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ABSTRACT: Hyperthermia (HT) has shown promising antitumor effects against various types of malignant tumors, and its pleiotropic effects support its combined use with radiotherapy and/or chemotherapy. However, HT is rendered less effective by the acquisition of thermoresistance in tumors, which arises through the elevation of heat shock proteins (HSPs) or other tumor responses. In mammals, the induction of HSPs is principally regulated at the transcriptional level by the activation of heat shock transcription factor 1 (HSF1). This transactivator has been shown to be abundantly expressed in a wide variety of tumors in humans. In addition, HSF1 participates in the initiation, proliferation and maintenance of tumors. Of note, HSF1 silencing has been shown to prevent the progression of tumors and to enhance their sensitivity to HT. Here, we review the physiological and pathological roles of HSF1 in cancer cells, and discuss its potential as a therapeutic target for HT therapy.
International Journal of Molecular Medicine 05/2013; · 1.98 Impact Factor
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ABSTRACT: Hyperthermia (HT) has been used as a possible treatment modality for various types of malignant tumors. Due to its pleiotropic effects, its combined use with radiotherapy and/or chemotherapy has proven to be beneficial. However, the molecular mechanisms underling the cellular responses to heat stress remain unclear. Therefore, the aim of this study was to identify common gene expression patterns responsive to mild HT (MHT) in human cancer cells. HeLa human cervical squamous cell carcinoma (SCC) and HSC-3 human oral SCC cells were exposed to MHT at 41˚C for 30 min, followed by culture at 37˚C for 0-24 h. MHT did not affect cell viability or the cell cycle. GeneChip microarray analysis clearly revealed that many probe sets were differentially expressed by a factor of ≥1.5 in both cell lines following exposure to MHT. Of the many differentially expressed probe sets, 114 genes were found to be commonly upregulated in both HeLa and HSC‑3 cells, and two significant gene networks were obtained from the commonly upregulated genes. Gene network A included several heat shock proteins, as well as BCL2-associated athanogene 3 (BAG3), and was found to be mainly associated with the biological functions of cellular function and maintenance. Gene network B included several anti-cell death genes, such as early growth response 1 (EGR1) and endothelin 1 (EDN1) and was found to be associated with the biological functions of cell death and survival. Real‑time quantitative polymerase chain reaction demonstrated that the gene expression patterns of the 12 genes selected were consistent with the microarray data in four cancer cell lines. These findings may provide further insight into the detailed molecular mechanisms of the MHT response in cancer cells.
International Journal of Molecular Medicine 04/2013; · 1.98 Impact Factor
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Kei-Ichiro Kitamura,
Koh Takahira,
Masato Inari,
Yusuke Satoh,
Kazuichi Hayakawa, Yoshiaki Tabuchi,
Kazuhiro Ohgai,
Takumi Nishiuchi,
Takashi Kondo,
Yuko Mikuni-Takagaki,
Wenxi Chen,
Atsuhiko Hattori,
Nobuo Suzuki
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ABSTRACT: Zebrafish scales consist of bone-forming osteoblasts, bone-resorbing osteoclasts, and calcified bone matrix. To elucidate the underlying molecular mechanism of the effects induced by dynamic and static acceleration, we investigated the scale osteoblast- and osteoclast-specific marker gene expression involving osteoblast-osteoclast communication molecules. Osteoblasts express RANKL, which binds to the osteoclast surface receptor, RANK, and stimulates bone resorption. OPG, on the other hand, is secreted by osteoblast as a decoy receptor for RANKL, prevents RANKL from binding to RANK and thus prevents bone resorption. Therefore, the RANK-RANKL-OPG pathway contributes to the regulation of osteoclastogenesis by osteoblasts. Semaphorin 4D, in contrast, is expressed on osteoclasts, and binding to its receptor Plexin-B1 on osteoblasts results in suppression of bone formation. In the present study, we found that both dynamic and static acceleration at 3.0 × g decreased RANKL/OPG ratio and increased osteoblast-specific functional mRNA such as alkaline phosphatase, while static acceleration increased and dynamic acceleration decreased osteoclast-specific mRNA such as cathepsin K. Static acceleration increased semaphorin 4D mRNA expression, while dynamic acceleration had no effect. The results of the present study indicated that osteoclasts have predominant control over bone metabolism via semaphorin 4D expression induced by static acceleration at 3.0 × g.
