Excess iron accumulation mediated senescence in diabetic kidney injury.

Cellular senescence and iron accumulation were separately observed in diabetic nephropathy (DN). Limited evidence supports that iron was significantly accumulated in senescent cells. We aimed to explore whether iron is involved in the pathogenesis role of senescence in DN. Renal cells were treated with high glucose (HG, 35 mM) for 10 or 15 days, and DN mice were induced by high-fat diet and streptozotocin. Gene ontology enrichment, gene set enrichment analysis analysis, ß-galactosidase staining, 5-ethynyl-2-deoxyuridine staining, and western blot depicted the upregulated senescence pathway in vitro and in vivo of DN. Lactate dehydrogenase (LDH) release was increased by HG and reversed by p16/p21 knockdown, and the supernatant of HG-treated cells caused increased LDH release from normal cells. Iron metabolism-related protein expression was disordered after HG exposure concomitant with senescence. Ferric ammonium citrate (50 µM) upregulated gamma-H2A.X variant histone and increased the senescence markers in HG-treated cells. The treatment of deferoxamine (0.5 µM) had the opposite effect. Compared to the non-DN individual, increased ferritin and senescence markers were verified in DN mice and patients, and the co-localization of ferritin and senescence markers was observed by immunofluorescence. These results suggested that accumulated iron was correlated with aggravated DNA damage and accelerated senescence, and revealed the role of iron in the cellular senescence of diseases.

Allyl isothiocyanate induces DNA damage and inhibits DNA repair-associated proteins in a human gastric cancer cells in vitro.

Allyl isothiocyanate (AITC) is abundant in cruciferous vegetables and it present pharmacological activity including anticancer activity in many types of human cancer cells in vitro and in vivo. Currently, no available information to show AITC affecting DNA damage and repair-associated protein expression in human gastric cancer cells. Therefore, in the present studies, we investigated AITC-induced cytotoxic effects on human gastric cancer in AGS and SNU-1 cells whether or not via the induction of DNA damage and affected DNA damage and repair associated poteins expressions in vitro. Cell viability and morphological changes were assayed by flow cytometer and phase contrast microscopy, respectively, the results indicated AITC induced cell morphological changes and decreased total viable cells in AGS and SNU-1 cells in a dose-dependently. AITC induced DNA condensation and damage in a dose-dependently which based on the cell nuclei was stained by 4', 6-diamidino-2-phenylindole present in AGS and SNU-1 cells. DNA damage and repair associated proteins expression in AGS and SNU-1 cells were measured by Western blotting. The results indicated AITC decreased nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), glutathione, and catalase, but increased superoxide dismutase (SOD (Cu/Zn)), and nitric oxide synthase (iNOS) in AGS cells, however, in SNU-1 cells are increased HO-1. AITC increased DNA-dependent protein kinase (DNA-PK), phosphorylation of gamma H2A histone family member X on Ser139 (γH2AXpSer139 ), and heat shock protein 90 (HSP90) in AGS cells. AITC increased DNA-PK, mediator of DNA damage checkpoint protein 1 (MDC1), γH2AXpSer139 , topoisomerase II alpha (TOPIIα), topoisomerase II beta (TOPIIß), HSP90, and heat shock protein 70 (HSP70) in SNU-1 cells. AITC increased p53, p53pSer15 , and p21 but decreased murine double minute 2 (MDM2)pSer166 and O6 -methylguanine-DNA methyltransferase (MGMT) in AGS cells; however, it has a similar effect of AITC except increased ataxia telangiectasia and Rad3 -related protein (ATR)pSer428 , checkpoint kinase 1 (CHK1), and checkpoint kinase 2 (CHK2) in SNU-1 cells. Apparently, both cell responses to AITC are different, nonetheless, all of these observations suggest that AITC inhibits the growth of gastric cancer cells may through induction off DNA damage in vitro.

Phenethyl isothiocyanate and irinotecan synergistically induce cell apoptosis in colon cancer HCT 116 cells in vitro.

