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The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1
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The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1 Wenjun Li a,1 , Minhong Zhang b,1 , Lei Xu a , Danmiao Lin a , Shaoxi Cai c , Fei Zou a,∗ a Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong Province 510515, China b Department of Occupational Health Assessment, Shenzhen Prevention and Treatment Center for Occupational Diseases, Shenzhen, Guangdong Province 518001, China c Department of Respiration, Chronic Airways Diseases Laboratory, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
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Article history: Received 14 February 2013 Accepted 19 April 2013 Keywords: Cisplatin Non-small cell lung cancer SOC STIM1 Calcium Apoptosis
a b s t r a c t Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most active antitumor agents used in human chemotherapy of non-small cell lung cancer. Cisplatin forms crosslinked DNA adducts and its cytotoxicity has been shown to be mediated by propagation of DNA damage recognition signals to downstream pathways prompting apoptosis. The steps involved in the process include changes in Ca2+ signaling with dysregulated tumor cell turn-over. Stromal interaction molecules 1 (STIM1), as one of the most potent tumor suppressor genes, are identified as the endoplasmic-reticulum (ER) Ca2+ sensor controlling storeoperated Ca2+ entry (SOCE) in non-excitable cells, which is main pathway to extracellular Ca2+ influx. Its role in STIM1 cisplatin-induced apoptosis of non-small cell lung cancer was the focus of study with focus on SOCE inhibitors 2-APB- and SKF96365-cisplatin-induced apoptosis in the non-small cell lung cancer (NSCLC) cell lines A549 and H460. In this experimental model, cisplatin-induced apoptosis and decreased concentration of intracellular Ca2+ was demonstrated. The expression of STIM1 was significantly higher in carcinoma tissue than in the adjacent non-neoplastic lung tissue. These findings support the conclusion that STIM1 may play an important role in the development of NSCLC which makes drugs that repress the expression of STIM1 to be a potential target for lung cancer therapy. © 2013 Elsevier GmbH. All rights reserved.
1. Introduction Lung cancer is one of the leading causes of cancer deaths in adults worldwide. There were 1.6 million individuals living with lung cancer out of which 1.4 million died in 2008 (Ferlay et al., 2010). More than 80% of lung cancer patients are non-small cell lung cancer (NSCLC), while the remaining 20% are small cell lung cancer (SCLC) (Monari et al., 2011). Notably, the incidence rate of lung cancer in the Chinese men and woman is higher than the rate among women in many European countries (Jemal et al., 2010). Cis-diamminedichloroplatinum (II) (cisplatin) is an important chemotherapeutic drug in the treatment of advanced cancer. Cisplatin and its derivative, carboplatin, are widely used in various cancers, including non-small cell lung cancer (Kelland,
∗ Corresponding author at: Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, No. 1838, North Guangzhou Road, Guangzhou, Guangdong Province 510515, China. Tel.: +86 20 6164 8301; fax: +86 20 6164 8324. E-mail address: [email protected] (F. Zou). 1 These authors contributed equally to this paper.
2007). Cisplatin exhibits its role as anticancer agent through binding to DNA and the associated DNA damage with resultant disruption of downstream cellular processes involved in the kinetics of cell turn-over (Siddik, 2003; Stewart, 2007). The molecular mechanisms involved in the antitumor mode of action of cisplatin are still incompletely elucidated and understood. STIM1 (stromal interaction molecule; also known as GOK) was identified as a novel human gene that maps to a region of chromosome 11p15.5 in 1995 (Parker et al., 1996). It was demonstrated that STIM1 has a growth-inhibitory function, which is specific to rhabdoid tumor and rhabdomyosarcoma cell lines. In vitro studies indicate that human STIM1 is one of the most potent tumor suppressor genes (Sabbioni et al., 1997). As the endoplasmic-reticulum (ER) Ca2+ sensor, STIM1 control store-operated Ca2+ entry (SOCE) in non-excitable cells. Any perturbation of intracellular Ca2+ homeostasis can cause cytotoxicity and may trigger either apoptotic or necrotic cell death (Orrenius et al., 2003). (Pinton et al., 2008; Pinton and Rizzuto, 2006; Schrodl et al., 2009). (Magnelli et al., 1994). Current evidence suggests that the expression of STIM1 in tumor tissues is higher than that in the adjacent non-neoplastic tissue (Jayadev et al., 1999).
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The objective of study was to extent the understanding the possible impact on intracellular Ca2+ -homeostasis in cisplatin-treated cells. The focus was on STIM1, factors that control tumor growth, from the tissue in the close proximity to the tumor, as to which extent they are modulated by cisplatin, and the involved molecular mechanisms.
2. Materials and methods 2.1. Materials Plasmids of STIM1 were generously provided by Dr. Worley (Johns Hopkins University School) and Dr Joe Yuan (University of
Fig. 1. Cisplatin induces apoptosis in human non-small lung cancer cells. A549 (A and C) and H460 (B and D) cells were treated for 24 h with indicated concentrations of cisplatin. For flow cytometry analysis (A–D), the cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. Panels C and D indicates the percentages of cells positive for Annexin V-FITC. (E and F) 15 M cisplatin induces the apoptosis of A549 (E) and H460 (F) cells as indicated by Hoechst 33342 staining. Light micrographs show the nuclear morphology of A549 and H460 cells. Arrows denote chromatin condensation and nuclear fragmentation. Scale bar, 10 m. Data are means ± SD of three independent experiments in triplicates (n = 3, N = 3). **P < 0.01 versus control.