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 04/2013; · 2.20 Impact Factor
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ABSTRACT: Hyperthermia (HT) is a widely used physical treatment for various cancers, but its effect is often insufficient because of cytoprotective effects of heat shock proteins. BAG3, a co-chaperone of the heat shock protein 70, is a stress-inducible protein and demonstrates a cytoprotective property against various stresses, including heat stress. Here, we examined the effects of silencing the BAG3 on the sensitivity to HT in human oral squamous cell carcinoma (OSCC) HSC-3 cells. HT (44˚C, 90 min) was significantly increased in apoptotic cells concomitant with the activations of caspase-3 and c-Jun N-terminal kinase (JNK) pathway. Furthermore, the sensitivity to HT was remarkably enhanced in BAG3-downregulated HSC-3 cells. Interestingly, the effects of this combination treatment were significantly enhanced in the cells pretreated with a JNK inhibitor, SP600125. These findings indicated that the disruption of functions of both BAG3 and the JNK pathway may become an option in HT therapy in OSCC cells.
Cancer letters 02/2013; · 4.86 Impact Factor
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Takashi Tanida,
Ken Tasaka,
Eiichi Akahoshi,
Mitsuko Ishihara-Sugano,
Michiko Saito,
Shigehisa Kawata,
Megumi Danjo,
Junko Tokumoto,
Youhei Mantani,
Daichi Nagahara, Yoshiaki Tabuchi,
Toshifumi Yokoyama,
Hiroshi Kitagawa,
Mitsuhiro Kawata,
Nobuhiko Hoshi
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ABSTRACT: Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000 ng kg(-1) body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg(-1) TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD). Copyright © 2013 John Wiley & Sons, Ltd.
Journal of Applied Toxicology 01/2013; · 2.48 Impact Factor
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ABSTRACT: Purpose: Heat stress induces complex cellular responses, and its detailed molecular mechanisms still remain to be clarified. The objective of this study was to investigate the molecular mechanisms underlying cellular responses to mild hyperthermia (MHT) in normal human fibroblastic (NHF) cells. Materials and methods: Cells were treated with MHT (41°C, 30 min) and then cultured at 37°C. Gene expression was determined by the GeneChip® system and bioinformatics tools. Results: Treatment of the NHF cell lines, Hs68 and OUMS-36, with MHT did not affect the cell viability or cell cycle. In contrast, many probe sets were differentially expressed by >1.5-fold in both cell lines after MHT treatment. Of the 1,196 commonly and differentially expressed probe sets analysed by k-means clustering, three gene clusters, Up-I, Down-I and Down-II, were observed. Interestingly, two gene networks were obtained from the up-regulated genes in cluster Up-I. The gene network E contained DDIT3 and HSPA5 and was mainly associated with the biological process of endoplasmic reticulum stress, while the network S contained HBEGF and LIF and was associated with the biological process of cell survival. Eighteen genes were validated by quantitative real-time polymerase chain reaction, consistent with the microarray data, in four kinds of NHF cells. Conclusions: Common genes that were differentially expressed and/or acted within a gene network in response to MHT in NHF cells were identified. These findings provide the molecular basis for a further understanding of the mechanisms of the MHT response in NHF cells.
