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...f apoptosis [24]. We have noted that both caspase 3 and caspase 9 are activated and cleaved. PARP cleavage is shown to have a role in DNA damage induced apoptosis and is the major target of caspase 3 =-=[25]-=-. We found that following ASHD treatment, caspase 3 activation leads to PARP cleavage. Further, we find that treatment of a pancaspase inhibitor on cells pretreated with ASHD resulted in a partial blo...

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...protein, BCL2 were downregulated leading to an increase in the ratio of proapoptotic to antiapoptotic proteins which has also been shown by others. Activation of caspases is the hallmark of apoptosis =-=[24]-=-. We have noted that both caspase 3 and caspase 9 are activated and cleaved. PARP cleavage is shown to have a role in DNA damage induced apoptosis and is the major target of caspase 3 [25]. We found t...

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...s that ASHD induces cell cycle arrest at least to a limited extent before triggering apoptosis. Apoptosis is a fundamental process essential for both development and maintenance of tissue homeostasis =-=[22]-=-. One of the early events in apoptosis is translocation of phosphatidylserine (PS), which is normally confined to the cytoplasmic face of the plasma membrane, to the cell surface. The appearance of th...

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...c pathways. In this respect, different apoptotic pathways leading to cytotoxicity have been studied extensively for many compounds PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e69103 =-=[2,3]-=-. These studies have become a focus of cancer chemotherapy and would shed light on the mechanism of action of candidate drugs. Since, defects in apoptotic pathways such as receptor- and mitochondrial-...

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...c pathways. In this respect, different apoptotic pathways leading to cytotoxicity have been studied extensively for many compounds PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e69103 =-=[2,3]-=-. These studies have become a focus of cancer chemotherapy and would shed light on the mechanism of action of candidate drugs. Since, defects in apoptotic pathways such as receptor- and mitochondrial-...

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...e is found in a variety of anticonvulsant drugs. In addition, they are being explored as aldose reductase inhibitors, antiarrhythmics, antimicrobials, CGRP receptor antagonists, and anticancer agents =-=[11,12]-=-. Previously, we have reported synthesis and characterization of a series of hydantoin derivatives [11,13]. Here, we show that the compound ASHD, an alkyl chain ester group containing hydantoin deriva...

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...reatment, an aqueous solution of [3H] thymidine (1 mCi) diluted with RPMI 1640 was added to each well. Following 48 h of incubation, the cells were pelleted, washed and processed as described earlier =-=[16]-=-. Radioactivity of the samples was expressed as disintegrations/min, which was proportional to the amount of [3H] thymidine incorporated into the DNA. Detection of mitochondrial transmembrane potentia...

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...ionic dye that exhibits potentialdependent accumulation in mitochondria, indicated by fluorescence emission shift from red (,590 nm) to green (,525 nm). MTP assay was carried out as described earlier =-=[17]-=-. In brief, Reh cells (0.756105 cells/ml) were treated with 10, 50, 100 and 250 mM of ASHD for 24, 48 and 72 h. Cells treated with 4 mM of 2,4-Dinitrophenyl hydrazine (Sigma-Aldrich, USA) for 24 h wer...

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...rn blotting analyses were repeated a minimum of two times with good agreement. Effect of a pan-caspase inhibitor on ASHD treated Reh cells The effect of a pan-caspase inhibitor, Z-VAD-FMK was studied =-=[18]-=- by incubating Reh cells with the inhibitor (50 mM) and ASHD (30 mM). Since ASHD and Z-VAD-FMK were dissolved in DMSO, cells with DMSO were used as control (equivalent to DMSO used in 30 mM concentrat...

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...ells/well. After 24 h, ASHD was added at a concentration of 10, 50, 100 or 250 mM. Following 48 or 72 h of treatment with the compounds, cells were collected and treated with MTT as described earlier =-=[15]-=-. In the case of 8E5, cells were harvested after 24, 48 and 72 h of ASHD treatment (10, 50 and 100 mM). The percentage of proliferating cells was calculated and depicted as a histogram. [3H] Thymidine...

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...obenzyl)-29,59-Dioxo-8-Azaspiro[Bicyclo[3.2.1] Octane-3,49-Imidazolidine]-19-yl) Acetate Induces Apoptosis in Human Leukemia Cells through Mitochondrial Pathway following Cell Cycle Arrest. PLoS ONE 8=-=(7)-=-: e69103. doi:10.1371/journal.pone.0069103 Editor: Kamyar Afarinkia, Univ of Bradford, United Kingdom Received April 26, 2013; Accepted June 6, 2013; Published July 26, 2013 Copyright: 2013 Kavitha ...

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...e is found in a variety of anticonvulsant drugs. In addition, they are being explored as aldose reductase inhibitors, antiarrhythmics, antimicrobials, CGRP receptor antagonists, and anticancer agents =-=[11,12]-=-. Previously, we have reported synthesis and characterization of a series of hydantoin derivatives [11,13]. Here, we show that the compound ASHD, an alkyl chain ester group containing hydantoin deriva...

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...inhibitors, antiarrhythmics, antimicrobials, CGRP receptor antagonists, and anticancer agents [11,12]. Previously, we have reported synthesis and characterization of a series of hydantoin derivatives =-=[11,13]-=-. Here, we show that the compound ASHD, an alkyl chain ester group containing hydantoin derivative (Fig. 1), can induce cytotoxicity in leukemia cells with remarkably high efficiency. Treatment with A...

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...[13]. Cell lines and culture conditions Human leukemia cell line, K562 and normal human T lymphocyte cell line, 8E5 were obtained from National Centre for Cell Science, Pune (India) and Reh cell line =-=[14]-=- was obtained as a gift from Prof. Michael Lieber, University of Southern California, Los Angeles (USA). All the cell lines were cultured in RPMI1640 (Sera Lab, UK) containing 10% FBS (Gibco BRL, USA)...

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...ed all morphological changes characteristic of apoptosis. In order to delineate the molecular details concerning cell death induced by ASHD, we investigated the possible molecular mechanisms involved =-=[23]-=-. Intrinsic pathway of apoptosis is one of the major pathways leading to cell death, which is regulated by BCL2 family of proteins. To test the role of these proteins in ASHD induced cell death, we me...