Upregulation of haeme oxygenase-1 by zinc in HCT-116 cells

Abstract

Haeme oxygenase-1 (HO-1) is often viewed as a cytoprotective gene. Toxic heavy metals induce HO-1, but it is unclear whether particular metal micronutrients also induce HO-1. Hence, the ability of exogenously-added copper, iron and zinc to influence HO-1 expression in HCT-116 cells was evaluated. Under the chosen experimental conditions, only zinc noticeably increased the expression of HO-1 mRNA and protein. Concurrently, zinc decreased non-protein thiol levels to a certain extent, but zinc did not increase the production of reactive oxygen species (ROS). Moreover, ascorbate and Trolox did not inhibit zinc-induced HO-1 upregulation. In contrast, deferoxamine blunted the induction of HO-1 mRNA, protein, and enzymatic activity caused by zinc. Additionally, N-acetylcysteine and Tiron inhibited zinc-induced HO-1 upregulation and also nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Collectively, these findings suggest that zinc at above normal levels upregulates HO-1 expression in HCT-116 cells in a ROS-independent manner.     

Zinc stabilizes adenomatous polyposis coli (APC) protein levels and induces cell cycle arrest in colon cancer cells

In the present study, we investigated the mechanisms by which zinc causes growth arrest in colon cancer cells. The results suggest that zinc treatment stabilizes the levels of the wild-type adenomatous polyposis coli (APC) protein at the post-translational level since the APC mRNA levels and the promoter activity of the APC gene were decreased in HCT-116 cells (which express the wild-type APC gene) after treatment with ZnCl2. Increased levels of wild-type but not truncated APC proteins were required for the ZnCl2-mediated G2/M phase arrest in different colon cancer cell lines. We further tested whether serum-stimulation, which induces cell cycle arrest in the S phase, can relieve ZnCl2-induced G2/M phase arrest of HCT-116 cells. Results showed that in the HCT-116 cells pretreated with ZnCl2, the serum-stimulation neither changed the distribution of G2/M phase arrested cells nor the increased levels of APC protein. The G2/M phase arrest correlated with retarded growth of HCT-116 cells. To further establish that wild-type APC protein plays a role in ZnCl2-induced G2/M arrest, we treated SW480 colon cancer cells that express truncated APC protein. We found that ZnCl2 treatment did not induce G2/M phase arrest in SW480 cells; however, the cell growth was retarded due to the loss of E-cadherin and alpha-tubulin levels. These results suggest that ZnCl2 inhibits the proliferation of colon cancer cells (which carry the wild-type APC gene) through stabilization of the APC protein and cell cycle arrest in the G2/M phase. On the other hand, ZnCl2 inhibits the proliferation of colon cancer cells (which carry the mutant APC gene) by disrupting cellular attachment and microtubule stability.     

Changes in Subcellular Localization of Visfatin in Human Colorectal HCT-116 Carcinoma cell Line After Cytochalasin-B Treatment

The aim of the study was to assess the expression and subcellular localization of visfatin in HCT-116 colorectal carcinoma cells after cytokinesis failure using Cytochalasin B (CytB) and the mechanism of apoptosis of cells after CytB. We observed translocation of visfatin’s antigen in cytB treated colorectal carcinoma HCT-116 cells from cytosol to nucleus. Statistical and morphometric analysis revealed significantly higher area-related numerical density visfatin-bound nano-golds in the nuclei of cytB-treated HCT-116 cells compared to cytosol. Reverse relation to visfatin subcellular localization was observed in un-treated HCT-116 cells. The total amount of visfatin protein and visfatin mRNA level in HCT-116 cells was also decreased after CytB treatment. Additionally, CytB significantly decreased cell survival, increased levels of G2/M fractions, induced bi-nuclei formation as well as increased reactive oxygen species (ROS) level in HCT-116 cells. CytB treatment showed cytotoxic effect that stem from oxidative stress and is connected with the changes in the cytoplasmic/nuclear amount of visfatin in HCT-116 cells.Key words: Visfatin, cytochalasin B, immunogold labeling, TEM, adipocytokines     

Cellular iron depletion weakens induction of heme oxygenase-1 by cadmium

Heme oxygenase-1 is an inducible cytoprotective gene, although its induction by environmental factors is not completely understood. This study aimed to ascertain if specific nutritive factors or related compounds influence heme oxygenase-1 expression. In HCT-116 cells, cadmium increased heme oxygenase-1 enzymatic activity. This effect of cadmium was weaker in cells made iron-deficient with the iron chelator, desferrioxamine, which was associated with repression of heme oxygenase-1 protein and mRNA expression. The repression by desferrioxamine of cadmium-induced heme oxygenase-1 upregulation was reversed upon iron replenishment of the cells. Additionally, it was found that thiol antioxidants inhibited the heme oxygenase-1 upregulation caused by cadmium and also by ethacrynic acid, which each decreased intracellular glutathione as did buthionine sulfoxamine. Interestingly, cadmium and ethacrynic acid increased nuclear translocation of Nrf2 and subsequent heme oxygenase-1 expression, but buthionine sulfoxamine did not. Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin, and a superoxide scavenger (Tiron) inhibited cadmium-induced upregulation of heme oxygenase-1. Diphenyleneiodonium was the most potent and inhibited NADPH-cytochrome P450 reductase as well, whereas apocynin and Tiron did not. It is concluded that adequate amounts of iron, which at the atomic level can serve as the pivotal element of heme in NADPH oxidase, must be present in cells to permit what appears to be thiol redox-sensitive, NADPH oxidase-dependent upregulation of heme oxygenase-1. Thus, these findings are significant because they suggest that cells without adequate iron would be unable to fully express the stress gene, heme oxygenase-1, when confronted with the toxic metal, cadmium.     

