Ulva compressa Linnaeus (UCL) is a green algae seaweed that performs photosynthesis and is used as a food material in some Asian regions including Korea. It is known to be the dominant species in copper ion-contaminated seas, and many studies on copper ion resistant mechanisms have been reported. UCL is known to have an excellent antioxidant effect, but limited information is available regarding its other physiological activities. In this study, we investigated the anticancer activity of 30% prethanol extracts of Ulva compressa Linnaeus (30% PeUCL) and the underlying mechanisms of its activity on human FaDu hypopharyngeal squamous carcinoma cells. The 30% PeUCL extracts suppressed FaDu cell viability without affecting normal cells (L929), as determined by MTT and viability assays. Furthermore, the 30% PeUCL extracts induced apoptosis, as determined by DAPI staining. The 30% PeUCL extracts inhibited colony formation effectively as well as wound-healing of FaDu cells, even at noncytotoxic concentrations. In addition, 30% PeUCL extracts induced apoptosis significantly through proteolytic cleavage of caspase-3, -7, and -9, and poly (ADP-ribose) polymerase, and by downregulation of Bcl-2 and upregulation of Bax in FaDu cells, as determined by Western blot analysis. Collectively, these results suggest that the inhibitory effect of 30% PeUCL extracts on the growth of oral cancer cells, colony formation and wound-healing may be mediated by caspase- and mitochondrial-dependent apoptotic pathways in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, 30% PeUCL extracts can be administered as a natural chemotherapeutic drug for the treatment of human oral cancers.

Humulus japonicus (HJ) is a widely used herbal medicine for pulmonary tuberculosis, hypertension, leprosy, and venomous wounds in Asia, particularly in China. Although HJ has certain physiological activities, such as longitudinal bone growth, antioxidation and alleviation of rheumatism, its anticancer activities, other than in colorectal and ovarian cancer, are yet to be studied. In this study, we investigated the anti-cancer activity and mechanism of methanol extracts of HJ (MeHJ) against human FaDu hypopharyngeal squamous carcinoma cells. MeHJ suppressed FaDu cell viability without affecting normal cells (L929), which was demonstrated using the MTT and Live & Dead assays. Furthermore, MeHJ effectively inhibited colony formation of FaDu cells, even at non-cytotoxic concentrations, and significantly induced apoptosis through the proteolytic cleavage of caspase-9, -3, -7, poly (ADP-ribose) polymerase and through the downregulation of BCL-2 and upregulation of BAX in FaDu cells, as determined by DAPI staining, flow cytometry, and western blot analyses. Collectively, these findings suggest that the inhibitory effects of MeHJ on the growth and colony formation of oral cancer cells may be mediated by caspase- and mitochondrial-dependent apoptotic pathways in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, MeHJ has the potential to be used as a natural chemotherapeutic drug against human oral cancer.

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.

Demethoxycurcumin (DMC), which is a curcuminoid found in turmeric, has anti-proliferative effects on cancer cells. However, the effect of DMC on osteosarcoma has not been established. The aim of this study was to examine the effects of DMC on cell growth and apoptosis induction in MG-63 human osteosarcoma cells. This study was investigated using 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromid assay, Live/Dead cell assay, 4’, 6-diamidino-2-phenylindole staining, and immunoblotting in MG-63 cells. DMC induced MG-63 cell death in a dosedependent manner, with an estimated IC50 value of 54.4 μM. DMC treatment resulted in nuclear condensation in MG-63 cells. DMC-induced apoptosis in MG-63 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting results showed that Bcl-2 and Bcl-xL were downregulated, while Bax and Bad were upregulated by DMC in MG-63 cells. These results indicated that DMC inhibits cell proliferation and induces apoptotic cell death in MG-63 human osteosarcoma cells via the death receptormediated extrinsic apoptotic pathway and mitochondria-mediated intrinsic apoptotic pathway.

