naphthoquinones and Retinoblastoma

We investigated the cytotoxic effects of plumbagin on metastatic retinoblastoma, using the highly metastatic cell line Y79.. Effect of plumbagin on cell growth was assessed with water-soluble tetrazolium 1 (WST-1) cell proliferation assay and automated hemocytometry with trypan blue-exclusion assay. Cell death was studied with acridine orange/ethidium bromide live-dead assay and annexin-V-fluorescein isothiocyanate/propidium iodide microscopy. Loss of mitochondrial membrane potential was studied with JC-10 dye and caspase activation was investigated using CellEvent Caspase-3/7 Green detection reagent.. Plumbagin highly significantly reduced the growth of Y79 cells treated for 24 h with 2.5 μM or more. Plumbagin also induced significantly high levels of cell death which was associated with loss of mitochondrial membrane potential and caspase activation.. At very low concentration (2.5 μM), plumbagin potently induced cytotoxicity in metastatic retinoblastoma cells via loss of mitochondrial membrane potential and caspase activation.

Topics: Antineoplastic Agents, Phytogenic; Caspases; Cell Death; Cell Line, Tumor; Cell Proliferation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Neoplasm Metastasis; Retinal Neoplasms; Retinoblastoma

Paclitaxel (PTX) and beta-lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 microM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p-Akt levels and in ps166-MDM2, the phosphorylated-MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt-MDM2-p53 complex and increased nuclear p53 levels. Our results suggest that phospho-Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho-Akt and inhibitor of apoptosis proteins.

Topics: Active Transport, Cell Nucleus; Androstadienes; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 6; Cell Line, Tumor; Cell Nucleus; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Drug Synergism; Humans; Inhibitor of Apoptosis Proteins; Lamin Type B; Naphthoquinones; Paclitaxel; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Protein Stability; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; Retinoblastoma; Time Factors; Transfection; Tumor Suppressor Protein p53; Wortmannin

To investigate the cytotoxicity of beta-lapachone, a potent agent that may selectively target tumour cells, in retinoblastoma (RB) cell lines.. Growth inhibitory effects of beta-lapachone were evaluated in Y79, WERI-RB1, and RBM human retinoblastoma cell lines. Pro-apoptotic effects of beta-lapachone were evaluated in Y79 cells by detection of caspase 3/7 activity, by enzyme-linked immunosorbent assay for nucleosome fragments, and by cellular morphological analysis.. Beta-lapachone induced significant dose-dependent growth inhibitory effects in all three retinoblastoma cell lines. The 50% growth inhibitory concentration (IC(50)) of this agent was 1.9 microM in Y79 cells, 1.3 microM in WERI-RB1 cells, and 0.9 microM in RBM cells. Beta-lapachone also induced proapoptotic effects in RB cells. Treatment of Y79 cells with 1.9 microM beta-lapachone (IC(50)) resulted in a peak, fourfold induction of caspase 3/7 activity at 72 h post-treatment; a peak, 5.6-fold increase in nucleosome fragments at 96 h post-treatment; and a peak, 1.7-fold increase in the frequency of apoptotic cells at 48 h post-treatment, relative to vehicle-treated controls.. Beta-lapachone induced potent cytotoxic effects in RB cell lines at low micromolar concentrations, suggesting this agent could be useful in the clinical management of RB.

Topics: Apoptosis; Caspase 3; Caspase 7; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Induction; Enzyme-Linked Immunosorbent Assay; Humans; Naphthoquinones; Retinal Neoplasms; Retinoblastoma; Reverse Transcriptase Inhibitors; Tumor Cells, Cultured