Irinotecan (IRI), an anticancer drug to treat colon cancer patients, causes cytotoxic effects on normal cells. Phenethyl isothiocyanate (PEITC), rich in common cruciferous plants, has anticancer activities (induction of cell apoptosis) in many human cancer cells, including colon cancer cells. However, the anticancer effects of IRI combined with PEITC on human colon cancer cells in vitro were unavailable. Herein, the aim of this study is to focus on the apoptotic effects of the combination of IRI and PEITC on human colon cancer HCT 116 cells in vitro. Propidium iodide (PI) exclusion and Annexin V/PI staining assays showed that IRI combined with PEITC decreased viable cell number and induced higher cell apoptosis than that of IRI or PEITC only in HCT 116 cells. Moreover, combined treatment induced higher levels of reactive oxygen species (ROS) and Ca2+ than that of IRI or PEITC only. Cells pre-treated with N-acetyl-l-cysteine (scavenger of ROS) and then treated with IRI, PEITC, or IRI combined with PEITC showed increased viable cell numbers than that of IRI or PEITC only. IRI combined with PEITC increased higher caspase-3, -8, and -9 activities than that of IRI or PEITC only by flow cytometer assay. IRI combined with PEITC induced higher levels of ER stress-, mitochondria-, and caspase-associated proteins than that of IRI or PEITC treatment only in HCT 116 cells. Based on these observations, PEITC potentiates IRI anticancer activity by promoting cell apoptosis in the human colon HCT 116 cells. Thus, PEITC may be a potential enhancer for IRI in humans as an anticolon cancer drug in the future.

Allyl isothiocyanate inhibits cell migration and invasion in human gastric cancer AGS cells via affecting PI3K/AKT and MAPK signaling pathway in vitro.

Metastasis is commonly occurred in gastric cancer, and it is caused and responsible for one of the major cancer-related mortality in gastric cancer patients. Allyl isothiocyanate (AITC), a natural product, exhibits anticancer activities in human many cancer cells, including gastric cancer. However, no available report shows AITC inhibits gastric cancer cell metastasis. Herein, we evaluated the impact of AITC on cell migration and invasion of human gastric cancer AGS cells in vitro. AITC at 5-20 µM did not induce significant cell morphological damages observed by contrast-phase microscopy but decreased cell viability assayed by flow cytometry. After AGS cells were further examined by atomic force microscopy (AFM), which indicated AITC affected cell membrane and morphology in AGS cells. AITC significantly suppressed cell motility examined by scratch wound healing assay. The results of the gelatin zymography assay revealed that AITC significantly suppressed the MMP-2 and MMP-9 activities. In addition, AITC suppressed cell migration and invasion were performed by transwell chamber assays at 24 h in AGS cells. Furthermore, AITC inhibited cell migration and invasion by affecting PI3K/AKT and MAPK signaling pathways in AGS cells. The decreased expressions of p-AKTThr308 , GRB2, and Vimentin in AGS cells also were confirmed by confocal laser microscopy. Our findings suggest that AITC may be an anti-metastasis candidate for human gastric cancer treatment.

Autophagy and exosomes coordinately mediate quercetin's protective effects on alcoholic liver disease.

