Role of the MAPK pathway in human lung epithelial-like A549 cells apoptosis induced by paraquat.

This study aims to investigate the value of mitogen-activated protein kinases (MAPKs) for paraquat (PQ)-induced apoptosis in human lung epithelial-like A549 cells and the specific mechanism. A549 cell apoptosis were induced by PQ. These cells were divided into six groups: control group (cells were cultured in RPMI-1640 medium); SP600125 group (cells were preconditioned with SP600125); SB203580 group (cells were preconditioned with SB203580); PQ group (cells were treated with PQ); SP600125+PQ group (cells were preconditioned with SP600125 following PQ); SB203580+PQ group (cells were preconditioned with SB203580 following PQ). The cell survival rate, apoptosis rate, and activities of caspase-3 and -9 were detected. When compared with the control group, both SP600125 and SB203580 groups had no significant difference in the detected indicators. When compared with PQ group, the cells in both SP600125+PQ group and SB203580+PQ group had significantly increased viability and level of anti-apoptotic protein Bcl-2; and had decreased apoptotic rates, decreased levels of caspase-3 and -9, and decreased level of pro-apoptotic protein Bax. The ratio of p-JNK/JNK protein expression in the SP600125+PQ group significantly decreased, while the ratio of the p-P38/P38 protein expression in the SB203580+PQ group decreased. PQ induced A549 cell apoptosis through the MAPKs pathway.

Neuropilin-1 regulated by miR-320 contributes to the growth and metastasis of cholangiocarcinoma cells.

BACKGROUND & AIMS: Neuropilin-1 (NRP-1) activates signalling pathways as multifunctional co-receptors in cancer cells. However, its role and how it is regulated by miRNAs in cholangiocarcinoma (CCA) have not yet been investigated. METHODS: The expression of NRP-1, miR-320 and key molecules involved in cell proliferation, migration and related signalling pathways were detected by immunohistochemistry, immunoblotting and qRT-PCR. Stable transfectants depleted of NRP-1 were generated. The regulatory effect of miR-320 on NRP-1 was evaluated by luciferase reporter assays. Cell proliferation, cell cycle distribution and migration were examined. Xenograft tumour models were established to assess tumourigenesis, tumour growth and lung metastasis. RESULTS: Cholangiocarcinoma tissues expressed higher levels of NRP-1 than adjacent normal biliary tissues, and its expression negatively correlated with miR-320. NRP-1 depletion inhibited cell proliferation and induced cell cycle arrest in the G1/S phase by upregulating p27, and downregulating cyclin E and cyclin-dependent kinase 2; and reduced cell migration by inhibiting the phosphorylation of focal adhesion kinase. NRP-1 depletion suppressed tumourigenesis, tumour growth and lung metastasis by inhibiting cell proliferation and tumour angiogenesis in experimental animals. Depletion of NRP-1 inhibited the activation of VEGF/VEGFR2, EGF/EGFR and HGF/c-Met pathways stimulated by respective ligands. MiR-320 negatively regulated the expression of NRP-1 by binding to the 3'-UTR of NRP-1 promoter, and miR-320 mimics inhibited cell proliferation and migration, and the growth of established tumours in animals by downregulating NRP-1. CONCLUSIONS: The present results indicate that NRP-1 is negatively regulated by miR-320, and both of them may be potentially therapeutic targets for CCA.

MicroRNA-21-Regulated Activation of the Akt Pathway Participates in the Protective Effects of H2S against Liver Ischemia-Reperfusion Injury.

