cytochrome-c-t and Lymphoma

Lymphoma is among the top 10 leading causes of cancer-related morbidity around the world in males, but currently, there is a lack of effective treatment strategies for this disease. Recently, we identified an alternatively spliced protein isoform, CMTM1-v5, which is significantly associated with tumor development and could serve as a potential therapeutic drug for lymphoma. Here, we showed that the overexpression of CMTM1-v5 in Raji cells or the addition of the CMTM1-v5 polypeptide to the cell culture medium induced apoptosis in vitro. During the in vivo experiments, most of the fluorescent CMTM1-v5 polypeptide converged within the tumor cells in Raji xenografts 24 h after treatment, and the injection of the polypeptide into the tail vein significantly extended survival in mice bearing Raji tumor cells. Mechanistically, the interaction between CMTM1-v5 and CAML (calcium-modulating cyclophilin ligand) negatively regulated the Ca

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Autophagy-Related Protein 5; Calcium; Caspases; Cell Line; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum; HEK293 Cells; HeLa Cells; Humans; K562 Cells; Lymphoma; Mice; Mice, Inbred NOD; Mice, SCID; Mitochondria; Peptides; U937 Cells

RU486 (mifepristone) exerts an anticancer effect on cancer cells via induction of apoptosis. However, the molecular mechanisms are not fully understood. Here, we investigated the effect of RU486 on the apoptosis of U937 human leukemia cells. RU486 markedly increased apoptosis in U937 cells as well as in MDA231 human breast carcinoma, A549 human lung adenocarcinoma epithelial and HCT116 human colorectal carcinoma cells. RU486 increased dose-dependent release of mitochondrial cytochrome c, and reduced the mitochondrial membrane potential (MMP, Δψm) in RU486-treated U937 cells. We also found that overexpression of Bcl-2 completely blocked RU486-mediated apoptosis. However, reactive oxygen species signaling had no effect on RU486-induced apoptosis. RU486 increased the phosphorylation of p38 MAPK and JNK, but p38 MAPK only was associated with RU486-mediated apoptosis. Taken together, RU486 induces apoptosis through reduction in the mitochondrial membrane potential and activation of p38 MAPK in U937 human leukemia cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Enzyme Activation; Female; HCT116 Cells; Hormone Antagonists; Humans; Leukemia; Lung Neoplasms; Lymphoma; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mifepristone; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Glucocorticoid; U937 Cells

3β,16β,17α-Trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1→3)-2-O-acetyl-α-L-arabinopyranoside (OSW-1) is a natural product with potent antitumor activity against various types of cancer cells, but the exact mechanisms of action remain to be defined. In this study, we showed that OSW-1 effectively killed leukemia cells at subnanomolar concentrations through a unique mechanism by causing a time-dependent elevation of cytosolic Ca(2+) prior to induction of apoptosis. A mechanistic study revealed that this compound inhibited the sodium-calcium exchanger 1 on the plasma membrane, leading to an increase in cytosolic Ca(2+) and a decrease in cytosolic Na(+). The elevated cytosolic Ca(2+) caused mitochondrial calcium overload and resulted in a loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3. Furthermore, OSW-1 also caused a Ca(2+)-dependent cleavage of the survival factor GRP78. Inhibition of Ca(2+) entry into the mitochondria by the uniporter inhibitor RU360 or by cyclosporin A significantly prevented the OSW-1-induced cell death, indicating the important role of mitochondria in mediating the cytotoxic activity. The extremely potent activity of OSW-1 against leukemia cells and its unique mechanism of action suggest that this compound may be potentially useful in the treatment of leukemia.

Topics: Biological Products; Calcium; Calcium Channels; Calpain; Caspase 3; Cell Death; Cell Line, Tumor; Cholestenones; Cyclosporine; Cytochromes c; Cytosol; Drug Screening Assays, Antitumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Activation; Extracellular Space; Heat-Shock Proteins; Homeostasis; Humans; Leukemia; Lymphoma; Membrane Potential, Mitochondrial; Mitochondria; Saponins; Sodium-Calcium Exchanger; Thapsigargin; Time Factors

