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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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.