Peroxynitrite induces HO-1 expression via PI3K/Akt-dependent activation of NF-E2-related factor 2 in PC12 cells

Peroxynitrite is a strong oxidant produced by rapid interaction between superoxide anion and nitric oxide radicals and induces oxidative stress and cell death. Treatment of PC12 cells with 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite, induced the expression of heme oxygenase-1 (HO-1), an antioxidant cytoprotective enzyme. Inhibition of the HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression with siRNA exacerbated the SIN-1-induced apoptosis. After SIN-1 treatment, there was a time-related increase in nuclear localization and subsequent binding of NF-E2-related factor 2 (Nrf2) to the antioxidant-responsive element (ARE). Transfection of PC12 cells with dominant-negative Nrf2 abolished the SIN-1-induced increase in Nrf2-ARE binding and subsequent upregulation of HO-1 expression, leading to enhanced cell death. Upon exposure of PC12 cells to SIN-1, the phosphatidylinositol 3-kinase (PI3K) activity was increased in a time-dependent manner. Pretreatment of cells with LY294002, a pharmacologic inhibitor of PI3K or transfection with the kinase-dead mutant Akt abrogated the SIN-1-induced Nrf2 activation and HO-1 expression. Taken together, these results suggest that peroxynitrite activates Nrf2 via PI3K/Akt signaling and enhances Nrf2-ARE binding, which leads to upregulation of HO-1 expression. The SIN-1-induced HO-1 upregulation may confer the adaptive survival response against nitrosative stress.     

Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mcl-1, an antiapoptotic Bcl-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mcl-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the G(2)-M phase. Western blot analyses demonstrated that there was decreased Bcl-2 and Mcl-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for alpha-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the G(2)-M phase of the cell cycle. We next determined the mechanism of CUGBP2-mediated reduction in Mcl-1 expression. Mcl-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mcl-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mcl-1 and luciferase mRNA containing the Mcl-1 3'-UTR. However, luciferase protein expression from the luciferase-Mcl-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mcl-1 expression by inhibiting Mcl-1 mRNA translation, resulting in driving the cells to apoptosis during the G(2) phase of the cell cycle.     

The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice

The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1.     

Upregulation of human glycolipid transfer protein (GLTP) induces necroptosis in colon carcinoma cells

Human GLTP on chromosome 12 (locus 12q24.11) encodes a 24 kD amphitropic lipid transfer protein (GLTP) that mediates glycosphingolipid (GSL) intermembrane trafficking and regulates GSL homeostatic levels within cells. Herein, we provide evidence that GLTP overexpression inhibits the growth of human colon carcinoma cells (HT-29; HCT-116), but spares normal colonic cells (CCD-18Co). Mechanistic studies reveal that GLTP overexpression arrested the cell cycle at the G1/S checkpoint via upregulation of cyclin-dependent kinase inhibitor-1B (Kip1/p27) and cyclin-dependent kinase inhibitor 1A (Cip1/p21) at the protein and mRNA levels, and downregulation of cyclin-dependent kinase-2 (CDK2), cyclin-dependent kinase-4 (CDK4), cyclin E and cyclin D1 protein levels. Assessment of the biological fate of HCT-116 cells overexpressing GLTP indicated no increase in cell death suggesting induction of quiescence. However, HT-29 cells overexpressing GLTP underwent cell death by necroptosis as revealed by phosphorylation of human mixed lineage kinase domain-like protein (pMLKL) via receptor-interacting protein kinase-3 (RIPK-3), elevated cytosolic calcium, and plasma membrane permeabilization by pMLKL oligomerization. Overexpression of W96A-GLTP, an ablated GSL binding site mutant, failed to arrest the cell cycle or induce necroptosis. Sphingolipid assessment (ceramide, monohexosylceramide, sphingomyelin, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate) of HT-29 cells overexpressing GLTP revealed large decreases (>5-fold) in sphingosine-1-phosphate with minimal change in 16:0-ceramide, tipping the ‘sphingolipid rheostat’ (S1P/16:0-Cer ratio) towards cell death. Depletion of RIPK-3 or MLKL abrogated necroptosis induced by GLTP overexpression. Our findings establish GLTP upregulation as a previously unknown suppressor of human colon carcinoma HT-29 cells via interference with cell cycle progression and induction of necroptosis.     

Critical role of p53 upregulated modulator of apoptosis in benzyl isothiocyanate-induced apoptotic cell death

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.