thapsigargin and Skin-Neoplasms

We have previously shown that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress-induced apoptosis, and this involves activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway and expression of the apoptosis repressor with caspase recruitment domain (ARC) protein in the cells. In the present study, we show that up-regulation of the antiapoptotic Bcl-2 family member Mcl-1 is another mechanism critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by small interference RNA (siRNA) rendered melanoma cells sensitive to apoptosis induced by the ER stress inducers thapsigargin and tunicamycin, but this sensitization was partially reversed by siRNA knockdown of PUMA or Noxa, as shown in Mcl-1-deficient melanoma cells. Both PUMA and Noxa were increased by ER stress through transcriptional up-regulation, but only up-regulation of Noxa was dependent on p53, whereas up-regulation of PUMA seemed to be mediated by a p53-independent mechanism(s). Up-regulation of Mcl-1 was also due to increased transcription that involved the IRE1alpha and activating transcription factor 6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway seemed to be necessary for optimal up-regulation of Mcl-1. Taken together, these results reveal the mechanisms of resistance of melanoma cells to apoptosis induction mediated by BH3-only proteins upon ER stress, and identify Mcl-1 as a target for the treatment of melanoma in combination with therapeutics that induce ER stress.

Topics: Activating Transcription Factor 6; Antiviral Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Endoplasmic Reticulum; Endoribonucleases; Enzyme Inhibitors; Flow Cytometry; Humans; Melanoma; Membrane Potential, Mitochondrial; Myeloid Cell Leukemia Sequence 1 Protein; Oxidative Stress; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Thapsigargin; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tunicamycin; Up-Regulation

Inhibition of the function of Bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy. G3139 (Genasense) is a phosphorothioate anti-Bcl-2 antisense oligonucleotide, but its mechanism of action is uncertain. The aim of the present work is to investigate inhibition of Bcl-2 expression in 518A2 melanoma cells, the cell line on which recent phase II and phase III clinical trials employing this agent were based.. We down-regulated the expression of Bcl-2 protein by two different strategies in these cells: one employing G3139 and controls, and the other using a small interfering RNA approach. Cell viability after treatment with oligonucleotides or small interfering RNA and cytotoxic agents including gemcitibine, DDP, docetaxel, and thapsigargin was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A 518A2 melanoma cell line stably overexpressing Bcl-2 protein was constructed and treated with either these cytotoxic agents or G3139.. The cytotoxic effects of either G3139 or small interfering RNA treatment of 518A2 melanoma cells are Bcl-2 independent. In addition, in the Bcl-2-overexpressing cells, only a modest increment in chemoresistance was observed, and treatment with G3139 not only did not down-regulate Bcl-2 expression but produced essentially identical toxicity as was observed in the wild-type or mock-transfected cells.. Our results suggest that the mechanism whereby G3139 produces drug-induced cytotoxicity in the 518A2 melanoma line is not dependent on levels of Bcl-2. These findings emphasize the nonsequence specific effects of this phosphorothioate oligonucleotide and call into question the validity of Bcl-2 as a target in this cell line.

Topics: Antineoplastic Agents; Apoptosis; Cisplatin; Deoxycytidine; Docetaxel; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gemcitabine; Humans; Melanoma; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Small Interfering; Skin Neoplasms; Taxoids; Thapsigargin; Thionucleotides; Tumor Cells, Cultured

Interleukin-1 receptor antagonist (IL-1Ra) is an endogenous inhibitor of interleukin-1. The expression of IL-1Ra and interleukin-1alpha (IL-1alpha) was measured in murine epidermis after treatment with tumor promoters and in tumor cell lines. A single treatment with three different tumor promoters (12-O-tetradecanoylphorbol-13-acetate (TPA), anthralin, and thapsigargin) induced IL-1Ra mRNA with different kinetics in mouse skin. The expression of IL-1Ra mRNA also was induced by TPA and IL-1alpha in a dose-related and time-dependent manner in cultured mouse keratinocytes. Expression of IL-1Ra mRNA peaked 6 h after treatment. Both IL-1Ra and IL-1alpha protein and IL-1Ra and IL-1alpha mRNA were measured in various keratinocyte tumor cell lines (C50, MT1/2, HEL30, JWF2, CH72, and BPCC2). The expression of IL-1alpha was increased in papilloma and squamous cell carcinoma cell lines. IL-1Ra protein also was increased in nontumorigenic and papilloma cell lines; however, the expression was dramatically reduced in some carcinoma cell lines. Finally, we detected IL-1alpha and IL-1Ra protein in mouse skin tumors by western blot analysis, and localization was assessed by immunohistochemical analysis. Positive staining for both IL-1alpha and IL-1Ra was observed in the cytoplasm and was most prominent in the suprabasal layer. Although IL-1Ra protein increased in papillomas and carcinomas, IL-1alpha protein was not significantly increased above basal level in most tumors.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Anthralin; Blotting, Northern; Blotting, Western; Carcinogens; Carcinoma, Squamous Cell; Cell Polarity; Cytoplasm; Disease Progression; Epidermis; Gene Expression Regulation, Neoplastic; Hyperplasia; Inflammation; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Keratinocytes; Mice; Mice, Inbred SENCAR; Neoplasm Proteins; Papilloma; Precancerous Conditions; RNA, Messenger; RNA, Neoplasm; Sialoglycoproteins; Skin Diseases; Skin Neoplasms; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