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Texas Southwestern Medical Center). An antibody against STIM1 was purchased from Cell Signaling; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Flow cytometric analysis of apoptosis was performed by staining cells with Annexin V-FITC and propidium iodide (PI) using the Annexin V Apoptosis detection kit (BD ParMingen San Diego, CA). STIM1 small interference (siRNA) and scrambled siRNA were purchased from GenePharma (Shanghai, China). Cisplatin was purchased from sigma. Lipofectamine 2000 reagent was purchased from Invitrogen (Carlsbad, CA). All other reagents were from Sigma.
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2.3. Biological samples Twenty-two tissue samples were obtained from patients who had undergone complete surgical resection of p-stage I primary NSCLC at Southern Chinese Hospitals (?) from May 2011 to July 2012. Histological type and grade of cell differentiation were determined according to the WHO classification system. Informed consent for participation in this study was obtained from all patients prior to the surgical operations. This study was reviewed and approved by the Ethics Committee of the Southern Medical University.
2.4. Cell transfection 2.2. Cell culture Stock cultures of human non-small lung cancer cell lines A549 and H460 were obtained from American type culture collection (ATCC). These cell lines were grown in RPMI medium 1640 (Life Technologies, Rockville, MD) containing glutamate (5 mM) and 10% fetal bovine serum; and they were cultured in the same conditions and maintained at 37 ◦ C in a humidified incubator with 5% CO2 .
The sequences of siRNA against the human STIM1: 5 -GGC UCU GGA UAC AGU GCU CTT-3 (sense) and 5 -GAG CAC UGU AUC CAG AGC CTT-3 (antisense); The scrambled control siRNA sequences were sense, 5 -UUC UCC GAA CGU GUC ACG UTT-3 and antisense, 5 -ACG UGA CAC GUU CGG AGA ATT-3 . The cells at ∼1 × 105 cells per dish were transfected with siRNA or plasmids using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The efficiency of siRNA to knockdown
Fig. 2. SOCE inhibitors attenuated the cisplatin-induced apoptosis. A549 (A and B) and H460 (C and D) cells were pre-incubated with 2-APB (75 M) or SKF-96365 (10 M) for 2 h followed by treatment with cisplatin for 24 h. The apoptosis of cells was determined by the Annexin V-FITC/PI assay. The right part (B and D) indicate percentages of cells positive for Annexin V. Data are means ± SD of three independent experiments in triplicates (n = 3, N = 3). *P < 0.05 versus control.
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the target protein and the efficacy of siRNA transfection were determined by western blot analysis. 2.5. Western analysis The A549 and H460 cells were washed twice with PBS and lysed for 30 min with ice-cold lysis buffer (50 mM Tris–HCl, pH
7.5, 0.5% cholate acid, 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% Triton X-100, and 10% glycerol) containing protease inhibitor cocktail (Roche, Mannheim, Germany). Total cell lysates were centrifuged at 14,000 × g for 15 min at 4 ◦ C. The protein concentration of the supernatant was determined by the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (50 g) were loaded and separated on a 10% SDS-PAGE and transferred to PVDF
Fig. 3. The influence of downregulation of STIM1 on the cisplatin-induced apoptosis. A549 (A, B, E and F) and H460 (C, D, G and H) cells were transfected with scrambled or STIM1 siRNA for 48 h and then 15 M cisplatin was added for another 24 h. STIM1 siRNA increased the apoptosis induced by cisplatin in A549 (A and B) and H460 (C and D) cells as determined by the Annexin V-FITC/PI assay. Panels B and D indicate percentages of cells positive for Annexin V-FITC. The effect of specific siRNA for STIM1 in A549 (C and F) and H460 (G and H) cells was observed by Western blot. Quantitative analysis of the protein levels of STIM1 normalized to those of GAPDH is shown (F and H). NC means negative control (transfected with scrambled). Results are the means ± SD of three independent experiments in triplicates (n = 3, N = 3). In (B and D), **P < 0.01, and in (F and H), *P < 0.05.
Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003
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Fig. 4. Overexpression of STIM1 increased cisplatin-induced apoptosis in A549 and H460 cells. A549 (A, B, E and F) and H460 (C, D, G and H) cells were transfected with wild-type STIM1 for overexpression of STIM1 for 72 h and then 15 M cisplatin was added for another 24 h. Overexpression of STIM1 attenuated the apoptosis induced by cisplatin in A549 (A and B) and H460 (C and D) cells as determined by the Annexin V-FITC/PI assay. Panels B and D indicate percentages of cells positive for Annexin V. The effect of wild-type STIM1 transfection in A549 (C and F) and H460 (G and H) cells was observed by Western blot. Quantitative analysis of the protein levels of STIM1 normalized to those of GAPDH is shown (F and H). Vector means transfected vector. Results are the means ± SD of three independent experiments in triplicates (n = 3, N = 3). In (B, D), **P < 0.01, and in (F and H), *P < 0.05.
