Protective effects of baicalin against phenylarsine oxide-induced cytotoxicity in human skin keratinocytes.

Phenylarsine oxide (PAO) is a known environmental pollutant and skin keratinocytes are most seriously affected. Baicalin (BCN) was reported to have antioxidant and anti-inflammatory effects, but its protective effect against PAO toxicity is unknown. This study aimed at exploring whether baicalin can reverse the toxicity of human epidermal keratinocytes that are subjected to PAO exposure and underlying mechanisms. In silico analysis from a publicly accessible HaCaT cell transcriptome dataset exposed to chronic Arsenic showed significant differential expression of several genes, including the genes related to DNA replication. Later, we performed in vitro experiments, in which HaCaT cells were exposed to PAO (500 nM) in the existence of BCN (10-50 µM). Treatment of PAO alone induces the JNK, p38 and caspase-3 activation, which were engaged in the apoptosis induction, while the activity of AKT was significantly inhibited, which was engaged in the suppression of apoptosis. PAO suppressed SIRT3 expression and induced intracellular reactive oxygen species (ROS), causing a marked reduce in cell viability and apoptosis. However, BCN treatment restored the PAO-induced suppression of SIRT3 and AKT expression, reduced intracellular ROS generation, and markedly suppressed both caspase-3 activation and apoptosis induction. However, the protective effect of BCN was significantly attenuated after pretreatment with nicotinamide, an inhibitor of SIRT3. These findings indicate that BCN protects against cell death induced by PAO via inhibiting excessive intracellular ROS generation via restoring SIRT3 activity and reactivating downstream AKT pathway. In this study, we firstly shown that BCN is an efficient drug to prevent PAO-induced skin cytotoxicity, and these findings need to be confirmed by in vivo and clinical investigations.

The molecular mechanism of a novel derivative of BTO-956 induced apoptosis in human myelomonocytic lymphoma cells.

Acute myeloid leukemia (AML) is a malignant cancer of the hematopoietic system. Although the effectiveness of arsenic compounds has been recognized and applied clinically, some patients are still found resistant to this chemotherapy. In this study, we investigated that a synthetic thyroid hormone analog (TA), 2-iodo-4-nitro-1-(o-tolyloxy) benzene, had a strong apoptosis effect on U937 cells. U937 cells were treated with TA, and examinted the generation of reactive oxygen species (ROS), dysfunction of mitochondria, expression of pro-apoptosis and anti-apoptosis, and cleavage of caspase-3 and Poly (ADP-ribose) polymerase (PARP). Further, it is also evaluated that insight molecular mechanism and signaling pathways involved in the study. It is found that TA significantly induced apoptosis in U937 cells through production of ROS, dysfunction of mitochondria, and activation of caspase cascade. It was also observed that MAPK signaling pathway including ERK, JNK, and P38 signals are involved in the induction of apoptosis. Moreover, marked activation of autophagy and ER stress markers such as LC3, P62, Beclin1 and GRP78, CHOP were observed, respectively. Pretreatment with ER stress inhibitor tauroursodeoxycholic acid (TUDCA) and autophagy inhibitor 3-Methyladenine (3-MA) have successfully attenuated and aggravated TA-induced apoptosis, respectively. We further confirmed the active involvement of ER stress and autophagy signals. In conclusion, TA induced apoptosis through ER stress and activation of autophagy, and the latter is not conducive to TA-induced cell death. Our results may provide a new insight into the strategic development of novel therapy for the treatment of AML.

Combination of RERG and ZNF671 methylation rates in circulating cell-free DNA: A novel biomarker for screening of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia, hence, identifying easily detectable biomarkers for NPC screening is essential for better diagnosis and prognosis. Using genome-wide and targeted analyses based on next-generation sequencing approaches, we previously showed that gene promoters are hypermethylated in NPC tissues. To confirm whether DNA methylation rates of genes could be used as biomarkers for NPC screening, 79 histologically diagnosed NPC patients and 29 noncancer patients were recruited. A convenient quantitative analysis of DNA methylation using real-time PCR (qAMP) was carried out, involving pretreatment of tissue DNA, and circulating cell-free DNA (ccfDNA) from nonhemolytic plasma, with methylation-sensitive and/or methylation-dependent restriction enzymes. The qAMP analyses revealed that methylation rates of RERG, ZNF671, ITGA4, and SHISA3 were significantly higher in NPC primary tumor tissues compared to noncancerous tissues, with sufficient diagnostic accuracy of the area under receiver operating characteristic curves (AUC). Interestingly, higher methylation rates of RERG in ccfDNA were statistically significant and yielded a very good AUC; however, those of ZNF671, ITGA4, and SHISA3 were not significant. Furthermore, the combination of methylation rates of RERG and ZNF671 in ccfDNA showed higher diagnostic accuracy than either of them individually. In conclusion, the methylation rates of specific genes in ccfDNA can serve as novel biomarkers for early detection and screening of NPC.

