Rosmarinic acid, a natural polyphenol, has a potential pro-oxidant risk via NADH-mediated oxidative DNA damage.

BACKGROUND: Rosmarinic acid (RA) has a wide range of beneficial effects on human health. On the other hand, RA has been reported to induce metal-mediated reactive oxygen species (ROS) generation and DNA damage. However, its mechanism remains unknown. In this study, to clarify the underlying mechanism, we analyzed metal-mediated DNA damage in isolated DNA treated with RA and its analog isorinic acid. RESULTS: RA plus Cu(II), but not Fe(III), significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage, in calf thymus DNA. Furthermore, a comparison of the 8-oxodG formation induced by RA and its analog isorinic acid suggested that the catechol groups in RA could be associated with their abilities to form 8-oxodG. Interestingly, the 8-oxodG formation induced by RA and isorinic acid plus Cu(II) was markedly enhanced by the addition of NADH, an endogenous reductant. To elucidate the mechanism of RA plus Cu(II)-induced oxidative DNA damage, we examined DNA damage in 32P-labeled DNA treated with RA in the presence of Cu(II). RA plus Cu(II) caused DNA cleavage, which was enhanced by piperidine treatment, suggesting that RA causes not only DNA strand breakage but also base modification. RA plus Cu(II)-induced DNA damage was inhibited by catalase (H2O2 scavenger), bathocuproine (Cu(I) chelator), and methional (scavenger of a variety of ROS other than •OH) but not by typical •OH scavengers and SOD, indicating the involvement of H2O2, Cu(I), and ROS other than •OH. DNA cleavage site analysis showing RA-induced site-specific DNA damage (frequently at thymine and some cytosine residues) supports the involvement of ROS other than •OH, because •OH causes DNA cleavage without site specificity. Based on these results, Cu(I) and H2O2 generation with concomitant RA autoxidation could lead to the production of Cu(I)-hydroperoxide, which induces oxidative DNA damage. o-Quinone and o-semiquinone radicals are likely to be again reduced to RA by NADH, which dramatically increases oxidative DNA damage, particularly at low concentrations of RA. CONCLUSIONS: In this study, physiologically relevant concentrations of RA effectively induced oxidative DNA damage in isolated DNA through redox cycle reactions with copper and NADH.

Oxidative DNA damage: Induction by fructose, in vitro, and its enhancement by hydrogen peroxide.

Sucrose and high-fructose corn syrup comprise nearly equal amounts of glucose and fructose. With the use of high-fructose corn syrup in the food industry, consumption of fructose, which may be a tumor promoter, has increased dramatically. We examined fructose-induced oxidative DNA damage in the presence of Cu(II), with or without the addition of H2O2. With isolated DNA, fructose induced Cu(II)-mediated DNA damage, including formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), to a greater extent than did glucose, and H2O2 enhanced the damage. In cultured human cells, 8-oxodG formation increased significantly following treatment with fructose and the H2O2-generating enzyme glucose oxidase. Fructose may play an important role in oxidative DNA damage, suggesting a possible mechanism for involvement of fructose in carcinogenesis.

Myricetin causes site-specific DNA damage via reactive oxygen species generation by redox interactions with copper ions.

Myricetin (MYR), found in tea and berries, may have preventive effects on diseases, including Alzheimer's disease and cancer. However, MYR is also a mutagen, inducing DNA damage in the presence of metal ions. We have studied the molecular mechanisms of DNA damage by MYR in the presence of Cu(II) (MYR+Cu). Using 32P-5'-end-labeled DNA fragments, we analyzed site-specific DNA damage caused by MYR+Cu. MYR+Cu caused concentration-dependent DNA strand breaks and base alterations, leading to cleavage of DNA at thymine, cytosine, and guanine nucleotides. Formation of the oxidative DNA damage indicator, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in calf thymus DNA was increased by MYR+Cu. The production of 8-oxodG in MYR-treated HL-60 cells was significantly higher than in HP100 cells, which are more resistant to H2O2 than are HL-60 cells. Reactive oxygen species (ROS) scavengers were used to elucidate the mechanism of DNA damage. DNA damage was not inhibited by typical free hydroxyl radical (•OH) scavengers such as ethanol, mannitol, or sodium formate. However, methional, catalase, and bathocuproine inhibited DNA damage induced by MYR+Cu. These results suggest that H2O2, Cu(I), and ROS other than •OH are involved in MYR+Cu-induced DNA damage. We conclude that the Cu(I)/Cu(II) redox cycle and concomitant H2O2 production via autoxidation of MYR generate a complex of H2O2 and Cu(I), probably Cu(I)-hydroperoxide, which induces oxidative DNA damage.