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membranes (Bio-Rad Laboratories, Hercules, CA) and incubated with primary antibodies at optimal dilution at 4 ◦ C overnight. The blots were washed and incubated for 1 h with horseradish peroxidase-conjugated anti-IgG antibody. Immunoreactive bands were developed using a chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific Pierce Protein Research Products) 2.6. Apoptosis assays For assessment of nuclear condensation, l × 106 cells were washed twice with PBS, stained with 5 g/ml Hoechst 33342 (Sigma, St. Louis, MO, USA) and incubated such cells at 37 ◦ C for 20 min, Fluorescence images were taken by using a scanning confocal microscope (FV-1000 Olympus, Tokyo, Japan). Phosphatidylserine externalization was assayed using the Annexin V/PI apoptosis detection Kit according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA). In brief, cells were washed twice with cold PBS and then re-suspend in 1 × Binding Buffer at a concentration of 1 × 106 cells/ml. Transfer 100 l of the solution (1 × 105 cells) to a 5 ml culture tube, added with 5 l of FITC Annexin V and 5 l PI. Cells were gently vortexed and then incubated for 15 min at RT (25 ◦ C) in the dark. Then 400 l of 1 × Binding Buffer was added to each tube. Subsequent flow cytometric analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA) with Cell Quest software (Becton Dickinson, Franklin Lakes, NJ). 2.7. Intracellular Ca2+ measurements Intracellular Ca2+ was monitored using Ca2+ sensitive fluorescent indicator, Fluo-4 acetoxymethyl ester (Fluo-4 AM; Invitrogen,
Carlsbad, CA) by an inverted laser scanning confocal microscope (Olympus, FV1000-IX71, Japan). Cells were loaded with 5 M Fluo4 AM at 37 ◦ C for 20 min in darkness with modified Hanks’ buffered salt solution (2 mM Ca2+ ). They were rinsed thrice with Hanks’ buffered salt solution and kept at room temperature for 20 min to allow de-esterification of Fluo-4 ester. Green fluorescence of Fluo4 was excited by a 10 mW multi-tune argon laser at 488 nm, and emitted fluorescence was recorded through a 525 nm channel. For imaging using Fluo-4, [Ca2+ ]i changes are presented as AF/Fo ratios after background subtraction, where AF was the change in fluorescence signal intensity and Fo was the baseline as calculated by averaging the 10 frames before stimulus application. 2.8. Statistical analysis The results are presented as the mean ± SEM. Data were analyzed by one-way ANOVA. All statistical tests were operated with the program SPSS version 13.0 and P < 0.05 was considered statistically significant. 3. Results 3.1. Cisplatin-induced the apoptosis of the non-small cell lung cancer cell lines (A549 and H460) In order to study of the role of STIM1 in non-small lung cancer, two non-small lung cancer cell lines A549 and H460 were treated with defined concentrations of cisplatin for 24 h. As shown in Fig. 1A–D, the apoptosis was induced in those cells treated with cisplatin, and a dose-dependent increase in Annexin V-positive
Fig. 5. Cisplatin inhibited the expression of STIM1. A549 (A and B) and H460 (C and D) cells were treated for 24 h with indicated 15 M of cisplatin. And Western blot was performed using anti-STIM1 or anti-GAPDH antibodies (A and C). GAPDH (A and C) served as the loading control. The right part (B and D) indicates quantitative analysis of western blot result. Data are means ± SD of three independent experiments in triplicates (n = 3, N = 3). **P < 0.01 versus control.
Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003
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Fig. 6. Cisplatin induces decreases in [Ca2+ ]i in A549 and H460 cells. In 2 mM Ca2 + , 15 M cisplatin evoked a slight decrease of Ca2 + in A549 cells (A) and H460 cells (B). Data are typical representatives of three independent experiments in triplicates (n = 10, N = 3). A549 (A) and H460 (B) cells were transfected with scrambled or STIM-1 siRNA for 48 h. In 2 mM Ca2+ , STIM1 siRNA attenuated the decrease of [Ca2+ ]i when treated with 15 M cisplatin in A549 (A) and H460 cells (B). A549 (C) and H460 (D) cells were transfected with wild-type STIM1 for overexpression of STIM1 for 72 h. In 2 mM Ca2+ , Overexpression of STIM1 caused no significant change in the decrease of [Ca2+ ]i when treated with 15 M cisplatin in A549 (C) and H460 (D) cells. Data are typical representatives of three independent experiments in triplicates (n = 10, N = 3).
staining was observed. The morphological characteristics of apoptosis are illustrated in Fig. 1E and F. 3.2. Pharmacological Inhibition of SOCE or depletion of STIM1 enhance the apoptosis-induced by cisplatin To investigate whether SOCE is involved in the cisplatin-induced apoptosis, A549 and H460 cells were pretreated with the pharmacological inhibitors of SOCE 2-APB (75 M) or SKF-96365 (10 M). As shown in Fig. 2, inhibited SOCE significantly enhanced the cisplatin-induced apoptosis in A549 cells (Fig. 2A and B) and H460 cells (Fig. 2C and D). Knockdown of STIM1 by RNAi, which reduced the protein level of STIM1 by 67% in A549 cells and 74% in H460 cells. The knocking down of STIM1 significantly enhanced the pre-apoptotic effects of cisplatin (Fig. 3). In contrast to the downregulation of STIM1, overexpression of STIM1 reduced apoptosis of non-small lung cancer cells (Fig. 4).
3.3. Cisplatin inhibition of protein expression of STIM1 in A549 and H460 cells To explore whether the apoptosis induced by cisplatin occurs through modulation of the expression of STIM1 in non-small lung cancer cells, the expression of STIM1 in A549 and H460 cells was examined. The results show that the expression of STIM1 was downregulated in s after cisplatin treatment (Fig. 5).
