Adipose Recruitment and Activation of Plasmacytoid Dendritic Cells Fuel Metaflammation.

In obese individuals, visceral adipose tissue (VAT) is the seat of chronic low-grade inflammation (metaflammation), but the mechanistic link between increased adiposity and metaflammation largely remains unclear. In obese individuals, deregulation of a specific adipokine, chemerin, contributes to innate initiation of metaflammation by recruiting circulating plasmacytoid dendritic cells (pDCs) into VAT through chemokine-like receptor 1 (CMKLR1). Adipose tissue-derived high-mobility group B1 (HMGB1) protein activates Toll-like receptor 9 (TLR9) in the adipose-recruited pDCs by transporting extracellular DNA through receptor for advanced glycation end products (RAGE) and induces production of type I interferons (IFNs). Type I IFNs in turn help in proinflammatory polarization of adipose-resident macrophages. IFN signature gene expression in VAT correlates with both adipose tissue and systemic insulin resistance (IR) in obese individuals, which is represented by ADIPO-IR and HOMA2-IR, respectively, and defines two subgroups with different susceptibility to IR. Thus, this study reveals a pathway that drives adipose tissue inflammation and consequent IR in obesity.

Cellular therapy by allogeneic macrophages against visceral leishmaniasis: role of TNF-α.

Tumor necrosis factor α (TNF-α) is an essential player in infection with Leishmania, controlling inflammatory lesion and parasite killing. We recently have shown the leishmanicidal activity of transmembrane form of TNF (mTNF) derived from allogeneic natural killer (NK) cells in experimental visceral leishmaniasis. Allogeneic macrophages and human monocytes derived mTNF has significantly higher antileishmanial activity compared to allogeneic NK cells. Unlike NK cells, syngeneic macrophages also possess antileishmanial activity, although degree of activity is significantly less compared to allogeneic macrophages. Cellular therapy by intravenous transfer of allogeneic macrophages enhances leishmanicidal effect against the established infection in susceptible animal by reducing the splenic parasite burden to 28.3 ± 4.71 × 10(5) compared to 256.00 ± 17.36 × 10(5) in control group. In vivo treatment with anti-mouse TNF-α reduces the therapeutic efficacy of the allogeneic macrophages by increasing the parasite load in spleen of infected mice. These results demonstrated that allogeneic and xenogeneic macrophages induce cytokine mediated protective mechanism against infected macrophages via TNF-α in vitro and, possibly in vivo. The macrophage mediated protective role in absence of T cell help demonstrate an unique property of the mononuclear phagocytes in controlling infection and inflammation in visceral leishmaniasis, despite being acts as a host cell for the same parasite.

Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development.

Superoxide activates mTOR-eIF4E-Bax route to induce enhanced apoptosis in leukemic cells.

Mammalian target of rapamycin (mTOR) is a central kinase that regulates cell survival, proliferation and translation. Reactive oxygen species (ROS) are second messengers with potential in manipulating cellular signaling. Here we report that two ROS generating phytochemicals, hydroxychavicol and curcumin synergize in leukemic cells in inducing enhanced apoptosis by independently activating both mitogen activated protein kinase (MAPK) (JNK and P(38)) and mTOR pathways. Low level transient ROS generated after co-treatment with these phytochemicals led to activation of these two pathways. Both mTOR and MAPK pathways played important roles in co-treatment-induced apoptosis, by knocking down either mTOR or MAPKs inhibited apoptosis. Activation of mTOR, as evident from phosphorylation of its downstream effector eukaryotic translation initiation factor 4E-binding protein 1, led to release of eukaryotic translation initiation factor 4E (eIF4E) which was subsequently phosphorylated by JNK leading to translation of pro-apoptotic proteins Bax and Bad without affecting the expression of anti-apoptotic protein Bcl-xl. Our data suggest that mTOR and MAPK pathways converge at eIF4E in co-treatment-induced enhanced apoptosis and provide mechanistic insight for the role of mTOR activation in apoptosis.

Synergistic apoptosis of CML cells by buthionine sulfoximine and hydroxychavicol correlates with activation of AIF and GSH-ROS-JNK-ERK-iNOS pathway.

BACKGROUND: Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism. MATERIALS AND METHODS: Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexin V-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action. RESULTS: Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells. CONCLUSION/SIGNIFICANCE: BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.

IL-15 activated human peripheral blood dendritic cell kill allogeneic and xenogeneic endothelial cells via apoptosis.