Knockdown of TFRC suppressed the progression of nasopharyngeal carcinoma by downregulating the PI3K/Akt/mTOR pathway.

BACKGROUND: The transferrin receptor (TfR) encoded by TFRC gene is the main cellular iron importer. TfR is highly expressed in many cancers and is expected to be a promising new target for cancer therapy; however, its role in nasopharyngeal carcinoma (NPC) remains unknown. METHODS: The TfR levels were investigated in NPC tissues and cell lines using immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. Knockdown of TFRC using two siRNA to investigate the effects on intracellular iron level and biological functions, including proliferation by CKK-8 assay, colony formation, cell apoptosis and cell cycle by flow cytometry, migration and invasion, and tumor growth in vivo by nude mouse xenografts. RNA sequencing was performed to find possible mechanism after TFRC knockdown on NPC cells and further verified by western blotting. RESULTS: TfR was overexpressed in NPC cell lines and tissues. Knockdown of TFRC inhibited cell proliferation concomitant with increased apoptosis and cell cycle arrest, and it decreased intracellular iron, colony formation, migration, invasion, and epithelial-mesenchymal transition in HK1-EBV cells. Western blotting showed that TFRC knockdown suppressed the levels of the iron storage protein FTH1, anti-apoptotic marker BCL-xL, and epithelial-mesenchymal transition markers. We confirmed in vivo that TFRC knockdown also inhibited NPC tumor growth and decreased Ki67 expression in tumor tissues of nude mouse xenografts. RNA sequencing and western blotting revealed that TFRC silencing inhibited the PI3K/Akt/mTOR signaling pathway. CONCLUSIONS: These results indicated that TfR was overexpressed in NPC, and TFRC knockdown inhibited NPC progression by suppressing the PI3K/Akt/mTOR signaling pathway. Thus, TfR may serve as a novel biomarker and therapeutic target for NPC.

Vegetable Oil-Peroxidation Product 'Hydroxynonenal' Causes Hepatocyte Injury and Steatosis via Hsp70.1 and BHMT Disorders in the Monkey Liver.

Hsp70.1 has a dual function as a chaperone protein and lysosomal stabilizer. In 2009, we reported that calpain-mediated cleavage of carbonylated Hsp70.1 causes neuronal death by inducing lysosomal rupture in the hippocampal CA1 neurons of monkeys after transient brain ischemia. Recently, we also reported that consecutive injections of the vegetable oil-peroxidation product 'hydroxynonenal' induce hepatocyte death via a similar cascade in monkeys. As Hsp70.1 is also related to fatty acid ß-oxidation in the liver, its deficiency causes fat accumulation. The genetic deletion of betaine-homocysteine S-methyltransferase (BHMT) was reported to perturb choline metabolism, inducing a decrease in phosphatidylcholine and resulting in hepatic steatosis. Here, focusing on Hsp70.1 and BHMT disorders, we studied the mechanisms of hepatocyte degeneration and steatosis. Monkey liver tissues with and without hydroxynonenal injections were compared using proteomics, immunoblotting, immunohistochemical, and electron microscopy-based analyses. Western blotting showed that neither Hsp70.1 nor BHMT were upregulated, but an increased cleavage was observed in both. Proteomics showed a marked downregulation of Hsp70.1, albeit a two-fold increase in the carbonylated BHMT. Hsp70.1 carbonylation was negligible, in contrast to the ischemic hippocampus, which was associated with ~10-fold increments. Although histologically, the control liver showed very little lipid deposition, numerous tiny lipid droplets were seen within and around the degenerating/dying hepatocytes in monkeys after the hydroxynonenal injections. Electron microscopy showed permeabilization/rupture of lysosomal membranes, dissolution of the mitochondria and rough ER membranes, and proliferation of abnormal peroxisomes. It is probable that the disruption of the rough ER caused impaired synthesis of the Hsp70.1 and BHMT proteins, while impairment of the mitochondria and peroxisomes contributed to the sustained generation of reactive oxygen species. In addition, hydroxynonenal-induced disorders facilitated degeneration and steatosis in the hepatocytes.