3.4. Cisplatin induce apoptosis of A549 and H460 cells through SOCE To determine whether the downregulation of STIM1 influence [Ca2+ ]i in non-small lung cancer cells as a result of cisplatin treatment, the [Ca2+ ]i in both cells lines was examined. [Ca2+ ]i was slightly decreased following cisplatin treatment as shown in Fig. 6A and B. Depletion of STIM1 by RNAi caused a slight decrease in
Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003
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Fig. 7. The expression of STIM1 in NSCLC. (A) The protein levels of STIM1 in the tissues were detected by western blot. Quantitative analysis of the protein levels of STIM1 normalized to those of GAPDH is shown (B).
[Ca2+ ]i, while the overexpression of STIM1 by transfected wild-type STIM1 had no effect. 3.5. The expression of STIM1 in non-small cell lung cancer (NSCLC) In order to explore the expression of STIM1 in non-small cell lung cancer (NSCLC), 22 cases of non-small lung cancer with interconnected tissues consisting of carcinoma and adjacent non-neoplastic lung tissues were analyzed by immunoblotting. Compared with that of non-neoplastic tissue, the level of STIM1 expression in tumor tissues was elevated in 16 cases (Fig. 7). This means that STIM1 expression in non-small cell lung cancer is higher than that in normal lung tissue in 72.72% of non-small lung cancer patients. 4. Discussion The Ca2+ signal regulate fundamental eukaryotic processes including secretion, muscle contraction, gene transcription, cell proliferation and apoptosis. The highly coordinated regulation of Ca2+ hemostasis in cell is depend on channels, ATPase (pumps) and exchangers, which tightly balance Ca2+ flux across the endoplasmic
reticulum (ER) and plasma membrane (PM). Recent year have seen a growing evidence show that Ca2+ signaling pathways are remodeled or deregulated in cancer, which is implicated in tumorigenesis and tumor progression. In non-excitable cell, Store-operated Ca2+ entry plays a significant role in extracellular Ca2+ homestasis. So we determine the quantity of STIM1, which is key component of SOCE, in neoplastic tissue and paired nonneoplastic tissue in 22 patients, As the results indicated that the level of STIM1 expression in tumor tissues is higher than those in paired nonneoplastic tissue. Those results are concordance with previous reports that STIM1 overexpression in cervical cancer tissue. Cisplatin is the most important and efficacious chemotherapeutic agent in the treatment of non-small-cell lung cancer. In order to determined whether the apoptosis of tumor cells induced by cisplatin through modulate the expression of STIM1. As a key component of SOCE, STIM1 is a single pass membrane protein located in ER which as a sensor of endoplasmic reticulum (ER) Ca2+ concentration and essential activator of SOCs channels, allowing extracellular Ca2+ influx (Abdullaev et al., 2008; Liu et al., 2011). We quantities the expression of STIM1 in non-small lung cancer cells followed by treated with cisplatin. These result indicated cisplatin suppress the expression of STIM1. To further elucidate the molecular mechanism of the apoptosis induced by cisplatin through SOCE, we downregulate the expression of STIM1 by siRNA in those cell
Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003
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line, followed by treated with cisplatin, which increase the apoptosis. Meanwhile we determined the intracellular Ca2+ followed the knockdown STIM1. The results show that the intracellular Ca2+ decrease in the STIM1-silencing NSCLC cells Furthermore, we transfected wild-type STIM1-expressing plasmids into the cells. After treatment of the cells expressing increased level of STIM1 with cisplatin, a reduced level of apoptosis was observed, while over expression through transfected wild STIM1 have no effect on the intracellular Ca2+ concentration. These results further supported that STIM1 is a negative regulator of apoptosis induced by cisplatin. It has been reported that cisplatin bound to DNA forms the intra- and inter-strand DNA cross-links, which is inhibited transcription of certain genes, triggered apoptosis of tumor cell. Our results suggested that cisplatin induced the apoptosis in NSCLC through modulate the expression of STIM1. Knockdown the STIM1 in NSCLC lead to enhance the apoptosis induced by cisplatin. The SOCE inhibitors, such as 2-APB and SKF96365 have same effect on the NSCLC cells treatment with cisplatin. As a main pathway of extracellular Ca2+ influx, SOCE play a key role in almost all cellular pathways (Liou et al., 2005; Roos et al., 2005). SOCE is important for proliferation and apoptosis of certain tumors and the imbalance of cell growth and cell death finally lead to cancer. Chen et al. proposed that STIM1 is important for cervical cancer cell proliferation, migration, and angiogenesis. In other studies, blocking STIM1-mediating Ca2+ influx impairs focal adhesion turn-over (Berridge et al., 2003). SOCE inhibition has been shown to arrest tumor cell cycle at G0/G1 phase and inhibiting tumor cell metastasis in various tumor cells (Yang et al., 2009). It is noticeable that previous research suggested that that STIM1 is a MT-Plus-End-Tracking Protein (EB1), which is involve in accurate chromosome segregation during mitosis and in the polarization of the microtubule cytoskeleton in migrating cells (Holmuhamedov et al., 2002; Vanden Abeele et al., 2003). So it is alternative molecular pathway maybe involved in cisplatin triggered apoptosis of NSCLE, which through inhibits the expression of STIM1, in turn to affect accurate chromosome segregation during mitosis and in the polarization of the microtubule cytoskeleton. Acknowledgments We thank Dr. Worley (Johns Hopkins University School) and Dr. Joe Yuan (University of Texas Southwestern Medical Center) for gift of plasmids. This work was supported by Natural Science Foundation of China (30971193) and National Basic Research Program of China (2012CB518200).
