cytochrome-c-t and Lymphoma--Non-Hodgkin

Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady-state levels of key antioxidant enzymes, with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. In this study, we examined the anti-proliferation effect of mimic of manganese superoxide dismutase (MnSODm) on human non-Hodgkin lymphoma Raji cells. The results showed that MnSODm significantly reduced the proliferation of Raji cells in a concentration and a time-dependent manner. By flow cytometric analysis, we found that MnSODm treatment resulted in an increased apoptosis in Raji cells. MnSODm also increased the production of ROS and the expression levels of cleaved caspase-9, caspase-3, poly (ADP-ribose) polymerase (PARP) and Bax in Raji cells. Moreover, the expression of Bcl-2 protein showed down-regulation in the MnSODm treatment group. In addition, MnSODm significantly elevated the level of cytochrome c in cytosol. These findings suggest that the activation of the mitochondrial pathway is involved in MnSODm-induced apoptosis in Raji cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; Humans; Lymphoma, Non-Hodgkin; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Superoxide Dismutase

Lym-1 was one of the first antibodies to be used successfully for the radioimmunotherapy of the human malignant lymphomas. This antibody, which recognizes the HLA-DR10 antigen preferentially expressed in B-cell lymphomas, was recently shown to induce apoptosis upon binding to lymphoma cells. In this study, Lym-1-induced apoptosis was studied to identify the potential molecular pathways of programmed cell death and to demonstrate the clinical potential of this antibody in the treatment of the human malignant lymphomas. Immunofluorescence microscopy revealed that Lym-1 stained focal areas of the cell surface, consistent with the fact that the HLA-DR10 antigen is associated with lipid rafts, a known prerequisite for apoptosis signaling. Likewise, Annexin V/propidium iodide staining and TUNEL assays demonstrated that both murine Lym-1 and chimeric Lym-1 induced both early and late apoptosis, respectively, unlike anti-CD20 rituximab. Furthermore, Lym-1 was found to produce a rapid loss of mitochondrial membrane potential and mitochondrial release of cytochrome C 14 hours post-Lym-1 treatment. Although it was found to activate caspase-3, inhibitors of caspase pathways showed that the Lym-1-induced apoptosis in lymphoma cell lines is independent of caspase induction. Finally, treatment studies in vivo demonstrated that, compared with murine anti-CD20 (2B8), Lym-1 was more effective in inducing the regression of human lymphoma xenografts. Based upon these results, chimeric Lym-1 should be especially effective in treating lymphoma patients, as, in addition to being able to elicit immune effector functions such as chimeric anti-CD20, it can also induce apoptosis directly upon cell binding.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; Enzyme Activation; HLA-DR Antigens; HLA-DR Serological Subtypes; Humans; Jurkat Cells; Lymphoma, Non-Hodgkin; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Protease Inhibitors; Rituximab; Transplantation, Heterologous

Gallium nitrate is a metallodrug with clinical efficacy in non-Hodgkin's lymphoma. Its mechanisms of antineoplastic action are not fully understood. In the present study, we investigated the roles of transferrin receptor (TfR) targeting and apoptotic pathways in gallium-induced cell death. Although DoHH2 lymphoma cells displayed a 3-fold lower number of TfRs than CCRF-CEM lymphoma cells, they were 3- to 4-fold more sensitive to gallium nitrate. Despite a lower TfR expression, DoHH2 cells had greater TfR cycling and iron and gallium uptake than CCRF-CEM cells. In other lymphoma cell lines, TfR levels per se did not correlate with gallium sensitivity. Cells incubated with gallium nitrate showed morphologic changes of apoptosis, which were decreased by the caspase inhibitor Z-VAD-FMK and by a Bax-inhibitory peptide. Cells exposed to gallium nitrate released cytochrome c from mitochondria and displayed a dose-dependent increase in caspase-3 activity. An increase in active Bax levels without accompanying changes in Bcl-2 or Bcl-X(L) was seen in cells incubated with gallium nitrate. The endogenous expression of antiapoptotic Bcl-2 was greater in DoHH2 cells than in CCRF-CEM cells, suggesting that endogenous Bcl-2 levels do not correlate with cell sensitivity to gallium nitrate. Gallium-induced apoptosis was enhanced by the proteasome inhibitor bortezomib. Our results suggest that TfR function rather than TfR number is important in gallium targeting to cells and that apoptosis is triggered by gallium through the mitochondrial pathway by activating proapoptotic Bax. Our studies also suggest that the antineoplastic activity of combination gallium nitrate and bortezomib warrants further investigation.