Social and family factors as determinants of exercise habits in Japanese elementary school children: a cross-sectional study from the Super Shokuiku School Project.

BACKGROUND: Many studies have already reported on the relationship between exercise habits and health among schoolchildren. However, few have examined social and/or family factors as determinants of exercise habits. METHODS: This study's participants included 1721 schoolchildren aged between 6 and 13 who were involved in the Super Shokuiku School Project in January 2016. A survey was conducted to assess gender, grade level, physical activity, lifestyle, overall health, enrichment of school life, social background, and parental lifestyles. Both dislike and lack of physical activity were used to measure poor exercise habits; correlates were analyzed using logistic regression. RESULTS: "Lack of close friends" had the strongest links with both dislike (adjusted odds ratio [OR] 5.30; 95% confidence interval [CI], 2.78-10.1) and lack of (adjusted OR 5.40; 95% CI, 2.81-10.4) physical activity. Further, children who engaged in long periods of screen time and lacked parental communication also tended to dislike and lack physical activity. Children with mothers who were unemployed (housewives) and had unhealthy lifestyles, as well as those with poor health, were also more likely to lack physical activity. CONCLUSION: Social and family factors (e.g., having close friends) may be determinants of exercise habits among schoolchildren, independent of their own lifestyle factors. Although a longitudinal study is needed to determine causality, substantial attention may thus be required to these factors when promoting physical activity in children.

Anti-Cancer Mechanisms of Taurine in Human Nasopharyngeal Carcinoma Cells.

Taurine displays anti-tumor activity in some kinds of human cancers. However, the underlying mechanisms are poorly understood. Epstein-Barr virus-related nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer in Southeast Asia with the highest incidence in South China. We examined an apoptosis-inducing effect of taurine against NPC cells (HK1 and HK1-EBV) to clarify the mechanisms of anti-tumor effects of taurine by immunocytochemical methods. We observed that taurine induced cleavage of caspase-9/3 in a concentration-dependent manner, suggesting the involvement of mitochondrial apoptotic signals. Both PTEN and p53 activation were detected in a dose-dependent manner after taurine treatment in NPC cells. In conclusion, taurine may play an anti-tumor role by activating tumor suppressor PTEN and p53.

Taurine exhibits an apoptosis-inducing effect on human nasopharyngeal carcinoma cells through PTEN/Akt pathways in vitro.

Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck malignancy with a high incidence in southern China. Previous studies have confirmed that taurine shows an anti-cancer effect on a variety of human tumors by inhibiting cell proliferation and inducing apoptosis. However, the underlying molecular mechanism of its anti-cancer effect on NPC is not well understood. To clarify these anti-cancer mechanisms, we performed cell viability and colony formation assays. Apoptotic cells were quantified by flow cytometry. The expression levels of apoptosis-related proteins were evaluated by Western blot. The results showed that taurine markedly inhibited cell proliferation in NPC cells, but only slightly in an immortalized normal nasopharyngeal cell line. Taurine suppressed colony formation and induced apoptosis of NPC cell lines in a dose-dependent manner. Furthermore, taurine increased the active form of caspase-9/3 in a dose-dependent manner. Taurine down-regulated the anti-apoptotic protein Bcl-xL and up-regulated the pro-apoptotic protein Bax and GRP78, a major endoplasmic reticulum (ER) chaperone. These results suggest the involvement of mitochondrial and ER stress signaling in apoptosis. In addition, taurine increased the levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10) and p53, and reduced phosphorylated Akt (protein kinase B). In conclusion, taurine may inhibit cell proliferation and induce apoptosis in NPC through PTEN activation with concomitant Akt inactivation.

