Proteasome Inhibitor PS-341 Induces Apoptosis in Cisplatin-resistant Squamous Cell Carcinoma Cells by Induction of Noxa (2006)

Cisplatin is one of the most common DNA-damaging agents used for treating patients with solid tumors such as squamous cell carcinoma (SCC). Unfortunately, significant levels of resistance in SCC cells emerge rapidly following cisplatin treatment. Here we report that the proteasome inhibitor PS-341, the representative of a new class of chemotherapeutic drugs, was capable of inducing apoptosis in cisplatin-resistant SCC cells via the endoplasmic reticulum stress. PS-341 stimulated the phosphorylation of PERK and the unfolded protein response, resulting in the induction of the transcription factor ATF-4. Importantly, the Bcl-2 homology domain 3-only (BH3-only) protein Noxa was found to be strongly induced in cisplatin-resistant SCC cells by PS-341 but not by cisplatin. The knock-down of Noxa using small interference RNA significantly abolished PS-341-mediated apoptosis in SCC cells. Using eIF2α mutant mouse embryonic fibroblasts, we found that functional eIF2α played an essential role in PS-341-induced Noxa expression. Taken together, our novel findings reveal a direct link between PS-341-induced endoplasmic reticulum stress and the mitochondria-dependent apoptotic pathway and suggest that PS-341 may be utilized for overcoming cisplatin-resistance in human SCC. Cisplatin is one of the most common DNA-damaging agents used for treating patients with solid tumors such as squamous cell carcinoma (SCC). Unfortunately, significant levels of resistance in SCC cells emerge rapidly following cisplatin treatment. Here we report that the proteasome inhibitor PS-341, the representative of a new class of chemotherapeutic drugs, was capable of inducing apoptosis in cisplatin-resistant SCC cells via the endoplasmic reticulum stress. PS-341 stimulated the phosphorylation of PERK and the unfolded protein response, resulting in the induction of the transcription factor ATF-4. Importantly, the Bcl-2 homology domain 3-only (BH3-only) protein Noxa was found to be strongly induced in cisplatin-resistant SCC cells by PS-341 but not by cisplatin. The knock-down of Noxa using small interference RNA significantly abolished PS-341-mediated apoptosis in SCC cells. Using eIF2α mutant mouse embryonic fibroblasts, we found that functional eIF2α played an essential role in PS-341-induced Noxa expression. Taken together, our novel findings reveal a direct link between PS-341-induced endoplasmic reticulum stress and the mitochondria-dependent apoptotic pathway and suggest that PS-341 may be utilized for overcoming cisplatin-resistance in human SCC. The proteasome inhibitor PS-341 (also known as Velcade or Bortezomib), represents a new class of chemotherapeutic drugs that has been demonstrated to induce apoptosis independently or synergistically with conventional cancer therapy in both solid and hematological tumors. PS-341 is a dipeptidyl boronic acid derivative that specifically inhibits the function of the 26 S proteasome (1Adams J. Nat. Rev. Cancer. 2004; 4: 349-360Crossref PubMed Scopus (1063) Google Scholar). The 26 S proteasome is a large multisubunit ATP-dependent threonine protease complex found in the cytoplasm and nucleus of all eukaryotic cells; its principle function is to degrade proteins via the ubiquitin pathway (2Maniatis T. Genes Dev. 1999; 13: 505-510Crossref PubMed Scopus (369) Google Scholar). Many proteasomal substrates are known to mediate pathways that are dysregulated in human cancers. Also, the 26 S proteasome pathway degrades the misfolded or unfolded proteins and thereby maintains normal cellular functions (1Adams J. Nat. Rev. Cancer. 2004; 4: 349-360Crossref PubMed Scopus (1063) Google Scholar). The inhibition of the 26 S proteasome by the proteasome inhibitors such as PS-341 may lead to the accumulation of the misfolded or unfolded proteins in ER, 2The abbreviations used are: ER, endoplasmic reticulum; BH3, Bcl-2-homology domain; MEF, mouse embryonic fibroblast; SCC, squamous cell carcinoma; UPR, unfolded protein response; eIF2, eukaryotic translation initiation factor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; siRNA, small interference RNA; GFP, green fluorescent protein; HNSCC, head and neck squamous cell carcinoma; PERK, RNA-dependent protein kinase-like ER kinase. 