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Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003
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The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1 Wenjun Li a,1 , Minhong Zhang b,1 , Lei Xu a , Danmiao Lin a , Shaoxi Cai c , Fei Zou a,∗ a Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong Province 510515, China b Department of Occupational Health Assessment, Shenzhen Prevention and Treatment Center for Occupational Diseases, Shenzhen, Guangdong Province 518001, China c Department of Respiration, Chronic Airways Diseases Laboratory, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
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Article history: Received 14 February 2013 Accepted 19 April 2013 Keywords: Cisplatin Non-small cell lung cancer SOC STIM1 Calcium Apoptosis
a b s t r a c t Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most active antitumor agents used in human chemotherapy of non-small cell lung cancer. Cisplatin forms crosslinked DNA adducts and its cytotoxicity has been shown to be mediated by propagation of DNA damage recognition signals to downstream pathways prompting apoptosis. The steps involved in the process include changes in Ca2+ signaling with dysregulated tumor cell turn-over. Stromal interaction molecules 1 (STIM1), as one of the most potent tumor suppressor genes, are identified as the endoplasmic-reticulum (ER) Ca2+ sensor controlling storeoperated Ca2+ entry (SOCE) in non-excitable cells, which is main pathway to extracellular Ca2+ influx. Its role in STIM1 cisplatin-induced apoptosis of non-small cell lung cancer was the focus of study with focus on SOCE inhibitors 2-APB- and SKF96365-cisplatin-induced apoptosis in the non-small cell lung cancer (NSCLC) cell lines A549 and H460. In this experimental model, cisplatin-induced apoptosis and decreased concentration of intracellular Ca2+ was demonstrated. The expression of STIM1 was significantly higher in carcinoma tissue than in the adjacent non-neoplastic lung tissue. These findings support the conclusion that STIM1 may play an important role in the development of NSCLC which makes drugs that repress the expression of STIM1 to be a potential target for lung cancer therapy. © 2013 Elsevier GmbH. All rights reserved.
1. Introduction Lung cancer is one of the leading causes of cancer deaths in adults worldwide. There were 1.6 million individuals living with lung cancer out of which 1.4 million died in 2008 (Ferlay et al., 2010). More than 80% of lung cancer patients are non-small cell lung cancer (NSCLC), while the remaining 20% are small cell lung cancer (SCLC) (Monari et al., 2011). Notably, the incidence rate of lung cancer in the Chinese men and woman is higher than the rate among women in many European countries (Jemal et al., 2010). Cis-diamminedichloroplatinum (II) (cisplatin) is an important chemotherapeutic drug in the treatment of advanced cancer. Cisplatin and its derivative, carboplatin, are widely used in various cancers, including non-small cell lung cancer (Kelland,
∗ Corresponding author at: Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, No. 1838, North Guangzhou Road, Guangzhou, Guangdong Province 510515, China. Tel.: +86 20 6164 8301; fax: +86 20 6164 8324. E-mail address: [email protected] (F. Zou). 1 These authors contributed equally to this paper.
2007). Cisplatin exhibits its role as anticancer agent through binding to DNA and the associated DNA damage with resultant disruption of downstream cellular processes involved in the kinetics of cell turn-over (Siddik, 2003; Stewart, 2007). The molecular mechanisms involved in the antitumor mode of action of cisplatin are still incompletely elucidated and understood. STIM1 (stromal interaction molecule; also known as GOK) was identified as a novel human gene that maps to a region of chromosome 11p15.5 in 1995 (Parker et al., 1996). It was demonstrated that STIM1 has a growth-inhibitory function, which is specific to rhabdoid tumor and rhabdomyosarcoma cell lines. In vitro studies indicate that human STIM1 is one of the most potent tumor suppressor genes (Sabbioni et al., 1997). As the endoplasmic-reticulum (ER) Ca2+ sensor, STIM1 control store-operated Ca2+ entry (SOCE) in non-excitable cells. Any perturbation of intracellular Ca2+ homeostasis can cause cytotoxicity and may trigger either apoptotic or necrotic cell death (Orrenius et al., 2003). (Pinton et al., 2008; Pinton and Rizzuto, 2006; Schrodl et al., 2009). (Magnelli et al., 1994). Current evidence suggests that the expression of STIM1 in tumor tissues is higher than that in the adjacent non-neoplastic tissue (Jayadev et al., 1999).
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The objective of study was to extent the understanding the possible impact on intracellular Ca2+ -homeostasis in cisplatin-treated cells. The focus was on STIM1, factors that control tumor growth, from the tissue in the close proximity to the tumor, as to which extent they are modulated by cisplatin, and the involved molecular mechanisms.
2. Materials and methods 2.1. Materials Plasmids of STIM1 were generously provided by Dr. Worley (Johns Hopkins University School) and Dr Joe Yuan (University of
Fig. 1. Cisplatin induces apoptosis in human non-small lung cancer cells. A549 (A and C) and H460 (B and D) cells were treated for 24 h with indicated concentrations of cisplatin. For flow cytometry analysis (A–D), the cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. Panels C and D indicates the percentages of cells positive for Annexin V-FITC. (E and F) 15 M cisplatin induces the apoptosis of A549 (E) and H460 (F) cells as indicated by Hoechst 33342 staining. Light micrographs show the nuclear morphology of A549 and H460 cells. Arrows denote chromatin condensation and nuclear fragmentation. Scale bar, 10 m. Data are means ± SD of three independent experiments in triplicates (n = 3, N = 3). **P < 0.01 versus control.
