No abstract

Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC 50 of 2.6 and 4.1 M respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs.

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. Hydroxychavicol (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 synthasemediated 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. (Cancer Sci 2012; 103: 88-99) I matinib (also known as STI571 or Gleevec), a small-molecule inhibitor of the Bcr-Abl kinase, has been used successfully to treat chronic myeloid leukemia,(1) but resistance has emerged against this drug. The T315I mutation is the most predominant among the mutations found in imatinib-resistant patients.(2) None of the available approved drugs have been effective in circumventing this T315I mutation. Recent reports suggest that the alcoholic extract of Piper betle (Piper betel L.) leaves induces apoptosis of imatinib-resistant cells (4) and shows activity against T315I tumor xenografts.The deep green heart-shaped leaves commonly referred to as ''betel leaves'' are traditionally consumed as a mouth freshener in Eastern Asia.(6) Hydroxychavicol (HCH), a phenolic compound of Piper betle leaves has been shown to have antimutagenic and anti-carcinogenic activity. (7,8) HCH possesses antimicrobial, antioxidant and anti-inflammatory properties.Recent studies also suggest apoptosis of oral (KB) carcinoma cells by HCH through induction of reactive oxygen species (ROS). None of the previous studies suggest any mechanisms downstream of ROS for HCH-induced apoptosis. (9) Reactive oxygen species are products of aerobic metabolism of cells. Tumor cells have higher levels of intracellular ROS than their normal counterparts.(10) This creates opposite effects upon augmentation of intracellular ROS on cell proliferation in normal cells versus cancer cells.(10) As the basal level of intracellular ROS is low in normal cells, its increase, to a certain extent, is associated with cell proliferati...

Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence of CD4+ T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial potential of antimony or vice versa.