Development of an Ex Vivo Porcine Eye Model for Exploring the Pathogenicity of Acanthamoeba.

Acanthamoeba, a widely distributed free-living amoeba found in various environments, is an opportunistic pathogen responsible for causing Acanthamoeba keratitis, a condition that may lead to blindness. However, identifying the pathogenicity of Acanthamoeba is challenging due to its complex life cycle, ability to adapt to different environments, variable virulence factors, and intricate interactions with the host immune system. Additionally, the development of an effective model for studying Acanthamoeba pathogenicity is limited, hindering a comprehensive understanding of the mechanisms underlying its virulence and host interactions. The aim of this study was to develop an ex vivo model for Acanthamoeba infection using porcine eyeballs and to evaluate the pathogenicity of the Acanthamoeba isolates. Based on slit lamp and biopsy analysis, the developed ex vivo model is capable of successfully infecting Acanthamoeba within 3 days. Histopathological staining revealed that clinical isolates of Acanthamoeba exhibited greater corneal stroma destruction and invasion in this model than environmental isolates. Our results highlight the importance of an ex vivo porcine eye model in elucidating the pathogenesis of Acanthamoeba infection and its potential implications for understanding and managing Acanthamoeba-related ocular diseases.

A gender-specific COMT haplotype contributes to risk modulation rather than disease severity of major depressive disorder in a Chinese population.

BACKGROUND: COMT rs4680 Val158 allele is associated with high MB-COMT protein expression and elevated activity compared to the Met158 allele in post-mortem brains. A meta-analysis study suggested the link between COMT SNPs and MDD risk; in addition, MB membrane-bound (MB-COMT) specific genetic variation was reported that influences predisposition to depression amongst females. METHODS: Four tagSNPs, including rs4680, were genotyped. 268 MDD subjects and 223 controls were enrolled. MDD severity was rated by HDRS. Total-COMT and MB-COMT mRNA were detected by quantitative PCR. COMT protein and activity were assayed by western blot and methyltransferase assay, respectively. RESULTS: Haplotype TG of rs4633-rs4680, rs4646312 C, and rs4633 T allele might be linked to MDD vulnerability. Haplotype TG may interact with gender and affect MDD risk, since female haplotype TG carriers were estimated for a 9.17-fold higher risk than counterparts. COMT SNPs were not associated with HDRS scores. Haplotype TG female controls had higher MB-COMT protein, whereas non-TG female controls had higher soluble cytoplasmic (S-COMT) protein than other groups. COMT activity was much higher in controls than in MDD subjects. LIMITATIONS: Restricted numbers of homozygous TG carriers were recruited and analyzed for COMT mRNA, protein and activity. Only peripheral blood samples were used. CONCLUSIONS: A female-specific haplotype (haplotype TG)-MDD vulnerability association was found. TG female controls had higher MB-COMT protein and S-COMT. Altogether, high COMT protein and activity in female TG controls may be predisposing factors for enhanced MDD risk, though not correlated to MDD severity as rated by HDRS.

Antifibrotic role of PGC-1α-siRNA against TGF-ß1-induced renal interstitial fibrosis.

Peroxisome proliferator-activated receptor coactivator-1 alpha (PGC-1α) is a transcriptional coactivator that regulates energy metabolism and mitochondrial biogenesis. Recently, mitochondrial dysfunction has been indicated as an established risk factor for the development of renal fibrosis. However, whether PGC-1α is involved in the pathogenesis of renal fibrosis is unknown. In this study, we treated NRK-49F (normal rat kidney fibroblast) cells with transforming growth factor-beta 1 (TGF-ß1) for 24 h to establish an in vitro fibrosis model. TGF-ß1 induced the upregulation of type I collagen, fibronectin, TGF-ß receptor I (TGFß-RI), TGFß-RII, Smad4, and pSmad2/3, as well as PGC-1α. NRK-49F cells transfected with pcDNA-PGC-1α showed significantly increased expression of fibronectin and type I collagen, as revealed by western blot assay. Interestingly, transfection with PGC-1α-siRNA caused a stark reversal of TGF-ß1-induced cellular fibrosis, with concomitant suppression of fibronectin and type I collagen, as revealed by western blot and immunofluorescence assays. Moreover, SB431542 (TGFß-RI), LY294002 (PI3K/Akt), and SB203580 (p38 MAPK), inhibitors of TGF-ß-associated pathways, markedly suppressed TGF-ß1-induced PGC-1α upregulation. These results implicate a role of PGC-1α in renal interstitial fibrosis mediated via the TGFß-RI, PI3K/Akt, and p38 MAPK pathways. Our findings that PGC-1α-siRNA downregulates fibronectin and type I collagen suggest that it can be used as a novel molecular treatment for renal fibrosis.

Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis.

BACKGROUND: Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. MATERIALS AND METHODS: In vitro fibrosis model was established by treatment with transforming growth factor-ß1 (TGF-ß1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis and fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). RESULTS: Under renal fibrosis conditions, TGF-ß1 (5ng/ml) increased ROS. Simultaneously, TGF-ß1-induced extracellular fibronectin by ELISA assay. In addition, TGF-ß1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2µg/ml) expression vector dramatically reverse TGF-ß1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2µg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. DISCUSSION: These finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells.

Treatment with cytokine thymic stromal lymphopoietin short hairpin RNA substantially reduces TGF-ß1-induced interstitial cellular fibrosis.

Thymic stromal lymphopoietin (TSLP) has previously been linked to allergic inflammatory diseases, and tissue fibrosis and organ dysfunction may also arise from such inflammation. It remains unclear, however, whether TSLP plays any role in the occurrence of renal fibrosis, so this study investigated that possibility. An in vitro fibrosis model was established by treating normal rat kidney fibroblast (NRK-49F) cells with transforming growth factor-ß1 (TGF-ß1), after which the levels of various fibrogenic markers (e.g., fibronectin) and downstream fibrogenic signal proteins (e.g., smad 7) were investigated. Also, TSLP shRNA was used to silence the effects of TSLP, while an ELISA was conducted to evaluate the fibronectin secretions. The level of fibronectin in the NRK-49F cells was dose- and time-dependently increased by the administration of exogenous TSLP (P<0.05). TSLP also significantly increased the level of fibrosis signaling, in addition to inducing a marked decrease in the down-regulation of Smad7. Interestingly, the application of TSLP shRNA caused a stark reversal of the TGF-ß1-induced cellular fibrosis while simultaneously leading to the suppression of fibronectin and fibrogenic signal proteins. Taken together, these observations provide insights into how extracellular matrices develop and could thus lead to potential therapeutic interventions for the suppression of renal fibrosis.

Apigenin, a dietary flavonoid, inhibits proliferation of human bladder cancer T-24 cells via blocking cell cycle progression and inducing apoptosis.

BACKGROUND: Apigenin is a nontoxic dietary flavonoid, and it may have chemopreventive and therapeutic potential as an anti-inflammatory, antioxidant, and anti-cancer agent. However, its role in bladder cancer remains poorly understood. The aim of this study was to investigate the anti-proliferative activity of apigenin in human bladder cancer T-24 cells. METHODS AND RESULTS: Apigenin inhibited T-24 cell proliferation in a dose-dependent manner. We demonstrated that apigenin-induced early and mid-apoptotic cell could be identified by Annexnin V-Alexa Fluor 488/PI apoptosis detection and TUNEL assay. Moreover, using a JC-1 staining assay, we found that apigenin may induce the loss of the mitochondrial membrane potential. By performing flow cytometry and Western blotting, apigenin-mediated subG1 phase acculmulation was also associated with an increase in the phospho-p53, p53, p21, and p27 levels, and with a decrease in the Cyclin A, Cyclin B1, Cyclin E, CDK2, Cdc2, and Cdc25C levels, thereby blocking cell cycle progression. ELISA showed that the subG1 phase acculmulation was due to the increase in the p53, p21, and p27 levels. In addition, apigenin increased the Bax, Bad, and Bak levels, but reduced the Bcl-xL, Bcl-2, and Mcl-1 levels, and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c and activation of caspase-9, caspase-3, caspase-7, and PARP). Further analysis demonstrated that apigenin increased the ROS levels and depleted GSH in T-24 cells at 12 h. CONCLUSIONS: The results suggested that apigenin inhibits T-24 cells proliferation via blocking cell cycle progression and inducing apoptosis. In addition, we discovered a potential anticancer activity of apigenin against T-24 cells.

