Korean J Obstet Gynecol.  2011 Apr;54(4):175-183. 10.5468/KJOG.2011.54.4.175.

The effect of cisplatin on endoplasmic reticulum stress of human cervical cancer cells

• Kim HY
• Kim BR
• Kim GD
• Kim HJ

• KMID: 2013231
• DOI: http://doi.org/10.5468/KJOG.2011.54.4.175

Abstract

OBJECTIVE
Cis-diamminedichloroplatinum (cisplatin) is a widely used chemotherapeutic agent. A number of evidences in cytotoxic mechanism of cisplatin, including perturbation of redox status, increase in lipid peroxydation, formation of DNA adduct, have been suggested. The author hypothesized that cisplatin would mediate apoptosis via endoplasmic reticulum (ER) stress in human cervical cancer cell.
METHODS
Human cervical cancer cell line (Hela cells) were treated with cisplatin and then ER stress-related response were performed using western blot, Flow cytometry and fluorescence analysis.
RESULTS
After addition of cisplatin to Hela cells, the author observed an expression of ER stress response genes through a gradual increase of nitric oxide and cytosolic Ca2+ concentration. Cisplatin-induced apoptosis can be inhibited by the inducible nitric oxide synthase inhibitor, 1400 W, and intracellular Ca2+ chelator, 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester (BAPTA-AM). These inhibitors also reduced mitochondrial apoptotic signals, such as mitochondrion membrane potential disruption, cytochrome c release and eventually reduced the death of Hela cells.
CONCLUSION
Taken together, ER would seem to contribute to cisplatin-induced apoptosis via both the early release of Ca2+ and the late amplification of mitochondria-mediated apoptotic signals.

Keyword

Cisplatin; Endoplasmic reticulum stress; Cervical cancer cells

MeSH Terms

Apoptosis
Blotting, Western
Cell Line
Cisplatin
Cytochromes c
Cytosol
DNA
Endoplasmic Reticulum
Endoplasmic Reticulum Stress
Flow Cytometry
Fluorescence
HeLa Cells
Humans
Imines
Membrane Potentials
Mitochondria
Nitric Oxide
Nitric Oxide Synthase Type II
Oxidation-Reduction
Uterine Cervical Neoplasms
Cisplatin
Cytochromes c
DNA
Imines
Nitric Oxide
Nitric Oxide Synthase Type II

Figure

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  Fig. 1 Cisplatin decreased the viability of Hela cells in a dose- and time-dependent manner. Cells were treated with various concentrations of cisplatin for 24 hr (A) and with 20 μM cisplatin for various periods (B). Then, cell viability was determined by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Data represents the mean±standard deviation of three independent experiments. aP <0.05 compared with control.

• 

  Fig. 2 Cisplatin increased intracellular nitric oxide and Calcium concentration in Hela cells. Cells were treated with cisplatin for various times. Then, western blot analysis was performed with specific antibodies for inducible nitric oxide synthase and ß-actin (A). Cells were treated with cisplatin in the absence or presence of 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester (BAPTA-AM) and 1400 W. Then, the cells were incubated with Fluo-3AM and subjected to flow cytometry (B).

• 

  Fig. 3 Effects of nitric oxide and calcium on cisplatin cytotoxicity. Cells were treated with cisplatin in the absence or presence of 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester (BAPTA-AM) and 1400 W. Cell viability was determined by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay (A). DNA fragmentation was determined by agarose-gel lectrophoresis. Extracted DNA was stained with ethidium bromime and visualized under ultraviolet (UV) light (B). After stimulation, cells were fixed with 4% paraformaldehyde and then stained with 4', 6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). Cells were observed under a fluorescent microscope, magnification ×100 (C). aP <0.05 compared with control.

• 

  Fig. 4 Effects of nitric oxide and calcium on cisplatin induced apoptotic population. Cells were treated with cisplatin in the absence or presence of 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester (BAPTA-AM) and 1400 W. Then, cells were stained with propidium iodide staining solution and analyzed by flow cytometry.

• 

  Fig. 5 Effects of nitric oxide and calcium on cisplatin induced endoplasmic reticulum (ER) stress. Cells were treated with cisplatin for various times. Then, Western blot analysis was performed with specific antibodies for activating transcription factor 6 (AFT6), X-box DNAbinding protein 1 (XBP1) (A), and C/EBP homologous protein (CHOP), B-cell lymphoma 2 (Bcl2), pro-caspase-3 and pro-caspase-9 (C). Cells were treated with cisplatin in the absence or presence of 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester (BAPTA-AM) and 1400 W. Then, Western blot analysis was performed with specific antibodies for AFT6, XBP-1 (B), and CHOP, Bcl2, pro-caspase-3 (D).

• 

  Fig. 6 Effects of nitric oxide and calcium on cisplatin induced the mitochondrial membrane potential transition. Cells were treated with cisplatin in the absence or presence of 1, 2-bis-(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA-AM) and 1400 W. Then, cells were stained with 5,5',6,6'-tetra chloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) for 30 min at 37℃. Membrane potential transition was visualized under a fluorescent microscope (×200).

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