Kriel 2015 Abstract MiPschool Cape Town 2015
|Quantification of mitochondrial fission and fusion rate through photoactivation in Glioblastoma Multiforme.|
Kriel J (2015)
Event: MiPschool Cape Town 2015
Glioblastoma Multiforme (GBM) is a grade IV astrocytoma known to be chemotherapeutically resistant. After treatment and surgical removal, the patient survival rate generally remains poor, stressing the need for new therapeutic approaches. Macroautophagy (hereafter referred to as autophagy), is a major protein degradation pathway. Autophagic inhibition through chloroquine treatment has shown to impair tumour growth in the clinical setting . Of note, the same has been shown with autophagic induction by means of rapamycin treatment in vivo. Furthermore, the clearance of damaged or depolarized mitochondria is mediated through autophagy, maintaining a healthy mitochondrial network. Mitochondrial fission and fusion serves to maintain mitochondrial membrane potential integrity in response to changing bioenergetic demand and therefore serves as a necessary response to excessive tumorigenic energy demands. Although an effective hydroxychloroquine concentration has been determined in the clinical setting , the extent to which autophagic modulation affects the bioenergetic capacity of tumours to survive is not well understood. Therefore, the quantification of mitochondrial fission and fusion events in response to autophagy modulation would substantially contribute to the development of accurate chemotherapeutic sensitization protocols.
Labels: MiParea: Instruments;methods, mt-Structure;fission;fusion, Patients
Organism: Human Tissue;cell: Nervous system, Other cell lines
This study therefore aimed to quantify fission and fusion though photoactivation in an in vitro model of GBM in response to autophagic modulation. Treatments were carried out using the U-118 MG (ATCC HTB15(TM)) human glioma cell line cultured in Dulbecco's Modified Eagle's Medium, incubated at 37°C and exposed to 5% CO2. 3-(4,5-dimethylthiazol-2-yl)-2,diphenyltetrazolium bromide (MTT) assays were conducted to assess reductive capacity impairment in relation to chloroquine (12.5 M, 25 M, 50 M, 100 M) and rapamycin (25 M) concentrations and treatment time points (6 and 12 hours). Cells were also transiently transfected with mitochondrial matrix targeted photoactivateable green fluorescent protein (mito-PA-GFP) according to the manufacturer's protocol. PA-GFP is a variant of Aequoravictoria GFP which increases green fluorescence 100-fold upon laser activation . Successfully transfected cells were photoactivated using a 405 nm laser and GFP as well as tetramethylrhodamine-ethyl ester (TMRE) signal was subsequently acquired with a 488 nm and 561 nm laser respectively using a Zeiss 780GAasp detector confocal platform. Staining with TMRE allowed for visualization of the mitochondrial network and co-localization with mito-PA-GFP signal. Mito-PA-GFP signal distribution was assessed by plotting the decrease in fluorescent intensity over time  allowing for the calculation of fission and fusion rates.
Results derived from MTT assays suggests that a 6 hour treatment period with 25 µM chloroquine decreases cell viability significantly. To our surprise, pre-treatment with rapamycin followed by chloroquine treatment affected reductive capacity to a greater extent than chloroquine treatment on its own. Fission and fusion rate was significantly decreased in both the chloroquine and rapamycin treatment groups when compared to the control. However, treatment with rapamycin prior to chloroquine administration impaired signal distributionand fission and fusion rates dramatically. When considering the ties between autophagic flux and fission and fusion events, the data generated indicate the importance of the precise quantification of these processes. The method set out here shows much promise for assessing the combined effect of autophagy modulation and chemotherapeutic treatment. Finally, the photoactivation protocol presented here is the first of its kind to be implemented in South Africa in the context of GBM and may allow for the development of more accurate chemotherapeutic sensitization protocols.
Dept Physiol Sci, Stellenbosch Univ, South Africa. - [email protected]
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