PCPE-1, a brown adipose tissue-derived cytokine, promotes obesity-induced liver fibrosis.

Metabolic dysfunction-associated steatohepatitis (MASH, previously termed non-alcoholic steatohepatitis (NASH)), is a major complication of obesity that promotes fatty liver disease. MASH is characterized by progressive tissue fibrosis and sterile liver inflammation that can lead to liver cirrhosis, cancer, and death. The molecular mechanisms of fibrosis in MASH and its systemic control remain poorly understood. Here, we identified the secreted-type pro-fibrotic protein, procollagen C-endopeptidase enhancer-1 (PCPE-1), as a brown adipose tissue (BAT)-derived adipokine that promotes liver fibrosis in a murine obesity-induced MASH model. BAT-specific or systemic PCPE-1 depletion in mice ameliorated liver fibrosis, whereas, PCPE-1 gain of function in BAT enhanced hepatic fibrosis. High-calorie diet-induced ER stress increased PCPE-1 production in BAT through the activation of IRE-1/JNK/c-Fos/c-Jun signaling. Circulating PCPE-1 levels are increased in the plasma of MASH patients, suggesting a therapeutic possibility. In sum, our results uncover PCPE-1 as a novel systemic control factor of liver fibrosis.

Differing impact of phosphoglycerate mutase 1-deficiency on brown and white adipose tissue.

Brown adipose tissue (BAT) is a metabolically active organ that contributes to the thermogenic response to cold exposure. In addition, other thermogenic cells termed beige adipocytes are generated in white adipose tissue (WAT) by cold exposure. Although activation of brown/beige adipose tissue is associated with mobilization of both glucose and lipids, few studies have focused on the role of glycolytic enzymes in regulating adipose tissue function. We generated mouse models with specific deletion of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) from adipose tissue. Deletion of Pgam1 from both BAT and WAT promoted whitening of BAT with beiging of visceral WAT, whereas deletion of Pgam1 from BAT alone led to whitening of BAT without beiging of WAT. Our results demonstrate a potential role of glycolytic enzymes in beiging of visceral WAT and suggest that PGAM1 would be a novel therapeutic target in obesity and diabetes.

Role of circulating molecules in age-related cardiovascular and metabolic disorders.

Studies analyzing heterochronic parabiosis mice models showed that molecules in the blood of young mice rejuvenate aged mice. Therefore, blood-based therapies have become one of the therapeutic approaches to be considered for age-related diseases. Blood includes numerous biologically active molecules such as proteins, metabolites, hormones, miRNAs, etc. and accumulating evidence indicates some of these change their concentration with chronological aging or age-related disorders. The level of some circulating molecules showed a negative or positive correlation with all-cause mortality, cardiovascular events, or metabolic disorders. Through analyses of clinical/translation/basic research, some molecules were focused on as therapeutic targets. One approach is the supplementation of circulating anti-aging molecules. Favorable results in preclinical studies let some molecules to be tested in humans. These showed beneficial or neutral results, and some were inconsistent. Studies with rodents and humans indicate circulating molecules can be recognized as biomarkers or therapeutic targets mediating their pro-aging or anti-aging effects. Characterization of these molecules with aging, testing their biological effects, and finding mimetics of young systemic milieu continue to be an interesting and important research topic to be explored.

Empagliflozin maintains capillarization and improves cardiac function in a murine model of left ventricular pressure overload.

Patients with type 2 diabetes treated with Sodium glucose transporter 2 (SGLT2) inhibitors show reduced mortality and hospitalization for heart failure (HF). SGLT2 inhibitors are considered to activate multiple cardioprotective pathways; however, underlying mechanisms are not fully described. This study aimed to elucidate the underlying mechanisms of the beneficial effects of SGLT2 inhibitors on the failing heart. We generated a left ventricular (LV) pressure overload model in C57BL/6NCrSlc mice by transverse aortic constriction (TAC) and examined the effects of empagliflozin (EMPA) in this model. We conducted metabolome and transcriptome analyses and histological and physiological examinations. EMPA administration ameliorated pressure overload-induced systolic dysfunction. Metabolomic studies showed that EMPA increased citrulline levels in cardiac tissue and reduced levels of arginine, indicating enhanced metabolism from arginine to citrulline and nitric oxide (NO). Transcriptome suggested possible involvement of the insulin/AKT pathway that could activate NO production through phosphorylation of endothelial NO synthase (eNOS). Histological examination of the mice showed capillary rarefaction and endothelial apoptosis after TAC, both of which were significantly improved by EMPA treatment. This improvement was associated with enhanced expression phospho-eNOS and NO production in cardiac endothelial cells. NOS inhibition attenuated these cardioprotective effects of EMPA. The in vitro studies showed that catecholamine-induced endothelial apoptosis was inhibited by NO, arginine, or AKT activator. EMPA activates the AKT/eNOS/NO pathway, which helps to suppress endothelial apoptosis, maintain capillarization and improve systolic dysfunction during LV pressure overload.

