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• W2155123561 abstract "Although the Chinese herb Gnetum cleistostachyum has been used as a remedy for cancers for hundred years, the active compounds and molecular mechanisms underlying its anti-cancer activity have not been explored. Recently a new derivative of stilbene compound, isorhapontigenin (ISO), was isolated from this Chinese herb. In the present study, we examined the potential of ISO in anti-cancer activity and the mechanisms involved in human cancer cell lines. We found that ISO exhibited significant inhibitory effects on human bladder cancer cell growth that was accompanied by marked apoptotic induction as well as down-regulation of the X-linked inhibitor of apoptosis protein (XIAP). Further studies have shown that ISO down-regulation of XIAP protein expression was only observed in endogenous XIAP, but not in constitutionally exogenously expressed XIAP in the same cells, excluding the possibility of ISO regulating XIAP expression at the level of protein degradation. We also identified that ISO down-regulated XIAP gene transcription via inhibition of Sp1 transactivation. There was no significant effect of ISO on apoptosis and colony formation of cells transfected with exogenous HA-tagged XIAP. Collectively, current studies, for the first time to the best of our knowledge, identify ISO as a major active compound for the anti-cancer activity of G. cleistostachyum by down-regulation of XIAP expression and induction of apoptosis through specific targeting of a SP1 pathway, and cast new light on the treatment of the cancer patients with XIAP overexpression. Although the Chinese herb Gnetum cleistostachyum has been used as a remedy for cancers for hundred years, the active compounds and molecular mechanisms underlying its anti-cancer activity have not been explored. Recently a new derivative of stilbene compound, isorhapontigenin (ISO), was isolated from this Chinese herb. In the present study, we examined the potential of ISO in anti-cancer activity and the mechanisms involved in human cancer cell lines. We found that ISO exhibited significant inhibitory effects on human bladder cancer cell growth that was accompanied by marked apoptotic induction as well as down-regulation of the X-linked inhibitor of apoptosis protein (XIAP). Further studies have shown that ISO down-regulation of XIAP protein expression was only observed in endogenous XIAP, but not in constitutionally exogenously expressed XIAP in the same cells, excluding the possibility of ISO regulating XIAP expression at the level of protein degradation. We also identified that ISO down-regulated XIAP gene transcription via inhibition of Sp1 transactivation. There was no significant effect of ISO on apoptosis and colony formation of cells transfected with exogenous HA-tagged XIAP. Collectively, current studies, for the first time to the best of our knowledge, identify ISO as a major active compound for the anti-cancer activity of G. cleistostachyum by down-regulation of XIAP expression and induction of apoptosis through specific targeting of a SP1 pathway, and cast new light on the treatment of the cancer patients with XIAP overexpression. Gnetum cleistostachyum, a Chinese herb that grows in the YunNan province of Southwestern of China, has been used for treatment of arthritis, bronchitis, cardiovascular system disease, and several cancers including bladder cancer for hundreds of years (1Huang K.S. Wang Y.H. Li R.L. Lin M. Stilbene dimers from the lianas of Gnetum hainanense.Phytochemistry. 