IL-15 is a pleotropic cytokine, which plays an important role in natural killer (NK) cell activity, T cell proliferation, and T cell cytotoxic activity. Dendritic cells (DCs) are the major antigen presenting cells in the immune system and presumed to play an important role in immune recognition of allo and xenotransplantation. We showed that IL-15 activated human peripheral blood DC is cytotoxic to human and porcine aortic endothelial cells. Unlike DCs, CD14+ monocytes show no cytotoxicity against the endothelial cells. This cytotoxic potential of IL-15 activated DC against endothelial cells is dose dependent and increases significantly upon treatment of endothelial cells with inflammatory cytokines like TNF-α or IFN-γ. The cytotoxic potential of IL-15 activated DC is associated with apoptosis of endothelial cells, as indicated by the increased Annexin V staining, caspase activation and loss of mitochondrial membrane potential. Further it was observed that DC mediated cytotoxicity against endothelial cell is mediated via granzyme B possibly secreted by the activated DCs.

ICB3E induces iNOS expression by ROS-dependent JNK and ERK activation for apoptosis of leukemic cells.

The role of c-Jun N terminal Kinase (JNK) has been well documented in various cellular stresses where it leads to cell death. Similarly, extracellular signal-regulated kinase (ERK) which was identified as a signalling molecule for survival pathway has been shown recently to be involved in apoptosis also. Recently we reported that ICB3E, a synthetic analogue of Piper betle leaf-derived apoptosis-inducing agent hydroxychavicol (HCH), possesses anti-chronic myeloid leukemia (CML) acitivity in vitro and in vivo without insight on mechanism of action. Here we report that ICB3E is three to four times more potent than HCH in inducing apoptosis of leukemic cells without having appreciable effects on normal human peripheral blood mononuclear cells, mouse fibroblast cell line NIH3T3 and monkey kidney epithelial cell line Vero. ICB3E causes early accumulation of mitochondria-derived reactive oxygen species (ROS) in K562 cells. Unlike HCH, ICB3E treatment caused ROS dependent activation of both JNK, ERK and induced the expression of iNOS leading to generation of nitric oxide (NO). This causes cleavage of caspase 9, 3 and PARP leading to apoptosis. Lack of cleavage of caspase 8 and inability of blocking chimera antibody to DR5 or neutralizing antibody to Fas to reverse ICB3E-mediated apoptosis suggest the involvement of only intrinsic pathway. Our data reveal a novel ROS-dependent JNK/ERK-mediated iNOS activation pathway which leads to NO mediated cell death by ICB3E.

Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings.

Natural products: promising resources for cancer drug discovery.

Natural products are important sources of anti-cancer lead molecules. Many successful anti-cancer drugs are natural products or their analogues. Many more are under clinical trials. The present review focuses on chemopreventive and anti-cancer activities of polar and non-polar extracts, semi purified fractions and pure molecules from terrestrial plants of India reported between 2005 and 2010 emphasizing possible mechanisms of action of pure molecules.

NPB001-05 inhibits Bcr-Abl kinase leading to apoptosis of imatinib-resistant cells.

The deregulated activity of the Bcr-Abl tyrosine kinase provides a rational basis for the development therapeutics in all phases of Chronic Myelogenous Leukemia (CML). Although a well studied imatinib therapy has clinical success against CML, resistance to imatinib due to mutations in the kinase domain, especially T315I poses a major problem for the ultimate success of CML therapy by this agent. Herein we describe an NPB001-05, derived from extract of Piper betle leafs, which is highly active in specifically inhibiting Bcr-Abl expressing cells. NPB001-05 inhibited the proliferation of BaF3 cells ectopically expressing wild type Bcr-Abl phenotype and 12 different imatinib-resistant mutations of clinical relevance (average IC50 5.7 microg/ml). Moreover, NPB001-05 was highly inhibitory to wild type P210(Bcr-Abl) and P210(Bcr-Abl-T315I) kinase activity and abrogated the autophosphorylating enzyme in time- and dose- dependent manner. NPB001-05 was non-toxic on normal cells, but was inhibitory to CML patient derived peripheral blood mononuclear cells. Treatment with NPB001-05 caused apoptosis induction and G0G1 cell cycle arrest in both Bcr-Abl wild type and T315I mutant cell lines.

Antioxidant and antibacterial activities of bark extracts from Commiphora berryi and Commiphora caudata.