Alcoholic liver disease (ALD), a spectrum of liver abnormalities induced by chronic alcohol abuse, continues to be the major cause of life-threatening liver disease in developed countries. Autophagy and exosomes were individually confirmed to be involved in the pathogenesis of ALD. Here, we sought to identify the role of autophagy and exosomes in the liver protective effects of quercetin. We observed decreased hepatic LC3II/LC3I and increased p62 level in ethanol-fed mice, and these changes were alleviated by quercetin. Meanwhile, nanoparticle tracking analysis (NTA) showed elevated serum exosomes numbers in ethanol-fed mice, which was combated by quercetin. Ethanol induced elevated LDH, ALT, and AST in HepG2 supernatant, which was alleviated by cytochalasin D (exosomes uptake inhibitor). Moreover, quercetin reduced ethanol-induced LDH and ALT elevation in vitro, and the effects of quercetin were reversed by Rab27a overexpression (induce exosomes release) or wortmannin treatment (autophagy inhibitor). Transcriptomic analysis supported that quercetin reversed the change of lysosome related genes disturbed by ethanol. Meanwhile, western blot analysis exhibited decreased hepatic expression of LAMP2 and ATPA6V1B2, and active Cathepsin B/Cathepsin B by quercetin treatment, indicating quercetin alleviated lysosome dysfunction in ethanol-fed mice. Baf A treatment or transfection of siTFEB offset quercetin's effects in ethanol-induced LDH and ALT elevation, exosomes release, and autophagy inhibition (LC3II/I and p62 accumulation). Taken together, quercetin coordinately activates autophagy and combats exosomes release by restoring lysosome function, and further mitigates ethanol-induced liver damage.

PW06 Triggered Fas-FADD to Induce Apoptotic Cell Death In Human Pancreatic Carcinoma MIA PaCa-2 Cells through the Activation of the Caspase-Mediated Pathway.

Pancreatic cancer has higher incidence and mortality rates worldwide. PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one] is a carbazole derivative containing chalcone moiety which was designed for inhibiting tumorigenesis in human pancreatic cancer. This study is aimed at investigating PW06-induced anticancer effects in human pancreatic cancer MIA PaCa-2 cells in vitro. The results showed PW06 potent antiproliferative/cytotoxic activities and induced cell morphological changes in a human pancreatic cancer cell line (MIA PaCa-2), and these effects are concentration-dependent (IC50 is 0.43 µM). Annexin V and DAPI staining assays indicated that PW06 induced apoptotic cell death and DNA condensation. Western blotting indicated that PW06 increased the proapoptotic proteins such as Bak and Bad but decreased the antiapoptotic protein such as Bcl-2 and Bcl-xL. Moreover, PW06 increased the active form of caspase-8, caspase-9, and caspase-3, PARP, releasing cytochrome c, AIF, and Endo G from mitochondria in MIA PaCa-2 cells. Confocal laser microscopy assay also confirmed that PW06 increased Bak and decreased Bcl-xL. Also, the cells were pretreated with inhibitors of caspase-3, caspase-8, and caspase-9 and then were treated with PW06, resulting in increased viable cell number compared to PW06 treated only. Furthermore, PW06 showed a potent binding ability with hydrophobic interactions in the core site of the Fas-Fas death domains (FADD). In conclusion, PW06 can potent binding ability to the Fas-FADD which led to antiproliferative, cytotoxic activities, and apoptosis induction accompanied by the caspase-dependent and mitochondria-dependent pathways in human pancreatic cancer MIA PaCa-2 cells.

Iron-frataxin involved in the protective effect of quercetin against alcohol-induced liver mitochondrial dysfunction.

Emerging evidence supports the beneficial effect of quercetin on liver mitochondrial disorders. However, the molecular mechanism by which quercetin protects mitochondria is limited, especially in alcoholic liver disease. In this study, C57BL/6N mice were fed with Lieber De Carli liquid diet (28% ethanol-derived calories) for 12 weeks plus a single binge ethanol and intervened with quercetin (100 mg/kg.bw). Moreover, HepG2CYP2E1+/+ were stimulated with ethanol (100 mM) and quercetin (50 µM) to investigate the effects of mitochondrial protein frataxin. The results indicated that quercetin alleviated alcohol-induced histopathological changes and mitochondrial functional disorders in mice livers. Consistent with increased PINK1, Parkin, Bnip3 and LC3II as well as decreased p62, TOM20 and VDAC1 expression, the inhibition of mitophagy by ethanol was blocked by quercetin. Additionally, quercetin improved the imbalance of iron metabolism-related proteins expression in alcohol-fed mice livers. Compared with ethanol-treated Lv-empty HepG2CYP2E1+/+ cells, frataxin deficiency further exacerbated the inhibition of mitochondrial function. Conversely, restoration of frataxin expression ameliorated the effect of ethanol. Furthermore, frataxin deficiency reduced the protective effects of quercetin on mitochondria disordered by ethanol. Attentively, ferric ammonium citrate (FAC) and deferiprone decreased or increased frataxin expression in HepG2CYP2E1+/+, respectively. Notably, we further found FAC reversed the increasing effect of quercetin on frataxin expression. Ultimately, silencing NCOA4 attenuated the inhibition of quercetin on LDH release and mitochondrial membrane potential increase, and similar results were observed by adding FAC. Collectively, these findings demonstrated quercetin increased frataxin expression through regulating iron level, thereby mitigating ethanol-induced mitochondrial dysfunction.

