Screening Cases of Suspected Early Stage Chronic Kidney Disease from Clinical Laboratory Data: The Comparison between Urine Conductivity and Urine Protein.

(1) Background: Chronic kidney disease (CKD) affects more than 800 million global population. Early detection followed by clinical management is among the best approaches for the affected individuals. However, a sensitive screening tool is not yet available. (2) Methods: We retrospectively reviewed 600 patients aged >20 years with a full range of estimated glomerular filtration rate (eGFR) for clinical assessment of kidney function between 1 January 2020, to 30 April 2021, at the Taichung Veterans General Hospital, Taichung, Taiwan. With stratified sampling based on the level of eGFR, participants were evenly grouped into training and validation sets for predictive modeling. Concurrent records of laboratory data from urine samples were used as inputs to the model. (3) Results: The predictive model proposed two formulae based on urine conductivity for detecting suspected early-stage CKD. One formula, P_male45, was for used male subjects aged ≥45 years, and it had a prediction accuracy of 76.3% and a sensitivity of 97.3%. The other formula, P_female55, was used for female subjects aged ≥55 years. It had a prediction accuracy of 81.9% and a sensitivity of 98.4%. Urine conductivity, however, had low associations with urine glucose and urine protein levels. (4) Conclusion: The two predictive models were low-cost and provided rapid detection. Compared to urine protein, these models had a better screening performance for suspected early-stage CKD. It may also be applied for monitoring CKD in patients with progressing diabetes mellitus.

Features of indeterminate results of QuantiFERON-TB Gold In-Tube test in patients with haematological malignancies.

BACKGROUND AND AIMS: The application of QuantiFERON-TB Gold in-Tube (QFT-GIT) in patients with haematological malignancies (HMs) has not been well studied. Therefore, we aimed to investigate the features of patients with HMs whose QFT-GIT results were indeterminate. METHODS: This study enrolled patients with HMs for the analysis of QFT-GIT tests and additional 2-year follow-up. The characteristics and predictors of QFT-GIT indeterminate results were identified. Mycobacterium tuberculosis (TB) incidence rate (IR) and incidence rate ratio (IRR) were also investigated. RESULTS: Of 89 participants, 27 (30.3%) had QFT-GIT indeterminate results. The QFT-GIT indeterminate patients were characterized with the diagnosis of leukaemia (63.0% versus 32.3%, p = 0.044), abnormal white blood count (WBC) (88.9% versus 14.5%, p = 0.001), abnormal lymphocyte percentage (81.5% versus 14.5%, p = 0.001) and lower lymphocyte count (×109/l) (0.5 versus 2.2, p = 0.000) when compared with those with determinate results. Meanwhile, abnormal WBC [odds ratios (OR): 15.18, p = 0.003] and lymphocyte percentage (OR: 6.90, p = 0.033) were predictors of indeterminate results. One patient with the QFT-GIT indeterminate status and high interferon-γ level of negative control result developed active TB with a TB IR of 18.5 per 1000 person-years and an IRR of 0.1 (95% confidence interval, 0.01-0.71) when compared with positive QFT-GIT patients without prophylaxis treatment. CONCLUSION: Abnormal ranges of WBC and lymphocyte differential count percentage were independent predictors useful to determine the optimal timing of implementing QFT-GIT test in patients with HMs.

Clinical Features and Antimicrobial Susceptibility of Pseudomonas aeruginosa and Acinetobacter baumannii Complex Isolates in Intensive Care Patients with Chronic Obstructive Pulmonary Disease and Community-Acquired Pneumonia in Taiwan.

