Nypa fruticans Wurmb (NFW) contains a large amount of phenolic acid and flavonoids, and is popular as a superfood in Myanmar. NFW has various biological activities, such as anti-inflammatory, anti-oxidant, and neuroprotective properties; however, the anti-cancer effect of NFW have not been reported. In this study, we investigated the anticancer activity of water extracts of NFW (WeNFW) and the underlying mechanism in human FaDu hypopharyngeal squamous carcinoma cells. The WeNFW inhibited FaDu cell growth in a dose-dependent manner without affecting normal cells (L929), as determined by an MTT assay and Live and Dead assay. In addition, the concentrations of WeNFW without cytotoxicity (0.025, 0.05, and 0.1 mg/mL) inhibited wound healing and colony formation. Furthermore, WeNFW significantly induced apoptosis through the proteolytic cleavage of caspase-3 and -9, poly (ADP-ribose) polymerase, and downregulation of Bcl-2 and upregulation of Bax in FaDu cells, as determined by DAPI staining, FACS analysis, and western blot analysis. Taken together, these results suggest that WeNFW exhibits potent anti-cancer effects by suppressing the growth of oral cancer cells, wound healing and colony formation activity. Via mitrochondrial-dependent apoptotic pathways in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, WeNFW can provide a natural chemotherapeutic drug for oral cancer in humans.

Asarum sieboldii Miq. (Aristolochiaceae) is a perennial herbaceous plant and has been used as traditional medicine for treating diseases, cold, fever, phlegm, allergies, chronic gastritis, and acute toothaches. Also, it has various biological activities, such as antiallergic, antiinflammatory, antinociceptive, and antifungal. However, the anticancer effect of A. sieboldii have been rarely reported, except anticancer effect on lung cancer cell (A549) of water extracts of A. sieboldii . This study investigated the anticancer activity of methanol extracts of A. sieboldii (MeAS) and the underlying mechanism in human FaDu hypopharyngeal squamous carcinoma cells. MeAS inhibited FaDu cells grown dose-dependently without affecting normal cells (L929), as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide and live and dead assay. In addition, concentration of MeAS without cytotoxicity (0.05 and 0.1 mg/mL) inhibited migration and colony formation. Moreover, MeAS treatment significantly induced apoptosis through the proteolytic cleavage of caspase-3, -7, -9, poly (ADP-ribose) polymerase, and downregulation of Bcl-2 and upregulation of Bax in FaDu cells, as determined by fluorescence-activated cell sorting analysis, 4`6-diamidino- 2-phenylindole stain, and western blotting. Altogether, these results suggest that MeAS exhibits strong anticancer effects by suppressing the growth of oral cancer cells and the migration and colony formation via caspase- and mitochondrial-dependent apoptotic pathways in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, MeAS can serve as a natural chemotherapeutic for human oral cancer.

Ficus carica L. (fig ) is one of the first cultivated crops and is as old as humans. This plant has been extensively used as a traditional medicine for treating diseases, such as cough, indigestion, nutritional anemia, and tuberculosis. However, the physiological activity of fig leaves on oral cancer is as yet unknown. In this study, we investigated the anticancer effect of methanol extracts of Ficus carica (MeFC) and the mechanism of cell death in human FaDu hypopharyngeal squamous carcinoma cells. MeFC decreased the viability of oral cancer (FaDu) cells but did not affect the viability of normal (L929) cells, as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and Live and Dead assay. In addition, MeFC induced apoptosis through the proteolytic cleavage of procaspase-3, -9, poly (ADP-ribose) polymerase (PARP), downregulation of Bcl-2, and upregulation of Bax, as determined by 4′,6-diamidino-2-phenylindole dihydrochloride staining and western blot analysis. Moreover, a concentration of MeFC without cytotoxicity (0.25 mg/mL) significantly suppressed colony formation, a hallmark of cancer development, and completely inhibited the colony formation at 1 mg/mL. Collectively, these results suggest that MeFC exhibits a potent anticancer effect by suppressing the growth of oral cancer cells and colony formation via caspase- and mitochondrial-dependent apoptotic pathways in FaDu human hypopharyngeal squamous carcinoma cells. Therefore, the methanol extract of Ficus carcica leaves provide a natural chemotherapeutic drug for human oral cancer.

Trifolium pratense leaves (red clover) has been used in Oriental and European folk medicine for the treatment of whooping cough, asthma, and eczema, and is now being used to treat and alleviate the symptoms, such as hot flushes, cardiovascular health effects that occur in postmenopausal women. However, relatively little scientific data is available on the physiological activity of this plant. Therefore, in this study, we investigated the anti-cancer activity of T. pratense leaves using methanol extract of T. pratense leaves (MeTP) on human FaDu hypopharyngeal squamous carcinoma cells. MeTP inhibited the viability of FaDu cells by inducing apoptosis through the cleavage of procaspase- 3, -7, and -9 and poly (adenosine diphosphate ribose-ribose) polymerase (PARP), downregulation of Bcl- 2, and upregulation of Bax, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Live & dead assay, 4’6-diamidino-2-phenylindole stain, fluorescence-activated cell sorting analysis, and Western blot analysis. In addition, colony formation was slightly inhibited when FaDu cells were treated with a non-cytotoxic concentration (0.125 mg/mL) of MeTP and almost completely inhibited when cells were treated with 0.25 mg/mL MeTP. Collectively, these results indicate that MeTP induced cell apoptosis via caspase- and mitochondrial-dependent apoptotic pathways, and inhibited colony formation of cancer cells in FaDu human hypopharyngeal squamous carcinoma cells. These findings suggest MeTP should be considered for clinical development as a chemotherapeutic option in oral cancer.

Codium fragile (Suringar) Hariot is an edible green seaweed that belong to the Codiaceae family and has been used in Oriental medicine for the treatment of enterobiasis, dropsy, and dysuria. Methanol extract of codium fragile has anti-oxidant, anti-inflammatory and anti-cancer properties, although the anti-cancer effect on oral cancer has not yet been reported. In this study, we investigated the anti-cancer activity and the mechanism of cell death by methanol extracts of Codium fragile (MeCF) on human FaDu hypopharyngeal squamous carcinoma cells. Our data showed that MeCF inhibits cell viability in a dose-dependent manner, and markedly induced apoptosis, as determined by the MTT assay, Live/Dead assay, and DAPI stain. In addition, MeCF induced the proteolytic cleavage of procaspase -3, -7, -9 and poly(ADP-ribose) polymerase(PARP), and upregulated or downregulated the expression of mitochondrial-apoptosis factor, Bax(pro-apoptotic factor), and Bcl-2(anti-apoptotic factor), . Futhermore, MeCF induced a cell cycle arrest at the G1/S phase through suppressing the expression of the cell cycle cascade proteins, p21, CDK4, CyclinD1, and phospho-Rb. Taken together, these results indicated that MeCF inhibits cell growth, and this inhibition is mediated by caspase- and mitochondrial-dependent apoptotic pathways through cell cycle arrest at the G1/S phase in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, methanol extracts of Codium fragile can be provided as a novel chemotherapeutic drug due to its growth inhibition effects and induction of apoptosis in human oral cancer cells.