Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells

Abstract

Abstract.De novo and acquired resistance to endocrinebased therapies in breast cancer occurs in parallel with epithelial to mesenchymal transition (EMT), which is associated with enhanced proliferative and metastatic potential, and poor clinical outcome. We have established several endocrine insensitive breast cancer lines by shRNA-induced depletion of estrogen receptor (ER) by transfection of MCF7 cells. All of these exhibit EMT. We have previously reported that brief exposure of specifically ER -breast cancer cells, to extracellular alkaline pH, results in cell rounding and segregation, and leads to enhanced invasive potential. In this study we describe more detailed morphological changes and compare these with cell exposure to acidic pH. Morphological changes and localization of various molecules critical for cell adhesion and motility, associated with pH effects, were assessed by live cell microscopy, electron microscopy, and immunofluorescence. Exposure of either ER -or ER + breast cancer cells to extracellular acidic pH did not induce significant changes in morphological appearance. Conversely, brief exposure of specifically ER silenced cells, to alkaline pH, resulted in cell contractolation and formation of bleb-like actin-rich structures which were evenly distributed on the outer membrane. Integrin α2, FAK, and JAM-1 were found in the cytoplasm streaming into the newly formed blebs. These blebs appear to be related to cell polarity and movement. Pre-treatment with cytochalasin-D or inhibitors of Rho or MLCK prevented both contractolation and bleb formation. Our data suggest that the effect of pH on the microenvironment of endocrine resistant breast cancer cells needs to be more extensively investigated. Alkaline, rather than acidic pH, appears to induce dramatic morphological changes, and enhances their invasive capabilities, through re-organization of cortical actin.

Involvement of Membrane Blebbing in Immunological Disorders and Cancer

“…Blebs have been observed in numerous cell types including fibroblasts [ 6 ], endothelial and mesenchymal cells [ 4 ], cancer cells [ 7 , 8 , 9 , 10 , 11 ], immune cells [ 12 , 13 , 14 ], germ cells [ 15 , 16 , 17 ], ameba [ 18 ], parasites [ 19 ] and bacteria [ 20 , 21 , 22 ]. It was originally assumed that blebbing was related solely to pathological conditions in response to nonspecific cellular insults such as lipid peroxidation [ 23 ], anoxia [ 24 ] and energy depletion [ 24 , 25 , 26 ], with motility/invasion [ 11 , 27 ] and multidrug resistance in tumor cells [ 28 ], or as a prerequisite for necrosis. However, it is now considered to be an important physiological process which occurs during cell blastulation in Fundulus deep cells [ 29 ], cytokinesis, cell spreading, virus infection, protective mechanisms against injury (either physical or chemical stress) [ 30 , 31 , 32 ] and as a hallmark of the execution stage of apoptosis [ 33 ].…”

Megakaryocyte emperipolesis mediates membrane transfer from intracytoplasmic neutrophils to platelets

“…After 2 hours, cells were either washed thoroughly to remove inhibitors and cultured overnight (upper photos) or left in culture in the presence of the inhibitors (lower photos). Images show a strong disruption of Factin in cells treated with latrunculin A, and bright actin aggregates distributed irregularly within the cells with cytochalasin D, as reported (Khajah et al, 2015;Rubtsova et al, 1998). B.…”

COL‑3 enhances the anti‑proliferative and pro‑apoptotic effects of paclitaxel in breast cancer cells

“…no. ab112146; Cambridge, UK), as previously described (25). pII cells were seeded (0.1x10 6 cells) into 6-well plates in culture medium at 37˚C and allowed to grow to 80% confluence.…”

“…where CI<1 indicates synergism, CI=1 indicates summation and CI>1 indicates antagonism (25,26). (D)1 and (D)2 represent the concentrations of paclitaxel and COL-3 in the combination that also inhibits cell growth by x%, respectively.…”