Ajay Rana - Academia.edu (original) (raw)

Papers by Ajay Rana

Apoptosis, 2001

Acute renal failure (ARF) can be defined as a sudden loss of renal function and is a common and s... more Acute renal failure (ARF) can be defined as a sudden loss of renal function and is a common and serious clinical problem. There are many causes of ARF but the most common cause results from injury to the renal tubular epi-thelial cells (RTECs). RTECs can be injured by schemia or by cytotoxic agents and, once injured, can die by necrosis or apotosis. In general, necrosis occurs in response to any severe injury, which leads to the biochemical collapse of the cell. Milder forms of the same types of injury cause apoptosis. At the cellular level there are fundamental differences between necrosis and apoptosis. Necrosis results from the additive effect of a number of independent biochemical events that are activated by severe depletion of cell energy stores. By contrast, apoptosis occurs via a coordinated, predictable and pre-determined pathway. These biochemical differences between apoptosis and necrosis have important therapeutic implications. Once a cell has been severely injured, necrosis is difficult to prevent. By contrast, the apoptotic pathway can potentially be modulated to maintain cell viability. The components of the apoptotic pathway that are potentially amenable to therapeutic modulation are discussed in detail in this review.

Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2008

Previous studies have shown that dietary micronutrient vanadium can protect neoplastic developmen... more Previous studies have shown that dietary micronutrient vanadium can protect neoplastic development induced by chemical carcinogens. Current investigation is an attempt to evaluate the role of vanadium (4.27 micro mol/l) in inhibiting 1,2 dimethyhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. We investigated the effect of vanadium against the formation of DMH-induced O(6)-methylguanine (O(6)-Meg) DNA adduct, a potent cytotoxic and mutagenic agent for colon cancer. Supplementation of vanadium significantly reduced the hepatic (P<0.05), and colonic (at three sequential time points; ANOVA, F=4.96, P<0.05) O(6)-Meg DNA adduct levels in rats, indicating vanadium's potency in limiting the initiation event of colon carcinogenesis. Removal of initiated and damaged precancerous cells by apoptosis can prevent tumorigenesis and further malignancy. DNA fragmentation study revealed the vanadium-mediated apoptotic induction in colon tumors. The increased value of apoptotic index (AI) (62.27%; P<0.01) in subsequent TUNEL assay further confirmed the apoptosis induction by vanadium. This paralleled the nuclear immunoexpression of p53. A significant positive correlation between p53 immunoexpression and AI (P=0.0026, r=0.83, r(2)=0.69) links its association with vanadium-mediated apoptotic induction. Vanadium treatment also abated the mRNA expression of iNOS (54.03%), reflecting its protective effect against nitric oxide-mediated genotoxicity and colon tumorigenesis. These studies cumulatively provide strong evidence for the inhibitory actions of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon.

Journal of Molecular Signaling, 2010

Background Ligands of Peroxisome proliferator-activated receptor gamma (PPARγ) can inhibit growth... more Background Ligands of Peroxisome proliferator-activated receptor gamma (PPARγ) can inhibit growth and promote apoptosis in various cancer cells, and thus have the potential to be utilized as anticancer drugs. This potential however, has been seriously challenged by observations that they can lead to tumor promotion in some cancer models, possibly due to activation of different signaling mechanisms in various tumor environments. Elucidation of the specific signaling events that modulate PPARγ ligand-mediated events is thus critical to increase their efficacy. The studies described here were designed to elucidate the signaling pathway(s) that modulate the apoptotic potential of Troglitazone (TRG), an artificial PPARγ ligand in hepatocellular carcinoma (HCC) cells. Results Our results indicate that the apoptotic potential of TRG was regulated by the presence or absence of serum in the media. When added in serum-containing media, TRG inhibited proliferation and cyclin D1 expression, but was unable to induce any apoptosis. However, TRG's apoptotic potential was induced significantly when added in serum deficient media, as indicated by increased PARP and Caspase-3 cleavage and results from apoptosis assay. Furthermore, TRG-induced apoptosis in serum deficient media was associated with a dramatic reduction in PI3Kinase downstream target AktSer473 and FoxO1Thr24/FoxO3aThr32 phosphorylation. On the contrary, there was an increase of PI3K-induced AktSer473 and FoxO1Thr24/FoxO3aThr32 phosphorylation involving Pak, when TRG was added in serum-containing media. Pharmacological inhibition of PI3Kinase pathway with LY294002 inhibited Aktser473 phosphorylation and sensitized cells towards apoptosis in the presence of serum, indicating the involvement of PI3K in apoptosis resistance. Interestingly, pharmacological inhibition or siRNA-mediated knockdown of Akt or inhibition of Pak was unable to sensitize cells towards TRG-induced apoptosis in the presence of serum. Similarly, TRG was unable to induce apoptosis in the Akt1-KO, Akt1&2-KO MEFs in serum-containing media. Conclusion These studies indicate that TRG-induced apoptosis is modulated by PI3K pathway in a novel Akt-independent manner, which might contribute to its tumor promoting effects. Since PI3K activation is linked with various cancers, combination therapy utilizing TRG and PI3K inhibitors has the potential to not only increase the efficacy of TRG as a chemotherapeutic agent but also reduce its off target effects.

