Treatment Of Hypoxic Cells Research Articles

Abstract P103: Apelin And Elabela Attenuate The Pro-inflammatory Response In Hypoxic HTR-8/SVneo Trophoblastic Cells

Preeclampsia is a significant pregnancy complication characterized by de novo hypertension, proteinuria, and further organ dysfunction. We previously demonstrated both cardio- and renoprotective effects by systemic activation of apelin receptor system (APJ) in preeclampsia. Although the APJ ligands apelin and elabela stimulate trophoblast cell proliferation and migration, the cellular mechanisms underlying these actions are not well understood, particularly in response to hypoxia. To examine the response of the APJ system to cellular hypoxia in human trophoblasts, HTR-8/SVneo trophoblastic cells were treated with cobalt chloride (CoCl 2: 200 μM, 24 hours) to induce hypoxic conditions. The cellular mRNA responses for sFlt-1 variants, VEGF, PLGF, IL-6, and IL-1β were determined by RT-qPCR for control and apelin- or elabela-treated cells with or without CoCl 2 . The presence of APJ mRNA was confirmed in HTR-8/SVneo cells. Hypoxia increased apelin content approximately 2-fold in both the cell lysate (723 ± 52 vs. 1216 ± 20 ng/mg protein, n=6, p<0.01) and the conditioned media (49 ± 5 vs. 102 ± 9 ng/ml, n=6, p<0.01). Elabela content also increased in the media of hypoxic cells (0.11 ± 0.01 vs. 0.14 ± 0.01 ng/ml, n=6, p<0.05), although elabela levels were 100-fold lower than apelin. Hypoxia upregulated hsFlt-1-i13 (2.6 fold), hsFlt-1-e15a (2.9 fold), VEGF (3.7 fold), PLGF (1.3 fold), and IL-6 (3.5 fold) mRNA levels. Treatment with (Pyr-1)-apelin-13, a stable APJ ligand (0.1 μM), decreased VEGF (1.2 fold; n=3, p<0.05) and IL-6 (1.4 fold, n=3, p<0.05) mRNA levels in cells exposed to COCl 2 , but had no effect in normoxic cells. Similarly, elabela (0.1 μM) downregulated VEGF (1.1 fold; n=3, p<0.05) and IL-6 (1.2 fold; n=3, p<0.05). We found no difference in sFlt-1 variants, PLGF, or IL-1β mRNAs following treatment of hypoxic cells with either peptide. We conclude that cobalt-induced hypoxia markedly increases various indices of inflammation, as well as stimulates the endogenous apelin and elabela systems in the HTR-8/SVneo cells. Furthermore, local activation of these peptidergic systems may be a compensatory response of the trophoblast cells to hypoxia as exogenous apelin and elabela treatment effectively ameliorate the hypoxia-induced pro-inflammatory milieu.

Alteration of Cellular Reduction Potential Will Change 64Cu-ATSM Signal With or Without Hypoxia.

Therapies targeting reductive/oxidative (redox) metabolism hold potential in cancers resistant to chemotherapy and radiation. A redox imaging marker would help identify cancers susceptible to redox-directed therapies. Copper(II)-diacetyl-bis(4-methylthiosemicarbazonato) (Cu-ATSM) is a PET tracer developed for hypoxia imaging that could potentially be used for this purpose. We aimed to demonstrate that Cu-ATSM signal is dependent on cellular redox state, irrespective of hypoxia. Methods: We investigated the relationship between 64Cu-ATSM signal and redox state in human cervical and colon cancer cells. We altered redox state using drug strategies and single-gene mutations in isocitrate dehydrogenases (IDH1/2). Concentrations of reducing molecules were determined by spectrophotometry and liquid chromatography-mass spectrometry and compared with 64Cu-ATSM signal in vitro. Mouse models of cervical cancer were used to evaluate the relationship between 64Cu-ATSM signal and levels of reducing molecules in vivo, as well as to evaluate the change in 64Cu-ATSM signal after redox-active drug treatment. Results: A correlation exists between baseline 64Cu-ATSM signal and cellular concentration of glutathione, nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NADH). Altering NADH and NADPH metabolism using drug strategies and IDH1 mutations resulted in significant changes in 64Cu-ATSM signal under normoxic conditions. Hypoxia likewise changed 64Cu-ATSM signal, but treatment of hypoxic cells with redox-active drugs resulted in a more dramatic change than hypoxia alone. A significant difference in NADPH was seen between cervical tumor orthotopic implants in vivo, without a corresponding difference in 64Cu-ATSM signal. After treatment with β-lapachone, there was a change in 64Cu-ATSM signal in xenograft tumors smaller than 50 mg but not in larger tumors. Conclusion:64Cu-ATSM signal reflects redox state, and altering redox state impacts 64Cu-ATSM metabolism. Our animal data suggest there are other modulating factors in vivo. These findings have implications for the use of 64Cu-ATSM as a predictive marker for redox therapies, though further in vivo work is needed.

