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Chronic arsenic exposure is known to produce arsenicosis and cancer. To ascertain whether perturbation of methylation plays a role in such carcinogenesis, the degree of methylation of p53 and p16 gene in DNA obtained from blood samples of people chronically exposed to arsenic and skin cancer subjects was studied. Methylation-specific restriction endonuclease digestion followed by polymerase chain reaction (PCR) of gene p53 and bisulfite treatment followed by methylation-sensitive PCR of gene p16 have been carried out to analyze the methylation status of the samples studied. Significant DNA hypermethylation of promoter region of p53 gene was observed in DNA of arsenic-exposed people compared to control subjects. This hypermethylation showed a dose-response relationship. Further, hypermethylation of p53 gene was also observed in arsenic-induced skin cancer patients compared to subjects having skin cancer unrelated to arsenic, though not at significant level. However, a small subgroup of cases showed hypomethylation with high arsenic exposure. Significant hypermethylation of gene p16 was also observed in cases of arsenicosis exposed to high level of arsenic. In man, arsenic has the ability to alter DNA methylation patterns in gene p53 and p16, which are important in carcinogenesis.

We report that chlorogenic acid (Chl) induces apoptosis of several Bcr-Ablpositive chronic myelogenous leukemia (CML) cell lines and primary cells from CML patients in vitro and destroys BcrAbl-positive K562 cells in vivo. In contrast, this compound has no effect on the growth and viability of Bcr-Abl-negative lymphocytic and myeloid cell lines and primary CML cells. Sodium chlorogenate (NaChl) exhibits 2-fold higher efficiency in killing K562 cells compared with Chl. NaChl also induces growth inhibition of squamous cell carcinoma (HSC-2) and salivary gland tumor cells (HSG), although at 50-fold higher concentration. NaChl inhibits autophosphorylation of p210 Bcr-Abl fusion protein rapidly. We demonstrate that p38 phosphorylation is increased in Bcr-Abl-positive cells after treatment with NaChl and closely paralleled the inhibition of Bcr-Abl phosphorylation. NaChl did not increase phosphorylation of p38 in Bcr-Abl-negative cells including HSC-2 and HSG that are responsive to this compound, indicating that p38 activation by NaChl is dependent on Bcr-Abl kinase inhibition. Inhibition of p38 activity by SB203580 significantly reduced NaChl-induced apoptosis of K562 cells, whereas activation of p38 by anisomycin augmented the apoptosis. These findings indicate that inhibition of BcrAbl kinase leading to activation of p38 mitogen-activated protein ( IntroductionChronic myelogenous leukemia (CML) is a malignant clonal disorder of hematopoietic stem cells leading to massive expansion of myeloid lineage cells. 1 The natural fate of CML is to progress from a benign chronic phase into the fatal blast crisis between approximately 3 and 5 years. Development of CML is associated with a specific chromosomal translocation known as the Philadelphia (Ph) chromosome that is detectable throughout the course of the disease. 2 Somatic mutation in Ph chromosome originates from reciprocal translocation between the long arms of chromosomes 9 and 22 and fuses Bcr with c-Abl genetic sequences. Both the Bcr-Abl fusion proteins p210 and p185 can cause CML or acute leukemia. 3,4 The p210 form of Bcr-Abl is seen in 95% of CML and in 20% of acute lymphocytic leukemia, whereas the p185 form is identified in about 10% of acute lymphocytic leukemia patients. 5,6 The Bcr-Abl fusion proteins are constitutively active non-receptor tyrosine kinases whose activity is essential for transforming abilities. 7 An almost universal presence of Bcr-Abl in CML patients made this fusion protein an attractive target for drug development. Bcr-Abl inhibitors, STI571, adaphostin, and PD173955, are capable of inducing a variable degree of apoptosis in human CML cells. [8][9][10] The signal transduction pathways involved in mediating apoptosis by Bcr-Abl inhibitors are poorly defined. In the current study, we describe a novel Bcr-Abl kinase inhibitor that triggers p38 mitogen-activated protein (MAP) kinase-dependent apoptosis of Bcr-Abl-positive CML cells. Materials and methods Cells and reagentsThe Ph chromosome ϩ CML cell line K562, 11 Ph chromosome-negative T-...

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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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BackgroundHydroxychavicol (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 MethodsAnti-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 annexinV-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.ResultsNon-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/SignificanceBSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.