Acquired resistance to drugs commonly used for lymphoma treatment poses a significant barrier to improving lymphoma patient survival. Previous work with a lymphoma tissue culture model indicates that selection for resistance to oxidative stress confers resistance to chemotherapy-induced apoptosis. This suggests that adaptation to chronic oxidative stress can contribute to chemoresistance seen in lymphoma patients. Oxidative stress-resistant WEHI7.2 cell variants in a lymphoma tissue culture model exhibit a range of apoptosis sensitivities. We exploited this phenotype to test for mitochondrial changes affecting sensitivity to apoptosis in cells made resistant to oxidative stress. We identified impaired release of cytochrome c, and the intermembrane proteins adenylate kinase 2 and Smac/DIABLO, indicating inhibition of the pathway leading to permeabilization of the outer mitochondrial membrane. Blunting of a glucocorticoid-induced signal and intrinsic mitochondrial resistance to cytochrome c release contributed to both points of resistance. The level of Bcl-2 family members or a difference in Bim induction were not contributing factors. The extent of cardiolipin oxidation following dexamethasone treatment, however, did correlate with apoptosis resistance. The differences found in the variants were all proportionate to the degree of resistance to glucocorticoid treatment. We conclude that tolerance to oxidative stress leads to mitochondrial changes that confer resistance to apoptosis.

Topics: Adaptation, Physiological; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cardiolipins; Cytochromes c; Immunoblotting; Lymphoma; Mice; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Thymus Neoplasms; Tumor Cells, Cultured

Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways.

Topics: Aminolevulinic Acid; Apoptosis; Calcium; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Endoplasmic Reticulum; Humans; Light; Lymphoma; Mitochondria; Photosensitizing Agents; Poly(ADP-ribose) Polymerases; Protoporphyrins; Sarcoplasmic Reticulum Calcium-Transporting ATPases

We examined the effect of apogossypolone (ApoG2), a new derivative from gossypol on cell cycle regulation in U937 human leukemic monocyte lymphoma cells in vitro. ApoG2 decreased the viability of U937 cells by inducing G1 arrest followed by apoptosis in a dose-dependent manner. The G0/G1 phase of the cell cycle is regulated by cyclin-dependent kinases (Cdk), cyclins and cyclin-dependent kinase inhibitors (Cdki). We show by western blot analysis, that the ApoG2-induced G1 arrest was mediated through the increased expression of Cdki proteins (p21cip1/waf1) with a simultaneous decrease in cdk2, cdk4, cyclin D1 and cyclin E expression. The induction of apoptosis after treatment with ApoG2 for 12, 24 and 48 h was demonstrated by flow cytometry analysis. ApoG2 also induced cytochrome c release and activation of caspase-3. To our knowledge, this is the first time that ApoG2 has been reported to potently inhibit the proliferation of human monocytic lymphoma U937 cells through G1 arrest. These findings suggest that ApoG2 may be a potential chemotherapeutic agent for the treatment of cancer.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Cycle; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Dose-Response Relationship, Drug; Gossypol; Humans; Lymphoma; Mitochondria; Proto-Oncogene Proteins c-myc; Time Factors; U937 Cells

Despite the significant implication of apoptosis in tumorigenesis, there is no biomarker to assess the extent of ongoing apoptosis in vivo for hematological malignancies. We investigated the potential of serum cytochrome c (cyto-c) as a biomarker for apoptosis. Cyto-c and lactate dehydrogenase (LD) were released into the culture medium from apoptotic cells induced by tumor necrosis factor-related apoptosis-inducing ligand in a time-dependent manner in vitro, with different kinetic patterns. Only one-third of 153 patients with hematological malignancies showed high levels of serum cyto-c (>20 ng/ml). Although serum cyto-c level was roughly correlated to serum LD activity, their different kinetic patterns from serial measurements indicated that serum cyto-c rather than LD is a more sensitive indicator for tracking changes of tumor status. Furthermore, serum cyto-c level stratified patients with acute adult T-cell leukemia into favorable and unfavorable subgroups with 5-year survival rates of 67%vs. 11%. In conclusion, serum cyto-c may provide a fast real-time biomarker for tracking changes of tumor status involved in apoptotic cell death, but lacking disease or cell-type specificity.