2000; 54: 875-881Crossref PubMed Scopus (58) Google Scholar). ISO is a new derivative of stilbene, recently isolated from the Gnetum cleistostachyum (2Huang K.S. Zhou S. Lin M. Wang Y.H. An isorhapontigenin tetramer and a novel stilbene dimer from Gnetum hainanense.Planta Med. 2002; 68: 916-920Crossref PubMed Scopus (30) Google Scholar). Increasing attention has been given to elucidating anti-cancer activity of natural oligostilbenes in the last 20 years because more and more of their multifaceted biological properties are being identified. For example, through attenuating the generation of reactive oxygen species and activation of the extracellular signal-regulated kinases (ERKs) pathway, ISO exhibits the inhibitory effect on oxidized low-density lipoprotein-induced proliferation and mitogenesis of bovine aortic smooth muscle cells (3Liu Y. Liu G. Isorhapontigenin and resveratrol suppress oxLDL-induced proliferation and activation of ERK1/2 mitogen-activated protein kinases of bovine aortic smooth muscle cells.Biochem. Pharmacol. 2004; 67: 777-785Crossref PubMed Scopus (49) Google Scholar). ISO also inhibits cardiac hypertrophy by antioxidative activity and attenuates oxidative stress-mediated signaling pathways, such as protein kinase C (PKC)-dependent phosphatidylinositol 3-kinases (PI3K)-AKT-GSK3/p70S6K pathway (4Li H.L. Wang A.B. Huang Y. Liu D.P. Wei C. Williams G.M. Zhang C.N. Liu G. Liu Y.Q. Hao D.L. Hui R.T. Lin M. Liang C.C. Isorhapontigenin, a new resveratrol analog, attenuates cardiac hypertrophy via blocking signaling transduction pathways.Free Radic. Biol. Med. 2005; 38: 243-257Crossref PubMed Scopus (95) Google Scholar). However, the potential anti-cancer activity of ISO has never been explored. As a potent and ubiquitous caspase inhibitor (5Augello C. Caruso L. Maggioni M. Donadon M. Montorsi M. Santambrogio R. Torzilli G. Vaira V. Pellegrini C. Roncalli M. Coggi G. Bosari S. Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma.BMC Cancer. 2009; 9: 125-134Crossref PubMed Scopus (135) Google Scholar), X-linked inhibitor of apoptosis protein (XIAP) 2The abbreviations used are: XIAPX-linked inhibitor of apoptosis proteinISOisorhapontigeninDMSOdimethyl sulfoxideERKextracellular signal-regulated kinasesFRA-1Fos-related antigen-1GAPDHglyceraldehyde 3-phosphate dehydrogenaseHSF-1heat shock factor-1IAPinhibitor of apoptosis proteinNF-ATnuclear factor of activated T cellsNF-κBnuclear factor-κBPARPpoly(ADP-ribose) polymeraseRhoGDIRho GDP dissociation inhibitorSP1specific protein 1. has garnered the most attention as a promising therapeutic target for overcoming drug resistance (6Wang Z.H. Chen H. Guo H.C. Tong H.F. Liu J.X. Wei W.T. Tan W. Ni Z.L. Liu H.B. Lin S.Z. Enhanced antitumor efficacy by the combination of emodin and gemcitabine against human pancreatic cancer cells via down-regulation of the expression of XIAP in vitro in vivo.Int. J. Oncol. 2011; 39: 1123-1131PubMed Google Scholar). Our most recent studies also demonstrate that there is a novel XIAP function that acts as a crucial regulator for controlling cancer cell motility and invasion via its RING domain interaction with the RhoGDP dissociation inhibitor (RhoGDI), and subsequent negative modulation of RhoGDI SUMOylation at Lys-138 (7Liu J. Zhang D. Luo W. Yu Y. Yu J. Li J. Zhang X. Zhang B. Chen J. Wu X.R. Rosas-Acosta G. Huang C. X-linked inhibitor of apoptosis protein (XIAP) mediates cancer cell motility via Rho GDP dissociation inhibitor (RhoGDI)-dependent regulation of the cytoskeleton.J. Biol. Chem. 2011; 286: 15630-15640Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). It was accepted that XIAP overexpression in cancer tissues is associated with cancer progression, metastasis, and resistance to cancer therapy such as immunotherapy, chemotherapy, and radiotherapy (8Dai Y. Lawrence T.S. Xu L. Overcoming cancer therapy resistance by targeting inhibitors of apoptosis proteins and nuclear factor-κB.Am. J. Transl. Res. 