Fabrication and characterization of UV-emitting nanoparticles as novel radiation sensitizers targeting hypoxic tumor cells

Radiation therapy is one of the primary therapeutic techniques for treating cancer, administered to nearly two-thirds of all cancer patients. Although largely effective in killing cancer cells, radiation therapy, like other forms of cancer treatment, has difficulty dealing with hypoxic regions within solid tumors. The incomplete killing of cancer cells can lead to recurrence and relapse.The research presented here is investigating the enhancement of the efficacy of radiation therapy by using scintillating nanoparticles that emit UV photons. UV photons, with wavelengths between 230 nm and 280 nm, are able to inactivate cells due to their direct interaction with DNA, causing a variety of forms of damage. UV-emitting nanoparticles will enhance the treatment in two ways: first by generating UV photons in the immediate vicinity of cancer cells, leading to direct and oxygen-independent DNA damage, and second by down-converting the applied higher energy X-rays into softer X-rays and particles that are more efficiently absorbed in the targeted tumor region. The end result will be nanoparticles with a higher efficacy in the treatment of hypoxic cells in the tumor, filling an important, unmet clinical need.Our preliminary experiments show an increase in cell death using scintillating LuPO4:Pr nanoparticles over that achieved by the primary radiation alone. This work describes the fabrication of the nanoparticles, their physical characterization, and the spectroscopic characterization of the UV emission. The work also presents in vitro results that demonstrate an enhanced efficacy of cell killing with x-rays and a low unspecific toxicity of the nanoparticles.

Abstract 1739: A novel HSP90 inhibitor induces metabolic oxidative stress in head and neck squamous cancers and prevents migration and stem cell EMT even under hypoxic conditions

Abstract INTRODUCTION: Advanced head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat. In HNSCC, hypoxia is a negative prognostic marker with altered metabolism, increased invasiveness, and resistance to therapy. Natural withanolides exhibit potent, highly selective anticancer effects in HNSCCs in vitro and in vivo through a novel anticancer mechanism. These drugs [withaferin A and the novel compound, withalongolide A-triacetate(WGA-TA)] selectively induce metabolic oxidative stress in cancer cells while inhibiting HSP90 chaperone function via disruption of CDC37 (co-chaperone) binding. We hypothesize that WGA-TA is a potent, novel therapeutic drug for HNSCC that will decrease tumor migration and cancer stem cell endothelial-mesenchymal transition (EMT) even in the setting of hypoxia. METHODS: Human HNSCC cell lines MDA-1986, UMSCC11A, 11B and 12 were grown either under normoxic or hypoxic conditions. CM-H2DCFDA level defined accumulation of intracellular reactive oxygen species. Effects on proliferation and apoptosis were evaluated by flow cytometry (FC), Western blotting (WB), and RT PCR. Viability was evaluated by MTS and clonigenic assay. Scratch wound-healing and Boyden chamber assays were used to follow cell migration. RESULTS: WGA-TA treatment of both hypoxic and normoxic cells showed dose dependent induction of oxidation (p<0.01 vs. controls). This effect was completely blocked by N-acetyl cysteine(NAC) under both conditions (p<0.001). Dose dependent changes in biomarkers of oxidative stress (200-400% increase in HSP32 and HSP70; 80-90% decrease in HSF-1) with functional inhibition of Akt, mTOR, the EMT marker vimentin (80-90%), and cancer stem cell marker (Bmi-1, CD44 & ALDH each 50-90% inhibition (p<0.01 vs. control) and modulation of glycolytic proteins (20-80%; p<0.01) was observed under both conditions after WGA-TA treatment. Oxidative stress effect was further enhanced by hypoxia or pretreatment with L-Buthione-[S,R]-sulfoxime (BSO), whereas NAC treatment completely abrogated this effect even at 500nM WGA-TA. Treatment of hypoxic cells with WGA-TA did not alter the IC50 values (50-100 nM) and NAC pretreatment did not inhibit proliferation, whereas BSO decreased IC50 values by 10-40% indicating the role of ROS in reducing cell proliferation and viability. Dose-dependent induction of apoptosis (starting at 500nM WGA-TA) on FC was confirmed by caspases 3 and PARP cleavage on WB and accelerated by BSO but blocked by NAC treatment. Finally 500-1000 nM WGA-TA concentrations prevented HNSCC cell migration by 80-90% (p<0.01 vs. control). CONCLUSION: WGA-TA potently induces glycolytic oxidative stress in HNSCC leading to decrease in CSC EMT as well as,induction of apoptosis and loss of tumor cell migration even in hypoxic conditions. These exciting findings warrant further evaluation in translational in vivo models. Citation Format: Chitra Subramanian, Qing Zhu, Eileen Brandes, Peter T. White, Barbara N. Timmermann, Mark S. Cohen. A novel HSP90 inhibitor induces metabolic oxidative stress in head and neck squamous cancers and prevents migration and stem cell EMT even under hypoxic conditions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2015-1739

