The relationship between HIF-1α and autophagy activity in the hypoxic environment of breast cancer
Stellenbosch : Stellenbosch University, 2013
Online
Hochschulschrift
Zugriff:
Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Introduction: Among the cancers that afflict females world-wide, neoplastic disease of breast tissue is the most frequently diagnosed form and the leading cause of cancer-related death. Conventional treatment entails the use of doxorubicin, an anticancer agent belonging to the anthracycline family of chemotherapeutic drugs. Cancer cells are becoming increasingly resistant to doxorubicin therapy. The existence of hypoxic zones, which is a common feature of solid tumours, has been shown to promote the selection of therapy resistant clones in proliferating cancer cells. By modifying cellular homeostasis, neoplastic cells are capable of tolerating the hypoxic insult and thriving within the hostile microenvironment of the tumour. This adaptation is known as ‘the hypoxic response’ and is mediated through the action of the transcriptional regulator, HIF-1. Its expression in cancer tissue has been associated with a dismal prognosis as it promotes the degree of malignancy to an advanced stage. Hypothesis & Aims: We hypothesized that the targeting of HIF-1α would circumvent the ‘protective’ hypoxic response conferred upon breast cancer and improve the cytotoxicity of doxorubicin treatment. In this study, the first aim was to identify the hypoxic conditions at which the MCF-7 breast cancer cell line manifests a doxorubicin-resistant phenotype. This was followed by examination of the molecular pathways contributing to the hypoxic resistance by elucidating the potential relationship with the hypoxic regulator HIF-1α. Once the involvement of HIF-1α was established, the next aim was to evaluate whether the attenuation of HIF-1α would terminate the resistant phenotype and sensitize the neoplastic MCF-7 cells to doxorubicin treatment. Finally, the reproducibility of the in vitro experiment and efficacy of treatments within an animal model was evaluated. 2-Methoxyestradiol is a naturally occurring metabolite originating from 17β-estradiol. It has recently been exploited as an anticancer agent due to its anti-proliferative and anti-angiogenic properties. Among its various mechanisms of action, this compound has been shown to inhibit the expression of HIF-1α. It is for this reason that this study employed 2-methoxyestradiol in the adjuvant therapeutic treatment, along with doxorubicin. Methods: The in vitro experimental model employed the use of the breast adenocarcinoma estrogen receptor (ER-positive cell line, MCF-7. These neoplastic cells were propagated under standard culture conditions until reaching ~70-80% confluency, after which treatment commenced. The treatment regime comprised a 12 hour exposure to the doxorubicin (1 μM) chemotherapeutic agent, either alone or in combination with HIF-1α inhibitors, 2-methoxyestradiol (10 μM) or siRNA duplex (400 nM), with parallel incubations under normoxic (21%) and hypoxic (~0.1%) conditions. To serve as a positive control for HIF-1α expression, cells were treated with CoCl2 (100 μM). Molecular techniques employed included the Caspase-Glo® 3/7 Assay, western blotting, and the bioreductive MTT Assay. Mitochondrial integrity was assessed by live cell imaging/fluorescent microscopy. Cellular viability was monitored at all times. The experiment was then translated into a pre-clinical in vivo model where C57BL/6 mice bearing E0771 xenografts (4 week growth) were allocated into the following treatment groups: (1) control (2) doxorubicin (5 mg.kg-1), (3) 2-methoxyestradiol (45 mg.kg-1), and (4) the combination of the two previously mentioned groups. Body weight and the rate of tumour growth were monitored throughout the experiment. Results: Treatment with CoCl2 effectively stabilized HIF-1α under normoxic conditions. 2-Methoxyestradiol was capable of attenuating HIF-1α expression under both normoxia and hypoxia as compared with siRNA transfection, which was only effective under normoxia. HIF-1α stabilization was accompanied by an increase in autophagy along with the morphological transformation of mitochondria from an elongated network to shorter disc-like forms. On the other hand, HIF-1α attenuation caused an induction in the expression of the apoptotic markers, cleaved caspase 3 and cleaved PARP, as well as the restoration of the normoxic morphology. The exposure of MCF-7 cells to 1 μM doxorubicin for 12 hours produced a differential effect in the bioreductive MTT assay between normoxic and hypoxic conditions (42.97 ± 3.095% vs. normoxic dox, p
