Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines

Background: It is more and more recognized that hypoxia plays a role in the resistance of cancer cells to chemotherapy. However, the mechanisms underlying this resistance still need deeper understanding. The aim of this study was to investigate the effect of hypoxia on this process since hypoxia is one of the hallmarks of tumor environment. Results: The effect of hypoxia on the apoptosis induced by etoposide, one drug commonly used in chemotherapy, was investigated using three different cancer cell lines. Gene expression changes were also studied in order to delineate the mechanisms responsible for the hypoxia-induced chemoresistance. We observed that hypoxia differentially influenced etoposide-induced cell death according to the cancer cell type. While hypoxia inhibited apoptosis in hepatoma HepG2 cells, it had no influence in lung carcinoma A549 cells and further enhanced it in breast cancer MCF-7 cells. Etoposide increased p53 activity in all cell lines while hypoxia alone decreased it only in HepG2 cells. Hypoxia had no influence on the etoposide-induced p53 activity in A549, increased p53 abundance in MCF-7 cells but markedly decreased p53 activity in HepG2 cells. Using low density DNA arrays to detect the expression of genes involved in the regulation of apoptosis, etoposide and hypoxia were shown to each influence the expression of numerous genes, many of the ones influenced by etoposide being p53 target genes. Again, the influence of hypoxia on the etoposideinduced changes was different according to the cell type. Conclusion: These results evidenced that there was a striking parallelism between the effect of hypoxia on the etoposide-induced p53 stabilization as well as p53 target gene expression and its effect on the etoposide-induced apoptosis according to the cell type. They are very interesting not only because they provide one possible mechanism for the induction of chemoresistance under hypoxic conditions in cells like HepG2 but also because they indicate that not all cell types respond the same way. This knowledge is of importance in designing adequate treatment according to the type of tumors

Activation of SK2 channels preserves ER Ca(2+) homeostasis and protects against ER stress-induced cell death

Alteration of endoplasmic reticulum (ER) Ca(2+) homeostasis leads to excessive cytosolic Ca(2+) accumulation and delayed neuronal cell death in acute and chronic neurodegenerative disorders. While our recent studies established a protective role for SK channels against excessive intracellular Ca(2+) accumulation, their functional role in the ER has not been elucidated yet. We show here that SK2 channels are present in ER membranes of neuronal HT-22 cells, and that positive pharmacological modulation of SK2 channels with CyPPA protects against cell death induced by the ER stressors brefeldin A and tunicamycin. Calcium imaging of HT-22 neurons revealed that elevated cytosolic Ca(2+) levels and decreased ER Ca(2+) load during sustained ER stress could be largely prevented by SK2 channel activation. Interestingly, SK2 channel activation reduced the amount of the unfolded protein response transcription factor ATF4, but further enhanced the induction of CHOP. Using siRNA approaches we confirmed a detrimental role for ATF4 in ER stress, whereas CHOP regulation was dispensable for both, brefeldin A toxicity and CyPPA-mediated protection. Cell death induced by blocking Ca(2+) influx into the ER with the SERCA inhibitor thapsigargin was not prevented by CyPPA. Blocking the K(+) efflux via K(+)/H(+) exchangers with quinine inhibited CyPPA-mediated neuroprotection, suggesting an essential role of proton uptake and K(+) release in the SK channel-mediated neuroprotection. Our data demonstrate that ER SK2 channel activation preserves ER Ca(2+) uptake and retention which determines cell survival in conditions where sustained ER stress contributes to progressive neuronal death.Cell Death and Differentiation advance online publication, 20 November 2015; doi:10.1038/cdd.2015.146.</p

Effect of hypoxia and/or etoposide on the HIF-1α protein level and HIF-1 DNA binding activity

<p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. , HIF-1α was detected in total cell extracts by western blotting. a-tubulin was used to assess the total amount of proteins loaded on the gel. , after the incubation, nuclear extracts were performed from three independent experiments and hybridized in the ELISA well containing specific DNA probes (TransAM assay). Detection was performed using an anti-HIF-1α antibody. Results are expressed in absorbance (means ± 1 SD, n = 3)

Gene expression profiling, for genes involved in regulating apoptosis, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions

<p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> Please refer to supplementary data [Additional file ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column