This paper aimed to investigate the effects of high mobility group box 1(HMGB1)-mediated pulmonary artery smooth muscle cell pyroptosis and immune imbalance on chronic obstructive pulmonary disease-associated pulmonary hypertension(COPD-PH) in rats and the intervening mechanism of Compound Tinglizi Decoction. Ninety rats were randomly divided into a normal group, a model group, low-dose, medium-dose, and high-dose Compound Tinglizi Decoction groups, and a simvastatin group. The rat model of COPD-PH was established by fumigation combined with lipopolysaccharide(LPS) intravascular infusion, which lasted 60 days. Rats in the low, medium, and high-dose Compound Tinglizi Decoction groups were given 4.93, 9.87, and 19.74 g·kg~(-1) Compound Tinglizi Decoction by gavage, respectively. Rats in the simvastatin group were given 1.50 mg·kg~(-1) simvastatin by gavage. After 14 days, the lung function, mean pulmonary artery pressure, and arterial blood gas of rats were analyzed. Lung tissues of rats were collected for hematoxylin-eosin(HE) staining to observe the pathological changes. Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR) was used to determine the expression of related mRNA in lung tissues, Western blot(WB) was used to determine the expression of related proteins in lung tissues, and enzyme linked immunosorbent assay(ELISA) was used to determine the levels of inflammatory factors in the lung tissues of rats. The ultrastructure of lung cells was observed by transmission electron microscope. The forced vital capacity(FVC), forced expiratory volume in 0.3 second(FEV_(0.3)), FEV_(0.3)/FVC, peek expiratory flow(PEF), respiratory dynamic compliance(Cdyn), arterial partial pressure of oxygen(PaO_2), and arterial oxygen saturation(SaO_2) were increased, and resistance of expiration(Re), mean pulmonary arterial pressure(mPAP), right ventricular hypertrophy index(RVHI), and arterial partial pressure of carbon dioxide(PaCO_2) were decreased by Compound Tinglizi Decoction in rats with COPD-PH. Compound Tinglizi Decoction inhibited the protein expression of HMGB1, receptor for advanced glycation end products(RAGE), pro caspase-8, cleaved caspase-8, and gasdermin D(GSDMD) in lung tissues of rats with COPD-PH, as well as the mRNA expression of HMGB1, RAGE, and caspase-8. Pulmonary artery smooth muscle cell pyroptosis was inhibited by Compound Tinglizi Decoction. Interferon-γ(IFN-γ) and interleukin-17(IL-17) were reduced, and interleukin-4(IL-4) and interleukin-10(IL-10) were incresead by Compound Tinglizi Decoction in lung tissues of rats with COPD-PH. In addition, the lesion degree of trachea, alveoli, and pulmonary artery in lung tissues of rats with COPD-PH was improved by Compound Tinglizi Decoction. Compound Tinglizi Decoction had dose-dependent effects. The lung function, pulmonary artery pressure, arterial blood gas, inflammation, trachea, alveoli, and pulmonary artery disease have been improved by Compound Tinglizi Decoction, and its mechanism is related to HMGB1-mediated pulmonary artery smooth muscle cell pyroptosis and helper T cell 1(Th1)/helper T cell 2(Th2), helper T cell 17(Th17)/regulatory T cell(Treg) imbalance.
Animals ; Rats ; Caspase 8 ; Pyroptosis ; HMGB1 Protein/genetics* ; Hypertension, Pulmonary/etiology* ; Pulmonary Disease, Chronic Obstructive/genetics*

