Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway
2009; Elsevier BV; Volume: 284; Issue: 17 Linguagem: Inglês
10.1074/jbc.m806268200
ISSN1083-351X
AutoresKuen‐Feng Chen, Pei-Yen Yeh, Chiun Hsu, Chih‐Hung Hsu, Yen‐Shen Lu, Hsing‐Pang Hsieh, Pei‐Jer Chen, Ann‐Lii Cheng,
Tópico(s)interferon and immune responses
ResumoHepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway. Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway. Hepatocellular carcinoma (HCC) 2The abbreviations used are: HCC, hepatocellular carcinoma; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; FADD, Fas-associated protein with death domain; c-FLIP, cellular FLICE-inhibitory protein; NF-κB, nuclear factor-κB; PI3K, phosphatidylinositol 3-kinase; PARP, poly(ADP-ribose) polymerase; DMEM, Dulbecco's modified Eagle's medium; DISC, death-inducing signaling complex; P-Akt, phospho-Akt; siRNA, small interfering RNA; PP2A, protein phosphatase 2A. 2The abbreviations used are: HCC, hepatocellular carcinoma; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; FADD, Fas-associated protein with death domain; c-FLIP, cellular FLICE-inhibitory protein; NF-κB, nuclear factor-κB; PI3K, phosphatidylinositol 3-kinase; PARP, poly(ADP-ribose) polymerase; DMEM, Dulbecco's modified Eagle's medium; DISC, death-inducing signaling complex; P-Akt, phospho-Akt; siRNA, small interfering RNA; PP2A, protein phosphatase 2A. is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1.Schwartz M. Roayaie S. Konstadoulakis M. Nat. Clin. Pract. Oncol. 2007; 4: 424-432Crossref PubMed Scopus (249) Google Scholar). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1.Schwartz M. Roayaie S. Konstadoulakis M. Nat. Clin. Pract. Oncol. 2007; 4: 424-432Crossref PubMed Scopus (249) Google Scholar). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2.Merino D. Lalaoui N. Morizot A. Solary E. Micheau O. Expert Opin. Ther. Targets. 2007; 11: 1299-1314Crossref PubMed Scopus (126) Google Scholar). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3.Takeda K. Stagg J. Yagita H. Okumura K. Smyth M.J. Oncogene. 2007; 26: 3745-3757Crossref PubMed Scopus (169) Google Scholar). Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4.Sayers T.J. Brooks A.D. Koh C.Y. Ma W. Seki N. Raziuddin A. Blazar B.R. Zhang X. Elliott P.J. Murphy W.J. Blood. 2003; 102: 303-310Crossref PubMed Scopus (209) Google Scholar, 5.Kim Y. Suh N. Sporn M. Reed J.C. J. Biol. Chem. 2002; 277: 22320-22329Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6.Burns T.F. El-Deiry W.S. J. Biol. Chem. 2001; 276: 37879-37886Abstract Full Text Full Text PDF PubMed Google Scholar, 7.LeBlanc H.N. Ashkenazi A. Cell Death Differ. 2003; 10: 66-75Crossref PubMed Scopus (753) Google Scholar), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8.Deng Y. Lin Y. Wu X. 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Cancer Res. 2008; 68: 6698-6707Crossref PubMed Scopus (111) Google Scholar). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway. Reagents and Antibodies—Bortezomib (Velcade®) was provided by Millennium Pharmaceuticals. For in vitro studies, Bortezomib at various concentrations was dissolved in DMSO and then added to the cells in 5% fetal bovine serum-containing Dulbecco's modified Eagle's medium (DMEM). The final DMSO concentration was kept at 0.1% after the addition to medium. Recombinant TRAIL and Paclitaxel were purchased from Biomol (Plymouth Meeting, PA). LY294002 and okadaic acid were purchased from Cayman Chemical (Ann Arbor, MI). Antibodies for immunoblotting, such as Akt1, Bad, Bak, Bax, Mcl-1, poly(ADP-ribose) polymerase (PARP), and PP2A-C, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other antibodies, such as