OBJECTIVE: To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. METHODS: We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). RESULTS: In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis ( < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 ( < 0.01) and Bax ( < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 ( < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. CONCLUSIONS ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.
Acetates ; pharmacology ; Apoptosis ; drug effects ; genetics ; Autophagy ; drug effects ; Autophagy-Related Proteins ; metabolism ; Benzopyrans ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation ; Humans ; MCF-7 Cells ; Morpholines ; pharmacology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; metabolism ; Pyrans ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Triazines ; pharmacology ; Ubiquitin-Conjugating Enzymes ; metabolism

The aim of this paper was to explore the mechanism of Shenxiong Glucose Injection antagonizing apoptosis of H9 c2 cells induced by H_2O_2. H9 c2 cells were pretreated with 1. 7%,3. 4% and 6. 8% Shenxiong Glucose Injection,and then H_2O_2 was introduced to induce apoptosis in vitro. Cell viability was detected by MTS assay,morphological changes of apoptosis were observed by AO/EB fluorescence staining,apoptosis rate was detected by Annexin/PI method,cell expression profile was detected by gene chip technology,the mRNA of PIK3 CA,Bcl-2,Bax,caspase-3 and GAPDH were detected by qRT-PCR,the protein expression levels of PIK3 CA,AKT,P-AKT,Bcl-2,Bax and caspase-3 were detected by Western blot,and the contents of LDH and MDA were detected by kit. The results showed that Shenxiong Glucose Injection of different concentrations significantly increased the viability of H9 c2 cells treated with H_2O_2( P<0. 01),and reversed H_2O_2-induced apoptosis( P< 0. 01). The microarray experiments showed that 138 genes were altered in H9 c2 cells after treatment with Shenxiong Glucose Injection. The differential expression fold of PIK3 CA associated with PI3 K/AKT pathway was 3. 59. The results of qRT-PCR and Western blot showed that Shenxiong Glucose Injection could down-regulate the mRNA and protein expression levels of caspase-3( P<0. 01),up-regulate the mRNA and protein expression level of PIK3 CA and Bcl-2( P<0. 01),and up-regulate the phosphorylation levels of AKT( P<0. 01) in H_2O_2-treated H9 c2 cells. The protective effect of Shenxiong Glucose Injection on H_2O_2 cells injury was significantly inhibited by LY294002,a PI3 K/AKT pathway inhibitor. The results suggested that Shenxiong Glucose Injection may inhibit H_2O_2-induced H9 c2 cells apoptosis by regulating PI3 K/AKT signaling pathway.
Animals ; Apoptosis ; Cell Line ; Chromones ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; Morpholines ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Signal Transduction

Prostaglandin (PG) E plays critical roles during pregnancy and parturition. Emerging evidence indicates that human labour is an inflammatory event. We sought to investigate the effect of PGE on the output of proinflammatory cytokines in cultured human uterine smooth muscle cells (HUSMCs) from term pregnant women and elucidate the role of subtypes of PGE receptors (EP, EP, EP and EP). After drug treatment and/or transfection of each receptor siRNA, the concentrations of inflammatory secreting factors in HUSMCs culture medium were detected by the corresponding ELISA kits. The results showed that, PGE increased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) output, decreased chemokine (c-x-c motif) ligand 8 (CXCL8) output in a dose-dependent manner, but had no effect on IL-1β and chemokine (c-c motif) ligand 2 (CCL-2) secretion of HUSMCs. EP/EP agonist 17-phenyl-trinor-PGE stimulated IL-6 and TNFα whilst suppressing IL-1β and CXCL8 output. The effects of 17-phenyl-trinor-PGE on IL-1β and CXCL8 secretion were remained whereas its effect on IL-6 and TNFα output did not occur in the cells with EP knockdown. The stimulatory effects of 17-phenyl-trinor-PGE on IL-6 and TNFα were remained whereas the inhibitory effects of 17-phenyl-trinor-PGE on IL-1β secretion was blocked in the cells with EP knockdown. Either of EP and EP agonists stimulated IL-1β and TNFα output, which was reversed by EP and EP siRNA, respectively. The inhibitors of phospholipase C (PLC) and protein kinase C (PKC) blocked EP/EP modulation of TNFα and CXCL8 output. PI3K inhibitor LY294002 and P38 inhibitor SB202190 blocked 17-phenyl-trinor-PGE-induced IL-1β and IL-6 output, respectively. The inhibitors of adenylyl cyclase and PKA prevented EP and EP stimulation of IL-1β and TNFα output, whereas PLC and PKC inhibitors blocked EP- and EP-induced TNFα output but not IL-1β output. Our data suggest that PGE receptors exhibit different effects on the output of various cytokines in myometrium, which can subtly modulate the inflammatory microenvironment in myometrium during pregnancy.
Cells, Cultured ; Chromones ; pharmacology ; Cytokines ; metabolism ; Female ; Humans ; Imidazoles ; pharmacology ; Inflammation ; Morpholines ; pharmacology ; Myocytes, Smooth Muscle ; cytology ; Myometrium ; cytology ; Phosphatidylinositol 3-Kinases ; Pregnancy ; Pyridines ; pharmacology ; Receptors, Prostaglandin E ; physiology

