Delicaflavone inhibits the invasion and migration of gefitinib-resistant lung cancer PC-9/GR cells by regulating epithelial-mesenchymal transition via PI3K/Akt/mTOR pathway / 中国临床药理学与治疗学

AIM: To study the effect and mechanism of Delicaflavone on migration and invasion of gefitinib-resistant lung cancer cell line PC-9/GR. METHODS: MTT assay was used to detect cell viability. Transwell and scratch assays were used to detect cell invasion and migration abilities. Western blotting was used to detect the expressions of MMP-9, MMP-2, E-cadherin, N-cadherin, Vimentin and PI3K/Akt/mTOR pathway-related proteins in PC-9/GR cells. RESULTS: Compared with control group, 20 mg/L Delicaflavone could significantly inhibit the viability of PC-9/GR cells for 24 h (P<0.05), while Delicaflavone below 10 mg/L had no significant effect on cell proliferation. The number of invasive cells and migrated cells were decreased significantly by Delicaflavone in a concentration-dependent way (P<0.05 and P<0.01). Delicaflavone could concentration-dependently reduce the expression of MMP-9, MMP-2, N-cadherin, vimentin (P<0.01), meanwhile up-regulate the expression of E-cadherin (P<0.01). In addition, Delicaflavone also decreased the expression of p-PI3K, p-Akt and p-mTOR in a concentration-dependent manner (P<0.01). CONCLUSION: Delicaflavone can inhibit the migration and invasion of PC-9/GR cells by regulating epithelial-mesenchymal transition via PI3K/Akt/mTOR pathway.

Enhancive effect of FGL1 gene silencing on sensitivity of human lung adenocarcinoma PC-9 cells to docetaxel / 中国药理学通报

Aim To investigate the effect of fibrinogenlike protein 1 (FGL1)) silencing on docetaxel sensitivity in human lung adenocarcinoma PC-9 cells. Methods Western blot was used to detect the expression of FGL1 protein in human normal bronchial epithelial cell line BEAS-2B and human lung adenocarcinoma cell line PC-9. The FGL1 gene in PC-9 cell line was silenced by siRNA. CCK-8 assay was used to detect the inhibitory effect of silencing FGL1 on PC-9 cell proliferation and its effect on docetaxel sensitivity. Results Compared with BEAS-2B cell line, FGL1 was highly expressed in PC-9 cell line, and the relative expression of FGL1 protein was 6. 5 times that of BEAS-2B cell line with statistically significant difference (P <0. 01). Silencing FGL1 by transfection with FGLlsiRNA could enhance the inhibitory effect of docetaxel on PC-9 cells. Compared with FGLlsiNC group, the IC50value of PC-9 cells in FGL1siRNA group was significantly reduced with statistically significant difference (P < 0. 01). Conclusions Specific silencing of FGL1 gene could inhibit the expression of FGL1 in human lung adenocarcinoma cell PC-9, inhibit the proliferation of PC-9 cells and increase the sensitivity to docetaxel.

CXCR4/SDF-1 axis regulates the effect of human lung adenocarcinoma PC-9 cells on function of in vitro blood-brain barrier model formed by Bends cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the influences of human lung adenocarcinoma PC-9 cells on tight junction proteins of blood brain barrier (BBB) under CXCR4/SDF-1 axis by establishing a model of BBB in vitro. Methods: The immortalized mouse brain microvascular endothelial Bends cells were used to establish a model of BBB in vitro by monolayer culture; Subsequently, transendothelial electric resistance (TEER) and fluorescein sodium permeability experiment were used to detect the function of in vitro BBB model and observe the effect of PC-9 cells on the function of BBB model, respectively. Western blotting was used to detect the effect of PC-9 cells on function of BBB model and expressions of endothelial tight junction proteins under the treatment of single or combined AMD3100 and SDF-1 (1 μg/ml AMD3100,100 ng/ml SDF-1, AMD3100+SDF-1). Transwell assay was used to detect the influence of CXCR4/SDF-1 axis on the ability of PC-9 cells transmigrating the cell layer of BBB model. Results: Monolayer culture of Bends cells can form tightly connected BBB withhighTEER,which reached (182.13±5.19) Ω.cm2 at the 96 h; in the meanwhile, fluorescein sodium permeability experiment showed that BBB had significantly lower permeability than that of control group ([40.31±2.43]% vs [150.10±3.17]%, P<0.05). The TEER of BBB decreased to (46.7±4.35) Ω·cm2 after coculture with PC-9 cells for 24 h, and at the same time the sodium fluorescein permeability of BBB significantly increased than that of pre-treatment ([136.32±4.93]% vs [50.24±6.21]%, P<0.05). PC-9 cells up-regulated the expressions of tight junction proteins of Bends cells under the treatment of AMD3100 (P<0.05). The number of PC-9 cells transmigrating the BBB inAMD3100 treatment group was significantly lower than that of CON group (43±2 vs 81±2, P<0.05). Conclusion: AMD3100 can reduce the ability of PC-9 cells destroying the tight junction of the BBB model established in vitro by Bends cells.

