (PKC) signaling pathway in the molecular mechanisms underlying the effects of EPO and MPSS was also investigated. The present results demonstrated that following treatment with EPO and MPSS, the mRNA expression levels of AQP4 were upregulated and cell viability was enhanced. EPO and MPSS effectively inhibited the oxygen and glucose deprivation­mediated downregulation of AQP4 following reperfusion. In addition, the combined treatment with EPO and MPSS exhibited higher AQP4 expression levels and cell viability compared with each treatment alone. Finally, the effects of EPO and MPSS on AQP4 expression were partially reversed by pretreatment with the PKC inhibitor Ro 31­8220. The present study indicated that EPO and MPSS had a synergistic effect on AQP4 expression following reperfusion, and suggest that they may be combined in the treatment of SCI.

Targeting hypoxia-mediated YAP1 nuclear translocation ameliorates pathogenesis of endometriosis without compromising maternal fertility.

Endometriosis is a highly prevalent gynaecological disease that severely reduces women's health and quality of life. Ectopic endometriotic lesions have evolved mechanisms to survive in the hypoxic peritoneal microenvironment by regulating the expression of a significant subset of genes. However, the master regulator controlling these genes remains to be characterized. Herein, by using bioinformatics analysis and experimental verification, we identified yes-associated protein 1 (YAP1) as a master regulator of endometriosis. Nuclear localization and transcriptional activity of YAP1 were up-regulated by hypoxia via down-regulation of LATS1, a kinase that inactivates YAP1. Disruption of hypoxia-induced YAP1 signalling by siRNA knockdown or inhibitor treatment abolished critical biological processes involved in endometriosis development such as steroidogenesis, angiogenesis, inflammation, migration, innervation, and cell proliferation. Treatment with a YAP1 inhibitor caused the regression of endometriotic lesions without affecting maternal fertility or the growth rate of offspring in the mouse model of endometriosis. Taken together, we identify hypoxia/LATS1/YAP1 as a novel pathway for the pathogenesis of endometriosis and demonstrate that targeting YAP1 might be an alternative approach to treat endometriosis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ZnCl2 sustains the adriamycin-induced cell death inhibited by high glucose.

Hyperglycemia, the condition of high blood glucose, is typical of diabetes and obesity and represents a significant clinical problem. The relationship between hyperglycemia and cancer risk has been established by several studies. Moreover, hyperglycemia has been shown to reduce cancer cell response to therapies, conferring resistance to drug-induced cell death. Therefore, counteracting the negative effects of hyperglycemia may positively improve the cancer cell death induced by chemotherapies. Recent studies showed that zinc supplementation may have beneficial effects on glycemic control. Here we aimed at evaluating whether ZnCl2 could counteract the high-glucose (HG) effects and consequently restore the drug-induced cancer cell death. At the molecular level we found that the HG-induced expression of genes known to be involved in chemoresistance (such as HIF-1α, GLUT1, and HK2 glycolytic genes, as well as NF-κB activity) was reduced by ZnCl2 treatment. In agreement, the adryamicin (ADR)-induced apoptotic cancer cell death was significantly impaired by HG and efficiently re-established by ZnCl2 cotreatment. Mechanistically, the ADR-induced c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) phosphorylation, inhibited by HG, was efficiently restored by ZnCl2. The JNK involvement in apoptotic cell death was assessed by the use of JNK dominant-negative expression vector that indeed impaired the ZnCl2 ability to restore drug-induced cell death in HG condition. Altogether, these findings indicate that ZnCl2 supplementation efficiently restored the drug-induced cancer cell death, inhibited by HG, by both sustaining JNK activation and counteracting the glycolytic pathway.

ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate.

In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

Polycomb YY1 is a critical interface between epigenetic code and miRNA machinery after exposure to hypoxia in malignancy.