International Journal of Hyperthermia 01/2013; 29(1):38-50. · 1.92 Impact Factor
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ABSTRACT: The inhibition of DNA damage response pathway seems to be an attractive strategy for cancer therapy. It was previously reported that in rodent cells exposed to heat stress, cell growth was promoted by the activity of DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair. The absence of a functioning DNA-PK was associated with down regulation of heat shock protein 70 (HSP70). The objective of this study is thus to investigate the role of DNA-PK inhibition in heat-induced apoptosis in human cell lines. The inhibitors of phosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser2056, such as NU7026 and NU7441, were utilized. Furthermore, knock down of DNA-PKcs was carried out using small interfering RNA (siDNA-PKcs). For heat exposure, cells were placed in water bath at 44°C for 60 min. Apoptosis was evaluated after 24 h incubation flow cytometrically. Proteins were extracted after 24 h and analyzed for HSP70 and HSP40 expression by Western blotting. Total RNA was extracted 6 h after treatment and analyzed using a GeneChip® microarray system to identify and select the up-regulated genes (≥1.5 fold). The results showed an enhancement in heat-induced apoptosis in absence of functioning DNA-PKcs. Interestingly, the expression levels of HSP70 and HSP40 were elevated in the absence of DNA-PKcs under heat stress. The results of genetic network analysis showed that HSPs and JUN genes were up-regulated independently of DNA-PKcs in exposed parent and knock out cells. In the presence of functioning DNA-PKcs, there was an observed up-regulation of anti-apoptotic genes, such as NR1D1, whereas in the absence of DNA-PKcs the pro-apoptotic genes, such as EGR2, were preferentially up-regulated. From these findings, we concluded that in human cells, the inactivation of DNA-PKcs can promote heat-induced apoptosis independently of heat-shock proteins.
PLoS ONE 01/2013; 8(3):e58325. · 4.09 Impact Factor
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Koji Yachiguchi,
Noriko Matsumoto,
Yuki Haga,
Motoharu Suzuki,
Chisato Matsumura,
Masahiro Tsurukawa,
Toshihiro Okuno,
Takeshi Nakano,
Kimi Kawabe,
Kei-Ichiro Kitamura, [......],
Masato Endo,
Atsuhiko Chiba,
Toshio Sekiguchi,
Masaki Nakano, Yoshiaki Tabuchi,
Takashi Kondo,
Shigehito Wada,
Hiroyuki Mishima,
Atsuhiko Hattori,
Nobuo Suzuki
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ABSTRACT: To analyze the effect of polychlorinated biphenyl (PCB) 118 on fish bone metabolism, we examined osteoclastic and osteoblastic activities, as well as plasma calcium levels, in the scales of PCB (118)-injected goldfish. In addition, effect of PCB (118) on osteoclasts and osteoblasts was investigated in vitro. Immature goldfish, in which the endogenous effects of sex steroids are negligible, were used. PCB (118) was solubilized in dimethyl sulfoxide at a concentration of 10 ppm. At 1 and 2 days after PCB (118) injection (100 ng/g body weight), both osteoclastic and osteoblastic activities, and plasma calcium levels were measured. In an in vitro study, then, both osteoclastic and osteoblastic activities as well as each marker mRNA expression were examined. At 2 days, scale osteoclastic activity in PCB (118)-injected goldfish increased significantly, while osteoblastic activity did not change significantly. Corresponding to osteoclastic activity, plasma calcium levels increased significantly at 2 days after PCB (118) administration. Osteoclastic activation also occurred in the marker enzyme activities and mRNA expressions in vitro. Thus, we conclude that PCB (118) disrupts bone metabolism in goldfish both in vivo and in vitro experiments.
Environmental Science and Pollution Research 12/2012; · 2.65 Impact Factor
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Katsunori Omori,
Shigehito Wada,
Yusuke Maruyama,
Atsuhiko Hattori,
Kei-Ichiro Kitamura,
Yusuke Sato,
Masayuki Nara,
Hisayuki Funahashi,
Koji Yachiguchi,
Kazuichi Hayakawa, [......],
Sadakazu Ejiri,
Wenxi Chen, Yoshiaki Tabuchi,
Yukihiro Furusawa,
Takashi Kondo,
Yuichi Sasayama,
Takumi Nishiuchi,
Masaki Nakano,
Tatsuya Sakamoto,
Nobuo Suzuki
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ABSTRACT: Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E₂ (PGE₂) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE₂(10⁻⁷ and 10⁻⁶ M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE₂ (10⁻⁹ to 10⁻⁶ M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE₂ (10(-7) to 10⁻⁶ M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE₂ treated-scales. Thus, PGE₂ acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE₂-injencted goldfish increased significantly. We conclude that, in teleosts, PGE₂ activates both osteoblasts and osteoclasts and participates in calcium metabolism.