Galangin, a novel dietary flavonoid, attenuates metastatic feature via PKC/ERK signaling pathway in TPA-treated liver cancer HepG2 cells.

BACKGROUND: Galangin (3,5,7-trihydroxyflavone) is a flavonoid compound found in high concentration in lesser galangal. The objective of this study was to investigate the ability of galangin to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced the invasion and metastasis of HepG2 liver cancer cells. RESULTS: First, using a cell-matrix adhesion assay, immunofluorescence assay, transwell-chamber invasion/migration assay, and wound healing assay, we observed that galangin exerted an inhibitory effect on TPA-induced cell adhesion, morphology/actin cytoskeleton arrangement, invasion and migration. Furthermore, the results of gelatin zymography and reverse transcriptase polymerase chain reaction (RT-PCR) assays showed that galangin reduced the TPA-induced enzyme activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in HepG2 cells; moreover, the messenger RNA level was downregulated. We also observed through a Western blotting assay that galangin strongly inhibited the TPA-induced protein expressions of protein kinase Cα (PKCα), protein kinase Cδ (PKCδ), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), the phospho-inhibitor of kappaBα (phospho-IκBα), c-Fos, c-Jun, and nuclear factor kappa B (NF-κB). Next, galangin dose-dependently inhibited the binding ability of NF-κB and activator protein 1 (AP-1) to MMP-2/MMP-9 promoters, respectively, resulting in the suppression of MMP-2/MMP-9 enzyme activity. CONCLUSIONS: The results revealed that galangin effectively inhibited the TPA-induced invasion and migration of HepG2 cells through a protein kinase C/extracellular signal-regulated kinase (PKC/ERK) pathway. Thus, galangin may have widespread applications in clinical therapy as an anti-metastatic medicament.

Andrographolide sensitizes the cytotoxicity of human colorectal carcinoma cells toward cisplatin via enhancing apoptosis pathways in vitro and in vivo.

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, has been shown to suppress the growth and invasion of human colorectal carcinoma (CRC) Lovo cells, and trigger apoptosis in vitro. The potential of Andro as a chemotherapeutic agent in CRC was evaluated by investigating its cytotoxic effects as a single agent or in coadministration with cisplatin (CDDP). Andro potentiated the cytotoxic effect of CDDP in Lovo cells through apoptosis. The molecular mechanism for these favorable cellular response was further investigated by analyzing the apoptotic profiles, protein levels, and mRNA expression patterns of several key genes after treatments of Andro or/and CDDP. Molecular results indicated that the effect of Andro alone might be mediated via both intrinsic and extrinsic apoptotic pathways in Lovo cells. The addition of Andro to CDDP induced synergistic apoptosis, which could be corroborated to the changes in protein and mRNA levels of Bax and Bcl-2, and the increased Fas/FasL association in these cells, resulting in increased release of cytochrome c, and activation of caspases. Pretreatment of Nok-1 monoclonal antibody, a Fas signaling inhibitor, or Bax inhibitor peptide V5 repressed the Andro-induced cleavage of procaspase and the sensitization to CDDP-induced apoptosis. Finally, the combination therapy of Andro with CDDP was evidenced by its synergistic inhibition on the growth of Lovo cells in xenograft tumor studies. The results indicate that Andro, in combination with chemotherapeutics, is likely to represent a potential therapeutic strategy for CRC.

Pinocembrin suppresses TGF-ß1-induced epithelial-mesenchymal transition and metastasis of human Y-79 retinoblastoma cells through inactivating αvß3 integrin/FAK/p38α signaling pathway.

