V. Vijaya Padma

Lutein protects HT-29 cells against Deoxynivalenol-induced oxidative stress and apoptosis: Prevention of NF-κB nuclear localization and down regulation of NF-κB and Cyclo-Oxygenase - 2 expression

Increasing evidence suggests that oxidative stress is closely linked to toxic responses in cells. The tricothecene mycotoxin, Deoxynivalenol (DON), primarily affects cells of the immune system and the GI tract. DONs cytotoxicity is closely linked to intracellular ROS, and it exerts its toxic effect by a mechanism known as ribotoxic stress response, which drives both cytokine expressions at low dosages and apoptosis at high dosages. Studies to alleviate DONs toxicity are sparsely reported in literature. In the present study, the cytoprotective effect of lutein, was tested in HT-29 cells against DON-induced oxidative stress and cytotoxicity. MTT assay revealed IC(20) values of DON at 250 ng/ml. Pre-treatment of cells with 10 microM lutein resulted in 95% cell viability. Lutein combated DON-induced oxidative stress and downregulated expression of inflammatory genes, NF-kappaB and COX-2. Lutein also prevented DON-induced migration of NF-kappaB into the nucleus, as measured by immunofluorescence. Morphological studies by Electron microscopy and Cell cycle analysis by flow cytometry indicated that lutein prevented DON-induced apoptosis. The results of the present study demonstrate for the first time that lutein exerts a cytoprotective role in DON-induced toxicity.

Protective effect of gallic acid against lindane induced toxicity in experimental rats

Lindane is an organochlorine pesticide that persists in the environment, bioaccumulate through food chain and has a risk of causing adverse effects to human health and the environment. It induces cell damage by producing free radicals and reactive oxygen species. The aim of the present study is to investigate the protective effect of gallic acid (a plant derived polyphenol) against lindane induced hepatic and renal toxicity in rats. Liver damage was assessed by hepatic serum marker enzymes like SGOT, SGPT and ALP and histopathological observation. Renal damage was observed by histopathological examination and serum markers like creatinine and urea. Treatment with lindane increased the levels of lipid peroxidation, serum marker enzyme activity with a concomitant decrease in GSH, CAT, SOD, GPx and GST. Histological alterations were also observed in kidney and liver tissue with lindane treatment. Co-treatment of gallic acid significantly prevented the lindane induced alterations in kidney and liver tissues with a decrease in LPO, serum marker enzyme activity and a significant increase in antioxidant levels. These results suggest that gallic acid has protective effect over lindane induced oxidative damage in rat liver and kidney.

Biological evaluation of new nickel(II) metallates: Synthesis, DNA/protein binding and mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hypergeneration and depletion of cellular antioxidant pool

A series of novel nickel(II) thiosemicarbazone complexes(1-4) have been prepared and characterized by various spectral, analytical techniques and X-ray crystallography. Further, their efficacy to interact with CT-DNA/BSA has been explored. From the binding studies, it is inferred that complex 4 found to be more active than other complexes. The complexes bound with CT-DNA by intercalation mode. Moreover, static quenching was observed for their interaction with BSA. The new complexes were tested for their in vitro cytotoxicity against human lung adenocarcinoma (A549) cell line. The results showed that the new complexes exhibited significant degree of cytotoxicity at given experimental condition. Further, the results of LDH and NO release supported the cytotoxic nature of the complexes. The observed cytotoxicity of the complexes may be routed through ROS-hypergeneration and lipid-peroxidation with subsequent depletion of cellular antioxidant pool (GSH, SOD, CAT, GPx and GST) resulted in the reduction of mitochondrial-membrane potential, caspase-3 activation and DNA fragmentation. Thus, the data from the present study disclose that the complexes could induce apoptosis in A549 cells through mitochondrial mediated fashion and inhibited the migration of lung cancer cells and by metastasis.

Synthesis, structure, DNA/protein binding and in vitro cytotoxicity of new ruthenium metallates

The reaction of [RuHCl(CO)(PPh3)3] with an equimolar amount of salicylaldehyde-4(N)-methylthiosemicarbazone [H2-(Sal-mtsc)] resulted in two entities, namely [Ru(H-Sal-mtsc)Cl(CO) (PPh3)2] (1) and [Ru(Sal-mtsc)(CO)(PPh3)2] (2) from a single tub. The new complexes were characterized by various spectro (IR, absorption and NMR), analytical and single crystal X-ray diffraction studies. From the crystallographic studies, it is confirmed that in complex 1, the ligand coordinated through the thiolate sulfur and the deprotonated hydrazinic nitrogen N(2), resulting in the formation of an unusual strained four membered chelate ring. The third potential donor, phenolic oxygen, remained uncoordinated. In complex 2, the ligand coordinated as an ONS chelate with the formation of more common five and six membered chelate rings. Complexes 1 and 2 have been tested for their DNA/protein binding properties by taking CT-DNA/lysozyme as models. From the protein binding studies, the alterations in the secondary structure of lysozyme by the ruthenium(II) complexes (1 and 2) were confirmed with synchronous and three-dimensional fluorescence spectroscopic studies. The in vitro cytotoxicity of the newly-synthesized complexes was carried out in two different human tumour cell lines, A549 and HepG2. The cytotoxicity studies showed that complex 2 exhibited higher activity than 1.

