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The protective effects of alpha-1 antitrypsin (AAT) in tacrolimus (TAC)-induced renal injury was evaluated in a rat model. The TAC group rats were subcutaneously injected with 2 mg/kg TAC every day for four weeks. The TAC with AAT group was cotreated with daily subcutaneous injections of TAC and intraperitoneal injections of AAT (80 mg/kg) for four weeks. The effects of AAT on TAC-induced renal injury were evaluated using serum biochemistry, histopathology, and Western blotting. The TAC injection significantly increased renal interstitial fibrosis, inflammation, and apoptosis as compared to the control treatment. The histopathological examination showed that cotreatment of TAC and AAT attenuated interstitial fibrosis (collagen, fibronectin, and α-SMA staining), and α-SMA expression in Western blotting was also decreased. Immunohistochemical staining for inflammation (osteopontin and ED-1 staining) revealed improved interstitial inflammation in the TAC with AAT group compared to that in the TAC group. The TAC treatment increased renal apoptosis compared to the control treatment, based on the results of increased immunohistochemical staining of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), increased caspase-3 activity, and lower Bcl-2 to Bad expression ratio. However, AAT cotreatment significantly changed these markers and consequently showed decreased apoptosis. AAT protects against TAC-induced renal injury via antifibrotic, anti-inflammatory, and antiapoptotic effects.

Background: The alpha-1 antitrypsin (AAT) protein has an important role in the anti-inflammatory and apoptotic response. AAT inhibits not only serine proteases but also cysteine and aspartic proteases. Apoptosis results from the sequential activation of cysteine proteases of the caspase family. This study aimed to evaluate the effect of AAT on formaldehyde-induced apoptosis of human peritoneal mesothelial cells (HPMCs)., Methods: HPMCs were cultured and treated with formaldehyde (250 µM) to induce apoptosis. In the AAT group, the cultured HPMCs were pretreated with AAT (2 mg/mL) for 1 h before formaldehyde treatment. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays to determine cell viability, and flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays to detect apoptosis. The MTT assays were used to find optimal concentrations of formaldehyde and AAT. We measured caspase-3 activity and used Western blotting to estimate Bcl-2 and Bad expression., Results: Flow cytometry and TUNEL assays revealed that formaldehyde exposure significantly increased apoptosis compared with the control treatment, but pretreatment with AAT significantly inhibited this effect. Compared with the control, caspase-3 activity was significantly increased and the ratio of Bcl-2 to Bad expression significantly decreased following treatment with formaldehyde. However, caspase-3 activity was significantly lower and the Bcl-2 to Bad expression ratio higher in the AAT group than in the formaldehyde-only group., Conclusion: AAT inhibits formaldehyde-induced apoptosis of HPMCs via a caspase-mediated pathway. These data support a potential use for AAT as a therapeutic agent for the inhibition of peritoneal cell apoptosis during peritoneal dialysis.

