Cellular geometry and epithelial-mesenchymal plasticity intersect with PIEZO1 in breast cancer cells

Differences in shape can be a distinguishing feature between different cell types, but the shape of a cell can also be dynamic. Changes in cell shape are critical when cancer cells escape from the primary tumor and undergo major morphological changes that allow them to squeeze between endothelial cells, enter the vasculature, and metastasize to other areas of the body. A shift from rounded to spindly cellular geometry is a consequence of epithelial-mesenchymal plasticity, which is also associated with changes in gene expression, increased invasiveness, and therapeutic resistance. However, the consequences and functional impacts of cell shape changes and the mechanisms through which they occur are still poorly understood. Here, we demonstrate that altering the morphology of a cell produces a remodeling of calcium influx via the ion channel PIEZO1 and identify PIEZO1 as an inducer of features of epithelial-to-mesenchymal plasticity. Combining automated epifluorescence microscopy and a genetically encoded calcium indicator, we demonstrate that activation of the PIEZO1 force channel with the PIEZO1 agonist, YODA 1, induces features of epithelial-to-mesenchymal plasticity in breast cancer cells. These findings suggest that PIEZO1 is a critical point of convergence between shape-induced changes in cellular signaling and epithelial-mesenchymal plasticity in breast cancer cells

Induction of Tumor Cell Death through Targeting Tubulin and Evoking Dysregulation of Cell Cycle Regulatory Proteins by Multifunctional Cinnamaldehydes

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G2/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G2 phase. G2 arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G2 to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G2 phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death

Transient receptor potential cation channel subfamily V and breast cancer

Transient receptor potential cation channel subfamily V (TRPV) channels play important roles in a variety of cellular processes. One example includes the sensory role of TRPV1 that is sensitive to elevated temperatures and acidic environments and is activated by the hot pepper component capsaicin. Another example is the importance of the highly Ca selective channels TRPV5 and TRPV6 in Ca absorption/reabsorption in the intestine and kidney. However, in some cases such as TRPV4 and TRPV6, breast cancer cells appear to overexpress TRPV channels. Moreover, TRPV mediated Ca influx may contribute to enhanced breast cancer cell proliferation and other processes important in tumor progression such as angiogenesis. It appears that the overexpression of some TRPV channels in breast cancer and/or their involvement in breast cancer cell processes, processes important in the tumor microenvironment or pain may make some TRPV channels potential targets for breast cancer therapy. In this review, we provide an overview of TRPV expression in breast cancer subtypes, the roles of TRPV channels in various aspects of breast cancer progression and consider implications for future therapeutic approaches

Dose-dependent effect of (a) CA, (b) BCA, (c) FHCA and (d) FBCA on cell cycle regulatory proteins.

<p>HCT 116 cells were treated with indicated concentrations of cinnamaldehydes for 12 h or 24 h. Cell lysates were analyzed by Western blotting for proteins controlling G₂ to mitosis transition (cdk1, phospho-cdk1, cdc25C, phospho-cdc25C, cyclin B1, phospho-cyclin B1), proteins regulating spindle assembly (mad2, cdc20, survivin) and apoptotic markers (full length and cleaved) caspase 3, and (full length and cleaved) PARP. Blots were also probed for β-actin to ensure equal protein loading.</p

Cinnamaldehydes cause dose-dependent accumulation of insoluble tubulin.

<p>(a) HCT 116 cells were treated with cinnamaldehydes at indicated concentrations for 16 h. Soluble (left panel) and insoluble (middle panel) fractions of collected cell lysates were analyzed by Western blotting with anti-β-tubulin antibody. (b) HCT 116 cells were treated with 40 µM cycloheximide (CHX) alone or 30 µM BCA+40 µM CHX and lysates containing both soluble and insoluble tubulin were collected at indicated timepoints. Total tubulin in the lysates was analyzed by western blotting with anti-β-tubulin antibody. Blots were probed for β-actin to ensure equal protein loading. Right panel of (a): band intensities quantified by densitometry and normalized to respective actin loading were expressed as percentage of insoluble tubulin per total tubulin. Right panel of (b): band intensities were quantified by densitometry and normalized to respective actin loading control. Results are presented as means±SD of at least two independent experiments; SD denoted by error bars. Statistically significant (<i>p</i><0.05 versus DMSO control) data points were boxed.</p

Chemical structures of cinnamaldehyde analogs and cinnamic acid (CAC).

<p>Chemical structures of cinnamaldehyde analogs and cinnamic acid (CAC).</p

Effect of FBCA on cell cycle regulatory proteins in cells pretreated with pan caspase inhibitor Z-VAD-FMK.

<p>HCT 116 cells were treated or untreated with 50 µM Z-VAD-FMK for 1 h, followed by addition of FBCA at indicated concentrations for another 12 h. Cell lysates were analyzed by Western blotting for proteins controlling G₂ to mitosis transition (cdk1, phospho-cdk1, cdc25C, phospho-cdc25C, cyclin B1, phospho-cyclin B1) and apoptotic markers (full length and cleaved) caspase 3, and (full length and cleaved) PARP. Blots were also probed for β-actin to ensure equal protein loading.</p

Antiproliferative activities of cinnamaldehyde analogs against HCT 116, MCF-7 and MRC-5 cells.

<p>Values are presented as means±SD from 3 independent experiments.</p>a<p>GI₅₀∶50% growth inhibition concentration;</p>b<p>LC₅₀∶50% lethal concentration;</p>c<p>Results for HCT 116 and MCF-7 cell lines were adopted from Ref 20.</p