• International Journal of Oral Biology
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• v.47 no.1
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• pp.9-15
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• 2022

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.

• International Journal of Oral Biology
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• v.47 no.3
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• pp.41-48
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• 2022

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.

• International Journal of Oral Biology
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• v.45 no.3
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• pp.107-114
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• 2020

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.

• International Journal of Oral Biology
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• v.46 no.2
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• pp.85-93
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• 2021

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.

• International Journal of Oral Biology
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• v.46 no.4
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• pp.160-167
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• 2021

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.

• International Journal of Oral Biology
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• v.45 no.1
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• pp.8-14
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• 2020

Bilobalide isolated from the leaves of Ginkgo biloba has several pharmacological activities such as neuroprotective, anti-inflammatory, and anticonvulsant. However, the effect of bilobalide on cancer has not been clearly established. The main purpose of this study was to investigate the effect of bilobalide on cell growth and apoptosis induction in FaDu human pharyngeal squamous cell carcinoma. This was examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, nuclear 4′,6-diamidino-2-phenylindole dihydrochloride staining, DNA fragmentation analysis, and immunoblotting. Bilobalide inhibited the growth of FaDu cells in dose- and time-dependent manners. Treatment with bilobalide resulted in nuclear condensation and DNA fragmentation in FaDu cells. Furthermore, it promoted the proteolytic cleavage of procaspase-3/-7/-8/-9 with increase in the amount of cleaved caspase-3/-7/-8/-9. Bilobalide-induced apoptosis in FaDu cells was mediated by the expression of Fas and the activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting revealed that the antiapoptotic mitochondrial protein Bcl-2 was downregulated, but the proapoptotic protein Bax was upregulated by bilobalide in FaDu cells. Bilobalide significantly increased Bax/Bcl-2 ratio. These results suggest that bilobalide inhibits cell proliferation and induces apoptosis in FaDu human pharyngeal squamous cell carcinoma via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondrial-mediated intrinsic apoptotic pathway.

• International Journal of Oral Biology
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• v.44 no.2
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• pp.43-49
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• 2019

Tannic acid (TA) is a water-soluble polyphenol compound found in various herbal plants. We investigated the chemopreventive effects of TA on FaDu hypopharyngeal squamous carcinoma cells. In an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, TA showed dose-dependent cytotoxicity with a half maximal inhibitory concentration (IC50) of 50 ?M. Cell cycle analysis and immunofluorescence imaging demonstrated that under low-dose ($25{\mu}M$) treatment, FaDu cells were arrested in G2/M phase, and as the dose of TA was increased, apoptosis was induced with the increase of cell population at sub-G1 phase. The expressions of various cyclins, including cyclin D1 and cyclin-dependent kinases (CDK-1 and CDK-2), were down-regulated at low doses of TA, whereas apoptotic effectors such as cleaved caspase 3, cleaved caspase 7, and poly (ADP-ribose) polymerase (PARP) were expressed in a dose-dependent manner in Western blotting. In addition, TA-induced apoptosis of FaDu cells might be mediated by the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway, with the upregulation of p-AKT/p-PKB (phosphorylated protein kinase B) and p-ERK. Overall, our data support the hypothesis that TA is a potential candidate agent for the treatment of hypopharyngeal cancer.

• International Journal of Oral Biology
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• v.35 no.4
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• pp.153-158
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• 2010

Angelica decursiva has been used in Korean traditional medicine as an antitussive, an analgesic, an antipyretic and a cough remedy. However, the anti-cancer properties of Angelica decursiva have not yet been well defined. In our current study the cytotoxic activity of ethanol extracts of Angelica decursiva root (EEAD) and the mechanism of cell death exhibited by EEAD were examined in FaDu human head and neck squamous cell carcinoma cells. The cytotoxic effects of EEAD upon the growth of FaDu cells were examined with an MTT assay. In addition, the mechanism of cell death induced by EEAD was evaluated by DNA fragmentation analysis, immunoblotting and caspase activation measurements. EEAD induced apoptotic cell death in FaDu cells in a concentration- and time-dependent manner, as determined by MTT assay and DNA fragmentation analysis. Furthermore, the proteolytic processing of caspase-3, -7 and -9 was increased by EEAD treatment of FaDu cells. In addition, the activation of caspase-3 and -7 was detected in living FaDu cells by fluorescence microscopy. These results suggest that EEAD can induce apoptosis and suppress cell growth in cancer cells and may have utility as a future anticancer therapy.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.6
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• pp.447-456
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• 2022

The present study was carried out to investigate the effect of Arctigenin on cell growth and the mechanism of cell death elicited by Arctigenin were examined in FaDu human pharyngeal carcinoma cells. To determine the apoptotic activity of Arctigenin in FaDu human pharyngeal carcinoma cells, cell viability assay, DAPI staining, caspase activation analysis, and immunoblotting were performed. Arctigenin inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Arctigenin-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was up-regulated by Arctigenin treatment. Moreover, caspase-8, a part of the extrinsic apoptotic pathway, was activated by Arctigenin treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria-dependent intrinsic apoptosis pathway, significantly decreased following Arctigenin treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9, and tumor suppressor -53 increased by Arctigenin treatments. In addition, Arctigenin activated caspase-3 and poly (ADP-ribose) polymerase (PARP) induced cell death. Arctigenin also inhibited the proliferation of FaDu cells by the suppression of p38, NF-κB, and Akt signaling pathways. These results suggest that Arctigenin may inhibit cell proliferation and induce apoptotic cell death in FaDu human pharyngeal carcinoma cells through both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway.

• International Journal of Oral Biology
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• v.44 no.3
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• pp.81-88
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• 2019

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.