Fumiaki Shinohara

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.

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.

Nrf2 Activation Attenuates Both Orthodontic Tooth Movement and Relapse

During orthodontic tooth movement, osteoclasts resorb the alveolar bone at the compress side of periodontium. Reactive oxygen species (ROS) works as intracellular signaling molecules of RANKL during osteoclastogenesis, although ROS has cytotoxicity against cells such as lipid oxidation. To deal with oxidative stress, cells have a defense system that is scavenging ROS by augmented antioxidative stress enzymes via transcriptional regulation with nuclear factor E2-related factor 2 (Nrf2). Previously, we reported that augmented antioxidative stress enzymes by Nrf2-gene transfer inhibited bone destruction. In the present study, we examined the effects of Nrf2 activation on osteoclastogenesis and, thereby, orthodontic tooth movement and orthodontic relapse. Mouse macrophage cell line RAW264.7 cells were used as osteoclast progenitor cells and stimulated with recombinant RANKL (100 ng/mL) with or without Nrf2 activator sulforaphane (SFN) and epigallocatechin gallate (EGCG) or ROS scavenger catechin. Osteoclastogenesis, resorption activity, and osteoclast marker gene expression were examined. Intracellular ROS was analyzed by flow cytometry. Maxillary first molars of C57BL6 male mice were moved palatally with 0.012-inch NiTi wire (100-mN force); SFN or EGCG was injected into the palatal gingiva once a week; and phosphate buffered saline was injected on the contralateral side. Tooth movement was monitored using a stone model with precise impression, and the amount of the tooth movement was compared among groups. SFN and EGCG significantly, but catechin weakly, inhibited RANKL-mediated osteoclastogenesis in vitro. Western blot analysis revealed that SFN and EGCG augmented the nuclear translocation of Nrf2 and the expression of anti-oxidative stress enzymes such as HO-1, although catechin did not. SFN and EGCG significantly, but catechin weakly, attenuated the intracellular ROS. Finally, animal experiment revealed that both SFN and EGCG successfully inhibited the orthodontic tooth movement. Additionally, SFN inhibited the relapse. These results suggest that Nrf2 activation could be therapeutic target for the anchorage enforcement in orthodontic treatment and pharmacologic retention against relapse.

Effects of Superantigen and Lipopolysaccharide on Induction of CD80 through Apoptosis of Human Monocytes

ABSTRACT To investigate the mechanisms underlying superantigen (SAg) stimulation, we analyzed the effect of SAg on monocyte responses with or without lipopolysaccharide (LPS). Addition of gamma interferon (IFN-γ) to unstimulated cultures induced a marked increase in the number of CD80+ monocytes, which was inhibited by LPS through the action of interleukin-10. However, CD80+monocytes began to increase before IFN-γ production, observed after 9 h of stimulation with staphylococcal enterotoxin B (SEB). SEB selectively increased the number of apoptotic CD80−monocytes, whereas LPS-treated monocytes were resistant to the apoptotic action of SEB. This SEB-induced killing was abrogated by anti-CD95 monoclonal antibody (MAb) ZB4 and anti-CD95 ligand (CD95L) MAb NOK2, suggesting a CD95-based pathway of apoptosis. Furthermore, the numbers of SEB-induced CD80+ monocytes were partially decreased by anti-CD119 (IFN-γ receptor) MAb and by anti-CD95L (NOK2) MAb. The CD30 expression of CD27high T cells induced by SEB was increased by agonistic anti-CD95 (CH11) MAb. Together, our findings showed that SEB-induced monocyte apoptosis is closely associated with the enrichment of CD80+ monocytes generated before IFN-γ production, followed by up-regulation of CD80 by IFN-γ, and that LPS has negative effects in both cases. These results also suggested that induction of monocyte apoptosis is an important mechanism by which SAg exerts its anti-inflammatory effects.

RANKL induces Bach1 nuclear import and attenuates Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular reactive oxygen species signaling and osteoclastogenesis in mice