International Journal of Cancer, 2007

In the present study, the authors evaluated the anticancer mechanism of vanadium, a dietary micro... more In the present study, the authors evaluated the anticancer mechanism of vanadium, a dietary micronutrient and an important pharmacological agent, on a defined model of chemically induced rat mammary carcinogenesis in vivo and on human breast cancer cell line MCF7 in vitro. Female Sprague-Dawley rats were treated with 7,12-dimethylbenz(α)anthracene (0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion to induce mammary preneoplasia. Vanadium (ammonium monovanadate) at a concentration of 0.5 ppm (4.27 μmol/l) was supplemented in drinking water and given ad libitum to the experimental groups for 24 weeks. Histological finding showed substantial repair of hyperplastic lesions. There was a significant reduction in incidence, multiplicity (34%, p < 0.01), size of palpable mammary tumors and delay in mean latency period of tumor appearance. Immunohistochemical analysis in vivo indicated a decrease in cell proliferation (24.68% p < 0.05) and an increase among the TUNEL-positive apoptotic cells along with strong expressions of p53 and Bax, and downregulation of Bcl2 proteins in the mammary tissue of vanadium-treated animals. Further, MCF7 cells were cultured in minimal essential medium and were treated with 100, 175 and 250 μM of vanadium (ammonium monovanadate) for 36 hr. Exposure of MCF7 cells to vanadium led to induction of apoptosis in a dose-dependent manner. It was found further that vanadium treatment brought about a prominent cell cycle arrest and chromosomal condensation, leading to apoptosis (42.62%, p < 0.05). Results of both the in vivo and in vitro study demonstrate that vanadium has the potential to be developed into an anti-breast cancer drug in the near future. © 2006 Wiley-Liss, Inc.

Life Sciences, 2007

Cell proliferation plays an important role in multistage chemical carcinogenesis. Again, several ... more Cell proliferation plays an important role in multistage chemical carcinogenesis. Again, several reports demonstrated that upregulation of metallothionein (MT) expression is associated with increased cell proliferation that may contribute to the pathogenesis of preneoplastic phenotype to frank malignancy. In this study, we evaluated the roles of early DNA damage, altered expressions of liver MT and Ki-67 nuclear antigen, and altered hepatic levels of zinc (Zn) and copper (Cu) on cell proliferation and the progression of hepatocarcinogenesis through premalignant, late premalignant and malignant transformation phases in male Sprague-Dawley rats. We have further studied the association between MT expression and cell proliferation in hepatocarcinogenesis. There was substantial induction of DNA single-strand breaks (SSBs) (P b 0.001) and development of hepatocellular premalignant lesions along with significant decrease in hepatic levels of Zn and increase in Cu content following a single, necrogenic, intraperitoneal (i.p.) injection (200 mg/Kg body weight) of diethylnitrosamine (DEN) at week 4 of the experimental protocol. Moreover, DEN + phenobarbital (PB)-treatment significantly elevated MT-, Ki-67-, and BrdU-immunoexpressions along with their immunolabeling indices. Furthermore, positive correlations between MT-and Ki-67-labeling (P = 0.0006) at various time intervals, as well as, between MT immunoreactivity and 5'-bromo-2'-deoxyuridine-labeling index (BrdU-LI) (P = 0.0007) indicate that, MT expression might be associated with Ki-67 expression and cell proliferation thereby. The study suggests that DEN treatment may lead to alteration of Zn and Cu levels resulting in early DNA damage along with elevation of MT expression that may ultimately lead to hepatic cell proliferation. The results thus provide evidence in support of the role of MT as a potential positive regulator of cell growth during the early stages of hepatocellular transformation in rats.