BACKGROUND: Acquired and primary resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is still the bottleneck of clinical treatment of advanced non-small cell lung cancer (NSCLC). STE029 is a novel anticancer drug which consists of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) inhibitor and novel cancer cell membrane targeting molecular. This study aimed to investigate the reversal mechanism of EGFR-TKI resistance by STE029 in lung adenocarcinoma. METHODS: CCK8 test was used to test the cell viability and survival rate of EGFR mutated PC9 cell (Gefitinib sensitive), PC9/BB4 cell (acquired Gefitinib resistant), and EGFR wild type A549 cell after treatment of STE029, Gefitinib or combination of both. EdU test was applied to detect changes in cell cycle and Hoechst 33258 was applied to detect apoptosis rate in overcoming the EGFR-TKI resistance. The activity of EGFR/PI3K/Akt, cell cycle and apoptosis signal pathways were examined. In vivo, nude mice were exposed to STE029, Gefitinib and STE029+Gefitinib for 5 wk. And the the tumor volume was measured and tumor weight was obtained on the last day. RESULTS: (1) PC9 cells was highly sensitive to Gefitinib, while PC9/BB4 and A549 cell showed significant resistance to Gefitinib treatment; (2) STE029+Gefitinib treatment could significantly decrease the 50% inhibitory concentrarion (IC₅₀) of Gefitinib in PC9, PC9/BB4 and A549 cells (P<0.05, respectively); (3) In PC9 and PC9/BB4 cells, STE029+Gefitinib can block cell cycle and inhibit cell proliferation (P<0.001), while there was no significant difference in apoptosis rate among three drug intervention groups (P>0.05); However, apoptosis rate was increased in STE029+Gefitinib group in A549 cell (P<0.01), while no significance detected in cell proliferation (P>0.05). (4) In PC9 and PC9/BB4 cells, the combination of STE029 and Gefitinib could downregulate p-EGFR, p-Akt, p-Cyclin D1 and Cyclin D1 (P<0.001), and upregulate the expression of GSK-3β (P<0.001), and the expression of cleaved caspase-8, caspase-8 cleaved caspase-9, caspase-9 showed no difference among groups (P>0.05). In A549 cells, the combination of STE029 and Gefitinib could downregulate p-Akt (P<0.001) and upregulate cleaved caspase-8 and cleaved caspase-9 (P<0.001); (5)In vivo, the combination of STE029 and Gefitinib effectively inhibited tumor development and progression compared to STE029 alone or Gefitinib alone, with significant difference (P<0.05) in PC9 and PC9/BB4 xenografted tumor. CONCLUSIONS STE029 could sensitize Gefitinib by inhibiting EGFR/PI3K/Akt pathway, blocking the tumor cell cycle and proliferation and inducing apoptosis through caspase-8 and caspase-9 dependent pathway. STE029 deserves further investigations in overcoming EGFR-TKI resistance in lung cancer.
Animals ; Mice ; Humans ; Gefitinib/pharmacology* ; Caspase 9 ; Caspase 8 ; Cyclin D1 ; Carcinoma, Non-Small-Cell Lung ; Glycogen Synthase Kinase 3 beta ; Mice, Nude ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Lung Neoplasms/genetics* ; Adenocarcinoma of Lung/drug therapy* ; Protein Kinase Inhibitors/pharmacology* ; ErbB Receptors/genetics*

OBJECTIVE: To investigate the relationships between caspase-8 (CASP8), fatty acid synthetase (Fas) gene polymorphisms and prognosis of non-Hodgkin's lymphoma patients in Han nationality. METHODS: The clinical data of 85 patients with non-Hodgkin's lymphoma were analyzed retrospectively. The polymorphisms of CASP8 and Fas gene were detected, and prognosis of the patients were analyzed. The polymorphisms of CASP8 and Fas gene in patients with different prognosis were compared, and the relationships between gene polymorphisms and the poor prognosis of the patients were investigated. RESULTS: The incidence rate of poor prognosis of the patients enrolled in the study was 65.88%. The polymorphisms of CASP8 and Fas genes in the patients with poor or good prognosis were in accordance with Hardy Weinberg's law of genetic balance. The frequencies of GG genotype and G allele at rs 1035142 of CASP8 gene, GA genotype and A allele at rs 1377 of Fas gene in patients with poor prognosis were lower than those of the patients with good prognosis (P<0.05). The frequencies of GT, TT and T alleles at rs 1035142 of CASP8 gene, GG and G alleles at rs 1377 of Fas gene in patients with poor prognosis were higher than those of the patients with good prognosis (P<0.05). The proportions of Ann Arbor stage III-IV and high malignancy in patients with poor prognosis were higher than those of the patients with good prognosis (P<0.05). Logistic multiple regression analysis showed that Ann Arbor stage III-IV, moderate malignant, high malignancy, CASP8 rs 1035142 GT genotype, CASP8 rs 1035142 TT genotype and Fas rs 1377 GG genotype were all the risk factors for the poor prognosis of the patients (P<0.05). CONCLUSION The poor prognosis rate of non-Hodgkin's lymphoma patients in Han nationality is relatively high, and the risk factors for the prognosis of the patients include Ann Arbor stage III-IV, moderate and high malignancy, CASP8 rs 1035142 GT genotype, CASP8 rs 1035142 TT genotype and Fas rs 1377 GG genotype.
Caspase 8/genetics* ; Ethnicity ; Fatty Acids ; Humans ; Ligases ; Lymphoma, Non-Hodgkin/genetics* ; Polymorphism, Genetic ; Prognosis ; Retrospective Studies ; fas Receptor

Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.
Animals ; Antioxidants/*metabolism/*pharmacology ; Apoptosis/drug effects ; Caspase 3/metabolism ; Caspase 8/metabolism ; Cell Line ; Cisplatin/*toxicity ; Cysteine/*analogs & derivatives/pharmacology ; Gene Expression Regulation/*drug effects ; Gene Knockdown Techniques ; Glutathione/*metabolism ; Heme Oxygenase-1/genetics ; Intracellular Space/metabolism ; Male ; Metabolic Detoxication, Phase II/genetics ; Mice ; NF-E2-Related Factor 2/genetics ; Nitric Oxide/biosynthesis ; Organ of Corti/*drug effects/*metabolism ; RNA Interference ; Rats ; Reactive Oxygen Species/metabolism ; Superoxide Dismutase/genetics

OBJECTIVEThe study aimed to test if Paridis Rhizoma total saponins (PRTS) could induce apoptosis of human gastric cancer cell MKN-45.

METHODBased on the previous researches, PRTS was set by different concentrations to treat human gastric cancer cell for 12 h (5, 10, 20 mg x L(-1)). Fluorescent staining methods were adopted to observe apoptotic morphological changes of MKN-45. The apoptosis rates were analyzed by flow cytometry with Annexin V-FITC/PI staining. The enzymatic activities of caspase-3 and caspase-8 were measured by ELISA. The protein levels of Fas and FasL were detected by Western blotting.

RESULTUnder a fluorescence microscope, MKN-45 treated by PRTS was seen typical apoptotic morphological features. PRTS significantly increased the rate of apoptosis. Compared with the control group, there exsited significant differences in apoptosis rate of PRTS concentration of 20 mg x L(-1) (P < 0.01); besides, the enzymatic activities of caspase-3 and caspase-8 were promoted obviously after the effect of PRTS on MKN-45 cells for 12 h (P < 0.01). The protein levels of Fas and FasL in the MKN-45 were upgraded significantly.

CONCLUSIONPRTS can induce apoptosis of human gastric cancer cell MKN-45 , which is concerned with caspase-3 and caspase-8 and upgraded Fas and FasL.

Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Caspase 8 ; genetics ; metabolism ; Cell Line, Tumor ; Drugs, Chinese Herbal ; pharmacology ; Fas Ligand Protein ; metabolism ; Humans ; Magnoliopsida ; chemistry ; Rhizome ; chemistry ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Stomach Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; fas Receptor ; metabolism

Various kinds of schiff base metal complexes have been proven to induce apoptosis of tumor cells. However, it remains largely unknown whether schiff base zinc complexes induce apoptosis in human cancer cells. Here, we synthesized a novel schiff base zinc coordination compound (SBZCC) and investigated its effects on the growth, proliferation and apoptosis of human osteosarcoma MG-63 cells. A novel SBZCC was synthesized by chemical processes and used to treat MG-63 cells. The cell viability was determined by CCK-8 assay. The cell cycle progression, mitochondrial membrane potential and apoptotic cells were analyzed by flow cytometry. The apoptosis-related proteins levels were determined by immunoblotting. Treatment of MG-63 cells with SBZCC resulted in inhibition of cell proliferation and cell cycle arrest at G1 phase. Moreover, SBZCC significantly reduced the mitochondrial membrane potential and induced apoptosis, accompanied with increased Bax/Bcl-2 and FlasL/Fas expression as well as caspase-3/8/9 cleavage. Our results demonstrated that the synthesized novel SBZCC could inhibit the proliferation and induce apoptosis of MG-63 cells via activating both the mitochondrial and cell death receptor apoptosis pathways, suggesting that SBZCC is a promising agent for the development as anticancer drugs.
Antineoplastic Agents ; chemical synthesis ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Caspase 8 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Coordination Complexes ; chemical synthesis ; pharmacology ; Fas Ligand Protein ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; pathology ; Osteoblasts ; drug effects ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Schiff Bases ; chemistry ; Signal Transduction ; Zinc ; chemistry ; bcl-2-Associated X Protein ; genetics ; metabolism ; fas Receptor ; genetics ; metabolism

OBJECTIVETo investigate the effects of trichloroethylene (TCE) toxicity on the normal human liver cells (L02 cells) and hepatocytes with CYP2E1 gene overexpression which was constructed through molecular cloning technology in our laboratory, then to explore the roles of CYP2E1 gene in TCE toxicity.

METHODSL02 cells and hepatocytes with CYP2E1 overexpression were treated with various doses of TCE (0,0.25, 0.5, 1.0, 2.0, 4.0 mmol/L) for 12h, the expression of apoptosis genes (Bcl-2, Caspase-3, Caspase-8, Caspase-9) and oncogenes (c-fos, c-myc, k-ras, p53) were determined by real-time fluorescent PCR.

