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.

Oxidative DNA Damage by N4-hydroxycytidine, a Metabolite of the SARS-CoV-2 Antiviral Molnupiravir.

Molnupiravir is an antiviral agent recently used for treating coronavirus disease 2019 (COVID-19). Here, we demonstrate that N4-hydroxycytidine (NHC), a molnupiravir metabolite, treated with cytidine deaminase (CDA) induced Cu(II)-mediated oxidative DNA damage in isolated DNA. A colorimetric assay revealed hydroxylamine generation from CDA-treated NHC. The site specificity of DNA damage also suggested involvement of hydroxylamine in the damage. Furthermore, Cu(I) and H2O2 play an important role in the DNA damage. We propose oxidative DNA damage via CDA-mediated metabolism as a possible mutagenic mechanism of NHC, highlighting the need for careful risk assessment of molnupiravir use in therapies for viral diseases, including COVID-19.

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.

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.

Chemoprevention by aspirin against inflammation-related colorectal cancer in mice.

Inflammation is a primary risk factor for cancer. Epidemiological studies previously demonstrated that aspirin decreased the incidence of cancer and specifically reduced the risk of colorectal cancer. However, the number of animal studies that have confirmed the efficacy of aspirin remains limited. Therefore, the purpose of the present study was to investigate the mechanisms by which aspirin prevents colorectal cancer in mice. ICR mice were treated with azoxymethane and the ulcerative colitis inducer, dextran sodium sulfate, to induce colorectal tumors. Aspirin was orally administered three times per week for 12 weeks. Aspirin significantly reduced the number and size of colorectal tumors. Aspirin also significantly decreased tumor necrosis factor alpha and reactive oxygen species (ROS) levels in the plasma. Immunohistochemical analyses and western blots showed that cyclooxygenase 2 (COX2), inducible nitric oxide synthase (iNOS), and the active form of Yes-associated protein 1 (YAP1), and cytosolic high mobility group box 1 (HMGB1) were strongly expressed at colorectal tumor sites and clearly suppressed by aspirin. An indicator of inflammation-related DNA damage, 8-nitroguanine, also accumulated in the colorectal tissues and was suppressed by aspirin. The present results suggest that the ingestion of aspirin suppressed carcinogenesis caused by inflammation through decreases in COX2 and ROS levels, resulting in reductions in DNA damage and oncogenic YAP1.

Glycyrrhizin and its derivatives promote hepatic differentiation via sweet receptor, Wnt, and Notch signaling.

The acute liver disease is involved in aberrant release of high-mobility group box 1 (HMGB1). Glycyrrhizin (GL), a traditional Chinese medicine for liver disease, binds to HMGB1, thereby inhibits tissue injury. However the mode of action of GL for chronic liver disease remains unclear. We investigated the effects of glycyrrhizin (GL) and its derivatives on liver differentiation using human iPS cells by using a flow cytometric analysis. GL promoted hepatic differentiation at the hepatoblast formation stage. The GL derivatives, 3-O-mono-glucuronyl 18ß-glycyrrhetinic acid (Mono) and 3-O-[glucosyl (1 â†’ 2)-glucuronyl] 18ß-glycyrrhetinic acid increased AFP+ cell counts and albumin+ cell counts. Glucuronate conjugation seemed to be a requirement for hepatic differentiation. Mono exhibited the most significant hepatic differentiation effect. We evaluated the effects of (±)-2-(2,4-dichlorophenoxy) propionic acid (DP), a T1R3 antagonist, and sucralose, a T1R3 agonist, on hepatic differentiation, and found that DP suppressed Mono-induced hepatic differentiation, while sucralose promoted hepatic differentiation. Thus, GL promoted hepatic differentiation via T1R3 signaling. In addition, Mono increased ß-catenin+ cell count and decreased Hes5+ cell count suggesting the involvement of Wnt and Notch signaling in GL-induced hepatic differentiation. In conclusion, GL exerted a hepatic differentiation effect via sweet receptor (T1R3), canonical Wnt, and Notch signaling.