2009; 1: 1-15PubMed Google Scholar). Thus, identifying a new anti-cancer drug targeting XIAP expression and function is one of the important priorities in the field of anti-cancer research. In the current study, the anti-cancer activity of ISO and the potential molecular mechanisms implicated in its anti-cancer activities were investigated in human cancer cells. X-linked inhibitor of apoptosis protein isorhapontigenin dimethyl sulfoxide extracellular signal-regulated kinases Fos-related antigen-1 glyceraldehyde 3-phosphate dehydrogenase heat shock factor-1 inhibitor of apoptosis protein nuclear factor of activated T cells nuclear factor-κB poly(ADP-ribose) polymerase Rho GDP dissociation inhibitor specific protein 1. cDNA contructs expressing HA-tagged XIAP and the pEBB empty vector were gifts from Dr. Colin S. Duckett (University of Michigan) (9Lewis J. Burstein E. Reffey S.B. Bratton S.B. Roberts A.B. Duckett C.S. Uncoupling of the signaling and caspase-inhibitory properties of X-linked inhibitor of apoptosis.J. Biol. Chem. 2004; 279: 9023-9029Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The transcription factor SP1 luciferase reporter, containing three consensus SP1 binding sites, was kindly provided by Dr. Peggy J. Farnham (McArdle Laboratory for Cancer Research, University of Wisconsin, Madison) (10Slansky J.E. Li Y. Kaelin W.G. Farnham P.J. A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.Mol. Cell. Biol. 1993; 13: 1610-1618Crossref PubMed Scopus (257) Google Scholar). Human XIAP promoter-driven luciferase reporter was gift from Dr. TaegKyu Kwon (Ajou University School of Medicine, Suwon, South Korea) (11Lee T.J. Jung E.M. Lee J.T. Kim S. Park J.W. Choi K.S. Kwon T.K. Mithramycin A sensitizes cancer cells to TRAIL-mediated apoptosis by down-regulation of XIAP gene promoter through Sp1 sites.Mol. Cancer Ther. 2006; 5: 2737-2746Crossref PubMed Scopus (63) Google Scholar). The antibodies against XIAP were purchased from Cell Signaling Technology (Boston, MA). The antibodies against c-FOS, FRA-1, JUN-D, P85, and SP1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies against AKT, BCL-2, BCL-xl, CASPASE-3, CIAP-1, c-JUN, GAPDH, NF-κB p65, P-AKT 473, P-AKT308, P-c-JUN (Ser-63), P-c-JUN (Ser-73), P-NF-κB p65, and poly(ADP-ribose) polymerase (PARP) were obtained from Cell Signaling Technology (Boston, MA). Antibodies against BAX and PKC-α were obtained from Upstate Biotechnology (Lake Placid, NY). Antibody against cIAP-2 was obtained from R & D Systems Inc. (Minneapolis, MN). The antibody against HA was obtained from Covance Antibody Service Inc. (Princeton, NJ). ISO with over 99% purity was provided by Dr. Qi Hou, Materia Medica of Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. The structure of ISO is shown in Fig. 1A. ISO was dissolved in dimethyl sulfoxide (DMSO) to make a stock concentration at 60 mm and further diluted in DMEM with a final DMSO concentration at 0.1% (v/v) for cell culture experiments. The same amount (0.1%, v/v) of DMSO was used as a negative control in all experiments. Human UMUC3 and RT112 bladder cancer cell lines were obtained from Dr. Xue-Ru Wu (Departments of Urology and Pathology, New York University School of Medicine) (12Huang H.Y. Shariat S.F. Sun T.T. Lepor H. Shapiro E. Hsieh J.T. Ashfaq R. Lotan Y. Wu X.R. Persistent uroplakin expression in advanced urothelial carcinomas. Implications in urothelial tumor progression and clinical outcome.Hum. Pathol. 2007; 38: 1703-1713Crossref PubMed Scopus (66) Google Scholar), and T24T was kindly provided by Dr. Dan Theodorescu (13Michael A. Harding D.T. RhoGDI signaling provides targets for cancer therapy.Eur. J. Cancer. 