Na-Young Song

Janus‐faced role of SIRT1 in tumorigenesis

Silent mating type information regulation 1 (Sirtuin 1; SIRT1) has been reported to regulate various physiological events, such as aging and metabolism, via deacetylation of histone and nonhistone proteins. Notably, cumulative evidence supports the notion that SIRT1 has a Janus‐faced role in tumorigenesis. SIRT1 contributes to anti‐inflammation, genomic stability, and cancer cell death, and hence it has tumor‐suppressor properties. On the other hand, SIRT1 can stimulate oncogenic signaling pathways and can create a tumor microenvironment favorable to growth and survival of cancer cells. Such dual functions of SIRT1 may be determined, at least in part, by its subcellular localization. Interestingly, SIRT1 displays differential localization in normal cells and cancer cells, which in turn may affect the substrate specificity for its deacetylase activity.

15-Deoxy-Δ12,14-prostaglandin J2 rescues PC12 cells from H2O2-induced apoptosis through Nrf2-mediated upregulation of heme oxygenase-1: Potential roles of Akt and ERK1/2

Oxidative stress induced by reactive oxygen intermediates has been implicated in a variety of human diseases including rheumatoid arthritis and neurodegenerative disorders. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a terminal dehydration product of prostaglandin D(2), is an endogenous ligand of peroxisome proliferator-activated receptor-gamma and exhibits a number of biological activities including the proapoptotic activity. Recent studies have revealed that this cyclopentenone prostaglandin, at non-toxic concentrations, can also exert antiapoptotic or cytoprotective effects. In this study, the underlying mechanisms involved in the protective effects of 15d-PGJ(2) on the H2O2-induced cytotoxicty were explored using cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with H2O2 underwent apoptosis, which was attenuated by pretreatment with non-toxic concentrations of 15d-PGJ(2). Treatment of the PC12 cells with 15d-PGJ(2) resulted in increased nuclear translocation, DNA-binding and transcriptional activity of NF-E2-related factor 2 (Nrf2), leading to upregulation of heme oxygenase-1 (HO-1) expression, which provided an adaptive survival response against the H2O2-derived oxidative cytotoxicity. Transfection of PC12 cells with dominant-negative Nrf2 gene abolished the 15d-PGJ(2)-derived induction of HO-1 expression. Moreover, the 15d-PGJ(2)-mediated increases in Nrf2-ARE binding and ARE luciferase activity were suppressed by the dominant-negative mutation as well as the pharmacological inhibition of Akt/protein kinase B or extracellular signal-regulated kinase 1/2 (ERK1/2). Taken together, these findings suggest that 15d-PGJ(2) augments cellular antioxidant defense capacity through activation of Akt and ERK signal pathways that leads to Nrf2 activation, and subsequently HO-1 induction, thereby protecting the PC12 cells from H2O2-induced oxidative cell death.

15-Deoxy-Δ12,14-prostaglandin J2 induces COX-2 expression through Akt-driven AP-1 activation in human breast cancer cells: a potential role of ROS

Recent studies suggest that inflammation is causally linked to carcinogenesis. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandins, is inappropriately expressed in various cancers and hence recognized as one of the hallmarks of chronic inflammation-associated malignancies. However, the mechanistic role of COX-2 as a link between inflammation and cancer remains undefined. Here, we report that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), one of the final products of COX-mediated arachidonic acid metabolism, upregulates the expression of COX-2 in the human breast cancer MCF-7 cell line. 15d-PGJ(2)-induced COX-2 expression was mediated by activation of Akt and subsequently activator protein-1 (AP-1). Furthermore, 15d-PGJ(2) formed reactive oxygen species, which led to increased phosphorylation of Akt, DNA binding of AP-1 and expression of COX-2. In contrast to 15d-PGJ(2), 9,10-dihydro-15d-PGJ(2) did not elicit any of effects induced by 15d-PGJ(2) in this study, suggesting that an electrophilic carbon center present in 15d-PGJ(2) is critical for COX-2 expression as well activation of upstream signal transduction induced by this cyclopentenone prostaglandin. Taken together, these observations suggest that 15d-PGJ(2) produced by COX-2 overexpression may function as a positive regulator of COX-2 in human breast cancer MCF-7 cells.

Multidrug resistance-associated protein 1 mediates 15-deoxy-Δ(12,14)-prostaglandin J2-induced expression of glutamate cysteine ligase expression via Nrf2 signaling in human breast cancer cells.