Taurine induces upregulation of p53 and Beclin1 and has antitumor effect in human nasopharyngeal carcinoma cells in vitro and in vivo.

Taurine is an amino acid that has several physiological functions. Previously, we reported the apoptosis-inducing effect of taurine in human nasopharyngeal carcinoma (NPC) cells in vitro. However, the effect of taurine on NPC cell growth in vivo has not been elucidated. Autophagy plays an important role in cell metabolism and exhibits antitumor effects under certain conditions. In this study, we investigated the effects of taurine on apoptosis- and autophagy-related molecules in NPC cells in vitro and in vivo. In our in vitro study, NPC cells (HK1-EBV) were treated with taurine, and Western blot and immunocytochemical analyses revealed that taurine co-upregulated Beclin 1 and p53, with autophagy upregulation. In the in vivo study, we used a nude mouse model with subcutaneous xenografts of HK1-EBV cells. Once the tumors reached 2-3 mm in diameter, the mice were provided with distilled water (control group) or taurine dissolved in distilled water (taurine-treated group) ad libitum (day 1) and sacrificed on day 13. The volume and weight of the tumors were significantly lower in the taurine-treated group. Using immunohistochemistry (IHC), we confirmed that taurine treatment reduced the distinct cancer nest areas. IHC analyses also revealed that taurine promoted apoptosis, as evidenced by an increase in cleaved caspase-3, accompanied by upregulation of p53. Additionally, taurine increased LC3B and Beclin 1 expression, which are typical autophagy markers. The present study demonstrated taurine-mediated tumor growth suppression. Therefore, taurine may be a novel preventive strategy for NPC.

Targeting fructose metabolism by glucose transporter 5 regulation in human cholangiocarcinoma.

Alterations in cellular metabolism may contribute to tumor proliferation and survival. Upregulation of the facilitative glucose transporter (GLUT) plays a key role in promoting cancer. GLUT5 mediates modulation of fructose utilization, and its overexpression has been associated with poor prognosis in several cancers. However, its metabolic regulation remains poorly understood. Here, we demonstrated elevated GLUT5 expression in human cholangiocarcinoma (CCA), using RNA sequencing data from samples of human tissues and cell lines, as compared to normal liver tissues or a cholangiocyte cell line. Cells exhibiting high-expression of GLUT5 showed increased rates of cell proliferation and ATP production, particularly in a fructose-supplemented medium. In contrast, GLUT5 silencing attenuated cell proliferation, ATP production, cell migration/invasion, and improved epithelial-mesenchymal transition (EMT) balance. Correspondingly, fructose consumption increased tumor growth in a nude mouse xenograft model, and GLUT5 silencing suppressed growth, supporting the tumor-inhibitory effect of GLUT5 downregulation. Furthermore, in the metabolic pathways of fructolysis-Warburg effect, the expression levels of relative downstream genes, including ketohexokinase (KHK), aldolase B (ALDOB), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 4 (MCT4), as well as hypoxia-inducible factor 1 alpha (HIF1A), were altered in a GLUT5 expression-dependent manner. Taken together, these findings indicate that GLUT5 could be a potential target for CCA therapeutic approach via metabolic regulation.

Copper-mediated DNA damage caused by purpurin, a natural anthraquinone.