Purpose: Little is known about the features and implications of Pseudomonas aeruginosa (PA) and Acinetobacter baumannii complex (ABC) isolates discovered in patients with chronic obstructive pulmonary disease (COPD) and community-acquired pneumonia (CAP) requiring invasive mechanical ventilation and admission to an intensive care unit. Thus, our study aimed to investigate the clinical characteristics and antimicrobial susceptibilities of PA and ABC isolates cultured from endotracheal aspirates (EAs) in such population. Patients and Methods: In this retrospective, cross-sectional study, clinical data from medical records were reviewed and collected for analysis. Results: Of the 262 participants, 17.2% (45/262), 11.5% (30/262), and 27.1% (71/262) had PA, ABC, and any of the two isolates discovered from EA cultures, respectively. Patients with PA isolates were associated with poorer lung function (the Global Initiative for Chronic Obstructive Lung Disease (GOLD) III+IV versus GOLD I+II, odds ratio (OR)=2.39, p= 0.022) and a lower body mass index (per increase of 1 kg/m2, OR= 0.93, p= 0.106) while the former was an independent predictor. Moreover, both subjects with ABC isolates and those with any of these two microorganisms were independently associated with a lower serum albumin level (per increase of 1 g/dL, OR= 0.44, p=0.009 and OR= 0.59, p=0.023, respectively). Participants with PA isolates were more likely to have failed weaning (62.2% versus 44.7%, p= 0.048) and death (28.9% versus 12.4%, p= 0.010) than those without PA isolates. The majority of the PA and ABC isolates were susceptible and resistant to all the tested antimicrobials, respectively, except that tigecycline had a reliable activity against ABC. Conclusion: Our findings provide important information to help intensivists make better treatment decisions in critically ill patients with COPD and CAP.

Enforced C-Src Activation Causes Compartmental Dysregulation of PI3K and PTEN Molecules in Lipid Rafts of Tongue Squamous Carcinoma Cells by Attenuating Rac1-Akt-GLUT-1-Mediated Sphingolipid Synthesis.

Pharmacologic intervention to affect the membrane lipid homeostasis of lipid rafts is a potent therapeutic strategy for cancer. Here we showed that gallic acid (GA) caused the complex formation of inactive Ras-related C3 botulinum toxin substrate 1 (Rac1)-phospho (p)-casein kinase 2 α (CK2α) (Tyr 255) in human tongue squamous carcinoma (TSC) cells, which disturbed the lipid raft membrane-targeting of phosphatidylinositol 3-kinase (PI3K)-Rac1-protein kinase B (Akt) signal molecules by inducing the association of p110α-free p85α with unphosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN) in lipid rafts. The effects on induction of inactive Rac1-p-CK2α (Tyr 255) complex formation and attenuation of p-Akt (Ser 473), GTP-Rac1, glucose transporter-1 (GLUT-1) lipid raft membrane-targeting, and cell invasive activity by GA were counteracted either by CK2α short hairpin RNA or cellular-Src (c-Src) inhibitor PP1. PP1 treatment, GLUT-1 or constitutively active Rac1 ectopic-expression blocked GA-induced decreases in cellular glucose, sphingolipid and cholesterol of lipid raft membranes, p85α-p110α-GTP-Rac1 complexes, glucosylceramide synthase activity and increase in ceramide and p110α-free p85α-PTEN complex levels of lipid raft membranes, which reversed the inhibition on matrix metalloproteinase (MMP)-2/-9-mediated cell invasion induced by GA. Using transient ectopic expression of nuclear factor-kappa B (NF-κB) p65, MMP-2/-9 promoter-driven luciferase, and NF-κB-dependent luciferase reporter genes and NF-κB specific inhibitors or Rac1 specific inhibitor NSC23766, we confirmed that an attenuation of Rac1 activity by GA confers inhibition of NF-κB-mediated MMP-2/-9 expression and cell invasion. In conclusion, GA-induced c-Src activation is a key inductive event for the formation of inactive Rac1-p-CK2α (Tyr 255) complexes, which disturbed lipid raft compartment of PI3K and PTEN molecules by impairing Akt-regulated GLUT-1-mediated sphingolipid synthesis, and finally resulting in inhibition of TSC cell invasion.

Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells.