Glycyrrhizin ameliorates melanoma cell extravasation into mouse lungs by regulating signal transduction through HMGB1 and its receptors.

Metastasis, which accounts for the majority of all cancer-related deaths, occurs through several steps, namely, local invasion, intravasation, transport, extravasation, and colonization. Glycyrrhizin has been reported to inhibit pulmonary metastasis in mice inoculated with B16 melanoma. This study aimed to identify the mechanism through which glycyrrhizin ameliorates the extravasation of melanoma cells into mouse lungs. Following B16 melanoma cell injection, mice were orally administered glycyrrhizin once every two days over 2 weeks; lung samples were then obtained and analyzed. Blood samples were collected on the final day, and cytokine plasma levels were determined. We found that glycyrrhizin ameliorated the extravasation of melanoma cells into the lungs and suppressed the plasma levels of interleukin-6, tumor necrosis factor-α, and transforming growth factor-ß. Furthermore, glycyrrhizin ameliorated the lung tissue expression of high mobility group box-1 protein (HMGB1), receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)-4, RAS, extracellular signal-related kinase, NF-κB, myeloid differentiation primary response 88, IκB kinase complex, epithelial-mesenchymal transition markers, and vascular endothelial growth factor-A. Our study demonstrates that glycyrrhizin ameliorates melanoma metastasis by regulating the HMGB1/RAGE and HMGB1/TLR-4 signal transduction pathways.

Glycyrrhizin Attenuates Carcinogenesis by Inhibiting the Inflammatory Response in a Murine Model of Colorectal Cancer.

Glycyrrhizin (GL), an important active ingredient of licorice root, which weakens the proinflammatory effects of high-mobility group box 1 (HMGB1) by blocking HMGB1 signaling. In this study, we investigated whether GL could suppress inflammation and carcinogenesis in an azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced murine model of colorectal cancer. ICR mice were divided into four groups (n = 5, each)-control group, GL group, colon cancer (CC) group, and GL-treated CC (CC + GL) group, and sacrificed after 20 weeks. Plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured using an enzyme-linked immunosorbent assay. The colonic tissue samples were immunohistochemically stained with DNA damage markers (8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxy-guanosine), inflammatory markers (COX-2 and HMGB1), and stem cell markers (YAP1 and SOX9). The average number of colonic tumors and the levels of IL-6 and TNF-α in the CC + GL group were significantly lower than those in the CC group. The levels of all inflammatory and cancer markers were significantly reduced in the CC + GL group. These results suggest that GL inhibits the inflammatory response by binding HMGB1, thereby inhibiting DNA damage and cancer stem cell proliferation and dedifferentiation. In conclusion, GL significantly attenuates the pathogenesis of AOM/DSS-induced colorectal cancer by inhibiting HMGB1-TLR4-NF-κB signaling.

Idarubicin, an Anthracycline, Induces Oxidative DNA Damage in the Presence of Copper (II).

BACKGROUND/AIM: The aim of the present study was to investigate whether idarubicin (IDR) induces oxidative DNA damage in the presence of copper (II). MATERIALS AND METHODS: DNA damage was evaluated by pBR322 plasmid DNA cleavage. The formation of oxidative stress markers [O2 •- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] was analysed. RESULTS: IDR induced DNA damage and O2 •- and 8-OHdG generation in the presence of copper (II). CONCLUSION: IDR induced oxidative DNA damage in the presence of copper (II). Since it has been reported that the concentration of copper in the serum of cancer patients is higher than that in healthy groups, IDR-induced oxidative DNA damage in the presence of copper (II) may play an important role in anticancer therapeutic strategies.

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.

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.

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.

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.

Oxidative DNA Damage and Apoptosis Induced by Aclarubicin, an Anthracycline: Role of Hydrogen Peroxide and Copper.