Yin Yang 1 (YY1) is a member of polycomb protein family involved in epigenetic modifications and transcriptional controls. We have shown that YY1 acts as positive regulator of tumor growth and angiogenesis by interfering with the VEGFA network. Yet, the link between polycomb chromatin complex and hypoxia regulation of VEGFA is still poorly understood. Here, we establish that hypoxia impairs YY1 binding to VEGFA mRNA 3'UTR (p<0.001) in bone malignancy. Moreover, RNA immunoprecipitation reveals the formation of triplex nuclear complexes among YY1, VEGFA DNA, mRNA, and unreached about 200 fold primiRNA 200b and 200c via Dicer protein. In this complex, YY1 is necessary to maintain the steady-state level of VEGFA expression while its silencing increases VEGFA mRNA half-life at 4 h and impairs the maturation of miRNA 200b/c. Hypoxia promotes histone modification through ubiquitination both of YY1 and Dicer proteins. Hypoxia-mediated down-regulation of YY1 and Dicer changes post-transcriptional VEGFA regulation by resulting in the accumulation of primiRNA200b/c in comparison to mature miRNAs (p<0.001). Given the regulatory functions of VEGFA on cellular activities to promote neoangiogenesis, we conclude that YY1 acts as novel critical interface between epigenetic code and miRNAs machinery under chronic hypoxia in malignancy.

Reduced serum and hypoxic culture conditions enhance the osteogenic potential of human mesenchymal stem cells.

UNLABELLED: Current protocols for inducing osteogenic differentiation in mesenchymal stem/stromal cells (MSCs) in culture for tissue engineering applications depend on the use of biochemical supplements. However, standard in vitro culture conditions expose cells to ambient oxygen concentrations and high levels of serum (21% O2, 10% FBS) that do not accurately recapitulate the physiological milieu. While we and others have examined MSC behavior under hypoxia, the synergistic effect of low serum levels, such as those present in ischemic injury sites, on osteogenic differentiation has not been clearly examined. We hypothesized that a concomitant reduction of serum and O2 would enhance in vitro osteogenic differentiation of MSCs by more accurately mimicking the fracture microenvironment. We show that serum deprivation, in conjunction with hypoxia, potentiates osteogenic differentiation in MSCs. These data demonstrate the role of serum levels in regulating osteogenesis and its importance in optimizing MSC differentiation strategies. HIGHLIGHTS: Serum levels, in addition to hypoxia, have a significant effect on MSC osteogenic differentiation. Both naïve and osteogenically induced MSCs exhibit higher osteogenic markers in reduced serum. MSCs deposit the most calcium under 5% O2 in osteogenic media supplemented with 5% FBS. Standard culture conditions (21% O2, 10% FBS) may not be optimal for MSC osteogenic differentiation.

Seminal and molecular evidence that sauna exposure affects human spermatogenesis.

STUDY QUESTION: What are the effects of continuous sauna exposure on seminal parameters, sperm chromatin, sperm apoptosis and expression of genes involved in heat stress and hypoxia? SUMMARY ANSWER: Scrotal hyperthermia by exposure to sauna can induce a significant alteration of spermatogenesis. WHAT IS KNOWN ALREADY: Several authors have evidenced that high temperature has dramatic effects on spermatogenesis. STUDY DESIGN, SIZE AND DURATION: A longitudinal time-course study. Data from 10 subjects exposed to Finnish sauna were collected before sauna (T0), after 3 months of sauna sessions (T1) and after 3 (T2) and 6 months (T3) from the end of sauna exposure. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Ten normozoospermic volunteers underwent two sauna sessions per week for 3 months, at 80-90°C, each lasting 15 min. Sex hormones, sperm parameters, sperm chromatin structure, sperm apoptosis and expression of genes involved in heat stress and hypoxia were evaluated at the start, at the end of sauna exposure and after 3 and 6 months from sauna discontinuation. Student's t-test for paired data was used for statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: At the end of sauna exposure, we found a strong impairment of sperm count and motility (P < 0.001), while no significant change in sex hormones was present. Decreases in the percentage of sperm with normal histone-protamine substitution (78.7 ± 4.5 versus 69.0 ± 4.1), chromatin condensation (70.7 ± 4.7 versus 63.6 ± 3.3) and mitochondrial function (76.8 ± 4.9 versus 54.0 ± 6.1) were also evident at T1, and strong parallel up-regulation of genes involved in response to heat stress and hypoxia was found. All these effects were completely reversed at T3. LIMITATIONS AND REASONS FOR CAUTION: Absence of subjects with abnormal sperm parameters was the major limitation of this study. WIDER IMPLICATIONS OF THE FINDINGS: Our data demonstrated for the first time that in normozoospermic subjects, sauna exposure induces a significant but reversible impairment of spermatogenesis, including alteration of sperm parameters, mitochondrial function and sperm DNA packaging. The large use of Finnish sauna in Nordic countries and its growing use in other parts of the world make it important to consider the impact of this lifestyle choice on men's fertility. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study and the authors have no conflict of interest to declare.