The specialized cholesterol/sphingolipid-rich membrane domains termed lipid rafts are highly dynamic in the cancer cells, which rapidly assemble effector molecules to form a sorting platform essential for oncogenic signaling transduction in response to extra- or intracellular stimuli. Density-based membrane flotation, subcellular fractionation, cell surface biotinylation, and co-immunoprecipitation analyses of bichalcone analog ((E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR4)-treated cancer cells showed dissociation between GRP78 and p85α conferring the recruitment of PTEN to lipid raft membranes associated with p85α. Ectopic expression of GRP78 could overcome induction of lipid raft membrane-associated p85α-unphosphorylated PTEN complex formation and suppression of GRP78PI3KAktGTP-Rac1-mediated and GRP78-regulated PERKNrf2 antioxidant pathway and cancer cell invasion by TSWU-BR4. Using specific inducer, inhibitor, or short hairpin RNA for ASM demonstrated that induction of the lipid raft membrane localization and activation of ASM by TSWU-BR4 is responsible for perturbing homeostasis of cholesterol and ceramide levels in the lipid raft and ER membranes, leading to alteration of GRP78 membrane trafficking and subsequently inducing p85α-unphosphorylated PTEN complex formation, causing disruption of GRP78PI3KAktGTP-Rac1-mediated signal and ER membrane-associated GRP78-regulated oxidative stress balance, thus inhibiting cancer cell invasion. The involvement of the enrichment of ceramide to lipid raft membranes in inhibition of NF-κB-mediated MMP-2 expression was confirmed through attenuation of NF-κB activation using C2-ceramide, NF-κB specific inhibitors, ectopic expression of NF-κB p65, MMP-2 promoter-driven luciferase, and NF-κB-dependent reporter genes. In conclusion, localization of ASM in the lipid raft membranes by TSWU-BR4 is a key event for initiating formation of ceramide-enriched lipid raft membrane platforms, which causes delocalization of GRP78 from the lipid raft and ER membranes to the cytosol and formation of p85α-unphosphorylated PTEN complexes to attenuate the GRP78-regulated oxidative stress balance and GRP78p85αAktGTP-Rac1NF-κBMMP-2-mediated cancer cell invasion.

Cardamonin induces immune responses and enhances survival rate in WEHI-3 cell-generated mouse leukemia in vivo.

Cardamonin, a monomeric alkaloid, is isolated from Alpinia conchigera Griff and other natural plants. Recently, it has been focused on its anticancer activities, and no information showing its immune effects on leukemia mice was reported. In this study, we investigated the immune effects of cardamonin on WEHI-3 cell-generated leukemia mice. Forty BALB/c mice were randomly divided into four groups: Group I mice were normal animals and groups II-IV were leukemia. Group II mice, as a positive control, were administered with normal diet, and group III and IV mice were treated with 1 and 5 mg/kg of cardamonin, respectively, by intraperitoneal injection every 2 days for 14 days. The population of white blood cells, macrophage phagocytosis, and the proliferations of T and B cells were analyzed by flow cytometry. Another forty mice were also separated randomly into four groups for the determination of survival rate. Results showed that cardamonin did not affect body weight. Cardamonin decreased CD3, CD11b, and Mac-3 cell populations but increased CD19 number. Cardamonin enhanced phagocytic abilities of macrophages from the peripheral blood mononuclear cells of leukemia mice. Furthermore, cardamonin at 1 mg/kg treatment improved the survival rate of leukemia mice in vivo. Therefore, cardamonin could be applied for a leukemia therapeutic reagent at a defined dose.

The association between airflow limitation and blood eosinophil levels with treatment outcomes in patients with chronic obstructive pulmonary disease and prolonged mechanical ventilation.