Synthesis, DNA/protein binding and in vitro cytotoxic studies of new palladium metallothiosemicarbazones

A series of four new thiosemicarbazone complexes of palladium have been synthesized, characterized and evaluated for their DNA/protein binding with CT-DNA and BSA, respectively. The new complexes bound to CT-DNA by intercalation mode and in protein binding studies, the complexes bound to BSA binding mechanism was found as static quenching. Among them the complex 4 had a strong binding affinity with BSA. In addition, in vitro cytotoxic studies were carried out on lung cancer (A549) and liver cancer (HepG2) cell lines and found that the complexes exhibited better activity than their parent thiosemicarbazone analogues. The complex 3 exhibited better activity than other complexes and this fact supported by the increased accumulation of the complexes in to the cancer cells which are evident from inter cellular uptake studies.

Synthesis and structural characterization of new ruthenium(II) complexes and investigation of their antiproliferative and metastatic effect against human lung cancer (A549) cells

Reaction of 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone [H2-(Nap-etsc)] with [RuHCl(CO)(PPh3)3] conferred two entities, namely [Ru(H-Nap-etsc)Cl(CO)(PPh3)2] (1) and [Ru(Nap-etsc)(CO)(PPh3)2]·Cl (2). The new complexes were characterized using various spectroanalytical and X-ray crystallographic techniques. In this reaction the dual coordination behaviour (thiolate/thione) of the ligand was observed. It acted as NS bidentate monobasic and ONS tridentate monobasic in entities 1 and 2 respectively. The binding ability of complexes (1 and 2) to calf thymus DNA (CT DNA)/BSA has been explored by absorption and emission titration methods. The cytotoxic nature of the complexes was evaluated on human lung cancer cells, A549. Complexes induced apoptotic cell death via ROS hypergeneration and mitochondrial membrane damage. In addition these newly synthesized ruthenium complexes inhibited A549 cell migration, as evidenced by wound healing assay. The activity of the complexes was found to be very high by comparing with cisplatin, a conventional standard. The time dependent release of the complexes from porous system was investigated by taking mesoporous silica as the host material. It was shown that the main portion of the embedded complexes was released after 20 h and reached a maximum after 96 h.

Tetramethylpyrazine Ameliorated Hypoxia- Induced Myocardial Cell Apoptosis via HIF-1α/JNK/p38 and IGFBP3/BNIP3 Inhibition to Upregulate PI3K/Akt Survival Signaling

Background: Hemorrhagic shock (HS) is the major cause of death from trauma. Hemorrhagic shock may lead to cellular hypoxia and organ damage. Our previous findings showed that HS induced a cardiac apoptosis pathway and synergistically caused myocardial cell damage in diabetic rats under trauma-induced HS. Tetramethylpyrazine (TMP) is a major biologically active ingredient purified from the rhizome of Ligusticum wallichii (called Chuang Xiong in Chinese). Chuan Xiong rescued cells from synergistic cardiomyoblast cell injury under high-glucose (HG) conditions plus hypoxia. TMP is one of the most important active ingredients that elevated the survival rate in ischemic brain injury and prevented inducible NO synthase expression to have anti-inflammatory effects against cell damage in different cell types. Method: Here, we further investigate whether TMP can protect against hypoxic (<1% oxygen) conditions in H9c2 cardiomyoblast cells for 24 hrs. Results: Our results showed that hypoxia mediated through HIF-1α/JNK/p38 activation significantly elevated the levels of the hypoxia-related proteins HIF-1α, BNIP3 and IGFBP3, further enhanced the pro-apoptotic protein Bak and upregulated downstream Caspase 9 and 3, resulting in cell death. All of these phenomena were fully recovered under TMP treatment. We observed that TMP exerted this effect by activating the IGF1 receptor survival pathway, dependent primarily on PI3K/Akt. When PI3K (class I) was blocked by specific siRNA, the hypoxia-induced activated caspase 3 and cell apoptosis could not be reversed by TMP treatment. Conclusion: Our results strongly suggest that TMP could be used to restore hypoxia-induced myocardial cell apoptosis and cardiac hypoxic damage.

