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

Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity

Background: it is now well established that hypoxia renders tumor cells resistant to radio- but also chemotherapy. However, few elements are currently available as for the mechanisms underlying this protection. Results: in this study, physiological hypoxia was shown to inhibit apoptosis induced in HepG2 cells by etoposide. Indeed, hypoxia reduced DNA fragmentation, caspase activation and PARP cleavage. The DNA binding activity of 10 transcription factors was followed while the actual transcriptional activity was measured using specific reporter plasmids. Of note is the inhibition of the etoposideinduced activation of p53 under hypoxia. In parallel, data from low density DNA microarrays indicate that the expression of several pro- and anti-apoptotic genes was modified, among which are Bax and Bak whose expression profile paralleled p53 activity. Cluster analysis of data unravels several possible pathways involved in the hypoxia-induced protection against etoposide-induced apoptosis: one of them could be the inhibition of p53 activity under hypoxia since caspase 3 activity parallels Bax and Bak expression profile. Moreover, specific downregulation of HIF-1α by RNA interference significantly enhanced apoptosis under hypoxia possibly by preventing the hypoxia mediated decrease in Bak expression without altering Bax expression. Conclusion: these results are a clear demonstration that hypoxia has a direct protective effect on apoptotic cell death. Moreover, molecular profiling points to putative pathways responsible for tumor growth in challenging environmental conditions and cancer cell resistance to chemotherapeutic agents

Etude des effets de l'hypoxie sur l'apoptose induite par différents agents chimiothérapeutiques dans différents types cellulaires cancéreux