AFRIKAANSE OPSOMMING: Inleiding: Borskanker is die mees algemeen gediagnoseerde kanker asook die hoof oorsaak van kanker-verwante sterftes in vrouens wêreldwyd. Konvensionele behandeling behels die toediening van doxorubicin, ‘n anti-kankermiddel wat aan die antrasiklien-familie van chemoterapeutiese middels behoort. Kankerselle begin egter toenemend weerstandbiedend raak teen doxorubicin behandeling. Daar is al bewys dat die voorkoms van hipoksiese sones, wat ‘n algemene eienskap van soliede tumore is, die seleksie vir weerstandbiedende klone van prolifererende kankerselle, veroorsaak. Neoplastiese selle kan hierdie hipoksiese toestande weerstaan en in hierdie ongunstige mikro-omgewing floreer deur sellulêre homeostase te modifiseer. Hierdie aanpassing staan bekend as die ‘hipoksiese respons’ en word bemiddel deur die aksies van die transkripsiefaktor reguleerder, HIF-1. Die verhoogde uitdrukking van HIF-1 in kankerweefsel word oor die algemeen geassosieer met ‘n swak prognose omdat dit die maligniteit vehoog. Hipotese en Doelwitte: Die hipotese van hierdie studie behels dus die volgende: Deur HIF-1α te inhibeer, sal die ‘beskermende’ hipoksiese respons wat in borskankerselle voorkom omseil kan word en sodoende die sitotoksisiteit van doxorubicin terapie verhoog. Die eerste doelwit van hierdie studie was dus om die hipoksiese kondisies te identifiseer waar MCF-7 selle ‘n doxorubicin-weerstandbiedende fenotipe vertoon. Daarna is die molekulêre paaie wat bydrae tot hierdie hipoksiese weerstand ondersoek asook hul moontlike verwantskap met die hipoksiese reguleerder, HIF-1α. Nadat die rol van HIF-1α bevestig is, was die volgende doelwit om te bepaal of die inhibisie van HIF-1α die weerstandbiedende fenotipe sal onderdruk en neoplastiese MCF-7 selle sal sensitiseer vir doxorubicin behandeling. Laastens is die herhaalbaarheid en effektiwiteit van behandeling in die in vitro eksperimente ook in ‘n diermodel getoets. 2-Methoxyestradiol is ‘n metaboliet van 17β-estradiol wat natuurlik in die liggaam voorkom. Dit is ook onlangs as ‘n anti-kanker middel geïdentifiseer as gevolg van die anti-verdelende en anti-angiogeniese eienskappe. Een van die eienskappe van 2-methoxyestradiol is dat dit ook die uitdrukking van HIF-1α kan onderdruk. Dit is dan ook vir hierdie rede dat 2-methoxyestradiol in hierdie studie as bykomende terapie saam met doxorubicin gebruik is. Metodes: Die in vitro eksperimentele model behels die gebruik van ‘n borsadenokarsinoom, estrogeenreseptor (ER)- positiewe sellyn, MCF-7. Hierdie neoplastiese selle is onder standaard weefselkultuur omstandighede gekweek totdat konfluensie van ~70-80% bereik is, waarna behandeling begin het. Die behandelingsprosedure behels ‘n 12 uur blootstelling aan doxorubicin (1 µM) chemoterapeutiese middel alleen of in kombinasie met die HIF-1α inhibitore, 2-methoxyestradiol (10 µM) of siRNA duplex (400 nM) in normoksiese (21% O2) en hipoksiese (~0.1% O2) toestande. Die selle is ook met CoCl2 behandel wat gedien het as ‘n positiewe kontrole vir HIF-1α uitdrukking. Molekulêre tegnieke wat tydens hierdie studie gebruik is, sluit die “Caspase-Glo® 3/7” bepaling in, asook die westelike kladtegniek en die MTT bepaling. Mitochondriale integriteit is bepaal deur middel van lewende sel afbeeldings/fluoresensie mikroskopie. Sellewensvatbaarheid is ten alle tye gemonitor. Hierdie eksperment is verder ook in ‘n pre-kliniese in vivo model uitgevoer waar C57BL/6 muise met E0771 xenografte (4 weke groei) geïnduseer is en in die volgende behandelingsgroepe verdeel is: (1) kontrole; (2) doxorubicin (5 mg.kg-1); (3) 2-methoxyestradiol (45 mg.kg-1); en (4) die kombinasie van laasgenoemde twee groepe. Die liggaamsgewig en die tempo van tumorgroei is tydens die hele eksperiment gemonitor. Resultate: CoCl2 behandeling het HIF-1α effektief gestabiliseer tydens normoksiese omstandighede. 2-Methoxyestradiol het HIF-1α uitdrukking tydens normoksiese en hipoksiese toestande onderdruk wanneer dit vergelyk is met siRNA transfeksie wat slegs tydens normoksiese toestande effektief was. HIF-1α stabilisering het gepaardgegaan met ‘n toename in autofagie asook morfologiese veranderinge in die mitochondria vanaf ‘n verlengde netwerk tot korter skyfagtige vorme. Aan die ander kant het HIF-1α onderdrukking ‘n toename in die apoptotiese merkers, nl kliewing in caspase-3 and PARP veroorsaak wat gepaard gegaan het met die herstel van die tubulêre mitochondriale netwerk. Die blootstelling van die MCF-7 selle aan 1 µM doxorubicin vir 12 ure het ‘n differensiële effek in die bioreduktiewe MTT bepaling tot gevolg gehad tussen normoksiese en hipoksiese toestande (42.97 ± 3.095%, p
CANSA and Marie Stander
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The relationship between HIF-1α and autophagy activity in the hypoxic environment of breast cancer
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Autor/in / Beteiligte Person: | Mills, Justin |
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Veröffentlichung: | Stellenbosch : Stellenbosch University, 2013 |
Medientyp: | Hochschulschrift |
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