Maintaining a certain level of hydrogen sulfide (H2S) in ischemia-reperfusion (I/R) is essential for limiting injury to the liver. Exogenous H2S exerts protective effects against this injury, but the mechanisms remain unclear. Liver injury was induced in Wistar rats undergoing hepatic I/R for 30 min, followed by a 3-h reperfusion. Administration of GYY4137 (a slow-releasing H2S donor) significantly attenuated the severity of liver injury and was reflected by reduced inflammatory cytokine production and cell apoptosis, the levels of which were elevated by I/R, while DL-propargylglycine (PAG, an inhibitor of cystathionine γ-lyase [CSE]) aggravated liver injury. Delivery of GYY4137 significantly elevated the plasma levels of H2S and upregulated the expression of microRNA-21 (miR-21), leading to the activation of the Akt pathway, in rat livers subjected to I/R. To further investigate the protective mechanisms of H2S during liver I/R injury, we established a cell model of hypoxia/reoxygenation (H/R) by incubating Buffalo rat liver (BRL) cells under hypoxia for 4 h followed by normoxia for 10 h. The regulatory effect of miR-21 on the Akt pathway by downregulating phosphatase and tensin homolog (PTEN) was validated by luciferase assays. Incubation of sodium hydrosulfide (NaHS), an H2S donor, increased the expression of miR-21, attenuated the reduced cell viability and the increased apoptosis by H/R, in BRL cells. Anti-miR-21 abolished the protective effects of NaHS by inactivating the Akt pathway. In conclusion, the present results indicate the activation of the Akt pathway regulated by miR-21 participates in the protective effects of H2S against I/R-induced liver injury.

2ME2 inhibits the activated hypoxia-inducible pathways by cabozantinib and enhances its efficacy against medullary thyroid carcinoma.

Cabozantinib is a multi-targeted tyrosine kinase inhibitor targeting vascular endothelial growth factor (VEGF) receptor (VEGFR)-2, MET (c-Met, also called hepatocyte growth factor (HGF) receptor), and other receptor tyrosine kinases. Cabozantinib has recently been approved for treating advanced medullary thyroid carcinoma (MTC), but its long-term benefit remains uncertain and dose-dependent adverse events are very common. The present study has demonstrated that 2-methoxyestradiol (2ME2), an inhibitor of hypoxia-inducible factors (HIFs) and a promising anticancer agent under investigation in clinical trials, strengthens anticancer activities of cabozantinib against MTC cells in vitro and in vivo. The activated hypoxia-inducible pathways, which are mainly regulated by HIF-1, contribute to the resistance of hypoxic MTC cells to cabozantinib. Cabozantinib upregulated HIF-1α expression at translational levels and increased the expression of the downstream factors including VEGF, lactate dehydrogenase A (LDHA), HGF, and MET. 2ME2 corrected the activated pathways by cabozantinib through downregulating HIF-1α expression and inhibiting its nuclear translocation in hypoxic MTC cells. Administration of 2ME2 enhanced the efficacy of cabozantinib in suppressing the growth of MTC cell line xenografts and patient-derived xenografts established in mice. Given that 2ME2 targets insensitive hypoxic cancer cells to cabozantinib and can inhibit the activated pathways by cabozantinib, the present results warrant further investigation of 2ME2, particularly in combination with cabozantinib, for the treatment of MTC.

Enhanced autophagy by everolimus contributes to the antirestenotic mechanisms in vascular smooth muscle cells.

OBJECTIVE: The present study aims to investigate the underlying mechanisms accounting for the activities of everolimus to inhibit the growth of vascular smooth muscle cells (VSMCs), which contributes to restenosis. METHODS: Primary VSMCs were cultured in media containing smooth muscle growth supplements and incubated with testing agents. Cell proliferation, cell cycle distribution, apoptosis and autophagy, and the key molecules involved, were examined. RESULTS: Everolimus inhibited the proliferation of VSMCs by inhibiting the activation of ribosomal protein S6 kinase and phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1, and downregulating proliferating cellular nuclear antigen. Everolimus induced cell cycle arrest at the G1 phase by downregulating cyclin D1 and upregulating p27, and increased apoptosis by downregulating Bcl-2, upregulating Bad and activating capsase-3 and poly ADP ribose polymerase. Everolimus enhanced autophagy by increasing the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II, and upregulating Beclin 1. Specific autophagy inhibitors, 3-methyladenine and bafilomycin A1, significantly attenuated the inhibition of cell proliferation, the increased apoptosis and the altered expression of the above key proteins induced by everolimus. CONCLUSIONS: Enhanced autophagy by everolimus contributes to its antirestenotic activity and its abilities to inhibit cell proliferation and to induce apoptosis of VSMCs.