BACKGROUND: Pinocembrin is the most abundant flavonoid in propolis. In this study, we investigated the antimetastatic effect of pinocembrin on TGF-ß1-induced epithelial-mesenchymal transition (EMT) and metastasis of human Y-79 retinoblastoma cells. RESULTS: Firstly, the results showed that pinocembrin significantly suppresses the TGF-ß1-induced abilities of the invasion and migration of Y-79 cells under non-cytotoxic concentration. Pinocembrin decreased TGF-ß1-induced expression of vimentin, N-cadherin, αv and ß3 integrin in Y-79 cells. Molecular data also showed pinocembrin inhibits the activation of focal adhesion kinase (FAK) and p38α signal involved in the downregulation of enzyme activities, protein and messenger RNA levels of matrix metalloproteinase-2/9 (MMP-2/-9) induced by TGF-ß1. Next, pinocembrin also strongly inhibited the degradation of inhibitor of kappaBα (IκBα) and the nuclear levels of nuclear factor kappa B (NF-κB). Also, a dose-dependent inhibition on the binding ability of NF-κB was further observed under pinocembrin treatment. CONCLUSIONS: Presented results indicated that pinocembrin inhibits TGF-ß1-induced epithelial-mesenchymal transition (EMT) and metastasis of Y-79 cells by inactivating the αvß3 integrin/FAK/p38α signaling pathway. Thus, our findings point to the anticancer potential of pinocembrin against retinoblastoma cells.

Factors associated with sex hormones and erectile dysfunction in male Taiwanese participants with obesity.

INTRODUCTION: Obesity has been receiving an increasing amount of attention recently, but investigations regarding the potential impact of obesity, sexual behaviors, and sex hormones on erectile dysfunction (ED) in men have not completely clarified the association. AIM: To identify the relationship between ED, sexual behavior, sexual satisfaction, sex hormones, and obesity in older adult males in Taiwan. METHODS: Data were obtained from a baseline survey of 476 older adult males (≧40 years old). Their demographic data, body mass index (BMI), sex hormones, sexual desire, sexual satisfaction, and ED status were assessed. MAIN OUTCOME MEASURES: The International Index of Erectile Function-5 (IIEF-5), Sexual Desire Inventory (SDI), and Sexual Satisfaction Scale (SSS) were used to assess ED, sexual desire, and sexual satisfaction. RESULTS: In all, 476 men were available for analysis. The mean age of the sample was 51.34 ± 7.84 years (range 40 to 70 years). The IIEF total score had a mean of 19.44 ± 4.98; 264 (55.5%) subjects had ED, 250 (52.9%) were currently obese (BMI ≧27), and 297 (62.4%) had metabolic syndrome. The results showed an increased risk of ED among obese men and subjects with lower levels of sex hormones and lower sexual desire. Testosterone levels were lower in subjects with obesity (P < 0.001). Among the predictors of ED, obesity (odds ratio [OR] = 1.62, 95% CI = 1.07-2.44, P = 0.021), abnormal high sensitivity C-reactive protein (hs-CRP) (OR = 10.59, 95% CI = 4.70-23.87, P < 0.001), and lower serum full testosterone (OR = 3.27, 95% CI = 2.16-4.93, P < 0.001) were significantly independent factors. CONCLUSIONS: This study supports the idea of a close relationship between low levels of sex hormones, sexual desire, sexual satisfaction, obesity, and ED, and also shows that low free testosterone and hs-CRP may predict ED, even in obese populations.

Nobiletin attenuates metastasis via both ERK and PI3K/Akt pathways in HGF-treated liver cancer HepG2 cells.

Hepatocyte growth factor (HGF), and its receptor, c-Met activation has recently been shown to play important roles in cancer invasion and metastasis in a wide variety of tumor cells. We use HGF as an invasive inducer of human HepG2 cells to investigate the effect of four flavones including apigenin, tricetin, tangeretin, and nobiletin on HGF/c-Met-mediated tumor invasion and metastasis. Among them, nobiletin markedly inhibited HGF-induced the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay and transwell-chamber invasion/migration assay under non-cytotoxic concentrations. Data also showed nobiletin inhibited HGF-induced cell scattering and cytoskeleton changed such as filopodia and lamellipodia. Furthermore, nobiletin could inhibit HGF-induced the membrane localization of phosphorylated c-Met, ERK2, and Akt, but not phosphorylated JNK1/2 and p38. Next, nobiletin significantly decreased the levels of phospho-ERK2 and phospho-Akt in ERK2 or Akt siRNA-transfected cells concomitantly with a marked reduction on cell invasion and migration. In conclusion, nobiletin attenuates HGF-induced HepG2 cells metastasis involving both ERK and PI3K/Akt pathways and are potentially useful as anti-metastatic agents for the treatment of hepatoma.

