Sib Sankar Roy

Analysis of T-cell proliferation and cytokine secretion in the individuals exposed to arsenic

Over six million people in nine districts of West Bengal, India are exposed to very high levels of arsenic primarily through their drinking water. More than 300,000 people showed arsenic-induced skin lesions in these districts. This is regarded as the greatest arsenic calamity in the world. Chronic arsenicosis causes varied dermatological signs ranging from pigmentation changes, hyperkeratosis to non-melanocytic cancer of skin, and also malignancies in different internal organs. Higher incidences of opportunistic infections are found in the arsenic-exposed individuals, indicating that their immune systems may be impaired somehow. We have thus investigated the effect of arsenic on T-cell proliferation and cytokine secretion in 20 individuals with arsenic-induced skin lesions and compared the results with 18 arsenic-unexposed individuals. A marked dose-dependent suppression of Concanavalin A (Con A) induced T-cell proliferation was observed in the arsenic-exposed individuals compared with the unexposed (P < 0.001) individuals. This correlated with a significant decrease in the levels of secreted cytokines by the T cells (TNF-α, IFN-γ, IL2, IL10, IL5, and IL4) in the exposed individuals (P < 0.001). Thus it can be inferred that arsenic exposure can cause immunosuppression in humans.

Molecular mechanism of insulin resistance

Free fatty acids are known to play a key role in promoting loss of insulin sensitivity, thereby causing insulin resistance and type 2 diabetes. However, the underlying mechanism involved is still unclear. In searching for the cause of the mechanism, it has been found that palmitate inhibits insulin receptor (IR) gene expression, leading to a reduced amount of IR protein in insulin target cells. PDK1-independent phosphorylation of PKCε causes this reduction in insulin receptor gene expression. One of the pathways through which fatty acid can induce insulin resistance in insulin target cells is suggested by these studies. We provide an overview of this important area, emphasizing the current status.

ETS-1 Protein Regulates Vascular Endothelial Growth Factor-induced Matrix Metalloproteinase-9 and Matrix Metalloproteinase-13 Expression in Human Ovarian Carcinoma Cell Line SKOV-3

Background: The mechanism of vascular endothelial growth factor (VEGF)-regulated expression of MMPs followed by cancer cell scattering/invasion is poorly understood. Results: VEGF induces MMP-9, MMP-13, and ETS-1 through PI3K/AKT and p38 MAPK pathways in SKOV-3 cells. Conclusion: VEGF induces ETS-1, which activates specific MMPS, leading to the invasion/scattering in SKOV-3 cells. Significance: This study provides useful information that reveals the molecular mechanism of ovarian cancer metastasis. Matrix metalloproteinase-mediated degradation of extracellular matrix is a crucial event for invasion and metastasis of malignant cells. The expressions of matrix metalloproteinases (MMPs) are regulated by different cytokines and growth factors. VEGF, a potent angiogenic cytokine, induces invasion of ovarian cancer cells through activation of MMPs. Here, we demonstrate that invasion and scattering in SKOV-3 cells were induced by VEGF through the activation of p38 MAPK and PI3K/AKT pathways. VEGF induced the expression of MMP-2, MMP-9, and MMP-13 and hence regulated the metastasis of SKOV-3 ovarian cancer cells, and the activities of these MMPs were reduced after inhibition of PI3K/AKT and p38 MAPK pathways. Interestingly, VEGF induced expression of ETS-1 factor, an important trans-regulator of different MMP genes. ETS-1 bound to both MMP-9 and MMP-13 promoters. Furthermore, VEGF acted through its receptor to perform the said functions. In addition, VEGF-induced MMP-9 and MMP-13 expression and in vitro cell invasion were significantly reduced after knockdown of ETS-1 gene. Again, VEGF-induced MMP-9 and MMP-13 promoter activities were down-regulated in ETS-1 siRNA-transfected cells. VEGF enriched ETS-1 in the nuclear fraction in a dose-dependent manner. VEGF-induced expression of ETS-1 and its nuclear localization were blocked by specific inhibitors of the PI3K and p38 MAPK pathways. Therefore, based on these observations, it is hypothesized that the activation of PI3K/AKT and p38 MAPK by VEGF results in ETS-1 gene expression, which activates MMP-9 and MMP-13, leading to the invasion and scattering of SKOV-3 cells. The study provides a mechanistic insight into the prometastatic functions of VEGF-induced expression of relevant MMPs.