Endothelial cell injury and dysfunction caused by reactive oxygen species (ROS) are implicated in the pathogenesis of vascular diseases. ROS are generated and hypoxanthine is degraded by xanthine oxidase. Smoking and alcohol consumption are associated with an increased level of hypoxanthine. We aimed to study the direct role of hypoxanthine in endothelial dysfunction in human umbilical vascular endothelial cells (HUVECs). Hypoxanthine induced cell death and production of ROS. Furthermore, hypoxanthine induced apoptosis through regulation of protein expression related to apoptosis. When cells were pretreated with N-acetylcysteine or a pancaspase inhibitor (Z-VAD-fmk) and stimulated with hypoxanthine, Z-VAD-fmk and N-acetylcysteine prevented hypoxanthine-induced apoptosis by inhibiting the ROS production and caspase pathway. Thus, an increased extracellular concentration of hypoxanthine induces endothelial dysfunction through ROS production and regulates expression of apoptosis-related proteins in HUVECs. These effects are expected to be associated with some vascular diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase that is highly expressed in Human Epidermal Growth Factor 2(+) (Her2(+)) breast cancers. Overexpression of PTK6 enhances anchorage-independent survival, proliferation, and migration of breast cancer cells. We hypothesized that PTK6 inhibition is an effective strategy to inhibit growth and survival of Her2(+) breast cancer cells, including those that are relatively resistant to Lapatinib, a targeted therapy for Her2(+) breast cancer, either intrinsically or acquired after continuous drug exposure., Methods: To determine the effects of PTK6 inhibition on Lapatinib-resistant Her2(+) breast cancer cell lines (UACC893R1 and MDA-MB-453), we used short hairpin ribonucleic acid (shRNA) vectors to downregulate PTK6 expression. We determined the effects of PTK6 downregulation on growth and survival in vitro and in vivo, as well as the mechanisms responsible for these effects., Results: Lapatinib treatment of "sensitive" Her2(+) cells induces apoptotic cell death and enhances transcript and protein levels of Bim, a pro-apoptotic Bcl2 family member. In contrast, treatment of relatively "resistant" Her2(+) cells fails to induce Bim or enhance levels of cleaved, poly-ADP ribose polymerase (PARP). Downregulation of PTK6 expression in these "resistant" cells enhances Bim expression, resulting in apoptotic cell death. PTK6 downregulation impairs growth of these cells in in vitro 3-D Matrigel(TM) cultures, and also inhibits growth of Her2(+) primary tumor xenografts. Bim expression is critical for apoptosis induced by PTK6 downregulation, as co-expression of Bim shRNA rescued these cells from PTK6 shRNA-induced death. The regulation of Bim by PTK6 is not via changes in Erk/MAPK or Akt signaling, two pathways known to regulate Bim expression. Rather, PTK6 downregulation activates p38, and pharmacological inhibition of p38 activity prevents PTK6 shRNA-induced Bim expression and partially rescues cells from apoptosis., Conclusions: PTK6 downregulation induces apoptosis of Lapatinib-resistant Her2(+) breast cancer cells by enhancing Bim expression via p38 activation. As Bim expression is a critical biomarker for response to many targeted therapies, PTK6 inhibition may offer a therapeutic approach to treating patients with Her2 targeted therapy-resistant breast cancers.

We aimed to evaluate whether methotrexate (MTX) in vitro induces apoptosis in synoviocytes obtained from rheumatoid arthritis patients and whether the apoptosis inducing effect of MTX to synoviocytes is correlated with the clinical responsiveness to MTX in patients with rheumatoid arthritis (RA). We evaluated 18 patients with RA taking MTX 15-20 mg/week as the subject group (nine responders and nine non-responders) and ten patients with osteoarthritis (OA) and nine patients with ankylosing spondylitis (AS) as the control group. Synoviocytes, cultured from the synovial fluid of the knee joint of each subject, were used for experiments between passages 4 and 6, and were treated with MTX. The induction of apoptosis was determined by the quantification of DNA hypoploidy by flow cytometry, nuclear morphology, caspases activation, DNA electrophoresis, and mitochondrial membrane potential measurements. The viability of synoviocytes treated with MTX was different between the MTX responders and nonresponders. MTX induced apoptosis in cultured synoviocytes by mitochondria- and caspase-dependent manners in the MTX responders but did not in the MTX non-responder, OA, and AS patients. The apoptotic responsiveness of the synoviocytes to MTX predicts the sensitivity to MTX treatment and provides a method determine the early application of an anti-tumor necrosis factor-α agent in RA treatment.