BACKGROUND: Chemotherapy is the most important method for cancer treatment. However, chemotherapy induced nausea and vomiting (CINV) has a profound effect on patients. In recent years, there have been new antiemetic drugs, such as aprepitant. We review the curative effect of aprepitant with tropisetron and dexamethasone for prevention of nausea and vomiting in patients receiving Cisplatin chemotherapy. METHODS: Observation is divided into three stages. Whole study phase (0-120 h after chemotherapy administration), acute phases (0-24 h), and delayed phase (24 h-120 h). The primary endpoints were complete response (CR) and complete prevention (CP) during the three different study phase. RESULTS: In the whole study phase, 86.02% of patients achieved CR; in acute phases and delayed phases were 89.25%, 87.1%, respectively. CP were 46.22%, 83.87%, 45.16%, respectively. Anti-CINV effect was significantly associated with age distribution (P=0.008). CONCLUSIONS Aprepitant with tropisetron and dexamethasone prevented effectively CNIV for patients receiving Cisplatin chemotherapy. This combination could improve the quality of life and the compliance of patient with chemotherapy.
Adult ; Aged ; Aged, 80 and over ; Antineoplastic Agents ; adverse effects ; Aprepitant ; Cisplatin ; adverse effects ; Female ; Humans ; Male ; Middle Aged ; Morpholines ; pharmacology ; Nausea ; chemically induced ; prevention & control ; Quality of Life ; Vomiting ; chemically induced ; prevention & control

OBJECTIVE: : To investigate the effect of nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 on the migration of human non-small cell cancer A549 cells and related mechanism. METHODS: : The inhibition effect of FK866 on A549 cells was tested by MTT assay. A549 cells were treated with 1.0 and 10.0 nmol/L FK866, and the cell migration was evaluated by modified wound scratch assay. The mRNA expression of E-cadherin and vimentin was detected by real-time RT-PCR, and the expression of ERK1/2 and pERK1/2 was determined by Western blotting. RESULTS: : FK866 inhibited the proliferation of A549 cells in a time-and concentration-dependent manner; after treatment for 72 h, the IC of FK866 was 9.55 nmol/L. When 1.0 nmol/L or 10.0 nmol/L FK866 was continuously applied 48 h before and 48 h after a scratch was made in wound scratch assay, the migration of A549 cells was significantly inhibited. However, when the FK866 was applied only 48 h after the scratch, the migration of A549 cells was inhibited by 10.0 nmol/L but not by 1.0 nmol/L FK866. The mRNA expression of E-cadherin and vimentin, and the activated ERK1/2 were significantly increased after 1.0 nmol/L FK866 treatment for 72 h. The pretreatment with nicotinamide adenine dinucleotide (NAD) precursor nicotinamide mononucleotide(1.0 mmol/L) or ERK1/2 inhibitor U0126 (10.0 μmol/L) reversed the up-regulation of E-cadherin and vimentin expression induced by FK866. CONCLUSIONS s: Low concentration of FK866 decreases the migration of A549 cells through the inhibition of NAD level, activation of ERK1/2 and up-regulation of E-cadherin expression. However, it also up-regulates the expression of vimentin, indicating that it may have dual effects on the migration of tumor cells.
A549 Cells ; Cadherins ; genetics ; Cell Movement ; drug effects ; Gene Expression Regulation ; drug effects ; Humans ; Morpholines ; pharmacology ; Neurokinin-1 Receptor Antagonists ; pharmacology ; Nicotinamide Phosphoribosyltransferase ; antagonists & inhibitors ; Piperazines ; pharmacology ; Vimentin ; genetics