Effects of Bezafibrate and Fenofibrate on the Proliferation of Lung Adenocarcinoma PC- 9 Cells and the Expression of c-myc / 中国药房

OBJECTIVE： To study the effects of bezafibrate （BEZ） and fenofibrate （FEN） on the proliferation of lung adenocarcinoma PC-9 cells and the expression of c-myc. METHODS： The effects of BEZ and FEN （12.5， 25， 50， 100， 200       μmol/L） on the survival rate of PC-9 cells were detected by CCK8 method. PC-9 cells were divided into administration group and control group. Administration group was given low， medium and high concentration （25， 50， 100 μmol/L） of BEZ and FEN； control group was treated with dimethyl sulfoxide for 48 h. Cell cycle distribution and apoptosis were detected by flow cytometry. qRT-PCR was used to detect mRNA relative expression of c-myc in cells. The protein relative expression of c-myc in cells were detected by Western blot assay. RESULTS： The survival rates of PC-9 cells were （80.76±3.2）％， （74.35±5.06）％， （62.8±1.23）％， （59.03±1.55）％， （39.8±1.01）％ under the action of above concentration of BEZ； and the survival rates of PC-9 cells were （74.46±1.30）％， （61.91±4.77）％， （48.95±2.8）％， （37.05±1.55）％， （32.49±1.36）％ under the action of FEN. Compared with control group， G1 phase cell ratio increased significantly in medium and high concentration groups of BEZ and FEN； the apoptotic rate of PC-9 cells was increased significantly in low， medium and high concentration groups of BEZ and FEN； mRNA and protein relative expression of c-myc were decreased significantly， with statistical significance （P＜0.05）. CONCLUSIONS： BEZ and FEN can inhibit the proliferation of PC-9 cells， and down-regulate c-myc expression.

lncRNA NEAT1 promotes cell proliferation of lung adenocarcinoma PC-9 cells through inhibiting DNAdamage / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effects of long non-coding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1) on the proliferation of lung adenocarcinoma PC-9 cells and to explore its mechanism. Methods: qPCR was used to detect the expression level of lncRNA NEAT1 in human lung adenocarcinoma PC-9 cells and human embryonic lung diploid 2BS cells. The sequence of small interfering RNA(siRNA) targeting lncRNANEAT1 gene was designed and synthesized, and then transfected into PC-9 cells by liposome method. The expression level of NEAT1 in PC-9 cells before and after transfection was detected by qPCR. MTT and flow cytometry were used to detect the effect of lncRNANEAT1 knockdown on proliferation and cell cycle distribution of PC-9 cells, respectively. WB assay was used to detect the expressions of DNA damage-related proteins, namely, double-stranded DNA breaks (DSBs) biomarker γ-H2AX and ataxia-telangiectasia mutated (ATM), before and after transfection. Results: Compared with 2BS cells, lncRNA NEAT1 was highly expressed in PC-9 cells (P<0.05). The PC-9 cells with lncRNA NEAT1 knock-down were successfully established. After being transfected with siRNA for 12 h, the proliferation of PC-9 cells in siNEAT1 group and siNEAT2 group significantly decreased as compared with the blank control group and the empty transfection group (P<0.05). In the interference groups, cell cycle was arrested in G1 phase ([88.97±2.64]%, [88.15±1.48]% vs [84.5±1.72]%, P<0.05) and G2/M phase ([8.35±2.02]%, [8.11± 1.36]% vs [4.28±1.28]%, P<0.05). The expression levels of DNA damage-related proteinsATM and γ-H2AX in the interference groups were significantly increased (all P<0.05). Conclusion: lncRNA NEAT1 is highly expressed in lung adenocarcinoma PC-9 cells. lncRNA NEAT1 inhibits DNA damage and causes cell cycle at G1/M phase switch, and thus promotes the proliferation of lung adenocarcinoma cells.