Study of the effects of hypoxia on the apoptosis induced by different chemotherapeutics agents in different cancer cell types Despite the significant progresses made in the field of cancer screening and treatment, this pathology is still presently accountable for the death of more than 7 million people each year in the world. Chemotherapy is frequently used to induce cancer cell death. However, resistance to this kind of treatment is not rare. Several causes are involved in inducing this resistance. Tumours can indeed express proteins responsible for drug extrusion, display mutations in tumour suppressor genes such as TP53, or contain hypoxic areas. Hypoxic areas are frequently observed inside tumours due to the important distance between the cancer cells and the blood vessels, as well as due to the disorganized tumour vessel architecture. Tumour hypoxia is associated with a bad prognosis for patients in many cancer types. Hypoxia can indeed increase tumour aggressiveness as well as it can protect it from cell death induced by chemotherapeutic agents. However, severe hypoxia has an opposite effect since it can induce cell death. It has moreover been shown that, at the same oxygen percentage, some cell types are protected against apoptosis induced by a chemotherapeutic molecule, while it is not the case for other cell types. The molecular causes for these intercellular differences are not known. During this work, we studied the effect of hypoxia on the cell death induced by five chemotherapeutic molecules in seven cancer cell types originating from different organs and harbouring wild-type, mutated or null p53 protein. In accordance with the data reported in the literature, we observed that hypoxia inhibits the cell death induced by chemotherapeutic agents in the majority of the studied cell types, while it was not the case in all cell types. We next tried to identify the cellular mechanisms induced by hypoxia that are responsible for chemoresistance. We first studied proteins directly involved in apoptosis, and more particularly proteins belonging to the BCL-2 family proteins. We observed, for example, that the abundance of many pro-apoptotic proteins of the BCL-2 family was decreased by hypoxia in HepG2 cells that are strongly protected by hypoxia against etoposide-induced cell death, while it was not the case in A549 cells that are not protected by hypoxia against apoptosis. Moreover, a simultaneous silencing of BIM and NOXA induced a decrease in etoposide-induced cell death, underlying a possible mechanism for chemoresistance. We secondly studied the effect of hypoxia on p53 and on the activation of the signalling pathways activated downstream the etoposide-induced DNA damages. We observed that hypoxia decreased p53 abundance as well as the activation of a part of this pathway in HepG2 cells, while it was not the case in A549 cells. The causes of the hypoxia-induced decrease in p53 abundance in HepG2 cells have been studied and our results suggest a decrease in the translation of this protein during hypoxia. Finally, preliminary results have been obtained with regard to the involvement of miRNAs in the response of hypoxic cells to etoposide-induced cell death. miRNAs are small molecules, recently discovered, that regulate the expression of numerous genes, among which those involved in apoptosis. In conclusion, our results show how different the regulation of cell death by hypoxia could be according to the cell type. Moreover we showed that the response of a cell to hypoxia is very complex and that multiple cellular pathways are modulated by hypoxia in order to induce chemoresistance. These results underline therefore the importance of a better understanding of the intercellular variations of responses to hypoxia in order to improve anti-cancer treatments.Malgré les progrès considérables réalisés dans le domaine du dépistage et des traitements des cancers, cette pathologie est, à l’heure actuelle, toujours responsable de la mort de plus de 7 million de personnes chaque année dans le monde. La chimiothérapie est fréquemment utilisée afin d’induire la mort des cellules cancéreuses. Cependant, une résistance à ce type de traitement n’est pas rare. Plusieurs causes sont responsables de cette résistance. Les tumeurs peuvent notamment exprimer des protéines permettant l’efflux des drogues, présenter des mutations dans des gènes suppresseurs de tumeur comme TP53, ou contenir une zone hypoxique. Une zone hypoxique est fréquemment observée au sein des tumeurs suite à la distance importante entre les cellules cancéreuses et les vaisseaux sanguins, ainsi que suite à l’architecture désorganisée des vaisseaux tumoraux. Cette hypoxie tumorale est associée à un mauvais pronostic pour le patient pour de nombreux types de cancers. L’hypoxie peut, en effet, rendre la tumeur plus agressive ainsi que la protéger contre la mort cellulaire induite par des agents chimiothérapeutiques. Cependant, l’hypoxie sévère a un effet opposé puisqu’elle peut engendrer la mort cellulaire. Il a, de plus, été observé que, à un même pourcentage d’oxygène, certains types cellulaires peuvent être protégés contre l’apoptose induite par une molécule chimiothérapeutique, alors que ce n’est pas le cas pour d’autres types cellulaires. Les causes moléculaires de ces différences intercellulaires ne sont pas connues. Au cours de ce travail, nous avons étudié l’effet de l’hypoxie sur la mort induite par cinq molécules chimiothérapeutiques dans sept types cellulaires cancéreux provenant de différents organes et exprimant une protéine p53 sauvage ou non. En accord avec les données rapportées dans la littérature, nous avons observé que l’hypoxie inhibe la mort cellulaire induite par des agents chimiothérapeutiques dans la majorité des types cellulaires, bien que cela ne soit pas le cas dans tous. Ensuite, nous avons cherché à identifier les mécanismes cellulaires mis en œuvre par la cellule se trouvant en hypoxie pour provoquer ou non cette chimiorésistance. Dans un premier temps, nous nous sommes intéressés aux protéines jouant un rôle direct dans l’apoptose, et plus particulièrement aux protéines de la famille BCL-2. Nous avons notamment observé que, dans les cellules HepG2 qui sont fortement protégées par l’hypoxie contre la mort cellulaire induite par l’étoposide, l’abondance de nombreuses protéines pro-apoptotiques de la famille BCL-2 est diminuée par l’hypoxie, alors que ce n’est pas le cas dans les cellules A549 qui ne sont pas protégées par l’hypoxie contre l’apoptose. Nous avons de plus montré qu’une diminution simultanée de l’abondance de BIM et de NOXA provoque une diminution de la mort cellulaire induite par l’étoposide, mettant ainsi en évidence un mécanisme possible de chimiorésistance. Dans un second temps, nous avons étudié l’impact de l’hypoxie sur p53 et sur l’activation de la voie de signalisation activée en aval des dommages à l’ADN créés par l’étoposide. Nous avons observé que l’hypoxie diminue l’abondance de p53 ainsi que l’activation d’une partie de cette voie dans les cellules HepG2, alors que ce n’est pas le cas dans les cellules A549. Les causes de la diminution de l’abondance de p53 par l’hypoxie dans les cellules HepG2 ont été étudiées et nos résultats suggèrent une diminution de la traduction de cette protéine en hypoxie. Enfin, des résultats préliminaires ont été obtenus quant à l’implication de miRNA dans la réponse de cellules hypoxiques à la mort cellulaire induite par l’étoposide. Les miRNA sont de petites molécules découvertes récemment qui régulent l’expression de nombreux gènes, dont ceux impliqués dans l’apoptose. En conclusion, nos résultats montrent à quel point la régulation de la mort cellulaire par l’hypoxie est différente selon le type cellulaire. De plus, nous avons montré que la réponse d’une cellule à l’hypoxie est très complexe et que de multiples voies cellulaires sont modulées par l’hypoxie afin de provoquer la chimiorésistance. Ces résultats mettent donc en évidence l’importance d’une meilleure compréhension des variations intercellulaires des réponses à l’hypoxie afin d’améliorer les traitements anti-cancéreux.(DOCSC03) -- FUNDP, 201

miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1

BACKGROUND: Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance. miRNAs have recently been shown to play important roles in tumorigenesis and drug resistance. Moreover, hypoxia also regulates the expression of a series of miRNAs. However, the interaction between chemoresistance, hypoxia and miRNAs has not been explored yet. The aim of this study is to understand the mechanisms activated/inhibited by miRNAs under hypoxia that induce resistance to chemotherapy-induced apoptosis. METHODS: TaqMan low-density array was used to identify changes in miRNA expression when cells were exposed to etoposide under hypoxia or normoxia. The effects of miR-196b overexpression on apoptosis and cell proliferation were studied in HepG2 cells. miR-196b target mRNAs were identified by proteomic analysis, luciferase activity assay, RT-qPCR and western blot analysis. RESULTS: Results showed that hypoxia down-regulated miR-196b expression that was induced by etoposide. miR-196b overexpression increased the etoposide-induced apoptosis and reversed the protection of cell death observed under hypoxia. By a proteomic approach combined with bioinformatics analyses, we identified IGF2BP1 as a potential target of miR-196b. Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level. The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia, probably because it is not the only target of miR-196b involved in the regulation of apoptosis. CONCLUSIONS: In conclusion, for the first time, we identified IGF2BP1 as a direct and functional target of miR-196b and showed that miR-196b overexpression reverses the chemoresistance induced by hypoxia. These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12943-015-0349-6) contains supplementary material, which is available to authorized users

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)