2010; 46: 1252-1259Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). These cell lines were maintained at 37 °C in a 5% CO2 incubator in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with 10% FBS, 2 μml-glutamine, and 25 μg/ml of gentamycin, respectively. The human colon cancer cell line HCT116 and XIAP−/− HCT116 cells were kind gifts from Dr. Bert Vogelstein (Howard Hughes Medical Institute and Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions) (14Cummins J.M. Kohli M. Rago C. Kinzler K.W. Vogelstein B. Bunz F. X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells.Cancer Res. 2004; 64: 3006-3008Crossref PubMed Scopus (184) Google Scholar), and HCT116 XIAP−/−(HA-XIAP) transfection was stably established by transfection with HA-XIAP as described in our previous studies (7Liu J. Zhang D. Luo W. Yu Y. Yu J. Li J. Zhang X. Zhang B. Chen J. Wu X.R. Rosas-Acosta G. Huang C. X-linked inhibitor of apoptosis protein (XIAP) mediates cancer cell motility via Rho GDP dissociation inhibitor (RhoGDI)-dependent regulation of the cytoskeleton.J. Biol. Chem. 2011; 286: 15630-15640Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Stable co-transfections were performed with specific cDNA constructs and/or pSUPER vector using PolyJetTM DNA In Vitro Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD) according to the manufacturer's instructions. For stable transfection selection, cultures were subjected to hygromycin selection for 4–6 weeks, and surviving cells were pooled as stable mass transfectants. Anchorage-independent growth (soft agar assay) in soft agar was carried out as described in our previous studies (15Zhang D. Li J. Costa M. Gao J. Huang C. JNK1 mediates degradation HIF-1 by a VHL-independent mechanism that involves the chaperones Hsp90/Hsp70.Cancer Res. 2010; 70: 813-823Crossref PubMed Scopus (81) Google Scholar). Briefly, 1 × 104 cells mixed with various concentrations of ISO in 10% FBS, β-mercaptoethanol containing 0.33% soft agar, was seeded over the bottom layer of 0.5% agar in 10% FBS, β-mercaptoethanol in each well of 6-well plates. The plates were incubated in 5% CO2 incubator at 37 °C for 3 weeks. Colonies were inspected under a microscope and only colonies with over 32 cells were counted. Total RNA was extracted with TRIzol reagent (Invitrogen Corp.) and cDNAs were synthesized with the Thermo-Script RT-PCR system (Invitrogen Corp.). Two oligonucleotides (5′-GATGATCTTGAGGCTGTTGTC-3′ and 5′-CAGGGCTGCTTTTAACTCTG-3′) were used as the specific primers to amplify human GADPH cDNA for loading controls. The human XIAP cDNA fragments were amplified by primers 5′-TTTCCAGATTGGGGCTCGGG-3′ and 5′-CCCTGCTCGTGCCAGTGTTGAT-3′. The HA-XIAP fragments were amplified by primers 5′-ACGTGCCTGACTATGCCAGCCT-3′ and 5′-ACCCTGCTCGTGCCAGTGTTG-3′. The human CIAP-1 fragments were amplified by primers 5′-ATCAGAATTGGCAAGAGCTGG-3′ and 5′-TAGCATCATCCTTTGGTTCCC-3′. The human CIAP-2 fragments were amplified by primers 5′-CTTTTGCTGTGATGGTGGACTC-3′ and 5′-CTGGCTTGAACTTGACGGATG-3′. The human SP1 fragments were amplified by primers 5′-CCAGGCCCTCCAAGCAGCAC-3′ and 5′-GCTGCTGGTCTGCCCCAAGG-3′. The PCR products were separated on 2% agarose gels and stained with ethidium bromide, and the images were visualized and scanned with UV lights with FluorChem SP imaging system (Alpha Innotech Inc.) as described previously (16Zhang D. Li J. Gao J. Huang C. c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells.Toxicol. Appl. Pharmacol. 