ADI-based Autophagy Induction in Prostate Cancer Cells: An Enzyme-based Technique to Measure Autophagic Response in Cells

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.

To begin, grow human prostate cancer cells expressing green fluorescent protein-coupled Light Chain 3 on 35-millimeter poly-D-lysine coated glass bottom culture dishes, as described in the text protocol.

Cells should be plated at sufficient density to facilitate rapid proliferation, but not so much that cells are overgrown and clumped by the time of imaging. Treat selected cell samples with arginine deaminase, or ADI, in PBS to deplete cells of free arginine and induce metabolic stress in the cancer cells.

Approximately one hour prior to imaging, dilute 1.5 microliters of LysoTracker Red with 20 milliliters of RPMI containing 10% FBS and 1% antibiotics. Prepare solutions with ADI for the selected samples that were treated.

After warming all media to 37 degrees Celsius, add the appropriate media to each culture dish. Incubate cells with RPMI containing LysoTracker Red for 15 to 45 minutes at 37 degrees Celsius.

Approximately 30 minutes prior to imaging, turn on the weather station environmental enclosure and allow equilibration to 37 degrees Celsius and 5% carbon dioxide. Wash cells with PBS and replace media with standard RPMI containing only 10% FBS and 1% antibiotics.

Add ADI to samples as indicated. Mount 35-millimeter cover glass bottom culture dishes in a customized adapter. Use immersion oil on the 60X magnification, 1.42 numerical aperture objective lens, and position the mounted culture dish on the microscope stage.

• Autophagy - An intracellular process that degrades unwanted cytoplasmic components.
• Argininosuccinate Synthase - An enzyme absent in prostate cancer cells essential for arginine synthesis.
• Arginine Deiminase (ADI) - Enzyme that hydrolyzes free arginine in media, causing cell stress.
• LC3 Proteins - Autophagic marker proteins that provide fluorescent tags to autophagosomes.
• Lysotracker Red DND-99 - A dye used to label lysosomes within cells during autophagy.

• Introduce Autophagy - A degradative process eliminating unwanted parts in cells (e.g., autophagy video).
• Outline Prostate Cancer Cells - Lacking an enzyme, they depend on external arginine sources (e.g., prostate enzyme).
• Explain Induction of Autophagy - ADI depletes arginine causing autophagy in the cells (e.g., autophagy induction).
• Connect to Experiment - The cells are simultaneously treated with lysotracker red DND-99 (e.g., autophagy dye).

• What is autophagy and how does it relate to prostate cancer cells (include ADI)?
• What are LC3 proteins and their role in autophagy?
• How does Arginine Deiminase initiate an autophagic response in cells?

• Practical Applications - Autophagy serves as a potential target for prostate cancer treatment (e.g., autophagy and prostate cancer).
• Industry Impact - Autophagy research benefits healthcare and pharmaceutical sectors (e.g., prostate cancer enzyme marker).
• Societal Importance - Observing real-time autophagy in cells can inform disease treatments (e.g., autophagy videos).
• Link to Scientific Advancements - Dyes like lysotracker red dnd-99 help visualize cellular processes.