2The abbreviations used are: ER, endoplasmic reticulum; BH3, Bcl-2-homology domain; MEF, mouse embryonic fibroblast; SCC, squamous cell carcinoma; UPR, unfolded protein response; eIF2, eukaryotic translation initiation factor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; siRNA, small interference RNA; GFP, green fluorescent protein; HNSCC, head and neck squamous cell carcinoma; PERK, RNA-dependent protein kinase-like ER kinase. resulting in ER stress (3Fribley A. Zeng Q. Wang C.Y. Mol. Cell. Biol. 2004; 24: 9695-9704Crossref PubMed Scopus (368) Google Scholar). The ER stress subsequently induces a coordinated cellular response called the unfolded protein response (UPR). Although the UPR inhibits general protein translation to reduce the protein loads in the ER lumen, it also specifically up-regulates a set of genes such as molecular chaperons to alleviate the ER stress. Most studies have focused on how the UPR as well as the signaling molecules in UPR activated by the ER stress provides a protection against apoptosis (4Kaufman R.J. J. Clin. Inv. 2002; 110: 1389-1398Crossref PubMed Scopus (1091) Google Scholar, 5Ron D. J. Clin. Inv. 2002; 110: 383-388Crossref Scopus (737) Google Scholar). For example, the deletion of PERK, an initiating kinase in the UPR, has been found to potentiate the ER stress-mediated apoptosis (6Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2364) Google Scholar). Similarly, the inhibition of the phosphorylation of the subunit of eukaryotic translation initiation factor 2 (eIF2α) rendered cells sensitive to glucose deprivation (7Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar). However, in ER stress-associated human chronic diseases, the ER stress eventually activates apoptotic signaling pathways to induce apoptosis, thereby resulting in tissue destruction. Although caspase-12 has been found to play a critical role in the ER stress-mediated apoptosis (4Kaufman R.J. J. Clin. Inv. 2002; 110: 1389-1398Crossref PubMed Scopus (1091) Google Scholar, 5Ron D. J. Clin. Inv. 2002; 110: 383-388Crossref Scopus (737) Google Scholar), the molecular signaling pathway that is associated with the ER stress-mediated apoptosis is not fully understood. We have recently found that the proteasome inhibitor PS-341 potently induced apoptosis in head and neck SCC cell lines through the induction of ER stress in addition to the inhibition of the pro-survival nuclear factor-kappa B (NF-κB) signaling pathway (3Fribley A. Zeng Q. Wang C.Y. Mol. Cell. Biol. 2004; 24: 9695-9704Crossref PubMed Scopus (368) Google Scholar). SCC cells comprise >90% of all malignancies diagnosed in the oral cavity and the head and neck region and is a tremendous public health challenge around the world. The 5-year survival rate for patients with head and neck cancer is one of the lowest of any major cancers and has remained un-improved over the last 20 years (8Patel V. Leethanakul C. Gutkind J.S. Crit. Rev. Oral Biol. Med. 2001; 12: 55-63Crossref PubMed Scopus (72) Google Scholar, 9Zeng Q. Li S. Chepeha D.B. Giodano T.J. Li J. Zhang H. Polverini P.J. Nor J. Kitajewski J. Wang C.-Y. Cancer Cell. 2005; 8: 13-23Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 10Ando N. Iizuka T. Ide H. Ishida K. Shinoda M. Nishimaki T. Takiyama W. Watanabe H. Isono K. Aoyama N. Makuuchi H. Tanaka O. Yamana H. Ikeuchi S. Kabuto T. Nagai K. Shimada Y. Kinjo Y. Fukuda H. J. Clin. Oncol. 2003; 21: 4592-4596Crossref PubMed Scopus (581) Google Scholar). Cisplatin has been a cornerstone chemotherapy treatment for patients with SCC. Unfortunately, significant numbers of resistant SCC cells emerge rapidly following initial cisplatin treatment. Thus, many retrospective clinical studies reported that cisplatin was unable to increase survival when administered as a monotherapy or in combination with surgery or radiotherapy (11Licitra L. Grandi C. Guzzo M. Mariani L. Lo Vullo S. Valvo F. Quattrone P. Valagussa P. Bonadonna G. Molinari R. Cantu G. J. Clin. Oncol. 