salubrinal and Carcinoma--Squamous-Cell

We have previously demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to induce tumor-specific apoptosis. The apoptotic effects of lovastatin were regulated in part by the integrated stress response (ISR) that regulates cellular responses to a wide variety of stress inducers. A key regulator of the ISR apoptotic response is activating transcription factor 3 (ATF3) and its target gene CHOP/GADD153. In our study, we demonstrate that in multiple lovastatin-resistant clones of the squamous cell carcinoma (SCC) cell line SCC9, lovastatin treatment (1-25 μM, 24 hr) in contrast to the parental line failed to significantly induce ATF3 expression. Furthermore, the SCC-derived cell lines SCC25 and HeLa that are sensitive to lovastatin-induced apoptosis also preferentially induce ATF3 expression compared to resistant breast (MCF-7) and prostate carcinoma (PC3)-derived cell lines. In HeLa cells shRNA targeting ATF3 expression as well as in ATF3-deficient murine embryonic fibroblasts, lovastatin-induced cytotoxicity and apoptosis were attenuated. In ex vivo HNSCC tumors, lovastatin also induced ATF3 mRNA expression in two of four tumors evaluated. Salubrinal, an agent that can sustain the activity of a key regulator of the ISR eIF2α, further increased the expression of ATF3 and demonstrated synergistic cytotoxicity in combination with lovastatin in SCC cells. Taken together, our results demonstrate preferential induction of ATF3 in lovastatin-sensitive tumor-derived cell lines that regulate lovastatin-induced apoptosis. Importantly, combining lovastatin with salubrinal enhanced ATF3 expression and induced synergistic cytotoxicity in SCC cells.

Topics: Activating Transcription Factor 3; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Membrane Permeability; Cell Proliferation; Cinnamates; Drug Resistance, Neoplasm; Drug Synergism; Embryo, Mammalian; Eukaryotic Initiation Factor-2; Female; Fibroblasts; Flow Cytometry; Head and Neck Neoplasms; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunoenzyme Techniques; Lovastatin; Male; Membrane Potential, Mitochondrial; Mice; Phosphorylation; Prostatic Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Thiourea; Tumor Cells, Cultured

An oral squamous cell carcinoma Ca9-22 cell line was treated with 11-dehydrosinulariolide, an active compound isolated from the soft coral Sinularia leptoclados, in order to evaluate the effect of this compound on cell growth and protein expression. Cell proliferation was strongly inhibited by 11-dehydrosinulariolide treatment. The 2-DE master maps of control and treated Ca9-22 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 23 proteins, of which 14 were upregulated and 9 were downregulated. The proteomic studies described here have identified some proteins, which are involved in the mitochondrial dysfunction and ER-stress pathway and imply that 11-dehydrosinulariolide induces cell apoptosis through either mitochondrial dysfunction-related or ER stress pathway. Based on this observation, several proteins related to apoptosis pathway were explored for the potential roles involved in this drug-induced cytotoxicity. Furthermore, Salubrinal, an ER stress inhibitor, is able to protect the cell from 11-dehydrosinulariolide-induced apoptosis in a physiological dosage. The significance of these studies illustrates the potential development of anticancer drugs from the natural derivatives of soft coral.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cinnamates; Diterpenes; Electrophoresis, Gel, Two-Dimensional; Endoplasmic Reticulum Stress; Humans; Mouth Neoplasms; Proteomics; Thiourea

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.. 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.. 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.. 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.

Topics: Apoptosis; Carcinoma, Squamous Cell; Caspase 12; Caspase Inhibitors; Caspases, Initiator; Cell Line, Tumor; Cinnamates; Cisplatin; Drug Synergism; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Female; Heat-Shock Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; In Situ Nick-End Labeling; Membrane Glycoproteins; Mouth Neoplasms; Phosphorylation; Protein Phosphatase 1; Proto-Oncogene Proteins c-akt; Thapsigargin; Thiourea; Tunicamycin; Valproic Acid; Vorinostat