Identifying Apoptosis-Evasion Proteins/Pathways in Human Hepatoma Cells via Induction of Cellular Hormesis by UV Irradiation Sen-Yung Hsieh,*,†,‡,§ Chih-Yun Hsu,†,‡ Jung-Ru He,‡ Chiung-Liang Liu,‡ Shao-Jung Lo,† Ying-Ching Chen,§ and Hui-Yu Huang‡ Liver Research Unit, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, Clinical Proteomics Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, and Graduate Institute of Basic Medical Science, School of Medicine, Chang Gung University, Taoyuan 333, Taiwan Received March 28, 2009
Evading apoptosis is pivotal in both of carcinogenesis and resistance to anticancer therapy. We investigated the molecules and pathways of apoptosis evasion in human hepatoma cells by irradiating hepatoma cells with optimized UV (so-called “hormetic responses”). Proteins and pathways related to hormetic responses were identified via proteomic approaches followed by reconstruction of functionnetworks. Of the 2326 defined protein spots, 42 distinct proteins significantly changed their expression. Eleven hormetic response proteins (HINT1, PHB, CTSD, ANXA1, LGASL1, TPT1, NPM, PRDX2, UCHL1, CERK, and C1QBP) were involved in 5 death-regulatory pathways, including the p53-dependent apoptotic pathway, protein ubiquinization, cellular redox, calcium-mediated signaling pathway, and sphingomyelin-metabolism pathway. Knockdown of HINT1 expression via RNA interference increased tumor cell resistance to apoptosis induction, while silencing NPM, UCHL1, or CERK greatly sensitized tumor cells to apoptosis induction. In conclusion, NPM, UCHL1, and CERK act as apoptosis-evasion proteins that may serve as therapeutic targets for hepatoma. Silencing their expression would increase therapeutic efficacy, thereby reducing the corresponding doses and side-effects of anticancer therapy. This model of induction of cellular hormetic responses to identify apoptosis-evasion molecules/pathways via proteomic approaches can be applied to other modalities of anticancer therapy. Keywords: proteomics • apoptosis evasion • hormesis • hepatoma • hepatocellular carcinoma • target therapy • molecular target
Introduction Apoptosis is a highly coordinated form of cell death with vital roles in embryonic development, cell homeostasis, and immune regulation in multicellular organisms.1 Evasion of apoptosis is the hallmark of cancer cells that is crucial in carcinogenesis and resistance to anticancer therapy.2 Indeed, activation of the apoptotic pathways is the key mechanism by which current anticancer therapies, including cytotoxic drugs, radiation, and immunotherapy, kill tumor cells. Defects or disruptions of apoptosis signaling contribute to tumor resistance to anticancer therapy.3 Therefore, understanding the regulation of apoptosis in tumor cells will provide molecular insight into the sensitivity or resistance of tumor cells and contribute to the development of better therapeutic strategies.4,5 Although apoptosis pathways have been thoroughly investigated, most studies have focused on elucidating death signaling, and few * Correspondence: Sen-Yung Hsieh, M.D., Ph.D., Liver Research Unit, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan. Phone: 886-33281200ext 8128. Fax: 886-3-3272236. E-mail:
[email protected];
[email protected]. † Liver Research Unit, Chang Gung Memorial Hospital. ‡ Clinical Proteomics Center, Chang Gung Memorial Hospital. § Chang Gung University. 10.1021/pr900289g CCC: $40.75
2009 American Chemical Society
have comprehensively addressed how cancer cells counteract apoptosis stimuli. Evolution has endowed biological organisms with the basic survival capacity to counteract or to adapt to environmental stress (homeodynamic or homeostatic ability). It is well documented that exposure to relatively low dose toxin or radiation induces a favorable biologic response for survival rather than a fatal outcome, a phenomenon known as “hormesis”.6,7 This study adopted the concept of “hormesis” using optimized doses of ultraviolet (UV) to irradiate human hepatoma cells to induce cellular hormetic mechanisms against apoptosis to identify molecular basis for apoptosis-evasion of cancer cells and molecular targets for future development of anticancer therapy.
Experimental Procedures Cells and UV-Irradiation. Human hepatoma cell lines, Mahlavu and Hep3B,8 were grown in 3-cm plates to about 70% confluence and treated with a defined dose (ranging from 10, 30, 65, 100, 140 to 180 mj/cm2) of UV-B (320-280 nm wavelength) as indicated in each experiment after removing medium and washing twice with PBS. Cells were then sequentially harvested for proteomic or functional assays at defined time points. Morphological features of membrane blebbing and cellular condensation or fragmentation, as well as the activation Journal of Proteome Research 2009, 8, 3977–3986 3977 Published on Web 06/22/2009
research articles of caspase 3/7 activity (Caspase 3/7 Assay, Promega) determined apoptosis. The percentages of apoptotic cells were assayed by trypan blue exclusion test. A cell suspension was mixed with equal volume of trypan blue dye and then visually examined to determine the percentages of cells taking up dye. Two-Dimensional Gel Electrophoresis. Cell pellets were resuspended in lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, and 1 mM sodium orthovanadate and sonicated on ice for 10 rounds of 10 s. Protein two-dimensional gel electrophoresis was performed as described previously.9 Briefly, 450 µg of each sample were applied to 17-cm IPG strips for isoelectric focusing by Protean IEF Cell (BioRad) according to the manufacturer’s instructions (BioRad). After treatment with 2.5% iodoacetamide, the strips were placed on a 12.5% polyacrylamide gel slab for separation in the second dimension at a current setting of 5 mA/gel for the initial 1 h and 10 mA/ gel thereafter. Protein Visualization and Image Analysis. Gels were fixed for 30 min in a buffer containing 10% methanol and 7.5% acetic acid, and then stained for three hours in SYPRO Ruby buffer (Molecular Probes, Eugene, OR). Protein patterns in the gels were recorded as digitalized images using a high-resolution scanner (Pro-Express, Perkin-Elmer, Boston, MA). Spot warping, matching, background subtraction, normalization, and filtering across gels were performed automatically using the Progenesis software package (Progenesis Discovery, Version 2005; Nonlinear Dynamics, Durham, NC) and verified manually. Protein spots with normalized volumes
