Maiko Suzuki

Differential apoptotic response of human cancer cells to organoselenium compounds

PurposeSelenium (Se) compounds are well known to inhibit cell proliferation and induce cell death in human cancer cells. Respective chemical forms of Se are intracellularly metabolized via complicated pathways, which target distinct molecules and exhibit varying degrees of anti-carcinogenicity in different cancer types; however, the precise mechanisms by which Se activates apoptosis remain poorly understood.MethodsThe effects of Se compounds, Se-methylselenocysteine (MSC), selenomethionine (SeMet), and selenite on cell proliferation, apoptosis and its pathway in established human carcinoma cell lines (HSC-3, -4, A549, and MCF-7) were investigated. Cancer cells were treated with each Se compound during different periods. Cell apoptosis, caspase activity and ER stress markers were analyzed by flow cytometric or immunoblotting analysis, respectively.ResultsWe examined four cell lines for their sensitivity to MSC and SeMet in comparison with selenite. SeMet increased apoptotic cells in p53-positive A549 cells, whereas MSC increased apoptotic cells in p53-mutated HSC-3 cells. High activities of caspase-3, -8 and -9 were observed during apoptosis, and a pan-caspase inhibitor, z-VAD-fmk, rescued the cell viability of HSC-3 cells exposed to MSC. In addition, the occurrence of endoplasmic reticulum (ER) stress was suggested by the observation that levels of phosphorylated eIF2α and caspase-12 activity are increased in Se-treated cells. Selenite and MSC were accompanied with the concurrent reduction of phosphorylated Akt levels, and the inhibitory effects of these Se compounds on vascular endothelial growth factor expression were observed with identical patterns.ConclusionThe present findings demonstrate that Se-induced apoptosis in carcinoma cells is basically a caspase-dependent process involving complicated mechanisms. Activation of both the intrinsic apoptotic pathway and ER stress pathway plays a major and concurrent role, while p53 activation seems to have only a functional role in SeMet.

Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling.

Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through c-Jun N-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125 ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0 ppm fluoride). These results suggest that fluoride-induced ROS generation causes mitochondrial damage and DNA damage, which may lead to impairment of ameloblast function. To counteract this impairment, SIRT1/autophagy is induced via JNK signaling to protect cells/ameloblasts from fluoride-induced oxidative damage that may cause dental fluorosis.

Sirtuin1 and autophagy protect cells from fluoride-induced cell stress

Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum (ER) stress and oxidative stress. Previously, we reported that fluoride induces ER-stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augment SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50, 100 and 125ppm) in drinking water for 6weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis.

Zebularine suppresses the apoptotic potential of 5-fluorouracil via cAMP/PKA/CREB pathway against human oral squamous cell carcinoma cells

PurposeDuring tumorigenesis, tumor suppressor and tumor-related genes are commonly silenced by aberrant DNA methylation in their promoter regions, which is one of the important determinants of susceptibility to 5-fluorouracil (5-FU) in oral squamous cell carcinoma (OSCC) cells. Here, we examine the chemotherapeutic efficacy of epigenetic agents on 5-FU cytotoxicity.MethodWe investigated the effect of a DNA methyltransferase (DNMT) inhibitor, zebularine (Zeb), on the chemosensitivity of 5-FU and cisplatin (CDDP) by MTT and TUNEL methods, and compared the molecular mechanism of action with those of a GSK3β inhibitor, LiCl, and an Hsp90 inhibitor, 17-AAG.ResultsA significant apoptotic effect by a combination of Zeb or 17-AAG was found in CDDP treatment; however, considerable suppression of 5-FU-induced apoptosis was observed after incubation with Zeb, 17-AAG, or LiCl. Zeb’s suppressive effects were associated with activation of the cAMP/PKA/CREB pathway, differing from mechanisms of 17-AAG and LiCl. Suppression of 5-FU-induced apoptosis by Zeb was not associated with increased Bcl-2 and Bcl-xL expressions dependent on transcription factor CREB, and with the expression level of thymidylate synthase.ConclusionsIn the present study, we identified a more detailed mechanism of action by which Zeb suppresses 5-FU-induced apoptosis. These results indicate that combination therapies have to be carefully investigated due to potential harmful effects in the clinical application of DNMT inhibitors.