OBJECTIVETo observe the protective effects of Tongxinluo (TXL) on apoptosis of rat cardiac microvascular endothelial cells (RCMECs) resulting from homocysteine (Hcy) induced endoplasmic reticulum stress (ERS), and to determine the signaling pathway behind its protection.

METHODSPrimary cultured RCMECs were isolated from neonatal rats using tissue explant method. The morphology of RCMECs was observed using inverted microscope, identified and differentiated by CD31 immunofluorescence method. Selected were well growing 2nd-4th generations of RCMECs. The optimal action time was determined by detecting the expression of glucose regulated protein 78 (GRP78) using immunofluorescence method. In the next experiment RCMECs were divided into 5 groups, i.e., the blank control group, the Hcy induced group (Hcy 10 mmol/L, 10 h), the Hcy + TXL group (Hcy 10 mmol/L + TXL 400 µg/mL), the Hcy +LY294002 group (Hcy 10 mmol/L + LY294002 5 µmol/L, LY294002 as the inhibitor of PI3K), the Hcy + LY294002 + TXL group (Hcy 10 mmol/L + LY294002 5 µmol/L + TXL 400 µg/mL). The apoptosis rate of RCMECs was detected by flow cytometry. mRNA and protein expressions of GRP78, C/ EBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase12) were detected by real-time reverse transcription PCR (RT-PCR) and Western blot respectively. Expression levels of phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T- P13K) , phosphorylation of kinase B (P-Akt) , and total kinase B (T-Akt) were detected by Western blot.

RESULTSTen hours Hcy action time was determined. Compared with the blank control group, the apoptosis rate was increased (22.77%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, protein expressions of P-PI3K and P-Akt,ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy induced group (P < 0.05, P < 0.01). Compared with the Hcy induced group, the apoptosis rate was decreased (10.17%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were decreased, and expression levels of P-PI3K, P-Akt, P-PI3K/T-PI3K, and P-Akt/T-Akt were increased in the Hcy + TXL group (P < 0.05, P < 0.01). Compared with the Hcy + TXL group, the apoptosis rate was increased (17.9%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, expression levels of P-PI3K and P-Akt, ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy + TXL + LY294002 group (P < 0.05, P < 0.01).

CONCLUSIONTXL could inhibit the apoptosis of RCMECs resulting from Hcy-induced ERS and its mechanism might be associated with activating PI3K/Akt signaling pathway.

Animals ; Apoptosis ; drug effects ; Caspase 12 ; metabolism ; Cells, Cultured ; Chromones ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum Stress ; Endothelial Cells ; drug effects ; Morpholines ; pharmacology ; Myocardium ; cytology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Signal Transduction ; Transcription Factor CHOP ; metabolism

OBJECTIVETo observe morphological changes of enteric nervous system (ENS)-interstitial cells of Cajal (ICC)-smooth muscle cell (SMC) structure injury in deep muscle nerve plexus offunctional dyspepsia (FD) rats, and the repair of Shuwei Decoction (SD) on it, and to explore its effecton FD.

