Joya Chandra

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis

Previous studies have demonstrated that Ca2+ is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca2+ pool in human PC-3 prostate cancer cells. This mobilization of ER Ca2+ was associated with a concomitant increase in mitochondrial Ca2+ levels, and neither ER Ca2+ mobilization nor mitochondrial Ca2+ uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca2+ release and mitochondrial Ca2+ uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca2+ uniporter (RU-360) attenuated mitochondrial Ca2+ uptake, cytochrome crelease, and DNA fragmentation, directly implicating the mitochondrial Ca2+ changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca2+ levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.

Immune Clearance of Phosphatidylserine-expressing Cells by Phagocytes THE ROLE OF β2-GLYCOPROTEIN I IN MACROPHAGE RECOGNITION

The function of β2-glycoprotein I (β2GPI), a 50-kDa serum glycoprotein, is not completely understood but has been suggested to be involved in the regulation of thrombosis (Brighton, T. A., Hogg, P. J., Dai, Y.-P., Murray, B. H., Choing, B. H., and Chesterman, C. N. (1996) Br. J. Haematol. 93, 185–194) and the clearance of phosphatidylserine (PS)-expressing cells (Chonn, A., Semple S. C., and Cullis P. R. (1995)J. Biol. Chem. 270, 25845–25849). To further understand the role of this protein, we characterized the ability of β2GPI to interact with PS vesicles and influence their uptake by macrophages in vitro. β2GPI bound to and precipitated vesicles containing anionic but not zwitterionic phospholipids in a gel diffusion assay. β2GPI also inhibited the procoagulant activity of PS liposomes. In vitro phagocytosis studies showed 20-fold greater uptake of PS liposomes over phosphatidylcholine liposomes. This enhanced uptake was maintained even after PS was “shielded” with β2GPI and further increased upon the addition of β2GPI antibodies. Similar to liposomes, PS-expressing apoptotic thymocytes and lipid symmetric red blood cell ghosts bound β2GPI. Macrophage uptake of these cells was also maintained or enhanced in the presence of β2GPI and further increased upon the addition of β2GPI antibodies. It is concluded that β2GPI can play a critical role in hemostasis by influencing both thrombosis and the clearance of PS-expressing cells.

Gemcitabine-induced programmed cell death (apoptosis) of human pancreatic carcinoma is determined by bcl-2 content

AbstractBackground: Gemcitabine is a new nucleoside analogue that produces a clinical response in 30% of patients with unresectable pancreatic carcinoma. The cytotoxic effects of many chemotherapeutic agents occur through induction of programmed cell death (apoptosis), which is controlled by the bcl-2 gene family. We determined whether induction of apoptosis by gemcitabine in pancreatic carcinoma is associated with cellular Bcl-2 content. Methods: Four pancreatic carcinoma cell lines (MIA-PaCa-2, AsPC-1, Panc-1, and Panc-48) were screened by Western blotting for Bcl-2 protein expression. Dose-response relationships for the cytotoxic effects of gemcitabine were determined using methylthiotetrazole assays, and induction of apoptosis was confirmed by fluorescence-activated cell sorting analysis. MIA-PaCa-2 cells transfected with human bcl-2 were also analyzed for gemcitabine-induced apoptosis. Results: Pancreatic cancer cell lines expressed varying amounts of Bcl-2, and the 50% lethal dose for gemcitabine-induced apoptosis was correlated with Bcl-2 content. Furthermore, Bcl-2 overexpression was associated with a significant increase in the 50% lethal dose for gemcitabine-induced apoptosis. Conclusions: Cellular Bcl-2 content was directly correlated with the cytotoxicity of gemcitabine in pancreatic carcinoma. Therefore, routine immunohistochemical analyses may be useful in predicting gemcitabine efficacy, and patients who would likely not benefit could be spared gemcitabine administration. Furthermore, the effectiveness of gemcitabine and other chemotherapeutic agents may be increased by gene therapy-mediated alteration of bcl-2 gene family members.