Prostaglandins Leukotrienes and Essential Fatty Acids, 2008

The present study investigated the chemopreventive effect of dietary fish oil (Maxepa), rich in e... more The present study investigated the chemopreventive effect of dietary fish oil (Maxepa), rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on induction of apoptosis in mammary carcinogenesis model. Mammary carcinogenesis was initiated by a single, tail vein injection of 7,12dimethylbenz(a)anthracene (DMBA) (0.5 mg/0.2 ml corn oil/100 g body weight) at 7 weeks of animal age. Ninety female Sprague-Dawley rats were divided into two parts: part one was used for histology and immunohistochemical study and part two for morphological analysis. Each part consists of three experimental groups having 15 animals, i.e., Group A (DMBA control), Group B (DMBA+fish oil) and Group C (DMBA+corn oil). Rats were fed either fish oil or corn oil (0.5 ml/day/rat) by oral gavage, 2 weeks prior to DMBA injection. Treatment was continued 25 weeks, studying histopathology, expression of Bax and Bcl-2 proteins by immunohistochemistry and apoptosis by TUNEL assay and morphological study at 36 weeks. Results showed that the fish oil-treated group exhibited a substantial increase in Bax (po0.05) immunolabelling and a reduction of Bcl-2 immunopositivity (po0.05), and increased TUNEL-positive apoptotic cells (po0.05); however, corn oil treatment did not show these beneficial effects toward mammary preneoplasia. We conclude that fish oil has the potential to play a significant role in limiting mammary tumourigenesis in vivo.

Biochimica Et Biophysica Acta-molecular Basis of Disease, 2007

Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molec... more Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molecular events in the initiation of chemical carcinogenesis and modulation of such events by chemopreventive agents could be an important step in limiting neoplastic transformation in vivo. Vanadium, a dietary micronutrient has been found to be effective in several types of cancers both in vivo and in vitro and also possesses profound anticarcinogenicity against rat models of mammary, colon and hepatocarcinogenesis. Presently, we report the chemopreventive potential of vanadium on diethylnitrosamine (DEN)-induced early DNA damages in rat liver. Hepatocarcinogenesis was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal (i.p.) injection of DEN (200 mg/kg body weight) at week 4. There was a significant induction of tissuespecific ethylguanines, steady elevation of modified DNA bases 8-hydroxy-2′-deoxyguanosines (8-OHdGs) (P < 0.0001; 89.93%) along with substantial increment of the extent of single-strand breaks (SSBs) (P < 0.0001) following DEN exposure. Supplementation of 0.5 ppm of vanadium throughout the experiment abated the formations of O 6 -ethylguanines and 7-ethylguanines (P < 0.0001; 48.71% and 67.54% respectively), 8-OHdGs (P < 0.0001; 81.37%), length:width (L:W) of DNA mass (P < 0.01; 62.12%) and the mean frequency of tailed DNA (P < 0.001; 53.58%), and hepatic nodulogenesis in preneoplastic rat liver. The study indicates that 0.5 ppm vanadium is potentially and optimally effective, as derived from dose-response studies, in limiting early molecular events and preneoplastic lesions, thereby modulating the initiation stage of hepatocarcinogenesis. Vanadium is chemopreventive against DEN-induced genotoxicity and resulting hepatocellular transformation in rats.

Journal of Molecular Signaling, 2010

Background: The gastrointestinal peptide hormone gastrin is known to regulate various cellular pr... more Background: The gastrointestinal peptide hormone gastrin is known to regulate various cellular processes including proliferation, migration and metastasis in gastrointestinal (GI) cells. The studies described here were undertaken to elucidate in detail the signaling pathways mediating the migratory responses of amidated gastrin (G17) and to understand the involvement of the serine/threonine kinase Glycogen Synthase Kinase-3 beta (GSK3β) in this.

Neurotoxicology, 2010

Parkinson's disease (PD) is a common neurodegenerative movement disorder characterized by extensi... more Parkinson's disease (PD) is a common neurodegenerative movement disorder characterized by extensive degeneration of dopaminergic neurons in the nigrostriatal system. Neurochemical and neuropathological analyses clearly indicate that oxidative stress, mitochondrial dysfunction, neuroinflammation and impairment of the ubiquitin-proteasome system (UPS) are major mechanisms of dopaminergic degeneration. Evidence from experimental models and postmortem PD brain tissues demonstrates that apoptotic cell death is the common final pathway responsible for selective and irreversible loss of nigral dopaminergic neurons. Epidemiological studies imply both environmental neurotoxicants and genetic predisposition are risk factors for PD, though the cellular mechanisms underlying selective dopaminergic degeneration remain unclear. Recent progress in signal transduction research is beginning to unravel the complex mechanisms governing dopaminergic degeneration. During 12 th International Neurotoxicology meeting, discussion at one symposium focused on several key signaling pathways of dopaminergic degeneration. This review summarizes two novel signaling pathways of nigral dopaminergic degeneration that have been elucidated using neurotoxicity models of PD. Dr. Anumantha Kanthasamy described a cell death pathway involving the novel protein kinase C delta isoform (PKCδ) in oxidative stress-induced apoptotic cell death in experimental models of PD. Dr. Ajay Rana presented his recent work on the role of mixed lineage kinase-3 (MLK3) in neuroinflammatory processes in neurotoxic cell death. Collectively, PKCδ and MLK3 signaling pathways provide new understanding of neurodegenerative processes in PD, and further exploration of these pathways may translate into effective neuroprotective drugs for the treatment of PD.

