Utilising autophagy modulation to combat cell death resistance in Glioblastoma Multiforme

Kriel, Jurgen Andries (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH SUMMARY: Introduction: Globally, Glioblastoma Multiforme (GBM) presents as both the most prevalent and invasive form of Central Nervous System (CNS) malignancy. Patient life expectancy has remained largely unchanged over the past three decades, with a mean survival time of only 15 months. Although many advances have been made in understanding the molecular pathogenesis of GBM, the clinical treatment regimen has not been improved upon. One major commonality between all gliomas is their excessive energy requirements to uphold proliferation. The GBM metabolic profile has been shown to comprise of both oxidative phosphorylation (OXPHOS) and aerobic glycolysis. Moreover, the protein degradation pathway known as autophagy has proved to uphold tumour survival in nutrient limiting conditions. Autophagy modulators have therefore shown much promise to act as adjuvants to chemotherapy, although determining the degree of inhibition and or induction necessary to achieve sensitisation remains a major challenge. We, therefore, hypothesised that glioma cells can be sensitised to undergo cell death by modulating autophagy in order to decrease bioenergetic efficiency and enhance susceptibility to chemotherapy. The aims were thus to: (i) determine the amount of autophagy modulation necessary to sensitise glioma cells to chemotherapy; (ii) assess mitochondrial bioenergetics in terms of topology, dynamics and electron transport system efficiency; (iii) determine the extent of diminished mitochondrial capacity necessary to achieve cell death sensitisation Methods: An in vitro model of GBM was employed utilising the U-118MG glioblastoma cell line. Chemical autophagy modulation was achieved by utilising Rapamycin (inducer) and Hydroxychloroquine (HCQ, inhibitor). Sensitisation was assessed through employing a combination of HCQ with Temozolomide (TMZ) (HT) and Rapamycin pre-treatment followed by HT (RHT). Effective drug concentrations were determined through WST-1 viability assays. Immunoblotting for LC3-II and p62 allowed the determination of autophagic activity, whilst MFN1, MFN2, OPA1 and DRP1 protein levels indicated mitochondrial morphology regulation. Live cell imaging was conducted to assess mitochondrial fission and fusion rate. Image processing algorithms were constructed in Wolfram Mathematica (v. 10.2) to quantify mitochondrial morphometric alterations. Finally, high-resolution respirometry was conducted to assess mitochondrial coupling efficiency and apoptosis onset was assessed by immunoblotting for cleaved caspase 3 and cleaved PARP. Results and Discussion: Autophagy induction with 50nM Rapamycin enhanced reductive capacity (135.0 ± 6.04 %), whilst inhibition with 50 μM HCQ resulted in a moderate decrease (86.22 ± 2.37 %). U-118MG cells proved sensitive to 24 hour TMZ (250 μM) treatment (80.19 ± 1.76 %), with reductive capacity decreased following HT (75.25 ± 3.493 %). Importantly, RHT treatment decreased viability to 52.65 % (± 1.06 %). This is further supported by significantly enhanced cleaved caspase 3 (146.00 ± 14.78 %) and cleaved PARP (157.60 ± 20.41 %) protein levels for the RHT group. Autophagic degradation was impaired in both the HT and RHT groups, as no significant increase in LC3-II expression was observed before and after Bafilomycin A1 treatment. Decreasedmitochondrial fusion rates were observed following HCQ, TMZ, HT and RHT treatment, however, a decrease in both DRP1 and OPA1 was observed for the RHT group only. OXPHOS and ETS capacity were significantly impaired following RHT treatment, with an intermediate amount of network connectivity observed compared to the control and TMZ treatment group. Of note, decreased lactate production was observed in both the HT and RHT treatment groups, indicating decreased glycolysis. Conclusion: Coordinated upregulation of autophagy followed by its inhibition prior to chemotherapy decreased OXPHOS capacity in GBM to the extent of enhancing apoptotic cell death onset. Impaired ETS coupling was associated with decreased OPA1 and DRP1 protein levels, which possibly contributed to intermediate mitochondrial network connectivity. Decreased lactate production in both modulation groups indicates that both glycolysis and OXPHOS was impaired in GBM cells. Therefore, reduced autophagic degradation likely impaired GBM metabolism and enhanced apoptosis onset.

