Correction: Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0059350.].

Calpain and reactive oxygen species targets Bax for mitochondrial permeabilisation and caspase activation in zerumbone induced apoptosis.

Fluorescent protein based signaling probes are emerging as valuable tools to study cell signaling because of their ability to provide spatio- temporal information in non invasive live cell mode. Previously, multiple fluorescent protein probes were employed to characterize key events of apoptosis in diverse experimental systems. We have employed a live cell image based approach to visualize the key events of apoptosis signaling induced by zerumbone, the active principle from ginger Zingiber zerumbet, in cancer cells that enabled us to analyze prominent apoptotic changes in a hierarchical manner with temporal resolution. Our studies substantiate that mitochondrial permeabilisation and cytochrome c dependent caspase activation dominate in zerumbone induced cell death. Bax activation, the essential and early event of cell death, is independently activated by reactive oxygen species as well as calpains. Zerumbone failed to induce apoptosis or mitochondrial permeabilisation in Bax knockout cells and over-expression of Bax enhanced cell death induced by zerumbone confirming the essential role of Bax for mitochondrial permeabilsation. Simultaneous inhibition of reactive oxygen species and calpain is required for preventing Bax activation and cell death. However, apoptosis induced by zerumbone was prevented in Bcl 2 and Bcl-XL over-expressing cells, whereas more protection was afforded by Bcl 2 specifically targeted to endoplasmic reticulum. Even though zerumbone treatment down-regulated survival proteins such as XIAP, Survivin and Akt, it failed to affect the pro-apoptotic proteins such as PUMA and BIM. Multiple normal diploid cell lines were employed to address cytotoxic activity of zerumbone and, in general, mammary epithelial cells, endothelial progenitor cells and smooth muscle cells were relatively resistant to zerumbone induced cell death with lesser ROS accumulation than cancer cells.

Effect of methanolic and water extract of Leucobryum bowringii Mitt. on growth, migration and invasion of MCF 7 human breast cancer cells in vitro.

Inhibitory effects of methanol and water extract of L. bowringii. on the adhesion, migration, invasion and matrix metalloproteinase (MMP) activities of MCF 7 human breast cancer cell line are reported. Cells were cultured with 10, 25, 50 microg/mL methanolic or water extract of L. bowringii. Culture medium containing 0.1% DMSO was used as a solvent control. Ultra structural analysis by electron microscopy revealed typical features of apoptosis. A remarkable dose-response parallelism was observed between methanolic extract with growth, migration and invasion of breast cancer cells. Fractionation of methanolic extract by RP-HPLC revealed a pool of phenolic acids. Hoechst 33342 staining assay reveals massive chromatin condensation and subsequent cleavage of structural components of nucleus. The results indicate that methanol extracts inhibit the growth of human breast cancer cells partially through the inhibition of metallo proteinases MMP-2 and MMP-9 activities. Methanolic extract has more anti-metastatic effects in cell based assay than water extract. Clinical application of L. bowringii extract as a bioactive chemopreventive compound may be helpful in limiting breast carcinoma invasion and metastasis.

Immortalized functional endothelial progenitor cell lines from umbilical cord blood for vascular tissue engineering.

Endothelial progenitor cells (EPCs) play a significant role in multiple biological processes such as vascular homeostasis, regeneration, and tumor angiogenesis. This makes them a promising cell of choice for studying a variety of biological processes, toxicity assays, biomaterial-cell interaction studies, as well as in tissue-engineering applications. In this study, we report the generation of two clones of SV40-immortalized EPCs from umbilical cord blood. These cells retained most of the functional features of mature endothelial cells and showed no indication of senescence after repeated culture for more than 240 days. Extensive functional characterization of the immortalized cells by western blot, flow cytometry, and immunofluorescence studies substantiated that these cells retained their ability to synthesize nitric oxide, von Willebrand factor, P-Selectin etc. These cells achieved unlimited proliferation potential subsequent to inactivation of the cyclin-dependent kinase inhibitor p21, but failed to form colonies on soft agar. We also show their enhanced growth and survival on vascular biomaterials compared to parental cultures in late population doubling. These immortalized EPCs can be used as a cellular model system for studying the biology of these cells, gene manipulation experiments, cell-biomaterial interactions, as well as a variety of tissue-engineering applications.

Identification of heat shock protein 90 inhibitors to sensitize drug resistant side population tumor cells using a cell based assay platform.

Current cancer therapeutics are identified based on initial tumor regression screens that mostly kill differentiated tumor cells, sparing the rare cancer stem cells (CSCs). Being rare and difficult to characterize, it remains a challenge to identify compounds active against them. Side population (SP) cells identified in multiple cancer cell line panels expressing mitochondrial Cytochrome C-EGFP were evaluated for identifying possible drug candidates utilizing high-throughput imaging. We identified heat shock protein 90 inhibitors as potential agents to sensitize SP cells to anticancer drugs. Hsp90 inhibitors induced down regulation of Akt leading to proteasomal degradation of survivin and consequent mitochondrial apoptosis. A successful screening platform for identifying compounds targeting drug resistant side population cells was developed.