The clinical implications of airflow limitation severity and blood eosinophil level in patients with chronic obstructive pulmonary disease (COPD) and prolonged mechanical ventilation (PMV) are unknown. Thus, this study aimed to identify whether or not these two indicators were significantly associated with short-term in-respiratory care center (RCC) treatment outcomes in this population. Of all participants (n = 181) in this retrospective cross-sectional study, 41.4%, 40.9%, 8.3%, and 52.5% had prolonged RCC admission (RCC length of stay >21 days), failed weaning, death, and any adverse outcomes of interest, respectively. Compared to participants without any adverse outcomes of interest, moderate (the Global Initiative for Chronic Obstructive Lung Disease (GOLD) II) and/or severe (GOLD III) airflow limitation were significantly associated with short-term in-RCC adverse outcomes in terms of failed weaning (for III versus I, OR = 15.06, p = 0.003) and having any adverse outcomes of interest (for II versus I, OR = 17.66, p = 0.002; for III versus I, OR = 37.07, p = 0.000) though the severity of airflow limitation did not have associations with prolonged RCC admission and death after adjustment. Meanwhile, blood eosinophilia defined by various cut-off values was not associated with any adverse outcomes. The findings have significant clinical implications and are useful in the management of patients with COPD and PMV.

Clinical features, bacteriology of endotracheal aspirates and treatment outcomes of patients with chronic obstructive pulmonary disease and community-acquired pneumonia in an intensive care unit in Taiwan with an emphasis on eosinophilia versus non-eosinophilia: a retrospective case-control study.

OBJECTIVES: The clinical implications of blood eosinophil level in patients with chronic obstructive pulmonary disease (COPD) and community-acquired pneumonia (CAP) requiring invasive mechanical ventilation (IMV) and intensive care unit (ICU) admission are still unknown. Thus, this study aimed to compare the features of such patients with and without blood eosinophilia. DESIGN: This was a retrospective case-control study. SETTING: An ICU of a medical centre in central Taiwan. PARTICIPANTS: A total of 262 patients with COPD and CAP requiring IMV and ICU admission. RESULTS: Of all participants (n=262), 32 (12.2%) had an eosinophil percentage (EP) >2% and 169 (64.5%) had an absolute eosinophil count (AEC) >300 cells/µL. Regardless of whether 2% or 300 cells/µL was used as a cut-off value, the eosinophilia group were slightly older (years) (82.9±5.4 vs 78.1±9.1, p=0.000 and 79.2±8.4 vs 77.6±9.6, p=0.246, respectively), and had a higher forced expiratory volume in 1 s/forced vital capacity (%) (56.0±8.0 vs 51.3±11.6, p=0.005 and 53.1±11.2 vs 49.5±11.2, p=0.013, respectively), less severe spirometric classification (p=0.008 and p=0.001, respectively), and lower white cell count 109/L (8.8±3.2 vs 11.1±4.9, p=0.009 and 10.3±4.4 vs 11.8±5.3, p=0.017, respectively) than the non-eosinophilia group. The bacteriology of endotracheal aspirates showed that Pseudomonas aeruginosa and other gram-negative bacilli were the most common organisms in all study groups. Participants with an EP >2% had a shorter ICU length of stay (OR=12.13, p=0.001) than those with an EP ≤2%, while an AEC >300 cells/µL was not associated with any in-ICUoutcomes. CONCLUSIONS: The results of this study have significant clinical implications and should be considered when making treatment decisions for the management of patients with COPD and CAP requiring IMV and ICU admission.

Quercetin induced cell apoptosis and altered gene expression in AGS human gastric cancer cells.

Quercetin is one of the natural components from natural plant and it induces cell apoptosis in many human cancer cell lines. However, no available reports show that quercetin induces apoptosis and altered associated gene expressions in human gastric cancer cells, thus, we investigated the effect of quercetin on the apoptotic cell death and associated gene expression in human gastric cancer AGS cells. Results indicated that quercetin induced cell morphological changes and reduced total viability via apoptotic cell death in AGS cells. Furthermore, results from flow cytometric assay indicated that quercetin increased reactive oxygen species (ROS) production, decreased the levels of mitochondrial membrane potential (ΔΨm ), and increased the apoptotic cell number in AGS cells. Results from western blotting showed that quercetin decreased anti-apoptotic protein of Mcl-1, Bcl-2, and Bcl-x but increased pro-apoptotic protein of Bad, Bax, and Bid. Furthermore, quercetin increased the gene expressions of TNFRSF10D (Tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain), TP53INP1 (tumor protein p53 inducible nuclear protein 1), and JUNB (jun B proto-oncogene) but decreased the gene expression of VEGFB (vascular endothelial growth factor B), CDK10 (cyclin-dependent kinase 10), and KDELC2 (KDEL [Lys-Asp-Glu-Leu] containing 2) that are associated with apoptosis pathways. Thus, those findings may offer more information regarding the molecular, gene expression, and signaling pathway for quercetin induced apoptotic cell death in human gastric cancer cells.