Adenovirus-mediated wild-type p53 tumor suppressor gene therapy induces apoptosis and suppresses growth of human pancreatic cancer

AbstractBackground: Thep53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-typep53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-typep53 tumor suppressor gene. Methods: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/β-gal. Cell proliferation was monitored using a3H-thymidine incorporation assay, Western blot analysis forp53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis.p53 gene therapy was tested in vivo in a subcutaneous tumor model. Results: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (β-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the β-gal gene. Adenovirus-mediatedp53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. Conclusions: Introduction of the wild-typep53 gene using an adenoviral vector in pancreatic cancer withp53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediatedp53 tumor suppressor gene therapy of human pancreatic cancer.

Synergistic Activity of the Src Family Kinase Inhibitor Dasatinib and Oxaliplatin in Colon Carcinoma Cells Is Mediated by Oxidative Stress

Chemotherapeutic regimens for the treatment of colorectal cancer generally include oxaliplatin, although inherent and acquired resistance is common. One potential mediator of oxaliplatin sensitivity is the nonreceptor protein tyrosine kinase, Src, the activity of which correlates with disease stage and patient survival. Therefore, we investigated the effects of Src inhibition using the tyrosine kinase inhibitor dasatinib on oxaliplatin sensitivity. We show that oxaliplatin acutely activates Src and that combination treatment with dasatinib is synergistic in a cell-line dependent manner, with the level of Src activation correlating with extent of synergy in a panel of six cell lines. Intracellular reactive oxygen species (ROS) are generated after oxaliplatin treatment, and ROS potently activates Src. Pretreatment with antioxidants inhibits oxaliplatin-induced Src activation. In oxaliplatin-resistant cell lines, Src activity is constitutively increased. In a mouse model of colorectal liver metastases, treatment with oxaliplatin also results in chronic Src activation. The combination of dasatinib and oxaliplatin results in significantly smaller tumors compared with single-agent treatment, corresponding with reduced proliferation and angiogenesis. Therefore, we conclude that oxaliplatin activates Src through a ROS-dependent mechanism. Src inhibition increases oxaliplatin activity both in vitro and in vivo. These results suggest that Src inhibitors combined with oxaliplatin may have efficacy in metastatic colon cancer and may provide the first indication of a molecular phenotype that might be susceptible to such combinations.

The proteasome inhibitor NPI-0052 is a more effective inducer of apoptosis than bortezomib in lymphocytes from patients with chronic lymphocytic leukemia

Proteasome inhibitors are potent inducers of apoptosis in isolated lymphocytes from patients with chronic lymphocytic leukemia (CLL). However, the reversible proteasome inhibitor bortezomib (PS-341; Velcade) did not display substantial antitumor activity in CLL patients. Here, we compared the effects of bortezomib and a new irreversible proteasome inhibitor (NPI-0052) on 20S chymotryptic proteasome activity and apoptosis in isolated CLL cells in vitro. Although their steady-state (3 hours) IC50s as proteasome inhibitors were similar, NPI-0052 exerted its effects more rapidly than bortezomib, and drug washout experiments showed that short exposures to NPI-0052 resulted in sustained (≥24 hours) 20S proteasome inhibition, whereas 20S activity recovered in cells exposed to even 10-fold higher concentrations of bortezomib. Thus, brief (15 minutes) pulses of NPI-0052 were sufficient to induce substantial apoptosis in CLL cells, whereas longer exposure times (≥8 hours) were required for commitment to apoptosis in cells exposed to equivalent concentrations of bortezomib. Commitment to apoptosis seemed to be related to caspase-4 activation, in that cells exposed to bortezomib or NPI-0052 could be saved from death by addition of a selective caspase-4 inhibitor up to 8 hours after drug exposure. Our results show that NPI-0052 is a more effective proapoptotic agent than bortezomib in isolated CLL cells and suggest that the chemical properties of NPI-0052 might also make it an effective therapeutic agent in CLL patients. [Mol Cancer Ther 2006;5(7):1836–43]

Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM.