ZOOLOGICAL SCIENCE 08/2012; 29(8):499-504. · 0.95 Impact Factor
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Takashi Kondo,
Toru Yoshida,
Ryohei Ogawa,
Mariame A. Hassan,
Yukihiro Furusawa,
Qing-Li Zhao,
Akihiko Watanabe,
Akihiro Morii,
Loreto B. Feril,
Katsuro Tachibana,
Hiroshi Kitagawa, Yoshiaki Tabuchi,
Ichiro Takasaki,
Mohammad H. Shehata,
Nobuki Kudo,
Kazuhiro Tsukada
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ABSTRACT: PurposeIn this study, the effects of low-intensity pulsed ultrasound (LIU) as an adjuvant to doxorubicin (DOX) treatment was further
investigated in comparison to hyperthermia as another widely used adjuvant. The effects were compared with respect to cell
killing and apoptosis induction in U937 cells. Human primary liver cancer (PLC) cells were also used to evaluate the effects
of the combinations. The use of an echo contrast agent was investigated for further enhancement of cytotoxicity. Finally,
the acoustic mechanisms involved were investigated.
MethodsThe effects of different treatment regimens on cell viability were determined using the Trypan blue dye-exclusion test. Apoptosis
induction was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. The mechanistic
study involved electron paramagnetic spin trapping for detecting free radical formation as an indicator of the occurrence
of inertial cavitation and spectrophotometry for sucrose hydrolysis as an indicator for noncavitational effects.
ResultsThe combination treatments exerted synergistic effects on cytotoxicity depending on the acoustic conditions used. The use
of LIU as an adjuvant to DOX treatment was shown to be superior to the use of hyperthermia as an adjuvant. Moreover, the combination
seems to be promising for other cancer types provided that the acoustic conditions are properly selected with respect to drug
concentration. The key ultrasound mechanism responsible for the synergism observed was shown to be the production of free
radicals by inertial cavitation. Non-cavitational forces were also shown to contribute to the effect.
ConclusionThis study is motivating to engage in in vivo research with various cancer types as a step toward clinical applicability and
is emphasizing on the importance of developing therapeutic protocols for setting LIU parameters with respect to other therapeutic
conditions.
Journal of Medical Ultrasonics 04/2012; 36(2):61-68. · 0.33 Impact Factor
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ABSTRACT: Low-intensity ultrasound (US) has been shown to induce death of cancer cells; however, the underlying mechanism remains unclarified. Here, we provide novel evidence that the inhibition of checkpoint kinase 1 (Chk1) by a selective inhibitor or small interfering RNA (siRNA) enhances US-induced apoptosis in Jurkat cells. Jurkat cells showed insignificant lysis immediately after US at any applied intensity, whereas approximately 70% of the cells were γH2AX-positive 30min after US at 0.4W/cm(2). Regarding DNA damage response (DDR), Chk1, known as a target of ataxia telangiectasia mutated (ATM) and rad3-related (ATR), was phosphorylated in cells after US exposure. An ATM inhibitor showed nearly no effect on Chk1 phosphorylation, whereas chemicals showing the ATR inhibitory effect markedly abrogated the phosphorylation, indicating that Chk1 phosphorylation is preferentially more dependent on ATR than on ATM in cells exposed to US. The pharmacological inhibition of Chk1 promoted caspase-3 cleavage and increased the percentage of cells in SubG1 after US exposure. siRNA targeting Chk1 abrogated approximately 55% of Chk1 expression and also promoted apoptosis, suggesting that Chk1 plays anti-apoptotic roles in response to US. These findings revealed, for the first time, that US activates Chk1 dependently on ATR and the activated Chk1 is involved in apoptosis of cells exposed to US. Moreover, we propose that Chk1 may be a promising target in US-aided therapy.