BACKGROUND: Purpurin (1,2,4-trihydroxy-9,10-anthraquinone), a natural red anthraquinone pigment, has historically been used as a textile dye. However, purpurin induced urinary bladder tumors in rats, and displayed a mutagenic activity in assay using bacteria and mammalian cells. Many carcinogenic dyes are known to induce bladder cancers via DNA adduct formation, but carcinogenic mechanisms of purpurin remain unknown. In this study, to clarify the mechanism underlying carcinogenicity of purpurin, copper-mediated DNA damage induced by purpurin was examined using 32P-labeled DNA fragments of human genes relevant to cancer. Furthermore, we also measured 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA. RESULTS: Purpurin plus Cu(II) cleaved 32P-labeled DNA fragments only under piperidine treatment, indicating that purpurin caused base modification, but not breakage of the DNA backbone. In the absence of Cu(II), purpurin did not induce DNA cleavage even with piperidine treatment. Purpurin plus Cu(II) caused piperidine-labile sites predominantly at G and some T residues. Bathocuproine, a Cu(I) chelator, completely prevented the occurrence of piperidine-labile sites, indicating a critical role of Cu(I) in piperidine-labile sites induced by purpurin plus Cu(II). On the other hand, methional, a scavenger of a variety of reactive oxygen species (ROS) and catalase showed limited inhibitory effects on the induction of piperidine-labile sites, suggesting that ROS could not be major mediators of the purpurin-induced DNA damage. Considering reported DNA adduct formation by quinone metabolites of several carcinogenic agents, quinone form of purpurin, which is possibly generated via purpurin autoxidation accompanied by Cu(I)/Cu(II) redox cycle, might lead to DNA adducts and piperidine-labile sites. In addition, we measured contents of 8-oxodG. Purpurin moderately but significantly increased 8-oxodG in calf thymus DNA in the presence of Cu(II). The 8-oxodG formation was inhibited by catalase, methional and bathocuproine, suggesting that Cu(I)-hydroperoxide, which was generated via Cu(I) and H2O2, caused oxidative DNA base damage. CONCLUSIONS: We demonstrated that purpurin induces DNA base damage possibly mediated by Cu(I)/Cu(II) redox cycle both with and without ROS generation, which are likely to play an important role in its carcinogenicity.

Moyamoya disease: diagnosis and interventions.

Moyamoya disease is a rare cause of stroke, radiologically characterised by progressive stenosis of the terminal portion of the internal carotid arteries and compensatory capillary collaterals. The discovery that RNF213, which encodes an unconventional E3 ubiquitin ligase, is the major susceptibility gene for moyamoya disease in people from east Asia has opened new avenues for investigation into the mechanisms of disease and potential treatment targets. The Arg4810Lys variant of the gene is most strongly associated with moyamoya disease, but the penetrance is lower than 1%, suggesting a synergistic relationship with additional environmental and genetic risk factors. White people carry less common non-Arg4810Lys variants of RNF213, which partly explains the lower prevalence of moyamoya disease in European countries and in the USA than in east Asian countries. Several monogenic moyamoya syndromes possess the radiological characteristics of moyamoya disease and have been associated with multiple genes and pathways involved in moyamoya angiopathy pathogenesis. Further clarification of the genetic and environmental factors that contribute to the emergence of moyamoya angiopathy could enable development of new treatment strategies for moyamoya disease.

Characterization of Moyamoya and Middle Cerebral Artery Diseases by Carotid Canal Diameter and RNF213 p.R4810K Genotype.

OBJECTIVES: It is sometimes difficult to differentiate middle cerebral artery disease from moyamoya disease because the two can present similarly yet have different treatment strategies. We investigated whether the presence of a narrow carotid canal and the RNF213 mutation can help differentiate between the two phenotypes. POPULATION AND METHODS: We analyzed 78 patients with moyamoya disease, 27 patients with middle cerebral artery disease, and 79 controls from 2 facilities. The carotid canal diameter was measured using computed tomography. The p.R4810K mutation was genotyped by TaqMan assay. A receiver operating characteristics analysis was performed to assess the significance of the carotid canal diameter for the accurate diagnosis of moyamoya disease. RESULTS: The carotid canal diameter was significantly narrower in patients with moyamoya disease than in controls. The optimal cutoff values were 5.0 mm for adult males and 4.5 mm for adult females and children (sensitivity: 0.82; specificity: 0.92). Among the patients with middle cerebral artery disease, 18.5% and 25.0% of the affected hemispheres had the p.R4810K mutation and narrow canal (i.e., below the cutoff), respectively, whereas only 3.1% of those had both. Contrastingly, 68.8% of the affected hemispheres in patients with moyamoya disease had both these characteristics. Among the patients with moyamoya disease, those with the p.R4810K mutation tended to have narrower carotid canals. CONCLUSIONS: Although the presence of a narrow carotid canal or the p.R4810K mutation alone could not be used to distinguish those with moyamoya disease from those with middle cerebral artery disease, the combination of these factors could better characterize the two phenotypes.

