Khaled Hossain

Glyoxal and methylglyoxal trigger distinct signals for map family kinases and caspase activation in human endothelial cells

Carbonyl compounds with diverse carbon skeletons may be differentially related to the pathogenesis of vascular diseases. In this study, we compared intracellular signals delivered into cultured human umbilical vein endothelial cells (HUVECs) by glyoxal (GO) and methylglyoxal (MGO), which differ only by a methyl group. Depending on their concentrations, GO and MGO promoted phosphorylations of ERK1 and ERK2, which were blocked by the protein-tyrosine kinase (PTK) inhibitors herbimycin A and staurosporine, thereby being PTK-dependent. GO and MGO also induced phosphorylations of JNK, p38 MAPK, and c-Jun, either PTK-dependently (GO) or -independently (MGO). Next, we found that MGO, but not GO, induced degradation of poly(ADP-ribose) polymerase (PARP) as the intracellular substrate of caspase-3. Curcumin and SB203580, which inhibit JNK and p38 MAPK signaling pathways, but not herbimycin A/staurosporine, prevented the MGO-induced PARP degradation. We then found that MGO, but not GO, reduced the intracellular glutathione level, and that cysteine, but not cystine, inhibited the MGO-mediated activation of ERK, JNK, p38 MAPK, or c-Jun more extensively than did lysine or arginine. In addition, all the signals triggered by GO and MGO were blocked by amino guanidine (AG), which traps carbonyls. These results demonstrated that GO and MGO triggered two distinct signal cascades, one for PTK-dependent control of ERK and another for PTK-independent redox-linked activation of JNK/p38 MAPK and caspases in HUVECs, depending on the structure of the carbon skeleton of the chemicals.

Arsenite Induces Apoptosis of Murine T Lymphocytes Through Membrane Raft-Linked Signaling for Activation of c-Jun Amino-Terminal Kinase

Because of its dual roles in acute toxicity and in therapeutic application in cancer treatment, arsenic has recently attracted a renewed attention. In this study, we report NaAsO2-induced signal cascades from the cell surface to the nucleus of murine thymic T lymphocytes that involve membrane rafts as an initial signal transducer. NaAsO2 induced apoptosis through fragmentation of DNA, activation of caspase, and reciprocal regulation of Bcl-2/Bax with the concomitant reduction of membrane potential. We demonstrated that NaAsO2-induced caspase activation is dependent on curcumin-sensitive c-Jun amino-terminal kinase and barely dependent on SB203580-sensitive p38 kinase or PD98059-sensitive extracellular signal-regulated kinase. Additionally, staurosporine, which severely inhibited the activation of mitogen-activated protein (MAP) family kinases and c-Jun, partially blocked the NaAsO2-mediated signal for poly(ADP-ribose) polymerase (PARP) degradation. Potentially as the initial cell surface event for intracellular signaling, NaAsO2 induced aggregation of GPI-anchored protein Thy-1 and superoxide production. This Thy-1 aggregation and subsequent activation of MAP family kinase and c-Jun and the degradation of PARP induced by NaAsO2 were all inhibited by DTT, suggesting the requirement of interaction between arsenic and protein sulfhydryl groups for those effects. β cyclodextrin, which sequestrates cholesterol from the membrane rafts, inhibited NaAsO2-induced activation of protein tyrosine kinases and MAP family kinases, degradation of PARP, and production of superoxide. In addition, β cyclodextrin dispersed NaAsO2-induced Thy-1 clustering. These results suggest that a membrane raft integrity-dependent cell surface event is a prerequisite for NaAsO2-induced protein tyrosine kinase/c-Jun amino-terminal kinase activation, superoxide production, and downstream caspase activation.