Bisdemethoxycurcumin suppresses human osteosarcoma U­2 OS cell migration and invasion via affecting the PI3K/Akt/NF­κB, PI3K/Akt/GSK3ß and MAPK signaling pathways in vitro.

The metastasis of human osteosarcoma (OS) shows a difficult­to­treat clinical scenario and results in decreased quality of life and diminished survival rates. Finding or developing novel treatments to improve the life quality of patients is urgent. Bisdemethoxycurcumin (BDMC), a natural product, was obtained from the rhizome of turmeric (Curcuma longa) and exerts antitumor activities in numerous human cancer cell lines. At present, there is no study showing BDMC effects on OS cell migration and invasion. In the present study, the effects of BDMC on cell migration and invasion of OS U­2 OS cells were investigated in vitro. Cell viability and proliferation were measured by flow cytometric and MTT assays, respectively. Cell motility, MMP­2 and ­9 activity, and cell migration and invasion were assayed by scratch wound healing, gelatin zymography, and Transwell chamber assays, respectively. The protein expression levels were measured by western blotting. BDMC at 20 and 40 µM significantly reduced total cell viability, and BDMC at 5 and 10 µM significantly inhibited cell motility in U­2 OS cells. BDMC significantly suppressed the activities of MMP­2 and MMP­9 in U­2 OS cells. BDMC suppressed cell invasion and migration after 24 h treatment in U­2 OS cells, and these effects were in a dose­dependently manner. Results from western blotting indicated that BDMC significantly decreased the protein expression levels of PI3K/Akt/NF­κB, PI3K/Akt/GSK3ß, and MAPK pathway in U­2 OS cells. Furthermore, BDMC inhibited uPA, MMP­2, MMP­9, MMP­13, N­cadherin, VE­cadherin, and vimentin but increased E­cadherin in U­2 OS cells. Based on these observations, it was suggested that BDMC may be a potential candidate against migration and invasion of human OS cells in the future.

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model.

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.

Phenethyl Isothiocyanate Suppresses the Proinflammatory Cytokines in Human Glioblastoma Cells through the PI3K/Akt/NF-κB Signaling Pathway In Vitro.

Phenethyl isothiocyanate (PEITC), extracted from cruciferous vegetables, showed anticancer activity in many human cancer cells. Our previous studies disclosed the anticancer activity of PEITC in human glioblastoma multiforme (GBM) 8401 cells, including suppressing the cell proliferation, inducing apoptotic cell death, and suppressing cell migration and invasion. Furthermore, PEITC also inhibited the growth of xenograft tumors of human glioblastoma cells. We are the first to investigate PEITC effects on the receptor tyrosine kinase (RTK) signaling pathway and the effects of proinflammatory cytokines on glioblastoma. The cell viability was analyzed by flow cytometric assay. The protein levels and mRNA expressions of cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), were determined by enzyme-linked immunosorbent assay (ELISA) reader and real-time polymerase chain reaction (PCR) analysis, respectively. Furthermore, nuclear factor-kappa B- (NF-κB-) associated proteins were evaluated by western blotting. NF-κB expression and nuclear translocation were confirmed by confocal laser microscopy. NF-κB binding to the DNA was examined by electrophoretic mobility shift assay (EMSA). Our results indicated that PEITC decreased the cell viability and inhibited the protein levels and expressions of IL-1ß, IL-6, and TNF-α genes at the transcriptional level in GBM 8401 cells. PEITC inhibited the binding of NF-κB on promoter site of DNA in GBM 8401 cells. PEITC also altered the protein expressions of protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and NF-κB signaling pathways. The inflammatory responses in human glioblastoma cells may be suppressed by PEITC through the phosphoinositide 3-kinase (PI3K)/Akt/NF-κB signaling pathway. Thus, PEITC may have the potential to be an anti-inflammatory agent for human glioblastoma in the future.

