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To prospectively evaluate the effect of benzalkonium chloride (BAK)-preserved latanoprost on ocular surface damage and identify the associated risk factors among treatment-naive glaucoma patients. The basal Schirmer’s test results, corneal Oxford staining score, non-invasive keratograph tear-breakup time, oculus hyperemia index score (objective metrics), and ocular surface disease index (OSDI) questionnaire (subjective metric) were evaluated at baseline, 1 month, and 4 months after receiving latanoprost eye drops. Associated risk factors were assessed by multivariate linear regression. Seventy-four eyes (44 patients) were enrolled. Basal Schirmer’s test tear-flow and Oxford scores gradually deteriorated (β = −0.14, P = 0.001 and β = 0.1, P < 0.001, respectively). The percentage of unstable tear-film (breakup time < 10 s) increased significantly at 4 months (6.21% vs 9.11%, P = 0.042). Hyperemic scores increased significantly at 1 month and normalized at 4 months (P = 0.01 and P = 0.16, respectively); total OSDI scores tended to improve (β = −0.76, P = 0.06). Older age was associated with additional corneal Oxford staining (P = 0.005); female sex was associated with increased unstable tear-film scores (P = 0.01). Artificial tear use was associated with a smaller decrease in basal Schirmer’s test values (P = 0.01) and a smaller increase in unstable tear-film scores (P = 0.02). Preserved latanoprost eye drops affected ocular surface changes in glaucoma patients through decreased basal tear secretion. Artificial tears represent an early intervention in vulnerable glaucoma patients with reduced tear secretion and impaired tear-film stability.

Persistent chronic liver diseases increase the scar formation and extracellular matrix accumulation that further progress to liver fibrosis and cirrhosis. Nevertheless, there is no antifibrotic therapy to date. The ketogenic diet is composed of high fat, moderate to low-protein, and very low carbohydrate content. It is mainly used in epilepsy and Alzheimer’s disease. However, the effects of the ketogenic diet on liver fibrosis remains unknown. Through ketogenic diet consumption, β-hydroxybutyrate (bHB) and acetoacetate (AcAc) are two ketone bodies that are mainly produced in the liver. It is reported that bHB and AcAc treatment decreases cancer cell proliferation and promotes apoptosis. However, the influence of bHB and AcAc in hepatic stellate cell (HSC) activation and liver fibrosis are still unclear. Therefore, this study aimed to investigate the effect of the ketogenic diet and ketone bodies in affecting liver fibrosis progression. Our study revealed that feeding a high-fat ketogenic diet increased cholesterol accumulation in the liver, which further enhanced the carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis. In addition, more severe liver inflammation and the loss of hepatic antioxidant and detoxification ability were also found in ketogenic diet-fed fibrotic mouse groups. However, the treatment with ketone bodies (bHB and AcAc) did not suppress transforming growth factor-β (TGF-β)-induced HSC activation, platelet-derived growth factor (PDGF)-BB-triggered proliferation, and the severity of CCl4-induced liver fibrosis in mice. In conclusion, our study demonstrated that feeding a high-fat ketogenic diet may trigger severe steatohepatitis and thereby promote liver fibrosis progression. Since a different ketogenic diet composition may exert different metabolic effects, more evidence is necessary to clarify the effects of a ketogenic diet on disease treatment.

The quantitative analysis of surface antigens on cells, especially red blood cells (RBCs), has attracted increasing attention due to the recognition of antigenic changes that can facilitate early diagnoses. This paper presents an alternative methodology developed using the optical cell-detachment technique to evaluate antibody-antigen interactions and quantitatively analyze the RBC surface antigen expression. RBC subtyping was used to verify the proposed detection principle based on a comparison of the bonding strengths between individual RBCs and antibody coatings. The bonding strengths were measured with serial antibody dilutions with gradually decreasing laser powers, for which a single cell was optically detached from the corresponding antibody-coated surface. With the quantitatively analysis, the proposed alternative methodology was verified as a highly sensitive technique for detecting antigen expression on the RBC surface.