Interpretable machine learning for the prediction of death risk in patients with acute diquat poisoning.

The aim of this study was to develop and validate predictive models for assessing the risk of death in patients with acute diquat (DQ) poisoning using innovative machine learning techniques. Additionally, predictive models were evaluated through the application of SHapley Additive ExPlanations (SHAP). A total of 201 consecutive patients from the emergency departments of the First Hospital and Shengjing Hospital of China Medical University admitted for deliberate oral intake of DQ from February 2018 to August 2023 were analysed. The initial clinical data of the patients with acute DQ poisoning were collected. Machine learning methods such as logistic regression, random forest, support vector machine (SVM), and gradient boosting were applied to build the prediction models. The whole sample was split into a training set and a test set at a ratio of 8:2. The performances of these models were assessed in terms of discrimination, calibration, and clinical decision curve analysis (DCA). We also used the SHAP interpretation tool to provide an intuitive explanation of the risk of death in patients with DQ poisoning. Logistic regression, random forest, SVM, and gradient boosting models were established, and the areas under the receiver operating characteristic curves (AUCs) were 0.91, 0.98, 0.96 and 0.94, respectively. The net benefits were similar across all four models. The four machine learning models can be reliable tools for predicting death risk in patients with acute DQ poisoning. Their combination with SHAP provides explanations for individualized risk prediction, increasing the model transparency.

Secretory clusterin contributes to oxaliplatin resistance by activating Akt pathway in hepatocellular carcinoma.

Secretory clusterin (sCLU) is expressed in numerous cancers and is associated with the resistance to chemotherapy. However, the role of sCLU in the resistance of hepatocellular carcinoma (HCC) to oxaliplatin (OXA), a recently used third-generation platinum agent, remains unclear. The stable transfectants that are depleted of or overexpress sCLU and OXA-resistant cells were generated using human HCC cells. Overexpression of sCLU abrogated OXA-induced inhibition of cell growth and cell apoptosis, but depletion of sCLU synergized with OXA to inhibit cell growth and enhance cell apoptosis, by regulating proteins involved in mitochondrial apoptosis pathways, such as Bcl-2, Bax, Bcl-xL and caspase-9, and affecting phosphorylation of Akt and GSK-3ß. Overexpression of sCLU in either OXA-resistant cells or stable transfectants that overexpress sCLU significantly increased phosphorylated Akt. However, specific inhibition of Akt enhanced sensitivity of sCLU-overexpressing cells to OXA, but had no effect on sCLU expression, suggesting that the regulatory effects between sCLU and pAkt may be in a one-way manner in HCC cells. The expression levels of sCLU affected the therapeutic efficacy of OXA to treat HCC tumors established in immunodeficiency mice. The results have demonstrated that sCLU contributes to OXA resistance by activating Akt pathway, indicating that sCLU may be a novel molecular target for overcoming OXA resistance in HCC.

Analysis of Human microRNA Expression Profiling During Diquat-Induced Renal Proximal Tubular Epithelial Cell Injury.

Background: We established a diquat-induced human kidney-2 cells (HK-2 cells) apoptosis model in this study to identify differentially expressed microRNAs (miRNAs) and signaling pathways involved in diquat poisoning via gene sequencing and bioinformatics analysis and explored the related therapeutic benefits. Methods: The effects of diquat on the viability and apoptosis of HK-2 cells were explored using the CCK-8 and Annexin V-FITC/PI double staining methods. Total RNAs were extracted using the TRizol method and detected by Illumina HiSeq 2500. Bioinformatics analysis was performed to explore differentially expressed (DE) miRNAs, their enriched biological processes, pathways, and potential target genes. The RT-qPCR method was used to verify the reliability of the results. Results: Diquat led to HK-2 cell injury and apoptosis played an important role, hence an HK-2 cell apoptosis model in diquat poisoning was established. Thirty-six DE miRNAs were screened in diquat-treated HK-2 cells. The enriched biological process terms were mainly cell growth, regulation of apoptotic signaling pathway, extrinsic apoptotic signaling pathway, and Ras protein signal transduction. The enriched cellular components were mainly cell-cell junction, cell-substrate junction, ubiquitin ligase complex, and protein kinase complex. The enriched molecular functions were mainly Ras GTPase binding, ubiquitin-like protein transferase activity, DNA-binding transcription factor binding, ubiquitin-protein transferase activity, nucleoside-triphosphatase regulator activity, transcription coactivator activity, and ubiquitin-like protein ligase binding. Signaling pathways such as MAPK, FoxO, Ras, PIK3-Akt, and Wnt were also enriched. Conclusion: These findings aid in understanding the mechanisms of diquat poisoning and the related pathways, where DE miRNAs serve as targets for gene therapy.

