Protective effects of Chlorella-derived peptide on UVB-induced production of MMP-1 and degradation of procollagen genes in human skin fibroblasts.

UV exposure is known to induce premature aging, which is mediated by matrix metalloproteinase-1 (MMP-1) activity. MMP-1 mRNA expression is up-regulated by elevated cysteine-rich 61 (CYR61) and monocyte chemoattractant protein-1 (MCP-1) via action of transcription factor AP-1. Collagen is degraded by MMP-1 activity but synthesized by transforming growth factor-ß (TGF-ß) signal. Chlorella has been shown to inhibit UVB-induced MMP-1 level, however its regulatory molecular mechanisms have not been studied. In this study, Chlorella derived peptide (CDP) was added to skin fibroblasts after UVB irradiation and the expression of MMP-1, CYR61, procollagen, c-fos, c-jun, and TGF-ß receptor (TbRII) mRNA and MCP-1 production were investigated. CDP (10 or 5mg/ml) diminished UVB-induced MMP-1 and CYR61 mRNA expression and MCP-1 production, whereas, UVB-suppressed procollagen and TbRII mRNA was restored by CDP treatment. UVB-induced c-fos and c-jun expressions were also inhibited by the CDP treatment. Taken together, CDP inhibits UVB-induced MMP-1 expression in skin fibroblasts by suppressing expression of AP-1 and CYR61 and MCP-1 production.

Safrole-DNA adduct in hepatocellular carcinoma associated with betel quid chewing.

Betel quid chewing, which contributes high concentration of safrole in saliva, is a popular oral habit in Taiwan. Safrole is a documented rodent hepatocarcinogen, yet its hepatocarcinogenic potential in human is not known. Here, we used LC/ESI-ITMS(n) and LC/QTOF-MS confirmed safrole-dGMP as reference standard to detect the safrole-DNA adduct in hepatic tissues from HBsAg-/HCV-seronegative hepatocellular carcinoma patients by (32)P-postlabeling. We first synthesized and confirmed safrole-dGMP by LC/MS. Two isomeric safrole-dGMPs were characterized as N(2)-(trans-isosafrol-3'-yl) deoxyguanosine and N(2)-(safrol-1'-yl) deoxyguanosine. This technique was able to detect hepatic safrole-DNA adduct in mice that were treated with safrole but not sensitive enough to detect safrole-DNA adduct in human samples. Using the nuclease P1 version of the (32)P-postlabeling technique, we detected the presence of safrole-DNA adduct in two out of 28 hepatic tissues from hepatocellular carcinoma patients, and only these two patients had a history of betel quid chewing lasting more than 10 years. From co-chromatography with the mass confirmed safrole-dGMPs, this safrole-DNA adduct was identified as N(2)-(trans-isosafrol-3'-yl) deoxyguanosine. These results suggest that betel quid-containing safrole might be involved in the pathogenesis of hepatocellular carcinoma in human beings and LC/MS has the potential to identify DNA adducts in clinical samples.

Mitochondrial dysregulation and glycolytic insufficiency functionally impair CD8 T cells infiltrating human renal cell carcinoma.

Cancer cells can inhibit effector T cells (Teff) through both immunomodulatory receptors and the impact of cancer metabolism on the tumor microenvironment. Indeed, Teff require high rates of glucose metabolism, and consumption of essential nutrients or generation of waste products by tumor cells may impede essential T cell metabolic pathways. Clear cell renal cell carcinoma (ccRCC) is characterized by loss of the tumor suppressor von Hippel-Lindau (VHL) and altered cancer cell metabolism. Here, we assessed how ccRCC influences the metabolism and activation of primary patient ccRCC tumor infiltrating lymphocytes (TIL). CD8 TIL were abundant in ccRCC, but they were phenotypically distinct and both functionally and metabolically impaired. ccRCC CD8 TIL were unable to efficiently uptake glucose or perform glycolysis and had small, fragmented mitochondria that were hyperpolarized and generated large amounts of ROS. Elevated ROS was associated with downregulated mitochondrial SOD2. CD8 T cells with hyperpolarized mitochondria were also visible in the blood of ccRCC patients. Importantly, provision of pyruvate to bypass glycolytic defects or scavengers to neutralize mitochondrial ROS could partially restore TIL activation. Thus, strategies to improve metabolic function of ccRCC CD8 TIL may promote the immune response to ccRCC.

Hydroxyl radical formation and oxidative DNA damage induced by areca quid in vivo.

