Rajdeep Chowdhury

Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2-mediated suppression of mTORC1.

Significance NO exposure triggered an ATM-mediated damage response in breast cancer cells involving activation of the LKB1 and TSC2 tumor suppressors, repression of mTORC1, ULK phosphorylation, and increased autophagic flux. The associated loss of cell viability indicates that autophagy can function as a cytotoxic response to nitrosative stress in tumor cells. Collectively, the data identify a nitrosative-stress signaling pathway that regulates autophagy. A more comprehensive understanding of signaling pathways regulating autophagy holds promise for developing new therapeutic approaches compromising prosurvival autophagic pathways that enable tumor cells to evade therapy, or promoting prodeath autophagic pathways that kill cancer cells. Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosome-dependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death.

Comparison of health effects between individuals with and without skin lesions in the population exposed to arsenic through drinking water in West Bengal, India

A study was conducted to explore the effect of arsenic causing conjunctivitis, neuropathy and respiratory illness in individuals, with or without skin lesions, as a result of exposure through drinking water, contaminated with arsenic to similar extent. Exposed study population belongs to the districts of North 24 Parganas and Nadia, West Bengal, India. A total of 725 exposed (373 with skin lesions and 352 without skin lesions) and 389 unexposed individuals were recruited as study participants. Participants were clinically examined and interviewed. Arsenic content in drinking water, urine, nail and hair was estimated. Individuals with skin lesion showed significant retention of arsenic in nail and hair and lower amount of urinary arsenic compared to the group without any skin lesion. Individuals with skin lesion also showed higher risk for conjunctivitis ((odds ratio) OR: 7.33, 95% CI: 5.05–10.59), peripheral neuropathy (OR: 3.95, 95% CI: 2.61–5.93) and respiratory illness (OR: 4.86, 95% CI: 3.16–7.48) compared to the group without any skin lesion. The trend test for OR of the three diseases in three groups was found to be statistically significant. Again, individuals without skin lesion in the exposed group showed higher risk for conjunctivitis (OR: 4.66, 95% CI: 2.45–8.85), neuropathy (OR: 3.99, 95% CI: 1.95–8.09), and respiratory illness (OR: 3.21, 95% CI: 1.65–6.26) when compared to arsenic unexposed individuals. Although individuals with skin lesions were more susceptible to arsenic-induced toxicity, individuals without skin lesions were also subclinically affected and are also susceptible to arsenic-induced toxicity and carcinogenicity when compared to individuals not exposed to arsenic.

Arsenic-induced cell proliferation is associated with enhanced ROS generation, Erk signaling and CyclinA expression.

Arsenic is a well-established human carcinogen; however molecular mechanisms to arsenic-induced carcinogenesis are complex and elusive. The present study identifies a potential biomarker of arsenic exposure, and redefines arsenic-induced signaling in stimulation of cell proliferation. The effect of arsenic exposure on gene expression was evaluated in PBMC of arsenic-exposed individuals selected from a severely affected district of West Bengal, India. A novel, un-documented biomarker of arsenic exposure, CyclinA was identified by microarray analysis from the study. Non-transformed cell lines HaCat and Int407 when exposed to clinically achievable arsenic concentration showed significant increase of CyclinA substantiating the clinical data. An associated increase in S phase population of cells in cell cycle, indicative of enhanced proliferation was also noticed. On further investigation of the pathway to arsenic-induced proliferation, we observed that arsenic resulted: ROS generation; activated Erk signaling; stimulated AP-1 activity, including immediate early genes, c-Jun and c-Fos. N-Acetyl-l-cysteine, a ROS quencher, blocked the arsenic-induced effects. Our study underlines a previously undefined mechanism by which arsenic imparts its toxicity and results in uncontrolled cell proliferation.

Arsenic induced apoptosis in malignant melanoma cells is enhanced by menadione through ROS generation, p38 signaling and p53 activation

IntroductionResistance to apoptosis is a prominent feature of melanoma. Pharmacological concentration of arsenic in combination with a widely known oxidant, menadione was explored in this study to synergistically sensitize malignant melanoma cells to apoptosis. The molecular mechanism of apoptosis and the signaling-pathways involved were thoroughly investigated.Materials methods and resultsMenadione synergized NaAsO2 to significantly increase ROS generation and facilitate the major apoptotic signaling events: alteration of mitochondrial membrane potential, cytochrome c release and anti-apoptotic protein Bcl-2 down-regulation and subsequent activation of caspase-9 and caspase-3 followed by poly-ADP-ribose polymerase-1 cleavage. Antioxidant N-acetyl-l-cysteine antagonized these events. Investigation of the signaling-pathway revealed significant suppression of AP-1 activity but not NF-κB upon NaAsO2 and menadione application. An increase in p38 phosphorylation and p53 protein expression did also dictate the apoptotic response. Suppression of p38 activation with SB203580 and inhibition of p53 expression by siRNA attenuated apoptosis. Transfection of p53, in p53 null HCT cells augmented the apoptotic events. Moreover, the treatment also led to tumor size reduction in BALB/c mice developed by intra-dermal B16 mouse melanoma cell injection; however, it had no detectable pro-proliferative or pro-apoptotic effect on non-tumor keratinocytes, normal fibroblasts or PBMC.ConclusionThis study thus provides an insight into innovative mechanisms of melanoma sensitization, a proper cure against which is still elusive. Taken together, our data also provides the first evidence of arsenic activity accentuation by menadione through modulation of specific signaling-pathways.