A local translation program regulates centriole amplification in the airway epithelium.

Biogenesis of organelles requires targeting of a subset of proteins to specific subcellular domains by signal peptides or mechanisms controlling mRNA localization and local translation. How local distribution and translation of specific mRNAs for organelle biogenesis is achieved remains elusive and likely to be dependent on the cellular context. Here we identify Trinucleotide repeat containing-6a (Tnrc6a), a component of the miRNA pathway, distinctively localized to apical granules of differentiating airway multiciliated cells (MCCs) adjacent to centrioles. In spite of being enriched in TNRC6A and the miRNA-binding protein AGO2, they lack enzymes for mRNA degradation. Instead, we found these apical granules enriched in components of the mRNA translation machinery and newly synthesized proteins suggesting that they are specific hubs for target mRNA localization and local translation in MCCs. Consistent with this, Tnrc6a loss of function prevented formation of these granules and led to a broad reduction, rather than stabilization of miRNA targets. These included downregulation of key genes involved in ciliogenesis and was associated with defective multicilia formation both in vivo and in primary airway epithelial cultures. Similar analysis of Tnrc6a disruption in yolk sac showed stabilization of miRNA targets, highlighting the potential diversity of these mechanisms across organs.

Downregulating hypoxia-inducible factor-2α improves the efficacy of doxorubicin in the treatment of hepatocellular carcinoma.

The hypoxic microenvironment inside solid tumors, including hepatocellular carcinoma (HCC), is a major cause of tumor resistance to chemotherapy. The recently identified hypoxia-inducible factor (HIF)-2 executes the hypoxia response. Its expression feature and transcriptional targets indicate a possible dominance of HIF-2 in regulating genes in HCC. The aim of the present study was to determine whether transfection of siRNA targeting HIF-2α could enhance the efficacy of doxorubicin, the most commonly used drug in the treatment of HCC. Transfection of HIF-2 siRNA into human HCC cells downregulated the expression of HIF-2α, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-α, and cyclin D1, but had little effect on the expression of HIF-1α, fms-related tyrosine kinase-1 (Flt-1), the glucose transporter (GLUT)-1, and lactate dehydrogenase A (LDHA). Doxorubicin itself only downregulated VEGF expression. Furthermore, HIF-2 siRNA inhibited proliferation, induced cell cycle arrest at the G(0)/G(1) phase, and acted synergistically with doxorubicin to inhibit the growth of human HCC cells in vitro. Transfection of HIF-2 siRNA also downregulated tumoral expression of HIF-2α, VEGF, TGF-α, and cyclin D1 in vivo, and acted synergistically with doxorubicin to suppress the growth of HepG2 tumors established in immunodeficient mice by inhibiting cell proliferation, tumor angiogenesis and microvessel perfusion. The results of the present study suggest that targeting HIF-2α with siRNA warrants investigation as a potential strategy to enhance the efficacy of doxorubicin in the treatment of HCC.

Analysis of microRNA expression profiling during paraquat-induced injury of murine lung alveolar epithelial cells.

