The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P<0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P<0.05,P<0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P<0.01, P<0.001). Drug administration, especially SFZY-H and YT, restored such changes(P<0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.
Female ; Animals ; Mice ; Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Choristoma ; Endometriosis/genetics* ; TOR Serine-Threonine Kinases/genetics* ; RNA, Messenger ; Signal Transduction ; Body Weight ; Mammals ; PTEN Phosphohydrolase/genetics*

OBJECTIVE: To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM. METHODS: HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected. RESULTS: CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance. CONCLUSION Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.
Humans ; Atorvastatin/pharmacology* ; PTEN Phosphohydrolase/pharmacology* ; Sincalide/metabolism* ; Drug Resistance, Neoplasm/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Leukemia, Promyelocytic, Acute/drug therapy* ; Doxorubicin/pharmacology* ; Apoptosis ; RNA, Small Interfering/pharmacology* ; Glycolysis ; Glucose/therapeutic use* ; Cell Proliferation

Objective: To explore the effect of growth arrest-specific5 (GAS5) inhibition on the proliferation, colony formation, invasion, migration andepithelial-mesenchymal transition(EMT), cancer cell stem of HCT-116 and its mechanism. Methods: The colorectal carcinoma (CRC) cell HCT116 was divided into blank control, negative control (NC), si-GAS5 and si-GAS5+ miR-21 inhibitor groups. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expressions of miR-21 and GAS5 at 48 h after transfection. The binding site of GAS5 and miR-21 was determined by luciferase reporter array. Cell proliferation ability was detected by CCK-8 assay. Cell colony ability was detected by colony formation assay. Cell invasion and migration abilities were detected by Transwell assay. Cell cycle and apoptosis were examined by flow cytometer (FCM). The protein levels of EMT associated factors including Snail, N-cadherin, vimentin, E-cadherin, stem cell related factors including CD44, SOX2, Oct2, and PTEN/Akt signal pathway associated factors were examined by western blotting. Results: The expression levels of miR-21 in blank, NC, si-GAS5 group were 1.00±0.10, 1.00±0.10, 1.80±0.20, the absorbance values were 0.51±0.02, 0.50±0.01 and 0.65±0.01, the cell clones were 90±4, 91±5, 200±8, the invaded cells were 118±3, 119±3, 150±4, the migrated cells were 110±2, 108±2, 127±2, the cell ratios in G(1) phase were (49.3±2.1)%, (50.1±2.0)% and (42.2±1.1)%, the cell ratios in S phase were (19.2±1.2)%, (20.2±1.1)% and (28.3±2.2)%, the cell apoptotic ratios were (14.4±2.2)%, (14.5±2.1)% and (7.2±1.3)%. These results indicated that inhibition of GAS5 up regulated the expression level of miR-21, promoted cell proliferation, invasion and migration, decreased G(1)-phase cells and increased S-phase cells, and suppressed cell apoptosis (P<0.05). Moreover, inhibition of GAS5 up regulated the expressions of Snail, N-cadherin, vimentin, Sox2, CD44, Oct2 and p-Akt in HCT-116 cells (P<0.05), while down regulated the expressions of E-cadherin and PTEN (P<0.05). Inhibition of miR-21 reversed the impact of GAS5 knockdown on PTEN/Akt signaling pathway (P<0.05). Conclusion: GAS5 can act as a competing endogenous RNA for miR-21, and down regulation of GAS5 can promote the development of CRC by activating the miR-21/PTEN/Akt signaling pathway and promoting the acquisition of EMT and tumor cell stemness.
Humans ; Cadherins/metabolism* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Colorectal Neoplasms/pathology* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; PTEN Phosphohydrolase/metabolism* ; Vimentin/metabolism*

Choriocarcinoma/pathology* ; Female ; Gene Deletion ; Humans ; PTEN Phosphohydrolase/genetics* ; Pregnancy ; Sequence Deletion ; Stomach Neoplasms/surgery*