Background: It has been demonstrated that phosphate uptake through the type III sodium-dependent phosphate co-transporter, Pit-1, induced apoptosis of aortic vascular smooth muscle cells and endothelial cells in vitro. However, the apoptotic effects of high phosphate (HP) level in human peritoneal mesothelial cells (HPMCs) are not known., Methods: To examine whether Pit-1 is expressed in HPMCs, we checked the Western blot assay of immunoreactive Pit-1 and the transcription of Pit-1 by reverse transcriptase PCR. We treated several different phosphate concentrations (1-4 mM) and calcium concentrations (1.8 and 2.8 mM) on HPMCs to assess the effects of concentration. MTT, TUNEL assays, and flow cytometry analysis using Annexin V and propidium iodide were performed to identify cell death and apoptosis. Bax and Bcl-2 by Western blot and caspase-3 activity were evaluated by colorimetric assay. In addition, phosphonoformic acid (PFA) and pan-caspase inhibitor, Z-VAD-FMK, were given to prevent phosphate-induced apoptosis., Results: Pit-1 expression on HPMCs was demonstrated. Apoptosis in HPMCs significantly increased with a high concentration of phosphate in a dose- and time-dependent manner, and was enhanced in the presence of 2.8 mM calcium. HP concentrations significantly decreased the anti-apoptotic Bcl-2/Bax ratio and increased caspase-3 activity. The treatment with PFA and Z-VAD-FMK prevented cell death by HP., Conclusion: Phosphate uptake through Pit-1 induces apoptosis in HPMCs by a caspase-related mechanism., (Copyright © 2011 S. Karger AG, Basel.)

Objective: Glucose degradation products (GDPs) are formed during heat sterilization and storage of peritoneal dialysis (PD) fluids. 3,4-dideoxyglucosone-3-ene (3,4-DGE) has been identified as the most bioreactive GDP. 3,4-DGE induces apoptosis in leukocytes and renal tubular epithelial cells. Our aim was to evaluate the apoptotic effects of 3,4-DGE on human peritoneal mesothelial cells (HPMCs)., Methods: Primary cultured HPMCs were treated with 25 or 50 micromol/L 3,4-DGE. MTT assay was used to determine cell viability. Apoptosis was measured using TUNEL assay and flow cytometry. Expressions of procaspase-3, Bax, and Bcl-2 were estimated by Western blot. Activity of caspase-3 was measured and the effect of the caspase inhibitor zVAD-fmk (Z-Val-Ala-DL-Asp-fluoromethylketone) was evaluated by TUNEL assay., Results: 3,4-DGE treatment accelerated cell death in HPMCs in a dose- and time-dependent manner. Treatment with 3,4-DGE (25 and 50 micromol/L) significantly increased apoptosis compared to control (p<0.05 and p<0.01 respectively) by TUNEL assay. Flow cytometry showed treatment with 50 micromol/L 3,4-DGE significantly increased apoptosis compared to control (p<0.05). Decreased expression of procaspase-3 and increased activity of caspase-3 were observed in the presence of 50 micromol/L 3,4-DGE compared to control and 25 micromol/L 3,4-DGE (p<0.05). 3,4-DGE-induced HPMC apoptosis was decreased after pretreatment with the pan-caspase inhibitor zVAD-fmk in the 50 micromol/L 3,4-DGE-treated group (p<0.001). The ratio of Bcl-2 to Bax expression was decreased in the 25 micromol/L and the 50 micromol/L 3,4-DGE-treated groups compared to control (p<0.05)., Conclusions: 3,4-DGE promotes apoptosis in HPMCs by a caspase-related mechanism.

Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways.

Contrast-induced acute kidney injury (CIAKI) is a leading cause of acute kidney injury following radiographic procedures. Intrarenal oxidative stress plays a critical role in CIAKI. Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidases (Noxs) are important sources of reactive oxygen species (ROS). Among the various types of Noxs, Nox4 is expressed predominantly in the kidney in rodents. Here, we evaluated the role of Nox4 and benefit of Nox4 inhibition on CIAKI using in vivo and in vitro models. HK-2 cells were treated with iohexol, with or without Nox4 knockdown, or the most specific Nox1/4 inhibitor (GKT137831). Effects of Nox4 inhibition on CIAKI mice were examined. Expression of Nox4 in HK-2 cells was significantly increased following iohexol exposure. Silencing of Nox4 rescued the production of ROS, downregulated pro-inflammatory markers (particularly phospho-p38) implicated in CIAKI, and reduced Bax and caspase 3/7 activity, which resulted in increased cellular survival in iohexol-treated HK-2 cells. Pretreatment with GKT137831 replicated these effects by decreasing levels of phospho-p38. In a CIAKI mouse model, even though the improvement of plasma blood urea nitrogen was unclear, pretreatment with GKT137831 resulted in preserved structure, reduced expression of 8-hydroxy-2’-deoxyguanosine (8OHdG) and kidney injury molecule-1 (KIM-1), and reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells. These results suggest Nox4 as a key source of reactive oxygen species responsible for CIAKI and provide a novel potential option for prevention of CIAKI. [ABSTRACT FROM AUTHOR]