2009; 235: 18-24Crossref PubMed Scopus (51) Google Scholar). The XIAP promoter-driven luciferase reporter stable transfection or SP1 luciferase reporter stable transfectant cells (1 × 104) were seeded into each well of 96-well plates and cultured in 5% CO2 incubator at 37 °C until 70–80% confluence. The cells were treated with ISO as indicated, then extracted with lysis buffer (25 mmol/liter of Tris phosphate (pH 7.8), 2 mm EDTA, 1% Triton X-100, and 10% glycerol). The luciferase activity was determined by a microplate luminometer LB 96V (Berthold GmbH & Co. KG, Bad Wildbad, Germany) using the luciferase assay system (Promega Corp., Madison, WI) as described (17Liu J. Zhang D. Mi X. Xia Q. Yu Y. Zuo Z. Guo W. Zhao X. Cao J. Yang Q. Zhu A. Yang W. Shi X. Li J. Huang C. p27 Suppresses Arsenite-induced Hsp27/Hsp70 Expression through Inhibiting JNK2/c-Jun- and HSF-1-dependent Pathways.J. Biol. Chem. 2010; 285: 26058-26065Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Preparation of nuclear extracts was assessed as previously described in Ref. 18Cai T. Li X. Ding J. Luo W. Li J. Huang C. A cross-talk between NFAT and NF-κB pathways is crucial for nickel-induced COX-2 expression in Beas-2B cells.Curr. Cancer Drug Targets. 2011; 11: 548-559Crossref PubMed Scopus (40) Google Scholar. For cell culture, cancer cells were plated into 10-cm culture dishes at 80% confluence, treated either with DMSO or 60 μm ISO for 12 h. The nuclear proteins were extracted according to the protocol of the Nuclear/Cytosol Fractionation Kit (BioVison Technologies, Mountain View, CA). Equal protein concentrations were determined using a protein quantification assay kit (Bio-Rad). Nuclear extracts were stored at −80 °C until they were used. Western blot assay was assessed as previously described in Ref. 19Ding J. Li J. Xue C. Wu K. Ouyang W. Zhang D. Yan Y. Huang C. Cyclooxygenase-2 induction by arsenite is through a nuclear factor of activated T-cell-dependent pathway and plays an antiapoptotic role in Beas-2B cells.J. Biol. Chem. 2006; 281: 24405-24413Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar. Cells were plated in 6-well plates and cultured in normal 10% serum medium until 70–80% confluence. The culture medium was replaced and starved with 0.1% FBS media. After being cultured for 24 h, the cells were exposed to the indicated amount of ISO or indicated time with 60 μm ISO. The cells were washed once with ice-cold phosphate-buffered saline and collected with cell lysis buffer (10 mm Tris-HCl (pH 7.4), 1% SDS, and 1 mm Na3VO4). 80 μg of protein sample from the cell extracts were separated on SDS-PAGE, transferred, and probed with the indicated antibody. The protein band that was specifically bound to the primary antibody was detected using an alkaline phosphatase-linked secondary antibody and an ECF Western blotting system (Amersham Biosciences). Flow cytometry assay was assessed as previously described in Ref. 20Luo W. Liu J. Li J. Zhang D. Liu M. Addo J.K. Patil S. Zhang L. Yu J. Buolamwini J.K. Chen J. Huang C. Anti-cancer effects of JKA97 are associated with its induction of cell apoptosis via a Bax-dependent and p53-independent pathway.J. Biol. Chem. 2008; 283: 8624-8633Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar. After the time periods indicated, the ISO-treated and control cells were harvested and fixed in 75% ethanol. The fixed cells were stained in the buffer containing 0.1% Triton X-100, 0.2 mg/ml of RNase A, and 50 μg/ml of propidium iodide at 4 °C for 1 h and then examined by flow cytometry utilizing an EpicsXL flow cytometer (Beckman Coulter Inc., Miami, FL) on the FL3 channel, and the gate was set to exclude debris and cellular aggregates. 20,000 events were counted for each analysis, and three independent experiments for each group were conducted. The subdiploid DNA peak, immediately adjacent to the G0/G1 peak, represented apoptotic cells and was quantified by histogram analyses. ChIP was performed using the EZ-CHIP kit (Millipore Technologies) according to the manufacturer's instructions as described in our previous publication (21Song L. Gao M. Dong W. Hu M. Li J. Shi X. Hao Y. Li Y. Huang C. p85α mediates p53 K370 acetylation by p300 and regulates its promoter-specific transactivity in the cellular UVB response.Oncogene. 