Topics: Antineoplastic Agents; bcl-2-Associated X Protein; Caspase 3; Caspase Inhibitors; Cell Death; Cytochromes c; Dose-Response Relationship, Drug; Gallium; Humans; Iron; Jurkat Cells; Lymphoma, Non-Hodgkin; Mitochondria; Proteasome Endopeptidase Complex; Receptors, Transferrin; Signal Transduction; Tumor Cells, Cultured

Previous studies have shown that unlike most apoptotic cells, Raji cells do not externalize phosphatidylserine (PS) upon apoptosis. Here we show that Raji cells are resistant to intrinsic apoptogenic agents, but sensitive to extrinsically triggered Fas-induced apoptosis. Treatment of intrinsic apoptosis-competent Jurkat cells with vitamin E implicated reactive oxygen species in intrinsic apoptosis because, like Raji cells, they became resistant to actinomycin D- but not Fas-triggered apoptosis. Oxidation of sulfhydryls in both cell types with N-ethylmaleimide resulted in rapid disruption of the mitochondrial membrane potential, release of cytochrome c from the mitochondria to the cytoplasm, and externalization of PS by a mechanism that was not inhibited by the pan caspase inhibiter zVAD-fmk. These results suggest that although cell death and PS externalization are both cytochrome c-dependent, they are distinct and separable processes.

Topics: Antibiotics, Antineoplastic; Antioxidants; Apoptosis; Cell Line, Tumor; Cell Membrane; Cytochromes c; Dactinomycin; Ethylmaleimide; fas Receptor; Humans; Jurkat Cells; Lymphoma, Non-Hodgkin; Membrane Potentials; Oxidation-Reduction; Phosphatidylserines; Reactive Oxygen Species; Sulfhydryl Compounds; Sulfhydryl Reagents; Vitamin E

The anti-CD20 monoclonal antibody rituximab (Rituxan, IDEC-C2B8) has shown promising results in the clinical treatment of a subset of patients with low grade or follicular non-Hodgkin's lymphoma (NHL). However, chemotherapy- and rituximab-refractory NHL patients may benefit from a regimen in which rituximab acts as a sensitizing agent. This study examined the apoptotic signaling mediated by rituximab on rituximab- and paclitaxel-resistant CD20(+) NHL B cell lines (Ramos, Raji, Daudi, and 2F7). Treatment with either rituximab (20 micro g/ml) or paclitaxel (0.1-1000 nM) inhibited viable cell recovery of NHL lines. Neither rituximab nor paclitaxel induced significant apoptosis, although the combination treatment resulted in synergy in apoptosis. Rituximab selectively down-regulated Bcl-xL and induced apoptosis protease activating factor 1 (Apaf-1) expressions in Ramos cells. Paclitaxel down-regulated the expression of Bcl-xL and inhibitor of apoptosis proteins (c-IAP-1) and up-regulated the expression of Bad and Apaf-1. The combination treatment resulted in the formation of truncated Bid, cytosolic accumulation of cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI, activation of caspase-9, caspase-7, caspase-3, and cleavage of poly(ADP-ribose) polymerase. The findings identify two potential novel intracellular targets of rituximab-mediated signaling in Ramos NHL cells (i.e., Bcl-xL and Apaf-1). Further, the findings show that both rituximab and paclitaxel selectively modify the expression pattern of proteins involved in the apoptosis signal transduction pathway and, through functional complementation, the combination results in synergy in apoptosis. The potential therapeutic significance of these findings is discussed.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-X Protein; Caspase 3; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, Non-Hodgkin; Paclitaxel; Proteins; Proto-Oncogene Proteins c-bcl-2; Rituximab; Signal Transduction