2003; 21: 327-333Crossref PubMed Scopus (198) Google Scholar, 12Foote R.L. Kasperbauer J.L. Okuno S.H. Nichols D.A. Olsen K.D. Brown P.D. Garces Y.I. Sargent D.J. Strome S.E. Cancer. 2005; 103: 559-568Crossref PubMed Scopus (23) Google Scholar, 13Akervall J. Guo X. Qian C.N. Schoumans J. Leeser B. Kort E. Cole A. Resau J. Bradford C. Carey T. Wennerberg J. Anderson H. Tennvall J. Teh B.T. Clin. Cancer Res. 2004; 10: 8204-8213Crossref PubMed Scopus (71) Google Scholar). Cisplatin is a platinum-containing compound that induces DNA damage by binding to DNA and forming adducts that impair de novo synthesis. Mechanisms of cisplatin resistance are known to be dependent on a myriad of factors, including loss of p53, aberrant levels of cell cycle regulators such as cyclin D1, and Bcl-2 family proteins (14Duffey D.C. Chen Z. Dong G. Ondrey F.G. Wolf J.S. Brown K. Siebenlist U. Van Waes C. Cancer Res. 1999; 59: 3468-3474PubMed Google Scholar, 15Akervall J. Kurnit D.M. Adams M. Zhu S. Fisher S.G. Bradford C.R. Carey T.E. Acta Otolaryngol. 2004; 124: 851-857Crossref PubMed Scopus (26) Google Scholar, 16Bradford C.R. Zhu S. Ogawa H. Ogawa T. Ubell M. Narayan A. Johnson G. Wolf G.T. Fisher S.G. Carey T.E. Head Neck. 2003; 25: 654-661Crossref PubMed Scopus (134) Google Scholar). Initial reductions in tumor burden are overshadowed by the ability of cisplatin-resistant cells to proliferate and emerge as recurrent or metastatic diseases for which the median survival plummets to ∼6 months (17Sturgis E.M. Wei Q. Spitz M.R. Semin. Oncol. 2004; 31: 726-733Crossref PubMed Scopus (170) Google Scholar, 18Torigoe T. Izumi H. Ishiguchi H. Yoshida Y. Tanabe M. Yoshida T. Igarashi T. Niina I. Wakasugi T. Imaizumi T. Momii Y. Kuwano M. Kohno K. Curr. Med. Chem. Anti-Cancer Agents. 2005; 5: 15-27Crossref PubMed Scopus (104) Google Scholar). Based on the unique ability of PS-341 to induce apoptosis in SCC cells (1Adams J. Nat. Rev. Cancer. 2004; 4: 349-360Crossref PubMed Scopus (1063) Google Scholar, 2Maniatis T. Genes Dev. 1999; 13: 505-510Crossref PubMed Scopus (369) Google Scholar, 3Fribley A. Zeng Q. Wang C.Y. Mol. Cell. Biol. 2004; 24: 9695-9704Crossref PubMed Scopus (368) Google Scholar), we were interested in investigating the potential of PS-341 to induce apoptosis in cisplatin-resistant SCC cell lines. Moreover, we wished that the studies might further help us to understand the molecular mechanism by which PS-341 induced apoptosis in SCC cells. We found that PS-341 was able to overcome cisplatin resistance and induce apoptosis in a panel of cisplatin-resistant SCC cell lines. Unlike cisplatin, PS-341 induced the markers of the ER stress, such as Gadd34, phosphorylation of the ER-resident stress-sensing kinase PERK, and a strong induction of the transcription factor ATF-4. Although the pro-apoptotic BH3-only protein Noxa was induced by both cisplatin and PS-341 in cisplatin-sensitive SCC cells, Noxa was strongly induced by PS-341, but not cisplatin, in cisplatin-resistant cells. The knock-down of Noxa by siRNA was able to significantly reduce the ability of PS-341 to induce apoptosis in cisplatin-resistant SCC cells. Taken together, our results elucidate a novel apoptotic signaling pathway that is activated by PS-341 through the ER stress and suggest that PS-341 may be utilized for treating cisplatin-resistant cancers. Reagents and Cell Culture—PS-341, kindly provided by Millennium Pharmaceuticals, Inc., was dissolved in Me2SO to a concentration of 10 mm and stored at –20 °C; the concentration of Me2SO in culture never exceeded 1:5000. Cisplatin (cis-diammineplatinum(II) dichloride) was purchased from Sigma. UMSCC cell lines were all obtained from Dr. Thomas Carey at The University of Michigan and were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin-streptomycin (Invitrogen). Wild-type (S/S) and mutant (A/A) eIF2-α mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin-streptomycin and supplemented with minimal essential medium amino acids solution and minimal essential medium non-essential amino acids solution (Invitrogen). Western Blot Analysis—Cells (2 × 106) were plated in 10-cm tissue culture dishes 16–24 h before treatment with PS-341, cisplatin, or vehicle control. Cells were treated, unless otherwise indicated, with 1.0 μm PS-341 or 50 μm cisplatin. Whole cell lysates were prepared using modified radioimmune precipitation assay buffer containing phenylmethylsulfonyl fluoride and protease inhibitors (Sigma-Aldrich). 