BACKGROUND/AIM: This study aimed to investigate aclarubicin (ACR)-induced oxidative DNA damage and apoptosis. MATERIALS AND METHODS: ACR-induced apoptosis was analyzed using HL-60 leukemia cells and HP100 cells, hydrogen peroxide (H2O2)-resistant cells derived from HL-60 cells. ACR-induced DNA damage was analyzed using plasmid DNA. RESULTS: HL-60 cells were more sensitive to ACR than HP100 cells. In HP100 cells, DNA ladder formation and caspase-3/7 activity induced by ACR were suppressed or delayed in comparison to those in HL-60 cells. ACR-induced DNA damage occurred in the presence of Cu(II), and scavenger experiments showed that the reactive species causing DNA damage appeared to be generated from H2O2 and Cu(I). Moreover, we detected intracellular Cu(I) induced by ACR in HL-60 cells, using CopperGREEN™, a fluorescent probe for detection of Cu(I) ion specifically. CONCLUSION: ACR-induced DNA damage and apoptosis can be accounted for by the involvement of H2O2 and Cu(I).

Mechanisms of DNA damage induced by morin, an inhibitor of amyloid ß-peptide aggregation.

Morin is a potential inhibitor of amyloid ß-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer's disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on 32P-5'-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical •OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H2O2 generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.

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.

Oxidative DNA Damage Induced by Pirarubicin, an Anthracycline Anticancer Agent, in the Presence of Copper(II).

BACKGROUND/AIM: One mechanism of the anticancer action of anthracyclines is believed to be oxidative DNA damage. Previously, we reported that doxorubicin induced oxidative DNA damage in the presence of Cu(II). However, the mechanism of pirarubicin-induced oxidative DNA damage has not been well clarified. MATERIALS AND METHODS: DNA damage by pirarubicin in the presence of Cu(II) was analyzed using pBR322 plasmid DNA. O2•- derived from pirarubicin in the presence of Cu(II) was detected by cytochrome c reduction. RESULTS: Pirarubicin induced DNA damage in the presence of Cu(II). Scavenger experiments suggest that reactive species are generated from H2O2 and Cu(I). Pirarubicin induced O2•- production in the presence of Cu(II). CONCLUSION: These findings suggest that pirarubicin plus Cu(II) induces oxidative DNA damage in a similar manner to doxorubicin, and Cu(II)-mediated oxidative DNA damage may serve as a common mechanism for antitumor effects of anthracyclines.

Mechanism of oxidative DNA damage induced by metabolites of carcinogenic naphthalene.

Naphthalene is a carcinogenic polycyclic aromatic hydrocarbon, to which humans are exposed as an air pollutant. Naphthalene is metabolized in humans to reactive intermediates such as 1,2-hydroxynaphthalene (1,2-NQH2), 1,4-NQH2, 1,2-naphthoquinone (1,2-NQ), and 1,4-NQ. We examined oxidative DNA damage by these naphthalene metabolites using 32P-labeled DNA fragments from human cancer-relevant genes. 1,2-NQH2 and 1,4-NQH2 induced DNA damage in the presence of Cu(II). The DNA-damaging activity of 1,2-NQH2 was significantly increased in the presence of the reduced form of nicotinamide adenine dinucleotide (NADH), whereas that of 1,4-NQH2 was not. In the presence of NADH, 1,2-NQ induced Cu(II)-dependent DNA damage, whereas 1,4-NQ did not. The calculated energy of the lowest unoccupied molecular orbital (LUMO), which corresponds to the reduction potential, was estimated to be -0.67 eV for 1,2-NQ and -0.75 eV for 1,4-NQ. These results suggest that 1,2-NQ was reduced more easily than 1,4-NQ. Furthermore, 1,2-NQH2, 1,4-NQH2, and 1,2-NQ plus NADH formed 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) as an oxidative DNA marker. Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 and Cu(I) were involved. These results indicate that NQH2s are oxidized to the corresponding NQs via semiquinone radicals, and that H2O2 and Cu(I) are generated during oxidation. 1,2-NQ is reduced by NADH to form the redox cycle, resulting in enhanced DNA damage. The formation of the corresponding semiquinone radicals was supported by an electron paramagnetic resonance (EPR) study. In conclusion, the redox cycle of 1,2-NQ/1,2-NQH2 may play a more important role in the carcinogenicity of naphthalene than that of 1,4-NQ/1,4-NQH2.