Myocardial infarction is the leading cause of morbidity and mortality worldwide. Although myocardial reperfusion after ischemia (I/R) is an effective method to save ischemic myocardium, it can cause adverse reactions, including increased oxidative stress and cardiomyocyte apoptosis. Mitochondrial fission and mitophagy are essential factors for mitochondrial quality control, but whether they play key roles in cardiac I/R injury remains unknown. New pharmacological or molecular interventions to alleviate reperfusion injury are currently considered desirable therapies. Vitamin D3 (Vit D3) regulates cardiovascular function, but its physiological role in I/R-exposed hearts, especially its effects on mitochondrial homeostasis, remains unclear. An in vitro hypoxia/reoxygenation (H/R) model was established in H9c2 cells to simulate myocardial I/R injury. H/R treatment significantly reduced H9c2 cell viability, increased apoptosis, and activated caspase 3. In addition, H/R treatment increased mitochondrial fission, as manifested by increased expression of phosphorylated dynein-related protein 1 (p-Drp1) and mitochondrial fission factor (Mff) as well as increased mitochondrial translocation of Drp1. Treatment with the mitochondrial reactive oxygen species scavenger MitoTEMPO increased cell viability and decreased mitochondrial fission. H/R conditions elicited excessive mitophagy, as indicated by increased expression of BCL2-interacting protein 3 (BNIP3) and light chain (LC3BII/I) and increased formation of autolysosomes. In contrast, Vit D3 reversed these effects. In a mouse model of I/R, apoptosis, mitochondrial fission, and mitophagy were induced. Vit D3 treatment mitigated apoptosis, mitochondrial fission, mitophagy, and myocardial ultrastructural abnormalities. The results indicate that Vit D3 exerts cardioprotective effects against I/R cardiac injury by protecting mitochondrial structural and functional integrity and reducing mitophagy.

Objectives Two nucleos(t)ide reverse-transcriptase inhibitors (NRTIs) plus 1 non-NRTI (nNRTI) remain the preferred or alternative combination antiretroviral therapy (cART) for antiretroviral-naive HIV-positive patients in Taiwan. The three most commonly used nNRTIs are nevirapine (NVP), efavirenz (EFV) and rilpivirine (RPV). This study aimed to determine the incidences of hepatotoxicity and skin rashes within 4 weeks of initiation of cART containing 1 nNRTI plus 2 NRTIs. Methods Between June, 2012 and November, 2015, all antiretroviral-naive HIV-positive adult patients initiating nNRTI-containing cART at 8 designated hospitals for HIV care were included in this retrospective observational study. According to the national HIV treatment guidelines, patients were assessed at baseline, 2 and 4 weeks of cART initiation, and subsequently every 8 to 12 weeks. Plasma HIV RNA load, CD4 cell count and aminotransferases were determined. The toxicity grading scale of the Division of AIDS (DAIDS) 2014 was used for reporting clinical and laboratory adverse events. Results During the 3.5-year study period, 2,341 patients initiated nNRTI-containing cART: NVP in 629 patients, EFV 1,363 patients, and RPV 349 patients. Rash of any grade occurred in 14.1% (n = 331) of the patients. In multiple logistic regression analysis, baseline CD4 cell counts (per 100-cell/μl increase, adjusted odds ratio [AOR], 1.125; 95% confidence interval [95% CI], 1.031-1.228) and use of NVP (AOR, 2.443; 95% CI, 1.816-3.286) (compared with efavirenz) were independently associated with the development of skin rashes. Among the 1,455 patients (62.2%) with aminotransferase data both at baseline and week 4, 72 (4.9%) developed grade 2 or greater hepatotoxicity. In multiple logistic regression analysis, presence of antibody for hepatitis C virus (HCV) (AOR, 2.865; 95% CI, 1.439-5.704) or hepatitis B surface antigen (AOR, 2.397; 95% CI, 1.150-4.997), and development of skin rashes (AOR, 2.811; 95% CI, 1.051-7.521) were independently associated with the development of hepatotoxicity. Conclusions The baseline CD4 cell counts and use of NVP were associated with increased risk of skin rashes, while hepatotoxicity was independently associated with HCV or hepatitis B virus coinfection, and development of skin rashes in antiretroviral-naive HIV-positive Taiwanese patients within 4 weeks of initiation of nNRTI-containing regimens.