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Texas Southwestern Medical Center). An antibody against STIM1 was purchased from Cell Signaling; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Flow cytometric analysis of apoptosis was performed by staining cells with Annexin V-FITC and propidium iodide (PI) using the Annexin V Apoptosis detection kit (BD ParMingen San Diego, CA). STIM1 small interference (siRNA) and scrambled siRNA were purchased from GenePharma (Shanghai, China). Cisplatin was purchased from sigma. Lipofectamine 2000 reagent was purchased from Invitrogen (Carlsbad, CA). All other reagents were from Sigma.
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2.3. Biological samples Twenty-two tissue samples were obtained from patients who had undergone complete surgical resection of p-stage I primary NSCLC at Southern Chinese Hospitals (?) from May 2011 to July 2012. Histological type and grade of cell differentiation were determined according to the WHO classification system. Informed consent for participation in this study was obtained from all patients prior to the surgical operations. This study was reviewed and approved by the Ethics Committee of the Southern Medical University.
2.4. Cell transfection 2.2. Cell culture Stock cultures of human non-small lung cancer cell lines A549 and H460 were obtained from American type culture collection (ATCC). These cell lines were grown in RPMI medium 1640 (Life Technologies, Rockville, MD) containing glutamate (5 mM) and 10% fetal bovine serum; and they were cultured in the same conditions and maintained at 37 ◦ C in a humidified incubator with 5% CO2 .
The sequences of siRNA against the human STIM1: 5 -GGC UCU GGA UAC AGU GCU CTT-3 (sense) and 5 -GAG CAC UGU AUC CAG AGC CTT-3 (antisense); The scrambled control siRNA sequences were sense, 5 -UUC UCC GAA CGU GUC ACG UTT-3 and antisense, 5 -ACG UGA CAC GUU CGG AGA ATT-3 . The cells at ∼1 × 105 cells per dish were transfected with siRNA or plasmids using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The efficiency of siRNA to knockdown
Fig. 2. SOCE inhibitors attenuated the cisplatin-induced apoptosis. A549 (A and B) and H460 (C and D) cells were pre-incubated with 2-APB (75 M) or SKF-96365 (10 M) for 2 h followed by treatment with cisplatin for 24 h. The apoptosis of cells was determined by the Annexin V-FITC/PI assay. The right part (B and D) indicate percentages of cells positive for Annexin V. Data are means ± SD of three independent experiments in triplicates (n = 3, N = 3). *P < 0.05 versus control.
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the target protein and the efficacy of siRNA transfection were determined by western blot analysis. 2.5. Western analysis The A549 and H460 cells were washed twice with PBS and lysed for 30 min with ice-cold lysis buffer (50 mM Tris–HCl, pH
7.5, 0.5% cholate acid, 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% Triton X-100, and 10% glycerol) containing protease inhibitor cocktail (Roche, Mannheim, Germany). Total cell lysates were centrifuged at 14,000 × g for 15 min at 4 ◦ C. The protein concentration of the supernatant was determined by the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (50 g) were loaded and separated on a 10% SDS-PAGE and transferred to PVDF
Fig. 3. The influence of downregulation of STIM1 on the cisplatin-induced apoptosis. A549 (A, B, E and F) and H460 (C, D, G and H) cells were transfected with scrambled or STIM1 siRNA for 48 h and then 15 M cisplatin was added for another 24 h. STIM1 siRNA increased the apoptosis induced by cisplatin in A549 (A and B) and H460 (C and D) cells as determined by the Annexin V-FITC/PI assay. Panels B and D indicate percentages of cells positive for Annexin V-FITC. The effect of specific siRNA for STIM1 in A549 (C and F) and H460 (G and H) cells was observed by Western blot. Quantitative analysis of the protein levels of STIM1 normalized to those of GAPDH is shown (F and H). NC means negative control (transfected with scrambled). Results are the means ± SD of three independent experiments in triplicates (n = 3, N = 3). In (B and D), **P < 0.01, and in (F and H), *P < 0.05.
Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003
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Fig. 4. Overexpression of STIM1 increased cisplatin-induced apoptosis in A549 and H460 cells. A549 (A, B, E and F) and H460 (C, D, G and H) cells were transfected with wild-type STIM1 for overexpression of STIM1 for 72 h and then 15 M cisplatin was added for another 24 h. Overexpression of STIM1 attenuated the apoptosis induced by cisplatin in A549 (A and B) and H460 (C and D) cells as determined by the Annexin V-FITC/PI assay. Panels B and D indicate percentages of cells positive for Annexin V. The effect of wild-type STIM1 transfection in A549 (C and F) and H460 (G and H) cells was observed by Western blot. Quantitative analysis of the protein levels of STIM1 normalized to those of GAPDH is shown (F and H). Vector means transfected vector. Results are the means ± SD of three independent experiments in triplicates (n = 3, N = 3). In (B, D), **P < 0.01, and in (F and H), *P < 0.05.