Suppression of RNF213, a susceptibility gene for moyamoya disease, inhibits endoplasmic reticulum stress through SEL1L upregulation.

RNF213, a susceptibility gene for moyamoya disease, is associated with stress responses to various stressors. We previously reported that Rnf213 knockout (KO) mitigated endoplasmic reticulum (ER) stress-induced diabetes in the Akita mouse model of diabetes. However, the role of RNF213 in ER stress regulation remains unknown. In the present study, RNF213 knockdown significantly inhibited the upregulation of ER stress markers (CHOP and spliced XBP1) by chemical ER stress-inducers in HeLa cells. Levels of SEL1L, a critical molecule in ER-associated degradation (ERAD), were increased by RNF213 knockdown, and SEL1L knockdown prevented the inhibitory effect of RNF213 suppression on ER stress in HeLa cells, indicating SEL1L involvement in this inhibition of ER stress. SEL1L upregulation was also confirmed in pancreatic islets of Rnf213 KO/Akita mice and in Rnf213 KO mouse embryonic fibroblasts. Additionally, RNF213 suppression increased levels of HRD1, which forms a complex with SEL1L to degrade misfolded protein in cells under ER stress. In conclusion, we demonstrate that RNF213 depletion inhibits ER stress possibly through elevation of the SEL1L-HRD1 complex, thereby promoting ERAD in vitro and in vivo.

Mechanism of reactive oxygen species generation and oxidative DNA damage induced by acrylohydroxamic acid, a putative metabolite of acrylamide.

Acrylamide is formed during the heating of food and is also found in cigarette smoke. It is classified by the International Agency for Research on Cancer as a probable human carcinogen (Group 2A). Glycidamide, an epoxide metabolite of acrylamide, is implicated in the mechanism of acrylamide carcinogenicity. Acrylamide causes oxidative DNA damage in target organs. We sought to clarify the mechanism of acrylamide-induced oxidative DNA damage by investigating site-specific DNA damage and reactive oxygen species (ROS) generation by a putative metabolite of acrylamide, acrylohydroxamic acid (AA). Our results, using 32P-5'-end-labeled DNA fragments, indicated that, although AA alone did not damage DNA, AA treated with amidase induced DNA damage in the presence of Cu(II). DNA cleavage occurred preferentially at T and C, and particularly at T in 5'-TG-3' sequences, and the DNA cleavage pattern was similar to that of hydroxylamine. The DNA damage was inhibited by methional, catalase, and Cu(I)-chelator bathocuproine, suggesting that H2O2 and Cu(I) are involved in the mechanism of DNA damage induced by AA treated with amidase. In addition, amidase-treated AA increased 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in calf thymus DNA, an indicator of oxidative DNA damage, in a dose-dependent manner. In conclusion, hydroxylamine, possibly produced from AA treated with amidase, was autoxidized via the Cu(II)/Cu(I) redox cycle and H2O2 generation, suggesting that oxidative DNA damage induced by ROS plays an important role in acrylamide-related carcinogenesis.

GDF10 inhibits cell proliferation and epithelial-mesenchymal transition in nasopharyngeal carcinoma by the transforming growth factor-ß/Smad and NF-κB pathways.

Growth differentiation factor-10 (GDF10) belongs to a member of the transforming growth factor-ß (TGF-ß) superfamily. Dysfunction of the TGF-ß pathway can lead to carcinoma progression. Previous studies have shown that GDF10 acts as a tumor suppressor gene in some cancers. However, the molecular mechanisms of the association between GDF10 and cell functions in nasopharyngeal carcinoma (NPC) remain unclear. In this study, the expression and methylation levels of GDF10 were studied in human subjects and cell lines. Furthermore, overexpression of GDF10 was used to explore its biological function and potential mechanism in NPC cell lines. GDF10 was downregulated in NPC owing to its aberrant promoter methylation. After treatment with 5-aza-2'-deoxycytidine, the expression of GDF10 in NPC cells was reversed. We also confirmed that the overexpression of GDF10 significantly inhibited cell proliferation and tumor growth both in vitro and in vivo, respectively. Additionally, GDF10 overexpression in NPC cells attenuated migration and invasion and inhibited epithelial-to-mesenchymal transition with a decrease in nuclear Smad2 and NF-κB protein accumulation. GDF10 was silenced owing to its promoter hypermethylation, and it might originally act as a functional tumor suppressor via TGF-ß/Smad and NF-κB signaling pathways in NPC.