This study investigates the in vitro antioxidant and antimicrobial activities of eight extracts obtained from the dried barks of Commiphora berryi and Commiphora caudata (Burseraceae). The radical scavenging activity was assessed by 1,1-diphenyl-2-picryl hydrazyl (DPPH) and nitric oxide assays. The methanolic extracts of C. berryi and C. caudata showed significant DPPH radical scavenging activity, with IC50 values of 26.92 and 21.16 µg mL⁻¹, respectively, and low radical scavenging activity against the nitric oxide assay. The antimicrobial activity of the plants was tested against the Gram-positive and Gram-negative bacteria. The ethyl acetate, chloroform and petroleum ether extracts of C. berryi showed good antibacterial activity against Pseudomonas aeruginosa, with a minimum inhibitory concentration (MIC) of 0.26 mg mL⁻¹, whereas the ethyl acetate and methanol extracts of C. caudata showed moderate antimicrobial activity with an MIC of more than 2.0 mg mL⁻¹ against P. aeruginosa compared to the petroleum ether and chloroform extracts, which showed an MIC of 1.1 mg mL⁻¹. The methanolic extracts of C. berryi and C. caudata also showed moderate cytotoxic activity against a human mammary carcinoma cell line (MCF-7), with values IC50 of 82.6 and 88.4 µg mL⁻¹, respectively.

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl+ CML cells.

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl(+) chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl(+) cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl(+) cells.

Innate immune defense in visceral leishmaniasis: cytokine mediated protective role by allogeneic effector cell.

Antileishmanial role of mouse splenic natural killer (NK) cell was studied in allogeneic condition. In vitro data indicates that NK cells of allogeneic (C57BL/6, H2(b)) non-leishmania exposed mouse have strong antileishmanial effect against Leishmania donovani infected BALB/c (H2(d)) macrophages. Physical contact between the effector (NK cell) and the target cells (infected macrophages) is essential in this system since; cell free supernatant generated after coculturing of effector cells with infected target cells fails to elicit any antileishmanial effect. Although NK cells from allogeneic mouse are strongly attached to the infected macrophages but unable to kill it in such interaction. The antileishmanial effect of allogeneic NK cells is mediated by TNF-alpha and not by IFN-gamma. In vivo cellular therapy of established infection with NK cells from non-leishmania exposed allogeneic mouse significantly reduces the total parasite burden in the spleen of infected animal.

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

INTRODUCTION: Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H(2)O(2), which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. MATERIALS AND METHODS: Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. CONCLUSION: NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

Arsenic-induced mitochondrial instability leading to programmed cell death in the exposed individuals.

In West Bengal, India, more than 6 million people in nine districts are exposed to arsenic through drinking water. It is regarded as the greatest arsenic calamity in the world. Arsenic is a well-documented human carcinogen, which does not induce cancer in any other animal model. Interestingly, at lower concentrations, arsenic is known to induce apoptosis in various cancer cell lines in vitro. We have studied apoptosis in human peripheral blood mononuclear cells (PBMC) of 30 arsenic exposed skin lesion individuals by annexin V-FITC staining and compared with 28 unexposed individuals. The percentage of apoptotic cells in individuals with skin lesions was significantly higher (p<0.001) in comparison to unexposed individuals. In the exposed individuals with skin lesions, there were elevated levels of intracellular reactive oxygen species (ROS), mitochondrial membrane permeability and increased cytochrome c release, leading to increased downstream caspase activity. Arsenic-induced DNA damage was confirmed by DNA ladder formation and confocal microscopy. We also observed that chronic arsenic exposure reduced Bcl-2/Bax ratio and also resulted in cell cycle arrest of PBMC in G0/G1 phase. All these observations indicate that mitochondria-mediated pathway may be responsible for arsenic-induced apoptosis.

Granulocyte-macrophage colony-stimulating factor drives monocytes to CD14low CD83+ DCSIGN- interleukin-10-producing myeloid cells with differential effects on T-cell subsets.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has long been found to have growth-promoting effects on multipotent haematopoietic lineages, specifically granulocytes and macrophages. GM-CSF combined with interleukin-4 (IL-4) drives monocytes to become myeloid dendritic cells (mDCs) in vitro. We report that culturing human monocytes with GM-CSF alone generates myeloid cells (GM-Mono) that have lower expression of CD14 than monocytes and that fail to express DC-SIGN. GM-Monos, however, express CD83 and the transcription factor PU.1, although at a lower level than the conventional mDCs generated in the presence of GM-CSF and IL-4. On stimulation with tumour necrosis factor-alpha, interferon-gamma and anti-CD40 monoclonal antibody, the GM-Monos predominantly produced IL-10 but were less efficient in IL-12 production. In a primary allogeneic mixed lymphocyte reaction, GM-Monos induced hyporesponsiveness and IL-10-biased cytokine production in CD4(+) T cells. In fresh mixed lymphocyte reaction, GM-Monos inhibited conventional mDC-induced allogeneic CD4(+) T-cell proliferation. GM-Mono-induced inhibition of allogeneic CD4(+) T-cell proliferation was partially attributed to IL-10. Interestingly, GM-Monos neither induced hyporesponsiveness in allogeneic CD8(+) T cells nor inhibited conventional mDC-induced allogeneic CD8(+) T-cell proliferation. Taken together, we characterize monocyte-derived CD14(low) CD83(+) cells generated by GM-CSF that can induce tolerance or stimulation of T cells depending on T-cell subsets.