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membranes (Bio-Rad Laboratories, Hercules, CA) and incubated with primary antibodies at optimal dilution at 4 ◦ C overnight. The blots were washed and incubated for 1 h with horseradish peroxidase-conjugated anti-IgG antibody. Immunoreactive bands were developed using a chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific Pierce Protein Research Products) 2.6. Apoptosis assays For assessment of nuclear condensation, l × 106 cells were washed twice with PBS, stained with 5 g/ml Hoechst 33342 (Sigma, St. Louis, MO, USA) and incubated such cells at 37 ◦ C for 20 min, Fluorescence images were taken by using a scanning confocal microscope (FV-1000 Olympus, Tokyo, Japan). Phosphatidylserine externalization was assayed using the Annexin V/PI apoptosis detection Kit according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA). In brief, cells were washed twice with cold PBS and then re-suspend in 1 × Binding Buffer at a concentration of 1 × 106 cells/ml. Transfer 100 l of the solution (1 × 105 cells) to a 5 ml culture tube, added with 5 l of FITC Annexin V and 5 l PI. Cells were gently vortexed and then incubated for 15 min at RT (25 ◦ C) in the dark. Then 400 l of 1 × Binding Buffer was added to each tube. Subsequent flow cytometric analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA) with Cell Quest software (Becton Dickinson, Franklin Lakes, NJ). 2.7. Intracellular Ca2+ measurements Intracellular Ca2+ was monitored using Ca2+ sensitive fluorescent indicator, Fluo-4 acetoxymethyl ester (Fluo-4 AM; Invitrogen,
Carlsbad, CA) by an inverted laser scanning confocal microscope (Olympus, FV1000-IX71, Japan). Cells were loaded with 5 M Fluo4 AM at 37 ◦ C for 20 min in darkness with modified Hanks’ buffered salt solution (2 mM Ca2+ ). They were rinsed thrice with Hanks’ buffered salt solution and kept at room temperature for 20 min to allow de-esterification of Fluo-4 ester. Green fluorescence of Fluo4 was excited by a 10 mW multi-tune argon laser at 488 nm, and emitted fluorescence was recorded through a 525 nm channel. For imaging using Fluo-4, [Ca2+ ]i changes are presented as AF/Fo ratios after background subtraction, where AF was the change in fluorescence signal intensity and Fo was the baseline as calculated by averaging the 10 frames before stimulus application. 2.8. Statistical analysis The results are presented as the mean ± SEM. Data were analyzed by one-way ANOVA. All statistical tests were operated with the program SPSS version 13.0 and P < 0.05 was considered statistically significant. 3. Results 3.1. Cisplatin-induced the apoptosis of the non-small cell lung cancer cell lines (A549 and H460) In order to study of the role of STIM1 in non-small lung cancer, two non-small lung cancer cell lines A549 and H460 were treated with defined concentrations of cisplatin for 24 h. As shown in Fig. 1A–D, the apoptosis was induced in those cells treated with cisplatin, and a dose-dependent increase in Annexin V-positive
Fig. 5. Cisplatin inhibited the expression of STIM1. A549 (A and B) and H460 (C and D) cells were treated for 24 h with indicated 15 M of cisplatin. And Western blot was performed using anti-STIM1 or anti-GAPDH antibodies (A and C). GAPDH (A and C) served as the loading control. The right part (B and D) indicates quantitative analysis of western blot result. Data are means ± SD of three independent experiments in triplicates (n = 3, N = 3). **P < 0.01 versus control.
Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003
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Fig. 6. Cisplatin induces decreases in [Ca2+ ]i in A549 and H460 cells. In 2 mM Ca2 + , 15 M cisplatin evoked a slight decrease of Ca2 + in A549 cells (A) and H460 cells (B). Data are typical representatives of three independent experiments in triplicates (n = 10, N = 3). A549 (A) and H460 (B) cells were transfected with scrambled or STIM-1 siRNA for 48 h. In 2 mM Ca2+ , STIM1 siRNA attenuated the decrease of [Ca2+ ]i when treated with 15 M cisplatin in A549 (A) and H460 cells (B). A549 (C) and H460 (D) cells were transfected with wild-type STIM1 for overexpression of STIM1 for 72 h. In 2 mM Ca2+ , Overexpression of STIM1 caused no significant change in the decrease of [Ca2+ ]i when treated with 15 M cisplatin in A549 (C) and H460 (D) cells. Data are typical representatives of three independent experiments in triplicates (n = 10, N = 3).
staining was observed. The morphological characteristics of apoptosis are illustrated in Fig. 1E and F. 3.2. Pharmacological Inhibition of SOCE or depletion of STIM1 enhance the apoptosis-induced by cisplatin To investigate whether SOCE is involved in the cisplatin-induced apoptosis, A549 and H460 cells were pretreated with the pharmacological inhibitors of SOCE 2-APB (75 M) or SKF-96365 (10 M). As shown in Fig. 2, inhibited SOCE significantly enhanced the cisplatin-induced apoptosis in A549 cells (Fig. 2A and B) and H460 cells (Fig. 2C and D). Knockdown of STIM1 by RNAi, which reduced the protein level of STIM1 by 67% in A549 cells and 74% in H460 cells. The knocking down of STIM1 significantly enhanced the pre-apoptotic effects of cisplatin (Fig. 3). In contrast to the downregulation of STIM1, overexpression of STIM1 reduced apoptosis of non-small lung cancer cells (Fig. 4).
3.3. Cisplatin inhibition of protein expression of STIM1 in A549 and H460 cells To explore whether the apoptosis induced by cisplatin occurs through modulation of the expression of STIM1 in non-small lung cancer cells, the expression of STIM1 in A549 and H460 cells was examined. The results show that the expression of STIM1 was downregulated in s after cisplatin treatment (Fig. 5).
