Inhibition of autophagy enhances cisplatin cytotoxicity through endoplasmic reticulum stress in human cervical cancer cells
Introduction
Cisplatin (cis-diamminedichloroplatinum II, CDDP) is one of the most effective chemotherapeutic agents and is widely used in the treatment of solid tumors, but its side effects, plus acquired resistance to the drug gained during the course of treatment, limits its usage [1], [2], [3]. It is generally considered to be a cytotoxic drug that kills cancer cells by damaging DNA and inhibiting DNA synthesis. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death, predominantly by apoptosis [4]. Recently, several research teams have found that cisplatin can induce endoplasmic reticulum (ER) stress and nucleus-independent apoptotic signaling [5], [6].
Various physiological and pathological conditions may cause an imbalance between ER protein folding load and capacity, leading to the accumulation of unfolded or misfolded proteins in the ER lumen, a condition referred to as “ER stress” [7], [8]. When misfolded proteins accumulate in the ER, the resulting stress activates the unfolded protein response (UPR) to induce the expression of chaperones and proteins involved in the recovery process. Severe ER stress can cause cell death, usually by activating intrinsic apoptosis [9]. Moreover, to clear the ER of accumulated terminally misfolded protein aggregates that cannot be degraded by the proteasome, the UPR may upregulate the autophagy machinery [10], [11].
Autophagy is an intracellular degradative system that plays important roles in regulating protein homeostasis, and is essential for survival when cells are faced with metabolic stress. Besides its role in cellular homeostasis, autophagy can be a form of programmed cell death, or may play a cytoprotective role in situations of nutrient starvation [12], [13]. Moreover, different anticancer treatments activate autophagy in tumor cells, a process which has been proposed to either enhance cancer cell death or act as a mechanism of resistance to chemotherapy [14], [15], [16], [17], [18], [19].
Here, by treating HeLa cells with cisplatin, we have tested the hypothesis that autophagy activation can alleviate ER stress, thus enabling the cell to avoid ER-mediated apoptosis while downregulating mitochondrial pathway-induced apoptosis. We found that cisplatin can induce ER stress by promoting formation of misfolded ubiquitinated proteins, and that both autophagy and apoptosis were activated. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) increased the level of ubiquitinated proteins, which elevated ER stress and resulted in a higher apoptotic rate of these cells when they were treated with cisplatin. We also found that combination of cisplatin treatment with an ER stress inducer, tunicamycin, increased cisplatin-induced apoptosis by inducing severe ER stress.
Section snippets
Cell culture
The human cervical cancer HeLa cell line was cultured at 37 °C in a 5% CO2 and 95% air atmosphere, in Iscove’s modified Dulbecco’s medium (IMDM) (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL Co., USA), 100 U/ml penicillin and 100 U/ml streptomycin.
Cell viability assays
Cell viability was determined by MTT assays. We seeded exponentially growing HeLa cells into 96-well culture plates in 100 μl medium at a density of 1 × 104 cells/well. The following day, varying
Cisplatin inhibits HeLa cell proliferation, and induces mitochondria-mediated apoptosis
We treated HeLa cells with increasing doses of cisplatin for 12 h and 24 h, and then examined growth inhibition using MTT assays. We found that cisplatin inhibited the proliferation of HeLa cells (Fig. 1A).
Based on MTT results and previous studies, we treated HeLa cells with 6 μg/ml cisplatin for 0 h and 12 h, and examined apoptotic chromatin condensation by Hoechst 33342 staining using confocal microscopy (Fig. 1B). Compared with controls, cisplatin-induced apoptotic chromatin condensation was
Discussion
Cisplatin is used as a first-line therapy for solid tumors, including cervical cancer, either alone or in combination with other anticancer agents [23]. The antitumor activity of cisplatin is believed to be due to its interaction with chromosomal DNA, where it forms intra- and inter-strand adducts leading to apoptotic cell death [24], [25], [26]. Recent data have shown that cisplatin may have important direct interactions with mitochondria, which can induce apoptosis [27], [28], [29]. The main
Acknowledgment
The research was supported by the Natural Science Foundation of Jilin Provincial Science and Technology Department (201015240).
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These authors contributed equally to this study.