Therapeutic Strategies to Enhance the Anticancer Efficacy of Histone Deacetylase Inhibitors

Histone acetylation is a posttranslational modification that plays a role in regulating gene expression. More recently, other nonhistone proteins have been identified to be acetylated which can regulate their function, stability, localization, or interaction with other molecules. Modulating acetylation with histone deacetylase inhibitors (HDACi) has been validated to have anticancer effects in preclinical and clinical cancer models. This has led to development and approval of the first HDACi, vorinostat, for the treatment of cutaneous T cell lymphoma. However, to date, targeting acetylation with HDACi as a monotherapy has shown modest activity against other cancers. To improve their efficacy, HDACi have been paired with other antitumor agents. Here, we discuss several combination therapies, highlighting various epigenetic drugs, ROS-generating agents, proteasome inhibitors, and DNA-damaging compounds that together may provide a therapeutic advantage over single-agent strategies.

Caspase-8 dependent histone acetylation by a novel proteasome inhibitor, NPI-0052: a mechanism for synergy in leukemia cells

Combination studies of histone deacetylase inhibitors (HDACi) and proteasome inhibitors are providing preclinical framework to build better strategies against hematologic malignancies. Our previous work found that a novel proteasome inhibitor, NPI-0052, and HDACi synergistically induce apoptosis in leukemia cells in a caspase-8- and oxidant-dependent manner. Here we extend those observations to primary leukemia cells and identify novel mechanisms of synergy. Because the proximal targets of NPI-0052 and HDACi are inhibition of proteasome activity and histone acetylation, we initially examined those biochemical events. Increased acetylation of histone-H3 was detected in Jurkat and CLL primary cells treated with NPI-0052, alone or in combination with various HDACi (MS/SNDX-275 or vorinostat). Hyperacetylation by NPI-0052 occurred to a lesser extent in caspase-8-deficient cells and in cells treated with an antioxidant. These results indicate that NPI-0052 is eliciting caspase-8 and oxidative stress-dependent epigenetic alterations. In addition, real-time PCR revealed that MS/SNDX-275 repressed expression of the proteasomal beta5, beta2, and beta1 subunits, consequently inhibiting respective enzymatic activities. Overall, our results suggest that crosstalk by NPI-0052 and HDACi are contributing, along with caspase-8 activation and oxidative stress, to their synergistic cytotoxic effects in leukemia cells, reinforcing the potential clinical utility of combining these 2 agents.

Redox Control of Leukemia: From Molecular Mechanisms to Therapeutic Opportunities

Reactive oxygen species (ROS) play both positive and negative roles in the proliferation and survival of a cell. This dual nature has been exploited by leukemia cells to promote growth, survival, and genomic instability-some of the hallmarks of the cancer phenotype. In addition to altered ROS levels, many antioxidants are dysregulated in leukemia cells. Together, the production of ROS and the expression and activity of antioxidant enzymes make up the primary redox control of leukemia cells. By manipulating this system, leukemia cells gain proliferative and survival advantages, even in the face of therapeutic insults. Standard treatment options have improved leukemia patient survival rates in recent years, although relapse and the development of resistance are persistent challenges. Therapies targeting the redox environment show promise for these cases. This review highlights the molecular mechanisms that control the redox milieu of leukemia cells. In particular, ROS production by the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidoreductase, and cytochrome P450 will be addressed. Expression and activation of antioxidant enzymes such as superoxide dismutase, catalase, heme oxygenase, glutathione, thioredoxin, and peroxiredoxin are perturbed in leukemia cells, and the functional consequences of these molecular alterations will be described. Lastly, we delve into how these pathways can be potentially exploited therapeutically to improve treatment regimens and promote better outcomes for leukemia patients.