thapsigargin and epigallocatechin-gallate

Direct scavenging of reactive oxygen species could not prevent ER stress-associated cytotoxicity of indomethacin or diclofenac in Caco-2 cells. This study investigated the effects of three polyphenolic antioxidants epigallocatechin gallate (EGCG), phyllanthin and hypophyllathin in non-steroidal anti-inflammatory drug-induced Caco-2 apoptosis.. Cells were treated with ER stressors (indomethacin, diclofenac, tunicamycin or thapsigargin) and the polyphenols for up to 72 h. Cell viability, apoptosis and mitochondrial function were monitored by MTT, Hoechst 33342 and TMRE assays, respectively. Protein expression was measured by Western blot analysis.. Epigallocatechin gallate suppressed increases in p-PERK/p-eIF-2α/ATF-4/CHOP and p-IRE-1α/p-JNK1/2 expression levels in the cells treated with any of the ER stressors, leading to inhibition of apoptosis. In contrast, phyllanthin increased apoptosis in the cells subsequently exposed to either diclofenac, tunicamycin or thapsigargin, but not in the indomethacin-treated cells. The potentiation effect of phyllanthin seen with the three ER stressors was related to suppression of survival p-Nrf-2/HO-1 expression, resulting in increased activation of the eIF-2α/ATF-4/CHOP pathway. On the other hand, hypophyllanthin had no significant effect on the ER stressor-induced apoptosis.. Epigallocatechin gallate, phyllanthin and hypophyllanthin displayed different effects in the ER stress-mediated apoptosis, depending upon their interaction with the specific unfolded protein response signalling.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Apoptosis; Caco-2 Cells; Catechin; Diclofenac; Endoplasmic Reticulum Stress; Humans; Indomethacin; Intestinal Mucosa; Lignans; Mitochondria; Oxidative Stress; Signal Transduction; Thapsigargin; Tunicamycin; Unfolded Protein Response

Endoplasmic reticulum (ER) stress-mediated apoptosis is a well-known factor in the pathogenesis of age-related macular degeneration (AMD). ER stress leads to accumulation of misfolded proteins, which in turn activates unfolded protein response (UPR) of the cell for its survival. The prolonged UPR of ER stress promotes cell death; however, the transition between adaptation and ER stress-induced apoptosis has not been clearly understood. Hence, the present study investigates the regulatory effect of (-)-epigallocatechin gallate (EGCG) on ER stress-induced by hydrogen peroxide (H

Topics: Animals; Antioxidants; Apoptosis; Calcium; Calcium Signaling; Catechin; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Humans; Hydrogen Peroxide; Macular Degeneration; Mice; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Thapsigargin; Unfolded Protein Response

Rapid growth of tumor cells coupled with inadequate vascularization leads to shortage of oxygen and nutrients. The unfolded protein response (UPR), a defense cellular mechanism activated during such stress conditions, is a complex process that includes upregulation of the endoplasmic reticulum chaperones, such as glucose-regulated protein 78 (GRP78). Due to its central role in UPR, GRP78 is overexpressed in many cancers; it is implicated in cancer cell survival through supporting of drug- and radioresistance as well as metastatic dissemination, and is generally associated with poor outcome. This is the reason why selective destruction of GRP78 could become a novel anticancer strategy. GRP78 is the only known substrate of the proteolytic A subunit (SubA) of a bacterial AB(5) toxin, and the selective SubA-induced cleavage of GRP78 leads to massive cell death. Targeted delivery of SubA into cancer cells via specific receptor-mediated endocytosis could be a suitable strategy for assaulting tumor cells. We fused SubA to epidermal growth factor (EGF), whose receptor (EGFR) is frequently overexpressed in tumor cells, and demonstrated that the resulting EGF-SubA immunotoxin is an effective killer of EGFR-positive tumor cells. Furthermore, because of its unique mechanism of action, EGF-SubA synergizes with UPR-inducing drugs, which opens a possibility for the development of mechanism-based combination regimens for effective anticancer therapy. In this chapter, we provide experimental protocols for the assessment of the effects of EGF-SubA on EGFR-positive cancer cells, either alone or in combination with UPR-inducing drugs.

Topics: Animals; Antineoplastic Agents; Catechin; Cell Line; Drug Screening Assays, Antitumor; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Heat-Shock Proteins; Humans; Neoplasms; Thapsigargin; Unfolded Protein Response

Although the consumption of tea has been associated with beneficial cardiovascular effects, (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin in this beverage has shown seemingly contradictory actions on vascular tissues, for example vasorelaxant activity that could contribute favourably to prevention of cardiovascular disease, and contractile activity that could act in the opposite direction. The purpose of the present work was to study the contractile effects of EGCG on isolated rat thoracic aorta rings and its effects on the cytosolic free [Ca(2+)] ([Ca(2+)](i)) measured with fura-2 in cultured rat aortic smooth muscle cell line. In partially depolarised (15 mM KCl) aortic rings EGCG (30-300 microM), (+/-)-BAY K 8644 (0.1 microM) and thapsigargin (1 microM) induced a Ca(2+)-dependent, endothelium-independent contraction associated with [Ca(2+)](i) elevation in RASMC. EGCG enhanced the responses elicited by (+/-)-BAY K 8644 and thapsigargin both in aortic rings and in RASMC. Nifedipine totally inhibited the (+/-)-BAY K 8644-induced contraction, but only partially blocked the contractile responses to EGCG and thapsigargin, while SKF 96365 abolished both responses. The effects of these channel blockers were associated with a decrease in [Ca(2+)](i) in RASMC. Re-introduction of Ca(2+) in the medium after depletion of intracellular Ca(2+) stores with thapsigargin in a Ca(2+)-free solution elicited a contraction of aortic rings and an increase in [Ca(2+)](i) in RASMC. In both cases, this response was partially sensitive to nifedipine, abolished by SKF 96365 and clearly enhanced by EGCG. These results suggest that EGCG induces a transient endothelium-independent contraction in the rat aorta, probably by increasing smooth vascular cell membrane permeability to Ca(2+) through both non-specific and dihydropyridine-sensitive Ca(2+) channels.

Topics: Animals; Antioxidants; Aorta; Azlocillin; Calcium; Catechin; Drug Synergism; Imidazolidines; Male; Muscle, Smooth, Vascular; Rats; Rats, Inbred WKY; Thapsigargin; Vasoconstriction; Vasodilator Agents