Alpha-lipoic acid (ALA) is a naturally occurring antioxidant and has been previously used to treat diabetes and cardiovascular disease. However, the autophagy effects of ALA against oxidative stress-induced dopaminergic neuronal cell injury remain unclear. The aim of this study was to investigate the role of ALA in autophagy and apoptosis against oxidative stress in the SH-SY5Y human dopaminergic neuronal cell line. We examined SH-SY5Y phenotypes using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (cell viability/proliferation), 4′,6-diamidino-2-phenylindole dihydrochloride nuclear staining, Live/Dead cell assay, cellular reactive oxygen species (ROS) assay, immunoblotting, and immunocytochemistry. Our data showed ALA attenuated hydrogen peroxide (H2O2)-induced ROS generation and cell death. ALA effectively suppressed Bax up-regulation and Bcl-2 and BclxL down-regulation. Furthermore, ALA increased the expression of the antioxidant enzyme, heme oxygenase-1. Moreover, the expression of Beclin-1 and LC-3 autophagy biomarkers was decreased by ALA in our cell model. Combined, these data suggest ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury by inhibiting autophagy and apoptosis.

Acacetin, which is present in damiana (Turnera diffusa ) and black locust (Robinia pseudoacacia ), has several pharmacologic activities such as antioxidant, anti-inflammatory, and anti-proliferative effects on cancer cells. However, the effect of acacetin on head and neck cancers has not been clearly established. This study aimed to examine the effects of acacetin on cell growth and apoptosis induction in FaDu human pharyngeal carcinoma cells. These were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, Live/Dead cell assay, 4′,6-diamidino-2-phenylindole dihydrochloride staining, caspase-3 and caspase-7 activation assay, and immunoblotting in FaDu cells. Acacetin induced FaDu cell death in a dose-dependent manner, with an estimated IC50 value of 41.9 µM, without affecting the viability of L-929 mouse fibroblasts as normal cells. Acacetin treatment resulted in nuclear condensation in the FaDu cells. It promoted the proteolytic cleavage of procaspase-3, -7, -8, and -9 with increasing amounts of the cleaved caspase isoforms in FaDu cells. Acacetin-induced apoptosis in FaDu cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting showed downregulation of the anti-apoptotic mitochondrial proteins Bcl-2 and Bcl-xL, but upregulation of the mitochondria-dependent pro-apoptotic proteins Bax and Badin FaDu cells after acacetin treatment. These findings indicate that acacetin inhibits cell proliferation and induces apoptotic cell death in FaDu human pharyngeal carcinoma cells via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondria-mediated intrinsic apoptotic pathway.

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.

Resveratrol (3,4',5,-trihydroxystilbene), a phytoalexin present in grapes, exerts a variety of actions to reduce superoxides, prevents diabetes mellitus, and inhibits inflammation. Resveratrol acts as a chemo-preventive agent and induces apoptotic cell death in various cancer cells. However, the role of resveratrol in odontoblastic cell differentiation is unclear. In this study, the effect of resveratrol on regulating odontoblast differentiation was examined in MDPC-23 mouse odontoblastic cells derived from mouse dental papilla cells. Resveratrol significantly accelerated mineralization as compared with the control culture in differentiation of MDPC-23 cells. Resveratrol significantly increased expression of ALP mRNA as compared with the control in differentiation of MDPC-23 cells. Resveratrol significantly accelerated expression of ColⅠmRNA as compared with the control in differentiation of MDPC-23 cells. Resveratrol significantly increased expressions of DSPP and DMP-1 mRNAs as compared with the control in differentiation of MDPC-23 cells. Treatment of resveratrol did not significantly affect cell proliferation in MDPC-23 cells. Results suggest resveratrol facilitates odontoblast differentiation and mineralization in differentiation of MDPC-23 cells, and may have potential properties for development and clinical application of dentin regeneration materials.

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.