Abstract: Retinoic acid (RA) is an important regulator of normal cellular proliferation and differentiation and suppressor of tumor growth by cell cycle arrest and apoptosis. Furthermore, RA showed a chemo-preventive activity in preclinical and/or clinical models of lung, head and neck, breast, and hepatocellular carcinoma (HCC). In this study, we examined the effect of RA on the proliferation of human HCC cells, in order to analyze its mode of action and, finally, we attempted to find a surrogate biomarker of RA for HCC chemopreventive treatment. Our findings suggested that the growth inhibition of RA in HCC cells differed according to G1 phase delay by CDK2 or 4, finally induction of apoptosis. No correlation was found between RA sensitivity and the expression of nuclear retinoid receptors, such as RARs or RXRs in HCC cells. RA treatment caused cell cycle arrest at G1 and decreased the expressions and activities of CDK2 or CDK4 in RA-sensitive HepG2 and SNU354 cells. On the other hand, RA-resistant Hep3B and SNU449 cells progressed into the S/G2 +M phase and showed increased CDK2 and CDK4 expression and activity. Since the inhibition of CDK2 or 4 activities resulted in sensitization of HCC cells to RA, the combination of RA and compounds of inhibiting CDKs such as UCN01 and flavopiridol might be a useful targeted therapy strategy for HCC. [Copyright &y& Elsevier]

Summary: Background: Transforming growth factor-β1 (TGF-β1) functions as a suppressor of tumor initiation by inhibiting cellular proliferation or by promoting cellular differentiation or apoptosis in the early phase of cancer development. Variations in the DNA sequence in the TGF-β1 gene may lead to altered TGF-β1 production and/or activity, and so this can modulate an individual''s susceptibility to lung cancer. To test this hypothesis, we investigated the association of the TGF-β1 −509C>T and 869T>C (L10P) polymorphisms and their haplotypes with the risk of lung cancer in a Korean population. Methods: The TGF-β1 genotypes were determined in 432 lung cancer patients and in 432 healthy control subjects who were frequency-matched for age and gender. The TGF-β1 haplotypes were predicted using a Bayesian algorithm in the Phase program. Results: Individuals with at least one −509T allele were at a significantly decreased risk of adenocarcinoma (AC) and small cell carcinoma (SM), as compared with carriers with the −509CC genotype [adjusted odds ratio (OR), 0.63; 95% confidence interval (CI), 0.42–0.96; P =0.04; and adjusted OR, 0.45; 95% CI, 0.27–0.76; P =0.002; respectively]. For the 869T>C polymorphism, the combined TC+CC genotype was associated with a significantly decreased risk of SM compared with the TT genotype (adjusted OR, 0.52; 95% CI, 0.31–0.88; P =0.01). Consistent with the results of the genotyping analyses, the −509T/869C haplotype was associated with a significantly decreased risk of AC and SM as compared with the −509C/869T haplotype (adjusted OR, 0.75; 95% CI, 0.57–0.98; P =0.04; and adjusted OR, 0.67; 95% CI, 0.47–0.96; P =0.02; respectively). Conclusion: The TGF-β1 −509C>T and 869T>C polymorphisms and their haplotypes may contribute to genetic susceptibility to AC and SM of the lung. [Copyright &y& Elsevier]