Interleukin-1β converting enzyme subfamily inhibitors prevent induction of CD86 molecules by butyrate through a CREB-dependent mechanism in HL60 cells

To investigate the underlying mechanism for induction of CD86 molecules, we analysed the ability of the histone deacetylase (HDAC) inhibitor, sodium butyrate (NaB), to induce CD86 at the transcriptional level in HL60 cells. Our studies showed that the expression of CD86 on the cell surface was increased by 24 hr of NaB treatment, and the enhancement of CD86 mRNA expression was observed by real‐time polymerase chain reaction. When we measured NF‐κB binding activity, significant activity was induced upon NaB stimulation, which was suppressed by the addition of pyrrolidine dithiocarbamate. Butyrate also induced phosphorylated cAMP response element‐binding protein (CREB), which bound to cAMP‐responsive elements. Dibutyryl (db) ‐cAMP induced active CREB and increased the levels of CD86 by 24 hr. These observations indicated that NF‐κB and/or CREB are crucial for butyrate‐dependent activation of CD86 gene expression. We examined the inhibitory effects of various caspase inhibitors on the expression of CD86 in cells treated with NaB, because NaB also induced apoptosis with slow kinetics. Intriguingly, our results demonstrated that inhibitors of the interleukin‐1β converting enzyme subfamily (caspase‐1, ‐4, ‐5 and ‐13) blocked the butyrate‐induced increase in level of CD86. These inhibitors interfered with CD86 gene transcription in the presence of activated NF‐κB, whereas phosphorylated CREB was down‐regulated in the reactions where these inhibitors were added to inhibit CD86 gene expression. These results suggested that butyrate not only acetylates histones on the CD86 promoter through the suppression of HDAC activity, but that butyrate also regulates CREB‐mediated transcription, possibly through the caspase activities triggered by NaB.

Fluoride Affects Enamel Protein Content via TGF-β1-mediated KLK4 Inhibition

Dental fluorosis is caused by chronic high-level fluoride (F–) exposure during enamel development, and fluorosed enamel has a higher than normal protein content. Matrix metalloproteinase 20 cleaves enamel matrix proteins during the secretory stage, and KLK4 further cleaves these proteins during the maturation stage so that the proteins can be reabsorbed from the hardening enamel. We show that transforming growth factor β1 (TGF-β1) can induce Klk4 expression, and we examine the effect of F– on TGF-β1 and KLK4 expression. We found that in vivo F– inhibits Klk4 but not Mmp20 transcript levels. LacZ-C57BL/6-Klk4+/LacZ mice have LacZ inserted in frame at the Klk4 translation initiation site so that the endogenous Klk4 promoter drives LacZ expression in the same temporal/spatial way as it does for Klk4. KLK4 protein levels in rat enamel and β-galactosidase staining in LacZ-C57BL/6-Klk4+/LacZ mouse enamel were both significantly reduced by F– treatment. Since TGF-β1 induces KLK4 expression, we tested and found that F– significantly reduced Tgf-β1 transcript levels in rat enamel organ. These data suggest that F–-mediated downregulation of TGF-β1 expression contributes to reduced KLK4 protein levels in fluorosed enamel and provides an explanation for why fluorosed enamel has a higher than normal protein content.

Uncoupling protein-2 is an antioxidant that is up-regulated in the enamel organ of fluoride-treated rats

Abstract Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F−) exposure generates reactive oxygen species (ROS) that can cause endoplasmic reticulum (ER)-stress. We therefore screened oxidative stress arrays to identify genes regulated by F− exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F−-treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F− (∼1.5 & 2.0 fold for the 50 or 100 ppm F− treatment groups, respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F− treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride-treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E-enriched diets within a given F− treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F− and activation of this gene may adversely affect ameloblast function.