SirT1 mediates hyperbaric oxygen preconditioning-induced ischemic tolerance in rat brain.

Our previous studies have shown that hyperbaric oxygen preconditioning (HBO-PC) induces tolerance to cerebral ischemia/reperfusion (I/R). This study aimed to investigate whether SirT1, a class III histone deacetylase, is involved in neuroprotection elicited by HBO-PC in animal and cell culture models of ischemia. Rats were subjected to middle cerebral artery occlusion for 120 minutes after HBO-PC (once a day for 5 days). Primary cultured cortical neurons were exposed to 2 hours of HBO-PC after 2 hours of oxygen-glucose deprivation (OGD). We showed that HBO-PC increased SirT1 protein and mRNA expression, promoted neurobehavioral score, reduced infarct volume, and improved morphology at 24 hours and 7 days after cerebral I/R. Neuroprotection of HBO-PC was attenuated by SirT1 inhibitor EX527 and SirT1 knockdown by short interfering RNA (siRNA), whereas it was mimicked by SirT1 activator resveratrol. Furthermore, HBO-PC enhanced SirT1 expression and cell viability and reduced lactate dehydrogenase release 24 hours after OGD/re-oxygenation. The neuroprotective effect of HBO-PC was emulated through upregulating SirT1 and, reversely, attenuated through downregulating SirT1. The modulation of SirT1 was made by adenovirus infection carrying SirT1 or SirT1 siRNA. Besides, SirT1 increased B-cell lymphoma 2 (Bcl-2) expression and decrease cleaved caspase 3. These results indicate that SirT1 mediates HBO-PC-induced tolerance to cerebral I/R through inhibition of apoptosis.

Differential expression of calcium transport channels in placenta primary cells and tissues derived from preeclamptic placenta.

Preeclampsia is a pregnancy-specific disease characterized by hypertension, proteinuria, and oxidative stress in the placenta. During the last trimester of gestation, calcium (Ca(2+)) transport from mother to fetus increases dramatically in response to the increased demand for Ca(2+) caused by bone mineralization in the fetus. Ca(2+) supplementation can significantly reduce the incidence and severity of preeclampsia or delay its onset. Ca(2+) transport channels (CTCs) include transient receptor potential vanilloid 6 (TRPV6), plasma membrane Ca(2+) ATPase (PMCA1), and Na(+)/Ca(2+) exchangers (NCKX3 or NCX1). We hypothesized that trans-placental Ca(2+) exchange in preeclamptic trophoblasts may be compensated for successful fetal bone mineralization. The roles of cell membrane channels (TRPV6, PMCA1, NCKX3 and NCX1) were examined in placental primary cells and in normotensive and preeclamptic placentas. The biomarker gene for preeclampsia, soluble fms-like tyrosine kinase-1 (sFLT1) or marker for oxygen-sensitive gene, hypoxia-sensitive inducible factor 1α (HIF-1α), were up-regulated in the preeclamptic placentas and hypoxic cells. The detection of sFLT1 and HIF-1α genes demonstrated that our experimental conditions were suitable to verify a preeclamptic condition. In women experiencing preterm labor, CTC expressions was found to be increased in the fetal and maternal regions of the preeclamptic placenta compared to the observed in normotensive placenta. During term labor, TRPV6 and PMCA1 were highly expressed in the fetal and maternal sections of preeclamptic placenta, while the expression of NCKX3 and NCX1 was reduced. In addition, the expression of CTCs was altered in hypoxia-stressed placental cells. Taken together, our findings demonstrated that the expression of CTCs was regulated by hypoxia stress in placenta tissues and cells, suggesting that our experimental in vitro hypoxic conditions were similar to those of preeclampsia. Furthermore, impaired Ca(2+) metabolism found in preeclamptic syncytiotrophoblasts was resulted from hypoxic stress, which may induce expression of Ca(2+) transport proteins in the placenta to maintain the balance between maternal and fetal Ca(2+) demand during pregnancy.