25- to 80-μg aliquots of lysates were resolved on 8% or 12% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (Immun-Blot, Bio-Rad) with a Bio-Rad semi-dry transfer apparatus. Membranes were blocked with 5% milk for 1 h at room temperature and probed with primary antibodies overnight at 4 °C. Antibodies were acquired from the following manufacturers: Puma, Phospho-PERK, Caspase-9, and Caspase-12 from Cell Signaling; Caspase-3, ATF-4 (CREB-2), Gadd34, and Gadd153/CHOP from Santa Cruz Biotechnology; Noxa from Abcam, Inc.; monoclonal α-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Sigma and Chemicon International, respectively. Following incubation with horseradish peroxidase-conjugated secondary antibodies, membranes were visualized by using an enhanced chemiluminescence reagent (Pierce). Northern Blot Analysis and Real-time Reverse Transcription-PCR—Total RNAs were harvested from cells using TRIzol (Invitrogen) to the For Northern to aliquots of RNAs were resolved on gels and transferred to a membrane The membranes were with and to The used to the Northern was using the (Invitrogen) the The human Noxa was obtained from Northern were with The human was a from at the Real-time using was with an The for mouse Noxa were and siRNA and mouse ATF-4 was provided by Ron at the University of or an was cells for 4 h with to the assay was and h following to the of cell a an containing green fluorescent protein was the cells For siRNA cells × were plated in tissue culture dishes the before siRNA was cells overnight with in (Invitrogen) to the h following cells were with 1.0 μm PS-341 as siRNA for and Noxa were at siRNA was also from DNA or cells were and with for 2 h at DNA was with and with were with and of was resolved on a PS-341 in SCC Cell from our demonstrated common chemotherapeutic drugs, PS-341 was able to induce ER stress and apoptosis in SCC cell lines (3Fribley A. Zeng Q. Wang C.Y. Mol. Cell. Biol. 2004; 24: 9695-9704Crossref PubMed Scopus (368) Google Scholar). Based on we that PS-341 might be able to induce apoptosis in cisplatin-resistant SCC cell lines. we utilized cell and both of which have been cultured to resistance with of cisplatin to the clinical of recurrent cisplatin-resistant SCC C.R. Zhu S. Ogawa H. Ogawa T. Ubell M. Narayan A. Johnson G. Wolf G.T. Fisher S.G. Carey T.E. Head Neck. 2003; 25: 654-661Crossref PubMed Scopus (134) Google Scholar). in a of cisplatin as as 50 μm induced cell both cell lines were sensitive to PS-341-induced we also found that cells that resistance to cisplatin were sensitive to 1 and that PS-341 induced cell in cells in a and Moreover, and and cell also that PS-341 strongly induced cell in cisplatin-resistant cells a we found that both cisplatin and PS-341 induce apoptosis in cisplatin-sensitive and cells, that cisplatin was functional PS-341 cisplatin-resistant cells via an apoptotic we harvested from SCC cells with PS-341 or cisplatin. in both cisplatin and PS-341 induced DNA in cisplatin-sensitive cells. the DNA was not in cells, PS-341 strongly induced DNA in cells. we found that and in SCC cells were activated by PS-341 but not by cisplatin Taken together, results suggest that PS-341 induce apoptosis in cisplatin-resistant SCC cells. PS-341 the ER in SCC on our findings that PS-341 induced the ER stress in SCC cells (3Fribley A. Zeng Q. Wang C.Y. Mol. Cell. Biol. 2004; 24: 9695-9704Crossref PubMed Scopus (368) Google Scholar), we that the ability of PS-341 to induce apoptosis in