Mitochondrial ROS-induced ERK1/2 activation and HSF2-mediated AT1R upregulation are required for doxorubicin-induced cardiotoxicity

Doxorubicin (DOX), one useful chemotherapeutic agent, is limited in clinical use because of its serious cardiotoxicity. Growing evidence suggests that angiotensin receptor blockers (ARBs) have cardioprotective effects in DOX‐induced cardiomyopathy. However, the detailed mechanisms underlying the action of ARBs on the prevention of DOX‐induced cardiomyocyte cell death have yet to be investigated. Our results showed that angiotensin II receptor type I (AT1R) plays a critical role in DOX‐induced cardiomyocyte apoptosis. We found that MAPK signaling pathways, especially ERK1/2, participated in modulating AT1R gene expression through DOX‐induced mitochondrial ROS release. These results showed that several potential heat shock binding elements (HSE), which can be recognized by heat shock factors (HSFs), located at the AT1R promoter region. HSF2 markedly translocated from the cytoplasm to the nucleus when cardiomyocytes were damaged by DOX. Furthermore, the DNA binding activity of HSF2 was enhanced by DOX via deSUMOylation. Overexpression of HSF2 enhanced DOX‐induced cardiomyocyte cell death as well. Taken together, we found that DOX induced mitochondrial ROS release to activate ERK‐mediated HSF2 nuclear translocation and AT1R upregulation causing DOX‐damaged heart failure in vitro and in vivo.

NFIL3 Suppresses Hypoxia-induced Apoptotic Cell Death by Targeting the Insulin-like Growth Factor 2 Receptor

The insulin‐like growth factor‐II/mannose 6‐phosphate receptor (IGF2R) over‐expression correlates with heart disease progression. The IGF2R is not only an IGF2 clearance receptor, but it also triggers signal transduction, resulting in cardiac hypertrophy, apoptosis and fibrosis. The present study investigated the nuclear factor IL‐3 (NFIL3), a transcription factor of the basic leucine zipper superfamily, and its potential pro‐survival effects in cardiomyocytes. NFIL3 might play a key role in heart development and act as a survival factor in the heart, but the regulatory mechanisms are still unclear. IGF2 and IGF2R protein expression were highly increased in rat hearts subjected to hemorrhagic shock. IGF2R protein expression was also up‐regulated in H9c2 cells exposed to hypoxia. Over‐expression of NFIL3 in H9c2 cardiomyoblast cells inhibited the induction of hypoxia‐induced apoptosis and down‐regulated IGF2R expression levels. Gel shift assay, double‐stranded DNA pull‐down assay and chromatin immune‐precipitation analyses indicated that NFIL3 binds directly to the IGF2R promoter region. Using a luciferase assay, we further observed NFIL3 repress IGF2R gene promoter activity. Our results demonstrate that NFIL3 is an important negative transcription factor, which through binding to the promoter of IGF2R, suppresses the apoptosis induced by IGF2R signaling in H9c2 cardiomyoblast cells under hypoxic conditions. J. Cell. Biochem. 116: 1113–1120, 2015.

p53-mediated miR-18 repression activates HSF2 for IGF-IIR-dependent myocyte hypertrophy in hypertension-induced heart failure

Hypertension-induced cardiac hypertrophy and attenuated cardiac function are the major characteristics of early stage heart failure. Cardiomyocyte death in pathological cardiac conditions is the primary cause of heart failure and mortality. Our previous studies found that heat shock factor 1 (HSF1) protected cardiomyocytes from death by suppressing the IGF-IIR signaling pathway, which is critical for hypertensive angiotensin II-induced cardiomyocyte apoptosis. However, the role of heat shock factor 2 (HSF2) in hypertension-induced cardiac hypertrophy is unknown. We identified HSF2 as a miR-18 target for cardiac hypertrophy. p53 activation in angiotensin II (ANG II)-stimulated NRVMs is responsible for miR-18 downregulation both in vitro and in vivo, which triggers HSF2 expression and the activation of IGF-IIR-induced cardiomyocyte hypertrophy. Finally, we provide genetic evidence that miR-18 is required for cardiomyocyte functions in the heart based on the gene transfer of cardiac-specific miR-18 via adenovirus-associated virus 2 (AAV2). Transgenic overexpression of miR-18 in cardiomyocytes is sufficient to protect against dilated cardiomyopathy during hypertension-induced heart failure. Our results demonstrated that the p53-miR-18-HSF2-IGF-IIR axis was a critical regulatory pathway of cardiomyocyte hypertrophy in vitro and in vivo, suggesting that miR-18 could be a therapeutic target for the control of cardiac functions and the alleviation of cardiomyopathy during hypertension-induced heart failure.