ADI-based Autophagy Induction in Prostate Cancer Cells: An Enzyme-based Technique to Measure Autophagic Response in Cells
Transcrição
Autophagy is an intracellular process that facilitates the degradation of unwanted cytoplasmic components and organelles within specialized digestive compartments called lysosomes. To assess autophagy, begin with an adherent culture of prostate cancer cells expressing autophagic marker proteins called LC3 coupled to green fluorescent proteins.
Inherently, prostate cancer cells lack the enzyme argininosuccinate synthase necessary for arginine amino acid synthesis. These cells are, therefore, dependent on external arginine supplementation through media for survival and proliferation.
Next, treat the cells with arginine deiminase or ADI. ADI hydrolyzes free arginine in the media, leading to arginine depletion. In consequence, the cells undergo metabolic stress, initiating an autophagic response internally. Simultaneously, treat the cells with a red fluorescent dye that labels lysosomes within the cells.
During autophagy, cup-shaped membranous structures called phagophores assemble around the damaged cytoplasmic components. Eventually, they expand and seal their cargo while conjugating with autophagic marker proteins LC3 to form double-membraned vesicles or autophagosomes. LC3 proteins also provide fluorescent tags to the autophagosomes. The vesicles then fuse with lysosomes to form autolysosomes. Lysosomal proteases within the autolysosomes degrade the engulfed cellular components.
In real-time, observe the bright fluorescence from autophagosomes and lysosomes to determine their distribution within the cells.
