INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 32: 439-447, 2013 MAGI2 enhances the sensitivity of BEL-7404 human hepatocellular carcinoma cells to staurosporine- induced apoptosis by increasing PTEN stability XIN LI1,4, ZENGXIA LI1-3, NA LI1, JINGJING QI1, KUN FAN1, PENG YIN1, CHAO ZHAO1, YONGLEI LIU1, WANTONG YAO1, XIUMEI CAI1-3, LIYING WANG1-3 and XILIANG ZHA1-3 1Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Fudan University; 2Key Laboratory of Glycoconjugate Research, Ministry of Health; 3Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai 200032; 4Department of Laboratory Medicine, Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508 P.R. China Received February 24, 2013; Accepted April 29, 2013 DOI: 10.3892/ijmm.2013.1411 Abstract. Adaptor proteins are involved in the assembly of of AKT. These results suggest that MAGI2 overexpression various intracellular complexes and the regulation of cellular enhances the sensitivity of cancer cells harboring ectopic functions. Membrane-associated guanylate kinase inverted 2 PTEN to STS-induced apoptosis. (MAGI2), also known as synaptic scaffolding molecule (S-SCAM), plays a critical role in signal transduction by assem- Introduction bling and anchoring its ligands. However, the role of MAGI2 in mediating apoptosis remains largely unknown. In the present Thousands of adaptor proteins are known in metazoans which study, BEL-7404 human hepatocellular carcinoma cells were are involved in the assembly of various intracellular complexes transfected with a plasmid containing myc-MAGI2 or an and the regulation of cellular functions (1). Cytoplasmic scaffold empty plasmid and cell viability was then determined using proteins may be defined as proteins that link two or more part- the Cell Counting kit-8. Apoptosis was also detected using an ners to enhance the efficiency of cellular signaling pathways (2). Annexin V apoptosis assay. The cells were then treated with Membrane-associated guanylate kinase inverted 2 (MAGI2), various doses of staurosporine (STS) for different periods of also known as synaptic scaffolding molecule (S-SCAM), acts as time. The overexpression of myc-MAGI2 was found to sensi- a central organizer of multi-component signaling proteins (3). tize the BEL-7404 cells to apoptosis in response to STS in a MAGI2 is highly expressed in brain tissue, which is associ- time- and dose-dependent manner. Our results demonstrated ated with the formation and maintenance of the vertebrate that MAGI2 enhanced STS-induced apoptosis by increasing central nervous system glutamatergic synapse (4). MAGI2 the protein expression of cytoplasmic phosphatase and tensin also exists in hepatocellular carcinoma cells as a tumor homologue deleted on chromosome 10 (PTEN) and decreasing suppressor (5). MAGI2 has multiple binding partners, including its protein degradation. The apoptotic sensitivity of the cells the β1-adrenergic receptor (3), N-methyl-D-aspartate (NMDA) caused by the overexpression of myc-MAGI2 was reversed glutamate receptors (6), atrophin-1 (7), β- and δ-catenin (8,9), by the silencing of PTEN expression by PTEN siRNA, thus membrane-associated guanylate kinase-interacting protein-1 revealing a momentous role of PTEN in the enhancement of the (MAGUIN-1) (10) and phosphatase and tensin homologue sensitivity of cancer cells to STS-induced apoptosis by MAGI2. deleted on chromosome 10 (PTEN) (11). MAGI2 is a multi- Finally, we observed that the MAGI-PTEN complex triggered domain adaptor protein containing a guanylate kinase (GUK) by MAGI2 overexpression reduced the phosphorylation levels domain, two WW domains and six PSD95/Dlg/ZO1 (PDZ) domains. PDZ domain proteins play a key role in mediating key cellular functions in cells and act as adapters for interac- tion with the C-terminal motifs of their ligands (12,13). It has been reported that PTEN interacts with MAGI2 via the PDZ-2 Correspondence to: Dr Liying Wang or Professor Xiliang Zha, domains of MAGI2 and the PDZ-binding motif of PTEN (14,15). Department of Biochemistry and Molecular Biology, Shanghai PTEN is composed of an N-terminal phosphatase catalytic Medical College, Fudan University, 138 Yixueyuan Road, Shanghai 200032, P.R. China domain and a C-terminal phospholipid-binding C2 domain. The E-mail: [email protected] integrity of both domains is required for full PTEN phospha- E-mail: [email protected] tase activity (16). The expression of PTEN has been shown to suppress the growth of several human malignancies (17). Key words: membrane-associated guanylate kinase inverted 2, PTEN classically converts phosphatidylinositol-3,4,5- staurosporine, phosphatase and tensin homologue, AKT, apoptosis trisphosphate (PIP3) in the cytoplasm to phosphatidylinositol- 4,5-bisphosphate (PIP2), thereby directly counteracting the 440 LI et al: MAGI2 ENHANCES THE SENSITIVITY OF CANCER CELLS TO STS-INDUCED APOPTOSIS activity of phosphatidylinositol 3 kinase (PI3K) (16). The cells grew to 90-95% confluence, the two plasmids were trans- inactivation of PTEN results in the constitutive activation of fected into the cells