Nutrition and Cancer-an International Journal, 2007

Previous studies from our laboratory have demonstrated the potential anticarcinogenicity of vanad... more Previous studies from our laboratory have demonstrated the potential anticarcinogenicity of vanadium, a dietary micronutrient in rat liver, colon, and mammary carcinogenesis models in vivo. In this paper, we have investigated further the antihepatocarcinogenic role of this essential trace element by studying several biomarkers of chemical carcinogenesis with special reference to cell proliferation and oxidative DNA damage. Hepatocarcinogenesis was induced in male Sprague-Dawley rats by chronic feeding of 2-acetylaminofluorene (2-AAF) at a dose of 0.05% in basal diet daily for 5 days a week. Vanadium in the form of ammonium metavanadate (0.5 ppm equivalent to 4.27 μ mol/l) was supplemented ad lib to the rats. Continuous vanadium administration reduced relative liver weight, nodular incidence (79.99%), total number and multiplicity (P < 0.001; 68.17%) along with improvement in hepatocellular architecture when compared to carcinogen control. Vanadium treatment further restored hepatic uridine diphosphate (UDP)-glucuronosyl transferase and UDP-glucose dehydrogenase activities, inhibited lipid peroxidation, and prevented the development of glycogen-storage preneoplastic foci (P < 0.01; 63.29%) in an initiation-promotion model. Long-term vanadium treatment also reduced BrdU-labelling index (P < 0.02) and inhibited cell proliferation during hepatocellular preneoplasia. Finally, short-term vanadium exposure abated the formations of 8-hydroxy-2′-deoxyguanosines (P < 0.001; 56.27%), length:width of DNA mass (P < 0.01), and the mean frequency of tailed DNA (P < 0.001) in preneoplastic rat liver. The study indicates the potential role of vanadium in suppressing cell proliferation and in preventing early DNA damage in vivo. Vanadium is chemopreventive against the early stages of 2-AAF-induced hepatocarcinogenesis in rats.

Biochimica Et Biophysica Acta-molecular Cell Research, 2003

Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kina... more Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kinase (MAPKKK) and cause neuronal cell death mediated through c-Jun, N-terminal kinase (JNK) pathway. Recently, genetic studies in Drosophila revealed the presence of an MLK termed slipper (slpr). However, its biochemical features like physiological substrate, role in different MAPK pathways and developmental and tissue-specific expression pattern were not reported. Here, we report cDNA cloning, expression analysis and biochemical characterization of a Drosophila mixed lineage kinase (dMLK) that is also known as slipper. The protein structure analysis of dMLK/slipper revealed, in addition to the conserved domains, a stretch of glutamine in the amino terminus and an asparagine -threonine stretch at the carboxy-terminus. In situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that dMLK is expressed in early embryonic stages, adult brain and thorax. Ectopic expression of dMLK either in Drosophila S2 or in mammalian HEK293 cells leads to activation of JNK, p38 and extracellular signal regulated kinase (ERK) pathways. Further, dMLK directly phosphorylates Hep, dMKK4 and also their mammalian counterparts, MKK7 and SEK1, in an in vitro kinase assay. Taken together, our results provide for the first time a comprehensive expression profile and new biochemical insight of dMLK/slipper. D

Cell Research, 2010

Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that i... more Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is activated by Tumor Necrosis Factor-α (TNF-α) and specifically activates c-Jun N-terminal kinase (JNK) upon TNF-α stimulation. The mechanism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adaptor molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose dependent manner, upon TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2 dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.

Molecular Cell, 2002

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell dea... more Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity remain unknown. Here, we demonstrate ceramide as the activator of Drosophila MLK (dMLK) and identify ceramide and TNF-α as agonists of mammalian MLK3. dMLK and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of dMLK and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-α also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of dMLK and MLK3, and MLK3 contributes to JNK activation induced by TNF-α.