Combination Treatment of Sorafenib and Bufalin Induces Apoptosis in NCI-H292 Human Lung Cancer Cells In Vitro.

BACKGROUND/AIM: Lung cancer notably contributes to tumor-associated mortality worldwide, and standard chemotherapy is used for lung cancer patients. However, its therapeutic efficacy remains unsatisfactory. This study aimed to evaluate the effects and molecular mechanisms of sorafenib and bufalin combination therapy on lung cancer cells in vitro. MATERIALS AND METHODS: NCI-H292 cells were treated with sorafenib, bufalin, and sorafenib in combination with bufalin. Cell viability, ROS production, Ca2+ release, and mitochondrial membrane potential were examined by flow cytometric assay. Annexin V/PI staining and chromatin condensation were examined by the apoptosis assays. Finally the molecular mechanism of apoptosis-associated protein expression was investigated by western blotting. RESULTS: NCI-H292 cells treated with sorafenib in combination with bufalin showed significantly decreased viability, enhanced cellular apoptosis, and DNA condensation when compared to that with sorafenib or bufalin alone. Moreover, the combination treatment exhibited higher reactive oxygen species (ROS) production and lower mitochondrial membrane potential (ΔΨm). The combined treatment resulted in higher expression of SOD but lower catalase compared to sorafenib treatment alone. Compared to sorafenib or bufalin treatment alone, the combination treatment resulted in lower Bcl-2 expression but higher Bax, Bad, APAF-1, caspase-3, and caspase-9. CONCLUSION: Sorafenib in combination with bufalin shows more potent cytotoxic effects and cell apoptosis than sorafenib or bufalin treatment alone in NCI-H292 cells. The combined treatment significantly enhanced apoptotic cell death in NCI-H292 lung cancer cells by activating ROS-, mitochondria-, and caspase-signaling pathways in vitro.

Association between the Oxidative Balance Score and Telomere Length from the National Health and Nutrition Examination Survey 1999-2002.

PURPOSE: Leukocyte telomere length (LTL) is an important biomarker of aging. The oxidative balance score (OBS) is used to assess the oxidative stress-related exposures of diet and lifestyle. This study is aimed at exploring if the OBS was associated with LTL. METHODS: 3220 adults were included in this study from the National Health and Nutrition Examination Survey (NHANES) 1999-2002. LTL was assayed from leukocyte DNA. Twenty dietary and lifestyle factors were selected to score the OBS. Survey-based multivariable linear regression was conducted to assess the association between the OBS and log-transformed LTL. RESULTS: The association between the OBS and log-transformed LTL was positive in females but not males. For females, compared with the lowest OBS category as a reference, the multivariable-adjusted beta estimate (95% confidence interval, CI) for the highest OBS category was 0.0701 (0.0205-0.1197) (p for trend < 0.01), and the multivariable-adjusted beta estimate (95% CI) of the continuous OBS was 0.0039 (0.0014-0.0065). There was also the gender difference in the correlations of the dietary OBS and the lifestyle OBS with log-transformed LTL. CONCLUSION: There was a positive association between the OBS and LTL in females. This result suggested that diet and lifestyle might affect LTL by regulating oxidative balance.

