Leishmanial lipid suppresses tumor necrosis factor alpha, interleukin-1beta, and nitric oxide production by adherent synovial fluid mononuclear cells in rheumatoid arthritis patients and induces apoptosis through the mitochondrial-mediated pathway.

OBJECTIVE: Leishmanial lipid is a strong immunosuppressor of host cells. Inhibition of the inflammatory responses of synovial cells through induction of apoptosis is one of the main targets of therapeutic intervention in rheumatoid arthritis (RA). This study was undertaken to examine the antiinflammatory and apoptosis-inducing effects of leishmanial lipid on adherent synovial fluid mononuclear cells (SFMCs) in patients with RA. METHODS: Lipid was extracted from a Leishmania donovani promastigote (MHO/IN/1978/UR6) by the Bligh and Dyer method. Nitric oxide (NO) was measured using the Griess reaction, and enzyme-linked immunosorbent assays for cytokines, NF-kappaB, and cytochrome c were performed. Levels of cytokines, inducible nitric oxide synthase, caspases, Bcl-2, Bax, t-Bid, and cytochrome c in the cell lysate and of NF-kappaB p65 in the nucleus were determined by Western blotting. Microscopic analysis, nuclear staining, DNA fragmentation assay, fluorescence-activated cell sorting, colorimetric assay for caspases, and fluorescent probe for measurement of mitochondrial membrane potential were used to study the leishmanial lipid-induced apoptotic pathway in SFMCs. RESULTS: Leishmanial lipid inhibited the release of tumor necrosis factor alpha, interleukin-1beta, and NO in the culture, decreased their cytosolic protein levels, and decreased NF-kappaB p65 levels in SFMCs, in a dose-dependent manner. It had the reverse effect on interleukin-10 levels. Leishmanial lipid-induced apoptosis involved the activation of caspase 3, caspase 9, and Bax, the release of cytochrome c, the alteration of mitochondrial membrane potential, and the down-regulation of Bcl-2. CONCLUSION: These results suggest that leishmanial lipid has strong antiinflammatory and apoptosis-inducing effects on SFMCs from patients with RA, and that apoptosis occurs via the mitochondrial pathway.

Attenuated Leishmanial sphingolipid induces apoptosis in A375 human melanoma cell via both caspase-dependent and -independent pathways.

A fraction of attenuated Leishmanial lipid (ALL) rich in sphingolipids, previously shown to have apoptosis inducing activity in mouse melanoma (B16F10) and human melanoma (A375) cells, was resolved to isolate the bioactive sphingolipid. The mechanism of apoptosis induction by this bioactive attenuated Leishmanial sphingolipid (ALSL) was studied in A375 cells. Apoptosis induced by ALSL in A375 cells was found to be dose and time-dependent. Exposure of cells to ALSL resulted in a rapid increase in reactive oxygen species generation. Pretreatment of cells with the antioxidant N-acetyl-cystein reduced ROS generation and attenuated apoptosis induced by ALSL. Again, ALSL sensitization resulted in the activation of caspase-3 and -9 but not caspase-8. However, inhibitors of these caspases could not protect the cells completely from ALSL-induced apoptosis. N-acetyl-cystein pretreatment was again found to attenuate the activation of caspase-3 and -9. ALSL treatment also resulted in the alteration of mitochondrial membrane potential, and release of pro-apoptotic factors such as cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Furthermore, c-Jun N-terminal kinase was activated that resulted in apoptosis of A375 cells, whereas p38 MAPK was activated to counteract the stress generated in cells in response to ALSL treatment. Taken together, our results indicate that ALSL-induced apoptosis of A375 cells is mediated by both mitochondrial caspase-dependent and -independent pathways and it involves ROS and JNK activation in the mitogen-activated protein kinase cascade.

A sphingolipid rich lipid fraction isolated from attenuated Leishmania donovani promastigote induces apoptosis in mouse and human melanoma cells in vitro.

Lipids, especially sphingolipids, are emerging as inducer of apoptosis in a wide range of immortal cells, potentiating their therapeutic application in cancer. In the present study, a sphingolipid rich lipid fraction (denoted here as ALL), isolated from an attenuated strain of Leishmania donovani promastigote, was tested for its tumoricidal activity taking melanoma, the dreaded form of skin cancer cells, as model. ALL was found to induce chromatin condensation, internucleosomal DNA fragmentation and phosphatidylserine externalization with enhanced cell population in sub-G1 region in both mouse and human melanoma systems, namely B16F10 and A375 respectively. These are the hallmarks of cells undergoing apoptosis. Further analysis demonstrated that ALL treated melanoma cells showed significant increase in ROS generation, mitochondrial membrane potential depolarization, release of cytochrome c, and caspase-3 activation, which are the events closely involved in apoptosis. These findings indicate that one or more bioactive sphingolipid(s)/ceramide(s) present in ALL could be the causative agent(s) for the induction of apoptosis in melanoma cells. Further studies are thus necessary to identify these specific bioactive sphingolipid(s)/ceramide(s) and to establish their mechanism of action, in order to explore their use as anticancer agents.