Influence of Epstein-Barr virus and human papillomavirus infection on macrophage migration inhibitory factor and macrophage polarization in nasopharyngeal carcinoma.

BACKGROUND: To assess the effects of Epstein-Barr virus (EBV) and human papillomavirus (HPV) infection on the tumor microenvironment, we examined the relationship between viral infection status, macrophage migration inhibitory factor (MIF), and tumor-associated macrophages in nasopharyngeal carcinoma (NPC). METHODS: A tissue microarray containing 150 cores from 90 patients with NPC and six with chronic inflammation was used. EBV and HPV status were detected using in situ hybridization with commercial EBER1 and HPV16/18 probes. Immunofluorescence double staining of MIF, pan-macrophage marker CD68, M1 macrophage marker CD11c, and M2 macrophage marker CD163 were analyzed using the same tissue microarray. The levels of these markers between NPC and inflammation cases and between tumor nests and stroma were compared. Correlations among these markers were analyzed. RESULTS: We found EBER1(+) cases in 90% of NPC patients, including 10% EBV/HPV co-infection. M1 macrophages mainly infiltrated the tumor nest, while M2 macrophages infiltrated the tumor stroma. We found a significant positive correlation between EBER1 levels and MIF levels in tumor nests and a significant positive correlation between HPV16/18 and CD11c(+) cell levels in NPC tissues. CONCLUSIONS: It is suggested that MIF is associated with EBV, and M1 macrophage infiltration is affected by HPV status in NPC.

Nitrative DNA damage in lung epithelial cells exposed to indium nanoparticles and indium ions.

Indium compounds have been widely used in manufacturing displays of mobile phones, computers and televisions. However, inhalation exposure to indium compounds causes interstitial pneumonia in exposed workers and lung cancer in experimental animals. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed under inflammatory conditions and may participate in indium-induced carcinogenesis. In this study, we examined 8-nitroG formation in A549 cultured human lung epithelial cells treated with indium compounds, including nanoparticles of indium oxide (In2O3) and indium-tin oxide (ITO), and indium chloride (InCl3). We performed fluorescent immunocytochemistry to examine 8-nitroG formation in indium-exposed A549 cells. All indium compounds significantly increased 8-nitroG formation in A549 cells at 5 ng/ml after 4 h incubation. 8-NitroG formation was largely reduced by 1400 W, methyl-ß-cyclodextrin (MBCD) and monodansylcadaverine (MDC), suggesting the involvement of nitric oxide synthase and endocytosis. 8-NitroG formation in A549 cells was also largely suppressed by small interfering RNA (siRNA) for high-mobility group box-1 (HMGB1), receptor for advanced glycation and end products (AGER, RAGE) and Toll-like receptor 9 (TLR9). These results suggest that indium compounds induce inflammation-mediated DNA damage in lung epithelial cells via the HMGB1-RAGE-TLR9 pathway. This mechanism may contribute to indium-induced genotoxicity in the respiratory system.

Taurine Attenuates Carcinogenicity in Ulcerative Colitis-Colorectal Cancer Mouse Model.