Paraquat (PQ) as a non-selective heterocyclic herbicide, has been applied worldwide for over a few decades. But PQ is very harmful to humans and rodents. The lung is the main target organ of PQ poisoning. It is an important event that lung epithelial cells are injured during PQ-induced acute lung injury and pulmonary fibrosis. As a regulator of mRNA expression, microRNA (miRNA) may play an important role in the progress. Our study was to investigate the mechanisms of PQ-induced injury of pulmonary epithelial cells through analyzing the profiling of miRNAs and their target genes. As a result, 11 differentially expressed miRNAs were screened, including 1 upregulated miRNA and 10 downregulated miRNAs in PQ-treated murine lung alveolar epithelial cells (MLE-12 cells). The bioinformatic analyses suggested that the target genes of these miRNAs were involved in mitochondrial apoptosis pathway and DNA methylation, and participated in the regulation of PI3K-Akt, mTOR, RAS, TNF, MAPK and other signal pathways which related to oxidative stress and apoptosis. This indicated that miRNAs were an important regulator of oxidative stress and apoptosis during PQ-induced injury of murine lung alveolar epithelial cells. The findings would deepen our understanding of the mechanisms of PQ-induced pulmonary injury and might provide new treatment targets for this disease.

Downregulation of developmentally regulated endothelial cell locus-1 inhibits the growth of colon cancer.

Developmentally regulated endothelial cell locus-1 (Del1) is an embryonic angiogenic factor expressed in early embryonic endothelial cells, but recently has been found to be expressed in some forms of cancers including colon and breast cancers, and melanoma, and human cancer cell lines. Overexpression of Del1 accelerates tumor growth by enhancing vascular formation, implying Del1 may be a potential target for anti-angiogenic cancer therapy. The study aims to investigate whether downregulation of Del1 could inhibit the growth of tumors established in nude Balb/c mice by subcutaneous implantation of human LS-174T colon cancer cells. The shRNA expression vectors targeting human Del1, and vascular endothelial growth factor (VEGF) were constructed. Gene transfection of Del1-shRNA downregulated expression of Del1 in LS-174T cells in vivo and in vitro, but did not alter the proliferative or survival properties of cells in vitro. Gene transfection of VEGF-shRNA downregulated expression of both VEGF and Del1 in LS-174T cells in vivo and in vitro. Both Del1-shRNA and VEGF-shRNA gene therapies exhibited anti-tumor activities and they also showed a synergistic effect in suppressing growth of colon tumors by anti-angiogenesis and anti-proliferation. Although further investigation to clarify the mechanisms explaining the role of Del1 in tumor growth, and the interaction between VEGF and Del1, is required, the results indicate that downregulation of Del1 presents a potent therapeutic strategy to combat colon cancer.

Protein kinase C is involved in arsenic trioxide-induced apoptosis and inhibition of proliferation in human bladder cancer cells.

OBJECTIVE: Arsenic trioxide (ATO) is a potent antitumor agent used to treat acute promyelocytic leukemia, and recently solid tumors including bladder cancers. However, a mechanism to explain its antitumor activity in bladder cancers is unclear. Here, we investigated the role of protein kinase C (PKC) in ATO-induced apoptosis and inhibition of proliferation in bladder cancer cells. METHODS: T24 human bladder carcinoma cells were incubated with different concentrations of ATO in the presence or absence of PMA (PKC activator) or H7 (PKC inhibitor). Cell proliferation was assessed by MTT assay, and apoptosis by TUNEL and electron microscopy. Flow cytometry was used to analyze cell cycle distribution, radioimmunoassay to measure PKC activity, and Western blot analysis to detect caspase-3. RESULTS: ATO inhibited proliferation and induced apoptosis of T24 cells in a dose-dependent manner, caused an increase of percentage of cells in the G(1) phase and a decrease in the S and G(2) phases, and upregulated the expression of activated caspase-3 and reduced PKC activity. These effects were abrogated by PMA, but enhanced by H7. CONCLUSIONS: PKC is involved in the anticancer activity of ATO for T24 bladder cancer cells, suggesting that targeting the PKC pathway may represent a potential approach to enhance the efficacy of ATO to treat bladder cancers.

Effect of endoplasmic reticulum calcium on paraquat­induced apoptosis of human lung type II alveolar epithelial A549 cells.

