Shun-Fa Yang

Cyanidin 3-Glucoside and Peonidin 3-Glucoside Inhibit Tumor Cell Growth and Induce Apoptosis In Vitro and Suppress Tumor Growth In Vivo

Abstract: Dietary polyphenols, including anthocyanins, are suggested to be involved in the protective effects of fruits and vegetables against cancer. However, anticancer effects of peonidin 3-glucoside have not been clearly demonstrated, with only limited studies being available concerning the inhibitory effect of cyanidin 3-glucoside for tumor cell growth. Therefore, in this study, we have isolated and identified the two bioactive compounds, peonidin 3-glucoside and cyanidin 3-glucoside, from Oryza sativa L. indica, to treat various cancer cells. The results showed that, among analyzed cell lines, HS578T was the most sensitive to peonidin 3-glucoside and cyanidin 3-glucoside. Treatment with peonidin 3-glucoside or cyanidin 3-glucoside resulted in a strong inhibitory effect on cell growth via G2/M arrest. Regarding cell cycle–related proteins, peonidin 3-glucoside treatment resulted in down-regulation of protein levels of cyclin-dependent kinase (CDK)-1, CDK-2, cyclin B1, and cyclin E, whereas cyanidin 3-glucoside could decrease the protein levels of CDK-1, CDK-2, cyclin B1, and cyclin D1. In addition, cyanidin 3-glucoside or peonidin 3-glucoside also induced caspase-3 activation, chromatin condensation, and cell death. Furthermore, anthocyanins from O. sativa L. indica were evidenced by their inhibition on the growth of Lewis lung carcinoma cells in vivo.

Silibinin inhibits the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2†

Cancer metastasis, involving multiple processes and various cytophysiological changes, is a primary cause of cancer death and may complicate the clinical management, even lead to death. Silibinin is a flavonoid antioxidant and wildly used for its antihepatotoxic properties and recent studies have revealed pleiotropic anticancer and antiproliferative capabilities of silibinin. In this study, we first observed that silibinin exerted a dose‐ and time‐dependent inhibitory effect on the invasion and motility, but hardly on the adhesion, of highly metastatic A549 cells in the absence of cytotoxicity. To look at the precise involvement of silibinin in cancer metastasis, A549 cells were treated with silibinin at various concentrations, up to 100 μM, for a defined period and then subjected to gelatin zymography, casein zymography and Western blot to investigate the impacts of silibinin on metalloproteinase‐2 (MMP‐2), urokinase plasminogen activator (u‐PA), and tissue inhibitor of metalloproteinase‐2 (TIMP‐2), respectively. The results showed that a silibinin treatment may decrease the expressions of MMP‐2 and u‐PA in a concentration‐ and time‐dependent manner and enhance the expression of TIMP‐2. Further analysis with semi‐quantitative RT‐PCR showed that silibinin may regulate the expressions of MMP‐2 and u‐PA on the transcriptional level while on the translational or post‐translational level for TIMP‐2.

Silibinin Inhibits Invasion of Oral Cancer Cells by Suppressing the MAPK Pathway

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity. Here, we provide molecular evidence associated with the anti-metastatic effect of silibinin by showing a marked inhibition of the invasion and motility of SCC-4 tongue cancer cells, with 89% and 66.4% of inhibition, respectively, by 100 μM of silibinin. This effect was associated with a reduced expression of MMP-2 and u-PA, together with an enhanced expression of TIMP-2 and PAI-1. Silibinin also exerted an inhibitory effect on the phosphorylation of ERK1/2. Additionally, pre-treatment of SCC-4 cancer cells with 10 and 20 μM of U0126, a specific MEK inhibitor, resulted in a reduced expression of MMP-2 (18.7 and 51.4%) and u-PA (19.2 and 48.9%) concomitantly with a marked inhibition of cell invasion (13.7 and 45.7%). Finally, silibinin was evidenced by its inhibition of the metastasis of Lewis lung carcinoma (LLC) cells in vivo. These results suggested that silibinin can reduce the invasion and metastasis of tumor cells, and such a characteristic may be of great value in the development of a potential cancer therapy.

