Activation of C-Jun N-Terminal Kinase Is Required for Mevastatin-Induced
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678 Preclinical report Activation of c-Jun N-terminal kinase is required for mevastatin-induced apoptosis of salivary adenoid cystic carcinoma cells Shan Zhanga, Xiao-Ling Wangb, Ye-Hua Gana and Sheng-Lin Lia Statins are inhibitors of 3-hydroxy-3-methylglutaryl mevastatin-induced JNK phosphorylation, but not coenzyme A reductase, originally developed for lowering p38 phosphorylation, and further decreased cholesterol. Statins also have pleiotropic effects, mevastatin-induced phosphorylation of ERK1/2. independent of cholesterol-lowering effects, including Taken together, the results suggest that the JNK induction of apoptosis in various cell lines. However, pathway was required for mevastatin-induced cell growth the mechanism underlying statin-induced apoptosis is inhibition and apoptosis in SACC cells. Statins could be still not fully understood. This study aims to explore the potential anticancer agents for SACC chemotherapy. proapoptotic effects and underlying mechanisms of statins Anti-Cancer Drugs 21:678–686 c 2010 Wolters Kluwer on human salivary adenoid cystic carcinoma (SACC). Health | Lippincott Williams & Wilkins. Exposure of SACC cells to mevastatin resulted in cell Anti-Cancer Drugs 2010, 21:678–686 growth inhibition and apoptosis in a dose-dependent manner, accompanied by the release of cytochrome Keywords: apoptosis, cell growth inhibition, mitogen-activated protein kinases, salivary adenoid cystic carcinoma, statin c and cleavage of caspase-3. A remarkable decrease in phosphorylation of extracellular signal-regulated kinase aCentral Laboratory and bDepartment of Preventive Dentistry, Peking University 1/2 (ERK1/2) and increase in phosphorylation of c-Jun School and Hospital of Stomatology, Zhongguancun Nandajie, Haidian District, N-terminal kinase (JNK) and p38 mitogen-activated kinase Beijing, PR China were observed. Furthermore, the JNK-specific inhibitor Correspondence to Dr Ye-Hua Gan or Dr Sheng-Lin Li, Central Laboratory, SP600125, but not the p38-specific inhibitor SB203580, Peking University School and Hospital of Stomatology, #22 Zhongguancun Nandajie, Haidian District, Beijing 100081, PR China abolished mevastatin-induced cell growth inhibition Tel: + 86 10 62179977 x2518; fax: + 86 10 62173402; and apoptosis in SACC cells. This was supported by e-mail: [email protected]; [email protected] results in which the JNK inhibitor efficiently blocked Received 19 November 2009 Revised form accepted 19 May 2010 Introduction kinases (MAPKs) play important roles in the response Statins are competitive inhibitors of 3-hydroxy-3-methyl- of tumor cells to statins [7,15]. The involved signaling glutaryl coenzyme A (HMG-CoA) reductase, a key rate- pathways in statin-induced apoptosis seem to be cell limiting enzyme in the biosynthesis of cholesterol [1]. type-specific or related to different biological features of In the last 20 years, statins have been used as first-line cell types. pharmacologic therapy for hypercholesterolemia. Inter- estingly, studies also show that statins have pleiotropic Salivary adenoid cystic carcinoma (SACC) is a malignancy effects independent of cholesterol-lowering effects, that arises in the major and minor salivary glands, and has presenting a potential benefit in different diseases such a tendency toward prolonged clinical course with local as atherosclerosis, osteoporosis, and even cancer [2,3]. recurrence and distant metastases on the lung [16]. SACC is usually treated with surgery and postoperative Statins have anticancer activity in several types of can- radiotherapy because the response rates of SACC to cer cells including malignant mesothelioma, thyroid cancer, chemotherapy or molecular therapies are generally poor breast cancer, C6 glioma, prostate cancer, pancreatic cancer, [17]. However, chemotherapy continues to play a role and HL60 cells [4–11]. Blockage of protein geranylger- in the treatment of metastasized SACC in the palliative anylation is believed to be the major cause of statin- setting. Therefore, it is still clinically required to test induced cell apoptosis [8,11,12]. However, the molecular potential anticancer agents for SACC to augment the mechanisms of proapoptotic effects of statins are still not current treatment or prevent SACC metastasis. Although fully understood. Several different signal pathways are statins have been tested in other tumor cell lines, their reported to be involved in the proapoptotic effects of proapoptotic effects and underlying molecular mechan- statins. The decrease in Bcl-2 expression, dephosphoryla- isms on SACC cells remain unexplored. tion of extracellular signal-regulated kinase 1/2 (ERK1/2), and increase of p21, p27, and proapoptotic Bax are In this study, we explored the proapoptotic effects and involved in statin-induced apoptosis [13,14]. Data show the underlying molecular mechanisms of mevastatin, an that signaling pathways of mitogen-activated protein inhibitor of HMG-CoA reductase, on SACC cells. 