Ultrasonics Sonochemistry 04/2012; 19(6):1246-51. · 3.57 Impact Factor
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ABSTRACT: Transforming growth factor-β-activated kinase 1 (TAK1) appears to play a role in inhibiting apoptotic death in response to multiple stresses. To assess the role of TAK1 in X-ray induced apoptosis and cell death, we irradiated parental and siRNA-TAK1-knockdown HeLa cells. Changes in gene expression levels with and without TAK1-knockdown were also examined after irradiation to elucidate the molecular mechanisms involved. After X-ray irradiation, cell death estimated by the colony formation assay increased in the TAK1-knockdown cells. Apoptosis induction, determined by caspase-3 cleavage, suggested that the increased radiosensitivity of the TAK1-knockdown cells could be partially explained by the induction of apoptosis. However, cell cycle analysis revealed that the percentage of irradiated cells in the G(2)/M-phase decreased, and those in the S- and SubG(1)-phases increased due to TAK1 depletion, suggesting that the loss of cell cycle checkpoint regulation may also be involved in the observed increased radiosensitivity. Interestingly, significant differences in the induction of NF-κB, p38 MAPK and ERK phosphorylation, the major downstream molecules of TAK1, were not observed in TAK1 knockdown cells compared to their parental control cells after irradiation. Instead, global gene expression analysis revealed differentially expressed genes after irradiation that bioinformatics analysis suggested are associated with cell cycle regulatory networks. In particular, CDKN1A (coding p21(WAF1)), which plays a central role in the identified network, was up-regulated in control cells but not in TAK1 knockdown cells after X-ray irradiation. Si-RNA knockdown of p21 decreased the percentage of cells in the G(2)/M phase and increased the percentage of cells in the S- and SubG(1)-phases after X-ray irradiation in a similar manner as TAK-1 knockdown. Taken together, these findings suggest that the role of TAK1 in cell death, cell cycle regulation and apoptosis after X irradiation is independent of NF-κB, p38 MAPK, and ERK phosphorylation, and dependent, in part, on p21 induction.
Radiation Research 04/2012; 177(6):766-74. · 2.68 Impact Factor
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ABSTRACT: Ultrasound (US) has been shown to induce cell death in cancer cells; however, the underlying mechanism remains elusive. Here, we report a set of novel findings on the molecular mechanism. We found that Akt (also known as protein kinase B), a substrate of DNA-dependent protein kinase (DNA-PK), was phosphorylated in U937 cells nullified with p53 or Molt-4 cells artificially abrogated with p53 after US exposure. On the contrary, Akt phosphorylation was transiently down-regulated then recovered in Molt-4 cells harboring wild-type p53 in US-exposed cells, possibly due to a mutual regulation between p53 and Akt. Inhibition of ataxia-telangiectasia mutated (ATM) or DNA-PK revealed that DNA-PK, rather than ATM, was preferentially involved in Akt phosphorylation and cell survival after US-exposure in all cell lines. These results indicate that DNA-PK plays a protective role against US-induced cell death regardless of p53 phenotype. In conclusion, our findings provide the first delineation of the role of DNA-PK in US-induced cell death and suggest that targeting DNA-PK might be a promising strategy to augment cancer eradication by US.
