Autophagosomal Sequestration of Mitochondria as an Indicator of Antiandrogen Therapy Resistance of Prostate Cancer (PCa)
Technical Report,01 Sep 2015,31 Oct 2019
The University of Texas MD Anderson Cancer Center Houston United States
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Purpose We are investigating if sequestration of metabolically dysfunctional mitochondria by the autophagosomes mitophagy imparts anti-androgen resistance and if this phenomenon can be applied in circulating tumor cells in patient blood samples as a biomarker for development of drug resistance. Method Effects of the anti-androgen enzalutamide on the autophagy and mitophagy of androgen-dependent LNCaP and -independent C4-2 and CWR22v1 cells are studied first. Autophagy is monitored by fluorescence of cells with anti-LC3B antibody. Cellular fluorescence due to Mitosox dye oxidation is used to identify mitochondria producing high superoxide O2-. Mitophagy is monitored using fluorescence resonance energy transfer FRET by visualization of FRET images and quantitation of FRET image intensities using a Leica Sp8 fluorescence STED confocal microscope and Image J software. Results and Discussion The degree of mitophagy is more in the surviving androgen-dependent LNCaP cells than in the -independent C4-2 cells, when grown in androgen-depleted media. Enzalutamide treatment induces mitophagy in both cell lines. However, the increase in mitophagy is significantly more in the enzalutamide resistant C4-2 than in the sensitive LNCaP cells. Mitosox fluorescence and mitophagy in circulating tumor cells CTCs isolated from patient blood samples are being quantitated to identify drug resistance.
- Medicine and Medical Research