cisplatin-resistant SCC cell lines might also be by the ER stress. in 2 and the of PS-341, but not cisplatin, induced the phosphorylation of the ER stress-sensing kinase PERK, a of ER stress (4Kaufman R.J. J. Clin. Inv. 2002; 110: 1389-1398Crossref PubMed Scopus (1091) Google Scholar, 5Ron D. J. Clin. Inv. 2002; 110: 383-388Crossref Scopus (737) Google Scholar), in cisplatin-resistant SCC cells. is known that activated PERK eIF2α to protein and to the of the genes the ER stress. Importantly, studies from have found that PERK is for translation of the transcription factor ATF-4 in response to the ER stress, which a critical role in the induction of genes (4Kaufman R.J. J. Clin. Inv. 2002; 110: 1389-1398Crossref PubMed Scopus (1091) Google Scholar, 5Ron D. J. Clin. Inv. 2002; 110: 383-388Crossref Scopus (737) Google Scholar). Western demonstrated that ATF-4 was induced by PS-341, but not cisplatin, in SCC cells and Also, the UPR protein was induced by PS-341 but not cisplatin and we found in to the was induced by PS-341 in SCC cells not Taken together, suggest that PS-341, but not cisplatin, is able to induce the ER stress in cisplatin-resistant SCC cells. PS-341 of the BH3-only Noxa in SCC that PS-341 was able to induce markers of ER stress in cisplatin-resistant SCC cells, we to for the induction of studies by L. T. 2003; PubMed Scopus Google have demonstrated that both and also played an essential role in the ER we found that the deletion of and also provided protection against PS-341-mediated apoptosis in However, both and were not induced by PS-341 in cisplatin-resistant cells not studies reported that the ER the BH3-only Bcl-2 family in cells by C. D. A. T. J. Cell Biol. 2003; PubMed Scopus Google Scholar, T. M. K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The BH3-only of the Bcl-2 family has at including Noxa and Puma, and is to induce apoptosis by the of from S. D.C. Adams J.M. 2003; PubMed Scopus Google Scholar, X. Genes Dev. 2001; PubMed Scopus Google Scholar). we was induced by Western found that PS-341 or induced in cisplatin-sensitive cells and cisplatin-resistant cells and cells with PS-341, cisplatin strongly induced in cisplatin-resistant cells, but not in cells B and results suggest that might not be a critical pro-apoptotic protein induced by BH3-only protein Noxa was reported to be apoptotic we also Noxa was induced by PS-341 in SCC cells. in Western found that Noxa was significantly induced by PS-341 in cisplatin-sensitive cells. Importantly, Noxa was also strongly induced by PS-341 in cisplatin-resistant and cells PS-341 might the of Noxa at the protein we also Northern to PS-341 induced Noxa expression. in and Noxa was rapidly induced by PS-341 in and cells. our results suggest that PS-341 might induce Noxa to overcome cisplatin-resistance in SCC cells. PS-341-induced UPR a in PS-341-induced Noxa the ER kinase PERK in response to the ER stress, it which to a general inhibition of protein (4Kaufman R.J. J. Clin. Inv. 2002; 110: 1389-1398Crossref PubMed Scopus (1091) Google Scholar, 5Ron D. J. Clin. Inv. 2002; 110: 383-388Crossref Scopus (737) Google Scholar). by (7Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1086) Google have found that eIF2α phosphorylation played a critical role in the induction of genes and cell survival in response to ER stress. further the role of UPR in PS-341-mediated apoptosis, we utilized that a mutant of eIF2α which be by we found that were significantly resistant to PS-341-mediated apoptosis with Although caspase-12 was in both cells, the of was significantly in we that PS-341 was unable to induce ATF-4 or the ER stress pro-apoptotic transcription factor Gadd153/CHOP in eIF2-α mutant cells with cells by Western were antibodies for mouse Noxa a for Noxa was in our an induction of Noxa in but not in a demonstrated that functional eIF2α and an UPR response were in for PS-341 to induce accumulation of Noxa a in PS-341-induced in further Noxa played a critical role in the induction of apoptosis in cisplatin-resistant SCC cells, we of Noxa induce apoptosis by in which