Cardiac mitofusin-1 is reduced in non-responding patients with idiopathic dilated cardiomyopathy.

Prognosis of severe heart failure remains poor. Urgent new therapies are required. Some heart failure patients do not respond to established multidisciplinary treatment and are classified as "non-responders". The outcome is especially poor for non-responders, and underlying mechanisms are largely unknown. Mitofusin-1 (Mfn1), a mitochondrial fusion protein, is significantly reduced in non-responding patients. This study aimed to elucidate the role of Mfn1 in the failing heart. Twenty-two idiopathic dilated cardiomyopathy (IDCM) patients who underwent endomyocardial biopsy of intraventricular septum were included. Of the 22 patients, 8 were non-responders (left ventricular (LV) ejection fraction (LVEF) of < 10% improvement at late phase follow-up). Electron microscopy (EM), quantitative PCR, and immunofluorescence studies were performed to explore the biological processes and molecules involved in failure to respond. Studies in cardiac specific Mfn1 knockout mice (c-Mfn1 KO), and in vitro studies with neonatal rat ventricular myocytes (NRVMs) were also conducted. A significant reduction in mitochondrial size in cardiomyocytes, and Mfn1, was observed in non-responders. A LV pressure overload with thoracic aortic constriction (TAC) c-Mfn1 KO mouse model was generated. Systolic function was reduced in c-Mfn1 KO mice, while mitochondria alteration in TAC c-Mfn1 KO mice increased. In vitro studies in NRVMs indicated negative regulation of Mfn1 by the ß-AR/cAMP/PKA/miR-140-5p pathway resulting in significant reduction in mitochondrial respiration of NRVMs. The level of miR140-5p was increased in cardiac tissues of non-responders. Mfn1 is a biomarker of heart failure in non-responders. Therapies targeting mitochondrial dynamics and homeostasis are next generation therapy for non-responding heart failure patients.

Casticin Induces DNA Damage and Affects DNA Repair Associated Protein Expression in Human Lung Cancer A549 Cells (Running Title: Casticin Induces DNA Damage in Lung Cancer Cells).

Casticin was obtained from natural plants, and it has been shown to exert biological functions; however, no report concerns the induction of DNA damage and repair in human lung cancer cells. The objective of this study was to investigate the effects and molecular mechanism of casticin on DNA damage and repair in human lung cancer A549 cells. Cell viability was determined by flow cytometric assay. The DNA damage was evaluated by 4',6-diamidino-2-phenylindole (DAPI) staining and electrophoresis which included comet assay and DNA gel electrophoresis. The protein levels associated with DNA damage and repair were analyzed by western blotting. The expression and translocation of p-H2A.X were observed by confocal laser microscopy. Casticin reduced total viable cell number and induced DNA condensation, fragmentation, and damage in A549 cells. Furthermore, casticin increased p-ATM at 6 h and increased p-ATR and BRCA1 at 6-24 h treatment but decreased p-ATM at 24-48 h, as well as decreased p-ATR and BRCA1 at 48 h. Furthermore, casticin decreased p-p53 at 6-24 h but increased at 48 h. Casticin increased p-H2A.X and MDC1 at 6-48 h treatment. In addition, casticin increased PARP (cleavage) at 6, 24, and 48 h treatment, DNA-PKcs and MGMT at 48 h in A549 cells. Casticin induced the expressions and nuclear translocation of p-H2AX in A549 cells by confocal laser microscopy. Casticin reduced cell number through DNA damage and condensation in human lung cancer A549 cells.

Gefitinib and curcumin-loaded nanoparticles enhance cell apoptosis in human oral cancer SAS cells in vitro and inhibit SAS cell xenografted tumor in vivo.

