Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3Ј-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (⌬⌿ m ). Because ⌬⌿ m is the driving force for mitochondrial ATP synthesis, loss of ⌬⌿ m results in depletion of cellular ATP level. The loss of ⌬⌿ m causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of ⌬⌿ m leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets.Apoptosis, a form of programmed cell death (PCD), is a genetically regulated active physiological process of cell suicide that causes cell deletion without inflammation, scarring, or release of cellular contents. Mitochondria of living cells play a pivotal role in controlling life and death (Green and Reed, 1998). Mitochondria are an important cellular source for the generation of reactive oxygen species (ROS) inside the cells (Halliwell and Gutteridge, 1990). Maintenance of proper mitochondrial transmembrane potential (⌬⌿ m ) is essential for the survival of the cell because it derives the synthesis of ATP and maintains oxidative phosphorylation (Gottlieb, 2001).3,3Ј-Diindolylmethane (DIM) is a major acid condensation product of indole-3-carbinol, a natural compound found in vegetables of the genus Brassica. It has an anticarcinogenic effect and inhibits the growth of human cancer cells. DIM-
DIM (3,3'-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.2 microM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73x10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). DIM stabilizes topoisomerase I-DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I-DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated 'cleavable complexes' in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1Delta39LS (N-terminal deletion of amino acids 1-39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 muM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Delta39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.
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...
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