Factors contributing to the disturbance of coagulation and fibrinolysis in dengue virus infection.

Hemorrhage is one of the hallmarks of dengue hemorrhagic fever. However, the mechanisms that cause hemorrhage are unclear. In this review we focus on the possible factors that may be involved in the disturbance of coagulation and fibrinolysis during dengue virus (DENV) infection. Factors such as autoantibodies and cytokines induced by DENV infection as well as hemostatic molecules expressed on DENV-infected cells, and DENV viral proteins may all contribute to the defect of hemostasis during DENV infection. It is the combination of these viral and host factors that may tilt the balance of coagulation and fibrinolysis toward bleeding in dengue patients.

CXCL12-G801A polymorphism modulates risk of colorectal cancer in Taiwan.

INTRODUCTION: The chemokine CXCL12, designated stromal cell-derived factor-1 (SDF-1), plays a significant role in many cancer metastases. Previous studies have shown that CXCL12-G801A, a single nucleotide polymorphism (SNP) in the 3' untranslated region, correlates with breast and lung cancer in Iran. The aim of this study was to evaluate the association of the gene variant CXCL12-G801A with colorectal cancer (CRC) in a Taiwanese cohort. MATERIAL AND METHODS: In this study, we used a denaturing high performance liquid chromatography (DHPLC) method to analyze the frequencies of CXCL12-G801A polymorphic variants between CRC patients (n = 258) and healthy controls (n = 300) in Taiwan. RESULTS: The SNP distribution was higher in CRC patients with TNM stage II (117/258) than healthy controls (52/300). We observed a significant increase in the G/A plus A/A genotype of the CXCL12-G801A polymorphism in CRC patients (45.35%) compared with healthy controls (17.33%). The analysis of allelic frequencies in both groups revealed that CRC patients have a higher frequency of A allele (23.45%) than healthy controls (8.67%). Furthermore, among older CRC patients, the frequency of the CXCL12-G801A genotype was significantly increased (p = 0.0148). CONCLUSIONS: Our observations suggest that the CXCL12-G801A genotype may be associated with some clinical manifestations in CRC patients in Taiwan.

Advanced glycation end products-mediated hypertrophy is negatively regulated by tetrahydrobiopterin in renal tubular cells.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease worldwide. The accumulation of advanced glycation end products (AGE) is a key mediator of renal tubular hypertrophy in DN. Elimination of tetrahydrobiopterin (BH(4)) and nitric oxide (NO) bioavailability may contribute to the aggravation of DN. The present study aims to explore any possible beneficial effect of exogenous BH(4) in alleviating the AGE-induced renal tubular hypertrophy in DN. Thus, renal tubular cells were treated with BH(4), BH(2), sepiapterin, or DAHP in the presence of AGE. We found that AGE (but not non-glycated BSA) markedly reduced NO production and increased hypertrophy index in these cells. Exogenous BH(4)/BH(2) and sepiapterin treatments attenuated AGE-inhibited the iNOS/NO/GTPCH I protein synthesis. Moreover, BH(4) and BH(2) significantly reversed AGE-enhanced the JAK2-STAT1/STAT3 activation. The abilities of BH(4) and BH(2) to inhibit AGE-induced renal cellular hypertrophy were verified by the observation that BH(4) and BH(2) inhibited hypertrophic growth and the protein synthesis of p27(Kip1) and α-SMA. These findings indicate for the first time that exogenous BH(4) and BH(2) attenuate AGE-induced hypertrophic effect at least partly by increasing the iNOS/GTPCH I synthesis and NO generation in renal tubular cells.

Metabolic outcome for diabetes shared care program outpatients in a veterans hospital of southern Taiwan.