Overexpression of CD44 Variant 9: A Novel Cancer Stem Cell Marker in Human Cholangiocarcinoma in Relation to Inflammation.

Various CD44 isoforms are expressed in several cancer stem cells during tumor progression and metastasis. In particular, CD44 variant 9 (CD44v9) is highly expressed in chronic inflammation-induced cancer. We investigated the expression of CD44v9 and assessed whether CD44v9 is a selective biomarker of human cholangiocarcinoma (CCA). The expression profile of CD44v9 was evaluated in human liver fluke Opisthorchis viverrini-related CCA (OV-CCA) tissues, human CCA (independent of OV infection, non-OV-CCA) tissues, and normal liver tissues. CD44v9 overexpression was detected by immunohistochemistry (IHC) in CCA tissues. There was a higher level of CD44v9 expression and IHC score in OV-CCA tissues than in non-OV-CCA tissues, and there was no CD44v9 staining in the bile duct cells of normal liver tissues. In addition, we observed significantly higher expression of inflammation-related markers, such as S100P and COX-2, in OV-CCA tissues compared to that in non-OV and normal liver tissues. Thus, these findings suggest that CD44v9 may be a novel candidate CCA stem cell marker and may be related to inflammation-associated cancer development.

Quantitation of DNA methylation in Epstein-Barr virus-associated nasopharyngeal carcinoma by bisulfite amplicon sequencing.

BACKGROUND: Epigenetic changes, including DNA methylation, disrupt normal cell function, thus contributing to multiple steps of carcinogenesis. Nasopharyngeal carcinoma (NPC) is endemic in southern China and is highly associated with Epstein-Barr virus (EBV) infection. Significant changes of the host cell methylome are observed in EBV-associated NPC with cancer development. Epigenetic marks for NPC diagnosis are urgently needed. In order to explore DNA methylation marks, we investigated DNA methylation of candidate genes in EBV-associated nasopharyngeal carcinoma. METHODS: We first employed methyl-capture sequencing and cDNA microarrays to compare the genome-wide methylation profiles of seven NPC tissues and five non-cancer nasopharyngeal epithelium (NNE) tissues. We found 150 hypermethylated CpG islands spanning promoter regions and down-regulated genes. Furthermore, we quantified the methylation rates of seven candidate genes using bisulfite amplicon sequencing for nine NPC and nine NNE tissues. RESULTS: All seven candidate genes showed significantly higher methylation rates in NPC than in NNE tissues, and the ratios (NPC/NNE) were in descending order as follows: ITGA4 > RERG > ZNF671 > SHISA3 > ZNF549 > CR2 > RRAD. In particular, methylation levels of ITGA4, RERG, and ZNF671 could distinguish NPC patients from NNE subjects. CONCLUSIONS: We identified the DNA methylation rates of previously unidentified NPC candidate genes. The combination of genome-wide and targeted methylation profiling by next-generation sequencers should provide useful information regarding cancer-specific aberrant methylation.

Crosstalk between DNA Damage and Inflammation in the Multiple Steps of Carcinogenesis.

Inflammation can be induced by chronic infection, inflammatory diseases and physicochemical factors. Chronic inflammation is estimated to contribute to approximately 25% of human cancers. Under inflammatory conditions, inflammatory and epithelial cells release reactive oxygen (ROS) and nitrogen species (RNS), which are capable of causing DNA damage, including the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-nitroguanine. We reported that 8-nitroguanine was clearly formed at the sites of cancer induced by infectious agents including Helicobacter pylori, inflammatory diseases including Barrett's esophagus, and physicochemical factors including asbestos. DNA damage can lead to mutations and genomic instability if not properly repaired. Moreover, DNA damage response can also induce high mobility group box 1-generating inflammatory microenvironment, which is characterized by hypoxia. Hypoxia induces hypoxia-inducible factor and inducible nitric oxide synthase (iNOS), which increases the levels of intracellular RNS and ROS, resulting DNA damage in progression with poor prognosis. Furthermore, tumor-producing inflammation can induce nuclear factor-κB, resulting in iNOS-dependent DNA damage. Therefore, crosstalk between DNA damage and inflammation may play important roles in cancer development. A proposed mechanism for the crosstalk may explain why aspirin decreases the long-term risk of cancer mortality.

Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation.

BACKGROUND: Carbon nanotube (CNT) is used for various industrial purposes, but exhibits carcinogenic effects in experimental animals. Chronic inflammation in the respiratory system may participate in CNT-induced carcinogenesis. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflammation. We have previously reported that multi-walled CNT (MWCNT) induced 8-nitroG formation in lung epithelial cells and this process involved endocytosis. To clarify the mechanism of CNT-induced carcinogenesis, we examined the role of Toll-like receptor (TLR) 9, which resides in endosomes and lysosomes, in 8-nitroG formation in human lung epithelial cell lines. METHODS: We performed immunocytochemistry to examine 8-nitroG formation in A549 and HBEpC cells treated with MWCNT with a length of 1-2 µm (CNT-S) or 5-15 µm (CNT-L) and a diameter of 20-40 nm. We examined inhibitory effects of endocytosis inhibitors, small interfering RNA (siRNA) for TLR9, and antibodies against high-mobility group box-1 (HMGB1) and receptor for advanced glycation end-products (RAGE) on 8-nitroG formation. The release of HMGB1 and double-stranded DNA (dsDNA) into the culture supernatant from MWCNT-treated cells was examined by ELISA and fluorometric analysis, respectively. The association of these molecules was examined by double immunofluorescent staining and co-immunoprecipitation. RESULTS: CNT-L significantly increased 8-nitroG formation at 0.05 µg/ml in A549 cells and its intensity reached a maximum at 1 µg/ml. CNT-L tended to induce stronger cytotoxicity and 8-nitroG formation than CNT-S. Endocytosis inhibitors, TLR9 siRNA and antibodies against HMGB1 and RAGE largely reduced MWCNT-induced 8-nitroG formation. MWCNT increased the release of HMGB1 and dsDNA from A549 cells into culture supernatant. The culture supernatant of MWCNT-exposed cells induced 8-nitroG formation in fresh A549 cells. Double immunofluorescent staining and co-immunoprecipitation showed that TLR9 was associated with HMGB1 and RAGE in lysosomes of MWCNT-treated cells. CONCLUSIONS: MWCNT induces injury or necrosis of lung epithelial cells, which release HMGB1 and DNA into the extracellular space. The HMGB1-DNA complex binds to RAGE on neighboring cells and then CpG DNA is recognized by TLR9 in lysosomes, leading to generation of nitric oxide and 8-nitroG formation. This is the first study demonstrating that TLR9 and related molecules participate in MWCNT-induced genotoxicity and may contribute to carcinogenesis.

DNA Damage in CD133-Positive Cells in Barrett's Esophagus and Esophageal Adenocarcinoma.

Barrett's esophagus (BE) caused by gastroesophageal reflux is a major risk factor of Barrett's esophageal adenocarcinoma (BEA), an inflammation-related cancer. Chronic inflammation and following tissue damage may activate progenitor cells under reactive oxygen/nitrogen species-rich environment. We previously reported the formation of oxidative/nitrative stress-mediated mutagenic DNA lesions, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-nitroguanine, in columnar epithelial cells of BE tissues and cancer cells of BEA tissues. We investigated the mechanisms of BEA development in relation to oxidative/nitrative DNA damage and stem cell hypothesis. We examined 8-nitroguanine and 8-oxodG formation and the expression of stem cell marker (CD133) in biopsy specimens of patients with BE and BEA by immunohistochemical analysis in comparison with those of normal subjects. CD133 was detected at apical surface of columnar epithelial cells of BE and BEA tissues, and the cytoplasm and cell membrane of cancer cells in BEA tissues. DNA lesions and CD133 were colocalized in columnar epithelial cells and cancer cells. Their relative staining intensities in these tissues were significantly higher than those in normal subjects. Our results suggest that BE columnar epithelial cells with CD133 expression in apical surface undergo inflammation-mediated DNA damage, and mutated cells acquire the property of cancer stem cells with cytoplasmic CD133 expression.

Inflammation-Related DNA Damage and Cancer Stem Cell Markers in Nasopharyngeal Carcinoma.