Induction of beta-cell resistance to hypoxia and technologies for oxygen delivery to transplanted pancreatic islets.

Hypoxia is believed to be a crucial factor involved in cell adaptation to environmental stress. Islet transplantation, especially with immunoisolated islets, interrupts vascular connections, resulting in the substantially decreased delivery of oxygen and nutrients to islet cells. Insulin-producing pancreatic beta cells are known to be highly susceptible to oxygen deficiency. Such susceptibility to hypoxia is believed to be one of the main causes of beta-cell death in the post-transplantation period. Different strategies have been developed for the protection of beta cells against hypoxic injury and for oxygen delivery to transplanted islets. The enhancement of beta-cell defense properties against hypoxia has been achieved using various techniques such as gene transfection, drug supplementation, co-culturing with stem cells and cell selection. Technologies for oxygen delivery to transplanted islets include local neovascularization of subcutaneous sites, electrochemical and photosynthetic oxygen generation, oxygen refuelling of bio-artificial pancreas and whole body oxygenation by using hyperbaric therapy. Progress in the field of oxygen technologies for islet transplantation requires a multidisciplinary approach to explore and optimize the interaction between components of the biological system and different technological processes. This review article focuses mainly on the recently developed strategies for oxygenation and protection from hypoxic injury - to achieve stable and long-term normoglycaemia in diabetic patients with transplanted pancreatic islets.

Cooperativity between inhibition of cytosolic K+ efflux and AMPK activation during suppression of hypoxia-induced cellular apoptosis.

Cellular potassium homeostasis has recently emerged as a critical regulator of apoptosis in response to variety of stimuli. However, functional hierarchy of this phenomenon in the apoptotic cascade and therefore, its significance as a pathway for intervention is not fully established. Chronic hypoxia, a known threat to cell survival, also modulates cellular potassium homeostasis. In this study, we tested if hypoxia-induced apoptosis in lymphocytes can be prevented by modulating cellular K+ homeostasis. We observed that chronic hypoxia accelerated the rate of apoptosis in resting murine splenocytes concomitant with cytosolic K+ efflux. We tested several modalities including elevated extracellular potassium besides various K+ channel inhibitors to curtail hypoxia-induced K+ efflux and interestingly, established that the supplementation of KCl in extracellular medium was most effective in preventing hypoxia-induced apoptosis in these cells. Subsequent mechanistic dissection of pathways underlying this phenomenon revealed that besides effectively inhibiting hypoxia-induced efflux of K+ ion and its downstream cell-physiological consequences; elevated extracellular KCl modulated steady state levels of cellular ATP and culminated in stabilization of AMPKα with pro-survival consequences. Also, interestingly, global gene expression profiling revealed that KCl supplementation down regulated a distinct p53-regulated cellular sub-network of genes involved in regulation of DNA replication. Additionally, we present experimental evidence for the functional role of AMPK and p53 activation during suppression of hypoxia-induced apoptosis. In conclusion, our study highlights a novel bimodal effect wherein cooperativity between restoration of K+ homeostasis and a sustainable 'metabolic quiescence' induced by AMPK activation appeared indispensible for curtailing hypoxia-induced apoptosis.

The novel Bcl-2 inhibitor ABT-737 is more effective in hypoxia and is able to reverse hypoxia-induced drug resistance in neuroblastoma cells.