Cancer letters 02/2012; 322(1):107-12. · 4.86 Impact Factor
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Katsunori Omori,
Shigehito Wada,
Yusuke Maruyama,
Atsuhiko Hattori,
Kei-Ichiro Kitamura,
Yusuke Sato,
Masayuki Nara,
Hisayuki Funahashi,
Koji Yachiguchi,
Kazuichi Hayakawa, [......],
Sadakazu Ejiri,
Wenxi Chen, Yoshiaki Tabuchi,
Yukihiro Furusawa,
Takashi Kondo,
Yuichi Sasayama,
Takumi Nishiuchi,
Maki Nakano,
Tatsuya Sakamoto,
Nobuo Suzuki
ZOOLOGICAL SCIENCE 01/2012; · 0.95 Impact Factor
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ABSTRACT: This study investigated the deleterious effects of the synthetic non-steroidal estrogen diethylstilbestrol (DES) on testicular Leydig cells and compared these effects with those of the natural estrogen 17β-estradiol (E2). For that purpose, we performed microarray analysis of a mouse Leydig cell line (TTE1) treated with these estrogens, followed by Gene Ontology (GO) analysis and parametric analysis of gene set enrichment (PAGE). Most notably, GO analysis revealed a significant decrease in the biological processes of the GO categories "DNA repair" and "apoptotic program" in DES-exposed cells. PAGE showed that "cell death," which is a superior GO category including apoptosis in the GO tree structure, significantly decreased in DES-exposed cells but significantly increased in E2-exposed cells. Interestingly, only 2 genes (Tia1 and Gas1) with altered expression patterns in the "cell death" category were common between DES- and E2-treated cells. The downregulation of apoptotic cell death pathways and DNA repair capability of DES-exposed cells implies that DES promotes carcinogenic processes more strongly than E2 does. These findings suggest that molecular events that occur following DES and E2 treatments differ substantially in Leydig cells, and that the effects of synthetic estrogen and natural estrogen differ more substantially than previously suspected.
The Journal of Toxicological Sciences 01/2012; 37(2):287-95. · 1.52 Impact Factor
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Ayaka Hosoki,
Shin-Ichiro Yonekura,
Qing-Li Zhao,
Zheng-Li Wei,
Ichiro Takasaki, Yoshiaki Tabuchi,
Li-Li Wang,
Shiga Hasuike,
Takaharu Nomura,
Akira Tachibana,
Kazunari Hashiguchi,
Shuji Yonei,
Takashi Kondo,
Qiu-Mei Zhang-Akiyama
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ABSTRACT: Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in HeLa S3/SOD2 and T-REx HeLa/SOD2 cells compared with those in the control cells. MitoSox(TM) Red assays showed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Furthermore, flow cytometry with a fluorescent probe (2',7'-dichlorofluorescein) revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. DNA microarray analysis revealed that, of 47,000 probe sets analyzed, 117 and 166 probes showed more than 2-fold changes after 5.5 Gy of γ-irradiation in control and HeLa S3/SOD2 cells, respectively. Pathway analysis revealed different expression profiles in irradiated control cells and irradiated SOD2-overexpressing cells. These results indicate that SOD2 protects HeLa cells against cellular effects of γ-rays through suppressing oxidative stress in irradiated cells caused by ROS generated in the mitochondria and through regulating the expression of genes which play a critical role in protection against ionizing radiation.
Journal of Radiation Research 01/2012; 53(1):58-71. · 1.68 Impact Factor
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ABSTRACT: Alkannin is an active constituent from the root extract of Alkanna tinctoria of the Boraginaceae family and it may have utility as a heat shock protein 70 (HSP70) inducer in living organisms. Here, the effects of alkannin-induced HSP70 on ultraviolet (UV) B (40 mJ/cm(2))-induced apoptosis were investigated in human keratinocyte HaCaT cells. Pretreatment of cells with alkannin (1 µM) caused significant inhibition of UVB-induced apoptosis and caspase-3 cleavage. On the other hand, the addition of KNK437 (HSP70 inhibitor) reversed the action of alkannin increasing UVB-induced apoptosis in a dose-dependent manner. In addition, differences in gene expression associated with the suppression of UVB-induced apoptosis in the presence of alkannin were investigated using Gene Chip assay. Our results indicate that alkannin suppresses UVB-induced apoptosis through the induction of HSP70 in human keratinocytes, and therefore, we suggest the usefulness of using alkannin as an antiaging agent.