using Lipofectamine 2000 according to the PI3K/AKT pathway and in cell cycle progression, migra- the manufacturer's instructions. After 7 h of transfection, the tion and survival (18,19). In our previous study, we showed that medium was changed with fresh medium containing 10% FBS PTEN plays a critical role in the MAGI2-induced inhibition and the cells were incubated at 37˚C for the appropriate amount of cell migration and proliferation in human hepatocellular of time. carcinoma cells (5). The PTEN-MAGI2 complex promotes a series of Cell viability assay. The cells were plated at a density of biological functions; however, the role of the complex in 2x105 cells/well in a 96-well plate and cultured for 12 h. mediating apoptosis is unknown. In present study, we found Following treatment with DMSO or 1 µM STS for different that cytoplasmic PTEN is involved in the enhancement of the periods of time, cell viability was determined using the CCK-8. sensitivity of BEL-7404 human hepatocellular carcinoma cells Subsequently, 5 µl of CCK-8 solution mixed with 100 µl culture to staurosporine (STS)-induced apoptosis by MAGI2. STS, medium were added to each well, and the cells were incubated a potent inhibitor of protein kinase C, is used in vitro as an for 0.5-1 h at 37˚C. The absorbance was then measured at initiator of apoptosis in a wide variety of cell types (20-22). 450 nm using an ELISA reader. The mechanism by which STS induces apoptosis has not yet been fully elucidated. In present study, we demonstrate that Annexin V apoptosis assay. The Annexin V-FITC Apoptosis MAGI2 enhances STS-induced apoptosis by increasing PTEN Detection kit was used to detect apoptosis. According to the stability and downregulating the phosphorylation level of AKT manufacturer's instructions, the cells were washed twice with in a hepatocellular carcinoma cell line. cold phosphate-buffered saline (PBS) and resuspended at a concentration of 1x105 cells/ml. After the addition of 195 µl Materials and methods of binding buffer, 5 µl Annexin V-FITC were added and the cells were incubated for 10 min at room temperature. After the Cell culture and cycloheximide (CHX) treatment. BEL-7404 addition of 190 µl of binding buffer, 10 µl propidium iodide human hepatocellular carcinoma cells were maintained (PI) were added and the cells were placed on ice in the dark. in Dulbecco's modified Eagle's medium (DMEM; Gibco, The samples were then analyzed by flow cytometry within 1 h. Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco) in a 37˚C incubator with 5% CO2. The cells RNA isolation and reverse transcription-polymerase chain were treated with CHX (100 µg/ml) for different periods of reaction (RT-PCR). Total RNA was isolated using TRIzol time and incubated for 10 h with different doses of CHX. reagent following the manufacturer's instructions. Oligo(dT)18 Cellular proteins were extracted, cell lysates were obtained, primer and M-MLV reverse transcriptase were used for the first total protein concentrations were determined using the Lowry strand synthesis. For semi-quantitative RT-PCR, the first primer method and western blot analysis was preformed. pair to PTEN was 5'-CAGAAAGACTTGAAGGCGTAT-3' and 5'-AACGGCTGAGGGAACTC-3'. The second primer pair Antibodies and reagents. Anti-PARP monoclonal antibody and to β-actin was 5'-TGATGATATCGCCGCGCTCGTCGT-3' LY294002 were purchased from Cell Signaling Technology, and 5'-CACAGCCTGGATAGCAACGTACAT-3'. β-actin was Inc. (Danvers, MA, USA). Anti-PTEN antibody was purchased used as the internal control. from BD Biosciences (Bedford, MA, USA). Anti-myc, anti- MAGI2 and the translation inhibitor, CHX, were obtained from siRNA transfection. The knockdown of PTEN was performed Sigma-Aldrich (St. Louis, MO, USA). STS was obtained from using siRNA (synthesized by GenePharma, Shanghai, China). Roche Applied Science (Indianapolis, IN, USA). Anti-β-actin, The target sequences were displayed as follows: PTEN sense, anti-α-tubulin monoclonal antibody, peroxidase-conjugated 5'-AGGCACAAGAGGCCCUAGAUU-3' and antisense, affinity goat anti-rabbit IgG and goat anti-mouse IgG secondary 5'-UCUAGGGCCUCUUGUGCCUUU-3'. Transfection was antibodies were purchased from Kangchen Biotech Co., Ltd. performed using Lipofectamine 2000 according to the manu- (Shanghai, China). The enhanced chemiluminescence (ECL) facturer's instructions. assay kit was commercially available from Tiangen Biotech Co., Ltd. (Shanghai, China). Alexa Fluor 488-conjugated goat Western blot analysis. The cells were lysed in 1X sodium anti-mouse IgG and Cy3-conjugated goat anti-rabbit IgG dodecyl sulfate (SDS) lysis buffer (50 mM Tris-HCl, pH 6.8, secondary antibodies were purchased from Beyotime (Jiangsu, 2% SDS, 10% glycerol, 100 mM Na3VO4 and 1 mM PMSF). China). DAPI (4',6-diamidino-2-phenylindole) was purchased Protein concentration was determined using the Lowry method. from Vector Laboratories. Lipofectamine™ 2000 was Equal amounts of protein were separated by SDS-PAGE and obtained from Invitrogen (Carlsbad, CA, USA). Opti-MEM electrophoretically transferred onto PVDF membranes.