2011; 30: 1360-1371Crossref PubMed Scopus (24) Google Scholar). Briefly, T24T cells were treated with 0.1% DMSO only or 60 μm ISO for 12 h. Then genomic DNA and the proteins were cross-linked with 1% formaldehyde. The cross-linked cells were pelleted, resuspended in lysis buffer, and sonicated to generate 200–500-bp chromatin DNA fragments. After centrifugation, the supernatants were diluted 10-fold and then incubated with anti-SP1 antibody or the control rabbit IgG at overnight at 4 °C. The immune complex was captured by protein G-agarose saturated with salmon sperm DNA, then eluted with the elution buffer. DNA-protein cross-linking was reversed by heating overnight at 65 °C. DNA was purified and subjected to PCR analysis. To specifically amplify the region containing the putative responsive elements on the human XIAP promoter, PCR was performed with the following pair of primers: 5′-TTTTACTTTATGACTTGAATGATG TGG-3′ (from −214 to −187) and 5′–TTCCTTATTGATGTCTGCAGGT-3′ (from +39 to +60). The PCR products were separated on 2% agarose gels and stained with ethidium bromide, the images were then scanned with a UV light. Student's t test was utilized to determine the significance of differences between different groups. The differences were considered to be significant at p < 0.05. The chemical structure of ISO is 4-methoxyresveratrol (Fig. 1A), with a molecular weight of 258. We first examined the effects of cell proliferation and colony formation by ISO on bladder cancer cells. The T24T bladder cancer cell line was selected for further experiments due to its genetic background and strong tendency of migration. As shown in Fig. 1, B and C, ISO significantly inhibited anchorage-independent growth (colony formation) in a dose-dependent manner (p < 0.01). These data demonstrate the anti-cancer effects of ISO on human bladder cancer cells. To assess the effect of ISO on cell viability, T24T was cultured with a range of ISO doses (20–60 μm) for 48 h. Cell viability of these cells was analyzed using the ATPase assay. A significant reduction of cell viability was observed in a dose-dependent manner (Fig. 2A). The IC50 of T24T cell lines was 55.2 ± 2.3 μm (n = 3). To determine whether ISO could inhibit cell viability due to apoptosis, the DNA cycle of T24T cells were examined. The T24T bladder cells were treated with ISO in concentrations of 0, 20, 40, and 60 μm for 48 h. High-resolution flow cytometric analysis of propidium iodide-stained nuclei revealed a substantial increase in sub-G1 DNA content (apoptotic peak) at 48 h compared with cells treated with 0.1% DMSO (v/v) as a negative control (Fig. 2B). PARP cleavage is often associated with apoptosis and has served as one of the hallmarks of apoptosis and caspase activation. Therefore, we determined whether induction of T24T cell apoptosis by ISO was mediated through activation of CASPASE-3 and cleavage of PARP proteins. Treatment of T24T with ISO for 24 h resulted in a dose-dependent increased cleavage of CASPASE-3 and PARP proteins compared with the DMSO-treated cells (Fig. 2C). Because the IC50 of the T24T cell line was 55.2 ± 2.3 μm, 60 μm ISO was used in the following experiments. The ISO-induced activation of CASPASE-3 and PARP were also determined and confirmed in a time-dependent manner from 3 to 36 h, with pre-treatment with ISO (Fig. 2D). Additionally, we determined the obvious cleavage of PARP in a dose-dependent manner with pre-treatment with ISO for 24 h in the other 3 cancer cell lines, RT112 bladder cancer (Fig. 2E), UMUC3 bladder cancer cell (Fig. 2F), and HCT116 colon cancer cell (Fig. 