Reactive oxygen species (ROS) play a role in intracellular signaling during osteoclastogenesis. We previously reported that transcriptional factor nuclear factor E2‐related factor 2 (Nrf2) was exported from the nucleus to the cytoplasm by receptor activator of nuclear factor‐κB ligand (RANKL), and that Nrf2 negatively regulated osteoclastogenesis via antioxidant enzyme up‐regulation. Knockout mice of BTB and CNC homology 1 (Bach1)‐the competitor for Nrf2 in transcriptional regulation‐was known to attenuate RANKL‐mediated osteoclastogenesis, although the mechanism remains unclear. Therefore, we hypothesized that RANKL could be involved in the nuclear translocation of Bach1, which would attenuate Nrf2‐mediated antioxidant enzymes, thereby augmenting intracellular ROS signaling in osteoclasts. RANKL induced Bach1 nuclear import and Nrf2 nuclear export. Induction of Bach1 nuclear export increased Nrf2 nuclear import, augmented antioxidant enzyme expression, and, thus, diminished RANKL‐mediated osteoclastogenesis via attenuated intracellular ROS signaling. Finally, an in vivo mouse bone destruction model clearly demonstrated that induction of Bach1 nuclear export inhibited bone destruction. In this study, we report that RANKL favors osteoclastogenesis via attenuation of Nrf2‐mediated antioxidant enzyme expression by competing with Bach1 nuclear accumulation. Of importance, induction of Bach1 nuclear export activates Nrf2‐dependent antioxidant enzyme expression, thereby attenuating osteoclastogenesis. Bach1 nuclear export might be a therapeutic target for such bone destructive diseases as rheumatoid arthritis, osteoporosis, and periodontitis.—Kanzaki, H., Shinohara, F., Itohiya, K., Yamaguchi, Y., Katsumata, Y., Matsuzawa, M., Fukaya, S., Miyamoto, Y., Wada, S., Nakamura, Y. RANKL induces Bach1 nuclear import and attenuates Nrf2‐mediated antioxidant enzymes, thereby augmenting intracellular reactive oxygen species signaling and osteoclastogenesis in mice. FASEB J. 31, 781–792 (2017). http://www.fasebj.org

Molecular regulatory mechanisms of osteoclastogenesis through cytoprotective enzymes

It has been reported that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, take part in osteoclast differentiation as intra-cellular signaling molecules. The current assumed signaling cascade from RANK to ROS production is RANK, TRAF6, Rac1, and then Nox. The target molecules of ROS in RANKL signaling remain unclear; however, several reports support the theory that NF-κB signaling could be the crucial downstream signaling molecule of RANKL-mediated ROS signaling. Furthermore, ROS exert cytotoxic effects such as peroxidation of lipids and phospholipids and oxidative damage to proteins and DNA. Therefore, cells have several protective mechanisms against oxidative stressors that mainly induce cytoprotective enzymes and ROS scavenging. Three well-known mechanisms regulate cytoprotective enzymes including Nrf2-, FOXO-, and sirtuin-dependent mechanisms. Several reports have indicated a crosslink between FOXO- and sirtuin-dependent regulatory mechanisms. The agonists against the regulatory mechanisms are reported to induce these cytoprotective enzymes successfully. Some of them inhibit osteoclast differentiation and bone destruction via attenuation of intracellular ROS signaling. In this review article, we discuss the above topics and summarize the current information available on the relationship between cytoprotective enzymes and osteoclastogenesis.

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.

Effects of zebularine on the apoptosis of 5-fluorouracil via cAMP/PKA/CREB pathway in HSC-3 cells

During 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 cells. We investigated the effect of a DNA methyltransferase (DNMT) inhibitor, zebularine (Zeb), on the chemosensitivity of 5-FU and cisplatin (CDDP), and compared the molecular mechanism of action with those of a GSK3β inhibitor, LiCl, and an Hsp90 inhibitor, 17-AAG. A 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.

Epigenetic regulation of susceptibility to anti-cancer drugs in HSC-3 cells

In this study, we investigated the effects of DNA methyltransferases inhibitor zebularine (ZEB) and histone deacetylases inhibitor suberoylanilide hydroxamic acid (SAHA) on the apoptosis induced by cisplatin (CDDP) or 5-fluorouracil (5-FU) in human oral squamous cell carcinoma (HSC)-3 cells. HSC-3 cells were incubated with CDDP (5 µ/ml) or 5-FU (250 µ/ml) with or without ZEB (120 µM) and/or SAHA (1.5 µM). CDDP or 5-FU alone induced apoptosis in about 30% of cells. The combination of CDDP/SAHA or CDDP/ZEB led to a significant increase in apoptotic cells up to 80% after 48 h incubation, and the triple combination of CDDP/SAHA/ZEB showed a synergetic effect on apoptosis induction. Although the combination of 5-FU/SAHA showed a moderate increase in apoptosis after 72 h, the combination of 5-FU/ZEB inhibited apoptosis rather than that of 5-FU alone. These results indicate that epigenetic active agents (ZEB and SAHA) could sensitize HSC-3 cells to apoptosis induced by these anti-cancer drugs, which may be an important characteristic of solid cancer treatment.