Hypoxia-Induced Autophagy Promotes Tumor Cell Survival and Adaptation to Antiangiogenic Treatment in Glioblastoma

Antiangiogenic therapy leads to devascularization that limits tumor growth. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. In this study, we explored the hypothesis that hypoxia caused by antiangiogenic therapy induces tumor cell autophagy as a cytoprotective adaptive response, thereby promoting treatment resistance. Hypoxia-induced autophagy was dependent on signaling through the hypoxia-inducible factor-1α (HIF-1α)/AMPK pathway, and treatment of hypoxic cells with autophagy inhibitors caused a shift from autophagic to apoptotic cell death in vitro. In glioblastomas, clinically resistant to the VEGF-neutralizing antibody bevacizumab, increased regions of hypoxia and higher levels of autophagy-mediating BNIP3 were found when compared with pretreatment specimens from the same patients. When treated with bevacizumab alone, human glioblastoma xenografts showed increased BNIP3 expression and hypoxia-associated growth, which could be prevented by addition of the autophagy inhibitor chloroquine. In vivo targeting of the essential autophagy gene ATG7 also disrupted tumor growth when combined with bevacizumab treatment. Together, our findings elucidate a novel mechanism of resistance to antiangiogenic therapy in which hypoxia-mediated autophagy promotes tumor cell survival. One strong implication of our findings is that autophagy inhibitors may help prevent resistance to antiangiogenic therapy used in the clinic.

Decay accelerating factor (CD55) protects neuronal cells from chemical hypoxia-induced injury

BackgroundActivated complement system is known to mediate neuroinflammation and neurodegeneration following exposure to hypoxic-ischemic insults. Therefore, inhibition of the complement activation cascade may represent a potential therapeutic strategy for the management of ischemic brain injury. Decay-accelerating factor (DAF, also known as CD55) inhibits complement activation by suppressing the function of C3/C5 convertases, thereby limiting local generation or deposition of C3a/C5a and membrane attack complex (MAC or C5b-9) production. The present study investigates the ability of DAF to protect primary cultured neuronal cells subjected to sodium cyanide (NaCN)-induced hypoxia from degeneration and apoptosis.MethodsCultured primary cortical neurons from embryonic Sprague-Dawley rats were assigned one of four groups: control, DAF treatment alone, hypoxic, or hypoxic treated with DAF. Hypoxic cultures were exposed to NaCN for 1 hour, rinsed, followed by 24 hour exposure to 200 ng/ml of recombinant human DAF in normal medium. Human DAF was used in the present study and it has been shown to effectively regulate complement activation in rats. Neuronal cell function, morphology and viability were investigated by measuring plateau depolarization potential, counting the number dendritic spines, and observing TUNEL and MTT assays. Complement C3, C3a, C3a receptor (R) production, C3a-C3aR interaction and MAC formation were assessed along with the generation of activated caspase-9, activated caspase-3, and activated Src.ResultsWhen compared to controls, hypoxic cells had fewer dendritic spines, reduced plateau depolarization accompanied by increased apoptotic activity and accumulation of MAC, as well as up-regulation of C3, C3a and C3aR, enhancement of C3a-C3aR engagement, and elevated caspase and Src activity. Treatment of hypoxic cells with 200 ng/ml of recombinant human DAF resulted in attenuation of neuronal apoptosis and exerted significant protection against neuronal dendritic spine loss and plateau depolarization reduction. Furthermore, treatment with DAF resulted in decreased accumulation of C3a, MAC, C3a-C3aR interaction, caspase-9, activated caspase-3, and pTyr416-Src (activated Src) tyrosine kinase.ConclusionDAF was found to reduce neuronal cell death and apoptosis in NaCN induced hypoxia. This effect is attributed to the ability of DAF to limit complement activation and inhibit the activity of Src and caspases 9 and 3. This study supports the inhibiting of complement as a neuroprotective strategy against CNS ischemia/reperfusion injury.