2G). These results indicated reproducible apoptotic effects of ISO on different human cancer cells lines. Having established that ISO could induce the apoptosis of T24T and other cancer cell lines in a dose- and time-dependent manner, we next focused on the molecular mechanisms of apoptosis induced by ISO. It has been reported that ISO exhibits anti-inflammation, antioxidant effects through suppressing the ERKs, p38, PKCα, NF-κB, and the PI3K/AKT pathway (4Li H.L. Wang A.B. Huang Y. Liu D.P. Wei C. Williams G.M. Zhang C.N. Liu G. Liu Y.Q. Hao D.L. Hui R.T. Lin M. Liang C.C. Isorhapontigenin, a new resveratrol analog, attenuates cardiac hypertrophy via blocking signaling transduction pathways.Free Radic. Biol. Med. 2005; 38: 243-257Crossref PubMed Scopus (95) Google Scholar). The activation and protein expression levels of those proteins upon ISO treatment were evaluated in T24T cells. As shown as Fig. 3A, there were no significant changes observed, indicating that these pathways may not be implicated in the anti-cancer activity of this ISO compound. IAPs family and BCL-2 family members are known to be regulators of CYTOCHROME c releasing from mitochondria during apoptosis. In addition to the BCL-2 family, IAP family proteins also regulate CASPASE activity, thus affecting apoptosis. Therefore, the expression of three IAP family proteins, XIAP, cIAP-1, and cIAP-2, were determined. ISO treatment down-regulated the XIAP protein level in dose- and time-dependent manners. However, it did not significantly affect cIAP-1 and cIAP-2 proteins, nor did it have a significant effect on BCL-2 family proteins, such as Bax, BCL-2, and BCL-Xl (Fig. 3, B and C). To elucidate the mechanism leading to XIAP down-regulation upon ISO treatment, the N terminally HA-tagged XIAP were transfected into RT112 and HCT116 XIAP−/− cells, respectively. The pEBB empty vector was transfected at the same time as the control. As shown as Fig. 4A, the exogenous HA-tagged XIAP inhibited the endogenous XIAP expression in RT112 (HA-XIAP) cells. Meanwhile, the results also showed that treatment with ISO did not affect the reconstituted expression of HA-tagged XIAP in RT112 (HA-XIAP) (Fig. 4A) and HCT116 XIAP−/− (HA-XIAP) cells (Fig. 4B), whereas it attenuated endogenous XIAP expression in the RT112 (vector) and HCT116 (vector) cells. These results strongly excluded the possibility that ISO regulated XIAP protein expression at the degradation level. To clarify the underlying mechanisms of ISO down-regulation of XIAP protein expression, we examined mRNA levels of XIAP in comparison to CIAP-1 and CIAP-2. Consistent with the results obtained at protein levels, ISO treatment led to marked reductions of XIAP mRNA in dose- and time-dependent manners, whereas it did not affect the CIAP-1 and CIAP-2 mRNA levels (Fig. 4, C and D). The results indicated that ISO treatment attenuated XIAP expression at the mRNA level. We further carried out experiments to confirm that XIAP was not down-regulated at mRNA stability by ISO using actinomycin D. The result showed that ISO did not affect the XIAP mRNA level in actinomycin D-treated T24T cells, whereas it reduced XIAP mRNA expression in T24T cells in the absence of actinomycin D treatment (Fig. 4E), suggesting that ISO did not regulate XIAP mRNA stability. We further compared the endogenous XIAP mRNA and exogenous HA-XIAP mRNA levels in both RT112 (HA-XIAP) and HCT116 XIAP−/−(HA-XIAP) cells. The results indicated that the exogenous HA-XIAP mRNA showed no observable changes after ISO treatment, whereas the endogenous XIAP mRNA was remarkably down-regulated in RT112 (Vector) and HCT116 (Vector) cells upon ISO treatment (Fig. 4, F and G). These results indicate that ISO regulated XIAP protein expression at the transcriptional