Paeoniflorin induces apoptosis of lymphocytes through a redox‐linked mechanism

Paeoniflorin (PF), isolated from paeony root, has been used as a herbal medicine for more than 1,200 years in China, Korea, and Japan for its anti‐allergic, anti‐inflamatory, and immunoregulatory effects. In this study, we found that PF induces apoptosis in both murine T‐lineage cells and human T‐cell leukemia Jurkat cells. This apoptosis was mediated through the reduction of mitochondrial membrane potential, activation of caspase, and fragmentation of DNA. Interestingly, PF induced generation of reactive oxygen species (ROS) and a reducing agent, dithiothreitol (DTT), and a ROS scavenger, N‐acetyl cysteine (NAC), successfully attenuated the PF‐induced apoptosis. Additionally, PF induced the phosphorylation of three mitogen‐activated protein (MAP) family kinases, extracellular signal‐regulated kinase, c‐Jun amino‐terminal kinase (JNK), and p38 MAP kinase. Curcumin, an anti‐oxidant and JNK inhibitor, inhibited PF‐induced apoptosis, suggesting the possible involvement of curcumin‐sensitive JNK or other redox‐sensitive elements in PF‐induced apoptosis. These results partially explain the action mechanism of PF‐containing paeony root as a herbal medicine.

c-Kit-Targeting Immunotherapy for Hereditary Melanoma in a Mouse Model

The role of c-Kit in the development of melanoma was studied in line 304/B6 of RET-transgenic mice, in which melanoma spontaneously develops. In Wv/Wv-RET (304/B6)-transgenic mice, in which c-Kit function was severely impaired, development of melanoma was strongly suppressed. Although 31 of the 44 original RET-transgenic mice died of rapidly growing melanoma within 12 months after birth, only 8 of the 44 Wv/Wv-RET-transgenic mice developed slowly growing melanocytic tumors with a greatly prolonged mean tumor-free period, 2 of which died of melanoma at a late stage. Even Wv/+-RET-transgenic mice had a clearly prolonged tumor-free period and definitely reduced frequency (6 of 61) of tumor death within 12 months after birth. Melanin production in the skin of these mice was not strongly impaired, suggesting that c-Kit affects the development of melanomas in these mice with only minor effects in melanin production. c-Kit expression in skin soon after birth was promoted in RET-transgenic mice, and c-Kit was expressed at high levels at the benign but not malignant stage of the tumor. A single injection of anti-c-Kit antibody (ACK2) into RET-transgenic mice soon after birth caused a surprisingly long-lasting suppression of development of melanoma, greatly prolonging the tumor-free period, and none of the 28 ACK2-treated RET-transgenic mice died from tumors at 12 months of age. The c-Kit function needed for melanin production was also suppressed for an unusually long time in ACK2-treated, RET-transgenic mice. These results suggest that c-Kit can be a unique target molecule for melanoma treatment.

Cepharanthine Activates Caspases and Induces Apoptosis in Jurkat and K562 Human Leukemia Cell Lines

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy‐provoked leukopenia. We here report that apoptosis was induced by low concentrations (1–5 μM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5–10 μM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr‐Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas‐independent pathway. CEP treatment induced activation of caspase‐9 and ‐3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase‐8 activation and Akt cleavage were observed only in Jurkat cells. The CEP‐induced apoptosis was completely blocked by zVAD‐fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr‐Abl protein in K562 cells, and this degradation was prevented partially by zVAD‐fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2–5 μM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr‐Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy. J. Cell. Biochem. 82: 200–214, 2001.

Increases in Oxidized Low-Density Lipoprotein and Other Inflammatory and Adhesion Molecules With a Concomitant Decrease in High-Density Lipoprotein in the Individuals Exposed to Arsenic in Bangladesh

