Examining the influence of hypoxia and normoxia on the expression of the H2S-producting enzymes CBS, CSE and MPS

Approximately 80% of renal cell carcinomas are of the clear cell subtype ccRCC, and common characteristics of this cancer include inactivation of the Von Hippel-Lindau tumor suppressor, leading to dysregulation of HIF-1 and 2α transcription factors and ultimately a pseudohypoxic tumor signature. Altered metabolism and upregulation of angiogenic factors result, and while therapies addressing individual such shifts are employed, an approach that targets the fundamental basis of the dysregulated survival and proliferation of ccRCC cells would increase effectivity while reducing systemic side effects.

HypOxystation users Sonke et al. in Canada examined the involvement of hydrogen sulfide H2S in angiogenesis, cytoprotection and metabolism in VHL-deficient ccRCC, where endogenous H2S accumulates as mitochondrial oxidation ceases under hypoxia and pseudo-hypoxia (“Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential; Nitric Oxide. 2015 Sep 15;49:26-39 “). The Sonke lab used the H85 HypOxystation by Don Whitley Scientific to examine the influence of hypoxia (1%) and normoxia on the expression of the H2S-producing enzymes CBS, CSE and MPS; cell viability under treatment with hydroxylamine HA, a CBS/CSE inhibitor, was also compared at normoxia and at hypoxia in the HypOxystation. The closed hypoxic environment created in the HypOxystation provides stable parameters favoring in vivo physiology of cancer cells. Using an avian chorioallantoic membrane xenograft system, they examined the inhibitory effects of H2S on neovascularization.

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Treating advanced cancers by implanting polymer-entrapped palladium nano particles within a tumor

Selective activation of a drug precursor in an orthogonal manner can reduce the adverse systemic effects of the therapy. In a 2015 paper published in Scientific Reports (Vol 5, 9329), HypOxystation users Weiss et al. describe a therapeutic strategy to treat advanced cancers by implanting polymer-entrapped palladium nanoparticles within a tumor in order to continuously induce the activation of propargylated Pro-FUdR to FUdr, a DNA synthesis-disrupting antimetabolite, which is administered enterally. In their paper “Palladium-mediated dealkylation of N-propargyl-floxuridine as a bioorthogonal oxygen-independent prodrug strategy”, they describe the balancing act necessary to design and synthesize a compound that is inactive but resistant to metabolic degradation outside of the tumor, reducing its cytotoxicity for the patient 6,250-fold, which can then be chemically activated by palladium, aided by the specific micro­environment present inside the tumor.

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Enhancing radiosensitivity to treat lung carcinoma at hypoxia and normoxia

Treatment of non-small cell lung cancer NSCLC, the leading cause of cancer-related death, relies heavily on radiation therapy, which however is frequently compromised by development of radioresistance. HypOxystation users Jiang et al. of the University of Oxford describe their study of olaparib, an inhibitor of Poly(ADP-ribose) polymerase 1 (PARP-1) which serves to enhance radiosensitivity, to treat lung carcinoma at hypoxia and normoxia ("Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality"; Int J Radiation Oncol Biol Phys, Vol. 95, No. 2, pp. 772e781, 2016). The lab uses a Don Whitley H35 HypOxystation to create stable, contiguous hypoxic conditions for their cell culture.

A combination of olaparib and radiation significantly increased DNA double strand breaks, and at hypoxia (1% oxygen), the radiation-sensitizing effect of olaparib was increased relative to normoxia (21% oxygen), as evidenced by a decrease in clonogenic survival rates. In vivo effects of oxygen availability were examined in subcutaneous Calu-6 and Calu-3 xenograft assays, where Calu-6 tumors comprise extensive hypoxic areas, and assays focusing on RAD51 protein expression showed that homologous recombination was considerably impacted by olaparib, specifically in hypoxic regions. CC3 staining of Calu-6 tumors as a measure of apoptosis also showed that hypoxic areas are more severely impacted by the combination of olaparib and radiation than normoxic areas. CA9 immunostaining served as a marker for hypoxia.