Bisdemethoxycurcumin Induces Cell Apoptosis and Inhibits Human Brain Glioblastoma GBM 8401/Luc2 Cell Xenograft Tumor in Subcutaneous Nude Mice In Vivo.

Bisdemethoxycurcumin (BDMC) has biological activities, including anticancer effects in vitro; however, its anticancer effects in human glioblastoma (GBM) cells have not been examined yet. This study aimed to evaluate the tumor inhibitory effect and molecular mechanism of BDMC on human GBM 8401/luc2 cells in vitro and in vivo. In vitro studies have shown that BDMC significantly reduced cell viability and induced cell apoptosis in GBM 8401/luc2 cells. Furthermore, BDMC induced apoptosis via inhibited Bcl-2 (anti-apoptotic protein) and increased Bax (pro-apoptotic proteins) and cytochrome c release in GBM 8401/luc2 cells in vitro. Then, twelve BALB/c-nude mice were xenografted with human glioblastoma GBM 8401/luc2 cancer cells subcutaneously, and the xenograft nude mice were treated without and with BDMC (30 and 60 mg/kg of BDMC treatment) every 3 days. GBM 8401/luc2 cell xenografts experiment showed that the growth of the tumors was significantly suppressed by BDMC administration at both doses based on the reduction of tumor size and weights. BDMC did not change the body weight and the H&E histopathology analysis of liver samples, indicating that BDMC did not induce systemic toxicity. Meanwhile, treatment with BDMC up-regulated the expressions of BAX and cleaved caspase-3, while it down-regulated the protein expressions of Bcl-2 and XIAP in the tumor tissues compared with the control group. This study has demonstrated that BDMC presents potent anticancer activity on the human glioblastoma GBM 8401/luc2 cell xenograft model by inducing apoptosis and inhibiting tumor cell proliferation and shows the potential for further development to the anti-GBM cancer drug.

Beyond Isocitrate Dehydrogenase Mutations: Emerging Mechanisms for the Accumulation of the Oncometabolite 2-Hydroxyglutarate.

2-Hydroxyglutarate (2-HG) is an unconventional oncometabolite of α-ketoglutarate. Isocitrate dehydrogenase mutation is generally acknowledged to be the main cause of 2-HG accumulation. In isocitrate dehydrogenase mutant tumors, 2-HG accumulation inhibits α-ketoglutarate/Fe(II)-dependent dioxygenases, resulting in epigenetic alterations. Recently, the increase of 2-HG has also been observed in the cases of mitochondrial dysfunction and hypoxia. In these cases, 2-HG not only inhibits α-ketoglutarate/Fe(II)-dependent dioxygenases to regulate epigenetics but also affects other cellular pathways, such as regulating hypoxia-inducible transcription factors and glycolysis. These provide a new perspective for the study of 2-HG.

Associations of blood and urinary heavy metals with rheumatoid arthritis risk among adults in NHANES, 1999-2018.

Heavy metals exposure has been widely recognized as a risk factor for human health. However, limited information is available about the impacts of heavy metals on rheumatoid arthritis (RA). Herein, we estimated the associations of 3 blood and 11 urinary metals with the risk of RA among 49830 U.S. adults from the National Health and Nutrition Examination Survey (NHANES), 1999-2018. In the single-exposure model, blood cadmium (Cd) and lead (Pb), urinary Cd, Pb, antimony (Sb), tungsten (Tu), and uranium (Ur) were identified to be positively associated with RA risk. Furthermore, weighted quantile sum (WQS) regression, quantile-based g computation (qgcomp), and Bayesian kernel machine regression (BKMR) analyses consistently showed that both blood and urinary metals-mixed exposure were positively correlated with the risk of RA, and highlighted that Cd and Pb were responsible for the outcomes. Such associations were more evident in the young and middle-aged population. These findings indicated that exposure to heavy metals increased RA risk, and advanced the identification of risk factors for RA.