Curcumin (Cur), a natural product, has been shown to have anti-tumor activities in many human cancer cells. Gefitinib (Gef) is a clinical drug for cancer patients. However, there is no available information to show whether Gef/Cur nanoparticles (NPs) increased cell apoptosis and anti-tumor effects on xenograft mice model in vivo. In this study, γ-polyglutamic acid-coated nanoparticles loaded with Gef and Cur (γ-PGA-Gef/Cur NPs) were developed and its physicochemical properties and antitumor effects were investigated in vitro and in vivo. The γ-PGA-Gef/Cur NPs showed 548.5 ±â€¯93.7 nm in diameter and -40.3 ±â€¯3.87 mV on surface charge. The loading efficiencies of Gef and Cur were 89.5 and 100%, respectively. γ-PGA-Gef/Cur NPs could be internalized into SAS cells and significantly decreased total cell viability of SAS cells. Western blotting results indicated that both free Gef/Cur and γ-PGA-Gef/Cur NPs induced apoptotic cell death via caspase- and mitochondria-dependent pathways. In vivo studies indicated that treatments of PLGA NPs, free Gef/Cur, and γ-PGA-Gef/Cur NPs did not significantly affect appearances and bodyweights of mice. But the γ-PGA-Gef/Cur NPs significantly suppressed tumor size when comparing to free Gef/Cur-treated group. The nanoparticles developed in this study may be used as a potential therapy for oral cancer.

Ursolic acid induces apoptosis and autophagy in oral cancer cells.

Oral squamous cell carcinoma (OSCC) is the fifth common cause of cancer mortality in Taiwan with high incidence and recurrence and needs new therapeutic strategies. In this study, ursolic acid (UA), a triterpenoid, was examined the antitumor potency in OSCC cells. Our results showed that UA inhibited the proliferation of OSCC cells in a dose- and time-dependent manner in both Ca922 and SCC2095 oral cancer cells. UA induced caspase-dependent apoptosis accompanied with the modulation of various biological biomarkers including downregulating Akt/mTOR/NF-κB signaling, ERK, and p38. In addition, UA inhibited angiogenesis as evidenced by abrogation of migration/invasion and blocking MMP-2 secretion in Ca922 cells. Interestingly, UA induced autophagy in OSCC cells, as manifested by LC3B-II conversion and increased p62 expression and accumulation of autophagosomes. Inhibition by autophagy inhibitor enhanced UA-mediated apoptosis in Ca922 cells. The experiment provides a rationale for using triterpenoid in the treatment of OSCC.

Casticin Induces DNA Damage and Impairs DNA Repair in Human Bladder Cancer TSGH-8301 Cells.

BACKGROUND/AIM: Casticin shows anti-cancer effects in many types of cancer. However, there is no information regarding its role in DNA damage in human bladder cancer. The aim of this study was to investigate the effects of casticin on TSGH-8301 cells in vitro. MATERIALS AND METHODS: Viability of cells was assayed by flow cytometry. DNA damage was assayed by DAPI staining, comet assay, and gel electrophoresis. Protein levels were examined by western blotting and confocal laser microscopy. RESULTS: Casticin decreased viability of cells and induced DNA damage. Furthermore, casticin decreased expression of p-ATM, p-ATR, MDC1 and MGMT levels after 48 h of treatment, however, it increased p-ATR and MGMT levels after 12 h. In contrast, casticin increased the levels of p-p53, p-H2A.X, and PARP after 48 h of treatment. As shown by confocal microscopy, casticin affected the translocation of DNA-PKcs and p-p53 to the nucleus of TSGH-8301 cells. CONCLUSION: Casticin decreased viability of human bladder cancer cells through DNA damage.

Etomidate Suppresses Invasion and Migration of Human A549 Lung Adenocarcinoma Cells.

BACKGROUND/AIM: Etomidate, an intravenous anesthetic, has been shown to have anticancer effects, including induction of cell-cycle arrest and apoptosis. However, to our knowledge, there are no reports about the anti-metastasis effects of etomidate on A549 human lung adenocarcinoma cells. MATERIALS AND METHODS: The cell viability, cell adhesion, gelatin zymography assay, transwell migration and invasion assay, and western blotting analysis were used to investigate the effects of etomidate on A549 cells. RESULTS: In our study, etomidate showed low cytotoxicity, inhibited cell adhesion, and suppressed the migration and invasion in A549 cells. The activity of matrix metallopeptidase 2 (MMP2) was reduced by 48 h treatment of etomidate. Results of western blotting analysis indicated that etomidate down-regulated the expression of protein kinase C, MMP7, MMP1, MMP9, and p-p-38, but up-regulated that of RAS, phosphoinositide 3-kinase, and phosphor-extracellular-signal related kinase after 24 and 48 h treatment, in A549 cells. CONCLUSION: Etomidate suppressed the migration and invasion of lung adenocarcinoma A549 cells via inhibiting the expression of MMP1, MMP2, MMP7 and MMP9, and provides potential therapeutic targets for lung cancer treatment.

