Journal: Marine Drugs

Article Title: Selective Inhibition of Liver Cancer Cells Using Venom Peptide

doi: 10.3390/md17100587

Figure Lengend Snippet: Potential mechanism of Tv1 antitumor activity inhibits COX2 and PGE2 function. In our model of Tv1 antitumor activity in liver cancer cells, overexpression of TRP channels (TRPC6 and V6) stimulates COX-2-dependent PGE2 production via enhanced [Ca 2+ ] dynamics. Influx of Ca 2+ can occur through voltage gated (VGC), receptor operated (ROC), and store operated (SOC) calcium channels. Transient receptor potential (TRP) channels contribute to store operated calcium (SOC) channels. Ca 2+ -dependent transcription factor NFAT is activated via dephosphorylation by calcineurin, which is activated upon binding of Ca 2+ /calmodulin. Ubiquitously present transcription factor NFAT regulates COX-2 expression and further prostaglandin E2 (PGE2) release in different cancer cells and its activation occurs through Ca 2+ influx associated with TRPC1-, TRPC3-, or TRPC6-associated SOC or ROC activities. PGE2 release plays multiple roles in cancer as shown. Upon Tv1 application to liver cancer (HCC) cells the pictured downstream pathways leading to proliferation inhibition and apoptosis are encountered.

Article Snippet: Rabbit polyclonal Anti-HERG (Kv11.1)-APC109, Anti TRPV6-ACC036, Anti TRPV2-ACC039, Anti TRPC1-ACC-118 were purchased from Alomone lab, Israel.

Techniques: Activity Assay, Over Expression, De-Phosphorylation Assay, Binding Assay, Expressing, Activation Assay, Inhibition

Journal: Cell Death Discovery

Article Title: Bcl-2 regulates store-operated Ca 2+ entry to modulate ER stress-induced apoptosis

doi: 10.1038/s41420-018-0039-4

Figure Lengend Snippet: a Control vector (C)-, wild-type Bcl-2 (WT)-, and Bcl-2 mutant (mt)-overexpressing MDCK cells were treated with DMSO or TG for 5 min. Subsequently, immunofluorescence staining was obtained to label STIM1 and nucleus. Representative fluorescence images were obtained using confocal microscopy (scale bar, 20 μm). Arrows indicate the translocation of STIM1 to the juxta-plasma membrane region. b , c Pre-incubation of cells with 2 μM fura-2/AM at 37 °C for 30 min for cytosolic Ca 2+ measurement using a single cell fluorimeter. Depletion of ER lumen-resident Ca 2+ was induced by treating cells in Ca 2+ -free buffer with 2 μM TG for 10 min. The subsequent elevation of Ca 2+ indicated that SOCE occurred during the exchange of Ca 2+ -free buffer to 2 mM-Ca 2+ buffer. b The data in representative curves for the measurement of SOCE are represented as mean ± SEM (where, n ≥ 60 cells) from three independent experiments. c Quantitative analysis of the changes in the peak Ca 2+ levels. All values are represented as mean ± SEM, and the data were found to be statistically significant at p < 0.001 (indicated by ***) compared to control vector (C)-overexpressing cells (Student’s t -test). d Western blotting of SOCE related molecules, such as ER Ca 2+ sensors (STIM1 and STIM2), plasma membrane Ca 2+ channels (Orai1, Orai2, Orai3, and TRPC1), and the internal control β-actin. e Cells were pre-incubated with 2 mM EGTA for 30 min and treated with DMSO or 2 μM TG for 24 h. Representative images of cells were obtained under a bright-field microscope (scale bar, 100 μm). f Bcl-2 mutant (mt)-overexpressing cells were pre-incubated with 20 μM BAPTA/AM, 2 mM EGTA, or 2 μM 2-APB for 30 min and treated with DMSO or 2 μM TG for 24 h. Quantitative analysis of the apoptosis ratio was assessed from the hypodiploid DNA peak of propidium iodide (PI)-stained cells from three independent experiments by flow cytometry. The data were found to be statistically significant at p < 0.01 (indicated by **) compared to the DMSO control (Student’s t -test)

Article Snippet: Cell lysates were harvested in RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris at pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and Complete TM ), and the lysates were analyzed by Western blotting using antibodies against Bcl-2 (DAKO, Grostrup, Denmark), Bax, Bak, calnexin, SERCA2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), porin (Molecular Probes, Eugene, OR), caspase-8, caspase-9, caspase-12, SERCA3, pSer70-Bcl-2, pThr167-Bax (Cell Signaling Technology, Beverly, MA), Grp78, STIM1, STIM2, IP3R3 (BD, Franklin Lakes, NJ), Orai1, Orai2, Orai3, and TRPC1 (ProSci, Poway, CA).

Techniques: Control, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining, Fluorescence, Confocal Microscopy, Translocation Assay, Clinical Proteomics, Membrane, Incubation, Western Blot, Microscopy, Flow Cytometry