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Interaction of DNA damage kinase ATM with the ATM interactor ATMIN at hypoxia

DNA repair pathways protecting against genomic instability are significantly repressed under the hypoxic conditions typical of many solid tumors. HypOxystation users Leszczynska et al. describe the interaction of DNA damage kinase ATM with the ATM interactor ATMIN at hypoxia as well as the downstream consequences for DNA repair (“Mechanisms and consequences of ATMIN  repression in hypoxic conditions: roles for p53 and HIF-1” , Scientific Reports 6:21698; 2016).

ATM activation is induced at severe hypoxia as a result of replication stress, and was thought to be dependent on the ATM interactor ATMIN, especially in the absence of DNA damage. Using ATMIN siRNA, ATM inhibitors, and knock-out cell lines, the authors were able to establish that ATMIN is not required for the activation of ATM in response to hypoxia-induced replication stress, and that ATMIN is repressed at hypoxia, an effect mediated by both p53 and HIF-1. The cells were exposed to varying degrees of hypoxia, from mild (2%) down to extreme (0.1%) in an H35 HypOxystation by Don Whitley Scientific. The closed cell culture environment created in the Don Whitley Scientific HypOxystation mimics physiological conditions with regard to oxygen, CO2, temperature, and humidity and enables cancer researchers to obtain a clearer picture of in vivo processes. qPCR analysis of cells in response to hypoxia and exposure to inhibitors of proteasomal degradation indicate that the repressive effect of hypoxia is due to inhibition of translation as opposed to transcription or altered stability of ATMIN.

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Targeting the adaptive hypoxic response in multiple myeloma


Patrick Frost is at the Department of Hematology-Oncology at the VA Hospital in Los Angeles. He has been using an H35 HypOxystation for almost 3 years, and just in March published his most recent findings on multiple myeloma cells cultured in the HypOxystation. At lower than 1% oxygen, the atmosphere inside the chamber mimics the hypoxic niche in the bone marrow microenvironment. Patrick Frost’s group investigated the effects of a polyamide capable of binding to the hypoxia response element HRE of genes such as VEGF that are up-regulated under low oxygen conditions. They found a significant cytotoxic effect of the polyamide against MM tumor cells which they traced to an inhibition of HIF-mediated gene transcription and decreased resistance to hypoxia-induced apoptosis. These results are described in “A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma Has Potent Antitumor Activity” (Mysore et al., 2016, Mol Cancer Res; 14(3); 253-66.)

The focus of our lab’s research is multiple myeloma, specifically with the aim of targeting the adaptive hypoxic response in myeloma cells. We have a number of interesting compounds, and we are studying HIF regulation to understand the mechanisms of what happens in these cells treated with the compounds. We have a polyamide, a synthetic molecule that recognizes the hypoxia response element HRE and binds to the minor groove of the DNA. We have to use relatively high concentrations of this compound but it is a good model to block the hypoxic response. In order to characterize the effect of the molecule, we used it in some reporter assays, in reporter cells, then we tested it to see whether it could overcome their resistance to hypoxia-mediated killing. There is some thought that hypoxia confers resistance to drugs, which we did not find in our particular experiments. We found that the drugs and the hypoxia actually synergize. I think there is a need to differentiate more between resistance and cell cycle arrest in this regard, as opposed to killing the cells. The HypOxystation is very good for placing the cells at hypoxic conditions, at different levels, and sampling at different time points. The time points especially are very useful with this chamber, because with other systems such as boxes, once the cells are inside and you start doing anything with them, you’ve re-set the system; it doesn’t take very long for those media to get re-oxygenated. So we can set up to do a 24 hour assay, using the interlock to get in and out, and take a sample of the growing cells every 24 hours. That really helps to get a nice time course. Once we have the cells inside the chamber, we can assay apoptosis, gene expression, protein expression, anything. The in vitro models are really very useful, we can cover a lot of ground quickly.

Read more: Targeting the adaptive hypoxic response in multiple myeloma