Apoptosis, 2001

Acute renal failure (ARF) can be defined as a sudden loss of renal function and is a common and s... more Acute renal failure (ARF) can be defined as a sudden loss of renal function and is a common and serious clinical problem. There are many causes of ARF but the most common cause results from injury to the renal tubular epi-thelial cells (RTECs). RTECs can be injured by schemia or by cytotoxic agents and, once injured, can die by necrosis or apotosis. In general, necrosis occurs in response to any severe injury, which leads to the biochemical collapse of the cell. Milder forms of the same types of injury cause apoptosis. At the cellular level there are fundamental differences between necrosis and apoptosis. Necrosis results from the additive effect of a number of independent biochemical events that are activated by severe depletion of cell energy stores. By contrast, apoptosis occurs via a coordinated, predictable and pre-determined pathway. These biochemical differences between apoptosis and necrosis have important therapeutic implications. Once a cell has been severely injured, necrosis is difficult to prevent. By contrast, the apoptotic pathway can potentially be modulated to maintain cell viability. The components of the apoptotic pathway that are potentially amenable to therapeutic modulation are discussed in detail in this review.

Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2008

Previous studies have shown that dietary micronutrient vanadium can protect neoplastic developmen... more Previous studies have shown that dietary micronutrient vanadium can protect neoplastic development induced by chemical carcinogens. Current investigation is an attempt to evaluate the role of vanadium (4.27 micro mol/l) in inhibiting 1,2 dimethyhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. We investigated the effect of vanadium against the formation of DMH-induced O(6)-methylguanine (O(6)-Meg) DNA adduct, a potent cytotoxic and mutagenic agent for colon cancer. Supplementation of vanadium significantly reduced the hepatic (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05), and colonic (at three sequential time points; ANOVA, F=4.96, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05) O(6)-Meg DNA adduct levels in rats, indicating vanadium&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s potency in limiting the initiation event of colon carcinogenesis. Removal of initiated and damaged precancerous cells by apoptosis can prevent tumorigenesis and further malignancy. DNA fragmentation study revealed the vanadium-mediated apoptotic induction in colon tumors. The increased value of apoptotic index (AI) (62.27%; P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.01) in subsequent TUNEL assay further confirmed the apoptosis induction by vanadium. This paralleled the nuclear immunoexpression of p53. A significant positive correlation between p53 immunoexpression and AI (P=0.0026, r=0.83, r(2)=0.69) links its association with vanadium-mediated apoptotic induction. Vanadium treatment also abated the mRNA expression of iNOS (54.03%), reflecting its protective effect against nitric oxide-mediated genotoxicity and colon tumorigenesis. These studies cumulatively provide strong evidence for the inhibitory actions of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon.

Journal of Molecular Signaling, 2010

Background Ligands of Peroxisome proliferator-activated receptor gamma (PPARγ) can inhibit growth... more Background Ligands of Peroxisome proliferator-activated receptor gamma (PPARγ) can inhibit growth and promote apoptosis in various cancer cells, and thus have the potential to be utilized as anticancer drugs. This potential however, has been seriously challenged by observations that they can lead to tumor promotion in some cancer models, possibly due to activation of different signaling mechanisms in various tumor environments. Elucidation of the specific signaling events that modulate PPARγ ligand-mediated events is thus critical to increase their efficacy. The studies described here were designed to elucidate the signaling pathway(s) that modulate the apoptotic potential of Troglitazone (TRG), an artificial PPARγ ligand in hepatocellular carcinoma (HCC) cells. Results Our results indicate that the apoptotic potential of TRG was regulated by the presence or absence of serum in the media. When added in serum-containing media, TRG inhibited proliferation and cyclin D1 expression, but was unable to induce any apoptosis. However, TRG's apoptotic potential was induced significantly when added in serum deficient media, as indicated by increased PARP and Caspase-3 cleavage and results from apoptosis assay. Furthermore, TRG-induced apoptosis in serum deficient media was associated with a dramatic reduction in PI3Kinase downstream target AktSer473 and FoxO1Thr24/FoxO3aThr32 phosphorylation. On the contrary, there was an increase of PI3K-induced AktSer473 and FoxO1Thr24/FoxO3aThr32 phosphorylation involving Pak, when TRG was added in serum-containing media. Pharmacological inhibition of PI3Kinase pathway with LY294002 inhibited Aktser473 phosphorylation and sensitized cells towards apoptosis in the presence of serum, indicating the involvement of PI3K in apoptosis resistance. Interestingly, pharmacological inhibition or siRNA-mediated knockdown of Akt or inhibition of Pak was unable to sensitize cells towards TRG-induced apoptosis in the presence of serum. Similarly, TRG was unable to induce apoptosis in the Akt1-KO, Akt1&2-KO MEFs in serum-containing media. Conclusion These studies indicate that TRG-induced apoptosis is modulated by PI3K pathway in a novel Akt-independent manner, which might contribute to its tumor promoting effects. Since PI3K activation is linked with various cancers, combination therapy utilizing TRG and PI3K inhibitors has the potential to not only increase the efficacy of TRG as a chemotherapeutic agent but also reduce its off target effects.