was utilized as a in of Noxa significantly induced apoptosis in cells h of Noxa played an essential role in PS-341-induced apoptosis in cisplatin-resistant SCC cells, we utilized siRNA to Noxa expression. Noxa siRNA, but not siRNA for significantly PS-341-induced Noxa by in and the knock-down of Noxa significantly provided protection against PS-341-mediated apoptosis in both cisplatin-resistant and cells. Moreover, we also the knock-down of PS-341-induced apoptosis in cells. in and the of not PS-341-mediated results suggest that the induction of Noxa by PS-341 a critical role in the induction of apoptosis in cisplatin-resistant SCC cells. we demonstrated that PS-341 potently induced cell in a panel of cisplatin-resistant SCC cell lines. of and and DNA were in cisplatin-resistant SCC cells with PS-341, the of the apoptotic with our that PS-341 induce ER stress in cisplatin-sensitive SCC cells (3Fribley A. Zeng Q. Wang C.Y. Mol. Cell. Biol. 2004; 24: 9695-9704Crossref PubMed Scopus (368) Google Scholar), we found that PS-341, but not cisplatin, was able to induce markers of ER stress in cisplatin-resistant SCC cells. We phosphorylation of the ER stress-sensing kinase PERK and an accumulation of ATF-4 and following treatment with Most we that Noxa played a critical role in the ER stress-mediated apoptosis induced by PS-341, a molecular link between the ER stress and of it has been demonstrated that the ER stress caspase-12 in which to be T. Zhu H. N. Li E. J. B. J. PubMed Scopus Google Scholar). we found that PS-341 induce caspase-12 in However, caspase-12 is not in human cells H. U. L. E. Res. 2002; PubMed Scopus Google Scholar). it is not is a activated by PS-341 in human SCC cells. Thus, we that PS-341 may also a pathway to induce apoptosis in SCC cells. studies by L. T. 2003; PubMed Scopus Google have found that and be in ER, and is a link between the ER stress and the The deletion of and provided protection against the Noxa has been found to function of and to from to the resulting in the of E. Wei S. T. Mol. Cell. 2001; 8: Full Text Full Text PDF PubMed Scopus Google Scholar, L. P. B. A. J.M. L. Mol. 2004; Google Scholar). of our results suggest that PS-341-induced ER stress the mitochondria-dependent apoptotic pathway to induce apoptosis in SCC cells. have reported the induction of pro-apoptotic BH3-only proteins in a of cell lines following treatment with drugs through an mechanism Chen L. G. Wei A. E. Adams J.M. Genes Dev. 2005; PubMed Scopus Google Scholar, E. Wei S. T. Mol. Cell. 2001; 8: Full Text Full Text PDF PubMed Scopus Google Scholar, L. P. B. A. J.M. L. Mol. 2004; Google Scholar, C. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). our Western with cell lysates prepared from cisplatin-sensitive SCC cells with PS-341 or cisplatin an accumulation of both Noxa and was or induced by PS-341 in and cells, we found that cisplatin induced accumulation in cells, cells are resistant to cisplatin, that may not be critical for PS-341 to overcome cisplatin it is that inhibitors of that may be in SCC cells the pro-apoptotic of we found that PS-341, but not cisplatin, strongly induced Noxa in cisplatin-resistant SCC cells, that the ability of PS-341 to Noxa might be an mechanism by which it was able to overcome cisplatin Moreover, the of Noxa by siRNA significantly PS-341-mediated apoptosis in cisplatin-resistant SCC cells, that Noxa is critical for PS-341-mediated However, the knock-down of Noxa not PS-341-induced was that PS-341 might induce apoptosis in SCC cells by inducing BH3-only Although we not that PS-341 induced BH3-only proteins by (3Fribley A. Zeng Q. Wang C.Y. Mol. Cell. Biol. 2004; 24: 9695-9704Crossref PubMed Scopus (368) Google Scholar), it is that PS-341 may induce the accumulation of BH3-only proteins by proteasomal in SCC cells. studies have reported that PS-341 induce the of proteins to apoptosis M. Johnson T. H. L. Adams J. Mol. Cancer 2005; 4: PubMed Google Scholar, H. Zhang L. Dong F. Guo W. S. F. P.J. B. 2005; 24: PubMed Scopus Google Scholar, D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, in the it be to PS-341 accumulation by its in SCC cells. Noxa was as a of apoptosis in R. H. J. T. T. T. T. Tanaka N. PubMed Scopus Google Scholar). the that Noxa is a of and that is by the 26 S proteasome C. