Schoepf 2014 Abstract MiP2014
|OXPHOS analysis in small prostate biopsies.|
In 2012, adenocarcinoma of the prostate was the most prevalent male cancer type in Western civilized countries, accounting for 9.5% of all cancer related deaths in Europe [1,2].
Numerous studies have addressed the metabolic characteristics and molecular pathways of the disease, primarily applying prostate cancer related cell lines as models. In contrast, very little is known about the metabolic properties of fresh prostate cancer tissue in terms of mitochondrial oxidative phosphorylation (OXPHOS) and ATP production. This lack of data is mainly due to the limited access to fresh prostate tissue biopsies and their small sample size. Therefore, in order to overcome this limitation, we developed a method which, for the first time, enables OXPHOS analysis of very small benign and malignant prostate tissue biopsies (2-5 mg wet weight per individual measurement). Samples were harvested immediately after radical prostatectomy and examined using high-resolution-respirometry (Oroboros Oxygraph-2k) and a sophisticated substrate-uncoupler-inhibitor titration (SUIT) protocol. For this purpose, prostate tissue was mechanically permeabilized which, as previously reported, represents a very good mitochondrial preparation alternative to isolated mitochondria, both reducing the amount of required sample material and largely preserving structural integrity of the cell . An artificial incubation with H2O2 was used to assess the mitochondrial response to cellular stress exerted by enhanced ROS production . In addition, an easy and fast assay for determination of CIV activity was applied adding ascorbate and the CIV substrate N,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD) followed by inhibition with sodium azide to correct for chemical background reactions. The SUIT protocol, including different substance combinations, allows measurement of flux control variables in different substrate and coupling states (Fig. 1).
Any sample biopsy (app. 6–10 mg of wet weight) was divided into two subsamples and mechanically permeabilized using two pairs of extra-sharp forceps . Each subsample was placed into one of the four chambers of two Oroboros Oxygraph-2k operated in parallel. Measurements were performed in MiR05 with creatine (MiR05Cr) at 37 °C. Parallel analyses of a paired sample consisting of one benign and one malign biopsy from a single patient could be conducted within little more than two hours, yielding high quality respirometry data while preserving tissue structure for subsequent tissue analysis and DNA extraction.
The first data evaluations revealed an overall decreased CI-linked OXPHOS capacity before H2O2 treatment, a higher vulnerability toward cellular stress mediated by H2O2 but a similar CI&II-linked OXPHOS capacity after H2O2 treatment in cancer compared to benign tissue (Fig. 2).
Labels: MiParea: Respiration, Patients Pathology: Cancer Stress:Oxidative stress;RONS Organism: Human Tissue;cell: Genital Preparation: Permeabilized tissue
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, CIV, NS HRR: Oxygraph-2k Event: A4, Oral MiP2014
1-Oroboros Instruments; 2-Oncotyrol – Center Personalized Cancer Medicine; 3-Div Genetic Epidemiology*; 4-Dep Urology*; 5-Dep Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab*, *Medical Univ Innsbruck; Innsbruck, Austria. - firstname.lastname@example.org
References and acknowledgements
Supported by Oncotyrol.
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