Tetrandrine Suppresses Human Brain Glioblastoma GBM 8401/luc2 Cell-Xenografted Subcutaneous Tumors in Nude Mice In Vivo.

Tetrandrine (TET), a bisbenzylisoquinoline (BBI) alkaloid, is isolated from the plant Stephania tetrandra S. Moore and has a wide range of biological activity, including anticancer properties in vitro and in vivo. At first, we established a luciferase-expressing stable clone that was named GBM 8401/luc2 cells. Herein, the primary results indicated that TET reduced the total cell viability and induced cell apoptosis in GBM 8401/luc2 human glioblastoma cells. However, there is no available information showing that TET suppresses glioblastoma cells in vivo. Thus, we investigated the effects and mechanisms of TET on a GBM 8401/luc2 cell-generated tumor in vivo. After the tumor volume reached 100-120 mm3 in subcutaneously xenografted nude mice, all of the mice were randomly divided into three groups: Group I was treated with phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 25 mg/kg of TET, and Group III with 50 mg/kg of TET. All mice were given the oral treatment of PBS or TET by gavage for 21 days, and the body weight and tumor volumes were recorded every 5 days. After treatment, individual tumors, kidneys, livers, and spleens were isolated from each group. The results showed that TET did not affect the body weights, but it significantly decreased the tumor volumes. The TET treatment at 50 mg/kg had a two-fold decrease in tumor volumes than that at 25 mg/kg when compared to the control. TET decreased the total photon flux, and treatment with TET at 50 mg/kg had a lower total photon flux than that at 25 mg/kg, as measured by a Xenogen IVIS imaging system. Moreover, the higher TET treatment had lower tumor volumes and weights than those of the lower dose. The apoptosis-associated protein expression in the tumor section was examined by immunohistochemical analysis, and the results showed that TET treatment reduced the levels of c-FLIP, MCL-1, and XIAP but increased the signals of cleaved-caspase-3, -8, and -9. Furthermore, the hematoxylin and eosin (H & E) staining of kidney, liver, and spleen tissues showed no significant difference between the TET-treated and control groups. Overall, these observations demonstrated that TET suppressed subcutaneous tumor growth in a nude-mice model via the induction of cell apoptosis.

Demethoxycurcumin Promotes Macrophage Cell Population and Phagocytosis in WEHI-3 Cell-generated Leukemia BALB/c Mice In Vivo.

BACKGROUND/AIM: Demethoxycurcumin (DMC), one of the components of curcuminoids, has antitumor activities in many human cancer cells and is known to induce apoptosis in human leukemia cells. However, there are no reports showing the effects of DMC on the immune response in leukemia mice in vivo. Herein, we evaluated the impact of DMC on immune responses in WEHI-3-generated leukemia mice in vivo. MATERIALS AND METHODS: Fifty male BALB/c mice were separated randomly into five groups. Group I is normal mice, and groups II-V mice of generated leukemia by WEHI-3 cells. Group II-V mice were intraperitoneally injected with dimethyl sulfoxide (DMSO, as the positive control), 15, 30, and 60 mg/kg of DMC, respectively, every two days for 14 days. The body weight, blood, peritoneal fluid, liver, and spleen were individually analyzed. RESULTS: DMC did not significantly affect animal appearance and body weight. It decreased liver and spleen weight at a high dose. DMC did not affect the cluster of differentiation 3 (CD3) and CD19 cell populations but induced decrease of CD11b at 30 mg/kg treatment. However, DMC at low dose significantly increased the cluster of macrophage (Mac-3) cell populations, but at high dose it decreased them. DMC increased macrophage phagocytosis from peripheral blood mononuclear cells at 15 mg/kg treatment and peritoneal cavity at 15, 30 and 60 mg/kg of DMC treatments. DMC did not significantly affect the cytotoxic activity of natural killer (NK) cells. Furthermore, DMC decreased B and T cell proliferation at high doses. CONCLUSION: DMC elevated macrophage phagocytosis in leukemia mice in vivo.