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), it is to that PS-341 might lead to of Noxa by cellular of However, SCC cell lines used in our studies were C.R. Zhu S. Ogawa H. Ogawa T. Ubell M. Narayan A. Johnson G. Wolf G.T. Fisher S.G. Carey T.E. Head Neck. 2003; 25: 654-661Crossref PubMed Scopus (134) Google Scholar). with our P. G. N. E. E. D. PubMed Scopus Google reported that PS-341 also induced Noxa in cells of were that PS-341 induce apoptosis in human cancer cells in the of L. Y. R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Cancer Rev. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, T. P. D. P.J. Adams J. Anderson Res. 2001; Google Scholar, T. C. M. T. D. P. R. K. N. Anderson 2003; PubMed Scopus Google Scholar). results reported suggest that PS-341 may induce apoptosis through the induction of most studies have focused on how the ER stress activates the UPR to protection against apoptosis (4Kaufman R.J. J. Clin. Inv. 2002; 110: 1389-1398Crossref PubMed Scopus (1091) Google Scholar, 5Ron D. J. Clin. Inv. 2002; 110: 383-388Crossref Scopus (737) Google Scholar). The of PERK or the inhibition of eIF2α phosphorylation has been found to the ER stress-mediated apoptosis (6Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2364) Google Scholar, D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar). However, are also studies that the UPR may induce pro-apoptotic genes to induce For example, the deletion of the ER transcription factor in a mouse has been found to apoptosis in via the UPR B. Li Y. Zhang D. H.P. M. G. Fisher D. I. Nat. Cell Biol. 2003; 5: PubMed Scopus Google Scholar). The has been found to be by the ER stress in cancer cells and cancer cells and T. Res. Scholar, H. K. Wang H. Res. 2003; Google Scholar). is well known that the of is dependent on ATF-4 (4Kaufman R.J. J. Clin. Inv. 2002; 110: 1389-1398Crossref PubMed Scopus (1091) Google Scholar, 5Ron D. J. Clin. Inv. 2002; 110: 383-388Crossref Scopus (737) Google Scholar). Although was strongly induced by PS-341 in it was or induced by PS-341 in SCC cells. we found that PS-341 strongly induced ATF-4 in SCC cells, that is not for PS-341-mediated apoptosis in cisplatin-resistant SCC cells. Using cells that have an at of the of activated PERK, we found that PS-341 was unable to induce ATF-4 and Importantly, we that PS-341 potently Noxa in but not in the of eIF2α phosphorylation to Noxa Moreover, we found that the knock-down of ATF-4 by siRNA also significantly the of Noxa by PS-341 Taken together, our results suggest the signaling pathway cell it also potently induce apoptosis by inducing the pro-apoptotic genes in response to the ER stress. Although initial reductions in tumor burden have been in patients cisplatin resistant cells proliferate and as or metastatic diseases, which to survival of months for head and neck cancer patients C.R. Zhu S. Ogawa H. Ogawa T. Ubell M. Narayan A. Johnson G. Wolf G.T. Fisher S.G. Carey T.E. Head Neck. 2003; 25: 654-661Crossref PubMed Scopus (134) Google Scholar, E.M. Wei Q. Spitz M.R. Semin. Oncol. 2004; 31: 726-733Crossref PubMed Scopus (170) Google Scholar, 18Torigoe T. Izumi H. Ishiguchi H. Yoshida Y. Tanabe M. Yoshida T. Igarashi T. Niina I. Wakasugi T. Imaizumi T. Momii Y. Kuwano M. Kohno K. Curr. Med. Chem. Anti-Cancer Agents. 2005; 5: 15-27Crossref PubMed Scopus (104) Google Scholar). The ability to or resistance to cisplatin has been in human including HNSCC, and cell which has an for novel the that the is a significant in cancer our results suggest that PS-341 may a novel for treating recurrent cancer we found that Noxa was induced by cisplatin in cisplatin-sensitive SCC cells, but not in cisplatin-resistant SCC cells, which that Noxa may play a critical role in Noxa may be an for the of cisplatin for patients with SCC. We Dr. Thomas Carey for SCC cell lines and for with