Effect of tunicamycin on cisplatin induced apoptosis of HeLa cells
- Author:
Ye XU
1
Author Information
1. Department of Histology and Embryology, Jilin Medical College
- Publication Type:Journal Article
- Keywords:
Apoptosis;
Cisplatin;
Endoplasmic reticulum stress;
Tunicamycin;
Uterine cervical neoplasms
- From:
Medical Journal of Chinese People's Liberation Army
2013;38(4):283-287
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of endoplasmic reticulum stress (ER stress) in cisplatin-induced apoptosis of human cervical cancer HeLa cells. Methods HeLa cells were used as the study object which were divided into four groups: TUNI (5mg/L) group, cisplatin (6mg/L) group, TUNI(5mg/L)+cisplatin(6mg/L) group, and negative control group (no drug treatment). MTT assay was employed to examine the growth status of the cells. Hoechst staining was used to observe the morphological change in the nucleus. Immunoblotting was used to detect the activation of apoptotic proteins, caspase-3 and caspase-4. Indirect immunofluorescence was used to assess the expression of the protein disulfide isomerase (PDI) and phosphorylated histone H2AX (γ-H2AX). Results MTT assay showed that the growth inhibition rates were 2.65%±2.71%, 19.60%±4.34%, 44.69%±7.07% and 0% in TUNI group, cisplatin group, TUNI+cisplatin group and control group, respectively (P<0.05). Cisplatin showed a significant inhibitory effect on the growth of HeLa cells, and TUNI enhanced the effect of cisplatin. Statistical significance was found between TUNI+cisplatin group and cisplatin group (P<0.05). Hoechst staining showed that the fluorescence of the nucleus in control group was weak and well-distributed. At 12h after treatment, the nuclei in some HeLa cells in cisplatin group and TUNI+cisplatin group diminished in size, thus showing dense hyperfluorescence, and some of them were broken. The proportion of karyorrhexis cells in TUNI+cisplatin group (44.5%±5.1%) was significantly higher than that in cisplatin group (22.7%±3.9%, P<0.05). Immunoblotting showed the expressions of activated caspase-3 and caspase-4 were up-regulated obviously in cisplatin group. Compared to cisplatin group, the expressions of those proteins significantly increased in TUNI+cisplatin group (P<0.05). Indirect immunofluorescence staining showed no PDI expression and weak fluorescence was found in control group. PDI proteins presented in granular form, distributing around the nuclei with strong fluorescence were found in TUNI group and cisplatin group. PDI proteins showed obviously stronger fluorescence in a large proportion of cells in TUNI+cisplatin group, and the fluorescence intensity was obviously higher than that in TUNI group and cisplatin group. No expression of γ-H2AX protein was found in the nucleus in either control group or TUNI group. However, obvious green fluorescence was observed in nuclei of a part of cells in cisplatin group and TUNI+cisplatin group, no obvious difference existed between the two groups. Conclusion Heightened ER stress by tunicamycin may increase the apoptosis of HeLa cells induced by cisplatin.