Ficus carica L. (common fig), one of the first plants cultivated by humans, originated in the Mediterranean basin and currently grows worldwide, including southwest Asia and South Korea. It has been used as a traditional medicine for treatment of metabolic, cardiovascular, and respiratory diseases as well as hemorrhoids and skin infections. Its pharmacological properties have recently been studied in detail, but research on the anti-cancer effect of its latex has been only been studied on a limited basis on several cell lines, such prostate cancer, breast cancer, and leukemia. In this study, we investigated the anti-cancer activity of the latex of Ficus carica L.and its underlying mechanism in FaDu human hypopharynx squamous carcinoma cells. (See Ed. note above) We confirmed through SDS-PAGE analysis and gelatinolytic activity analysis that the latex of Ficus carica contains cysteine protease ficin. Our data showed that the latex inhibited cell growth in a dose-dependent manner. In addition, the latex treatment markedly induced apoptosis in FaDu cells as determined by FACS analysis, elevated expression level of cleaved caspase-9, -3 and PARP (poly (ADP-ribose) polymerase), and. increased the expression of Bax (pro-apoptotic factor) while decreasing the expression of Bcl-2 (anti-apoptotic factor). Taken together, these results suggested that latex containing the ficin inhibited cell growth and induced apoptosis by caspase and the Bcl-2 family signaling pathway in FaDu human hypopharynx squamous carcinoma cells. These findings point to the potential of latex of Ficus carica to provide a novel chemotherapeutic drug due to its growth inhibition effects and induction of apoptosis in human oral cancer cells.

Anthriscus sylvestris (L.) Hoffm. is a perennial herb found widely distributed in various regions of Korea, Europe, and New Zealand. The root of A. sylvestris have been extensively used in the treatment for antitussive, antipyretic, cough remedy in Oriental medicine, but the physiologically active function of the leaf of A. sylvestris is as yet unknown. In this study, we investigated the anti-cancer activity and the mechanism of cell death of water extracts of leaf of Anthriscus sylvestris (WELAS), on human FaDu hypopharyngeal squamous carcinoma cells. Our data showed that WELAS treatment inhibited cell viability in a concentration- and time-dependent manner. In addition, the treatment of WELAS markedly induced apoptosis in FaDu cells, as determined by the viability assay, DAPI stain and FACS analysis. WELAS also increased the proteolytic cleavage of procaspase-3, -9 and PARP (poly(ADP-ribose) polymerase). In addition, exposure to WELAS decreased the expression of Bcl-2 (an anti-apoptotic factor), but increased the expression of Bax (a pro-apoptotic factor), suggesting that mitochondria-dependent apoptotic pathways are mediated in WELAS-induced apoptosis. Taken together, these results indicate that water extracts of leaf of A. sylvestris inhibits cell growth and induces apoptosis via the mitochondrial-dependent apoptotic pathway in FaDu human hypopharyngeal squamous carcinoma cells. Therefore, we propose that the water extracts of leaf of A. sylvestris is a novel chemotherapeutic drug, having growth inhibitory properties and induction of apoptosis in human oral cancer cells.

Metformin (1,1-dimethylbiguanide hydrochloride), derived from French lilac (Galega officinalis), is a first-line anti-diabetic drug prescribed for patients with type 2 diabetes. However, the role of metformin in odontoblastic cell differentiation is still unclear. This study therefore undertook to examine the effect of metformin on regulating odontoblast differentiation in MDPC-23 mouse odontoblastic cells derived from mouse dental papilla cells. As compared to controls, metformin significantly accelerated the mineralization, significantly increased and accelerated the expressions of ALP and Col I mRNAs, and significantly increased the accelerated expressions of DSPP and DMP-1 mRNAs, during differentiation of MDPC-23 cells. There was no alteration in cell proliferation of MDPC-23 cells, on exposure to metformin. These results suggest that the effect of metformin on MDPC-23 mouse odontoblastic cells derived from mouse dental papilla cells, facilitates the odontoblast differentiation and mineralization, without altering the cell proliferation.