Thapsigargin (Tg), applied twice weekly to the backs of CD-1 mice initiated with 7,12-dimethylbenz[a]anthracene, promoted papillomas on the skin of approximately 50% of the mice. Tg alone induced papillomas in 10% of the mice. Although Tg and 12-O-tetradecanoylphorbol-13-acetate (TPA) are synergistic in a keratinocyte co-culture assay for promotion, skin tumor promotion by TPA was inhibited by co-treatment with Tg. Treatment of cultured keratinocytes with non-toxic doses of Tg increased the level of intracellular free Ca(2+)-induced stratification. Tg blocked expression of the spinous layer differentiation marker keratin 1 (K1), while inducing cornification, a marker of differentiation in the granular layer/stratum corneum. In medium with 1.4 mM Ca2+, Tg prolonged keratinocyte proliferation and selected foci of monolayer cells. A Tg-independent cell line, TK-1, was developed from a single dish in which foci continued to expand after Tg removal. Grafting TK-1 cells on to the backs of nude mice as part of a reconstituted skin resulted in the development of dysplastic papillomas with a high rate of progression to squamous cell carcinomas.

Topics: Animals; Calcium; Carcinogens; Cell Differentiation; Cell Division; Cells, Cultured; Drug Synergism; Female; Hyperplasia; Keratinocytes; Mice; Phenotype; Skin; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin

A method is described for measuring rapid, specific, and saturable binding of the skin irritant and tumour-promoting secretagogue thapsigargin (sesquiterpene lactone) to the microsomal fraction from mouse brain. Employing the tritium-labelled compound its apparent dissociation constant, Kd, and the maximal amount of binding Bmax are shown to be 9.8 nM and 1.9 pmol/mg protein respectively. Such a Kd for thapsigargin is similar to (a) its IC50 value for inhibiting Ca2+ uptake in the microsomal fraction from rat brain and (b) its EC50 values for inducing a rise in the cytoplasmic Ca2+ concentration of human platelets and histamine release from rat peritoneal mast cells. A positive correlation is found between the binding affinities of thapsigargin, thapsitranstagin, and trilobolide, their potencies as secretagogues and their lipophilicities. This correlation does not extend to the skin-irritant activities of the compounds thus emphasizing that their mechanism of action is unlike that of 12-O-tetradecanoylphorbol 13-acetate.

Topics: Animals; Brain; Calcium-Transporting ATPases; Carcinogens; Cold Temperature; Filtration; In Vitro Techniques; Mice; Microsomes; Skin Neoplasms; Terpenes; Thapsigargin

Thapsigargin, a hexaoxygenated tetraacylated sesquiterpene lactone, induced irritation of mouse ear and histidine decarboxylase (HDC) activity in mouse skin, but it did not induce ornithine decarboxylase in mouse skin or adhesion of human promyelocytic leukemia (HL-60) cells. Although thapsigargin did not give consistent positive results in a short-term screening system for tumor promoters, it was tested in a two-stage carcinogenesis experiment on mouse skin. The potency of thapsigargin to induce HDC in mouse skin was used to determine the dose in this experiment. Application of 10 micrograms (17 nmol) thapsigargin induced HDC activity of 139 pmol CO2/mg protein per 60 min. Tumors were found in the skin of 53.5% of the mice treated with DMBA plus 5 micrograms (8.5 nmol) thapsigargin in week 22, in none of those treated with thapsigargin alone by week 30. One tumor appeared in 1 of 15 mice treated with DMBA alone in week 21. Thapsigargin cannot bind to the phorbol ester receptor in the particulate fraction of mouse skin and so is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA) type tumor promoter. It is a new tumor promoter differing in many respects from the well-defined TPA type tumor promoters. Several naturally occurring analogues of thapsigargin, such as thapsigargicin and thapsitranstagin, might also be new non-TPA type tumor promoters, because thapsigargicin and thapsitranstagin induced irritation of mouse ear and HDC activity in mouse skin.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Dose-Response Relationship, Drug; Enzyme Induction; Female; Histamine Release; Histidine Decarboxylase; Irritants; Lactones; Mice; Mice, Inbred Strains; Ornithine Decarboxylase; Plant Extracts; Sesquiterpenes; Skin Neoplasms; Tetradecanoylphorbol Acetate; Thapsigargin; Tritium

Ten new tumor promoters which are structurally different from TPA but of similar biological activity were found. Based on their binding to the phorbol ester receptors of cell membranes, these new tumor promoters were classified as TPA-type tumor promoters, teleocidin and aplysiatoxin, which like TPA, activated protein kinase C in vitro, whereas two non-TPA-type tumor promoters, palytoxin and thapsigargin did not induce ODC activity in mouse skin, adhesion of HL-60 cells or activation of protein kinase C, but did show tumor-promoting activity in a two-stage carcinogenesis experiment. Although these two types of tumor promoter exert their tumor-promoting activities through different pathways, production of prostaglandin E2 by rat macrophages was induced by both the TPA-type and non-TPA-type promoters. Therefore, stimulation of arachidonic acid metabolism is suggested to be one of the important biological activities for tumor promotion.

Topics: Acrylamides; Animals; Arachidonic Acid; Arachidonic Acids; Carcinogens; Cnidarian Venoms; Enzyme Activation; Humans; Lyngbya Toxins; Mice; Neoplasms, Experimental; Phorbols; Plant Extracts; Protein Kinase C; Receptors, Cell Surface; Skin Neoplasms; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Thapsigargin