Elevated exposure to arsenic has been suggested to be associated with atherosclerosis leading to cardiovascular disease (CVD). However, biochemical events underlying the arsenic-induced atherosclerosis have not yet been fully documented. The aim of this study was to investigate the associations of circulating molecules involved in atherosclerosis with arsenic exposure in the individuals exposed to arsenic in Bangladesh. A total of 324 study subjects, 218 from arsenic-endemic areas and 106 from nonendemic areas in Bangladesh, were recruited. Drinking water, hair, nail, and blood samples were collected from the study subjects for analysis. Total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels were lower in arsenic-endemic subjects than those of nonendemic subjects. Oxidized LDL (Ox-LDL), C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) levels were significantly higher in arsenic-endemic subjects than those in nonendemic subjects. All these circulating molecules showed significant correlations with arsenic exposure (water, hair, and nail arsenic concentrations), and all these relations were significant before and after adjusting for relevant covariates. Among the circulating molecules tested in this study, HDL, Ox-LDL, and CRP showed dose-response relationships with arsenic exposure. Ox-LDL/HDL ratios were increased with the increasing concentrations of arsenic in the water, hair, and nails. Furthermore, non-HDL cholesterol and TC/HDL ratios were significantly correlated with arsenic exposure before and after adjusting for relevant covariates. Thus, all the observed associations may be the major features of arsenic exposure-related atherosclerosis leading to CVD.

Sustained activation of c-jun-terminal kinase (JNK) is closely related to arsenic trioxide-induced apoptosis in an acute myeloid leukemia (M2)-derived cell line, NKM-1

High concentrations (greater than 5 μM) of arsenic trioxide (As2O3) have been reported to be able to induce apoptosis in several malignant cells. We explored cell lines in which apoptosis was induced with a therapeutic concentration (1–2 μM) of As2O3, and found that 1 μM of As2O3 induced apoptosis in the NKM-1 cell line, which was established from a patient with acute myeloid leukemia (M2). Apoptosis induced by 1 μM of As2O3 in NKM-1 cells was accompanied by an increased cellular content of H2O2, a decreased mitochondrial membrane potential (Δψm), and activation of caspase-3. C-Jun-terminal kinase (JNK) was activated only in NKM-1 cells and arsenic-sensitive NB4 cells, but not in arsenic-insensitive HL-60 cells. Activation of JNK in NKM-1 was sustained from 6 to 24 h after As2O3 treatment, and preceded changes in cellular H2O2, Δψm, and caspase-3 activation. Moreover, addition of a JNK inhibitor reduced the percentage of apoptotic cells after the As2O3 treatment. Taken together, in the M2 cell line NKM-1, 1 μM of As2O3 induced sustained activation of JNK and apoptosis. This finding may provide a basis to select a subgroup other than acute promyelocytic leukemia, which can benefit from As2O3 treatment.

Association between arsenic exposure and plasma cholinesterase activity: a population based study in Bangladesh

BackgroundArsenic is a potent pollutant that has caused an environmental catastrophe in certain parts of the world including Bangladesh where millions of people are presently at risk due to drinking water contaminated by arsenic. Chronic arsenic exposure has been scientifically shown as a cause for liver damage, cancers, neurological disorders and several other ailments. The relationship between plasma cholinesterase (PChE) activity and arsenic exposure has not yet been clearly documented. However, decreased PChE activity has been found in patients suffering liver dysfunction, heart attack, cancer metastasis and neurotoxicity. Therefore, in this study, we evaluated the PChE activity in individuals exposed to arsenic via drinking water in Bangladesh.MethodsA total of 141 Bangladeshi residents living in arsenic endemic areas with the mean arsenic exposure of 14.10 ± 3.27 years were selected as study subjects and split into tertile groups based on three water arsenic concentrations: low (< 129 μg/L), medium (130-264 μg/L) and high (> 265 μg/L). Study subjects were further sub-divided into two groups (≤50 μg/L and > 50 μg/L) based on the recommended upper limit of water arsenic concentration (50 μg/L) in Bangladesh. Blood samples were collected from the study subjects by venipuncture and arsenic concentrations in drinking water, hair and nail samples were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PChE activity was assayed by spectrophotometer.ResultsArsenic concentrations in hair and nails were positively correlated with the arsenic levels in drinking water. Significant decreases in PChE activity were observed with increasing concentrations of arsenic in water, hair and nails. The average levels of PChE activity in low, medium and high arsenic exposure groups were also significantly different between each group. Lower levels of PChE activity were also observed in the > 50 μg/L group compared to the ≤50 μg/L group. Moreover, PChE activity was significantly decreased in the skin (+) symptoms group compared to those without (-).ConclusionsWe found a significant inverse relationship between arsenic exposure and PChE activity in a human population in Bangladesh. This research demonstrates a novel exposure-response relationship between arsenic and PChE activity which may explain one of the biological mechanisms through which arsenic exerts its neuro-and hepatotoxicity in humans.