Topics: Apoptosis; Biomarkers, Tumor; Cytochromes c; Humans; L-Lactate Dehydrogenase; Leukemia; Lymphoma

Arsenic trioxide (As2O3) is an effective therapy in acute promyelocytic leukemia (APL), but its use in other malignancies is limited by the higher concentrations required to induce apoptosis. We have reported that trolox, an analogue of alpha-tocopherol, increases As2O3-mediated apoptosis in a variety of APL, myeloma and breast cancer cell lines, while non-malignant cells may be protected. In the present study, we extended previous results to show that trolox increases As2O3-mediated apoptosis in the P388 lymphoma cell line in vitro, as evidenced by decrease of mitochondrial membrane potential and release of cytochrome c. We then sought to determine whether this combination can enhance antitumor effects while protecting normal cells in vivo. In BDF1 mice, trolox treatment decreased As2O3-induced hepatomegaly, markers of oxidative stress and hepatocellular damage. In P388 tumor-bearing mice, As2O3 treatment prolonged survival, and the addition of trolox provided a further significant increase in lifespan. In addition, the combination of As2O3 and trolox inhibited metastatic spread, and protected the tumor-bearing mice from As2O3 liver toxicity. Our results suggest, for the first time, that trolox might prevent some of the clinical manifestations of As2O3-related toxicity while increasing its pro-apoptotic capacity and clinical efficacy in hematological malignancies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Line, Tumor; Chromans; Cytochromes c; Drug Synergism; Humans; Liver; Lymphoma; Membrane Potential, Mitochondrial; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Oxides

Nur77 is reported to undergo translocation to mitochondria in response to apoptotic signaling in a variety of cancer cell lines. It was shown that on the mitochondrial membrane, Nur77 interacts with Bcl-2, leading to the conversion of this protein from a protector to a killer with subsequent release of cytochrome c to the cytosol. Here it is shown that in thymic lymphoma cells resistant to calcium-mediated apoptosis, cytochrome c release is abolished despite of Nur77 mitochondrial targeting. However, cytochrome c release and apoptosis can be restored by treatment with FK506. Hence, the molecular target regulation of the sensitivity of lymphoma cells to calcium signaling is associated with cytochrome c release and is FK506 sensitive. These results provide new insight into the role of FK506-sensitive factors as a critical link between calcium signaling and resistance of lymphoma cells to death.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Nucleus; Cytochromes c; Cytoplasm; DNA-Binding Proteins; Immunosuppressive Agents; Lymphoma; Mice; Mitochondria; Nuclear Receptor Subfamily 4, Group A, Member 1; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Signal Transduction; Tacrolimus; Transcription Factors

Phosphatidylinositol 3'-kinase (PI3'-kinase) can be activated by the K1 protein of Kaposi sarcoma-associated herpes virus (KSHV). However, the role of PI3'-kinase in KSHV-associated primary effusion lymphoma (PEL) is not known. To assess this, we studied survival and apoptosis in PEL cell lines following inhibition of PI3'-kinase.. Constitutive activation of several targets of PI3-kinase and apoptotic proteins were determined by Western blot analysis using specific antibodies. We used LY294002 to block PI3'-kinase/AKT activation and assess apoptosis by flow cytometric analysis.. Blocking PI3'-kinase induced apoptosis in PEL cells, including BC1, BC3, BCBL1, and HBL6, whereas BCP1 was refractory to LY294002-induced apoptosis. LY294002-induced apoptosis did not seem to involve Fas/Fas-L but had an additive effect to CH11-mediated apoptosis. We also show that AKT/PKB is constitutively activated in all PELs and treatment with LY294002 causes complete dephosphorylation in all cell lines except BCP1 where a residual AKT phosphorylation remained after 24 hours of treatment. FKHR and GSK3 were also constitutively phosphorylated in PELs and treatment with LY294002 caused their dephosphorylation. Although inhibition of PI3'-kinase induced cleavage of BID in all cell lines, cytochrome c was released from the mitochondria and caspase-9 and caspase-3 were activated in LY294002-induced apoptotic BC1 but not in resistant BCP1. Similarly, XIAP, a target of AKT, was down-regulated after LY294002 treatment only in sensitive PEL cells.. Our data show that the PI3'-kinase pathway plays a major role in survival of PEL cells and suggest that this cascade may be a promising target for therapeutic intervention in primary effusion lymphomas.