Allyl Isothiocyanate Induces DNA Damage and Impairs DNA Repair in Human Breast Cancer MCF-7 Cells.

BACKGROUND/AIM: Ally lisothiocyanate (AITC), a constituent of naturally occurring isothiocyanates (ITCs) found in some Brassica vegetables, has been previously demonstrated to have anti-carcinogenic activity. However, there is no available information showing that AITC induces DNA damage and alters DNA damage repair proteins in human breast cancer MCF-7 cells. MATERIALS AND METHODS: In the present study, we investigated the effects of AITC on DNA damage and repair responses in human breast cancer MCF-7 cells in vitro. Cell viability was measured by flow cytometric assay. DNA condensation (apoptotic cell death) and DNA fragmentation (laddered DNA) were assayed by DAPI staining and DNA gel electrophoresis assays, respectively. Furthermore, DNA damage (comet tail) was measured by the comet assay. Western blotting was used to measure the expression of DNA damage- and repair-associated proteins. RESULTS: AITC decreased cell viability in a dose-dependent and induced apoptotic cell death (DNA condensation and fragmentation) and DNA damage in MCF-7 cells. AITC increased p-ATMSer1981, p-ATRSer428, p53, p-p53Ser15, p-H2A.XSer139, BRCA1, and PARP at 10-30 µM at 24 and 48 h treatments. However, AITC decreased DNA-PK at 24 and 48 h treatment, and decreased MGMT at 48 h in MCF-7 cells. CONCLUSION: AITC induced cytotoxic effects (decreased viable cell number) through induction of DNA damage and condensation and altered DNA damage and repair associated proteins in MCF-7 cells in vitro.

Ouabain Suppresses Cell Migration and Invasion in Human Gastric Cancer AGS Cells Through the Inhibition of MMP Signaling Pathways.

BACKGROUND/AIM: Ouabain has been shown to induce human cancer cell death via apoptosis. Still, its anti-metastatic effect on cell migration and invasion of human gastric cancer cells has not been addressed. MATERIALS AND METHODS: Cell proliferation and viability were measured by the MTT assay and flow cytometry, respectively. Cell motitlity was analysed by wound healing assay. Cell migration and invasion were analysed by the transwell system. Protein expression was assayed by western blotting. RESULTS: Ouabain decreased AGS cell proliferation, cell viability, and motility. In addition, ouabain inhibited AGS cell migration and invasion. Furthermore, ouabain decreased matrix metalloproteinase-2 (MMP-2) activity at 48 h. Ouabain reduced the levels of proteins associated with PI3K/AKT and p38/MAPK pathways. In addition, ouabain decreased the expressions of N-cadherin, tissue inhibitor of metalloproteinases-1 (TIMP-1), urokinase-type plasminogen activator (c-uPA), and MMP-2 at 48 h. CONCLUSION: Ouabain suppresses cell metastasis through multiple signaling pathways in AGS cells.

Melittin suppresses epithelial-mesenchymal transition and metastasis in human gastric cancer AGS cells via regulating Wnt/BMP associated pathway.

Gastric cancer has a poor prognosis; once cancer has metastasized, it can easily lead to patient death. Melittin is one of the major components extracted from the bee venom. It has been shown that melittin emerges antitumor activities against many human cancer cell lines. Our results indicated that melittin at 0.2-0.5 µm significantly reduced total cell viability in human gastric cancer AGS cells. At low concentrations (0.05-0.15 µm), melittin displayed antimetastasis effects and inhibited cell adhesion and colony formation. Besides, it inhibited cell motility and suppressed cell migration and invasion. Melittin inhibited the activities of MMP-2 and MMP-9 and the integrity of cell membrane in AGS cells. Furthermore, Western blotting results showed that melittin decreased the protein expressions of Wnt/BMP and MMP-2 signaling pathways. Based on these observations, melittin inhibited cell migration and invasion of AGS cells through multiple signaling pathways. It may be used to treat metastasized gastric cancers in the future.