Dual role of NF-κB in apoptosis of THP-1 cells during treatment with etoposide and lipopolysaccharide

One of the mechanisms repressing apoptosis in tumor cells can involve the expression of anti-apoptotic NF-kappaB target genes. In this study, we demonstrated that a potent NF-kappaB inhibitor, Nalpha-tosyl-L-lysinyl chloromethyl ketone (TLCK), inhibits apoptosis of THP-1 cells triggered by etoposide (VP16), and actinomycin D (ACT D) or cycloheximide inhibits apoptosis. However, persistent activation of NF-kappaB by lipopolysaccharide (LPS) led to the survival of leukemic cells against VP16-induced apoptosis. Thus, the molecular events (Bax/X-chromosome-linked IAP (XIAP)) occurring downstream of NF-kappaB activation during VP16 and/or LPS stimulation may become important to understand the multiple effects of NF-kappaB.

Murine matrix metalloproteinase-20 overexpression stimulates cell invasion into the enamel layer via enhanced Wnt signaling

Matrix metalloproteinase-20 (MMP20) is expressed by ameloblasts in developing teeth and MMP20 mutations cause enamel malformation. We established a stably transfected Tet-Off Mmp20-inducible ameloblast-lineage cell line and found that MMP20 expression promoted cell invasion. Previously, we engineered transgenic mice (Tg) that drive Mmp20 expression and showed that Mmp20+/+Tg mice had soft enamel. Here we asked if Mmp20 overexpression disrupts ameloblast function. Incisors from Mmp20+/+ mice expressing the Mmp20 Tg had a striking cell infiltrate which nearly replaced the entire enamel layer. A thin layer of enamel-like material remained over the dentin and at the outer tooth surface, but between these regions were invading fibroblasts and epithelial cells that surrounded ectopic bone-like calcifications. Mmp20+/+Tg mice had decreased enamel organ cadherin levels compared to the Mmp20 ablated and WT mice and, instead of predominantly locating adjacent to the ameloblast cell membrane, β-catenin was predominantly present within the nuclei of invading cells. Our data suggest that increased cadherin cleavage by transgenic MMP20 in the WT background releases excess β-catenin, which translocates to ameloblast nuclei to promote cell migration/invasion. Therefore, we conclude that MMP20 plays a role in normal ameloblast migration through tightly controlled Wnt signaling and that MMP20 overexpression disrupts this process.

Appropriate real-time PCR reference genes for fluoride treatment studies performed in vitro or in vivo

OBJECTIVE Quantitative real-time PCR (qPCR) is routinely performed for experiments designed to identify the molecular mechanisms involved in the pathogenesis of dental fluorosis. Expression of reference gene(s) is expected to remain unchanged in fluoride-treated cells or in rodents relative to the corresponding untreated controls. The aim of this study was to select optimal reference genes for fluoride experiments performed in vitro and in vivo. DESIGN Five candidate genes were evaluated: B2m, Eef1a1, Gapdh, Hprt and Tbp. For in vitro experiments, LS8 cells derived from mouse enamel organ were treated with 0, 1, 3 and/or 5mM sodium fluoride (NaF) for 6 or 18 h followed by RNA isolation. For in vivo experiments, six-week old rats were treated with 0 or 100 ppm fluoride as NaF for six weeks at which time RNA was isolated from enamel organs. RNA from cells and enamel organs were reverse-transcribed and stability of gene expression for the candidate reference genes was evaluated by qPCR in treated versus non-treated samples. RESULTS The most stably expressed genes in vitro according to geNorm were B2m and Tbp, and according to Normfinder were Hprt and Gapdh. The most stable genes in vivo were Eef1a1 and Gapdh. Expression of Ddit3, a gene previously shown to be induced by fluoride, was demonstrated to be accurately calculated only when using an optimal reference gene. CONCLUSIONS This study identifies suitable reference genes for relative quantification of gene expression by qPCR after fluoride treatment both in cultured cells and in the rodent enamel organ.