The present study aimed to explore the role of endoplasmic reticulum calcium (ER Ca2+) in the apoptosis of human lung type II alveolar epithelial A549 cells induced by paraquat (PQ) in vitro. PQ significantly elevated the intracellular Ca2+ concentration. Treatment with the Ca2+­ATPase inhibitor thapsigargin significantly increased PQ­induced cytotoxicity, elevated the intracellular level of Ca2+, and increased the apoptosis rate, the protein expression of glucose­regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), and the activities of caspase­7 and caspase­12 in PQ­treated cells. By contrast, treatment with heparin, an inositol 1,4,5­triphosphate receptor inhibitor, remarkably attenuated cytotoxicity and decreased the intracellular level of Ca2+, the apoptosis rate and the expression levels of GRP78, CHOP and Caspases. In conclusion, PQ impaired the regulating function of ER Ca2+ and resulted in an excessive increase of intracellular Ca2+. Therefore, influencing the Ca2+ signaling in the ER influenced the apoptosis of A549 cells via the ER stress pathway.

LncRNA SNHG1 contributes to sorafenib resistance by activating the Akt pathway and is positively regulated by miR-21 in hepatocellular carcinoma cells.

BACKGROUND: Acquired resistance to sorafenib greatly limits its therapeutic efficiency in the treatment of hepatocellular carcinoma (HCC). Increasing evidence indicates that long noncoding RNAs (lncRNAs) play important roles in the resistance to anti-cancer drugs. The present study aims to explore the involvement of lncRNA SNHG1 (small nucleolar RNA host gene 1) in sorafenib resistance and how SNHG1 is associated with overexpressed microRNA-21 (miR-21) and the activated Akt pathway, which have been demonstrated to mediate this resistance in HCC cells. METHODS: Sorafenib-resistant HCC (SR-HCC) cells were generated and their sorafenib-resistant properties were confirmed by cell viability and apoptosis assays. Potential lncRNAs were screened by using multiple bioinformatics analyses and databases. The expression of genes and proteins was detected by qRT-PCR, Western blot and in situ hybridization. Gene silencing was achieved by specific siRNA or lncRNA Smart Silencer. The effects of anti-SNHG1 were evaluated in vitro and in experimental animals by using quantitative measures of cell proliferation, apoptosis and autophagy. The binding sites of miR-21 and SNHG1 were predicted by using the RNAhybrid algorithm and their interaction was verified by luciferase assays. RESULTS: The Akt pathway was highly activated by overexpressed miR-21 in SR-HCC cells compared with parental HCC cells. Among ten screened candidates, SNHG1 showed the largest folds of alteration between SR-HCC and parental cells and between vehicle- and sorafenib-treated cells. Overexpressed SNHG1 contributes to sorafenib resistance by activating the Akt pathway via regulating SLC3A2. Depletion of SNHG1 enhanced the efficacy of sorafenib to induce apoptosis and autophagy of SR-HCC cells by inhibiting the activation of Akt pathway. Sorafenib induced translocation of miR-21 to the nucleus, where it promoted the expression of SNHG1, resulting in upregulation of SLC3A2, leading to the activation of Akt pathway. In contrast, SNHG1 was shown to have little effect on the expression of miR-21, which downregulated the expression of PTEN, leading to the activation of the Akt pathway independently of SNHG1. CONCLUSIONS: The present study has demonstrated that lncRNA SNHG1 contributes to sorafenib resistance by activating the Akt pathway and its nuclear expression is promoted by miR-21, whose nuclear translocation is induced by sorafenib. These results indicate that SNHG1 may represent a potentially valuable target for overcoming sorafenib resistance for HCC.

Adeno-associated virus-mediated expression of kallistatin suppresses local and remote hepatocellular carcinomas.