0959-4973 c 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/CAD.0b013e32833c4b3b Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Mevastatin induces apoptosis in SACC Zhang et al. 679 Materials and methods Rockford, Illinois, USA). Equal amounts of samples were Cell culture and reagents subjected to 12% sodium dodecyl sulfate-polyacrylamide Both SACC83 and ACC2 cell lines were established gel electrophoresis and transferred onto a polyvinylidene from human SACC in our laboratory [18] and in the fluoride membrane (Millipore, Billerica, Massachusetts, Shanghai Second Medical University Department of USA). The membrane was blocked with 5% fat-free milk in Oral and Maxillofacial Surgery, respectively [19]. The TBS-T (50 mmol/l Tris, pH 7.5, 150 mmol/l NaCl, 0.05% cells were cultured in RPMI-1640 (GIBCO, Grand Island, Tween 20) for 1 h. After incubation with primary antibodies New York, USA) supplemented with 10% fetal bovine diluted 1 : 1000 in TBS-T containing 1% milk overnight at serum and incubated at 371C in a humidified atmosphere 41C, the membrane was washed extensively with TBS-T of 5% CO2. For cell treatment, the reagents were added and then incubated with a secondary antibody conjugated singly or combinatorially into the culture media; the same with horseradish peroxidase for 1 h at room temperature. volume of solvent was added into the culture media as After extensive washes with TBS-T, the membrane was the corresponding controls. visualized with enhanced chemiluminescence reagents kit (Applygen Technologies, Inc., Beijing, China). For internal Mevastatin, SP600125, and SB203580 were purchased controls of equal loading, the blots were also stripped with from Sigma-Aldrich (St Louis, Missouri, USA). Mevasta- stripping buffer (100 mmol/l 2-mercaptoethanol, 2% SDS, tin was chemically activated by alkaline hydrolysis before 62.5 mmol/l, Tris pH 6.8) and reprobed with anti-ERK1/2, use as described earlier [20]. anti-JNK, anti-p38, or b-actin antibodies. Assessment of cell viability and apoptosis Viable cells were determined by trypan blue exclusion Immunocytochemistry assay. After treatment, the cells were harvested, resus- SACC83 cells cultured on glass coverslips were treated pended in D-Hanks’ solution, and mixed with an equal with or without 40 mmol/l mevastatin for 24 h. After amount of 0.4% trypan blue. The viable cells (from trypan fixation with 75% methanol and 25% acetic acid, the cells blue exclusion) were counted using a Fuchs-Rosenthal were permeabilized with 0.1% Triton X-100 and blocked hemocytometer (Shanghai Precision Instruments Co., with 10% goat serum at room temperature for 30 min, Ltd., Shanghai, China) under a microscope and calculated followed by incubation with anticytochrome c antibody in as mean ± SD of at least three independent experiments, PBS overnight at 41C. After washing three times with and presented as a percentage of controls. PBS, the cells were incubated with the secondary antibody conjugated with fluorescein isothiocyanate for 30 min at Apoptotic cells were determined by 40,6-diamidino-2- room temperature in the dark. After extensive washes phenylindole dihydrochloride (DAPI) staining. The cells with PBS, the cells were stained with 5 mg/ml DAPI for were fixed with 10% formaldehyde in phosphate-buffered 3 min at room temperature in the dark, and then imaged saline (PBS) for 5 min and incubated with 5 mg/ml DAPI under ultraviolet or excitation at 488 nm using confocal in the dark for 3 min at room temperature. After washing laser scanning microscopy (Zeiss LSM 510, Carl Zeiss, with PBS, the cells were examined under a fluorescence Jena, Germany). microscope (Nikon Eclipse TE2000-U, Tokyo, Japan). The cells presenting features of nuclear condensation and Statistical analysis fragmentation were identified as apoptotic cells and Statistical analysis was performed with SPSS 13.0 for counted within the randomly selected fields. The rate of Windows (SPSS, Inc., Chicago, Illinois, USA). All data apoptotic cells was presented as mean ± SD of at least were presented as mean ± SD. Differences were analyzed three independent experiments. by one-way analysis of variance, and a P value of less than 0.05 was regarded as statistically significant. Western blot The primary antibodies used in this assay were anti- cytochrome c, anti-ERK, anti-phospho-ERK (Thr202/Tyr204), Results anti-c-Jun N-terminal kinase (JNK), anti-phospho-JNK Induction of cell growth inhibition and apoptosis (Thr183/Tyr185), anti-p38, anti-phospho-p38