METHODSTotally 72 rats were randomly divided into the control group, the model group, the lowdose SD group, the medium dose SD group, and the high dose SD group, the Mosapride group, 12 ineach group. Rats in the low dose SD group, the medium dose SD group, and the high dose SD group were intragastrically fed with SD at 0.767, 1.534, 3.068 g/mL, respectively. Rats in the Mosapride group were intragastrically fed with Mosapride (1.37 mg/kg). FD rat model with Gan depression Pi deficiency syndrome (GDPDS) was established using complex pathogenic factors. Corresponding liquors were respectively administered to rats in corresponding groups from the 3rd day after modeling. Distilled water(10 mL/kg) was administered to rats in the control group and the model group, once per day for 14 successive days. Rats were sacrificed and small intestine tissues collected for observing ENS-ICC-SMC structure injury using immunofluorescence double labeling, laser scanning confocal microscope, and transmission electron microscope at day 15. Repair of SD on it was also observed.

RESULTSENS-ICC SMC structure was incomplete, with obvious injury in mutual link of ICC, ICC, SMC, and connecting structure. ENS-ICC-SMC structure was more complete in high, medium, and low dose SD groups, with close link of ICC and SMO. Their connecting structures were in good conditions.

CONCLUSIONSD could keep the integrity of ENS-ICC-SMC structure by promoting regeneration and morphology of ICC, thereby, improving gastrointestinal movement disorder and showing therapeutic effect on FD.

Animals ; Benzamides ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Dyspepsia ; drug therapy ; Enteric Nervous System ; drug effects ; Interstitial Cells of Cajal ; drug effects ; Morpholines ; pharmacology ; Muscle, Smooth ; drug effects ; Random Allocation ; Rats

Cancer stem cells (CSCs) have tumor initiation, self-renewal, metastasis and chemo-resistance properties in various tumors including colorectal cancer. Targeting of CSCs may be essential to prevent relapse of tumors after chemotherapy. Phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signals are central regulators of cell growth, proliferation, differentiation, and apoptosis. These pathways are related to colorectal tumorigenesis. This study focused on PI3K and mTOR pathways by inhibition which initiate differentiation of SW620 derived CSCs and investigated its effect on tumor progression. By using rapamycin, LY294002, and NVP-BEZ235, respectively, PI3K and mTOR signals were blocked independently or dually in colorectal CSCs. Colorectal CSCs gained their differentiation property and lost their stemness properties most significantly in dual-blocked CSCs. After treated with anti-cancer drug (paclitaxel) on the differentiated CSCs cell viability, self-renewal ability and differentiation status were analyzed. As a result dual-blocking group has most enhanced sensitivity for anti-cancer drug. Xenograft tumorigenesis assay by using immunodeficiency mice also shows that dual-inhibited group more effectively increased drug sensitivity and suppressed tumor growth compared to single-inhibited groups. Therefore it could have potent anti-cancer effects that dual-blocking of PI3K and mTOR induces differentiation and improves chemotherapeutic effects on SW620 human colorectal CSCs.
AC133 Antigen/genetics/metabolism ; Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Cell Differentiation/*drug effects ; Cell Line, Tumor ; Cell Survival/drug effects ; Chromones/pharmacology/therapeutic use ; Colorectal Neoplasms/drug therapy/metabolism/pathology ; Humans ; Imidazoles/pharmacology/therapeutic use ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Morpholines/pharmacology/therapeutic use ; Neoplastic Stem Cells/cytology/drug effects/metabolism ; Paclitaxel/pharmacology/therapeutic use ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors/metabolism ; Quinolines/pharmacology/therapeutic use ; SOXB1 Transcription Factors/genetics/metabolism ; Signal Transduction/*drug effects ; Sirolimus/pharmacology/therapeutic use ; TOR Serine-Threonine Kinases/*antagonists & inhibitors/metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the impact of NU7026 and Wortmannin, inhibitors of DNA-dependent protein kinase (DNA-PK), in HL60 cells apoptosis induced by 1, 4-benzoquinone (1, 4-BQ).

