In addition, DNA laddering and FITC-annexin V/PI assays were carried out to determine the cell death mode induced by DAM and NDAM
In addition, DNA laddering and FITC-annexin V/PI assays were carried out to determine the cell death mode induced by DAM and NDAM. DAM and NDAM was detected using mitochondrial membrane potential, Cytochrome c, and caspases assays. Finally, the effect of DAM and NDAM on cell cycle phase distribution of OSCC cells was detected by circulation cytometry. In the present study, DAM and NDAM showed cytotoxicity towards OSCC cell lines and the maximum growth inhibition for both compounds was observed in H400 cells with IC50 value of 1 1.9 and 6.8?g/ml, respectively, after 72?h treatment. The results also exhibited the inhibition of H400 OSCC cells proliferation, internucleosomal cleavage of DNA, activation of intrinsic apoptosis pathway, and cell cycle arrest caused by DAM and NDAM. Therefore, these findings suggest that DAM and NDAM can be potentially used as antitumor brokers for oral malignancy therapy. L., commonly known as noni, belongs to the Rubiaceae family. It is native to the Pacific islands, Hawaii, Caribbean, Asia and Australia. Damnacanthal (DAM) and nordamnacanthal (NDAM) are a part of a general class of athraquinone derivatives which are isolated from species. Both DAM and NDAM incorporate some unique chemical and biological characteristics (Alitheen et al. 2010). DAM displayed cytotoxic activity against breast malignancy cell lines along with small cell lung malignancy cell lines (Kanokmedhakul et al. 2005). In addition, it was documented that DAM isolated from the root of noni acted as an inhibitor associated with ras function, which is considered to be linked to the transmission transduction in various human cancers including colon, lungs and leukaemia (Hiramatsu et al. 1993). NDAM has also featured many biological properties, which include antioxidant activities, cytotoxic properties and anti-cancer effects on human B-lymphoblastoid cell lines (Jasril et al. 2003). Apoptosis, or programmed cell death, is usually a sophisticated and highly intricate mechanism which consists of two unique pathways; intrinsic (mitochondrial) and extrinsic (death receptor) (Elmore 2007). Mitochondria perform crucial functions in apoptotic cell death and it is becoming one of the important targets in screening treatment brokers against malignancy (Kumar et al. 2009). The objectives of this study were to evaluate the anti-proliferative or DCPLA-ME cytotoxic activity and induction DCPLA-ME of apoptosis capability of DAM and NDAM on the most common type of oral cancer, oral squamous cell carcinoma (OSCC) cells. To achieve these objectives numerous assays were carried out. MTT assay was performed to detect the cytotoxicity or cell growth inhibition effect of DAM and NDAM. In addition, DNA laddering and FITC-annexin V/PI assays were carried out to determine the cell death mode induced by DAM and NDAM. Moreover, the molecular mechanism of apoptosis induced by DAM and NDAM against OSCC cell lines was decided using mitochondrial membrane potential, Cytochrome c and caspases assays. Furthermore, cell cycle analysis was performed to investigate the effect of DAM and NDAM on cell cycle phase distribution of OSCC cells. Materials and methods Damnacanthal and nordamnacanthal The damnacanthal and nordamnacanthal (Fig.?1) were kindly supplied by Prof. Dr. Nor Hadiani Ismail from Universiti Teknologi MARA (UiTM, Shah Alam Selangor, Malaysia) were isolated from your roots of (Ismail et al. 1997). The compounds in powdered-form were dissolved in dimethylsulphoxide (DMSO) (Vivantis Technologies Sdn. Bhd, Subang Jaya, Malaysia) to get a stock answer of 10?mg/mL, which was then stored at ?20?C in aliquots for future use. Open in a separate windows Fig.?1 The molecular structure of Damnacanthal (a) and Nordamnacanthal (b) Cell lines and culture conditions The human oral squamous cell carcinoma cell lines used in this study, H103, H400, H413, H357, H376 and H314, were kindly provided by Professor. Dr. Ian Charles Paterson (University or college of Malaya, Kuala Lumpur, Malaysia) (Table?1). OSCC cell lines were routinely cultured in DMEM/Hams F-12 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10?% foetal bovine serum (J R Scientific, Inc., Woodland, CA, USA), 100 Models/mL penicillin and 100 g/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37?C in a humidified atmosphere of 5?% CO2. In the current study, 3T3 (normal mouse fibroblast) (ATCC, Manassas, VA, USA) cells were used as normal cell line. Growth and morphology of the cells were regularly monitored and the culture medium was renewed 2C3 occasions weekly. Table?1 Human OSCC cell lines and the sites from which the cell lines have been derived test. One-way analysis of variance (ANOVA) was also utilized for multiple comparisons, where 1?kb DNA marker, control cells, Treatment with IC50 concentration for 24?h, Treatment with IC75 concentration for 24?h, Treatment with IC50 concentration Tmeff2 for 48?h, Treatment with IC75 concentration for 48?h, Treatment with IC50 concentration for 72?h, Treatment with IC75 concentration for 72?h Circulation cytometric analysis of apoptosis by FITC-annexin V/PI To confirm apoptosis induced by DAM DCPLA-ME and NDAM, the phosphatidylserine (PS) externalisation, which was an early event in the apoptosis pathway, was measured using annexin V-FITC and propidium iodide double staining. Treatment of H400 OSCC cells at.