Higher stroke incidence in the patients with pancreatic cancer: A nation-based cohort study in Taiwan.

No study has investigated the role of pancreatic cancer in the stroke risk using population data. We used claims data obtained from a universal health insurance program of Taiwan to evaluate the stroke risk in pancreatic cancer patients.From the catastrophic disease registry of the insurance, we identified 7479 patients with pancreatic malignancy without stroke history from 2000 to 2009. The comparison cohort consisted of 29,916 individuals identified from 1 million insured people without cancer and stroke history, matching with the cancer cohort by propensity score. We followed each selected individual until stroke was diagnosed or until being censored for death or withdrawal from insurance, or for a maximum of 3 follow-up years, or the end of 2011.The pancreatic cancer cohort had a 2.3-fold greater incident stroke than comparisons had (28.5 vs 12.3 per 1000 person-years), with an adjusted hazard ratio (aHR) of 2.74 (95% confidence interval (CI) = 2.31-3.24) after controlling for covariates, or a subdistribution hazard ratio (SHR) of 2.04 (95% CI = 1.74-2.40) accounting for the competing risk of deaths. During the follow-up period, stroke events occurred constantly in comparisons, but declined rapidly in the cancer cohort. The pancreatic cancer cohort had a stroke incidence of 46.6 per 1000 person-years within 6 months postdiagnosis, with an aHR of 4.37 (95% CI = 3.45-5.54) and a SHR of 3.87 (95% CI = 3.08-4.86), relative to comparisons.Our study suggests that patients with pancreatic cancer are at an elevated risk of stroke, patients deserve sufficient follow-up care, particularly in the first 6 months after the diagnosis of the cancer, and for those with comorbidities.

Fisetin Induces Apoptosis of HSC3 Human Oral Cancer Cells Through Endoplasmic Reticulum Stress and Dysfunction of Mitochondria-mediated Signaling Pathways.

BACKGROUND/AIM: Oral cancer has been reported to be one of the major cancer-related diseases in human populations and the treatment of oral cancer is still unsatisfied. Fisetin, is a flavonoid from plants and has several biological activities such as antioxidant, anti-inflammatory and anticancer function, but its cytotoxicity in human oral cancer cells is unknown. In the present study, we investigated fisetin-induced cytotoxic effects on HSC3 human oral cancer cells in vitro. Materials and Methods/Results: We used flow cytometric assay to show fisetin induced apoptotic cell death through increased reactive oxygen species and Ca2+, but reduced the mitochondrial membrane potential and increased caspase-8, -9 and -3 activities in HSC3 cells. Furthermore, we also used 4' 6-diamidino-2-phenylindole staining to show that fisetin induced chromatin condensation (apoptotic cell death), and Comet assay to show that fisetin induced DNA damage in HSC3 cells. Western blotting was used to examine the levels of apoptotic-associated protein and results indicated that fisetin increased expression of pro-apoptotic proteins such as B-cell lymphoma 2 (BCL2) antagonist/killer (BAK) and BCL2-associated X (BAX) but reduced that of anti-apoptotic protein such as BCL2 and BCL-x, and increased the cleaved forms of caspase-3, -8 and -9, and cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (ENDO G) in HSC3 cells. Confocal microscopy showed that fisetin increased the release of cytochrome c, AIF and ENDO G from mitochondria into the cytoplasm. CONCLUSION: Based on these observations, we suggest that fisetin induces apoptotic cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.

Antrodia Cinnamomea Reduces Carbon Tetrachloride-induced Hepatotoxicity In Male Wister Rats.