BACKGROUND: The current treatments for hepatocellular carcinoma (HCC) are poor, particularly for metastatic HCC. Intraportal transfusion of adeno-associated virus (AAV) leads to long-term and persistent transgenic expression in livers. Kallistatin, a novel angiogenesis inhibitor, exhibits anti-tumor activity. The aim of the study was to investigate whether intraportal injection of AAV-kallistatin could suppress local and metastatic HCC in mice. METHODS: An AAV vector encoding kallistatin was constructed, and its transduction efficiency by intraportal transfusion in livers was examined by RT-PCR, immunohistochemical and Western blotting analysis. The anti-tumor activity was tested in three HCC models including hepatic and subcutaneous human Hep3B HCC tumors in BALB/c athymic (nu/nu) mice, and subcutaneous mouse BNL HCC tumors in BALB/c mice. Tumor cell proliferation in situ was examined by anti-Ki-67 staining, and apoptosis by TUNEL. RESULTS: Gene transfection by rAAV-kallistatin inhibited proliferation of human umbilical vein endothelial cells and HCC cells in vitro. Intraportal injection of rAAV-kallistatin resulted in persistent and specific expression of kallistatin in livers detected by RT-PCR and immunohistochemical analysis, and kallistatin protein in circulation detected by Western blotting analysis. Intraportal injection of rAAV-kallistatin significantly suppressed angiogenesis and growth of hepatic Hep3B tumors. The kallistatin released by hepatocytes into the circulation suppressed remote Hep3B and BNL tumors established subcutaneously. The rAAV-kallistatin gene therapy significantly inhibited tumor cell proliferation and induced apoptosis. CONCLUSIONS: Intraportal injection of rAAV-kallistatin suppressed hepatic and subcutaneous HCC tumors, relying on its anti-angiogenic and anti-proliferative activities.

Taurine attenuates multiple organ injury induced by intestinal ischemia reperfusion in rats.

BACKGROUND: Intestinal ischemia reperfusion (II/R) is a serious clinical condition associated with simultaneous multiple organ dysfunction. The purpose of this study was to investigate whether taurine, an anti-oxidative, anti-inflammatory, and anti-apoptotic agent, could attenuate multiple organ injury induced by II/R. MATERIALS AND METHODS: Taurine was intravenously injected to Wistar rats 30 min before II/R; physiological saline and sham operation served as controls. II/R was produced by occlusion of superior mesenteric artery for 60 min. Rats were randomly sacrificed 1.5, 3, 12, and 36 h after II/R; blood samples were collected for assessing alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr), and tumor necrosis factor-alpha (TNF-alpha), and intestines, livers, kidneys, and lungs were removed for histological examination of scoring injury severity and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling analysis. The amount of lipid peroxides (LPO) was measured in intestinal tissues, and expression of caspase-3 was detected in all of the tissues with Western blot analysis. RESULTS: II/R resulted in injury to intestines as well as livers, kidneys, and lungs, evidenced by morphological alteration, increased cell apoptosis, and elevated serum levels of ALT, AST, BUN, and Cr. The damage reached peak 3 h after II/R. The intestinal LPO and serum levels of TNF-alpha were increased after II/R. Pre-administration of taurine significantly attenuated multiple organ injury as the histological score, apoptosis index, LPO, and levels of ALT, AST, BUN, Cr, and TNF-alpha were significantly lower compared with saline controls. CONCLUSIONS: Taurine attenuates multiple organ injury induced by II/R. Although the mechanism needs further investigation, taurine inhibits production of intestinal LPO, release of TNF-alpha, cell apoptosis, and expression of caspase-3.

Protective Effects of Chymostatin on Paraquat-Induced Acute Lung Injury in Mice.