Neuroblastoma is a common solid tumor of childhood and advanced disease carries a poor prognosis despite intensive multimodality therapy. Hypoxia is a common feature of solid tumors because of poorly organized tumor-induced neovasculature. Hypoxia is associated with advanced stage and poor outcome in a range of tumor types, and leads to resistance to clinically relevant cytotoxic agents in neuroblastoma and other pediatric tumors in vitro. Resistance to apoptosis is a common feature of tumor cells and leads to pleiotropic drug resistance, mediated by Bcl-2 family proteins. ABT-737 is a novel small-molecule inhibitor of Bcl-2 and Bcl-x(L) that is able to induce apoptosis in a range of tumor types. Neuroblastoma cell lines are relatively resistant to ABT-737-induced apoptosis in normoxia, but in contrast to the situation with conventional cytotoxic agents are more sensitive in hypoxia. This sensitization is because of an increase in ABT-737-induced apoptosis and is variably dependent upon the presence of functional hypoxia-inducible factor 1 (HIF-1) α. In contrast to the situation in colon carcinoma and non-small cell lung cancer cells, hypoxia does not result in downregulation of the known ABT-737 resistance factor, Mcl-1, nor any other Bcl-2 family proteins. ABT-737 sensitizes neuroblastoma cells to clinically relevant cytotoxic agents under normal levels of oxygen, and importantly, this sensitization is maintained under hypoxia when neuroblastoma cells are resistant to these agents. Thus rational combinations of ABT-737 and conventional cytotoxics offer a novel approach to overcoming hypoxia-induced drug resistance in neuroblastoma.

Hyperthermia conditioned astrocyte-cultured medium protects neurons from ischemic injury by the up-regulation of HIF-1 alpha and the increased anti-apoptotic ability.

It has been demonstrated that conditioned medium from astrocytes challenged by in vitro ischemia (oxygen-glucose deprivation, OGD) improved neuronal survival. In addition, preconditioning stimuli can be cross-tolerant, safeguarding against other types of injury. We therefore hypothesized that hyperthermia-conditioned astrocyte-cultured medium (ACM) might also have protective effect on neurons against ischemic injury. The cultured-media, named 38ACM and 40ACM respectively, were collected after astrocytes had been incubated at 38 °C or 40 °C for 6h, followed by incubation at 37 °C for 24h. It was found that ischemia for 6h induced a significant reduction in the number of neuronal cells and cell-viability, and an increase in lactate dehydrogenase (LDH) release and the percentage of apoptotic nuclei in neurons. Pre-treatment with 38ACM or 40ACM for 24h significantly diminished ischemia injury, enhanced cell viability, reduced LDH release and reversed apoptosis. Western blot analysis showed that treatment with 38ACM or 40ACM for 24h led to a significant increase in hypoxia-inducible factor-1 (HIF-1) alpha expression. The EMSA demonstrated that the ACM increased the binding activity of HIF-1 in ischemic neurons. The data implied that hyperthermia-conditioned ACM protects neurons from ischemic injury by up-regulating HIF-1 alpha, and the increased binding activity of HIF-1 and anti-apoptotic ability.

Putative cancer stem cells in malignant pleural mesothelioma show resistance to cisplatin and pemetrexed.

Malignant pleural mesothelioma (MPM) is a lethal cancer of the mesothelium with high chemotherapeutic resistance via unknown mechanisms. A prevailing hypothesis states that cancer stem cells (CSCs) persist in tumors causing relapse after chemotherapy, thus, rendering these cells as critical targets responsible for tumor resistance and recurrence. We selected candidate CSC markers based on expansion under hypoxic conditions, a hallmark for the selection of chemoresistant cells; and investigated the expression of CSC markers: CD133, Bmi-1, uPAR and ABCG2 in three MPM cell lines and normal mesothelial cells by quantitative RT-PCR. Furthermore, we evaluated the chemotherapeutic resistance associated with each CSC marker by determining the change in CSC marker-mRNA levels as an index of drug-resistance following treatment with either cisplatin or pemetrexed. We demonstrate the expression of CSC markers: CD133, Bmi-1, uPAR and ABCG2 in both normal and MPM cell lines. Bmi-1+, uPAR+ and ABCG2+ cells show a distinct role in conferring chemoresistance to cisplatin and pemetrexed in the malignant setting. By contrast, these markers have no apparent participation in chemoresistance to drug treatments in normal mesothelial cells. Intriguingly, CD133 revealed chemoresistant properties in both normal mesothelial and malignant pleural mesothelioma cells. This study provides evidence of putative CSCs conferring drug-resistance to cisplatin and pemetrexed in MPM cell lines. Specific targeting of these drug-resistant cells, while considering the functional heterogeneity of the MPM subtypes, may contribute to more focused and effective chemotherapeutic regimens for malignant pleural mesothelioma.

