探讨秋水仙碱对人口腔癌细胞钙处理及其相关生理学的影响。

Exploration of the effect of the alkaloid colchicine on Ca handling and its related physiology in human oral cancer cells.

机构信息

Department of Anesthesiology, Kaohsiung Medical University Hospital, Kaohsiung, 80708, Taiwan; Department of Anesthesiology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, 80756, Taiwan.

Yuh-Ing Junior College of Health Care & Management, Kaohsiung, 80776, Taiwan.

出版信息

Arch Oral Biol. 2019 Jun;102:179-185. doi: 10.1016/j.archoralbio.2019.04.017. Epub 2019 Apr 29.

DOI:10.1016/j.archoralbio.2019.04.017

PMID:31059912

Abstract

OBJECTIVE

Colchicine, extracted from plants of the genus Colchicum, is a commonly prescribed drug for inflammatory diseases. It has been shown that colchicine affected various physiological responses in different models. However, the effect of colchicine on cytosolic free Ca levels ([Ca]) and its related physiology in human oral cancer cells is unknown. This study examined whether colchicine altered Ca homeostasis and caused cytotoxicity in OC2 human oral cancer cells.

METHODS

The Ca-sensitive fluorescent dye fura-2 was used to measure [Ca]. Cell viability was measured by the fluorescent reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay.

RESULTS

Colchicine at concentrations of 250-650 μM induced [Ca] rises concentration-dependently. The response was reduced by approximately 40% by removing extracellular Ca. In Ca-free medium, treatment with the endoplasmic reticulum Ca pump inhibitor thapsigargin inhibited colchicine-evoked [Ca] rises. Conversely, treatment with colchicine inhibited thapsigargin-evoked [Ca] rises. Inhibition of phospholipase C (PLC) with U73122 abolished colchicine-induced Ca release. In Ca-containing medium, colchicine-induced Ca entry was supported by Mn-caused quenching of fura-2 fluorescence and the entry was partly inhibited by protein kinase C (PKC) modulators (phorbol 12-myristate 13 acetate, PMA; and GF109203X) and by three modulators of store-operated Ca channels (nifedipine, econazole and SKF96365). Colchicine at 250-650 μM decreased cell viability, which was not reversed by pretreatment with the Cachelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM).

CONCLUSIONS

In OC2 cells, colchicine induced [Ca] rises by evoking PLC-dependent Ca release from the endoplasmic reticulum and Ca entry via PKC-sensitive store-operated Ca entry. Furthermore, colchicine caused cell death that was not triggered by preceding [Ca] rises.

摘要

目的

秋水仙碱是从秋水仙属植物中提取的一种常用药物，用于治疗炎症性疾病。研究表明，秋水仙碱在不同模型中影响多种生理反应。然而，秋水仙碱对人口腔癌细胞胞浆游离钙水平([Ca])及其相关生理学的影响尚不清楚。本研究旨在探讨秋水仙碱是否改变了 Ca 稳态并导致 OC2 人口腔癌细胞发生细胞毒性。

方法

使用 Ca 敏感荧光染料 fura-2 测量[Ca]。通过荧光试剂 4-[3-[4-碘苯基]-2-[4-（4-硝基苯基）-2H-5-四唑基]-1,3-苯二磺酸钠]水溶性四唑盐-1（WST-1）测定细胞活力。

结果

浓度为 250-650μM 的秋水仙碱诱导[Ca]浓度依赖性升高。去除细胞外 Ca 后，反应减少约 40%。在无 Ca 培养基中，内质网 Ca 泵抑制剂 thapsigargin 处理抑制秋水仙碱诱导的[Ca]升高。相反，秋水仙碱处理抑制 thapsigargin 诱导的[Ca]升高。用 U73122 抑制磷脂酶 C（PLC）可消除秋水仙碱诱导的 Ca 释放。在含有 Ca 的培养基中，Mn 引起的 fura-2 荧光猝灭支持秋水仙碱诱导的 Ca 内流，蛋白激酶 C（PKC）调节剂（佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）和 GF109203X）和三种钙库操纵型钙通道调节剂（硝苯地平、克霉唑和 SKF96365）部分抑制 Ca 内流。浓度为 250-650μM 的秋水仙碱降低细胞活力，但先用 Cachelator 1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸-乙氧甲酰甲酯（BAPTA/AM）预处理不能逆转这种降低。