Enhancement of cisplatin cytotoxicity by SAHA involves endoplasmic reticulum stress-mediated apoptosis in oral squamous cell carcinoma cells.
OBJECTIVE
The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), enhances cisplatin [cis-diammine dichloroplatinum (II)] (CDDP)-induced apoptosis in the oral squamous cell carcinoma (OSCC) cell line by complex, multifunctional mechanisms. We investigated the role of endoplasmic reticulum (ER) stress in the enhancing effect of SAHA on CDDP, compared with the ER stressor thapsigargin.


METHODS
We chose OSCC cell line HSC-3 to ascertain the mechanism of SAHA-enhanced cytotoxicity among various cell lines. HSC-3 cells were incubated with CDDP/SAHA for 48 h, followed by the assessment of cell chemosensitivity to CDDP with MTT and TUNEL assays. Western blot analysis was used to detect the expressions of ER-related molecules, and flow cytometry was used to monitor caspase activity.


RESULTS
Treatment with CDDP/SAHA potently induced apoptosis in HSC-3 cells with a significant increase in caspase-4 and -12 functions. For example, 60% of cells became apoptotic after 48 h of treatment with CDDP/SAHA. In addition, SAHA alone rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)alpha, which is up-regulated during ER stress. Inhibition of ER stress by salubrinal, an inhibitor of eIF2alpha dephosphorylation, abrogated SAHA's enhancement of CDDP cytotoxicity. Levels of phospho-Akt are decreased in SAHA-treated cells, and this is in turn associated with increased activity of protein phosphatase 1 (PP1) by SAHA, the phosphatase upstream of Akt.


CONCLUSIONS
These data indicate that up-regulation of specific-ER stress-associated events is an integral part of the mechanism by which SAHA enhances CDDP-induced apoptosis, and PP1 up-regulation followed by Akt dephosphorylation plays an important role in SAHA-enhanced CDDP apoptosis.
Suzuki
Maiko
M
Department of Microbiology and Immunology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Endo
Manabu
M
Shinohara
Fumiaki
F
Echigo
Seishi
S
Rikiishi
Hidemi
H
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D053454:Caspase 12 / Q000378:metabolism; D061945:Caspase Inhibitors; D053471:Caspases, Initiator / Q000378:metabolism; D045744:Cell Line, Tumor; D002934:Cinnamates / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology* / Q000633:toxicity; D004357:Drug Synergism; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000502:physiology; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D006360:Heat-Shock Proteins / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D008562:Membrane Glycoproteins / Q000378:metabolism; D009062:Mouth Neoplasms / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D014635:Valproic Acid / Q000494:pharmacology; D000077337:Vorinostat
D061945:Caspase Inhibitors; D002934:Cinnamates; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; D015852:Eukaryotic Initiation Factor-2; D006360:Heat-Shock Proteins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D008562:Membrane Glycoproteins; C053387:endoplasmin; C496827:salubrinal; D014415:Tunicamycin; D000077337:Vorinostat; D014635:Valproic Acid; D019284:Thapsigargin; D051057:Proto-Oncogene Proteins c-akt; D054645:Protein Phosphatase 1; C093516:CASP4 protein, human; D053454:Caspase 12; D053471:Caspases, Initiator; D013890:Thiourea; D002945:Cisplatin