International Journal of Cancer, 2007

In the present study, the authors evaluated the anticancer mechanism of vanadium, a dietary micro... more In the present study, the authors evaluated the anticancer mechanism of vanadium, a dietary micronutrient and an important pharmacological agent, on a defined model of chemically induced rat mammary carcinogenesis in vivo and on human breast cancer cell line MCF7 in vitro. Female Sprague-Dawley rats were treated with 7,12-dimethylbenz(α)anthracene (0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion to induce mammary preneoplasia. Vanadium (ammonium monovanadate) at a concentration of 0.5 ppm (4.27 μmol/l) was supplemented in drinking water and given ad libitum to the experimental groups for 24 weeks. Histological finding showed substantial repair of hyperplastic lesions. There was a significant reduction in incidence, multiplicity (34%, p < 0.01), size of palpable mammary tumors and delay in mean latency period of tumor appearance. Immunohistochemical analysis in vivo indicated a decrease in cell proliferation (24.68% p < 0.05) and an increase among the TUNEL-positive apoptotic cells along with strong expressions of p53 and Bax, and downregulation of Bcl2 proteins in the mammary tissue of vanadium-treated animals. Further, MCF7 cells were cultured in minimal essential medium and were treated with 100, 175 and 250 μM of vanadium (ammonium monovanadate) for 36 hr. Exposure of MCF7 cells to vanadium led to induction of apoptosis in a dose-dependent manner. It was found further that vanadium treatment brought about a prominent cell cycle arrest and chromosomal condensation, leading to apoptosis (42.62%, p < 0.05). Results of both the in vivo and in vitro study demonstrate that vanadium has the potential to be developed into an anti-breast cancer drug in the near future. © 2006 Wiley-Liss, Inc.

Life Sciences, 2007

Cell proliferation plays an important role in multistage chemical carcinogenesis. Again, several ... more Cell proliferation plays an important role in multistage chemical carcinogenesis. Again, several reports demonstrated that upregulation of metallothionein (MT) expression is associated with increased cell proliferation that may contribute to the pathogenesis of preneoplastic phenotype to frank malignancy. In this study, we evaluated the roles of early DNA damage, altered expressions of liver MT and Ki-67 nuclear antigen, and altered hepatic levels of zinc (Zn) and copper (Cu) on cell proliferation and the progression of hepatocarcinogenesis through premalignant, late premalignant and malignant transformation phases in male Sprague-Dawley rats. We have further studied the association between MT expression and cell proliferation in hepatocarcinogenesis. There was substantial induction of DNA single-strand breaks (SSBs) (P b 0.001) and development of hepatocellular premalignant lesions along with significant decrease in hepatic levels of Zn and increase in Cu content following a single, necrogenic, intraperitoneal (i.p.) injection (200 mg/Kg body weight) of diethylnitrosamine (DEN) at week 4 of the experimental protocol. Moreover, DEN + phenobarbital (PB)-treatment significantly elevated MT-, Ki-67-, and BrdU-immunoexpressions along with their immunolabeling indices. Furthermore, positive correlations between MT-and Ki-67-labeling (P = 0.0006) at various time intervals, as well as, between MT immunoreactivity and 5'-bromo-2'-deoxyuridine-labeling index (BrdU-LI) (P = 0.0007) indicate that, MT expression might be associated with Ki-67 expression and cell proliferation thereby. The study suggests that DEN treatment may lead to alteration of Zn and Cu levels resulting in early DNA damage along with elevation of MT expression that may ultimately lead to hepatic cell proliferation. The results thus provide evidence in support of the role of MT as a potential positive regulator of cell growth during the early stages of hepatocellular transformation in rats.