Benzyl Isothiocyanate Induces Apoptotic Cell Death Through Mitochondria-dependent Pathway in Gefitinib-resistant NCI-H460 Human Lung Cancer Cells In Vitro.

BACKGROUND/AIM: Gefitinib is used to treat patients with lung cancer, but in some patients, the disease becomes gefitinib-resistant. Benzyl isothiocyanate (BITC), found in cruciferous vegetables, has shown anticancer activity in many human cancer cell lines. However, the effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells in vitro have not been investigated. MATERIALS AND METHODS: The effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells were investigated in vitro. Flow cytometric assay was used for determining the total viable cell number, apoptotic cell death, the production of reactive oxygen species (ROS) and Ca2+, mitochondriaI membrane potential (Ψm) and caspase-3, -8 and -9 activities. Furthermore, 4', 6-diamidino-2-phenylindole staining was used to examine chromatin condensation in NCI-H460 and NCI-H460/G cells. RESULTS: BITC reduced total viable cell number via the induction of apoptotic cell death, that was also confirmed by annexin V/propidium iodide double staining assay. BITC increased ROS and Ca2+ production, reduced Ψm and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting assay also showed that BITC increased expression of cleaved caspase-3 and -9, cytochrome c, BCL2-associated X protein, endonuclease G, poly (ADP-ribose) polymerase, growth arrest and DNA-damage protein 153, caspase-7 and activating transcription factor 6 alpha, but reduced apoptosis-inducing factor and caspase-9, BH3-interacting domain death agonist, calpain 1, glucose-regulated protein 78 and inositol requiring enzyme 1 alpha in NCI-H460/G cells. CONCLUSION: BITC-induced apoptotic cell death appears to occur via caspase- and mitochondria-dependent pathways in both cell lines.

Curcuminoids Induce Reactive Oxygen Species and Autophagy to Enhance Apoptosis in Human Oral Cancer Cells.

Numerous studies support the use of herbal medicine or natural products for chemotherapy in human cancers. Reports have associated curcumin (CUR), dimethoxy curcumin (DMC) and bisdemethoxycurcumin (BDMC) with numerous biological activities including anticancer activities, but no available information have shown that these induced apoptotic cell death and autophagy in human oral cancer cells. In the present study, we investigated the effect of CUR, DMC and BDMC on the cell viability, apoptotic cell death, reactive oxygen species (ROS), Ca[Formula: see text], mitochondria membrane potential (MMP) and caspase activities using flow cytometry assay and autophagy by monodansylcadaverine (MDC) and acridine orange (AO) staining in human oral cancer SAS cells. Results indicated that CUR, DMC and BDMC decreased total viable cell number through the induction of cell autophagy and apoptosis in SAS cells. Cells were pretreated with N-acetyl-cysteine (NAC), 3-methyladenine (3MA), rapamycin and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoro-methylketone (Z-VAD-fmk) and then were treated with CUR, DMC and BDMC that led to increased total viable cell number when compared to CUR, DMC and BDMC treatments only. Results indicated induced apoptotic cell death through ROS, mitochondria-dependent pathway and induction of cell autophagy. Based on those observations, we suggest that CUR, DMC and BDMC could be used as a potential anticancer agent in human oral cancer.

Curcuminoids combined with gefitinib mediated apoptosis and autophagy of human oral cancer SAS cells in vitro and reduced tumor of SAS cell xenograft mice in vivo.

