감귤폐기물에 의한 인체 흑색종 세포사멸 및 알킬아닐린 유발독성 억제효과

Abstract

Inducible nitric oxide synthase (iNOS) is associated with disease progression in human malignant melanoma. We therefore tested the hypothesis that targeted iNOS inhibition would interfere with cell growth and thereby induce apoptosis in human malignant melanoma cells. Release of nitric oxide (NO•) induced by iNOS expression was inhibited by a specific iNOS inhibitor, N-[(3-aminomethyl) benzyl]acetamidine (1400W), and/or an NO• scavenger carboxy PTIO in A375 (p53 wildtype) and SK mel-28 (p53 mutant) cell lines, and subsequently led to p53-dependent apoptosis via sub-G1 cell cycle arrest, as indicated by Annexin V/propidium iodide and DNA fragmentation assays. Human malignant melanoma cell apoptosis induced by iNOS inhibition was blocked by an exogenous NO• donor, sodium nitroprusside. Comparison of control and 1400W and/or carboxy-PTIO-treated human malignant melanoma cells by quantitative RT-PCR analysis revealed that inhibition of NO• synthesis modulated levels of inhibitors of apoptosis family (XIAP, cIAP-1 and -2, and survivin). Collectively, these data show that endogenous NO• accelerates cancer cell proliferation activity, and iNOS expression may have predictive value for tumor progression in human malignant melanoma cells. Citrus wastes such as premature fruit drop and citrus juice processing waste material cause many economic and environmental problems. Citrus wastes were evaluated for their bioactive compounds and antioxidant capacity. The contents of total phenolic and flavonoid pigments were measured using colorimetric method. In addition, free radicals (DPPH, O2-, H2O2 and NO•) scavenging assays were used to determine the antioxidant capacity. The highest contents of total phenolic and flavonoid pigments were found in citrus wastes. These contents were more prevalent in premature fruit drops than the level found in mature fruits. 2 All citrus wastes possess an evident antioxidant capacity. The results showed that citrus wastes could be economic and readily accessible source of natural antioxidants and as a possible pharmaceutical supplement. We investigated the role of nitric oxide (NO•) and influence of p53 status during apoptosis induced by these agents in A375 (p53 wildtype) and SK mel-28 (p53 mutant) cells. Citrus wastes inhibited cell growth and promoted apoptosis by the elevation of NO•; treatment with citrus juice processing waste in p53 wildtype cell was most effective. Increased NO• production after treatment with citrus juice processing waste modulated levels of gene products involved in the apoptosis pathway such as Bax, DR4, Bcl-2, PARP, caspase-3, and inhibitors of apoptosis family (XIAP, cIAP-1 and -2, and survivin), dependently of p53 status. These findings support the hypothesis that citrus wastes are effective apoptosis-promoting agent in human malignant melanoma cells and indicate that NO• could be considered a potential target for improvement of the effectiveness of human malignant melanoma treatment. In this work the influence of citrus wastes on nitric oxide (NO•) level and on the apoptotic processes in human malignant melanoma melanin-free A375 and melanin-producing SK mel-100 cells were investigated. Citrus wastes inhibited cell growth and promoted apoptosis by the elevation of NO•; treatment with citrus juice processing waste in melanin-free cell was most effective. Gene product levels of inhibitors of apoptosis family were depressed, but increases in p53, PTEN, caspase-3 and PARP occurred, dependently of melanin status. The data suggests that citrus wastes may be beneficial in combating human malignant melanoma and nitric oxide and melanin can be considered as a target for human malignant melanoma chemotherapy. Exposure to monocyclic aromatic alkylanilines namely o-toluidine, m-toluidine, 2 ethylaniline and 3-ethylaniline, was significantly and independently associated with bladder cancerincidence. Chinese hamster ovary AS52 cells were exposed to o-toluidine, m-toluidine, 3 2-ethylaniline and 3-ethylaniline for 6 h in the presence and absence of a metabolic activator (human S9 mix). Cell survival was determined by trypan blue exclusion 24 h after treatment, and 6-thioguanine-resistant mutants at the xanthine-guanine phosphoribosyltransferase (gpt) gene locus were assessed. Chinese hamster ovary AS52 cells exhibited a dose-dependent increase in cytotoxicity, mutant fraction and nitrite production on treatment of o-toluidine, m toluidine, 2-ethylaniline and 3-ethylaniline. Supplementation with immature citrus extract reduced toxicity induced by o-toluidine, m-toluidine, 2-ethylaniline and 3-ethylaniline. Immature citrus extract also provided protection in oxidant status of o-toluidine, m-toluidine, 2 ethylaniline and 3-ethylaniline-treated cells. Our results indicate the importance of immature citrus extract as a public health concern and its importance with respect to the high probability of alkylaniline exposures and their toxic effects.