RESULTSBcl-2 mRNA expression levels increased significantly in normal liver cells and CYP2E1-overexpressing cells after TCE treatment, Bcl-2 levels were 20%∼50%higher in CYP2E1-overexpressing cells than in L02 liver cells at doses of 0.25∼2.0 mmol/L TCE. Caspase-3, Caspase-8 and caspase-9 mRNA expression increased by 30%∼600% in CYP2E1-overexpressing cells at doses of 0.5∼4.0 mmol/L TCE when compared with L02 cells (P < 0.01). Additionally, c-fos, k-ras and c-myc mRNA expression levels were 25%∼120% higher in CYP2E1-overexpressing cells than in L02 cells (P < 0.01), p53 mRNA expression levels were lower 10%∼50% in CYP2E1-overexpressing cells than in L02 cells (P < 0.05 or P < 0.01).

CONCLUSIONSThere were significant differences for apoptosis gene and oncogene expression levels between normal liver cells and CYP2E1-overexpressing cells after they were treated with TCE, these findings indicated that CYP2E1 might play an important role in TCE metabolism in vivo.

Apoptosis Regulatory Proteins ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cytochrome P-450 CYP2E1 ; genetics ; Gene Expression ; Hepatocytes ; drug effects ; Humans ; Liver ; Proto-Oncogenes ; genetics ; RNA, Messenger ; Trichloroethylene ; toxicity

OBJECTIVETo explore the effect of RNA interference of human I2PP2A gene on the proliferation and apoptosis of retinoic acid-resistant human acute promyelocytic leukemia (APL) cell line NB4-R1.

METHODSDesigned and constructed a RNA interference lentiviral vector I2PP2A-shRNA which targeted against I2PP2A gene, then transfected it into NB4-R1 via polybrene mediation. The I2PP2A expression levels before and after transfection were detected by qRT-PCR and Western blot, respectively. Meanwhile, the proliferation and apoptosis rates of each group were determined by CCK-8 and flow cytometry assay. The protein expressions of caspase-8 and PARP were detected by Western blot.

RESULTSBoth qRT-PCR and Western blot data showed the I2PP2A expression level was significantly downregulated in the transfection group. The I2PP2A mRNA expression level decreased by (70.0 ± 9.6)% and (64.0 ± 6.2)% respectively, compared with blank control and negative control group, and the I2PP2A protein expression level showed a consistent trend. CCK-8 proliferation assay indicated the NB4-R1 cell proliferation rate in I2PP2A-shRNA transfection group significantly reduced compared to blank control group (P<0.05). Flow cytometry results showed that NB4-R1 apoptosis rate in I2PP2A-shRNA transfection group increased by (6.30 ± 0.67) times and (6.04 ± 0.56) times, respectively (P<0.01). After inhibition of I2PP2A, the total caspase-8 and total PARP expressions decreased by (44.0 ± 3.1)% and (57.0 ± 4.0)%, respectively; Meanwhile, the cleaved caspase-8 (p43) and cleaved PARP (p89) increased by (36.0 ± 2.5)% and (45.0 ± 4.8)%, respectively compared with blank control group (P<0.05).

CONCLUSIONI2PP2A gene silenced by RNA interference could inhibit the proliferation and promote the apoptosis of NB4-R1, which may be regulated through caspase-8-induced exogenous apoptosis pathway.

Apoptosis ; genetics ; Caspase 8 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Drug Resistance, Neoplasm ; Histone Chaperones ; genetics ; metabolism ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; RNA Interference ; Transcription Factors ; genetics ; metabolism ; Tretinoin ; pharmacology

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.
Animals ; Caspase 8 ; biosynthesis ; genetics ; Cell Line ; Cyclin D1 ; biosynthesis ; genetics ; G1 Phase ; physiology ; Histones ; genetics ; metabolism ; Ki-67 Antigen ; biosynthesis ; genetics ; Male ; Mice ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Resting Phase, Cell Cycle ; physiology ; Spermatogonia ; cytology ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; genetics

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.
Antineoplastic Agents ; chemistry ; pharmacology ; Apoptosis ; drug effects ; genetics ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Caspase 8 ; genetics ; metabolism ; Caspases ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cell Size ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cytochromes c ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Fucus ; chemistry ; G1 Phase Cell Cycle Checkpoints ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; MCF-7 Cells ; Microscopy, Fluorescence ; Molecular Structure ; Polysaccharides ; chemistry ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; bcl-2-Associated X Protein ; genetics ; metabolism