This study aims to evaluate the role of chymostatin in paraquat-induced acute lung injury. Institute of Cancer Research mice were randomly distributed into the NS, DMSO, chymostatin, paraquat or chymostatin treatment groups. Six mice from each group were intraperitoneally injected with chloral hydrate at 0, 1, 2, 4, 8, 12, 24 and 48 h after treatment administration. Blood samples were collected through cardiac puncture. Lung tissues were stained with haematoxylin and eosin for the observation of lung histology. The degree of pulmonary oedema was determined on the basis of lung wet-to-dry ratio (W/D). The serum activity of cathepsin G was determined through substrate fluorescence assay. The serum levels of endothelial cell-specific molecule-1 (endocan), tumour necrosis factor-a (TNF-a), interleukin-1ß (IL-1ß), IL-6 and high-mobility group box protein 1 (HMGB1) were determined through enzyme-linked immunosorbent assay. The expression levels of endocan and nuclear NF-κBp65 in the lung were quantified through Western blot. Chymostatin alleviated the pathological changes associated with acute alveolitis in mice; decreased the lung W/D ratio, the activity of cathepsin G and the serum concentrations of TNF-a, IL-1ß, IL-6 and HMGB1; and increased the serum concentration of endocan. Western blot results revealed that chymostatin up-regulated endocan expression and down-regulated nuclear NF-κBp65 expression in the lung. Chymostatin reversed the inflammatory effects of paraquat-induced lung injury by inhibiting cathepsin G activity to up-regulate endocan expression and indirectly inhibit NF-κBp65 activity.

MnTMPyP inhibits paraquat-induced pulmonary epithelial-like cell injury by inhibiting oxidative stress.

OBJECTIVE: To investigate the protective effect and underlying mechanism of the superoxide dismutase mimic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), on paraquat (PQ)-induced lung alveolar epithelial-like cell injury. METHODS: Lung alveolar epithelial-like cells (A549) were pretreated with 10 µM MnTMPyP for 1.5 hr and then cultured with or without PQ (750 uM) for 24 hr. Cell survival was determined using the MTT assay. Apoptosis, mitochondrial transmembrane potential, reactive oxygen species (ROS) production, and Ca2+ levels were measured using flow cytometry. Glutathione reductase activity (GR activity) and caspase-3 activation were determined using spectrophotometry. Expression of the apoptosis proteins, Bcl-2 and Bax, and the endoplasmic reticulum (ER) stress proteins, glucose regulatory protein 78 (Grp78) and C/EBP homologous protein (CHOP), was measured using Western blot analysis. RESULTS: Cell viability, mitochondrial membrane potential, GR activity, and Bcl-2 expression were decreased, but apoptosis, ROS production, caspase-3 activity, cytoplasmic Ca2+ levels, and Bax, Grp78 and CHOP expression were all increased in the PQ group compared to the control group. There were no statistically significant changes in the MnTMPyP group. Cell viability, GR activity, mitochondrial membrane potential, and Bcl-2 protein expression were all increased, while apoptosis, ROS production, cytoplasmic Ca2+ levels, caspase-3 activity, and Bax, Grp78 and CHOP expression were all significantly reduced in the MnTMPyP group compared to PQ group. CONCLUSION: MnTMPyP effectively reduced PQ-induced lung epithelial-like cell injury, and the underlying mechanism is related to antagonism of PQ-induced oxidative stress.

Involvement of PINK1/Parkin-mediated mitophagy in paraquat- induced apoptosis in human lung epithelial-like A549 cells.

Paraquat (PQ) is one of the most popular herbicides and has been widely used all over the world over the past several decades. However, PQ exposure can cause multiple organ failure, especially acute lung injury in humans as well as in rodent animals. Mitochondrial dysfunction plays a crucial role in PQ-induced lung cell damage. Mitophagy, defined as the selective autophagic elimination process of mitochondria, is a significant mechanism controlling mitochondrial quality. In this study, we investigated PINK1/Parkin-mediated mitophagy activated in the process of the PQ-induced cell apoptosis by using human lung epithelial-like A549 cells. We showed that PQ inhibited cell viability and induced mitochondrial damage as well as cell apoptosis in A549 cells. During this process, PQ induced PINK1/Parkin-mediated mitophagy. Knocking down the expression of Parkin gene by the transient transfection of Parkin small interfering RNA mitigated PQ-induced mitophagy and worsened A549 cell apoptosis. On the contrary, overexpression of Parkin attenuated PQ-induced cell injury by promoting mitophagy. These results indicated PINK1/Parkin-mediated mitophagy played a protective role in PQ-induced A549 cell damage and provided a potential therapeutic strategy for enhancing mitophagy against PQ poisoning.