Bcl-2, Bcl-xL, Bid, Bik, Bim, caspase-3, caspase-8, caspase-9, FADD, c-FLIP, and P-Akt (Ser473), were from Cell Signaling (Danvers, MA). DR4 and DR5 were from Biolegend (flow cytometry) and Diaclone (Western blot). Cell Culture—The Huh-7 HCC cell line was obtained from the Health Science Research Resources Bank (Osaka, Japan; JCRB0403). The Sk-Hep-1, and Hep3B cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate and 25 μg/ml amphotericin B in a 37 °C humidified incubator under an atmosphere of 5% CO2 in air. Western Blot Analysis—Lysates of Huh-7, Sk-hep1, and Hep3B cells treated with bortezomib and/or TRAIL at the indicated concentrations for 24 h were prepared for immunoblotting of caspase-9, caspase-3, PARP, P-Akt, Akt, Bcl-2 family, DR4, DR5, FADD, c-FLIP, etc. Western blot analysis was performed as previously reported (32.Kulp S.K. Yang Y.T. Hung C.C. Chen K.F. Lai J.P. Tseng P.H. Fowble J.W. Ward P.J. Chen C.S. Cancer Res. 2004; 64: 1444-1451Crossref PubMed Scopus (222) Google Scholar). Apoptosis Analysis—The following three methods were used to assess drug-induced apoptotic cell death: the measurement of apoptotic cells by flow cytometry (sub-G1) and Western blot analysis of caspase activations and PARP cleavage. HCC cells were treated with bortezomib and/or TRAIL at the indicated concentrations and time. The procedure was performed as described previously (32.Kulp S.K. Yang Y.T. Hung C.C. Chen K.F. Lai J.P. Tseng P.H. Fowble J.W. Ward P.J. Chen C.S. Cancer Res. 2004; 64: 1444-1451Crossref PubMed Scopus (222) Google Scholar). NF-κB Binding Activity—A Trans-AM NF-κB p65 transcription factor assay kit (Active Motif North America, Carlsbad, CA) was used to determine the activity of NF-κB binding in bortezomib-treated HCC cells. The entire procedure was done in accordance with the manufacturer's manual. Briefly, HCC cells were exposed to bortezomib at the indicated doses for 4, 8, and 24 h, and cell lysates were prepared. NF-κB binding to related DNA fragments was determined by incubation with primary antibody, anti-p65, and quantified at 450 nm after incubation in anti-IgG-horseradish peroxidase conjugate as described previously (31.Chen K. Yeh P.Y. Yeh K.H. Lu Y.S. Huang S.Y. Cheng A.L. Cancer Res. 2008; 68: 6698-6707Crossref PubMed Scopus (111) Google Scholar). Detection of Surface TRAIL Receptors—The procedure for antibody staining and analysis by flow cytometry has been described previously. Briefly, HCC cells were exposed to bortezomib at 100 nm for 24 h and then incubated with DR4 or DR5 antibodies for 30 min. Antibodies and kit were purchased from Biolegend (San Diego, CA), and the procedure was done according to the manufacturer's instructions (23.Liu X. Yue P. Chen S. Hu L. Lonial S. Khuri F.R. Sun S.Y. Cancer Res. 2007; 67: 4981-4988Crossref PubMed Scopus (145) Google Scholar). Gene Knockdown Using siRNA—Smart pool siRNAs, including control (D-001810-10), DR4 (L-008090-00), DR5 (L-004448-00), Akt1 (M-003000-02), and PP2A-C (L-003598-01), were all purchased from Dharmacon Inc. (Chicago, IL). The procedure has been described previously. Briefly, HCC cells were transfected with siRNA (the final concentration was 100 nm) in 6-well plates using the Dharma-FECT4 transfection reagent (Dharmacon) according to the instructions in the manufacturer's manual. After 48 h, the medium was replaced, and HCC cells were incubated for an additional 24 h with bortezomib and/or TRAIL for Western blot analysis and apoptosis analysis by flow cytometry, as described previously (31.Chen K. Yeh P.Y. Yeh K.H. Lu Y.S. Huang S.Y. Cheng A.L. Cancer Res. 2008; 68: 6698-6707Crossref PubMed Scopus (111) Google Scholar). Huh-7 with Constitutive Active Akt1—Constitutive active (myristoylated) Akt1 construct was a