Taurine (2-aminoethane-sulfonic acid) is a type of amino acids and has numerous physiological and therapeutic functions, including anti-inflammation. However, there are few studies on the anticancer action of taurine. Our previous studies have demonstrated that taurine exhibits an apoptosis-inducing effect on human nasopharyngeal carcinoma cells in vitro. In this study, we have investigated whether taurine has an anticancer effect, using azoxymethane (AOM)/sulfate sodium (DSS)- induced mouse model for colon carcinogenesis. All mice, except those in control group, received a single intraperitoneal injection of AOM and DSS in the drinking water for 7 days twice, with 1-week interval. After the first DSS treatment, mice were given distilled water (model group) or taurine in the drinking water (taurine group) ad libitum. No tumor was observed in the control group. Taurine significantly suppressed AOM+DSS-induced tumor formation. Histopathological examination revealed AOM/DSS treatment induced colon cancer in all mice (8/8, 100%), and taurine significantly inhibited the progression of colon cancer (4/9, 44.4%). Taurine significantly attenuated cell proliferation in cancer tissues detected by Ki-67 staining. Taurine significantly increased the levels of an apoptosis marker cleaved caspase-9 and tumor suppressor protein PTEN. This is the first study that demonstrated that taurine significantly reduced carcinogenicity in vivo using AOM/DSS-induced colon cancer mouse model.

CD44v9 Induces Stem Cell-Like Phenotypes in Human Cholangiocarcinoma.

Background: Our previous study demonstrated an overexpression of CD44 variant 9 (CD44v9) in human cholangiocarcinoma (CCA) tissues that was associated with inflammation-related tumor development. However, the participation of CD44v9 in cholangiocarcinogenesis remains poorly understood. Therefore, in this study, we examined the potential roles of CD44v9 in CCA cells to understand the carcinogenic mechanism. Methods: Using normal cholangiocytes (MMNK1) and CCA cells (KKU213), the expression levels of CD44v9 and its related molecules were quantified through RT-qPCR and immunofluorescence (IF) staining. To evaluate its biological functions, we performed CD44v9 (exon 13) silencing using siRNA transfection, and assessed cell proliferation through MTT assay, cell migration and invasion by transwell technique, and carried out cell cycle analysis by flow cytometry. In vivo tumor growth was assessed by nude mouse xenografts, and histological and molecular changes were determined. Results: KKU213 exhibited higher protein expression levels of CD44v9 than those of MMNK1 through IF staining. RT-qPCR analysis revealed that the mRNA expression level of CD44v9 was predominantly elevated in CCA cells along with its neighboring exons such as variant 8 and 10, minimally affecting the standard form of CD44. CD44v9 silencing could regulate redox system in CCA cells by reducing the expression levels of SOD3 and cysteine transporter xCT. CD44v9 silencing suppressed the CCA cell proliferation by induction of apoptosis and cell cycle arrest. Migration and invasion were decreased in CD44v9 siRNA-treated CCA cells. CD44v9 downregulation inhibited CCA tumor growth in mouse xenografts. IF analysis demonstrated the histological changes in xenograft tissues such as an increase in connective tissues through collagen deposition and reduction of hyaluronic acid synthesis through CD44v9 silencing. CD44v9 knockdown in vitro and in vivo increased E-cadherin and reduced vimentin expression levels, resulting in reduction of epithelial-mesenchymal transition (EMT) process. Moreover, CD44v9 modulated Wnt10a and ß-catenin in tumorigenesis. Conclusion: Our results indicate that CD44v9 plays a potential role in CCA development by the regulation of cell proliferation and redox balancing. CD44v9 silencing may suppress tumor growth, migration and invasion through EMT: a finding that could potentially be applied in the development of targeted cancer therapy.

Polyphenols with Anti-Amyloid ß Aggregation Show Potential Risk of Toxicity Via Pro-Oxidant Properties.

Alzheimer's disease (AD) is the most common form of dementia among older people. Amyloid ß (Aß) aggregation has been the focus for a therapeutic target for the treatment of AD. Naturally occurring polyphenols have an inhibitory effect on Aß aggregation and have attracted a lot of attention for the development of treatment strategies which could mitigate the symptoms of AD. However, considerable evidence has shown that the pro-oxidant mechanisms of polyphenols could have a deleterious effect. Our group has established an assay system to evaluate the pro-oxidant characteristics of chemical compounds, based on their reactivity with DNA. In this review, we have summarized the anti-Aß aggregation and pro-oxidant properties of polyphenols. These findings could contribute to understanding the mechanism underlying the potential risk of polyphenols. We would like to emphasize the importance of assessing the pro-oxidant properties of polyphenols from a safety point of view.

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.