15-Deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a representative J-series cyclopentenone prostaglandin bearing an electrophilic α,β-unsaturated carbonyl group. In the present study, treatment of human breast cancer MCF-7 cells with 15d-PGJ(2) caused the up-regulation of the glutamate cysteine ligase catalytic (GCLC) subunit, the rate-limiting enzyme in glutathione (GSH) synthesis. 15d-PGJ(2) treatment caused nuclear translocation and transactivation of Nrf2, a redox-sensitive transcription factor responsible for induced expression of antioxidant and other cytoprotective genes. siRNA knockdown of Nrf2 abrogated 15d-PGJ(2)-induced GCLC expression. Following 15d-PGJ(2) treatment, the intracellular GSH level was initially diminished but eventually enhanced even above the basal level. The reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) abolished the 15d-PGJ2-induced Nrf2 activation and GCLC expression. Pharmacologic inhibition or siRNA knockdown of Akt, the target of phosphoinositide 3-kinase (PI3-K), attenuated 15d-PGJ(2)-induced Nrf2 activation and GCLC expression, and NAC treatment inhibited phosphorylation of Akt, and subsequently Nrf2 activation and GCLC upregulation. 9,10-Dihydro-15-PGJ2, a nonelectrophilic analogue of 15d-PGJ(2) that lacks the ability to form a conjugate with GSH, failed to induce activation of Akt and Nrf2 as well as ROS generation. These findings, taken all together, suggest that intracellular accumulation of ROS formed as a consequence of initial depletion of GSH can activate Akt, which in turn induces Nrf2 activation and subsequently the expression of GCLC, leading to the restoration of GSH. Interestingly, the extracellular GSH level was increased, concomitantly with the depletion of the intracellular GSH following 15d-PGJ(2) treatment. However, 15d-PGJ(2) was unable to influence both intra- and extra-cellular GSH levels when multidrug resistance-associated protein 1 (MRP1), the efflux pump for GSH conjugates, was blocked by its antagonist, MK571. Moreover, 15d-PGJ(2)-induced GCLC expression was attenuated by the MK571 and also by siRNA knockdown of MRP1, suggesting that MRP1 contributes to 15d-PGJ(2)-mediated up-regulation of GCLC by pumping out the 15d-PGJ(2)-GSH conjugate. It is speculated that 15d-PGJ(2), once effluxed through MRP, liberates from the GSH conjugate, and the free 15d-PGJ(2) re-enters the cell and forms the GSH conjugate again. In conclusion, MRP1 mediates Nrf2-dependent up-regulation of GCLC in 15d-PGJ(2)-treated MCF-7 cells, possibly via a putative recycling loop of 15d-PGJ(2)-GSH conjugation.

15‐Deoxy‐Δ12,14‐prostaglandin J2 Induces Upregulation of Multidrug Resistance‐Associated Protein 1 via Nrf2 Activation in Human Breast Cancer Cells

15‐Deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2), a representative J‐series cyclopentenone prostaglandin, exerts cytoprotective effects that are mainly mediated by Nrf2. Nrf2 is a major transcription factor involved in the transactivation of genes encoding many phase 2 detoxifying and antioxidant enzymes via interaction with the antioxidant response element (ARE). Recently it has been reported that expression of phase 3 efflux transporters, such as multidrug resistance‐associated proteins (MRPs), is also regulated by Nrf2. It is well known that cancer cells overexpressing MRPs are more resistant to anticancer drugs. In the present study we have found that 15d‐PGJ2 induces the expression of MRP1, one of the phase 3 efflux transporters, in human breast cancer cells (MCF‐7). In addition, treatment of MCF‐7 cells with 15d‐PGJ2 resulted in nuclear translocation and DNA binding of Nrf2. In contrast to 15d‐PGJ2, 9,10‐dihydro‐15d‐PGJ2, an analogue of 15d‐PGJ2 that lacks an electrophilic cyclopentenone ring moiety, failed to induce not only Nrf2 activation but also MRP1 upregulation in MCF‐7 cells. 15d‐PGJ2‐induced MRP1 overexpression was abrogated by Nrf2 gene knockdown, using RNA interference. These results, taken together, suggest that 15d‐PGJ2 induces MRP1 upregulation via Nrf2–ARE signaling.