This study investigated the correlations between consumption of antifungal agents and species distribution in candidaemia. The incidence of patients with community-acquired candidaemia (CAC) (per 1000 admissions) and healthcare-associated candidaemia (HCAC) (per 1000 admissions) as well as data on annual consumption [defined daily doses (DDD)/1000 patient-days] of various antifungal agents at a medical centre in Taiwan from 2000 to 2010 were evaluated. A total of 2682 episodes of candidaemia were identified, including 2468 HCAC (92.0%) and 214 CAC (8.0%). The most prevalent species was Candida albicans (53.3%), followed by Candida tropicalis (20.5%), Candida glabrata (15.4%), Candida parapsilosis (14.1%), Candida guilliermondii (1.7%) and Candida krusei (1.5%). The overall incidence of candidaemia remained stable, whereas that of candidaemia due to C. parapsilosis and C. guilliermondii decreased significantly with time. Significant negative correlations were found between the use of echinocandins and voriconazole and the incidence of C. parapsilosis candidaemia and between the use of caspofungin and the incidence of C. guilliermondii candidaemia. In contrast, there were significant positive correlations between the use of echinocandins and the incidence of C. tropicalis candidaemia, the use of azoles and the incidence of C. glabrata and non-albicans Candida candidaemia, and the use of itraconazole and the incidence of C. parapsilosis and C. guilliermondii candidaemia. Increased use of fluconazole was associated with an increased incidence of HCAC due to non-albicans Candida spp. In conclusion, the impact of consumption of antifungal agents on the incidence of candidaemia caused by different Candida spp. varies and warrants further studies to confirm these findings.

Nobiletin, a compound isolated from citrus fruits, is a polymethoxylated flavone derivative shown to have anti-inflammatory, antitumor, and neuroprotective properties. This study has investigated that nobiletin exerted inhibitory effects on the cell adhesion, invasion, and migration abilities of a highly metastatic AGS cells under non-cytotoxic concentrations. Data also showed nobiletin could inhibit the activation of focal adhesion kinase (FAK) and phosphoinositide-3-kinase/Akt (PI3K/Akt) involved in the downregulation of the enzyme activities, protein expressions, messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-2 (MMP-9). Also, our data revealed that nobiletin inhibited FAK/PI3K/Akt with concurrent reduction in the protein expressions of Ras, c-Raf, Rac-1, Cdc42, and RhoA by western blotting, whereas the protein level of RhoB increased progressively. Otherwise, nobiletin-treated AGS cells showed tremendously decreased in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, nobiletin significantly decreased the levels of phospho-Akt and MMP-2/9 in Akt1-cDNA-transfected cells concomitantly with a marked reduction in cell invasion and migration. These results suggest that nobiletin can reduce invasion and migration of AGS cells, and such a characteristic may be of great value in the development of a potential cancer therapy.

Lung cancer is the leading cause of death among cancers worldwide and non-small cell lung cancer (NSCLC) comprises more than 80% of lung cancer cases. Treatment options for patients with advanced NSCLC have evolved in the last decade with the advent of novel biological agents. Andrographolide, a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to have the potential to be developed as a chemotherapeutic agent. In order to understand the anti-cancer properties of andrographolide, we examined its effect on migration and invasion in human NSCLC A549 cells. The results of wound-healing assay and in vitro transwell assay revealed that andrographolide inhibited dose-dependently the migration and invasion of A549 cells under non-cytotoxic concentrations. Molecular data showed that the effect of andrographolide in A549 cells might be mediated via sustained inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt signal involved in the up-regulation of matrix metalloproteinases (MMPs). Our results showed that andrographolide exerted an inhibitory effect on the activity and the mRNA and protein levels of MMP-7, but not MMP-2 or MMP-9. The andrographolide-inhibited MMP-7 expression or activity appeared to occur via activator protein-1 (AP-1) because of its DNA binding activity was suppressed by andrographolide. Additionally, the transfection of Akt over-expression vector (Akt1 cDNA) to A549 cells could result in an increase expression of MMP-7 concomitantly with a marked induction on cell invasion. These findings suggested that the inhibition on MMP-7 expression by andrographolide may be through suppression on PI3K/Akt/AP-1 signaling pathway, which in turn led to the reduced invasiveness of the cancer cells.

AIM To investigate the inhibitory effects of plumbagin (5-hydroxy-2 methyl-1,4-naphthoquinone) on the invasion and migration and its correlation with matrix metalloproteinase-2 (MMP-2) and urokinase-plasminogen activator (u-PA) in liver cancer HepG2 cells under non-cytotoxic concentrations. METHODS The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The adhesion, migration and invasion were measured by cell-matrix adhesion assay and Boyden chamber assay. The MMP-2 and u-PA activities were estimated by gelatin and casein-plasminogen zymography. The mRNA and protein levels of MMP-2, u-PA, urokinase-plasminogen activator receptor (u-PAR), tissue inhibitor of metalloproteinase-2 (TIMP-2), plasminogen activator inhibitor-1 (PAI-1), nuclear factor kappa B (NF-kappaB), c-Fos and c-Jun were evaluated by semi-quantitative reverse transcription polymerase chain reaction and western blotting. Also, the binding abilities of NF-kappaB and activator protein-1 (AP-1) were analyzed by electrophoretic mobility shift assay (EMSA). RESULTS In this study, plumbagin had exhibited an inhibitory effect on the abilities of adhesion, migration and invasion. The results from zymography showed plumbagin treatment may decrease the activities of MMP-2 and u-PA. Further, the mRNA and protein levels of MMP-2, u-PA and u-PAR were significantly reduced, while TIMP-2 and PAI-1 were elevated by plumbagin treatment. Next, plumbagin significantly decreased the nuclear levels of NF-kappaB, c-Fos and c-Jun. Also, treating HepG2 cells with plumbagin leads to dose-dependent inhibition on the binding abilities of NF-kappaB and AP-1. CONCLUSION We demonstrated the inhibitory effects of plumbagin on the invasion, migration and adhesion of HepG2 cells, while plumbagin treatment may decrease the expressions of MMP-2 and u-PA and enhance the expressions of TIMP-2 and PAI-1.