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.
Adenoviridae/metabolism* ; Animals ; Carrier Proteins ; Cell Proliferation ; Cortactin ; Filamins/genetics* ; Hepatic Stellate Cells/metabolism* ; PTEN Phosphohydrolase/metabolism* ; RNA, Small Interfering/genetics* ; Rats ; Vinculin/genetics*

The purpose of our study is to investigate the prognostic value of phosphatase and tensin homolog on chromosome 10 (PTEN) expression in patients with de novo metastatic castration naïve prostate cancer (mCNPC). A total of 205 patients with mCNPC at Fudan University Shanghai Cancer Center (Shanghai, China) were retrospectively examined. Immunohistochemical staining of PTEN was performed on prostate biopsy samples of these patients. Associations among clinicopathological features, patient survival and PTEN protein expression were analyzed. PTEN loss occurred in 58 of 205 (28.3%) patients. Loss of PTEN was significantly correlated with high metastatic volume (P = 0.017). No association between PTEN expression and Gleason score was observed. Patients with PTEN loss had significantly shorter progression-free survival (PFS, P < 0.001) and overall survival (OS, P < 0.001) compared with patients with intact PTEN expression. Multivariate analysis showed that elevated alkaline phosphatase, high metastatic volume and PTEN loss were independent poor prognostic factors for PFS. The Eastern Cooperative Oncology Group performance status (ECOG PS)#8805; 2 and PTEN loss were independent poor prognostic factors for OS. The adjusted hazard ratio of PTEN loss for PFS and OS was 1.67 (95% confidence interval [CI]: 1.14-2.43, P = 0.008) and 1.95 (95% CI: 1.23-3.10, P = 0.005), respectively. PTEN loss was also significantly associated with shorter PFS (P = 0.025) and OS (P < 0.001) in patients with low-volume metastatic disease. Our data showed that PTEN loss is an independent predictor for shorter PFS and OS in patients with de novo mCNPC.
China/epidemiology* ; Humans ; Male ; PTEN Phosphohydrolase/genetics* ; Prognosis ; Prostatic Neoplasms ; Retrospective Studies

This study explored the effects of resveratrol(Res) on the expression of phosphatase and tensin homolog deleted on chromosome ten(PTEN) and the fibrosis of rat renal tubular epithelial cells in a high-glucose environment and the possible mechanism underlying the fibrosis reduction. After the pretreatment of rat renal tubular epithelial cells(NRK-52 E) cultured in a high-glucose condition with Res or PTEN inhibitor SF1670, they were divided into several groups, i.e., normal glucose(NG), normal glucose + SF1670(NS), high glucose(HG), high glucose + SF1670(HS), high glucose + Res at different concentrations(5, 10, 25 μmol·L~(-1)). The expression and distribution of E-cadherin and α-SMA in renal tubular epithelial cells were observed by immunofluorescence cytochemistry. The protein expression levels of PTEN, E-cadherin, α-SMA, p-Akt~((Thr308)) and collagen Ⅳ were determined by Western blot. Real-time PCR was employed to detect the expression of PTEN mRNA. Compared with the NG group, the HG group witnessed the reduced expression of PTEN mRNA, PTEN protein and E-cadherin protein, but saw the increased expression of α-SMA, p-Akt~((Thr308)) and collagen Ⅳ proteins. Besides, with the increase in Res concentration, the expression levels of PTEN mRNA, PTEN protein and E-cadherin protein gradually increased, while those of α-SMA, collagen Ⅳ, p-Akt~((Thr308)) proteins gradually decreased in the Res groups, showing a dose-effect dependence, compared with the HG group. No distinct difference was found between the NS group and the NG group. The expression level of E-cadherin was even lower and those of α-SMA, p-Akt~((Thr308)), and collagen Ⅳ were higher in the HS group than in the HG group, with no marked difference shown in the two groups in terms of PTEN mRNA and protein. Although the PTEN inhibitor did not affect PTEN, the expression changes of the other proteins were opposite to the results after Res treatment and the fibrosis was aggravated, which suggested that SF1670 promoted the fibrosis by inhibiting PTEN, activating Akt and increasing the synthesis of collagen Ⅳ and other extracellular matrix. The results show that Res can antagonize the high glucose-mediated fibrosis of renal tubular epithelial cells. This may be achieved via the up-regulation of PTEN and the inhibition of PI3 K/Akt signaling pathway.
Animals ; Epithelial Cells ; Fibrosis ; Glucose ; PTEN Phosphohydrolase/genetics* ; Rats ; Resveratrol/pharmacology*