15-Deoxy-Δ12,14-prostaglandin J2induces p53 expression through Nrf2-mediated upregulation of heme oxygenase-1 in human breast cancer cells

Abstract Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that has antioxidant and cytoprotective functions. However, HO-1 has oncogenic functions in cancerous or transformed cells. In the present work, we investigated the effects of HO-1 on the expression of p53 induced by 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in human breast cancer (MCF-7) cells. Treatment of MCF-7 cells with 15d-PGJ2 led to time-dependent increases in the expression of p53 as well as HO-1. Upregulation of p53 expression by 15d-PGJ2 was abrogated by si-RNA knock-down of HO-1. In MCF-7 cells transfected with HO-1 si-RNA, 15d-PGJ2 failed to induce expression of p53 as well as HO-1. In addition, HO-1 inducers enhanced the p53 expression. We speculated that iron, a by-product of HO-1-catalyzed reactions, could mediate 15d-PGJ2–induced p53 expression. Upregulation of p53 expression by 15d-PGJ2 was abrogated by the iron chelator desferrioxamine in MCF-7 cells. Iron released from heme by HO-1 activity is mostly in the Fe2+ form. When MCF-7 cells were treated with the Fe2+-specific chelator phenanthroline, 15d-PGJ2–induced p53 expression was attenuated. In addition, levels of the Fe-sequestering protein H-ferritin were elevated in 15d-PGJ2-treated MCF-7 cells. In conclusion, upregulation of p53 and p21 via HO-1 induction and subsequent release of iron with accumulation of H-ferritin may confer resistance to oxidative damage in cancer cells frequently challenged by redox-cycling anticancer drugs.

Docosahexaenoic acid inhibits insulin-induced activation of sterol regulatory-element binding protein 1 and cyclooxygenase-2 expression through upregulation of SIRT1 in human colon epithelial cells

Multiple lines of compelling evidence from clinical and population-based studies support that hyperinsulinemia often accompanying obesity-associated insulin insensitivity promotes colon carcinogenesis. Insulin can acetylate, thereby activating sterol regulator element-binding protein 1 (SREBP-1), a prime transcription factor responsible for expression of genes involved in lipogenesis. Moreover, SREBP-1 upregulates cyclooxygenase-2 (COX-2), a key player in inflammatory signaling. Docosahexaenoic acid (DHA), a representative omega-3 polyunsaturated fatty acid, has been known to negatively regulate SREBP-1, but the underlying molecular mechanism is not fully clarified yet. This prompted us to investigate whether DHA could inhibit insulin-induced activation of SREBP-1 and COX-2 expression in the context of its potential protective effect on obesity-induced inflammation and carcinogenesis. SIRT1, a NAD(+)-dependent histone/non-histone protein deacetylase, has been reported to inhibit intracellular signaling mediated by SREBP-1 through deacetylation of this transcription factor. We found that DHA induced SIRT1 expression in CCD841CoN human colon epithelial cells. DHA abrogated insulin-induced acetylation as well as expression of SREBP-1 and COX-2 upregulation. Insulin-induced stimulation of CCD841CoN cell migration was also inhibited by DHA. These effects mediated by DHA were attenuated by pharmacologic inhibition of SIRT1. Hyperinsulinemia or insulin resistance is considered to be associated with obesity-associated inflammation. Genetically obese (ob/ob) mice showed higher colonic expression levels of both SREBP-1 and COX-2 than did normal lean mice. Likewise, expression of SREBP-1 and COX-2 was elevated in human colon tumor specimens compared with surrounding normal tissues. In conclusion, DHA may protect against obesity-associated inflammation and colon carcinogenesis by suppressing insulin-induced activation of SREBP-1 and expression of COX-2 through up-regulation of SIRT1.

Abstract 1250: Curcumin inhibits migration and growth of human colon cancer cells through covalent modification of oncogenic SIRT1: Cysteine 67 as a potential binding site