Gefitinib has been used for cancer patients and curcumin (CUR), demethoxycurcumin (DMC), or bisdemethoxycurcumin (BDMC) also shown to induce cancer cell apoptosis. However, no report shows the combination of gefitinib with, CUR, DMC, or BDMC induce cell apoptosis and autophagy in human oral cancer cells. In this study, we investigated the effects of gefitinib with or without CUR, DMC, or BDMC co-treatment on the cell viability, apoptotic cell death, autophagy, mitochondria membrane potential (MMP), and caspase-3 activities by flow cytometry assay and autophagy by acridine orange (AO) staining in human oral cancer SAS cells. Results indicated that gefitinib co-treated with CUR, DMC, or BDMC decreased total viable cell number through the induction of cell apoptosis and autophagy and decreased the levels of MMP and increased caspase-3 activities in SAS cells. Western blotting indicated that gefitinib combined with CUR, DMC, or BDMC led to decrease Bcl-2 protein expression which is an antiapoptotic protein and to increase ATG5, Beclin 1, p62/SQSTM1, and LC3 expression that associated with cell autophagy in SAS cells. Gefitinib combined with CUR and DMC led to significantly reduce the tumor weights and volumes in SAS cell xenograft nude mice but did not affect the total body weights. Based on those observations, we suggest that the combination of gefitinib with CUR, DMC, and BDMC can be a potential anticancer agent for human oral cancer in future.

Phenethyl Isothiocyanate Induces Apoptotic Cell Death Through the Mitochondria-dependent Pathway in Gefitinib-resistant NCI-H460 Human Lung Cancer Cells In Vitro.

BACKGROUND/AIM: Some lung cancer patients treated with gefitinib develop resistance to this drug resulting in unsatisfactory treatment outcomes. Phenethyl isothiocyanate (PEITC), present in our common cruciferous vegetables, exhibits anticancer activities in many human cancer cell lines. Currently, there is no available information on the possible modification of gefitinib resistance of lung cancer in vitro by PEITC. Thus, the effects of PEITC on gefitinib resistant lung cancer NCI-H460 cells were investigated in vitro. MATERIALS AND METHODS: The total cell viability, apoptotic cell death, production of reactive oxygen species (ROS) and Ca2+, levels of mitochondria membrane potential (ΔΨm) and caspase-3, -8 and -9 activities were measured by flow cytometry assay. PEITC induced chromatin condensation was examined by DAPI staining. RESULTS: PEITC-induced cell morphological changes, decreased total viable cell number and induced apoptotic cell death in NCI-H460 and NCI-H460/G cells. PEITC decreased ROS production in NCI-H460 cells, but increased production in NCI-H460/G cells. PEITC increased Ca2+ production, decreased the levels of ΔΨm and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting was used to examine the effect of apoptotic cell death associated protein expression in NCI-H460 NCI-H460/G cells after exposure to PEITC. Results showed that PEITC increased expression of cleaved caspase-3, PARP, GADD153, Endo G and pro-apoptotic protein Bax in NCI-H460/G cells. CONCLUSION: Based on these results, we suggest that PEITC induces apoptotic cell death via the caspase- and mitochondria-dependent pathway in NCI-H460/G cells.

Deguelin Impairs Cell Adhesion, Migration and Invasion of Human Lung Cancer Cells through the NF-[Formula: see text]B Signaling Pathways.

Deguelin, a rotenoid, is isolated from a natural plant species, and has biological activities including antitumor function. In the present study, we investigated the effect of deguelin on the cell adhesion, migration and invasion of NCI-H292 human lung cancer cells in vitro. Cell viability was analyzed by using flow cytometer. Cell adhesion was determined by using the cell-matrix adhesion assay. Wound healing assay was used to examine cell migration. Cell migration and invasion were investigated using a Boyden chamber assay. The protein expression was measured by Western blotting and confocal laser microscopy. The electrophoretic mobility shift assay was used to measure NF-[Formula: see text]B p65 binding to DNA.We selected the concentrations of deguelin at 0, 0.5, 1.0, 1.5, 2.0 and 2.5[Formula: see text][Formula: see text]M and we found that those concentrations of deguelin did not induce significant cytotoxic effects on NCI-H292 cells. Thus, we selected those concentrations of deguelin for metastasis assay. We found that deguelin inhibited cell adhesion, migration and invasion in dose-dependent manners that was assayed by wound healing and transwell methods, respectively. Deguelin decreased the expression of MMP-2/-9, SOS 1, Rho A, p-AKT (Thr308), p-ERK1/2, p-p38, p-JNK, NF-[Formula: see text]B (p65) and uPA in NCI-H292 cells. Deguelin suppressed the expression of PI3K, SOS 1, NF-[Formula: see text]B (p65), but did not significantly affect PKC and Ras in the nuclei of NCI-H292 cells that were confirmed by confocal laser microscopy. We suggest that deguelin may be used as a novel anticancer metastasis of lung cancer in the future.