Objective: To investigate if microRNA (miR) -23a knockdown could attenuate angiotensin Ⅱ(AngⅡ) induced cardiac hypertrophy by activating phosphatase and tensin homolog deleted on chromosome ten(PTEN) and AMP-activated protein kinase(AMPK) pathway. Methods: Rat H9c2 cells were cultured in DMEM high glucose medium and put in 5% CO(2) incubator at 37 ℃(normal group). After 48 hours of culture, H9c2 cells were stimulated with 10 nmol/L AngⅡ to establish cell hypertrophy model (AngⅡgroup). The H9c2 cells were inoculated in a 6-well cell culture plate and cultured in an incubator at 37 ℃. When the confluence degree of cell growth was about 70%, the cells were transfected with different reagents, and 24 hours after transfection, 10 nmol/L AngⅡ was used to interfere with the cells. The H9c2 cells were divided into different groups according to the reagents, namely AngⅡ+anti-miR group(transfected with miR-23a inhibitor), Ang Ⅱ+NC group(transfected with miR-23a inhibitor negative control), Ang Ⅱ+anti-miR+si-PTEN group(cotransfected with miR-23a inhibitor and PTEN small interference RNA(siRNA)), and AngⅡ+anti-miR+si-NC group(cotransfected with miR-23a inhibitor and PTEN siRNA negative control). The surface area of single cell was measured by Image J software.The mRNA expression levels of α-actin 1 (ACTA1) and β-myosin heavy chain (β-MHC) and miR-23a were detected by quantitative real-time PCR(qRT-PCR). The expression levels of PTEN and AMPK signal pathway related proteins were detected by Western blot. In order to verify whether miR-23a targets PTEN gene, double luciferase reporter gene experiment was performed. The luciferase reporter gene vector recombinant plasmids of wild type pGL-WT-PTEN and mutant pGL-MUT-PTEN were constructed and prepared after normal sequencing. H9c2 cells was inoculated into 24-well cell culture plate and cultured overnight in 37 ℃ incubator. The cells were co-transfected with miR-23a mimic or miR-23a mimic negative control and wild type or mutant reporter gene recombinant plasmid. Forty-eight hours after transfection, firefly luciferase activity and sea kidney luciferase activity were measured, and the ratio of them was recorded as relative luciferase activity. Results: Compared with the normal group, the cell surface area, the mRNA expression levels of ACTA1, β-MHC and miR-23a were significantly higher, while the protein expression levels of PTEN and p-AMPK were significantly lower in the Ang Ⅱ group(all P<0.05). The results of double luciferase reporter gene assay showed that the relative luciferase activity of cells co-transfected with miR-23a mimic and wild-type reporter gene recombinant plasmid was lower than that of miR-23a mimic negative control (P<0.05), and PTEN served as the target gene of miR-23a. In AngⅡ+anti-miR group the mRNA expression levels of miR-23a, ACTA1 and β-MHC were lower, and the cell surface area was smaller, while the protein expression levels of PTEN and p-AMPK were higher than that in AngⅡ group and AngⅡ+NC group(all P<0.05). Compared with AngⅡ+anti-miR group, the cell surface area was bigger, the expression of ACTA1 and β-MHC mRNA was up-regulated, and the protein expression levels of PTEN and p-AMPK were down-regulated in Ang Ⅱ+anti-miR+si-PTEN group(all P<0.05). Conclusion: Inhibition of miR-23a can attenuate Ang Ⅱ-induced hypertrophy in H9c2 cells through targeting PTEN and activating AMPK signaling pathway.
AMP-Activated Protein Kinases ; Angiotensin II ; Animals ; Cardiomegaly ; Cell Line ; Cell Proliferation ; MicroRNAs/genetics* ; PTEN Phosphohydrolase ; Rats ; Signal Transduction