Prostaglandins Leukotrienes and Essential Fatty Acids, 2008

The present study investigated the chemopreventive effect of dietary fish oil (Maxepa), rich in e... more The present study investigated the chemopreventive effect of dietary fish oil (Maxepa), rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on induction of apoptosis in mammary carcinogenesis model. Mammary carcinogenesis was initiated by a single, tail vein injection of 7,12dimethylbenz(a)anthracene (DMBA) (0.5 mg/0.2 ml corn oil/100 g body weight) at 7 weeks of animal age. Ninety female Sprague-Dawley rats were divided into two parts: part one was used for histology and immunohistochemical study and part two for morphological analysis. Each part consists of three experimental groups having 15 animals, i.e., Group A (DMBA control), Group B (DMBA+fish oil) and Group C (DMBA+corn oil). Rats were fed either fish oil or corn oil (0.5 ml/day/rat) by oral gavage, 2 weeks prior to DMBA injection. Treatment was continued 25 weeks, studying histopathology, expression of Bax and Bcl-2 proteins by immunohistochemistry and apoptosis by TUNEL assay and morphological study at 36 weeks. Results showed that the fish oil-treated group exhibited a substantial increase in Bax (po0.05) immunolabelling and a reduction of Bcl-2 immunopositivity (po0.05), and increased TUNEL-positive apoptotic cells (po0.05); however, corn oil treatment did not show these beneficial effects toward mammary preneoplasia. We conclude that fish oil has the potential to play a significant role in limiting mammary tumourigenesis in vivo.

Biochimica Et Biophysica Acta-molecular Basis of Disease, 2007

Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molec... more Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molecular events in the initiation of chemical carcinogenesis and modulation of such events by chemopreventive agents could be an important step in limiting neoplastic transformation in vivo. Vanadium, a dietary micronutrient has been found to be effective in several types of cancers both in vivo and in vitro and also possesses profound anticarcinogenicity against rat models of mammary, colon and hepatocarcinogenesis. Presently, we report the chemopreventive potential of vanadium on diethylnitrosamine (DEN)-induced early DNA damages in rat liver. Hepatocarcinogenesis was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal (i.p.) injection of DEN (200 mg/kg body weight) at week 4. There was a significant induction of tissuespecific ethylguanines, steady elevation of modified DNA bases 8-hydroxy-2′-deoxyguanosines (8-OHdGs) (P < 0.0001; 89.93%) along with substantial increment of the extent of single-strand breaks (SSBs) (P < 0.0001) following DEN exposure. Supplementation of 0.5 ppm of vanadium throughout the experiment abated the formations of O 6 -ethylguanines and 7-ethylguanines (P < 0.0001; 48.71% and 67.54% respectively), 8-OHdGs (P < 0.0001; 81.37%), length:width (L:W) of DNA mass (P < 0.01; 62.12%) and the mean frequency of tailed DNA (P < 0.001; 53.58%), and hepatic nodulogenesis in preneoplastic rat liver. The study indicates that 0.5 ppm vanadium is potentially and optimally effective, as derived from dose-response studies, in limiting early molecular events and preneoplastic lesions, thereby modulating the initiation stage of hepatocarcinogenesis. Vanadium is chemopreventive against DEN-induced genotoxicity and resulting hepatocellular transformation in rats.

Journal of Molecular Signaling, 2010

Background: The gastrointestinal peptide hormone gastrin is known to regulate various cellular pr... more Background: The gastrointestinal peptide hormone gastrin is known to regulate various cellular processes including proliferation, migration and metastasis in gastrointestinal (GI) cells. The studies described here were undertaken to elucidate in detail the signaling pathways mediating the migratory responses of amidated gastrin (G17) and to understand the involvement of the serine/threonine kinase Glycogen Synthase Kinase-3 beta (GSK3β) in this.

Neurotoxicology, 2010

Parkinson's disease (PD) is a common neurodegenerative movement disorder characterized by extensi... more Parkinson's disease (PD) is a common neurodegenerative movement disorder characterized by extensive degeneration of dopaminergic neurons in the nigrostriatal system. Neurochemical and neuropathological analyses clearly indicate that oxidative stress, mitochondrial dysfunction, neuroinflammation and impairment of the ubiquitin-proteasome system (UPS) are major mechanisms of dopaminergic degeneration. Evidence from experimental models and postmortem PD brain tissues demonstrates that apoptotic cell death is the common final pathway responsible for selective and irreversible loss of nigral dopaminergic neurons. Epidemiological studies imply both environmental neurotoxicants and genetic predisposition are risk factors for PD, though the cellular mechanisms underlying selective dopaminergic degeneration remain unclear. Recent progress in signal transduction research is beginning to unravel the complex mechanisms governing dopaminergic degeneration. During 12 th International Neurotoxicology meeting, discussion at one symposium focused on several key signaling pathways of dopaminergic degeneration. This review summarizes two novel signaling pathways of nigral dopaminergic degeneration that have been elucidated using neurotoxicity models of PD. Dr. Anumantha Kanthasamy described a cell death pathway involving the novel protein kinase C delta isoform (PKCδ) in oxidative stress-induced apoptotic cell death in experimental models of PD. Dr. Ajay Rana presented his recent work on the role of mixed lineage kinase-3 (MLK3) in neuroinflammatory processes in neurotoxic cell death. Collectively, PKCδ and MLK3 signaling pathways provide new understanding of neurodegenerative processes in PD, and further exploration of these pathways may translate into effective neuroprotective drugs for the treatment of PD.