Tetrandrine Induces Apoptosis in Human Nasopharyngeal Carcinoma NPC-TW 039 Cells by Endoplasmic Reticulum Stress and Ca2+/Calpain Pathways.

Tetrandrine is an alkaloid extracted from a traditional China medicine plant, and is considered part of food therapy as well. In addition, it has been widely reported to induce apoptotic cell death in many human cancer cells. However, the mechanism of Tetrandrine on human nasopharyngeal carcinoma cells (NPC) is still questioned. In our study, we examined whether Tetrandrine can induce apoptosis of NPC-TW 039 cells. We found that cell morphology was changed after treatment with different concentrations of Tetrandrine. Further, we indicated that the NPC-TW 039 cells viability decreased in a Tetrandrine dose-dependent manner. We also found that tetrandrine induced cell cycle arrest in G0/G1 phase. Tetrandrine induced DNA condensation by DAPI staining as well. In addition, we found that Tetrandrine induced Ca2+ release in the cytosol. At the same time, endoplasmic reticulum (ER) stress occurred. Then we used western blotting to examine the protein expression which is associated with mitochondria-mediated apoptotic pathways and caspase-dependent pathways. To further examine whether Ca2+ was released or not with Tetrandrine induced-apoptosis, we used the chelator of Ca2+ and showed that cell viability increased. At the same time, caspase-3 expression was decreased. Furthermore, confocal microscopy examination revealed that Tetrandrine induced expression of ER stress-related proteins GADD153 and GRP78. Our results indicate that Tetrandrine induces apoptosis through calcium-mediated ER stress and caspase pathway in NPC-TW 039 cells. In conclusion, Tetrandrine may could be used for treatment of human nasopharyngeal carcinoma in future.

Phenethyl Isothiocyanate (PEITC) and Benzyl Isothiocyanate (BITC) Inhibit Human Melanoma A375.S2 Cell Migration and Invasion by Affecting MAPK Signaling Pathway In Vitro.

BACKGROUND/AIM: Numerous evidence has shown that PEITC and BITC inhibit cancer cell migration and invasion. In this study, we investigated the anti-metastatic mechanisms of PEITC and BITC in human melanoma cancer A375.S2 cells in vitro. MATERIALS AND METHODS: We used a cell viability assay, an in-vitro scratch wound healing assay, a transwell assay for cell migration and invasion, a gelatin zymography assay, western blotting and EMSA to examine the anti-metastatic mechanisms of PEITC and BITC in A375.S2 cells. RESULTS: Sublethal concentrations of PEITC (0, 1, 2 and 2.5 µM) and BITC (0, 0.5, 1 and 2 µM) inhibited mobility, migration and invasion of A375.S2 cells that were assayed by wound healing and Transwell filter. PEITC and BITC inhibited MMP-2 activity in A375.S2 cells, as assessed by gelatin zymography assay. Results from western blotting indicated that PEITC (2.5 µM) and BITC (2 µM) decreased the levels of p-p38 following 24 and 48 h treatment. PEITC (1-2.5 µM) reduced the levels of p-JNK1/2 proteins following 48-h treatment but BITC increased p-JNK1/2 levels following 24-h treatment. PEITC (2.5 µM) reduced the levels of p-ERK1/2 proteins following 48-h treatment but BITC (0.5-2 µM) increased p-ERK1/2 levels following 24- and 48-h treatment. PEITC and BITC affect cell migration and invasion of A375.S2 cells via MAPK pathway. PEITC and BITC inhibited MMP-2 activity. PEITC increased NF-κB expression but BITC decreased NF-κB expression in the nucleus. Furthermore, NF-κB p65 binding to DNA was decreased following 2.5 µM PEITC treatment, but increased following treatment with 1-2 µM. However, 0.5-2 µM BITC treatment decreased the binding of NF-κB to DNA in A375.S2 cells, as assessed by electrophoretic mobility shift (EMSA) assay. CONCLUSION: Based on these observations, we suggest that PEITC and BITC can be used as anti-metastastic agents of human melanoma cells in the future.

Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro.

Benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC) have been demonstrated to induce anticancer function in many human cancer cells and also inhibit cancer cell migration and invasion. However, there are no studies that show BITC and PEITC to inhibit cell migration and invasion in mouse melanoma B16F10 cells. In this study, we investigated anti-metastasis effects of BITC and PEITC in melanoma cancer cells in vitro. Under sub-lethal concentrations (from 1, 2.5 up to 5 µM), BITC and PEITC significantly inhibited cell mobility, migration and invasion nature of B16F10 cells. Gelatin zymography assay also showed that BITC and PEITC inhibited matrix metalloproteinase-2 (MMP-2) activity in B16F10 cells. PEITC reduced MAPK signaling associated proteins such as p-ERK1/2, p-p38 and p-JNK1/2 but BITC increased those MAPK signaling associated proteins. BITC and PEITC both suppressed the expression of RhoA, Ras, and SOS-1, however, PEITC increased FAK and GRB2 but BITC increased FAK at 48 h. Furthermore, PEITC decreased the expression of MMP-2 and tissue inhibitors of matrix metalloproteinases (TIMP) but BITC increased them. PEITC inhibited NF-κB protein levels and DNA binding which was confirmed by electrophoretic mobility shift (EMSA) assay. Based on these observations, we suggest that BITC and PEITC can be used in anti-metastasis of melanoma cells in the future.

Bufalin induces apoptosis in vitro and has Antitumor activity against human lung cancer xenografts in vivo.

Bufalin has been shown to be effective against a variety of cancer cells, but its role in lung cancer has never been studied in an animal model. In this study, we evaluated bufalin effects in a human lung cancer cell line NCI-H460 both in vitro and in vivo. Bufalin caused significant cytotoxicity in NCI-H460 cells at a concentration as low as 1 µM. DNA condensation was observed in bufalin-treated cells in a dose-dependent manner. Mitochondrial membrane potential (ΔΨm ) was reduced and reactive oxygen species (ROS) were increased in bufalin-treated NCI-H460 cells. Levels of several proapoptotic proteins such as Fas, Fas-ligand, cytochrome c, apoptosis protease activating factor-1, endonuclease G, caspase-3 and caspase-9 were increased after bufalin treatment. At the same time, anti-apoptotic B-cell lymphoma 2 protein levels were reduced. Bufalin decreased glucose regulated protein-78 gene expression but increased growth arrest- and DNA damage-inducible 153 gene expression. Bufalin injected intraperitoneally in a dose-dependent manner reduced tumor size in BALB/C nu/nu mice implanted with NCI-H460 cells. Bufalin injection did not produce significant drug-related toxicity in experimental animals except at a high dose (0.4 mg kg-1 ). In conclusion, low concentrations of bufalin can induce apoptosis in the human lung cancer cell line NCI-H460 in vitro. Bufalin also reduced tumor size in mice injected with NCI-H460 cells without significant drug-related toxicity. These results indicate that bufalin may have potential to be developed as an agent for treating human non-small cell lung cancer. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1305-1317, 2017.

Bufalin inhibits gefitinib resistant NCI-H460 human lung cancer cell migration and invasion in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Bufalin, a component of Chan Su (frog), has been shown to have biological activities including anti-tumor effects. Gefitinib has been used as an anti-cancer drug in lung cancer patients; however, some patients eventually become gefitinib resistant. AIM OF THE STUDY: In this study, we investigated anti-metastasis effects of bufalin in gefitinib resistant NCI-H460 lung cancer cells. MATERIALS AND METHODS: The effects of the bufalin in gefitinib resistant NCI-H460 lung cancer cells were investigated on cell viability using flow cytometry. The adhesion capacity, wound healing assay, invasion and migration assay, and Western blot analysis were used to understand the molecular mechanisms in this study RESULTS: Under sub-lethal concentrations (from 2.5 up to 10nM), bufalin significantly inhibits cell adhension, migration and invasion nature of gefitinib resistant H460 cells. Western blotting assay revealed that bufalin depressed some of the key metastasis-related proteins, such as SOS-1, MMP-2 and Rho A underwent significant reduction. Phosphorylated Focal adhesion kinase (p-FAK), phosphorylated extracellular signal-regulated kinase (p-ERK1/2), Ras and E-cadherin were significantly reduced at 48h treatment. However, phosphorylated p38 (p-p38), phosphorylated c-Jun NH2-terminal kinase (p-JNK1/2) and NF-κBp65 were increased. CONCLUSIONS: Based on these observations, we suggest that bufalin can be used in anti-metastasis of gefitinib resistant NCI-H460 lung cancer cells in the future.