gift from Dr. Tushar Patel (Ohio State University, Columbus, OH). Briefly, following transfections, cells were incubated in the presence of G418 at 0.78 mg/ml. After 8 weeks of selection, surviving colonies (i.e. those arising from stably transfected cells) were selected and individually amplified. Huh-7 cells with stable expression of constitutive Akt (Huh7-akt) were then treated with various doses of bortezomib for Western blot analysis as described previously (32.Kulp S.K. Yang Y.T. Hung C.C. Chen K.F. Lai J.P. Tseng P.H. Fowble J.W. Ward P.J. Chen C.S. Cancer Res. 2004; 64: 1444-1451Crossref PubMed Scopus (222) Google Scholar). PP2A Phosphatase Activity—The protein phosphatase activity of total cellular lysate was determined by measuring the generation of free phosphate from the threonine phosphopeptide using the malachite green-phosphate complex assay, as described by the manufacturer (Upstate Biotechnology, Inc.). Cell lysates were prepared in a low detergent lysis buffer (1% Nonidet P-40, 10 mm HEPES, 150 mm NaCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 5 mm benzamidine, and 10 μg/ml leupeptin). The phosphatase assay was performed in a PP2A-specific reaction buffer (Upstate Biotechnology) using 750 μm phosphopeptide substrate. After 10 min of incubation at 30 °C, malachite dye was added, and free phosphate was measured by optical density at 650 nm. To avoid variability among different immunoprecipitated samples, the phosphatase activities were normalized with the amount of PP2A immunoprecipitated, as detected and quantified by immunoblot analysis of each treatment group. Co-immunoprecipitation Assay—Cells were harvested and lysed on ice for 30 min in lysis buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 0.5% Nonidet P-40, 50 mm NaF, 1 mm Na3VO4, 5 mm sodium pyrophosphate, and protease inhibitor tablet). The cell lysates were centrifuged at 14,000 × g for 15 min, and the supernatants were recovered. Supernatants containing equal amounts of proteins were incubated with 2.5 mg of primary antibodies overnight at 4 °C. The immunoprecipitates were harvested by protein G PLUS-agarose beads (Santa Cruz Biotechnology). The beads were washed once with regular washing buffer (50 mm Tris-HCl, 100 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-40), twice with high salt washing buffer (50 mm Tris-HCl, 500 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-40), and another time with regular washing buffer. Immunoprecipitates were then eluted by 5-min boiling of the beads in SDS-PAGE sample buffer and characterized by Western blotting with appropriate antibodies (5.Kim Y. Suh N. Sporn M. Reed J.C. J. Biol. Chem. 2002; 277: 22320-22329Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Statistical Analysis—Comparisons of mean values were performed using the independent sample t test in SPSS for Windows version 11.5 software (SPSS, Inc., Chicago, IL) (32.Kulp S.K. Yang Y.T. Hung C.C. Chen K.F. Lai J.P. Tseng P.H. Fowble J.W. Ward P.J. Chen C.S. Cancer Res. 2004; 64: 1444-1451Crossref PubMed Scopus (222) Google Scholar). Bortezomib Enhances TRAIL-induced Apoptosis in Resistance HCC Cells—To investigate the anti-tumor effect of TRAIL on HCC cells, we first examined the apoptotic effect of TRAIL in a panel of three human HCC cell lines, including Huh-7, Sk-Hep1, and Hep3B, at the clinical relevant concentrations. Apoptotic cells (sub-G1) were determined by flow cytometry after 24-h treatment. Our results showed that HCC cells were quite resistant to TRAIL, and TRAIL alone was unable to induce apoptosis in all three types of cells even at concentrations up to 1000 ng/ml. However, combing bortezomib at 50 nm with TRAIL reversed the resistance in three cell