Leishmania donovani infection of human myeloid dendritic cells leads to a Th1 response in CD4+ T cells from healthy donors and patients with kala-azar.

The role played by dendritic cells (DCs) in Leishmania donovani infection is poorly understood. Here, we report that L. donovani amastigotes efficiently infect human peripheral-blood monocyte-derived DCs. Opsonization with normal human serum enhanced the infectivity of amastigotes and promastigotes only marginally. Surface attachment versus internalization was distinguished by incubation of DCs with live, fluorescein isothiocyanate-labeled parasites, followed by quenching with crystal violet. Infection with amastigotes was accompanied by DC maturation, as was evident from the up-regulation of maturation-associated cell-surface markers, the nuclear translocation of RelB, and the release of cytokines. Amastigote-primed DCs produced inflammatory cytokines in response to subsequent treatment with interferon- gamma or anti-CD40 monoclonal antibody. When cocultured, amastigote-infected DCs induced T helper cell type 1 (Th1) responses both in naive allogeneic CD4(+) T cells and in autologous CD4(+) T cells from patients with kala-azar and up-regulated the expression of T-bet. Our data reveal that infection with L. donovani amastigotes induces a Th1 cytokine milieu in both DCs and T cells.

Interaction of Leishmania parasites with dendritic cells and its functional consequences.

Interaction between dendritic cells (DC) and T cells is essential for the generation of cell mediated immunity and thus DC play a critical role in the initiation of immune responses against Leishmania parasites. Although macrophages (Mphi) are the major targets of all species of Leishmania, a number of studies demonstrated the infection of DC by Leishmania. DC specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), has been reported to be the receptor for Leishmania amastigotes. The functional consequences in DC after Leishmania infections appear to depend on species of Leishmania. Some species of Leishmania enhance the surface expression of co-stimulatory molecules and CD40-ligand-induced IL-12 production in DC. On the other hand other species down-regulate co-stimulatory molecules and inhibit IL-12 production. The intrinsic differences among Leishmania species with regard to alteration of cell surface molecules and IL-12 production in DC may contribute to the healing and non-healing forms of the disease.

Camptothecin-induced imbalance in intracellular cation homeostasis regulates programmed cell death in unicellular hemoflagellate Leishmania donovani.

Leishmania, a unicellular trypanosomatid protozoan parasite, causes a wide range of human diseases ranging from the localized self-healing cutaneous lesions to fatal visceral leishmaniasis. However, it undergoes a process of programmed cell death during treatment with the topoisomerase I poison camptothecin (CPT). The present study shows that CPT-induced formation of reactive oxygen species increases the level of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (DeltaPsim), which is followed by a significant decrease in intracellular pH levels. CPT-induced oxidative stress also causes impairment of the Na+ - K+ -ATPase pump and subsequently decreases the intracellular K+ level in leishmanial cells. A decrease in both intracellular pH and K+ levels propagates the apoptotic process through activation of caspase 3-like proteases by rapid formation of cytochrome c-mediated apoptotic complex. In addition to caspase-like protease activation, a lower level of intracellular K+ also enhances the activation of apoptotic nucleases at the late stage of apoptosis. This suggests that the physiological level of pH and K+ are inhibitory for apoptotic DNA fragmentation and caspase-like protease activation in leishmanial cells. Moreover, unlike mammalian cells, the intracellular ATP level gradually decreases with an increase in the number of apoptotic cells after the loss of DeltaPsim. Taken together, the elucidation of biochemical events, which tightly regulate the process of growth arrest and death of Leishmania donovani promastigotes, allows us to define a more comprehensive view of cell death during treatment with CPT.

Involvement of Leishmania donovani major surface glycoprotein gp63 in promastigote multiplication.

The major surface glycoprotein gp63 of the kinetoplastid protozoal parasite Leishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector in L. donovani promastigotes, gp63-deficient transfectants were obtained. Reduction of the gp63 level resulted in increased generation times, altered cell morphology, accumulation of cells in the G2/M phase of the cell cycle, and increased numbers of binucleate cells with one or two kinetoplasts. Growth was stimulated, and the number of binucleate cells reduced, by addition to the culture of a bacterially expressed fusion protein containing the fibronectin-like SRYD motif and the zinc-binding (metalloprotease) domain of gp63. These observations support an additional role of gp63 in promastigote multiplication; the fibronectin-like properties of gp63 may be important in this process