Nitrative and oxidative DNA damage plays an important role in inflammation-related carcinogenesis. To investigate the involvement of stem cells in Epstein-Barr virus infection-related nasopharyngeal carcinoma (NPC), we used double immunofluorescence staining to examine several cancer stem/progenitor cell markers (CD44v6, CD24, and ALDH1A1) in NPC tissues and NPC cell lines. We also measured 8-nitroguanine formation as an indicator of inflammation-related DNA lesions. The staining intensity of 8-nitroguanine was significantly higher in cancer cells and inflammatory cells in the stroma of NPC tissues than in chronic nasopharyngitis tissues. Expression levels of CD44v6 and ALDH1A1 were significantly increased in cancer cells of primary NPC specimens in comparison to chronic nasopharyngitis tissues. Similarly, more intense staining of CD44v6 and ALDH1A1 was detected in an NPC cell line than in an immortalized nasopharyngeal epithelial cell line. In the case of CD24 staining, there was no significant difference between NPC and chronic nasopharyngitis tissues. 8-Nitroguanine was detected in both CD44v6- and ALDH1A1-positive stem cells in NPC tissues. In conclusion, CD44v6 and ALDH1A1 are candidate stem cell markers for NPC, and the increased formation of DNA lesions by inflammation may result in the mutation of stem cells, leading to tumor development in NPC.

Metal-mediated oxidative DNA damage induced by methylene blue.

BACKGROUND: Methylene blue (MB) is used for various clinical purposes, including chromoendoscopy and methemoglobinemia treatment. However, MB induces tumors of pancreatic islets and small intestine in experimental animals. This finding raises a possibility that MB induces carcinogenicity in these organs via light-independent mechanisms, although MB is known to cause light-dependent DNA damage. METHODS: We investigated the mechanism of MB-induced DNA damage using (32)P-5'-end-labeled DNA fragments of human tumor-relevant genes. We investigated the redox reaction of MB by UV-visible spectrometry. RESULTS: MB induced DNA damage at the 5'-ACG-3' sequence, a hot spot of the p53 gene, in the presence of NADH and Cu(II). DNA damage was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator. MB induced DNA damage at every nucleotide in the presence of NADH and Fe(III)-ethylenediaminetetraacetic acid, which was inhibited by OH scavengers and catalase. MB significantly increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative DNA lesion, in the presence of NADH and metal ions. UV-visible spectrometry revealed that the absorbance of oxidized form of MB at 668nm was decreased by NADH, and the addition of metal ions attenuated the spectral change. CONCLUSIONS: MB undergoes NADH-dependent reduction followed by metal ion-mediated reoxidation. Reduced metal ions [Cu(I) and Fe(II)] interact with H2O2, generated during the redox reaction, to produce Cu(I)OOH and OH that cause DNA damage, respectively. These findings suggest that metal-mediated DNA damage contributes to MB-mediated carcinogenesis. GENERAL SIGNIFICANCE: This study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.

Oxidative stress and its significant roles in neurodegenerative diseases and cancer.

Reactive oxygen and nitrogen species have been implicated in diverse pathophysiological conditions, including inflammation, neurodegenerative diseases and cancer. Accumulating evidence indicates that oxidative damage to biomolecules including lipids, proteins and DNA, contributes to these diseases. Previous studies suggest roles of lipid peroxidation and oxysterols in the development of neurodegenerative diseases and inflammation-related cancer. Our recent studies identifying and characterizing carbonylated proteins reveal oxidative damage to heat shock proteins in neurodegenerative disease models and inflammation-related cancer, suggesting dysfunction in their antioxidative properties. In neurodegenerative diseases, DNA damage may not only play a role in the induction of apoptosis, but also may inhibit cellular division via telomere shortening. Immunohistochemical analyses showed co-localization of oxidative/nitrative DNA lesions and stemness markers in the cells of inflammation-related cancers. Here, we review oxidative stress and its significant roles in neurodegenerative diseases and cancer.

Taurine reduces microglia activation in the brain of aged senescence-accelerated mice by increasing the level of TREM2.

