Browsing by Author "Kriel, Jurgen Andries"

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    Utilising autophagy modulation to combat cell death resistance in Glioblastoma Multiforme
    (Stellenbosch : Stellenbosch University, 2016-12) Kriel, Jurgen Andries; Loos, Benjamin; Kristian, Muller-Nedebock; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    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.