To investigate the effect of Parkinson's disease related protein DJ-1 on the cell proliferation, apoptosis, invasion and migration in human osteosarcoma cells and the underlying molecular mechanisms. 
 Methods: The protein expression levels of DJ-1 were detected in human osteosarcoma cell lines (MG-63, Saos-2, and U2OS) and human osteoblast cell line hFOB1.19 with or without deficiency in phosphatase and tensin homolog deleted from chromosome 10 (PTEN) were detected by Western blot. Osteosarcoma cells were treated with DJ-1 siRNA, and then the protein expression levels of DJ-1 were detected by Western blot. Cell survival rate of osteosarcoma cells was detected by cell counting kit-8 (CCK-8) assay. Cell apoptosis of osteosarcoma cells was measured by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method. Cell invasive and migration ability of osteosarcoma cells were examined by transwell invasion and migration assay. 
 Results: Compared with that of human osteoblast cell line (hFOB1.19), the protein expression level of DJ-1 was significantly upregulated in human osteosarcoma cell lines (MG-63, Saos-2, and U2OS) (all P<0.05), and U2OS had the highest level of DJ-1 when compared with the other three cell lines (P<0.01). DJ-1 siRNA could significantly down-regulate the DJ-1 protein expression in U2OS cells, and also diminish the cell survival rate. Moreover, DJ-1 down-regulation of DJ-1 could promote cell apoptosis, suppress the ability of cell invasion and migration, and increase the PTEN protein expression level (all P<0.05). In addition, the protein expression level of PTEN was markedly up-regulated in human osteosarcoma cell lines when compared with that in the hFOB1.19 cells (P<0.05). 
 Conclusion: DJ-1 can promote the cell proliferation, inhibit cell apoptosis, and decrease the ability of cell invasion and migration, and the potential underlying mechanisms may be associated with the up-regulation of PTEN protein expression.
Apoptosis ; genetics ; Cell Line, Tumor ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasm Invasiveness ; genetics ; PTEN Phosphohydrolase ; genetics ; Parkinson Disease ; physiopathology ; Protein Deglycase DJ-1 ; genetics ; metabolism ; RNA, Small Interfering ; genetics

The aim of this paper was to observe the effect of gambogenic acid on angiogenesis of lung cancer and its preliminary mechanism. After culturing lung adenocarcinoma A549 cells, the conditioned medium was treated with gambogenic acid and then used to culture human umbilical vein endothelial cells (HUVECs) to establish the indirect contact cell co-culture system. A two-dimensional culture model of HUVEC was established with matrigel to observe the effect of gambogenic acid on angiogenesis. DAPI staining was used to observe the morphological changes in HUVEC cells after treatment with gambogenic acid under the fluorescence microscope. Annexin V-FITC/PI staining and flow cytometry analysis were used to determine gambogenic acid's effect on HUVEC cell apoptosis rate. The protein expressions of PI3K, p-PI3K, Akt, p-Akt were measured by Western blot. PTEN-siRNA was transfected into cells, and RT-PCR was used to detect the expression levels of PI3K and Akt genes. Gambogenic acid can significantly inhibit angiogenesis, and its inhibitory effect was dose-dependent. DAPI staining showed apoptotic morphological features of HUVEC cells under fluorescence microscope. Annexin V-FITC/PI staining showed that gambogenic acid induced apoptosis in HUVECs. The results of Western blot showed that the expressions of p-PI3K and p-Akt protein were down-regulated with gambogenic acid, while the expressions of PI3K and Akt protein was insignificant. The results of RT-PCR indicated that the expressions of PI3K and Akt protein were up-regulated by PTEN siRNA. Gambogenic acid can inhibit angiogenesis in lung cancer in vitro, and the mechanism of inhibiting angiogenesis may be related to the PI3K/Akt signaling pathway.
A549 Cells ; Apoptosis ; Coculture Techniques ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Neovascularization, Pathologic ; pathology ; PTEN Phosphohydrolase ; genetics ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Transfection ; Xanthenes ; pharmacology