Chewing areca quid (AQ) has been implicated as a major risk factor for the development of oral squamous-cell carcinoma (OSCC). Recent studies have suggested that AQ-generated reactive oxygen species (ROS) is one of the contributing factors for oral carcinogenesis. However, the AQ used in Taiwan is different from that used in other countries. This study is designed to test whether ROS are generated and the consequent effects in locally prepared AQ in vivo. We measured the hydroxyl radical formation, as represented by the presence of o- and m-tyrosine in saliva from volunteers who chewed AQ containing 20 mg phenylalanine. Their saliva contained significantly higher amounts (p < .05) of o- and m-tyrosine as compared to the controls. In addition, chewing AQ containing Piper betle inflorescence generated higher amounts of m-tyrosine, but not o-tyrosine, in saliva than did chewing AQ containing betel leaf. We further tested the oxidative DNA damaging effect of the reconstituted AQ, as evidenced by the elevation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels, in hamster buccal pouch. Following daily painting for 14 d, the 8-OH-dG level in hamster buccal pouch is significantly elevated (p < .05) in the AQ-treated group versus the controls. These findings demonstrate that ROS, such as hydroxyl radical, are formed in the human oral cavity during AQ chewing, and chewing such prepared AQ might cause oxidative DNA damage to the surrounding tissues.

Anti-melanogenic effects of δ-tocotrienol are associated with tyrosinase-related proteins and MAPK signaling pathway in B16 melanoma cells.

Tocotrienols are known to possess potent antioxidant, anticancer, and cholesterol lowering activities. Being able to rapidly penetrate the skin, these vitamin E isoforms have been explored for potential treatment against melanoma. This study aimed to elucidate the mechanism involved in the anti-melanogenic effects of δ-tocotrienol (δT3) in B16 melanoma cells. Results showed that at 20 µM of δT3 significantly inhibited melanin formation and ROS generation. Treatment with δT3 also effectively suppressed the expression of melanogenesis-related proteins, including MC1R, MITF, TYRP-1, and TYRP-2. More importantly, we observed that the mitogen-activated protein kinase (MAPK) pathway was involved in mediating δT3's inhibitory effect against melanin production. Specifically, δT3 treatment markedly induced the activation of extracellular signal-regulated kinases (ERK). The use of ERK activation inhibitor (PD98059) abrogated the δT3-mediated downregulation expression melanogenesis-related proteins and restored melanin production. Furthermore, siRNA targeting ERK effectively blocked the δT3-induced repression of tyrosinase and TYRP-1 expression. These results suggest that δT3's inhibitory effect against melanogenesis is mediated by the activation of ERK signaling, thereby resulting in downstream repression of melanogenesis-related proteins and the subsequent melanin production. These data provide insight to δT3's effect and the targeting of ERK signaling for treatment against melanogenesis.

Increased oxidative damage to peripheral blood leukocyte DNA in chronic peritoneal dialysis patients.

This study focuses on the extent of oxidative DNA damage in peripheral blood leukocytes of chronic peritoneal dialysis (CPD) patients. 8-Hydroxy 2'-deoxyguanosine (8-OHdG) contents in peripheral leukocyte DNA were measured by an HPLC-electrochemical detection method in 24 age- and sex-matched healthy subjects, 22 nondialyzed patients with advanced renal failure, and 42 CPD patients. Mean 8-OHdG content was the highest in CPD patients, followed by the nondialyzed patients, and then by the healthy subjects (19.4 versus 11.9 versus 8.3/10(6) dG; ANOVA P < 0.001). In nondialyzed subjects, peripheral leukocyte 8-OHdG contents inversely correlated with renal creatinine clearance (r = -0.772; P < 0.001). Deficiency of blood antioxidants in CPD and nondialyzed patients was expressed by the lower plasma levels of ascorbate, cholesterol-standardized alpha-tocopherol and whole-blood reduced glutathione, and the higher levels of whole-blood oxidized glutathione as compared with healthy subjects (ANOVA P < 0.05). Mean serum ferritin and iron levels and transferrin saturation were higher in the CPD patients than those in the nondialyzed patients and controls (ANOVA P < 0.05). Flow cytometric analyses of intracellular reactive oxygen species production of peripheral leukocytes showed that spontaneous production by granulocytes, as well as phorbol-12-myristate-13-acetate (PMA)-induced production by granulocytes, lymphocytes and monocytes, were the highest from CPD patients, followed by nondialyzed patients, and then by the healthy subjects (ANOVA P < 0.05). Forward stepwise multiple regression disclosed that uremia, PD treatment, spontaneous and PMA-induced reactive oxygen species production in leukocytes, and serum iron were the independent determinants of peripheral leukocyte 8-OHdG content (R(2) = 0.769; P < 0.001). In conclusion, profound increased 8-OHdG levels in peripheral leukocyte DNA occur in the course of chronic renal failure, gradually increase with its progression, and are further exacerbated by PD treatment.