Involvement of novel PKC isoforms in FFA induced defects in insulin signaling

Involvement of novel PKCs (nPKCs) in the negative regulation of insulin-signaling pathway is a current interest of many workers investigating the cause for insulin resistance and type 2 diabetes. Free fatty acids (FFAs) are recently shown to be the major players in inducing insulin resistance in insulin target cells. They are also found to be involved in activating nPKCs associated with the impairment of insulin sensitivity. In this overview, we describe PKC delta, theta and epsilon linked to the FFA induced damage of insulin-signaling molecules.

Lipid induced overexpression of NF-κB in skeletal muscle cells is linked to insulin resistance

Lipid induced NF-kappaB activation is known to be associated with insulin resistance and type2 diabetes. Here we show that incubation of L6 skeletal muscle cells with palmitate significantly increased NF-kappaB p65 and NF-kappaB p50 expression along with their phosphorylation. NF-kappaB p65 siRNA inhibited palmitate induced overexpression of NF-kappaB p65 indicating palmitate effect on transcriptional activation. RT-PCR and real time PCR experiments also showed a significant increase in NF-kappaB p65 gene expression due to palmitate. Overexpression of NF-kappaB p65 by palmitate was linked to impairment of insulin activity. Palmitate effect on NF-kappaB gene and protein expression was found to be mediated by phospho-PKCepsilon as calphostin C (an inhibitor of PKC) and epsilonV1 (PKCepsilon translocation inhibitor) significantly reduced NF-kappaB expression. To understand the underlying mechanism, we purified NF-kappaB and pPKCepsilon from palmitate incubated skeletal muscle cells and their interaction in cell free system demonstrated the transfer of phosphate from PKCepsilon to NF-kappaB. This prompted us to transduct pPKCepsilon to the skeletal muscle cells. These cells showed increased amount of pNF-kappaB and NF-kappaB. Excess of NF-kappaB p65 pool thus created in the cells made them insulin resistant. Addition of NF-kappaB p65 siRNA and SN50 inhibited palmitate induced NF-kappaB p65 expression indicating NF-kappaB regulation of its gene expression. Increase of NF-kappaB did not affect the activation of IKK/IkappaB indicating NF-kappaB p65 expression to be a distinct effect of palmitate. Since NF-kappaB p65 is linked to several diseases, including type2 diabetes, this report may be important in understanding the pathogenicity of these diseases.

Inhibition of Insulin Receptor Gene Expression and Insulin Signaling by Fatty Acid: Interplay of PKC Isoforms Therein

Fatty acids are known to play a key role in promoting the loss of insulin sensitivity causing insulin resistance and type 2 diabetes. However, underlying mechanism involved here is still unclear. Incubation of rat skeletal muscle cells with palmitate followed by I125- insulin binding to the plasma membrane receptor preparation demonstrated a two-fold decrease in receptor occupation. In searching the cause for this reduction, we found that palmitate inhibition of insulin receptor (IR) gene expression effecting reduced amount of IR protein in skeletal muscle cells. This was followed by the inhibition of insulin-stimulated IRβ tyrosine phosphorylation that consequently resulted inhibition of insulin receptor substrate 1 (IRS 1) and IRS 1 associated phosphatidylinositol-3 kinase (PI3 Kinase), phosphoinositide dependent kinase-1 (PDK 1) phosphorylation. PDK 1 dependent phosphorylation of PKCζ and Akt/PKB were also inhibited by palmitate. Surprisingly, although PKCΕ phosphorylation is PDK1 dependent, palmitate effected its constitutive phosphorylation independent of PDK1. Time kinetics study showed translocation of palmitate induced phosphorylated PKCΕ from cell membrane to nuclear region and its possible association with the inhibition of IR gene transcription. Our study suggests one of the pathways through which fatty acid can induce insulin resistance in skeletal muscle cell.

