Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 microM curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. We conclude that curcumin-induced cytotoxicity was due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes.

Cells that form the corneal epithelium, the outermost layer of the cornea, are exposed to shear stress through blinking during waking hours. In this in vitro study, the effect of fluid shear stress on human corneal epithelial cells (HCECs) was investigated. Following exposure to shear stresses of 4 and 8 dyn/cm2, HCECs showed cytoskeletal rearrangement with more prominent, organized and elongated filamentous actin. Cytoskeletal changes were time-dependent, and were most significant after 24 hours of shear stress. Higher rates of migration and proliferation, as evaluated by a scratch assay, were also observed following 24 hours of low shear stress exposure (4 dyn/cm2). This result contrasted the poor migration observed in samples scratched before shear exposure, indicating that shear-induced cytoskeletal changes played a key role in improved wound healing and must therefore precede any damage to the cell layer. HCEC cytoskeletal changes were accompanied by an upregulation in integrin β1 and downregulation of ICAM-1. These results demonstrate that HCECs respond favourably to flow-induced shear stress, impacting their proliferation and migration properties as well as phenotype.

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Abstract. 2-Chloro-2'-deoxyadenosine (CdA; cladribine) is a chemotherapeutic agent used in the treatment of certain leukemias. However, the signalling events that govern CdAmediated cytotoxicity in leukemia cells remain unclear. We show here that CdA treatment caused Jurkat human T leukemia cells to die via apoptosis in a dose-and time-dependent fashion. Bcl-2 overexpression protected Jurkat T leukemia cells from CdA-induced apoptosis and loss of mitochondrial transmembrane potential (ΔΨ m ). Furthermore, mitochondria that were isolated from Jurkat T leukemia cells and then exposed to CdA showed a loss of ΔΨ m , indicating that CdA directly compromised outer mitochondrial membrane integrity. CdA treatment of Jurkat T leukemia cells resulted in the activation of caspase-3, -8, and -9, while inhibition of these caspases prevented the CdA-induced loss of ΔΨ m , as well as DNA fragmentation. In addition, caspase-3 inhibition prevented caspase-8 activation while caspase-8 inhibition prevented caspase-9 activation. Death receptor signalling was not involved in CdA-induced apoptosis since cytotoxicity was not affected by FADD-deficiency or antibody neutralization of either Fas ligand or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Taken together, these data suggested that CdA-induced apoptosis in Jurkat T leukemia cells was mediated via a caspase-3-dependent mitochondrial feedback amplification loop. CdA treatment also increased p38 mitogen-activated protein (MAPK) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in Jurkat T leukemia cells. Although ERK1/2 inhibition did not affect CdA-mediated cytotoxicity, inhibition of p38 MAPK had an enhancing effect, which suggested a cytoprotective function for p38 MAPK. Agents that inhibit p38 MAPK might therefore increase the effectiveness of CdA-based chemotherapy.

Abstract. 2-Chloro-2'-deoxyadenosine (CdA; cladribine) is a chemotherapeutic agent used in the treatment of certain leukemias. However, the signalling events that govern CdAmediated cytotoxicity in leukemia cells remain unclear. We show here that CdA treatment caused Jurkat human T leukemia cells to die via apoptosis in a dose-and time-dependent fashion. Bcl-2 overexpression protected Jurkat T leukemia cells from CdA-induced apoptosis and loss of mitochondrial transmembrane potential (ΔΨ m ). Furthermore, mitochondria that were isolated from Jurkat T leukemia cells and then exposed to CdA showed a loss of ΔΨ m , indicating that CdA directly compromised outer mitochondrial membrane integrity. CdA treatment of Jurkat T leukemia cells resulted in the activation of caspase-3, -8, and -9, while inhibition of these caspases prevented the CdA-induced loss of ΔΨ m , as well as DNA fragmentation. In addition, caspase-3 inhibition prevented caspase-8 activation while caspase-8 inhibition prevented caspase-9 activation. Death receptor signalling was not involved in CdA-induced apoptosis since cytotoxicity was not affected by FADD-deficiency or antibody neutralization of either Fas ligand or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Taken together, these data suggested that CdA-induced apoptosis in Jurkat T leukemia cells was mediated via a caspase-3-dependent mitochondrial feedback amplification loop. CdA treatment also increased p38 mitogen-activated protein (MAPK) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in Jurkat T leukemia cells. Although ERK1/2 inhibition did not affect CdA-mediated cytotoxicity, inhibition of p38 MAPK had an enhancing effect, which suggested a cytoprotective function for p38 MAPK. Agents that inhibit p38 MAPK might therefore increase the effectiveness of CdA-based chemotherapy.