benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and beta-thujaplicin

Tropolones, such as β-thujaplicin, are small lead-like natural products that possess a variety of biological activities. While the β-substituted natural products and their synthetic analogs are potent inhibitors of human cancer cell growth, less is known about their α-substituted counterparts. Recently, we synthesized a series of α-substituted tropolones including 2-hydroxy-7-(naphthalen-2-yl)cyclohepta-2,4,6-trien-1-one (α-naphthyl tropolone). Here, we evaluate the antiproliferative mechanisms of α-naphthyl tropolone and the related α-benzodioxinyl analog. The α-substituted tropolones inhibit growth of lymphocytic leukemia cells, but not healthy blood cells, with nanomolar potency. Treatment of leukemia cell lines with the tropolone dose-dependently induces apoptosis as judged by staining with annexin V and propidium iodide and Western blot analysis of cleaved caspase 3 and 7. Moreover, pre-treatment of cells with the caspase inhibitor Z-VAD-FMK inhibited the apoptotic effects of the tropolone in two lymphocytic lines. Caspase inhibition also blocked elevated histone acetylation caused by the tropolone, indicating that its effects on histone acetylation are potentiated by caspases. In contrast, α-naphthyl tropolone upregulated p53 expression and phosphorylation of Akt and mTOR in a manner that was not rescued by caspase inhibition. The effects of tropolone were blocked by co-incubation with high levels of free extracellular iron but not by pre-loading with iron. Additionally, dose and time dependent reduction in ex vivo viability of cells from leukemia patients was observed. Taken together, we demonstrate that α-substituted tropolones upregulate DNA damage repair pathways leading to caspase-dependent apoptosis in malignant lymphocytes.

Topics: Acetylation; Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Cell Line; Cell Proliferation; Cell Survival; DNA Repair; Histones; Humans; Leukemia; Leukocytes, Mononuclear; Monoterpenes; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Tropolone; Tumor Suppressor Protein p53; Up-Regulation

Hinokitiol, a potent iron chelator, has been reported to induce differentiation in teratocarcinoma F9 cells with a reduction of viable cells. In this study, we examined the steps leading to eventual cell death by hinokitiol during differentiation. Hinokitiol induced DNA fragmentation of F9 cells in a concentration- and time-dependent manner. This effect was also observed in a cell-free system using the nuclei from intact cells and the cytosols from hinokitiol-treated cells. In contrast, hinokitiol methyl ether and hinokitiol-Fe (III) complex, which are deficient in iron-chelating activity, showed no DNA fragmentation activity in both cell culture and cell-free systems. These results suggest that iron deprivation by hinokitiol may be involved in the induction of apoptosis of F9 cells. Caspase-3, one of the key enzymes in the apoptotic cascade, was specifically activated by hinokitiol treatment, but not by the other two derivatives. In addition, its specific inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, strongly blocked hinokitiol-induced DNA fragmentation. These results indicate that iron deprivation by hinokitiol can induce apoptosis of F9 cells through the activation of caspase-3.

Topics: Amino Acid Chloromethyl Ketones; Aniline Compounds; Apoptosis; Caspase 3; Caspases; Cell-Free System; Cysteine Proteinase Inhibitors; DNA Fragmentation; Embryonal Carcinoma Stem Cells; Enzyme Activation; Iron Chelating Agents; Monoterpenes; Neoplastic Stem Cells; Oligopeptides; Teratocarcinoma; Tropolone