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      Antiproliferation and Induction of Apoptosis in Ca9-22 Oral Cancer Cells by Ethanolic Extract of Gracilaria tenuistipitata

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          Abstract

          The water extract of Gracilaria tenuistipitata have been found to be protective against oxidative stress-induced cellular DNA damage, but the biological function of the ethanolic extracts of G. tenuistipitata (EEGT) is still unknown. In this study, the effect of EEGT on oral squamous cell cancer (OSCC) Ca9-22 cell line was examined in terms of the cell proliferation and oxidative stress responses. The cell viability of EEGT-treated OSCC cells was significantly reduced in a dose-response manner ( p < 0.0001). The annexin V intensity and pan-caspase activity of EEGT-treated OSCC cells were significantly increased in a dose-response manner ( p < 0.05 to 0.0001). EEGT significantly increased the reactive oxygen species (ROS) level ( p < 0.0001) and decreased the glutathione (GSH) level ( p < 0.01) in a dose-response manner. The mitochondrial membrane potential (MMP) of EEGT-treated OSCC cells was significantly decreased in a dose-response manner ( p < 0.005). In conclusion, we have demonstrated that EEGT induced the growth inhibition and apoptosis of OSCC cells, which was accompanied by ROS increase, GSH depletion, caspase activation, and mitochondrial depolarization. Therefore, EEGT may have potent antitumor effect against oral cancer cells.

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          Most cited references36

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          Role of reactive oxygen species (ROS) in apoptosis induction.

          Reactive oxygen species (ROS) and mitochondria play an important role in apoptosis induction under both physiologic and pathologic conditions. Interestingly, mitochondria are both source and target of ROS. Cytochrome c release from mitochondria, that triggers caspase activation, appears to be largely mediated by direct or indirect ROS action. On the other hand, ROS have also anti-apoptotic effects. This review focuses on the role of ROS in the regulation of apoptosis, especially in inflammatory cells.
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            Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader.

            Oxidative stress (OS) has been implicated in various degenerative diseases in aging. In an attempt to quantify OS in a cell model, we examined OS induced by incubating for 30 min with various free radical generators in PC12 cells by using the dichlorofluorescein (DCF) assay, modified for use by a fluorescent microplate reader. The nonfluorescent fluorescin derivatives (dichlorofluorescin, DCFH), after being oxidized by various oxidants, will become DCF and emit fluorescence. By quantifying the fluorescence, we were able to quantify the OS. Our results indicated that the fluorescence varied linearly with increasing concentrations (between 0.1 and 1 mM) of H2O2 and 2,2'-azobios(2-amidinopropane) dihydrochloride (AAPH; a peroxyl radical generator). By contrast, the fluorescence varied as a nonlinear response to increasing concentrations of 3-morpholinosydnonimine hydrochloride (SIN-1; a peroxynitrite generator), sodium nitroprusside (SNP; a nitric oxide generator), and dopamine. Dopamine had a biphasic effect; it decreased the DCF fluorescence, thus acting as an antioxidant, at concentrations <500 microM in cells, but acted as a pro-oxidant by increasing the fluorescence at 1 mM. While SNP was not a strong pro-oxidant, SIN-1 was the most potent pro-oxidant among those tested, inducing a 70 times increase of fluorescence at a concentration of 100 microM compared with control. Collectively, due to its indiscriminate nature to various free radicals, DCF can be very useful in quantifying overall OS in cells, especially when used in conjunction with a fluorescent microplate reader. This method is reliable and efficient for evaluating the potency of pro-oxidants and can be used to evaluate the efficacy of antioxidants against OS in cells.
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              Oral squamous cell carcinoma overview.

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                Author and article information

                Journal
                Molecules
                Molecules
                molecules
                Molecules
                MDPI
                1420-3049
                11 September 2012
                September 2012
                : 17
                : 9
                : 10916-10927
                Affiliations
                [1 ]Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
                [2 ]Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan
                [3 ]Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung 811, Taiwan
                [4 ]Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
                [5 ]Department of Radiation Oncology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
                [6 ]Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan
                [7 ]Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan
                Author notes
                [* ] Authors to whom correspondence should be addressed; Email: aaronfrc@ 123456kmu.edu.tw (F.-R.C.) changhw@ 123456kmu.edu.tw (H.-W.C.); Tel.: +886-7-312-1101 (ext. 2691) (H.-W.C.); Fax: +886-7-312-5339 (H.-W.C.).
                Article
                molecules-17-10916
                10.3390/molecules170910916
                6269058
                22968475
                7f2a6679-510d-4f37-968b-5e2f0be60823
                © 2012 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                : 20 August 2012
                : 06 September 2012
                : 07 September 2012
                Categories
                Article

                oral cancer,apoptosis,ros,glutathione,mitochondrial membrane potential,marine natural product

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