Melatonin protects against lipid‐induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice

Lipid generates reactive oxygen species (ROS) in consequence to mitochondrial fission followed by inflammation in propagating hepatic fibrosis. The interaction of SIRT1/Mitofusin2 is critical for maintaining mitochondrial integrity and functioning, which is disrupted upon excess lipid infiltration during the progression of steatohepatitis. The complex interplay between hepatic stellate cells and steatotic hepatocytes is critically regulated by extracellular factors including increased circulating free fatty acids during fibrogenesis. Melatonin, a potent antioxidant, protects against lipid‐mediated mitochondrial ROS generation. Lipotoxicity induces disruption of SIRT1 and Mitofusin2 interaction leading to mitochondrial morphological disintegration in hepatocytes. Further, fragmented mitochondria leads to mitochondrial permeability transition pore opening, cell cycle arrest and apoptosis and melatonin protects against all these lipotoxicity‐mediated dysfunctions. These impaired mitochondrial dynamics also enhances the cellular glycolytic flux and reduces mitochondrial oxygen consumption rate that potentiates ROS production. High glycolytic flux generates metabolically unfavorable milieu in hepatocytes leading to inflammation, which is abrogated by melatonin. The melatonin‐mediated protection against mitochondrial dysfunction was also observed in high‐fat diet (HFD)‐fed mice through restoration of enzymatic activities associated with respiratory chain and TCA cycle. Subsequently, melatonin reduces hepatic fat deposition and inflammation in HFD‐fed mice. Thus, melatonin disrupts the interaction between steatotic hepatocyte and stellate cells, leading to the activation of the latter to abrogate collagen deposition. Altogether, the results of the current study document that the pharmacological intervention with low dose of melatonin could abrogate lipotoxicity‐mediated hepatic stellate cell activation and prevent the fibrosis progression.

Wnt/β-Catenin Pathway Is Regulated by PITX2 Homeodomain Protein and Thus Contributes to the Proliferation of Human Ovarian Adenocarcinoma Cell, SKOV-3

Background: Wnt pathway and homeodomain proteins are associated with cancer, but their interaction in ovarian cancer cells has not been studied. Results: PITX2 itself and through inducing Wnt ligands activates the canonical Wnt pathway and cell proliferation. Down-regulation of Frizzled receptors limits further Wnt activation. Conclusion: PITX2 enhances proliferation of SKOV-3 cells by inducing canonical Wnt signaling. Significance: This study will help understand the mechanism of proliferation in ovarian cancer cells. Pituitary homeobox-2 (PITX2) plays a substantial role in the development of pituitary, heart, and brain. Although the role of PITX2 isoforms in embryonic development has been extensively studied, its possible involvement in regulating the Wnt signaling pathway has not been reported. Because the Wnt pathway is strongly involved in ovarian development and cancer, we focused on the possible association between PITX2 and Wnt pathway in ovarian carcinoma cells. Remarkably, we found that PITX2 interacts and regulates WNT2/5A/9A/6/2B genes of the canonical, noncanonical, or other pathways in the human ovarian cancer cell SKOV-3. Chromatin immunoprecipitation and promoter-reporter assays further indicated the significant association of PITX2 with WNT2 and WNT5A promoters. Detailed study further reveals that the PITX2 isoform specifically activates the canonical Wnt signaling pathway either directly or through Wnt ligands. Thus, the activated Wnt pathway subsequently enhances cell proliferation. Moreover, we found the activation of Wnt pathway reduces the expression of different FZD receptors that limit further Wnt activation, demonstrating the existence of an auto-regulatory feedback loop. In contrast, PITX2 could not activate the noncanonical pathway as the Wnt5A-specific ROR2 receptor does not express in SKOV-3 cells. Collectively, our findings demonstrated that, despite being a target of the canonical Wnt signaling pathway, PITX2 itself induces the same, thus leading to the activation of the cell cycle regulating genes as well as the proliferation of SKOV-3 cells. Collectively, we highlighted that the PITX2 and Wnt pathway exerts a positive feedback regulation, whereas frizzled receptors generate a negative feedback in this pathway. Our findings will help to understand the molecular mechanism of proliferation in ovarian cancer cells.