BACKGROUND/AIM: Antrodia cinnamomea is found with polysaccharides, lipids, vitamins, fibers and ash (minerals) and is well known in Taiwan as a traditional Chinese medicine. Its biological activities have been reported to have anti-inflammatory, anti-fatigue, anti-tumor and immunomodulatory effects, but its protective effects on liver function are still unclear. MATERIALS AND METHODS: We determined if Antrodia cinnamomea was hepatoprotective against carbon tetrachloride (CCl4) toxicity in Wistar rats. Six groups were used in the study: 1) control (no induction by CCl4); 2) negative control (CCl4-induction and no treatment); 3) positive control (silymarin treatment); 4) groups 4-6 were treated with CC14 and different concentrations (350 mg/kg, 1,400 mg/kg, 3,150 mg/kg) of Antrodia cinnamomea. Blood and liver samples of rats were harvested and then detected by biochemical and tissue histochemical analysis. Activity of the antioxidative enzymes glutathione peroxidase, superoxide dismutase and catalase in the liver were also monitored. RESULTS: Only the high-dose treatment was able to decrease serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels and improve liver function. High and medium doses increased total liver protein and reduced hydroxyproline. It was also observed that the high dose treatment reduced lipid peroxidation. Liver sections of CC14 treated animals receiving Antrodia cinnamomea showed less fibrosis compared to the CCl4 control group. CONCLUSION: This finding suggested that Antrodia cinnamomea can either enhance liver recovering from CCl4 damage or attenuate CCl4 toxicity in rats.

Ouabain impairs cell migration, and invasion and alters gene expression of human osteosarcoma U-2 OS cells.

Ouabain, the specific Na+ /K+ -ATPase blocker, has biological activity including anti-proliferative and anti-metastasis effects in cancer cell. There is no study to show ouabain inhibiting cell migration and invasion in human osteosarcoma U-2 OS cells. Thus, we investigated the effect of ouabain on the cell migration and invasion of human osteosarcoma U-2 OS cells. Results indicated that ouabain significantly decreased the percentage of viable cells at 2.5-5.0 µM, thus, we selected 0.25-1.0 µM for inhibiting studies. Ouabain inhibited cell migration, invasion and the enzymatic activities of MMP-2, and also affected the expression of metastasis-associated protein in U-2 OS cells. The cDNA microarray assay indicated that CDH1, TGFBR3, SHC3 and MAP2K6 metastasis-related genes were increased, but CCND1, JUN, CDKN1A, TGFB1, 2 and 3, SMAD4, MMP13, MMP2 and FN1 genes were decreased. These findings provide more information regarding ouabain inhibited cell migration and invasion and associated gene expressions in U-2 OS cells after exposed to ouabain.

Increased risk of stroke in contact dermatitis patients: A nationwide population-based retrospective cohort study.

Dermatologic diseases are not traditional risk factors of stroke, but recent studies show atopic dermatitis, psoriasis, and bullous skin disease may increase the risk of stroke and other cardiovascular diseases. No previous studies have focused on the association between contact dermatitis and stroke.We established a cohort comprised of 48,169 contact dermatitis patients newly diagnosed in 2000-2003 and 96,338 randomly selected subjects without the disorder, frequency matched by sex, age, and diagnosis year, as the comparison cohort. None of them had a history of stroke. Stroke incidence was assessed by the end of 2011 for both cohorts.The incidence stroke was 1.1-fold higher in the contact dermatitis cohort than in the comparison cohort (5.93 vs 5.37 per 1000 person-years, P < 0.01). The multivariable Cox method analyzed adjusted hazard ratios (aHRs) were 1.12 (95% confidence interval [CI], 1.05-1.19) for all stroke types and 1.12 (95% CI, 1.05-1.20) for ischemic stroke and 1.11 (95% CI, 0.94-1.30) for hemorrhagic stroke. The age-specific aHR of stroke for contact dermatitis cohort increased with age, from 1.14 (95% CI, 1.03-1.27) for 65 to 74 years; to 1.27 (95% CI, 1.15-1.42) for 75 years and older. The aHR of stroke were 1.16 (95% CI, 1.07-1.27) and 1.09 (95% CI, 1.00-1.18) for men and women, respectively.This study suggests that patients with contact dermatitis were at a modestly increased risk of stroke, significant for ischemic stroke but not for hemorrhagic stroke. Comorbidity, particularly hypertension, increased the hazard of stroke further.