PLoS ONE 01/2012; 7(10):e47903. · 4.09 Impact Factor
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ABSTRACT: Ultrasonic technologies pervade the medical field: as a long established imaging modality in clinical diagnostics; and, with the emergence of targeted high intensity focused ultrasound, as a means of thermally ablating tumours. In parallel, the potential of [non-thermal] intermediate intensity ultrasound as a minimally invasive therapy is also being rigorously assessed. Here, induction of apoptosis in cancer cells has been observed, although definitive identification of the underlying mechanism has thus far remained elusive. A likely candidate process has been suggested to involve sonochemical activity, where reactive oxygen species (ROS) mediate the generation of DNA single-strand breaks. Here however, we provide compelling new evidence that strongly supports a purely mechanical mechanism. Moreover, by a combination of specific assays (neutral comet tail and staining for γH2AX foci formation) we demonstrate for the first time that US exposure at even moderate intensities exhibits genotoxic potential, through its facility to generate DNA damage across multiple cancer lines. Notably, colocalization assays highlight that ionizing radiation and ultrasound have distinctly different signatures to their respective γH2AX foci formation patterns, likely reflecting the different stress distributions that initiated damage formation. Furthermore, parallel immuno-blotting suggests that DNA-PKcs have a preferential role in the repair of ultrasound-induced damage.
PLoS ONE 01/2012; 7(1):e29012. · 4.09 Impact Factor
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ABSTRACT: Environmental stress induces damage that activates an adaptive response in any organism. The cellular stress response is based on the induction of cytoprotective proteins, the so-called stress or heat shock proteins (HSPs). HSPs are known to function as molecular chaperones which are involved in the therapeutic approach of many diseases. Therefore in the current study we searched nontoxic chaperone inducers in chemical compounds isolated from medicinal plants. Screening of 80 compounds for their Hsp70-inducing activity in human lymphoma U937 cells was performed by western blotting. Five compounds showed significant Hsp70 up-regulation among them shikonin was most potent. Shikonin was able to induce Hsp70 at 0.1 µM after 3 h without activation of heat shock transcription factor 1 (HSF-1). It also induces significant reactive oxygen species generation. The expression level of genes responsive to shikonin was studied using global-scale microarrays and computational gene expression analysis tools. Significant increase in the nuclear factor erythroid 2-related factor 2 (Nrf2, NFEL2L2) -mediated oxidative stress response was observed that leads to the activation of HSP. The results of gene chip analysis were further confirmed by real-time qPCR assay. In short, the detailed mechanisms of Hsp70 induction by shikonin is not fully understood, Nrf2 and its target genes might be involved in the Hsp70 up-regulation in U937 cells.
International Journal of Hyperthermia 01/2012; 28(1):1-8. · 1.92 Impact Factor
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ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is increasingly recognized as a condition in which excess fat accumulates in hepatocytes. Nonalcoholic steatohepatitis (NASH), a severe form of NAFLD in which inflammation and fibrosis in the liver are noted, may eventually progress to end-stage liver disease. Galectin-3, a β-galactoside-binding animal lectin, is a multifunctional protein. This protein is involved in inflammatory responses and carcinogenesis. We investigated whether galectin-3 is involved in the development of NASH by comparing galectin-3 knockout (gal3(-/-)) mice and wild-type (gal3(+/+)) mice with choline-deficient L-amino-acid-defined (CDAA) diet-induced NAFLD/NASH. Hepatic injury was significantly more severe in the gal3(-/-) male mice, as compared to the gal3(+/+) mice. Data generated by microarray analysis of gene expression suggested that galectin-3 deficiency causes alterations in the expression of various genes associated with carcinogenesis and lipid metabolism. Through canonical pathway analysis, involvement of PDGF and IL-6 signaling pathways was suggested in galectin-3 deficiency. Significant increase of CD14, Fos, and Jun, those that were related to lipopolysaccharide-mediated signaling, was candidate to promote hepatocellular damages in galectin-3 deficiency. In conclusion, galectin-3 deficiency in CDAA diet promotes NAFLD features. It may be caused by alterations in the expression profiles of various hepatic genes including lipopolysaccharide-mediated inflammation.
TheScientificWorldJOURNAL 01/2012; 2012:959824. · 1.66 Impact Factor
