Abstract:
Objective: I aimed to investigate the potential immunomodulatory role of complex Autophagy, Toll-like Receptors and PI3-Akt signaling networks/cross-talks in single cell immunobiology by targeting circulating tumor cells in benign prostate hyperplasia and prostate cancer for eventual design of promising patient-friendly cost-effective predictive and prognostic biomarkers and/or pharmacological scaffolds for future immunotherapeutically potent combinatorial drugs with minimal adverse effects in the robotic prostatectomy era
Material and Methods: Cell-viability MTT-proliferation and cytotoxicity assays were performed under basal and Glucose-deprived metabolic/physiological conditions in TLR-4 agonist Lipopolysaccharide (LPS/endotoxin)-treated AR+/- prostate cancer cells in culture, primarily LNCaP and PC3 cells.
Autophagy-flux was monitored by assessing the relative ratios of LC3-II vs LC3-I in 0-12-24-48 hours’ time-course in GD-triggered cells in absence and/or presence of TLR-4 agonist LPS followed by protein isolation by RIPA-method, protein estimation by Bradford’s assay, Western blotting (primary antibodies: LC3, Beclin1, Bcl-2, Atg 2/5, HMGB1 from Cell Signaling Tech./Santa Cruz Biotech., CA, USA). Immunoblots (primary antibody 1:10) were subjected to densitometric scans and relative protein expressions/fold-changes determined (Kodak Imaging software); Glyceraldehyde-3-Phosphate-Dehydrogenase (GAPDH) and/or beta-actin were used as internal controls.
Tumor biopsies were collected from clinically diagnosed/confirmed patients of prostate cancer (early vs advanced as per Prostate Specific Antigen (PSA)-Gleason grade/TNM stage) of North American ethnicity (American Whites, African Americans, Caucasians, Hispanics) from New York State, USA undergoing radical robotic prostatectomy and circulating tumor cells (CTCs) were evaluated for phenotypic differential expression of target genes involved in autophagy/apoptosis/proliferation/necrosis using high-throughput precision-based single cell analysis (SCA). Further, clinical follow-ups of robotic prostatectomy patients (tobacco users/non-users) post-surgery was planned for future bio-bank development in prostate cancer.
Results: LNCaP and PC3 prostate cancer cells were ≥80% viable under basal and Glucose-deprived metabolic/physiological conditions in TLR-4 agonist LPS/endotoxin-stimulated sterile culture in vitro conditions; autophagy-flux was significant in GD-triggered/starvation and/or hypoxic conditions with relatively higher expression levels of LC3-II (14 kDa) vs LC3-I (16 kDa) in 0-12-24-48 hours’ time-course. TLR4 agonist LPS-modulated autophagic flux was significant in LNCaP cells in 48 hours with differential LC3-II vs LC3-I expression levels; protein expression patterns of LC3-II isoform were significantly higher than LC3-1 over 0-12-24-48 hours in AR+/- prostate cancer cells (p≤0.05).
Hypoxic, vascular insufficient, necrotic tumor cores were assessed for CTCs and frozen for prostate cancer/oncofertility biobank/biorepository. Robotic prostatectomy CTCs-SCA clinical research data-sets in North American patients of New York State, USA yielded promising outcomes for predictive and prognostic biomarkers-development (Autophagy-TLR-PI3Akt) for subgroup-stratification of susceptible early vs advanced prostate cancer patients (tobacco users vs non-users) with differential survival trends (Risk Ratios/Hazard Ratios/Odds Ratios) post-surgery. Moreover, necrotopic marker High-Mobility-Group-Box-1 protein, apoptotic marker Bcl-2, and autophagy-signature: Atg2-Atg5, Atg7, Atg-10 and Beclin1 protein expression levels were relatively higher in TLR agonist/GD-triggered prostate cancer cells.
Conclusions: My promising translational research study strongly highlights the emerging immunotherapeutic potential of Autophagy/Toll-like Receptors/PI3-Akt cross-talks/signaling-networks in DAMPs-mediated AR+/- Prostate Cancer for future large sample size-based pharmacogenetics/genomics/transcriptomics/metabolomics-based CTCs-single cell biology-public health oriented meaningful multicentric large sample-size based epidemiology studies (prospective/retrospective) in ethnically disparate population-subsets (tobacco users/non-users) of States of New York/Texas, USA as well as Asia-Pacific region (North+South India). Further, bio-banking in prostate cancer and development of oncofertility-biorepositories may prove to be a boon in DAMPs-mediated “prostate cancer-infertility” men’s health/urology-oncology-reproductive medicine research globally.
