Ca<sup>2+</sup>在顺铂诱导人卵巢癌SKOV3细胞自噬反应中的作用

曾林川; 邓慧敏; 陈　君

doi:10.3969/j.issn.1007-3969.2016.04.005

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Ca²⁺在顺铂诱导人卵巢癌SKOV3细胞自噬反应中的作用

更新时间：2025-12-31

- Ca²⁺在顺铂诱导人卵巢癌SKOV3细胞自噬反应中的作用
- The effect of cytoplasmic Ca2+ on cisplatin-induced autophagy in ovarian carcinoma SKOV3 cells and its mechanism
- 中国癌症杂志 2016年26卷第4期页码：313-319
- 作者机构：
  
  1. 吉林医药学院公共卫生学院,吉林,吉林,132013
  2. 吉林医药学院医学科研实验室,吉林,吉林,132013
- 作者简介：
- 基金信息：
  
  国家自然科学基金面上项目(81372793);吉林省教育厅十三五科技项目(2016237);2014吉林省大学生创新创业项目(2014001)。
- DOI：10.3969/j.issn.1007-3969.2016.04.005
  中图分类号：
- 网络出版：2016-06-16，
  
  纸质出版：2016-06-16
- 稿件说明：
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曾林川,邓慧敏,陈　君,等. Ca

曾林川, 邓慧敏, 陈　君. The effect of cytoplasmic Ca2+ on cisplatin-induced autophagy in ovarian carcinoma SKOV3 cells and its mechanism[J]. China Oncology, 2016, 26(4): 313-319.
曾林川,邓慧敏,陈　君,等. Ca DOI： 10.3969/j.issn.1007-3969.2016.04.005.

曾林川, 邓慧敏, 陈　君. The effect of cytoplasmic Ca2+ on cisplatin-induced autophagy in ovarian carcinoma SKOV3 cells and its mechanism[J]. China Oncology, 2016, 26(4): 313-319. DOI： 10.3969/j.issn.1007-3969.2016.04.005.

摘要

背景与目的：Ca

在维持细胞生物活性方面扮演着很重要的角色，其在细胞内的储存、释放和摄取主要受内质网调节，细胞内Ca

浓度的稳态是维持细胞生物能量代谢、蛋白质折叠和分泌的基础条件。本研究探讨Ca

在顺铂诱导SKOV3细胞内质网应激-自噬反应中的作用机制。方法：取人卵巢癌SKOV3细胞系为研究对象，按以下步骤分组：① 探讨顺铂诱导内质网应激与自噬反应，用6 μg/mL的顺铂处理SKOV3细胞0、6、12和24 h；② 了解顺铂和毒胡萝卜内酯(thapsigargin

TG)诱导内质网应激释放的Ca

与自噬的关系，分别用TG和顺铂处理SKOV3细胞0、9和12 h；③ 探究Ca

对自噬的作用机制，分成对照组、顺铂组、TG组、BAPTA-AM组、顺铂联合BAPTA-AM组和TG联合BAPTA-AM组。用蛋白［质］印迹法(Western blot)检测内质网应激相关蛋白GRP78和自噬标志性蛋白LC3蛋白的表达水平；用Fluo-4钙离子荧光探针检测细胞质中的Ca

浓度变化；间接免疫荧光染色后，用共聚焦显微镜检测LC3蛋白的表达情况。结果：SKOV3细胞经6 μg/mL顺铂作用6 h时GRP78灰度值(1.393±0.004)与其对照组(0.679±0.011)相比显著提高(t=113.2，P=0.000)，在12 h时LC3灰度值(0.072±0.002)与其对照组(0.038±0.000)相比显著提高(t=25.5，P=0.000)。间接免疫荧光结果显示，顺铂(6 μg/mL)组和TG(3 μmol/L)组随作用时间的延长，细胞内LC3荧光斑点会逐渐增多，并伴随着细胞质Ca

浓度上升。后经钙离子络合剂BAPTA-AM干预后，细胞内LC3荧光强度进一步增强。Westren blot结果显示，顺铂组LC3灰度值(0.039±0.000)小于顺铂联合BAPTA-AM组(0.071±0.001)，TG组(0.035±0.001)小于TG联合BAPTA-AM组(0.065±0.001)，差异有统计学意义(P=0.000)。结论：顺铂诱导SKOV3细胞内质网应激和自噬的发生，并伴随着细胞质内Ca

浓度的上升。络合细胞质内Ca

能增强顺铂诱导的自噬反应。

Abstract

Background and purpose: Ca

plays a very important role in the maintenance of cell biological functions. The storage

release and

uptake capacity of Ca

is controlled by endoplasmic reticulum (ER). Ca

homeostasis is essential for cellular energy metabolism and proper protein folding. This study aimed to investigate the effect of cytoplasmic Ca

on cisplatin induced ER stress-mediated autophagy in ovarian carcinoma SKOV3 and its underlying mechanism. Methods: The ovarian cancer SKOV3 was used as a study object. The experiment consisted of three parts: ① To explore the possible relationship between cisplatin-induced ER stress and autophagy

SKOV3 cells were treated with cisplatin for 0

12 and 24 h

respectively; ② To explore the possible relationship between ER stress induced Ca

efflux and autophagy

SKOV3 cells were treated with cisplatin for 0

9 and 12 h

respectively

and TG was used as a positive control; ③ To explore the effects of blocking calcium efflux on autophagy

SKOV3 cells were divided into control group

cisplatin group

TG group

BAPTA-AM group

cisplatin combined with BAPTA-AM group and TG combined with BAPTA-AM group. Western blot was used to detect the protein levels of GRP78 and LC3. Fluo-4 calcium fluorescent probe was used to examine cytoplasmic Ca

levels. Confocal microscopy was used to detect LC3 level by immunoflurescence staining. Results: Compared to control group (0.679±0.011)

GRP78 was significantly accumulated at 6

12 and 24 h after cisplatin treatment and reached the maximum value at 6 h (1.393±0.004

P=0.000). Similarly

compared to control group (0.038±0.000)

LC3 puncta were clearly seen after cisplatin treatment and reached the maximum value at 12 h (0.072±0.002

P=0.000). Using confocal microscopy

we found that cisplatin and TG increased LC3 punctate accumulation and cytoplasmic Ca

levels in a time-dependent manner. Immunofluorescent method showed that treatment with cisplatin combined with BAPTA-AM or TG combined with BAPTA-AM increased LC3 punctate accumulation induced by cisplatin or TG. The results of Western blot showed

that cisplatin combined with BAPTA-AM (0.071±0.001) or TG combined with BAPTA-AM (0.065±0.001) significantly increased LC3Ⅱ/LC3Ⅰ ratio induced by cisplatin (0.039±0.000

P=0.000) or TG (0.035±0.001

P=0.000). Conclusion: Cisplatin induces intracellular ER stress and autophagy in SKOV3 cells

accompanied by increased cytoplasmic Ca

levels. Chelating cytoplasmic Ca

enhances cisplatin-induced autophagy.

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Ca2+在顺铂诱导人卵巢癌SKOV3细胞自噬反应中的作用

The effect of cytoplasmic Ca2+ on cisplatin-induced autophagy in ovarian carcinoma SKOV3 cells and its mechanism

DOI：10.3969/j.issn.1007-3969.2016.04.005

摘要

Abstract

关键词

Keywords

references

Ca²⁺在顺铂诱导人卵巢癌SKOV3细胞自噬反应中的作用