lines and induced significant apoptosis in a dose-dependent manner starting from TRAIL at a concentration of 100 ng/ml (Fig. 1A, left). We then examined the effect of TRAIL on various doses of bortezomib in three cell lines and found that TRAIL enhanced bortezomib-induced apoptosis in a dose-dependent manner. These results indicate that combinational effects of bortezomib and TRAIL on apoptosis exist in HCC cells (Fig. 1A, right). To examine the apoptotic pathways, we used two types of approaches. First, we performed a dose escalation analysis of bortezomib on TRAIL-induced apoptosis in Huh-7 cells and found that the combination of bortezomib and TRAIL induced activation of caspase-8, followed by the cleavage of Bid and activation of caspase-9 and caspase-3 and PARP cleavage in a dose-dependent manner. Second, we examined combinational effects of TRAIL and bortezomib in two different periods of time in Sk-Hep1 cells. Our data showed that the combination of TRAIL at 100 ng/ml and bortezomib at 50 nm induced apoptosis in association with the cleavage of Bid and activation of caspase-9. These results indicate the importance of the intrinsic mitochondria pathway in TRAIL-induced apoptosis (Fig. 1B). Bortezomib Does Not Affect NF-κB/Nuclear p65 DNA Binding Activity in HCC Cells—NF-κB has been proposed as a major target of bortezomib in myeloma and lymphoma (9.Voorhees P.M. Orlowski R.Z. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 189-213Crossref PubMed Scopus (219) Google Scholar). NF-κB plays a key role in cell proliferation, apoptosis, metastasis, invasiveness, angiogenesis, and metastasis (33.Orlowski R.Z. Baldwin Jr., A.S. Trends Mol. Med. 2002; 8: 385-389Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). Bortezomib as a proteasome inhibitor sequestered NF-κB in the cytoplasm and reduced its transcriptional activity through inhibiting the reduction of its inhibitor IκB. Notably, NF-κB activation induced by TRAIL has been reported to be responsible for the resistance of TRAIL, and numerous reports have shown that bortezomib enhanced the anti-tumor activity of TRAIL through the inhibition of NF-κB activation in pancreatic cancer (30.Khanbolooki S. Nawrocki S.T. Arumugam T. Andtbacka R. Pino M.S. Kurzrock R. Logsdon C.D. Abbruzzese J.L. McConkey D.J. Mol. Cancer Ther. 2006; 5: 2251-2260Crossref PubMed Scopus (123) Google Scholar), non-small cell lung cancer (24.Voortman J. Resende T.P. Abou El Hassan M.A. Giaccone G. Kruyt F.A. Mol. Cancer Ther. 2007; 6: 2103-2112Crossref PubMed Scopus (106) Google Scholar), and non-Hodgkin lymphoma (12.Smith M.R. Jin F. Joshi I. Clin. Cancer Res. 2007; 13: 5528s-5534sCrossref PubMed Scopus (38) Google Scholar). In this study, we examined the NF-κB binding activity in HCC cells. Cells were exposed to TRAIL and/or bortezomib for the indicated time. As shown in Fig. 2, A–C, neither TRAIL nor bortezomib changed the NF-κB binding activity in three HCC cell lines, indicating that the NF-κB pathway was not a major target in mediating the effect of bortezomib on TRAIL-treated cells. Notably, bortezomib plus TRAIL reversed the degradation of IκB-α induced by tumor necrosis factor-α and subsequently inhibited NF-κB activation in Sk-Hep1 cells (Fig. 2D). Effects of Bortezomib on TRAIL Receptors Are Not Essential in Mediating Its Effects on TRAIL-induced Apoptosis—To understand the effect of bortezomib on TRAIL-induced apoptosis, we next examined TRAIL receptors and TRAIL DISC proteins, including FADD and c-FLIP, in bortezomib-treated cells. HCC cells were exposed to bortezomib at 100 nm for 24 h and then harvested for analysis of surface expression of TRAIL receptors by immunoflu
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