Silent mating type information regulator 2 homolog 1 (SIRT1), an NAD+-dependent histone/protein deacetylase, has diverse physiological functions, including metabolic regulation and stress response. Despite extensive research, however, the role of SIRT1 in tumorigenesis remains controversial. Recent studies have demonstrated that SIRT1 is abnormally overexpressed in several human malignancies, and elevated levels of SIRT1 are correlated with the tumor invasion and metastasis. Curcumin (diferuloymethane), a major component of the spice turmeric (Curcuma longa L.), has been reported to possess anti-inflammatory and anti-carcinogenic properties. In the present study, we found that SIRT1 is predominantly overexpressed in the cytoplasm of several colorectal cancer cells as well as colon tumors. Curcumin abrogated migration and colony forming capability of human colon cancer (HCT-116) cells. This prompted us to investigate the effect of curcumin on the expression of SIRT1 and underlying molecular mechanisms in the context of its inhibition of the migration and growth of these cells. When HCT-116 cells were treated with curcumin, the protein expression of SIRT1 was significantly reduced, but the level of its mRNA transcript remained unchanged. The curcumin-induced suppression of SIRT1 protein expression was abrogated by the proteasomal inhibitor, MG-132. When HCT-116 cells were treated with curcumin, ubiquitination of SIRT1 was elevated. Notably, tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,β-unsaturated carbonyl moiety, failed to ubiquitinate and degrade SIRT1. Nano-LC-ESI-MS/MS analysis revealed the modification of the SIRT1 cysteine 67 residue. In line with this observation, the protein stability of a mutant SIRT1 in which cysteine 67 was replaced by alanine (SIRT1-C67A) was unaffected by curcumin treatment. Furthermore, migration and anchorage-independent growth of cells expressing SIRT1-C67A were barely inhibited by curcumin compared with those in cells harbouring wild-type SIRT1. Lysates of HCT-116 cells incubated with curcumin-conjugated Sepharose 4B beads exhibited covalent binding of curcumin to SIRT1. However, such direct interaction was markedly reduced in cells with SIRT1 cysteine 67 mutation. Taken together, these findings suggest that curcumin exerts inhibitory effects on progression of colon cancer through destabilization of oncogenic SIRT1. The electrophilic α,β-unsaturated carbonyl group present in curcumin can covalently modify SIRT1, preferentially at the cysteine 67 residue, facilitating its degradation via the ubiquitin-proteasome pathway in HCT-116 cells. Citation Format: Yeon-Hwa Lee, Na-Young Song, Do-Hee Kim, Hye-Kyung Na, Young-Joon Surh. Curcumin inhibits migration and growth of human colon cancer cells through covalent modification of oncogenic SIRT1: Cysteine 67 as a potential binding site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1250. doi:10.1158/1538-7445.AM2017-1250

Leptin induces SIRT1 expression through activation of NF-E2-related factor 2: Implications for obesity-associated colon carcinogenesis

&NA; Leptin, a representative adipokine secreted from the white adipose tissue, is considered as a potential linker between obesity and cancer. SIRT1 is an NAD+‐dependent histone/protein deacetylase speculated to function as an oncogene. In the present study, we found that leptin signaling‐defective ob/ob and db/db mice had lower colonic expression of SIRT1 compared with leptin signaling‐intact C57BL/6J mice, implying that leptin signaling is crucial for SIRT1 expression in vivo. Moreover, leptin induced up‐regulation of SIRT1 in human colon cancer (HCT‐116) cells. Leptin stimulated migration and invasion of cultured HCT‐116 cells and tumor growth in the xenograft assay, and these effects were abrogated by a SIRT1 inhibitor sirtinol, suggesting that SIRT1 plays a role in leptin‐induced colon carcinogenesis. Leptin‐induced SIRT1 expression was regulated by the redox‐sensitive transcription factor NF‐E2‐related factor 2 (Nrf2). Leptin stimulated nuclear accumulation of Nrf2 as well as its binding to the antioxidant response elements located in the SIRT1 promoter. Moreover, siRNA knockdown of Nrf2 abrogated the leptin‐induced SIRT1 expression. Notably, SIRT1 was significantly reduced in colon tissues of Nrf2‐null mice, lending further support to Nrf2‐dependent SIRT1 expression. Expression of leptin, Nrf2 and SIRT1 was coordinately increased in human colon tumor tissues. In conclusion, leptin might play a role in colon carcinogenesis by inducing Nrf2‐dependent SIRT1 overexpression. Graphical abstract Figure. No caption available.

Curcumin suppresses oncogenicity of human colon cancer cells by covalently modifying the cysteine 67 residue of SIRT1

SIRT1, an NAD+-dependent histone/protein deacetylase, has diverse physiological actions. Recent studies have demonstrated that SIRT1 is overexpressed in colorectal cancer, suggesting its oncogenic potential. However, the molecular mechanisms by which overexpressed SIRT1 induces the progression of colorectal cancer and its inhibition remain largely unknown. Curcumin (diferuloymethane), a major component of the spice turmeric derived from the plant Curcuma longa L., has been reported to exert chemopreventive and anti-carcinogenic effects on colon carcinogenesis. In the present study, we found that curcumin reduced the expression of SIRT1 protein without influencing its mRNA expression in human colon cancer cells, suggesting posttranslational regulation of SIRT1 by this phytochemical. Notably, ubiquitination and subsequent proteasomal degradation of SIRT1 were induced by curcumin treatment. Results of nano-LC-ESI-MS/MS revealed the direct binding of curcumin to cysteine 67 of SIRT1. In line with this result, the protein stability and clonogenicity of a mutant SIRT1 in which cysteine 67 was substituted by alanine were unaffected by curcumin. Taken together, these observations suggest that curcumin facilitates the proteasomal degradation of oncogenic SIRT1 through covalent modification of SIRT1 at the cysteine 67 residue.