V-ATPase roles in Prostate Cancer

Vacuolar ATPase (V-ATPase) is responsible for maintaining the acidic pH of the endomembrane system in eukaryotic cells. V-ATPase active transport of protons generates the differential luminal pH in lysosomes, endosomes, and the Golgi. In addition to intracellular V-ATPase, cancer cells have V-ATPase at the plasma membrane. Plasmalemmal V-ATPase acidifies the extracellular milieu and enhances cell motility and invasion, evidence that V-ATPase contributes to tumorigenic phenotypes. We studied V-ATPase cellular functions in prostate cancer (PCa), the most commonly diagnosed cancer for men in the United States. V-ATPase inhibitors decreased invasion and migration of PCa cells. In aggressive PCa cell lines, C4-2B and PC3, V-ATPase was detected in plasma membrane enriched extracts and near the leading edge of migrating cells, respectively. V-ATPase was very abundant in the Golgi and clathrin-coated vesicles (CCV) of all the cell lines studied (LAPC4, LNCaP, C4-2B and PC3). V-ATPase inhibition increased the endo-lysosomal pH and altered F-actin organization and membrane traffic. Accrued cytosolic vesicles included CCV, recycling endosomes, and secretory vesicles. Prostate Specific Antigen (PSA) accumulated in the cytosol and its secretion of was reduced. PSA mRNA expression was reduced as well. The androgen receptor (AR) that controls PSA transcription was inhibited and AR protein and mRNA reduced by 50% or more. Moreover, V-ATPase inhibition increased cellular levels of the subunit α of hypoxia inducible factor 1 (HIF-1α), a transcription factor that regulates tumorigenesis. V-ATPase-dependent HIF-1α accumulation repressed AR expression that was rescued by iron in LNCaP and LAPC4 cells. Thus, V-ATPase-dependent pH and iron homeostasis can be altered to target androgen-AR signaling to inhibits cell proliferation in prostate tumors

Beta-Alanine Suppresses Malignant Breast Epithelial Cell Aggressiveness Through Alterations In Metabolism and Cellular Acidity In Vitro

Background: Deregulated energetics is a property of most cancer cells. This phenomenon, known as the Warburg Effect or aerobic glycolysis, is characterized by increased glucose uptake, lactate export and extracellular acidification, even in the presence of oxygen. beta-alanine is a non-essential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. Because of this buffering capacity, we investigated the effects of beta-alanine on the metabolic cancerous phenotype. Methods: Non-malignant MCF-10a and malignant MCF-7 breast epithelial cells were treated with beta-alanine at 100 mM for 24 hours. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate (OCR). mRNA of metabolism-related genes was quantified by qRT-PCR with corresponding protein expression quantified by immunoblotting, or by flow cytometry which was verified by confocal microscopy. Mitochondrial content was quantified using a mitochondria-specific dye and measured by flow cytometry. Results: Cells treated with beta-alanine displayed significantly suppressed basal and peak ECAR (aerobic glycolysis), with simultaneous increase in glucose transporter 1 (GLUT1). Additionally, cells treated with beta-alanine exhibited significantly reduced basal and peak OCR (oxidative metabolism), which was accompanied by reduction in mitochondrial content with subsequent suppression of genes which promote mitochondrial biosynthesis. Suppression of glycolytic and oxidative metabolism by beta-alanine resulted in the reduction of total metabolic rate, although cell viability was not affected. Because beta-alanine treatment reduces extracellular acidity, a constituent of the invasive microenvironment that promotes progression, we investigated the effect of beta-alanine on breast cell viability and migration. beta-alanine was shown to reduce both cell migration and proliferation without acting in a cytotoxic fashion. Moreover, beta-alanine significantly increased malignant cell sensitivity to doxorubicin, suggesting a potential role as a co-therapeutic agent. Conclusion: Taken together, our results suggest that beta-alanine may elicit several anti-tumor effects. Our observations support the need for further investigation into the mechanism(s) of action and specificity of beta-alanine as a co-therapeutic agent in the treatment of breast tumors

F-actin reorganization by V-ATPase inhibition in prostate cancer

The vacuolar ATPase (V-ATPase) proton pump sustains cellular pH homeostasis, and its inhibition triggers numerous stress responses. However, the cellular mechanisms involved remain largely elusive in cancer cells. We studied V-ATPase in the prostate cancer (PCa) cell line PC-3, which has characteristics of highly metastatic PCa. V-ATPase inhibitors impaired endo-lysosomal pH, vesicle trafficking, migration, and invasion. V-ATPase accrual in the Golgi and recycling endosomes suggests that traffic of internalized membrane vesicles back to the plasma membrane was particularly impaired. Directed movement provoked co-localization of V-ATPase containing vesicles with F-actin near the leading edge of migrating cells. V-ATPase inhibition prompted prominent F-actin cytoskeleton reorganization. Filopodial projections were reduced, which related to reduced migration velocity. F-actin formed novel cytoplasmic rings. F-actin rings increased with extended exposure to sublethal concentrations of V-ATPase inhibitors, from 24 to 48 h, as the amount of alkalinized endo-lysosomal vesicles increased. Studies with chloroquine indicated that F-actin rings formation was pH-dependent. We hypothesize that these novel F-actin rings assemble to overcome widespread traffic defects caused by V-ATPase inhibition, similar to F-actin rings on the surface of exocytic organelles

A high-throughput imaging and quantification pipeline for the EVOS imaging platform.

Self-contained imaging systems are versatile instruments that are becoming a staple in cell culture laboratories. Many of these machines possess motorized stages and on-stage incubators that permit programmable imaging of live cells that make them a sensible tool for high-throughput applications. The EVOS imaging system is such a device and is capable of scanning multi-well dishes and stitching together multiple adjacent fields to produce coherent individual images of each well. Automated batch analysis and quantification of these tiled images does however require off-loading files to other software platforms. Our initial attempts to quantify tiled images captured on an EVOS device was plagued by some expected-and other unforeseeable-issues that arose at nearly every stage of analysis. These included: high background, illumination and stitching artifacts, low contrast, noise, focus inconsistencies, and image distortion-all of which negatively impacted processing efficiency. We have since overcome these obstacles and have created a rigorous cell counting pipeline for analyzing images captured by the EVOS scan function. We present development and optimization of this automated pipeline and submit it as an effective and facile tool for accurately counting cells from tiled images