level, rather than mRNA stability. This notion was fully supported by the results obtained from T24T stably transfected with the XIAP promoter-driven luciferase reporter. Treatment of T24T XIAP promoter-luciferase cells with ISO resulted in inhibition of XIAP promoter transcription activity in a time-dependent manner (Fig. 4H). To identify the transcription factor responsible for ISO down-regulation of XIAP transcription, TFANSFAC® Transcription Factor Binding Sites Software (Biological Database, Wolfenbüttel, Germany) was used for bioinformatics analysis of the XIAP promoter region. The results revealed that the promoter region of the human XIAP gene contains the putative DNA-binding site of cellular oncogene Fos (c-FOS), c-JUN, FOS-related antigen-1 (FRA-1), heat shock factor-1 (HSF-1), Jun-D, nuclear factor of activated T cells (NF-AT), nuclear factor κB (NF-κB), and specific proteins 1 (SP1) (Fig. 5A). We next examined changes in the nuclear translocation of related transcription factors upon ISO treatment for 12 h. As shown in Fig. 5B, inhibition of SP1 protein expression clearly occurred in the nuclear protein extract pretreated with 60 μm ISO for 12 h. In contrast, there was no significant suppression of the transcription factors of c-FOS, c-JUN, FRA-1, HSF-1, JUN-D, NF-AT, and NF-κB. To further confirm the mechanism of down-regulation of SP1 by ISO, we tested the effects of ISO on the protein and mRNA levels of SP1 in T24T cells. The results indicated that the SP1 mRNA level was not reduced even though its protein level was profoundly down-regulated upon ISO treatment (Fig. 5, C and D), suggesting that ISO regulates SP1 protein expression at levels of either protein translation and/or degradation. To confirm that down-regulation of XIAP by ISO was mediated by SP1, we tested the effects of ISO on SP1 transactivation in T24T cells stably transfected with SP1-luciferase reporter containing three SP1 consensus binding sites. The results, as expected, showed that ISO treatment significantly blocked SP1 transactivation in a time-dependent manner in T24T cells (Fig. 5E). To study whether down-regulation of the SP1 level by ISO is associated with SP1 sites in the XIAP promoter in vitro, we performed a CHIP assay with T24T cells followed by PCR with primers specifically targeting the SP1 binding region from −214 to +60 in the XIAP promoter. As shown in Fig. 5F, compared with the DMSO control, ISO treatment could suppress the binding of SP1 to the XIAP promoter region between −214 and +60 (Fig. 5F). Considering that there are two SP1 binding sites between −214 and +60 (−144 and −25) and that the previous report (11Lee T.J. Jung E.M. Lee J.T. Kim S. Park J.W. Choi K.S. Kwon T.K. Mithramycin A sensitizes cancer cells to TRAIL-mediated apoptosis by down-regulation of XIAP gene promoter through Sp1 sites.Mol. Cancer Ther. 2006; 5: 2737-2746Crossref PubMed Scopus (63) Google Scholar) showed that XIAP promoter activity is significantly decreased by double mutation of two SP1 sites at −144 and −25, we suggest that ISO down-regulation of XIAP transcription is med" @default.
• W2155123561 created "2016-06-24" @default.
• W2155123561 creator A5001171800 @default.
• W2155123561 creator A5005318671 @default.
• W2155123561 creator A5020089499 @default.
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• W2155123561 creator A5049560731 @default.
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• W2155123561 date "2012-10-01" @default.
• W2155123561 modified "2023-10-17" @default.
• W2155123561 title "The Chinese Herb Isolate Isorhapontigenin Induces Apoptosis in Human Cancer Cells by Down-regulating Overexpression of Antiapoptotic Protein XIAP" @default.
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