Alzheimer's disease (AD), a chronic neurodegenerative disorder, is the leading cause of dementia. Over-activated microglia is related to amyloid-beta (Aß) and phosphorylated tau (phospho-tau) accumulation in the AD brain. Taurine is an amino acid with multiple physiological functions including anti-inflammatory effects, and has been reported to be neuroprotective in AD. However, the role of taurine in microglia-mediated AD remains unclear. Here, we examined the effects of taurine on the brains of senescence-accelerated mouse prone 8 (SAMP8) mice by comparing those administered 1% taurine water with those administered distilled water (DW). We observed increased levels of taurine and taurine transporter (TAUT) in the brains of the taurine-treated mice compared with those of control mice. Immunohistochemical and Western blot analyses revealed that taurine significantly reduced the number of activated microglia, levels of phospho-tau and Aß deposit in the hippocampus and cortex. Triggering receptors expressed on myeloid cells-2 (TREM2) are known to protect against AD pathogenesis. Taurine upregulated TREM2 expression in the hippocampus and cortex. In conclusion, the present study suggests that taurine treatment may upregulate TREM2 to protect against microglia over-activation by decreasing the accumulation of phospho-tau and Aß; providing an insight into a novel preventive strategy in AD.

Plasma hydroxyproline, MMP-7 and collagen I as novel predictive risk markers of hepatobiliary disease-associated cholangiocarcinoma.

Chronic opisthorchiasis caused by Opisthorchis viverrini infection is characterized by advanced periductal fibrosis leading to hepatobiliary diseases (HBD), including cholangiocarcinoma (CCA). We aimed to determine fibrotic markers to differentiate HBD status including opisthorchiasis, benign biliary disease (BBD) and CCA. Candidate fibrotic markers in plasma of healthy individuals (n = 14) and patients with opisthorchiasis (n = 32, pre- and post-treatment with praziquantel), BBD (n = 31), CCA (n = 37) and other types of tumors (n = 14) were measured by ELISA and zymography. Plasma levels of hydroxyproline (HYP), collagen I, MMP-7 and TIMP2 in opisthorchiasis patients were significantly higher than those in healthy individuals, and MMP9/TIMP2 balance may be associated with tissue resorption after praziquantel treatment. HYP and TIMP-2 levels were significantly correlated with periductal fibrosis status evaluated by ultrasonography. Plasma HYP level of CCA patients was the highest among HBD patients (p < 0.05). ROC curves revealed HYP, MMP-7 and collagen I levels significantly distinguished opisthorchiasis, BBD and CCA (p < 0.001). Odd ratio (OR) analysis demonstrated these markers in opisthorchiasis were predictable for BBD risk (p < 0.05; OR = 28.50, 10.12 and 4.63 for collagen I, MMP-7 and HYP, respectively), and the risk was reduced by praziquantel treatment. Interestingly, only plasma HYP level in BBD was predictable for CCA risk (OR = 3.69; p = 0.020). In conclusion, plasma HYP, collagen I and MMP-7 may be useful as novel predictive markers of opisthorchiasis-related BBD, and HYP may be used as a diagnostic marker for CCA.

Proton pump inhibitors suppress iNOS-dependent DNA damage in Barrett's esophagus by increasing Mn-SOD expression.

Barrett's esophagus (BE), an inflammatory disease, is a risk factor for Barrett's esophageal adenocarcinoma (BEA). Treatment of BE patients with proton pump inhibitors (PPIs) is expected to reduce the risk of BEA. We performed an immunohistochemical study to examine the formation of nitrative and oxidative DNA lesions, 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxygaunosine (8-oxodG), in normal esophageal, BE with pre- and post-treatment by PPIs and BEA tissues. We also observed the expression of an oxidant-generating enzyme (iNOS) and its transcription factor NF-κB, an antioxidant enzyme (Mn-SOD), its transcription factor (Nrf2) and an Nrf2 inhibitor (Keap1). The immunoreactivity of DNA lesions was significantly higher in the order of BEA>BE>normal tissues. iNOS expression was significantly higher in the order of BEA>BE>normal tissues, while Mn-SOD expression was significantly lower in the order of BEA

Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells.