METHODSHL60 cells were divided into three groups according to the exposures: the poisoned groups which were treated with 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ for 24 h, respectively, the NU7026 groups which were preincubated with 10 µmol/L NU7026 for 1h prior to the 24h treatment of 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ and the Wortmannin groups which were preincubated with 25 µmol/L Wortmannin for 1h prior to the 24 h treatment of 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ. Then we detected the apoptosis via flowcytometry Annexin V-FITC/PI and the DNA Ladder, respectively. We also tested the expressions of Bax mRNA with Real-Time PCR in HL60 cells which were exposed to 10 µmol/L NU7026 for 24 h, 25 µmol/L Wortmannin 24 h, 10 µmol/L 1, 4-BQ 24 h, 10 µmol/L NU7026 1h+10 µmol/L 1, 4-BQ 24 h and 25 µmol/L Wortmannin 1 h+10 µmol/L 1, 4-BQ 24 h, as well as null (control). We also examed the expressions of p53 in HL60 cells with Western blot.

RESULTSAnnexin V-FITC/PI apoptosis tests suggested that apoptosis rates of NU7026+10 µmol/L 1, 4-BQ group and Wortmannin +10 µmol/L 1, 4-BQ were 17.6±1.19% and 15.2±1.22%, respectively. Both of results were higher than that of 10 µmol/L 1, 4-BQ group (6.3±1.04%); Apoptosis of NU7026+25 µmol/L 1, 4-BQ group was 46.2±3.55%, and Wortmannin +25 µmol/L 1, 4-BQ group 26.9±2.62%. Both of results were also higher than that of 25 µmol/L 1, 4-BQ group (14.1±1.54%); Apoptosis of NU7026+50 µmol/L 1, 4-BQ group (61.8±1.78%) was higher than that of 50 µmol/L 1, 4-BQ group (35.9±4.51%). The above results were all statistically significant (P < 0.05).

RESULTSof DNA-Ladder were basically consistent with those of Annexin V-FITC/PI apoptosis tests. In addition, both NU7026 and Wortmannin pretreatment elicited the higher expression of Bax mRNA in HL60 treated by 1, 4-benzoquinone with statistically significance (P < 0.05). However, p53 protein was not detected in HL60 cells as the western blot indicated.

CONCLUSIONInhibitors of DNA-PK, NU7026 and Wortmannin, promote p53-independent apoptosis induced by 1, 4-benzoquinone in HL60 cells.

Androstadienes ; pharmacology ; Apoptosis ; drug effects ; Benzoquinones ; toxicity ; Chromones ; pharmacology ; DNA-Activated Protein Kinase ; antagonists & inhibitors ; Flow Cytometry ; HL-60 Cells ; Humans ; Morpholines ; pharmacology ; RNA, Messenger ; Tumor Suppressor Protein p53

To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether the extracellular signal PI3K/AKT pathway was involved in the Pue-induced PASMC apoptosis. With the serum starvation group (SD group) as the control group, the MTT colorimetry method, Annexin V-FITC apoptosis detection kit and Western blot were used to detect Pue's effect on apoptosis of rat PASMCs. The protein immunoblot assay was used to detect whether PI3K/AKT pathway was involved in the inhibition of hypoxia-induced PASMC apoptosis process. The results show that under normoxic conditions, Pue had no effect on PASMC apoptosis; Under hypoxia conditions, Pue can inhibit PASMC apoptosis; Under normoxic and hypoxic conditions, Pue had no effect on TNF-α expression. Pue can reverse hypoxia-induced Bcl-2 (P <0.01), up-regulate it and down-regulated Bax (P <0.01). Under normoxic conditions, Pue had no effect on P-AKT expression. Both LY294002 and Pue can inhibit hypoxia-induced Bcl-2, up-regulation of P-AKT expression and down-regulation of Bax expression. Compared with the hypoxia + Pue group or the hypoxia + LY294002 group, the hypoxia + Pue + LY294002 group showed more significantly changes in Bcl-2, Bax, P-AKT expressions. The results show that, Pue can inhibit the hypoxic-induced PASMC apoptosis, which may be regulated through PI3K/AKT pathway.
Animals ; Apoptosis ; drug effects ; Cells, Cultured ; Chromones ; pharmacology ; Isoflavones ; pharmacology ; Morpholines ; pharmacology ; Myocytes, Smooth Muscle ; drug effects ; Phosphatidylinositol 3-Kinases ; physiology ; Proto-Oncogene Proteins c-akt ; physiology ; Pulmonary Artery ; cytology ; drug effects ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects