Connexin Hemichannels: Methods for Dye Uptake and Leakage.

It is now clear that connexin-based, gap junction "hemichannels" in an undocked state are capable of opening and connecting cytoplasm to the extracellular milieu. Varied studies also suggest that such channel activity plays a vital role in diverse cell processes and abnormal hemichannel activity contributes to pathogenesis. To pursue fundamental questions in this area, investigators require methods for studying hemichannel permeability and dynamics that are quantitative, sensitive, versatile, and available to most cellular and molecular laboratories. Here we first provide a theoretical background for this work, including the role of cellular membrane potentials. We then describe in detail our computer-assisted methods for both dye uptake and leakage along with illustrative results from different cell systems. A key feature of our protocol is the inclusion of a mechanical stimulation step. We describe dye uptake, interpreted as connexin dependent, that is shown to be enhanced with reduced extracellular Ca2+, mechanically responsive, inhibited by TPA, inhibited by EL186 antibodies for Cx43 and sustained for more than 15 min following mechanical stimulation. We describe dye leakage that displays these same properties, with estimates of hemichannel numbers per cell being derived from leakage rates. We also describe dye uptake that is shown to be unaffected by a reduction in external Ca2+, insensitive to EL186 antibodies and relatively short-lived following mechanical stimulation; this uptake may occur via pannexin 1 channels expressed in the cells studied here. It is unlikely that cell damage plays a significant role in dye uptake following mechanical stimulation, given compelling results from various control experiments.

Triptolide and its prodrug minnelide suppress Hsp70 and inhibit in vivo growth in a xenograft model of mesothelioma.

Malignant mesothelioma is a devastating disease with a poor prognosis for which there is a clear need for more successful therapeutic approaches. Triptolide, a diterpenoid triepoxide, is a highly effective agent against several cancer types in animal models. Owing to triptolide's poor solubility in water, a water-soluble analog, minnelide, was synthesized. Minnelide is a prodrug of triptolide and is activated by exposure to phosphatases that are found in all body tissues, including blood. Mesothelioma cells were treated in vitro with minnelide or its parent compound, triptolide. Minnelide and triptolide were both found to significantly reduce mesothelioma cell viability and induce apoptosis. The level of Hsp70, a protein that promotes cancer cell survival, was measured in mesothelioma cells before and after treatment with triptolide. Hsp70 levels were decreased in a dose-dependent manner. In addition, triptolide sensitized cells to gemcitabine and pemetrexed as measured by cell viability. Mice bearing mesothelioma flank tumors were treated with daily injections (28 d) of minnelide or saline solution and xenograft tumor growth recorded. Mice displayed significantly reduced tumor burden. These findings support the clinical evaluation of minnelide therapy for mesothelioma.

Minnelide: a novel therapeutic that promotes apoptosis in non-small cell lung carcinoma in vivo.

BACKGROUND: Minnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear. METHODS: Cell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA, APAF-1) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections. RESULTS: In this study, we investigated in vitro and in vivo antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA) and up-regulated pro-apoptotic APAF-1 gene, in part, via attenuating the NF-κB signaling activity. CONCLUSION: In conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.