Increased tissue inhibitor of metalloproteinase-1 expression and inhibition of gelatinase A activity in buccal mucosal fibroblasts by arecoline as possible mechanisms for oral submucous fibrosis

Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). To date, there has been little research about the role of tissue inhibitors of metalloproteinase (TIMPs) and matrix metalloproteinases (MMPs) in the pathogenesis of OSF. In the present study, we examined the activity of TIMPs from cells cultured from OSF and normal buccal mucosa. OSF specimens were found to have higher TIMP-1 expression than normal buccal mucosal fibroblasts (BMFs) by Western blots. To verify whether arecoline, a major areca nut alkaloid, could affect TIMP or MMP production by human BMFs, Western blots and gelatine zymography were used. Arecoline was found to elevate TIMP-1 expression at the concentration level under 20 microg/ml in a dose-dependent manner. The amount of TIMP-1 was about 2.7 fold at a concentration level of 10 microg/ml compared with control. From gelatin zymograms, the main gelatinolytic proteinase secreted by the human BMFs was MMP-2, and only minimal amounts of MMP-9 could be detectable from zymogram. In addition, arecoline was found to inhibit MMP-2 secretion and production at the concentration level of 40 microg/ml. The gelatinolytic activity of MMP-2 was about 54% at a concentration level of 80 microg/ml compared with control. Taken together, it was found that arecoline acted not only as an inhibitor on gelatinolytic activity of MMP-2, but also a stimulator for TIMP-1 activity. These synergistic effects may contribute to the ECM components accumulation in the areca quid associated OSF.

Platelet-rich Fibrin Increases Proliferation and Differentiation of Human Dental Pulp Cells

INTRODUCTION Platelet-rich fibrin (PRF) by Choukrouns technique is derived from an autogenous preparation of concentrated platelets without any manipulation. When delicately pressed between 2 gauzes, the PRF clot becomes a strong membrane with high potential in clinical application. However, the effect of PRF on dental pulp cells (DPCs) remains to be elucidated. This study was to determine the biological effects of PRF on DPCs. METHODS PRF samples were obtained from 6 healthy volunteers. Human DPCs were derived from healthy individuals undergoing extraction for third molars. Cell proliferation resulting from PRF was evaluated by colorimetric assay. Western blot was used to evaluate the expression of osteoprotegerin (OPG). Alkaline phosphatase (ALP) activity was examined by substrate assay. RESULTS PRF did not interfere with cell viability of DPCs (P > .05). DPCs were observed to attach at the edges of PRF by phase-contrast microscopy. PRF was found to increase DPC proliferation during 5-day incubation period (P < .05). PRF was found to increase OPG expression in a time-dependent manner (P < .05). ALP activity was also significantly up-regulated by PRF (P < .05). CONCLUSIONS PRF was demonstrated to stimulate cell proliferation and differentiation of DPCs by up-regulating OPG and ALP expression. These findings might serve as a basis for preclinical studies that address the role of PRF in reparative dentin formation.

Antimetastatic effects of Terminalia catappa L. on oral cancer via a down-regulation of metastasis-associated proteases

The incidence and mortality of oral cancer in Taiwan have been increased during the last decade, which could be mainly resulted from the difficulty in treatment related to metastasis. As a potential and popular folk medicine, Terminalia catappa leaves have been proven to possess various biological benefits including anti-cancer activities. However, the detailed effects and molecular mechanisms of T. catappa leaves on the metastasis of oral cancer cells were still unclear. Thus, SCC-4 oral cancer cells were subjected to a treatment with ethanol extracts of T. catappa leaves (TCE) and then analyzed for the effect of TCE on the migration and invasion. Modified Boyden chamber assays revealed that TCE treatment significantly inhibited the cell migration/invasion capacities of SCC-4 cells. Furthermore, results of zymography and western blotting showed that activities and protein levels of MMP-2, MMP-9 and u-PA were all inhibited by TCE. Further studies indicated that TCE may inhibit phosphorylation of ERK1/2, JNK1/2 and Akt while the expression of nuclear protein NF-kappaB, c-Jun and c-Fos were inhibited as well. EMSA assay revealed that the DNA-binding activity with AP-1 and NF-kappaB was also decreased by TCE. In conclusion, TCE may serve as a powerful chemopreventive agent against oral cancer metastasis.

Antimetastatic effects of norcantharidin on hepatocellular carcinoma by transcriptional inhibition of MMP-9 through modulation of NF-Kb activity

Background The rate of morbidity and mortality of hepatocellular carcinoma (HCC) in Taiwan has not lessened because of difficulty in treating tumor metastasis. Norcantharidin (NCTD) is currently used as an anticancer drug for hepatoma, breast cancer, and colorectal adenocarcinoma. NCTD possesses various biological anticancer activities, including apoptosis. However, detailed effects and molecular mechanisms of NCTD on metastasis are unclear. Thus, HCC cells were subjected to treatment with NCTD and then analyzed to determine the effects of NCTD on cell metastasis. Methodology/Principal Findings Modified Boyden chamber assays revealed that NCTD treatment inhibited cell migration and invasion capacities of HCC cells substantially. Results of zymography and western blotting showed that activities and protein levels of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (u-PA) were inhibited by NCTD. Western blot analysis showed that NCTD inhibits phosphorylation of ERK1/2. Testing of mRNA level, quantitative real-time PCR, and promoter assays evaluated the inhibitory effects of NCTD on MMP-9 and u-PA expression in HCC cells. The chromatin immunoprecipitation (ChIP) assay for analyzing the genomic DNA sequences bound to these proteins was reactive to the transcription protein nuclear factor (NF)-kappaB, which was inhibited by NCTD. The expression of NF-kappa B was measured by western blot analysis, which revealed decreased nuclear-factor DNA-binding activity after NCTD treatment. Conclusions NCTD inhibited MMP-9 and u-PA expression through the phosphorylation of ERK1/2 and NF-kappaB signaling pathway which serves as a powerful chemopreventive agent in HCC cell metastasis.