NaCN-induced chemical hypoxia is associated with altered gene expression.

Sodium cyanide (NaCN)-induced chemical hypoxia is known to increase intracellular free calcium concentration and reduce cell survival, but its effect on gene expression has not been studied. In this study, we designed primers to conduct a rapid and reliable assay for the expression of mRNA of inducible nitric oxide synthase (iNOs), tumor suppressor protein p53, Bcl-2, heat shock protein 70 (HSP-70), and beta-actin in human intestinal epithelial T84 cells and Jurkat T cells. NaCN-induced chemical hypoxia increased iNOs and HSP-70 mRNA in both types of cells, whereas p53 and Bcl-2 mRNA were singularly induced in T84 cells and Jurkat T cells, respectively. In both cell types, treatment of hypoxic cells with a reversible selective iNOs inhibitor, Now-nitro-L-arginine (LNNA), blocked iNOs, Bcl-2, and HSP-70 mRNA, but increased p53. The NaCN-induced hypoxia was also found to increase caspase-3 cellular activity in both cell types. Treatment with LNNA alone decreased the basal caspase-3 cellular activity. A prior treatment of LNNA significantly inhibited the NaCN-induced increase in the cellular activity of this apoptotic enzyme. This is the first report to show that NaCN-induced chemical hypoxia alters both stress-related gene expression and caspase-3 cellular activity and can be regulated by the iNOs inhibitor LNNA. Since NaCN has been included in the 'National chemical terrorism threat' list, by the US Department of Defense, our studies provide useful insight in the development of molecular sensors to detect early exposure to this chemical terrorism threat.

Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells

Purpose : To examine how hypoxia influences ionizing irradiation-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. Materials and methods : Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. Results : In HL60 cells, apototic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-strand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. Conclusion : These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.

Oxygen-dependent Regulation of in VivoReplication of Simian Virus 40 DNA Is Modulated by Glucose

Simian virus 40 (SV40)-infected CV1 cells exposed to hypoxia show an inhibition of viral replication. Reoxygenation after several hours of hypoxia results in new initiations followed by a nearly synchronous round of SV40 replication. In this communication, we examined the effect of glucose on inhibition of viral DNA replication under hypoxia. We found that glucose stimulated SV40 DNA replication under hypoxia in two different ways. First, the rate of DNA synthesis, i.e. the fork propagation rate, increased. This effect seemed to be mediated by inhibition of mitochondrial respiration by glucose (Crabtree effect). Inhibition of mitochondrial respiration probably resulted in a higher intracellular oxygen concentration and an activation of oxygen-dependent ribonucleotide reductase, which provides the precursors for DNA synthesis. This glucose effect was consequently strongly dependent on the strength of hypoxia and the extent of intracellular respiration; hypoxic gassing with 10 ppm instead of 200-400 ppm O(2) or treatment of hypoxic cells with a mitochondrial uncoupler (carbonyl cyanide m-chlorophenylhydrazone) reduced the glucose effect on replication, whereas antimycin A, an inhibitor of respiration, increased it. The second effect of glucose concerned initiation, i.e. stimulation of unwinding of the viral origin. This effect was not influenced by the strength of hypoxia or the extent of cellular respiration and seemed, therefore, not to be mediated through a Crabtree effect. No evidence for a direct correlation between the cellular ATP concentration and the extent of SV40 replication under hypoxia was found. The effect of glucose on replication under hypoxia was not restricted to SV40-infected CV1 cells but was also detectable in HeLa cells. This suggests it to be a mechanism of more general validity.