This study first investigates the anti-metastatic effect of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMPs and u-PA expressions in human lung cancer cells, A549. First, the result demonstrated plumbagin could inhibit TPA induced the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay and Boyden chamber assay. Data also showed plumbagin could inhibit the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) involved in the down-regulating enzyme activities, protein and messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and urokinase-type plasminogen activator (u-PA) induced by TPA. Next, plumbagin also strongly inhibited TPA-induced phosphorylation and 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 plumbagin treatment was further observed. Further, the treatment of specific inhibitor for ERK (U0126) to A549 cells could inhibit TPA-induced MMP-2 and u-PA expressions along with an inhibition on cell invasion and migration. Presented data reveals that plumbagin is a novel, effective, anti-metastatic agent that functions by down-regulating MMP-2 and u-PA gene expressions.

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.

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.

This study first investigates the anti-metastatic effect of alpha-tomatine in the human lung adenocarcinoma cell line: A549. In this study, we first noted alpha-tomatine inhibited A549 cells invasion and migration by wound-healing assay and Boyden chamber assay. The data also showed alpha-tomatine could inhibit phosphorylation of Akt and extracellular signal-regulated kinase 1 and 2 (ERK1/2), which is involved in the up-regulating matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) or urokinase-type plasminogen activator (u-PA), whereas it did not affect phosphorylation of c-Jun N-terminal kinase (JNK) and p38. Next, alpha-tomatine significantly decreased the nuclear levels of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun. Also, treating A549 cells with alpha-tomatine also leads to a dose-dependent inhibition on the binding abilities of NF-kappaB and activator protein-1 (AP-1). Further, the treatment of inhibitors specific for PI3K (Wortmannin) or ERK (U0126) to A549 cells could cause reduced activities of MMP-2, MMP-9, and u-PA. These results showed alpha-tomatine could inhibit the metastatic ability of A549 cells by reducing MMP-2, MMP-9, and u-PA activities through suppressing phosphoinositide 3-kinase/Akt (PI3K/Akt) or ERK1/2 signaling pathway and inhibition NF-kappaB or AP-1 binding activities. These findings proved alpha-tomatine might be an anti-metastatic agent against human lung adenocarcinoma.

Gaseous nitrogen oxide (gNO) is an important indoor and outdoor air pollutant. Many studies have indicated gNO causes lung tissue damage by its oxidation properties and free radicals. However, there are considerably few data on the association between lung cancer and gNO exposure. The purpose of this study was to examine whether gNO could contribute to the process of malignant progression of lung cancer. The results of wound-healing assay and in vitro transwell assay revealed that gNO-induced dose and time dependently the migration and invasion of A549 cells, a human lung cancer cell line, under noncytotoxic concentrations. gNO was able to induce release of NO from A549 cells, an effect that was mediated via the activation of inducible nitric oxide synthases (iNOS), but not constitutive isoforms, during the same treatment period. An increased expression of matrix metalloproteinase (MMP) and a coincided reduction in repress tissue inhibitors of metalloprotease-2 were observed upon the treatment of gNO. The gNO-mediated MMP-2 induction appeared to be a consequence of nuclear factor kappa B and activation protein-1 activation, because that their DNA binding activity was enhanced by gNO. All these influences of gNO were efficiently repressed by the pretreatment of a NOS inhibitor (N(G)-nitro-L-arginine methyl ester). Using a mouse model, we showed that gNO promoted A549 metastasis to the lung through a mechanism involving the iNOS-dependent MMP-2 activity. Our data imply that gNO exposure, which in turn led to iNOS activation and the enhancement of MMP-mediated cellular events, was related to lung cancer development.

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.

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.