Roles of endoplasmic reticular Ca2+-store in regulating the cellular proliferation and survival

• Main Authors: Chen Yu-Jen, 陳有任
• Other Authors: Lin-Shiau Shoei-Yn
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/51367788586724256431

博士 === 國立臺灣大學 === 毒理學研究所 === 88 === The intracellular free calcium played a vital role in regulating the cellular physiological and biochemical functions. The dynamic homeostasis of intracellular free calcium concentration is mainly due to a balance between the influx and efflux on the plasma membrane and the intracellular calcium stores. The purpose of this study is aimed to use thapsigargin (TG), a endoplasmic reticulum (ER)-Ca2+ ATPase inhibitor, to increase intracellular calcium concentration and deplete their Ca2+ store for investigating the influences on cellular functions. The mechanism of depleting ER-Ca2+ store involved in regulating cellular proliferation In C6 glioma cells, we investigated the role of intracellular calcium store in modulating the cellular proliferation and the expression of cell cycle regulatory proteins. We found that TG irreversibly and DBHQ reversibly depleted the Ca2+ store accompanied with the induction of G0/G1 arrest, an increase in GFAP expression and morphological changes from a round flat shape to a differentiated spindle. The machinery underlying these changes induced by Ca2+-store depletion was investigated. The results indicated that Ca2+-store depletion caused an increased expression of p21 and p27 proteins (CDK inhibitors), with unchanged mutant p53 protein of C6 cells but reduced amounts of the cell cycle regulators: CDK2, cdc2, cyclin C, cyclin D1, cyclin D3 and PCNA in a time-dependent manner. These findings indicate a new function of ER-Ca2+ store in regulating cellular proliferation rate through altering the expression of p21 and p27 proteins. Moreover, cellular differentiation as revealed by spindle morphology and induced GFAP expression were also tended to be modulated by ER Ca2+ store. The implication of this finding is that the abnormal growth of cancer cells such as C6 glioma cells may be derived from a signalling of ER which can be manipulated by depleting the Ca2+ store. Studies on the influence of ER-Ca2+ store on the cell viability and TNF- production In RAW 264.7 cell, we investigated the effects of TG on cell viability and TNF- production in murine macrophage RAW264.7 cell line. We found that treatment with TG (10-800nM) induced the apoptosis of RAW 264.7 cells in a dose-dependent manner (IC50, 200nM). LPS (1g/ml) markedly potentiated low concentrations of TG (10-75nM) in inducing apoptosis (IC50, 20nM). On the agarose electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition, Hoechst staining studies showed the formation of apoptotic nuclei and flowcytometry also showed the increase of hypodiploid population. The inactivated form of Caspase 1 (ICE cysteine protease) is converted to activated form of p20 unit in Western blotting analysis. Polymycin B (a LPS receptor antagonist) inhibited the cytotoxic effect induced by LPS plus TG. Although all of TG, A23187 and ionomycin definitely increased the intracellular Ca2+ concentration, neither A23187 nor ionomycin mimicked TG in inducing apoptosis events in LPS-activated RAW 264.7 cells. But all of TG, A23187 and ionomycin reduced the LPS-induced nitric oxide (NO) production. Moreover, the production of TNF-induced by LPS was profoundly potentiated by TG but not by A23187 nor by ionomycin. Taken together, we conclude from these results that TG-sensitive ER-Ca2+ store plays a pivotal role in modulating the cell viability and TNF-production. The mutual potentiations between LPS receptor signaling pathway and the depletion of ER-Ca2+ store imply the cross-talk of these multi-regulatory mechanisms existed in this murine macrophage RAW 264.7 cell line. We used another murine macrophage cell line: J774 cell to investigated the cell viability during the same treatment. We found that J774 cell was resistant to TG-induced cytotoxicity. However, when treatment the cells with TG and combined with LPS would also induce cell death. This cell death is also belonged to apoptosis. But the production of NO induced by TG plus LPS was increased in J774 cell line. We also isolated the peritoneal macrophages (PM) after treatment the mice with thioglycollate. PMs were found to be resistant to TG whether with or without LPS. The production of NO was significantly increased by TG plus LPS in PMs. Studies of the cytotoxicity induced by TG plus curcumin on various cell lines We investigated the effect of TG combined with curcumin (an anti-cancer drug) on the cellular viability of the cultured cells. We found that TG combined with curcumin profoundly increased cytotoxicity on TG-sensitive cell lines: RAW 264.7 and HL 60 cell lines. The cell death was due to the apoptotic process as revealed by DNA ladder. The TG-resistant cell lines (J774, L929 and C6 cells) were also resistant to the cytotoxic effect of combined TG plus curcumin. Although both A23187 and ionomycin were capable of increasing intracellular calcium concentration, neither of them combined with curcumin could mimic TG in enhancing apoptosis. Besides, the TG-resistant cell lines were also resistant to high concentration of curcumin. The major degradation products of curcumin (ferulic acid and vanillin) could not mimick curcumin in augmenting cell death induced by TG. Treatment with BAPTA-AM to chelate the intracellular free calcium also increased the cytotoxicity induced by TG plus curcumin. Neither CHX and Acti. D could prevent the cell death induced by TG plus curcumin, but ruthenium red (a mitochondial Ca2+ uptake inhibitor) significantly reduced the cytotoxicity induced by TG plus curcumin. From these findings, we suggested that the increased cytotoxicity induced by TG plus curcumin is mediated by the depletion of calcium store by TG coupled with the dysfunction mitochondrial buffering calcium system.