Inhibition of histone deacetylase attenuates hypoxia-induced migration and invasion of cancer cells via the restoration of RECK expression.

Hypoxia is a strong signal for cell migration and invasion in cancer. The reversion-inducing cysteine-rich protein with Kazal motif (RECK), a tumor suppressor, inhibits cancer cell migration and invasion and is frequently silenced in aggressive tumor cells by histone deacetylases (HDAC). However, the effect of RECK silencing in several cancer cells in a hypoxic microenvironment has not been fully delineated. In this report, we investigated whether hypoxia suppressed RECK expression and used HDAC inhibitor (HDACI) inhibition to restore RECK expression to inhibit cancer cell migration and invasion. HDACIs, including trichostatin A (TSA), completely rescued RECK expression, which was suppressed by hypoxia, in the H-Ras-transformed human breast MCF10A and the HT1080 cell lines (human fibrosarcoma). TSA suppressed the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9, induced by hypoxia, and significantly inhibited hypoxia-stimulated migration and invasion of both cancer cells. RECK overexpression significantly inhibited the migration and invasion of cancer cells induced by hypoxia. The hypoxic effect on the migration and invasion of cells was equivalent to the effect seen using the small interfering RNA (siRNA) of RECK under normoxia, suggesting an inhibitory role for RECK in hypoxic conditions. We also showed that siRNA silencing of HDAC1 suppressed hypoxia-induced RECK downregulation and inhibited the migration and invasion of cancer cells. In conclusion, the inhibition of HDAC successfully restored the expression of RECK under hypoxic conditions. This resulted in the inhibition of cancer cell migration and invasion through the repression of MMP-2 and MMP-9 activity.

HIPK2-a therapeutical target to be (re)activated for tumor suppression: role in p53 activation and HIF-1α inhibition.

Oncosuppressor p53 is often inactivated by either mutations or deregulation of regulatory proteins. These include the homeodomain-interacting protein kinase 2 (HIPK2) that, by phosphorylating p53 at Ser46 modulates p53 response to DNA damage by inducing pro-apoptotic transcription. There is compelling evidence that HIPK2 is also involved in the response to hypoxia by acting as co-suppressor of hypoxia inducible factor 1α (HIF-1α), a major factor in cancer progression that activates the transcription of genes involved in angiogenesis, glucose metabolism and invasion. Hence conditions that induce HIPK2 deregulation would end up in a multifactorial response leading to tumor chemoresistance by affecting p53 activity on one hand and to angiogenesis and cell proliferation by affecting HIF-1 activity on the other hand. For these reasons, HIPK2 protein is a promising target for anti-cancer therapies. HIPK2 can be inhibited by hypoxia. In this respect, we have recently shown that hypoxia-driven HIPK2 downregulation is not irreversible. We found that, zinc supplementation reactivates the hypoxia-inhibited HIPK2, leading to repression of the HIF-1 pathway and restoration of p53Ser46 apoptotic activity. Here, we discuss about these findings and the potential relevance of zinc supplementation to chemotherapy in cancer treatment. The results will be also discussed in light of recent findings showing that cancer treatment with antiangiogenic agents may result in hypoxia and selection of cancer cells with increased tumor aggressiveness and metastasis.

Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL.

OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.