BACKGROUND: To evaluate the metabolic outcomes of the Diabetes Shared Care Program (DSCP) for Type 2 diabetes after completion of 1 year and 3 years of intervention. METHODS: Total 162 Type 2 diabetes (average age 67.14 years with 62.35% men and 37.65% women) in 2004 were referred to the diabetes educator for DSCP. Parameters related to diabetes among these patients were inquired, and biochemical data were compared before and after the DSCP by using SPSS 12.0 software. RESULTS: These patients had 3.1% emergency utilization rate and 1.9% hospitalization utilization rate; significant improvement in diastolic blood pressure (DBP), body weight after one year; and significant improvement in systolic blood pressure, DBP, body weight, total cholesterol, high-density lipoproteins cholesterol (HDL-C) and low-density lipoproteins cholesterol (LDL-C) levels after three years. But only 4.84% and 8.87% met all the A1C, blood pressure, and LDL-C target values after the 1- and 3-year interventions, respectively. CONCLUSION: The A1C, blood pressure, and LDL-C achievement rate of DSCP in our hospital is low. DSCP is suggestive to patients with lower duration of diabetes, high baseline A1C, systolic blood pressure, DBP, LDL-C, and low baseline high-density lipoproteins cholesterol levels. Furthermore public health efforts are needed to control risk factors for vascular disease among diabetes.

α-Tomatine suppresses invasion and migration of human non-small cell lung cancer NCI-H460 cells through inactivating FAK/PI3K/Akt signaling pathway and reducing binding activity of NF-κB.

α-Tomatine, isolated from Lycopersicon esculentum Linn., is a naturally occurring steroidal glycoalkaloid in immature green tomatoes. Some reports demonstrated that α-tomatine had various anticarcinogenic properties. The purpose of this study is to investigate the anti-metastatic effect of α-tomatine in NCI-H460 human non-small cell lung cancer cells. First, the results showed that α-tomatine significantly suppressed the abilities of the adhesion, invasion, and migration of NCI-H460 cells under non-cytotoxic concentrations. Molecular data also showed α-tomatine could inhibit the activation of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K)/Akt signal involve in the downregulation the enzyme activities, protein and messenger RNA levels of matrix metalloproteinase-7 (MMP-7). Next, α-tomatine also strongly inhibited the degradation of inhibitor of kappaBα (IκBα) and the nuclear levels of nuclear factor kappa B (NF-κB). Also, a dose-dependent inhibition on the binding ability of NF-κB by α-tomatine treatment was further observed. Furthermore, α-tomatine significantly decreased the levels of phospho-Akt and MMP-7 in Akt1-cDNA-transfected cells concomitantly with a marked reduction on cell invasion and migration. Presented results indicated α-tomatine might be further application for treating cancer metastasis.

alpha-Mangostin, a novel dietary xanthone, suppresses TPA-mediated MMP-2 and MMP-9 expressions through the ERK signaling pathway in MCF-7 human breast adenocarcinoma cells.

This study first investigates the anti-metastatic effect of alpha-mangostin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions in human breast adenocarcinoma cells, MCF-7. First, the result demonstrated alpha-mangostin could inhibit TPA-induced abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay and Boyden chamber assay. Data also showed alpha-mangostin could inhibit the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) involved in the downregulation the enzyme activities, protein, and messenger RNA levels of MMP-2 and MMP-9 induced by TPA. Next, alpha-mangostin also strongly inhibited TPA-induced degradation of inhibitor of kappaBalpha (IkappaBalpha) and the nuclear levels of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun. Also, a dose-dependent inhibition on the binding abilities of NF-kappaB and activator protein-1 (AP-1) by alpha-mangostin treatment was further observed. Further, the treatment of specific inhibitor for ERK (U0126) to MCF-7 cells could inhibit TPA-induced MMP-2 and MMP-9 expressions along with an inhibition on cell invasion and migration. Presented data reveal that alpha-mangostin is a novel, effective, antimetastatic agent that functions by downregulating MMP-2 and MMP-9 gene expressions.

Andrographolide could inhibit human colorectal carcinoma Lovo cells migration and invasion via down-regulation of MMP-7 expression.