Anthricin (Deoxypodophyllotoxin), a naturally occurring flavolignan, has well known anti-cancer properties in several cancer cells, such as prostate cancer, cervical carcinoma and pancreatic cancer. However, the effects of Anthricin are currently unknown in oral cancer. We examined the anticancer effect and mechanism of action of Anthricin in human FaDu hypopharyngeal squamous carcinoma cells. Our data showed that Anthricin inhibits cell viability in a dose- and time-dependent manner (IC50 50 nM) in the MTT assay and Live & Dead assay. In addition, Anthricin treated FaDu cells showed marked apoptosis by DAPI stain and FACS. Furthermore, Anthricin activates anti-apoptotic factors such as caspase-3, -9 and poly (ADP-ribose) polymerase (PARP), suggesting that caspase-mediated pathways are involved in Anthricin- induced apoptosis. Anthricin treatment also leads to accumulation of the pro-apoptotic factor Bax, followed by inhibition of cell growth. Taken together, these results indicate that Anthricn-induced cell death of human FaDu hypopharyngeal squamous carcinoma cells is mediated by mitochondrial-dependent apoptotic pathway. In summary, our findings provide a framework for further exploration on Anthricin as a novel chemotherapeutic drug for human oral cancer.

Tyrosol, a phenylethanoid and a derivative of phenethyl alcohol, possesses various biological properties, such as anti-oxidative and cardioprotective activity. Olive oil is the principal source of tyrosol in the human diet. However, so far the anti-cancer activity of tyrosol has not yet been well defined. This study therefore undertakes to examine the cytotoxic activity and the mechanism of cell death exhibited by tyrosol in KB human oral cancer cells. Treatment of KB cells with tyrosol induced the cell growth inhibition in a concentration- and a time-dependent manner. Furthermore, the treatment of tyrosol induced nuclear condensation and fragmentation of KB cells. Tyrosol also promoted proteolytic cleavage of procaspase-3, -7, -8 and –9, increasing the amounts of cleaved caspase-3, -7, -8 and –9. In addition, tyrosol increased the levels of cleaved PARP in KB cells. These results suggest that tyrosol induces the suppression of cell growth and cell apoptosis in KB human oral cancer cells, and is therefore a potential candidate for anti-cancer drug discovery.

β-carotene is present in carrots, pumpkins, and sweet potatoes. It suppresses many types of cancers by regulating cellular proliferation and apoptosis through a variety of mechanisms. However, the effects of β -carotene on oral cancer cells have not been clearly established. The main goal of this study was to investigate the effects of β-carotene on cell growth and apoptosis in oral cancer cells. Our results demonstrate that treatment with β-carotene induced inhibition of cell growth, and that the effect was dependent on β-carotene treatment time and concentration in KB cells. Furthermore, treatment with β-carotene induced nuclear condensation and fragmentation in KB cells. β-carotene promoted proteolytic cleavage of procaspase-3, -7, -8 and –9 with associated increases in the concentration of cleaved caspase-3, -7, -8 and –9. In addition, the level of cleaved PARP was increased by β-carotene treatment in KB cells. These results suggest that β-carotene can suppress cell growth and induce apoptosis in KB human oral cancer cells, and that it may have potential usefulness in anti-cancer drug discovery efforts.

Curcumin (diferuloylmethane), a constituent of turmeric powder derived from the rhizome of Curcuma longa, has been shown to inhibit the growth of various types of cancer cells by regulating cell proliferation and apoptosis. However, a need exists to design more effective analogs because of curcumin's poor intestinal absorption. EF-24 (diphenyl difluoroketone), the monoketone analog of curcumin, has shown good efficacy in anticancer screens. However, the effects of curcumin and EF-24 on salivary gland epidermoid carcinoma cells are not clearly established. The main goal of this study was to investigate the effects of curcumin and EF-24 on cell growth and induction of apoptosis in human salivary gland epidermoid carcinoma cells. Our studies showed that curcumin and EF-24 inhibited the growth of HTB-41 cells in a dose- and time-dependent manner, and the potency of EF-24 was > 34-fold that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in HTB-41 cells, whereas the control HTB-41 cell nuclei retained their normal regular and oval shape. Curcumin and EF-24 promoted proteolytic cleavages of procaspase-3/-7/-9, resulting in an increase in the amount of cleaved caspase-3/-7/-9 in the HTB-41 cells. Caspase-3 and -7 activities were detected in viable HTB-41 cells treated with curcumin or EF-24. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptosis in HTB-41 human salivary gland epidermoid carcinoma cells, and that they may have potential properties as an anti-cancer drug therapy.