3.4. Cisplatin induce apoptosis of A549 and H460 cells through SOCE To determine whether the downregulation of STIM1 influence [Ca2+ ]i in non-small lung cancer cells as a result of cisplatin treatment, the [Ca2+ ]i in both cells lines was examined. [Ca2+ ]i was slightly decreased following cisplatin treatment as shown in Fig. 6A and B. Depletion of STIM1 by RNAi caused a slight decrease in
Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003
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Fig. 7. The expression of STIM1 in NSCLC. (A) The protein levels of STIM1 in the tissues were detected by western blot. Quantitative analysis of the protein levels of STIM1 normalized to those of GAPDH is shown (B).
[Ca2+ ]i, while the overexpression of STIM1 by transfected wild-type STIM1 had no effect. 3.5. The expression of STIM1 in non-small cell lung cancer (NSCLC) In order to explore the expression of STIM1 in non-small cell lung cancer (NSCLC), 22 cases of non-small lung cancer with interconnected tissues consisting of carcinoma and adjacent non-neoplastic lung tissues were analyzed by immunoblotting. Compared with that of non-neoplastic tissue, the level of STIM1 expression in tumor tissues was elevated in 16 cases (Fig. 7). This means that STIM1 expression in non-small cell lung cancer is higher than that in normal lung tissue in 72.72% of non-small lung cancer patients. 4. Discussion The Ca2+ signal regulate fundamental eukaryotic processes including secretion, muscle contraction, gene transcription, cell proliferation and apoptosis. The highly coordinated regulation of Ca2+ hemostasis in cell is depend on channels, ATPase (pumps) and exchangers, which tightly balance Ca2+ flux across the endoplasmic
reticulum (ER) and plasma membrane (PM). Recent year have seen a growing evidence show that Ca2+ signaling pathways are remodeled or deregulated in cancer, which is implicated in tumorigenesis and tumor progression. In non-excitable cell, Store-operated Ca2+ entry plays a significant role in extracellular Ca2+ homestasis. So we determine the quantity of STIM1, which is key component of SOCE, in neoplastic tissue and paired nonneoplastic tissue in 22 patients, As the results indicated that the level of STIM1 expression in tumor tissues is higher than those in paired nonneoplastic tissue. Those results are concordance with previous reports that STIM1 overexpression in cervical cancer tissue. Cisplatin is the most important and efficacious chemotherapeutic agent in the treatment of non-small-cell lung cancer. In order to determined whether the apoptosis of tumor cells induced by cisplatin through modulate the expression of STIM1. As a key component of SOCE, STIM1 is a single pass membrane protein located in ER which as a sensor of endoplasmic reticulum (ER) Ca2+ concentration and essential activator of SOCs channels, allowing extracellular Ca2+ influx (Abdullaev et al., 2008; Liu et al., 2011). We quantities the expression of STIM1 in non-small lung cancer cells followed by treated with cisplatin. These result indicated cisplatin suppress the expression of STIM1. To further elucidate the molecular mechanism of the apoptosis induced by cisplatin through SOCE, we downregulate the expression of STIM1 by siRNA in those cell
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line, followed by treated with cisplatin, which increase the apoptosis. Meanwhile we determined the intracellular Ca2+ followed the knockdown STIM1. The results show that the intracellular Ca2+ decrease in the STIM1-silencing NSCLC cells Furthermore, we transfected wild-type STIM1-expressing plasmids into the cells. After treatment of the cells expressing increased level of STIM1 with cisplatin, a reduced level of apoptosis was observed, while over expression through transfected wild STIM1 have no effect on the intracellular Ca2+ concentration. These results further supported that STIM1 is a negative regulator of apoptosis induced by cisplatin. It has been reported that cisplatin bound to DNA forms the intra- and inter-strand DNA cross-links, which is inhibited transcription of certain genes, triggered apoptosis of tumor cell. Our results suggested that cisplatin induced the apoptosis in NSCLC through modulate the expression of STIM1. Knockdown the STIM1 in NSCLC lead to enhance the apoptosis induced by cisplatin. The SOCE inhibitors, such as 2-APB and SKF96365 have same effect on the NSCLC cells treatment with cisplatin. As a main pathway of extracellular Ca2+ influx, SOCE play a key role in almost all cellular pathways (Liou et al., 2005; Roos et al., 2005). SOCE is important for proliferation and apoptosis of certain tumors and the imbalance of cell growth and cell death finally lead to cancer. Chen et al. proposed that STIM1 is important for cervical cancer cell proliferation, migration, and angiogenesis. In other studies, blocking STIM1-mediating Ca2+ influx impairs focal adhesion turn-over (Berridge et al., 2003). SOCE inhibition has been shown to arrest tumor cell cycle at G0/G1 phase and inhibiting tumor cell metastasis in various tumor cells (Yang et al., 2009). It is noticeable that previous research suggested that that STIM1 is a MT-Plus-End-Tracking Protein (EB1), which is involve in accurate chromosome segregation during mitosis and in the polarization of the microtubule cytoskeleton in migrating cells (Holmuhamedov et al., 2002; Vanden Abeele et al., 2003). So it is alternative molecular pathway maybe involved in cisplatin triggered apoptosis of NSCLE, which through inhibits the expression of STIM1, in turn to affect accurate chromosome segregation during mitosis and in the polarization of the microtubule cytoskeleton. Acknowledgments We thank Dr. Worley (Johns Hopkins University School) and Dr. Joe Yuan (University of Texas Southwestern Medical Center) for gift of plasmids. This work was supported by Natural Science Foundation of China (30971193) and National Basic Research Program of China (2012CB518200).
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Please cite this article in press as: Li W, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol (2013), http://dx.doi.org/10.1016/j.etp.2013.04.003