AFRIKAANSE OPSOMMING: Inleiding: Multivormige Glioblastoma (GBM) staan internasionaal bekend as die mees algemeen en kwaadaardige sentrale senuwee stelsel (SSS) gewas. Die gemiddelde oorlewingstydperk vir GBM pasiënte is slegs 15 maande en het vir die afgelope 30 jaar onveranderd gebly. Alhoewel baie vordering gemaak is in terme van die molekulêre patogenese van GBM, is daar min verandering gemaak aan kliniese behandelingstrategieë. ‘n Algemene ooreenkoms tussen alle GBM gewasse is hul oormatige energie vereistes om aanhoudende groei te verseker. Die metaboliese profiel van GBM bestaan uit beide oksidatiewe fosforilasie (OKSFOS) en aërobiese glikoliese. Met betrekking tot metabolisme is die proteïen afbraakproses, bekend as autofagie, bewys om kankermetabolisme te ondersteun tydens ‘n tekort aan voedingstowwe. Die vermoë van autofagie moduleerders om op te tree as bevorderingsmiddele vir chemoterapie is onlangs bestudeer, alhoewel die mate van inhibisie of induksie benodig vir effektiewe bevordering van seldood steeds onbepaald bly. Ons hipotese is dus dat autofagie modulasie die bioenergetika van glioomselle tot so ‘n mate sal belemmer dat dit seldood deur middel van chemoterapie sal bevorder. Hierdie studie beoog dus om: (i) die mate van autofagie modulasie wat benodig is vir seldood bevordering te bepaal; (ii) mitochondriese bioenergetika te evalueer in terme van topologie, dinamika en effektiwiteit van die elektronoordragsketting; (iii) te bepaal tot watter mate mitochondriese kapasiteit belemmer moet word om seldood te bevorder. Metodes: ‘n In vitro model van GBM was saamgestel deur gebruik te maak van U-118MG glioblastoma selle. Chemiese autofagie modulasie was uitgevoer deur gebruik te maak van Rapamycin (induseerder) en hydroksiechloroquine (HCQ, inhibitor). Seldood bevordering was geëvalueer deur gebruik te maak van ‘n kombinasie van HCQ en Temozolomide chemoterapie (TMZ) (HT) asook vooraf behandeling met Rapamycin gevolg deur die HT behandelingskedule. Effektiewe dwelmkonsentrasies is bepaal deur middel van die WST-1 sellewensvatbaarheid toets. Proteïen uitdrukking van LC3-II en p62 het die evalueering van autofagiese aktiwiteit moontlik gemaak, terwyl die uitdrukking van MFN1, MFN2, OPA1 en DRP1 gedien het as aanwysers vir mitochondriese morfologiese regulasie en was bepaal deur die westelike kladtegniek. Die spoed van mitokondriese fisie en samesmelting is bepaal deur lewendige sell mikroskopie en beeldverwerking algoritmes is saamgestel in Wolfram Mathemica (v. 10.2) om morfologiese verandering te kwantifiseer. Hoë resolusie respirometrie was toegepas om mitochondriese suurstof verbruik te evalueer. Laastens is proteïen uitdrukking van gekleefde caspase 3 en gekleefde PARP nagegaan om apoptotiese seldood bevordering te evalueer en is bepaal deur middel van die westelike kladtegniek. Resultate en bespreking: Autofagie induksie deur middel van 50nM Rapamycin het reduktiewe kapasiteit vermeerder (135.0 ± 6.04%), terwyl inhibisie met 50 μM HCQ ‘n matige vermindering in sellewensvatbaarheid veroorsaak het (86.22 ± 2.37 %). U-118MG selle was nie weerstandig teen 24 uur se inkubasie met TMZ (250 μM) nie (80.19 ± 1.76 %), met ‘n verdere afname in reduktiewe kapasiteit veroorsaak deur HT behandeing. Van groot belang is dat RHT behandeling sellewensvatbaarheid tot 52.65 % (± 1.06 %) laat daal het. Hierdie bevinding is verder ondersteun deur ‘n insiggewende vermeerdering in beide gekleefde caspase 3 (146.00 ± 14.78 %) en gekleefde PARP (157.60 ± 20.41 %) proteïenvlakke vir die RHT behnadelingsgroep. Die afbraak vermoë van autofagie was belemmer in beidie die HT en RHT groepe, aangesien geen insiggewende toename in LC3-II uitdrukking opgemerk is voor en na Bafilomycin A1 behandeling nie. ‘n Aansienlike afname in die spoed van mitochondriese samesmelting was aanskou vir die HCQ, TMZ, HT en RHT groepe, alhoewel verminderde proteïen uitdrukking slegs gevind was vir die RHT groep. OKSFOS en elektronoordragstketting kapasiteit was beduidend verminder na RHT behandling, met ‘n intermediêre hoeveelheid netwerkverbindings in vergelyking met die kontrole en TMZ groepe. Laktaat produksie was ook beduidend minder in the HT en RHT behandelingsgroepe, wat ‘n aanduiding is van ‘n belemmerde glikolise. Gevolgtrekkings: Gekoӧrdineerde induksie van autofagie gevolg deur inhibisie voor blootstelling aan chemoterapie het OKSFOS kapasiteit in GBM selle belemmer en apoptotiese seldood bevorder. Verswakte elektronoordragstketting aktiwiteit was geassosieer met verlaage OPA1 en DRP1 proteïen uitdrukking wat moontlik bygedra het tot die intermediêre hoeveelheid netwerkverbindings. Verlaagde laktaat produksie in beide modulasie groepe dui op verswatke glikolise en oksidatiewe fosforilasie. Die moontlikheid bestaan dus dat verminderde autofagiese aktiwiteit bygedra het tot verswakte GBM metaboliese kapasiteit en het sodoende apoptose veroorsaak.

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