Fisetin induces apoptosis in human cervical cancer HeLa cells through ERK1/2-mediated activation of caspase-8-/caspase-3-dependent pathway

Fisetin is a naturally occurring flavonoid that has been reported to inhibit the proliferation and to induce apoptotic cell death in several tumor cells. However, the apoptosis-inducing effect of fisetin on tumor cell lines was investigated besides HeLa cells. In this study, we found that fisetin induced apoptosis of HeLa cells in a dose- and time-dependent manner, as evidenced by nuclear staining of 4′-6-Diamidino-2-phenylindole (DAPI), flow cytometry assay, and Annexin-V/PI double-labeling. In addition, fisetin triggered the activations of caspases-3 and -8 and the cleavages of poly (ADP-ribose) polymerase, resulting in apoptosis induction. Moreover, treatment of HeLa cells with fisetin induced a sustained activation of the phosphorylation of ERK1/2, and inhibition of ERK1/2 by PD98059 (MEK1/2 inhibitor) or transfection with the mutant ERK1/2 expression vector significantly abolished the fisetin-induced apoptosis through the activation of caspase-8/-3 pathway. The in vivo xenograft mice experiments revealed that fisetin significantly reduced tumor growth in mice with HeLa tumor xenografts. In conclusion, our results indicated that fisetin exhibited anti-cancer effect and induced apoptosis in HeLa cell lines both in vitro and in vivo.

Luteolin Induces Apoptosis in Oral Squamous Cancer Cells

Oral squamous cell carcinoma is the most common malignancy of the oral cavity, and treatment approaches are inadequate. Luteolin, a natural flavonoid compound, has been shown to have anti-tumorigenic properties on various types of tumors. Therefore, we hypothesized that luteolin has anti-tumorigenic properties for oral squamous cell carcinoma, and may provide effective chemotherapy. Results revealed that luteolin reduced the viability of SCC-4 cells and induced apoptosis by decreasing the expression of cyclin-dependent kinase (CDKs), cyclins, and phosphor- retinoblastoma (p-Rb) anti-apoptotic protein, but increased the expression of pro-apoptotic proteins and activated caspase 9 and 3, with a concomitant increase in the levels of cleaved poly-ADP-ribose polymerase (PARP). Combination treatment of luteolin with paclitaxel enhanced the cytotoxic effect of paclitaxel in SCC-4 cells, and continuous administration of luteolin suppressed the growth of xenograft tumors in nude mice. These results suggest that luteolin could be an effective chemotherapeutic agent for the treatment of oral squamous cell carcinoma.

Stimulation of matrix metalloproteinases by black-pigmented Bacteroides in human pulp and periodontal ligament cell cultures.

Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes capable of degrading most components of the extracellular matrix. Recently, evidence has shown that MMPs may play a role in tissue degradation in inflamed dental pulp. To date very little is known regarding the mechanism of extracellular matrix destruction at the site of bacterial infection. The purpose of this study was to determine the effects of the supernatants from Porphyromonas endodontalis and Porphyromonas gingivalis on the production and secretion of MMPs by primary human pulp and periodontal ligament (PDL) cell cultures in vitro. The results were evaluated by substrate gel zymography from long-term cultures. The main gelatinase secreted by human pulp and PDL cells migrated at 72 kDa and represented MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. After an 8-day culture period, P. endodontalis and P. gingivalis were found to elevate MMP-2 production both in human pulp and PDL cell cultures. In addition, the stimulation was in a dose- and time-dependent manner. Both human pulp and PDL cells, however, treated with either P. endodontalis or P. gingivalis had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. These results indicate that black-pigmented Bacteroides species play an important role in tissue destruction and disintegration of extracellular matrix in pulpal and periapical diseases. Thus, activation of MMPs may be one of the distinct host degradative pathways in the pathogenesis of microbial-induced pulpal and periapical lesion. An understanding of the actions of these black-pigmented Bacteroides species on pulp and PDL cells may result in new therapies to augment current treatment of pulpal and periapical lesions.