Carbon nanotube (CNT) has a promising usage in the field of material science for industrial purposes because of its unique physicochemical property. However, intraperitoneal administration of CNT was reported to cause mesothelioma in experimental animals. Chronic inflammation may contribute to carcinogenesis induced by fibrous materials. 8-Nitroguanine is a mutagenic DNA lesion formed during inflammation and may play a role in CNT-induced carcinogenesis. In this study, we examined 8-nitroguanine formation in A549 human lung alveolar epithelial cells treated with multi-walled CNT (MWCNT) by fluorescent immunocytochemistry. Both MWCNTs with diameter of 20-30 nm (CNT20) and 40-70 nm (CNT40) significantly induced 8-nitroguanine formation at 5 and 10 µg/ml (p<0.05), which persisted for 24h, although there was no significant difference in DNA-damaging abilities of these MWCNTs. MWCNTs significantly induced the expression of inducible nitric oxide synthase (iNOS) for 24 h (p<0.05). MWCNTs also significantly increased the level of nitrite, a hydrolysis product of oxidized NO, in the culture supernatant at 4 and 8 h (p<0.05). MWCNT-induced 8-nitroguanine formation and iNOS expression were largely suppressed by inhibitors of iNOS (1400 W), nuclear factor-κB (Bay11-7082), actin polymerization (cytochalasin D), caveolae-mediated endocytosis (methyl-ß-cyclodextrin, MBCD) and clathrin-mediated endocytosis (monodansylcadaverine, MDC). Electron microscopy revealed that MWCNT was mainly located in vesicular structures in the cytoplasm, and its cellular internalization was reduced by MBCD and MDC. These results suggest that MWCNT is internalized into cells via clathrin- and caveolae-mediated endocytosis, leading to inflammatory reactions including iNOS expression and resulting nitrative DNA damage, which may contribute to carcinogenesis.

Nuclear localization of COX-2 in relation to the expression of stemness markers in urinary bladder cancer.

Inflammation may activate stem cells via prostaglandin E2 (PGE2) production mediated by cyclooxygenase-2 (COX-2) expression. We performed an immunohistochemical analysis of the expression of stemness markers (Oct3/4 and CD44v6) and COX-2 in urinary bladder tissues obtained from cystitis and cancer patients with and without Schistosoma haematobium infections. Immunoreactivity to Oct3/4 was significantly higher in S. haematobium-associated cystitis and cancer tissues than in normal tissues. CD44v6 expression was significantly higher in bladder cancer without S. haematobium than in normal tissues. COX-2 was located in the cytoplasmic membrane, cytoplasm, and nucleus of the cancer cells. Interestingly, the nuclear localization of COX-2, which was reported to function as a transcription factor, was significantly associated with the upregulation of Oct3/4 and CD44v6 in bladder cancer tissues with and without S. haematobium infection, respectively. COX-2 activation may be involved in inflammation-mediated stem cell proliferation/differentiation in urinary bladder carcinogenesis.

Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways.

Cholangiocarcinoma (CCA) is a tumor with poor prognosis that is resistant to all currently available treatments. Whether curcumin, a nutraceutical derived from turmeric (Curcuma longa), has potential therapeutic activity against human CCA was investigated using three CCA cell lines (KKU100, KKU-M156 and KKU-M213). Examination of mitochondrial dehydrogenase activity, phosphatidylserine externalization, esterase staining, caspase activation and poly-adenosine diphosphate ribose polymerase cleavage demonstrated that curcumin inhibited proliferation of and induced apoptosis in these biliary cancer cells. Colony-formation assay confirmed the growth-inhibitory effect of curcumin on CCA cells. When examined for the mechanism, curcumin was found to activate multiple cell signaling pathways in these cells. First, all CCA cells exhibited constitutively active nuclear factor (NF)-κB, and treatment with curcumin abolished this activation as indicated by DNA binding, nuclear translocation and p65 phosphorylation. Second, curcumin suppressed activation of signal transducer and activator of transcription-3 as indicated by decreased phosphorylation at both tyrosine(705) and serine(727) and inhibition of janus kinase-1 phosphorylation. Third, curcumin induced expression of peroxisome proliferator-activated receptor gamma. Fourth, curcumin upregulated death receptors, DR4 and DR5. Fifth, curcumin suppressed the Akt activation pathway. Sixth, curcumin inhibited expression of cell survival proteins such as B-cell lymphoma-2, B-cell leukemia protein xL, X-linked inhibitor of apoptosis protein, c-FLIP, cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2 and survivin and proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Seventh, the growth inhibitory effect of curcumin was enhanced in the IκB kinase-deficient cells, the enzyme required for nuclear factor-kappaB activation. Overall, our results indicate that curcumin mediates its antiproliferative and apoptotic effects through activation of multiple cell signaling pathways, and thus, its activity against CCA should be further investigated.