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to have potent anti-cancer activity against human colorectal carcinoma Lovo cells by inhibiting cell-cycle progression. To further investigate the mechanism for the anti-cancer properties of Andro, it was used to examine the effect on migration and invasion of Lovo cells. The results of wound-healing assay and in vitro transwell assay revealed that Andro inhibited dose-dependently the migration and invasion of Lovo cells under non-cytotoxic concentrations. Using zymographic assay and RT-PCR, the results revealed that Andro diminished the activity and the mRNA and protein levels of MMP-7, but not MMP-2 or MMP-9. The down-regulation of MMP-7 appeared to be via the inactivation of activator protein-1 (AP-1) since the treatment with Andro suppressed the nuclear protein level of AP-1, which was accompanied by a decrease in DNA-binding level of the factor. Taken together, these results indicated that Andro reduces the MMP-7-mediated cellular events in Lovo cells, and provided a new mechanism for its anti-cancer activity.

Myricetin suppresses invasion and migration of human lung adenocarcinoma A549 cells: possible mediation by blocking the ERK signaling pathway.

Cancer metastasis, involving multiple processes and various cytophysiological changes, is a primary cause of cancer death and may complicate clinical management, even leading to death. Myricetin (3,5,7,3',4',5'-hexahydroxyflavone), a naturally occurring flavonoid, has various anticancer activities. This is the first study to explore the antimetastatic effect of myricetin in human adenocarcinoma A549 cells in vitro. First, myricetin exerted a dose- and time-dependent inhibitory effect on the adhesion, invasion, and migration of A549 cells in the absence of cytotoxicity. Gelatin or casein zymography assays showed that myricetin inhibited the matrix metalloproteinase-2 (MMP-2) and urokinase-plasminogen activator (u-PA) activities of A549 cells. Moreover, myricetin also exerted an inhibitory effect on the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and inhibition of activation of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun. Treatment with myricetin of A549 cells also led to a dose-dependent effect on the binding abilities of NF-kappaB and AP-1. Furthermore, the ERK inhibitor (U0126) could result in reduced activities of MMP-2 and u-PA concomitantly with a marked inhibition on cell invasion and migration. These results demonstrated that the inhibition of MMP-2 and u-PA expression by myricetin may be through a suppression on ERK1/2 phosphorylation and inhibit A549 cells invasion and migration. As shown by the above results, myricetin may be a powerful candidate in developing preventive agents for cancer metastasis.

Inhibition of cell-cycle progression in human colorectal carcinoma Lovo cells by andrographolide.

In recent years, attention has been focused on the anti-cancer properties of pure components, an important role in the prevention of disease. Andrographolide (Andro), the major constituent of Andrographis paniculata (Burm. F.) Nees plant, is implicated towards its pharmacological activity. To investigate the mechanism basis for the anti-tumor properties of Andro, Andro was used to examine its effect on cell-cycle progression in human colorectal carcinoma Lovo cells. The data from cell growth experiment showed that Andro exhibited the anti-proliferation effect on Lovo cells in a time- and dose-dependent manner. This event was accompanied the arrest of the cells at the G1-S phase by Andro at the tested concentrations of 0-30 microM. Cellular uptake of Andro and Andro was confirmed by capillary electrophoresis analysis and the intracellular accumulation of Andro (0.61+/-0.07 microM/mg protein) was observed when treatment of Lovo cells with Andro for 12h. In addition, an accumulation of the cells in G1 phase (15% increase for 10 microM of Andro) was observed as well as by the association with a marked decrease in the protein expression of Cyclin A, Cyclin D1, Cdk2 and Cdk4. Andro also inducted the content of Cdk inhibitor p21 and p16, and the phosphorylation of p53. Further immunoprecipitation studies found that, in response to the treatment, the formation of Cyclin D1/Cdk4 and Cyclin A/Cdk2 complexes had declined, preventing the phosphorylation of Rb and the subsequent dissociation of Rb/E2F complex. These results suggested Andro can inhibit Lovo cell growth by G1-S phase arrest, and was exerted by inducing the expression of p53, p21 and p16 that, in turn, repressed the activity of Cyclin D1/Cdk4 and/or Cyclin A/Cdk2, as well as Rb phosphorylation.