Topics: Antibodies, Monoclonal; Apoptosis; Cell Line, Tumor; Chromones; Cytochromes c; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; fas Receptor; Flow Cytometry; Forkhead Box Protein O1; Forkhead Transcription Factors; Glycogen Synthase Kinase 3; Humans; Immunoblotting; Lymphoma; Mitochondria; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Pleural Effusion, Malignant; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Time Factors; Transcription Factors; X-Linked Inhibitor of Apoptosis Protein

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Carcinoma, Small Cell; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Drug Synergism; Humans; Lymphoma; Magnetic Resonance Spectroscopy; Mice; Mitochondria; Models, Molecular; Neoplasms; Nitrophenols; Paclitaxel; Piperazines; Proto-Oncogene Proteins c-bcl-2; Structure-Activity Relationship; Sulfonamides; Survival Rate

Sapphyrins are pentapyrrolic, metal-free, expanded porphyrins. In the present study, the activity of sapphyrins as anticancer agents in hematopoietic-derived tumor cells was explored. It was found that a dihydroxylated water-soluble sapphyrin derivative (PCI-2000) is a potent inducer of apoptosis in a wide variety of tumor cell lines including lymphoma (Ramos, DHL-4, and HF-1), leukemia (Jurkat and HL-60), and myeloma (8226/S, 1-310, C2E3, and 1-414). PCI-2000 triggers an apoptotic pathway in these tumor cells as shown by release of cytochrome c from mitochondria; activation of caspases 9, 8, and 3; cleavage of the caspase substrate poly(ADP-ribose) polymerase; and Annexin V binding. Apoptosis can be partially inhibited by overexpression of the antiapoptotic protein Bcl-2 or treatment with benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethylketone, a cell-permeable caspase inhibitor. Both PCI-2000 and PCI-2010, a tetrahydroxy bis-carbamate derivative of PCI-2000, result in increased levels of phosphorylated p38 mitogen-activated protein kinase. Inhibition of p38 mitogen-activated protein kinase phosphorylation resulted in a synergistic increase of PCI-2000 cytotoxicity. PCI-2010 showed less toxicity in mice than PCI-2000 and was active in slowing the growth of Ramos and HL-60 tumor xenografts in nude mice. These results provide preclinical rationale for the further study of sapphyrins for potential use in the treatment of hematopoietic-derived tumors.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Caspases; Cell Line, Tumor; Cytochromes c; Cytoplasm; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Leukemia; Lymphoma; Mice; Mice, Nude; Mitochondria; Molecular Structure; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Porphyrins; Proto-Oncogene Proteins c-bcl-2; Xenograft Model Antitumor Assays

The mechanisms that regulate induction of the antiapoptotic state and mitogenic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we found that curcumin (diferuloylmethane), a natural compound isolated from the plant Curcuma Ionga, inhibits cell proliferation and induces apoptosis in a dose dependent manner in several PEL cell lines. Such effects of curcumin appear to result from suppression of the constitutively active STAT3 through inhibition of Janus kinase 1 (JAK1). Our data also demonstrate that curcumin induces loss of mitochondrial membrane potential with subsequent release of cytochrome c and activation of caspase-3, followed by polyadenosin-5'-diphosphate-ribose polymerase (PARP) cleavage. Altogether, our findings suggest a novel function for curcumin, acting as a suppressor of JAK-1 and STAT3 activation in PEL cells, leading to inhibition of proliferation and induction of caspase-dependent apoptosis. Therefore, curcumin may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of STAT3.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Proliferation; Curcumin; Cytochromes c; DNA-Binding Proteins; Enzyme Activation; Humans; Janus Kinase 1; Lymphoma; Membrane Potentials; Mitochondria; Pleural Effusion, Malignant; Poly(ADP-ribose) Polymerases; Protein-Tyrosine Kinases; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents. We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases. To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis. CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential. The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe. The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein. These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis. The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Caspases; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cell Survival; Cytochromes c; DNA Mutational Analysis; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Activation; Green Fluorescent Proteins; Humans; Hydrolysis; Hydroxamic Acids; Idarubicin; Lymphoma; Mitosis; Mutation; Retroviridae; Structure-Activity Relationship; Time Factors; Vincristine

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.

Topics: Animals; Antigen-Presenting Cells; Apoproteins; B-Lymphocytes; Bone Marrow Cells; Cell Line; Clone Cells; Cytochrome c Group; Cytochromes c; Epitopes; Lymphocyte Activation; Lymphokines; Lymphoma; Macrophages; Mice; Mice, Inbred BALB C; Spleen; T-Lymphocytes