Bufalin Induces Apoptosis of Human Osteosarcoma U-2 OS Cells through Endoplasmic Reticulum Stress, Caspase- and Mitochondria-Dependent Signaling Pathways.

Bone cancer is one of the cancer-related diseases, and there are increased numbers of patients with bone cancer worldwide. Therefore the efficacy of treatment of bone cancer is considered extremely vital. Bufalin has been showed to have biological activities including anticancer activities in vitro and in vivo. However, the exact associated mechanisms for bufalin induced apoptosis in human bone cancer cells are still unclear. In the present study, we investigated the effect of bufalin on the cytotoxic effects in U-2 OS human osteosarcoma cells. For examining apoptotic cell deaths, we used flow cytometry assay, Annexin V/PI double staining, and TUNNEL assay. Reactive oxygen species (ROS), Ca2+, mitochondrial membrane potential (ΔΨm), and caspase-8, -9 and -3 activities were measured by flow cytometry assay. Furthermore, western blotting and a confocal laser microscopy examination were used for measuring the alterations of apoptotic associated protein expression and translocation, respectively. The results indicated that bufalin induced cell morphological changes, decreased the viable cell number, induced apoptotic cell death, and increased the apoptotic cell number, and affected apoptotic associated protein expression in U-2 OS cells. Bufalin increased apoptotic proteins such as Bak, and decreased anti-apoptotic proteins such as Bcl-2 and Bcl-x in U-2 OS cells. Furthermore, bufalin increased the protein levels of cytochrome c (Cyto c), AIF (Apoptosis inducing factor) and Endo G (Endonuclease G) in cytoplasm that were also confirmed by confocal microscopy examination. Based on those findings, bufalin induced apoptotic cell death in U-2 OS cells may be via endoplasmic reticulum (ER) stress, caspase-, and mitochondria-dependent pathways; thus, we may suggest that bufalin could be used as an anti-cancer agent for the treatment of osteosarcoma in the future, and further in vivo studies are needed.

Sulforaphane-induced apoptosis in human leukemia HL-60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray.

Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN-induced cytotoxic effects and whether or not they went through cell-cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL-60 cells. Cell viability, cell-cycle distribution, sub-G1 (apoptosis), reactive oxygen species (ROS) and Ca2+ production, levels of mitochondrial membrane potential (ΔΨm ), and caspase-3, -8, and -9 activities were assayed by flow cytometry. Apoptosis-associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub-G1 phase development. Furthermore, SFN increased ROS and Ca2+ production and decreased the levels of ΔΨm and activated caspase-3, -8, and -9 activities in HL-60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas-L, caspase-8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl-x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase -8, -3, -4, -6, and -7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL-60 cells via Fas- and mitochondria-dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311-328, 2017.

Anti-inflammatory effect of longan seed extract in carrageenan stimulated Sprague-Dawley rats.

