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.
Arch Oral Biol. 2019 Jun;102:179-185. doi: 10.1016/j.archoralbio.2019.04.017. Epub 2019 Apr 29.
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.
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.
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).
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.
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。
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。
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。
在 OC2 细胞中，秋水仙碱通过触发 PLC 依赖性内质网 Ca 释放和 PKC 敏感钙库操纵型 Ca 内流引起[Ca]升高。此外，秋水仙碱引起的细胞死亡不是由先前的[Ca]升高引发的。
