thapsigargin and Mouth-Neoplasms

Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44(high)EpCAM(low/-) CD24(+) cell surface marker profile. Treatment with TGFβ and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; CD24 Antigen; Cell Line; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial Cell Adhesion Molecule; Epithelial-Mesenchymal Transition; Female; Humans; Hyaluronan Receptors; Male; Mice; Mice, Inbred NOD; Mice, SCID; Mouth Neoplasms; Neoplastic Stem Cells; Phenotype; Thapsigargin; Transforming Growth Factor beta; Tretinoin

The effect of [6]-shogaol (1) on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) and viability has not been explored previously in oral epithelial cells. The present study has examined whether 1 alters [Ca(2+)](i) and viability in OC2 human oral cancer cells. Compound 1 at concentrations > or = 5 microM increased [Ca(2+)](i) in a concentration-dependent manner with a 50% effective concentration (EC(50)) value of 65 microM. The Ca(2+) signal was reduced substantially by removing extracellular Ca(2+). In a Ca(2+)-free medium, the 1-induced [Ca(2+)](i) elevation was mostly attenuated by depleting stored Ca(2+) with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). The [Ca(2+)](i) signal was inhibited by La(3+) but not by L-type Ca(2+) channel blockers. The elevation of [Ca(2+)](i) caused by 1 in a Ca(2+)-containing medium was not affected by modulation of protein kinase C activity, but was inhibited by 82% with the phospholipase A2 inhibitor aristolochic acid I (20 microM). U73122, a selective inhibitor of phospholipase C, abolished 1-induced [Ca(2+)](i) release. At concentrations of 5-100 microM, 1 killed cells in a concentration-dependent manner. These findings suggest that [6]-shogaol induces a significant rise in [Ca(2+)](i) in oral cancer OC2 cells by causing stored Ca(2+) release from the thapsigargin-sensitive endoplasmic reticulum pool in an inositol 1,4,5-trisphosphate-dependent manner and by inducing Ca(2+) influx via a phospholipase A2- and La(3+)-sensitive pathway.

Topics: Calcium; Calcium Channels, L-Type; Catechols; Cell Line, Tumor; Cytosol; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Estrenes; Humans; Inositol 1,4,5-Trisphosphate; Molecular Structure; Mouth Neoplasms; Phospholipase A2 Inhibitors; Protein Kinase C; Pyrrolidinones; Thapsigargin

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

The effect of fendiline on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) and proliferation has not been explored in human oral cancer cells. This study examined whether fendiline altered Ca(2+) levels and caused cell death in OC2 human oral cancer cells. [Ca(2+)](i) and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Fendiline at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The fendiline-induced Ca(2+) influx was sensitive to blockade of L-type Ca(2+) channel blockers. In Ca(2+)-free medium, after pretreatment with 50 microM fendiline, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor)-induced [Ca(2+)](i) rises were inhibited; and conversely, thapsigargin pretreatment nearly abolished fendiline-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change fendiline-induced [Ca(2+)](i) rises. At concentrations between 5 and 25 microM, fendiline killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM fendiline was not reversed by prechelating cytosolic Ca(2+) with BAPTA/AM. Collectively, in OC2 cells, fendiline induced [Ca(2+)](i) rises by causing Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx from L-type Ca(2+) channels. Furthermore, fendiline-caused cytotoxicity was not via a preceding [Ca(2+)](i) rise.

Topics: Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Death; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Estrenes; Fendiline; Fluorescent Dyes; Fura-2; Humans; Mouth Neoplasms; Nifedipine; Phosphodiesterase Inhibitors; Pyrrolidinones; Tetrazolium Salts; Thapsigargin

The effect of thimerosal on cytosolic free Ca(2+) concentrations ([Ca(2+)](i) ) in human oral cancer cells (OC2) is unclear. This study explored whether thimerosal changed basal [Ca(2+)](i) levels in suspended OC2 cells using fura-2. Thimerosal at concentrations between 1and 50 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca( 2+). Thimerosal-induced Ca(2+) influx was not blocked by L-type Ca(2+) entry inhibitors and protein kinase C modulators (phorbol 12-myristate 13-acetate [PMA] and GF109203X). In Ca(2+)-free medium, 50 microM thimerosal failed to induce a [Ca(2+)](i) rise after pretreatment with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). Inhibition of phospholipase C with U73122 did not change thimerosal-induced [Ca(2+)](i) rises. At concentrations between 5 and 10 microM, thimerosal killed cells in a concentration-dependent manner. The cytotoxic effect of 8 muM thimerosal was potentiated by prechelating cytosolic Ca(2+) with the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate/acetomethyl (BAPTA/ AM). Flow cytometry data suggested that 1-7 microM thimerosal-induced apoptosis in a concentration-dependent manner. Collectively, in OC2 cells, thimerosal-induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx through non-L-type Ca(2+) channels. Thimerosal killed cells in a concentration-dependent manner through apoptosis.

Topics: Apoptosis; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Cell Line, Tumor; Cell Survival; Chelating Agents; Dose-Response Relationship, Drug; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Estrenes; Humans; Mouth Neoplasms; Phosphodiesterase Inhibitors; Protein Kinase C; Pyrrolidinones; Thapsigargin; Thimerosal; Type C Phospholipases

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) and cell viability in OC2 human oral cancer cells. [Ca(2+)](i) and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The tamoxifen-induced Ca(2+) influx was sensitive to blockade of L-type Ca(2+) channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca(2+)-free medium, after pretreatment with 1 muM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), tamoxifen-induced [Ca(2+)](i) rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change tamoxifen-induced [Ca(2+)](i) rises. At concentrations between 10 and 50 microM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 microM tamoxifen was not reversed by prechelating cytosolic Ca(2+) with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca(2+)](i) rises, in a nongenomic manner, by causing Ca(2+) release from the endoplasmic reticulum, and Ca(2+) influx from L-type Ca(2+) channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca(2+)](i) rise.

Topics: Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Death; Cell Line, Tumor; Cell Survival; Cytosol; Dose-Response Relationship, Drug; Estrogen Receptor Modulators; Humans; Mouth Neoplasms; Protein Kinase C; Tamoxifen; Thapsigargin

Buccal mucosa carcinoma-derived cell line, HO-1-N-1, epithelial-like cells, was obtained in order to investigate the characteristics of oral cancer cells and examine the [Ca2+]i responses to stimulants, such as bradykinin (BK), histamine (HIST), thapsigargin (TG), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha ). Intracellular Ca2+ influx was observed by all stimulants that enhanced the [Ca2+]i response. However, intracellular Ca2+ release was not observed in response to growth factors. The [Ca2+]i response of BK (100 nM) was inhibited by 10 micro M of the BKB2 antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-BK, and HIST (1 mM) was completely inhibited by 100 nM of the H1 antagonist, (+)-chlorpheniramine, in the presence and absence of extracellular Ca2+ (1.5 mM).

Topics: Bradykinin; Calcium; Calcium Signaling; Carcinoma; Epidermal Growth Factor; Epithelial Cells; Histamine; Histamine H1 Antagonists; Humans; Mouth Mucosa; Mouth Neoplasms; Receptors, Cell Surface; Stimulation, Chemical; Thapsigargin; Transforming Growth Factor alpha; Tumor Cells, Cultured