OBJECTIVES: Longan seeds have been used as a folk medicine in China. Longan seed extract (LSE) is known for antioxidative, antiproliferative, hypoglycemic, and hypouremic effects. However, its anti-inflammatory effect has not been shown. MATERIALS AND METHODS: In this study, Sprague-Dawley (SD) rats were given LSE orally (vehicle, 10, and 30 mg/kg) for 3 days to its test anti-inflammatory effect by injecting λ-carrageenan (CARR) in the right hind paw or lipopolysaccharide (LPS), IP. For the positive control, animals were given aspirin (20 mg/kg) orally and treated likewise. Serum or tissue samples from treated rats were collected after 3 hr of stimulation. Regarding the in vitro study, BV2 microglial cells were stimulated with LPS in the presence of LSE or normal saline for 10 min or 24 hr for Western blot and ELISA assay, respectively. RESULTS: LSE reduced CARR-induced edema in the experimental animals. LSE also reduced LPS/CARR-induced nitric oxide (NO), interleukin-1ß (IL1ß), IL6, and COX2 productions. These inflammatory factors were also reduced dose dependently by LSE in LPS-stimulated BV2 cells. Furthermore, Western blot analysis revealed that LSE inhibited LPS activated c-Jun NH2-terminal protein kinase (JNK), extracellular signal-regulated kinases (ERKs), and p38 MAP kinases signaling pathways, caspase-3, inducible NO synthase, and COX2 expressions. CONCLUSION: LSE pretreatment suppressed CARR- and LPS-induced inflammations and these effects might be through the inhibition of MAP kinases signaling pathways and inflammatory factors.

Sulforaphane promotes immune responses in a WEHI­3­induced leukemia mouse model through enhanced phagocytosis of macrophages and natural killer cell activities in vivo.

Sulforaphane (SFN) is an isothiocyanate, inducing cytotoxic effects in various human cancer cells, including leukemia cells through cell cycle arrest and apoptosis. However, the effect of SFN on the immune responses in a leukemia mouse model remains to be investigated. The present study investigated whether SFN has an effect on the immune responses in a WEHI­3­induced leukemia mouse model in vivo. Normal BALB/c mice were injected with WEHI­3 cells to generate the leukemia mouse model, and were subsequently treated with placebo or SFN (0, 285, 570 and 1,140 mg/kg) for 3 weeks. Following treatment, all mice were weighted and blood samples were collected. In addition, liver and spleen samples were isolated to determine cell markers, phagocytosis and natural killer (NK) cell activities, and cell proliferation was examined using flow cytometry. The results indicated that SFN treatment had no significant effect on the spleen weight, however it decreased liver and body weight. Furthermore, SFN treatment increased the percentage levels of CD3 (T cells) and CD19 (B cell maker), however had no effect on the levels of CD11b (monocytes) or Mac­3 (macrophages), compared with the WEHI­3 control groups. The administration of SFN increased the phagocytosis of macrophages from peripheral blood mononuclear cells and peritoneal cavity, and increased the activity of NK cells from splenocytes. Administration of SFN promoted T and B cell proliferation following stimulation with concanavalin A and lipopolysaccharide, respectively.

The Prognostic Role of STEAP1 Expression Determined via Immunohistochemistry Staining in Predicting Prognosis of Primary Colorectal Cancer: A Survival Analysis.

STEAP1 (six transmembrane epithelial antigen of the prostate 1) is a transmembrane protein that functions as a potential channel or transporter protein. It is overexpressed in certain cancers and is viewed as a promising therapeutic target. However, the prognostic role of STEAP1 is still controversial, and no role for STEAP1 has yet been indicated in colorectal cancer. The aim of this study was to investigate the possible association of STEAP1 expression with colorectal cancer prognosis. STEAP1 expression was analyzed by immunohistochemical staining of a tissue array of 165 cancer specimens from primary colorectal cancer patients. The mean and medium follow-up times after surgery were 5.1 and 3.9 years, respectively. A total of 139 patients died during the 13 years of follow-up in the survey period. The prognostic value of STEAP1 with respect to overall survival was analyzed by Kaplan-Meier analysis and Cox proportional hazard models. In total, 164 samples displayed detectable STEAP1 expression in the cytoplasm and membrane. Low STEAP1 expression was correlated with poor overall survival (five-year survival: 33.7% vs. 57.0%, low expression vs. high expression, p = 0.020). Accordingly, multivariate analysis identified low STEAP1 expression as an independent risk factor (hazard ratio = 1.500, p = 0.018), especially in elderly patients or those with late stage cancers, late T values, and early N values. We suggest that analysis of STEAP1 expression by immunohistochemical staining could serve as an independent prognostic marker for colorectal patients. This finding should be validated by other investigative groups.