FGF16 Promotes Invasive Behavior of SKOV-3 Ovarian Cancer Cells through Activation of Mitogen-activated Protein Kinase (MAPK) Signaling Pathway

Background: The mechanism of FGF16-mediated invasions in ovarian cancer cells was not reported. Results: Through the activation of FGFR-MAPK-pathway, FGF16 regulates SNAI1/CDH1/MMP2/-MMP9, promoting invasion of ovarian cancer cells. Wnt-signaling and PITX2 synergistically regulates FGF16 expression. Conclusion: The oncogenic potential of FGF16 in ovarian cancer was established. Significance: Growth factor/signaling pathways leading to increased proliferation/invasion of ovarian cancer were identified. Uncontrolled cell growth and tissue invasion define the characteristic features of cancer. Several growth factors regulate these processes by inducing specific signaling pathways. We show that FGF16, a novel factor, is expressed in human ovary, and its expression is markedly increased in ovarian tumors. This finding indicated possible involvement of FGF16 in ovarian cancer progression. We observed that FGF16 stimulates the proliferation of human ovarian adenocarcinoma cells, SKOV-3 and OAW-42. Furthermore, through the activation of FGF receptor-mediated intracellular MAPK pathway, FGF16 regulates the expression of MMP2, MMP9, SNAI1, and CDH1 and thus facilitates cellular invasion. Inhibition of FGFR as well as MAPK pathway reduces the proliferative and invasive behavior of ovarian cancer cells. Moreover, ovarian tumors with up-regulated PITX2 expression also showed activation of Wnt/β-catenin pathway that prompted us to investigate possible interaction among FGF16, PITX2, and Wnt pathway. We identified that PITX2 homeodomain transcription factor interacts with and regulates FGF16 expression. Furthermore, activation of the Wnt/β-catenin pathway induces FGF16 expression. Moreover, FGF16 promoter possesses the binding elements of PITX2 as well as T-cell factor (Wnt-responsive), in close proximity, where PITX2 and β-catenin binds to and synergistically activates the same. A detail study showed that both PITX2 and T-cell factor elements and the interaction with their binding partners are necessary for target gene expression. Taken together, our findings indicate that FGF16 in conjunction with Wnt pathway contributes to the cancer phenotype of ovarian cells and suggests that modulation of its expression in ovarian cells might be a promising therapeutic strategy for the treatment of invasive ovarian cancers.

Arsenic exposure through drinking water leads to senescence and alteration of telomere length in humans: A case‐control study in West Bengal, India

Arsenic (As) induces pre‐malignant and malignant dermatological lesions, non‐dermatological health effects and cancers in humans. Senescence involves telomere length changes and acquisition of senescence‐associated secretory phenotype (SASP), which promotes carcinogenesis. Though in vitro studies have shown that As induces senescence, population based studies are lacking. We investigated the arsenic‐induced senescence, telomere length alteration and its contribution towards development of As‐induced skin cancer. The study participants included 60 each of As‐exposed individuals with skin lesion (WSL), without skin lesions (WOSL) and 60 unexposed controls. Exposure assessment of drinking water and urine was done. SA β‐gal activity, ELISA, and quantification of senescence proteins, alternative lengthening of telomere (ALT) associated proteins and telomerase activity were performed. Relative telomere length (RTL) was determined by qPCR. A significantly higher number of senescent cells, over‐expression of p53 and p21 were observed in the As‐exposed individuals when compared to unexposed. SASP markers, MMP‐1/MMP‐3 were significantly higher in the WSL but not IL‐6/IL‐8. A significant increase of RTL was observed in the WSL group, which was telomerase‐independent but exhibited an over‐expression of ALT associated proteins TRF‐1 and TRF‐2 with higher increase in TRF‐2. An increased risk for developing As‐induced skin lesions was found for individuals having RTL greater than 0.827 (odds ratio, 13.75; 95% CI: 5.66–33.41; P < 0.0001). Arsenic induces senescence in vivo, but the SASP markers are not strictly over‐expressed in the As‐induced skin lesion group, whereas telomerase‐independent elongation of telomere length might be useful for predicting the risk of development of As‐induced skin lesions.