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin.

Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

HnRNP E2 is downregulated in human oral cancer cells and the overexpression of hnRNP E2 induces apoptosis.

Human hnRNP genes have been reported to be involved in human malignancies and several hnRNPs are promising biomarkers of lung, head and neck, colon, breast, and pancreatic cancers. The present study investigated the clinicopathologic and biological significance of hnRNP E2 gene expression in oral cancer. Human hnRNP E2 was significantly downregulated in oral cancer tissues compared to normal one (P < 0.0001) as determined by quantitative real‐time reverse transcription PCR. The expression of hnRNP E2 is correlated with histology, being lower in moderate and poorly differentiated squamous cell carcinoma (SCC) compared to well‐differentiated SCC. Transient transfection of hnRNP E2 in cancerous cell lines resulted in reduced cell viability and increased apoptotic nuclei. Compared to control transfectants, cells with higher expression showed an increase in the number of apoptotic cells by annexin‐PI staining and an increase in caspase activity. The present study thus implicates downregulation of hnRNP E2 as a novel mechanism to enhance the resistance of cancer cells to apoptosis.

Nitric Oxide Produced Endogenously Is Responsible for Hypoxia- Induced HIF-1α Stabilization in Colon Carcinoma Cells

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, the cellular HIF-1α level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation, and proteasomal degradation. During hypoxia, degradation decreases, and its intracellular level is increased. Exogenously administered nitric oxide (NO)-donor drugs stabilize HIF-1α; thus, NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1α stabilization has not been defined. Here, we demonstrate that NO and reactive oxygen species (ROS) produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1α accumulation in hypoxia. The antioxidant N-acetyl-L-cysteine (NAC) and NO synthase inhibitor N(G)-monomethyl L-arginine (L-NMMA) effectively reduced HIF-1α stabilization and decreased HIF-1α hydroxylation. These effects suggested that endogenous NO and ROS impaired PHD activity, which was confirmed by reversal of L-NMMA- and NAC-mediated effects in the presence of dimethyloxaloylglycine, a PHD inhibitor. Thiol reduction with dithiothreitol decreased HIF-1α stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1α stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypoxia. Our results support a regulatory mechanism of HIF-1α during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.

Isolation and characterization of an arsenic-resistant bacterium from a bore-well in West Bengal, India.

An arsenic-resistant bacterium, strain KRPC10YT, was isolated from arsenic-infested bore-well of West Bengal, India. The bacterium was resistant to exceeding concentrations of arsenate (30 mM) and arsenite (20 mM). The bacterium was Gram-positive, rod-shaped, motile and yellowish to orange-pigmented. The major fatty acids were anteiso-C15:0, iso-C15∶0. The DNA G+C content was 49 mol %. Based on its phenotypic, chemotaxonomic and phylogenetic characteristics, it was identified as a member of the genusPlanococcus and is the first knownPlanococcus resistant to arsenic. KRPC10YTT was positive for indole, catalase, tolerated up to 12.0% NaCl and exhibited phenotypic differences with other type strains of genusPlanococcus. Strain KRPC10YT thus could be a novel species of the genusPlanococcus. The type strain is KRPC10YT (= MTCC7758T, =JCM 13947T).

Rare observation of ‘aggregation induced emission’ in cyclometalated platinum(II) complexes and their biological activities

Three strong solid state emissive cyclometalated platinum(II) complexes [Pt(C⁁N) (CH⁁N) (Cl)] (1) (C⁁N/CH⁁N = 2-phenylpyridine, C⁁N = bidentate and CH⁁N = monodentate), [Pt(C⁁N) (P⁁P)]Cl [P⁁P = bis(diphenylphosphino)ethane (2) and cis-1,2-bis(diphenylphosphino)ethene (3)] were reported. These were identified as ‘Aggregation Induced Emission (AIE)’ active complexes based on controlled experiments. Cytotoxicity and cell imaging have been studied for the complex 2.

‘Aggregation induced emission’ active iridium(III) complexes with applications in mitochondrial staining

Two new bis-cyclometalated iridium(III) complexes, [Ir(F2ppy)2(L)] and [Ir(ppy)2(L)], where F2ppy = 2-(2′,4′-difluoro)phenylpyridine, ppy = 2-phenylpyridine and L = 1,2-((pyridin-2-ylimino)methyl)phenol, have been designed and synthesized by a convenient route. We have univocally characterized their structure by 1H NMR, 19F NMR, HRMS and SXRD. Both complexes exhibit strong ‘Aggregation Induced Emission (AIE)’ activity, which has been investigated using spectroscopy measurements, ab initio quantum chemical calculations and by analysing their crystal packing. One of the complexes has been shown to have a potential application as a non-toxic bio-imaging probe for mitochondrial staining.