Redox-Linked Cell Surface-Oriented Signaling for T-Cell Death

T-cell death, which occurs either for ontogenic T-cell selection or for activated T-cell elimination, is normally induced through binding of a specific ligand to cell-surface T-cell receptor for crosslinkage. Heavy metals and carbonyl compounds that bind to protein-reactive groups such as cysteine sulfhydryl groups and lysine epsilon-amino groups may also induce crosslinkage of cell-surface proteins, in part replacing or modifying the ligand-mediated action. This chemical event has been found to accompany clustering of membrane rafts, to which signal-transducing elements such as glycosylphosphatidylinositol-anchored proteins and Src family protein tyrosine kinases (PTKs) are attached, and to trigger the signal transduction for apoptotic T-cell death, inducing mitochondrial membrane potential reduction, caspase activation and DNA fragmentation. As signals potentially upstream of this signaling, activations of PTKs and mitogen-activated protein (MAP) family kinases and production of reactive oxygen species (ROS) were induced following the cell-surface event, and crucial roles of activation of c-Jun amino-terminal kinase and apoptosis signal-regulating kinase 1 by a redox-linked mechanism in the cell-death signaling were demonstrated. Intriguingly, ROS production as well as PTK/MAP family kinase activation occurred in a membrane raft integrity-dependent manner. The redox-linked and cell surface-oriented signal delivery pathway demonstrated here may play an important role in induction of immune disorders by protein reactive group-binding chemicals.

Acrolein induces activation of the epidermal growth factor receptor of human keratinocytes for cell death

Acrolein, which is a highly reactive formaldehyde generated by lipid peroxidation, can affect skin and cause various disorders. The effect of exposure of human keratinocytes to acrolein on cell surface‐oriented signal transduction into cells was examined. Incubation of human keratinocytes with a relatively low concentration (50 μM) of acrolein caused a prompt and selective induction of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) as a 180‐kDa molecule during the period from 5–30 min after the start of incubation. This early event was followed by an increase in the density and number of phosphotyrosine‐containing proteins during the period from 60–120 min after the start of incubation. The catalytic activity of EGFR as measured by the levels of autophorphorylation and phosphorylation of an exogenously added substrate, casein, in in vitro kinase assay, greatly increased as early as 1 min after the start of incubation and then decreased gradually 30 min later. MAP family kinases, including ERK, JNK, and p38 kinase, and the potentially downstream transcription factor c‐Jun were all promoted for phosphorylation/activation during a period of 5–30 min. Selective prompt phosphorylation/activation of EGFR followed by phosphorylation of MAP family kinases and c‐Jun and their blockade by a specific EGFR inhibitor, AG1478, suggested that activation of EGFR is the major, and possibly single, cell surface element for intracellular signal transduction in acrolein‐treated cells. Incubation of human keratinocytes with 50 μM of acrolein induced atypical apoptosis with morphologic apoptotic features with low‐grade oligonucleoside‐sized DNA fragmentation. Partial inhibition of such a cytopathic effect of acrolein on human keratinocytes by preincubation with AG1478 suggests the involvement of an EGFR‐mediated signal pathway for atypical apoptosis. These results provide new information on acrolein‐induced cell surface‐oriented signal transduction to human keratinocytes, and this information may be useful for understanding the pathogenesis of a number of skin diseases in response to environmental acrolein and acrolein‐generating ultraviolet irradiation. J. Cell. Biochem. 81: 679–688, 2001.