Nutrition and Cancer-an International Journal, 2007

Previous studies from our laboratory have demonstrated the potential anticarcinogenicity of vanad... more Previous studies from our laboratory have demonstrated the potential anticarcinogenicity of vanadium, a dietary micronutrient in rat liver, colon, and mammary carcinogenesis models in vivo. In this paper, we have investigated further the antihepatocarcinogenic role of this essential trace element by studying several biomarkers of chemical carcinogenesis with special reference to cell proliferation and oxidative DNA damage. Hepatocarcinogenesis was induced in male Sprague-Dawley rats by chronic feeding of 2-acetylaminofluorene (2-AAF) at a dose of 0.05% in basal diet daily for 5 days a week. Vanadium in the form of ammonium metavanadate (0.5 ppm equivalent to 4.27 μ mol/l) was supplemented ad lib to the rats. Continuous vanadium administration reduced relative liver weight, nodular incidence (79.99%), total number and multiplicity (P < 0.001; 68.17%) along with improvement in hepatocellular architecture when compared to carcinogen control. Vanadium treatment further restored hepatic uridine diphosphate (UDP)-glucuronosyl transferase and UDP-glucose dehydrogenase activities, inhibited lipid peroxidation, and prevented the development of glycogen-storage preneoplastic foci (P < 0.01; 63.29%) in an initiation-promotion model. Long-term vanadium treatment also reduced BrdU-labelling index (P < 0.02) and inhibited cell proliferation during hepatocellular preneoplasia. Finally, short-term vanadium exposure abated the formations of 8-hydroxy-2′-deoxyguanosines (P < 0.001; 56.27%), length:width of DNA mass (P < 0.01), and the mean frequency of tailed DNA (P < 0.001) in preneoplastic rat liver. The study indicates the potential role of vanadium in suppressing cell proliferation and in preventing early DNA damage in vivo. Vanadium is chemopreventive against the early stages of 2-AAF-induced hepatocarcinogenesis in rats.

Biochimica Et Biophysica Acta-molecular Cell Research, 2003

Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kina... more Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kinase (MAPKKK) and cause neuronal cell death mediated through c-Jun, N-terminal kinase (JNK) pathway. Recently, genetic studies in Drosophila revealed the presence of an MLK termed slipper (slpr). However, its biochemical features like physiological substrate, role in different MAPK pathways and developmental and tissue-specific expression pattern were not reported. Here, we report cDNA cloning, expression analysis and biochemical characterization of a Drosophila mixed lineage kinase (dMLK) that is also known as slipper. The protein structure analysis of dMLK/slipper revealed, in addition to the conserved domains, a stretch of glutamine in the amino terminus and an asparagine -threonine stretch at the carboxy-terminus. In situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that dMLK is expressed in early embryonic stages, adult brain and thorax. Ectopic expression of dMLK either in Drosophila S2 or in mammalian HEK293 cells leads to activation of JNK, p38 and extracellular signal regulated kinase (ERK) pathways. Further, dMLK directly phosphorylates Hep, dMKK4 and also their mammalian counterparts, MKK7 and SEK1, in an in vitro kinase assay. Taken together, our results provide for the first time a comprehensive expression profile and new biochemical insight of dMLK/slipper. D

Cell Research, 2010

Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that i... more Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is activated by Tumor Necrosis Factor-α (TNF-α) and specifically activates c-Jun N-terminal kinase (JNK) upon TNF-α stimulation. The mechanism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adaptor molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose dependent manner, upon TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2 dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.

Molecular Cell, 2002

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell dea... more Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity remain unknown. Here, we demonstrate ceramide as the activator of Drosophila MLK (dMLK) and identify ceramide and TNF-α as agonists of mammalian MLK3. dMLK and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of dMLK and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-α also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of dMLK and MLK3, and MLK3 contributes to JNK activation induced by TNF-α.