Effect of axitinib on the proliferation and apoptosis of human lung adenocarcinoma PC9 cells and its mechanism / 肿瘤研究与临床

Objective To study the effect of axitinib on the proliferation and apoptosis of human lung adenocarcinoma PC9 cells and its mechanism. Methods PC90 cells were treated with different concentrations (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 μmol/L) of axitinib for 72 h, and half-inhibitory concentration (IC50) was calculated. The cell proliferation ability was detected by CCK-8 method. Plate cloning experiments were performed to observe the effect of axitinib on the formation of PC9 cell clones. The mitochondrial membrane potential and apoptosis of PC9 cells were detected by flow cytometry. The expression of cleaved-Caspase-3 protein in PC9 cells was detected by Western blot. Results Asitinib inhibited the proliferation of PC9 cells in a concentration-dependent manner. The IC50 at 72 h was 10.18μmol/L. The clone formation rates of PC9 cells were (100.0±3.2)％, (58.6±2.7)％, (29.3±3.3)％, and (10.9±3.0)％10 d after treatment with 0, 1, 2 and 4 μmol/L axitinib, and the difference was statistically significant (F= 316.922, P< 0.01). The apoptotic rate of PC9 cells at early and late stages increased after treatment with different concentrations of axitinib for 48 h, and the differences were statistically significant (both P< 0.01). After treatment with 0, 4, 8 and 16 μmol/L axitinib for 24 h, the percentage of PC9 cells with low mitochondrial membrane potential was (11.9±1.9)％, (38.5±2.3)％, (56.3±2.7)％, and (76.9±3.1)％, and the difference was statistically significant (F=234.320, P<0.01). The expression level of cleaved-Caspase-3 protein in PC9 cells increased, and the relative expression levels were 1.00±0.04, 1.26±0.09, 1.78±0.12, and 2.10±0.11, respectively, and the difference was statistically significant (F=55.670, P<0.01). Conclusions Axitinib could inhibit the proliferation of human lung adenocarcinoma PC9 cells. Axitinib induces the apoptosis of PC9 cells possibly through decreasing the mitochondrial membrane potential of PC9 cells.

Imperatorin increased sensitivity of lung cancer CD133 +cell subsets to gefitinib by down-regulating c-met expression / 中国病理生理杂志

AIM:To investigate the role of imperatorin in reversing the resistance of the PC 9 CD133+cell sub-sets to gefitinib.METHODS:MTT assay was performed to evaluate the viability of PC 9 cells treated with imperatorin and gefitinib.The expression of c-met, activation of caspases and phosphorylation of epidermal growth factor receptor (EGFR), PI3K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot .The percentage of CD133 +cell subsets population and the apoptotic rate of the PC 9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry .RESULTS:The sensitivity of the PC 9 CD133 +cell subsets to gefitinib was significantly lower than that of the PC9 CD133 -cell subsets.Treatment with gefitinib alone significantly inhibited the protein levels of EGFR /PI3K/AKT in the PC9 CD133 -cell subsets but not the PC 9 CD133 +cell subsets .Treatment with gefitinib alone increased the percentage of CD133 +cell subsets population in the PC9 cells.However, combination of gefitinib with imperatorin signifi-cantly inhibited the enrichment of CD 133 +cell subsets population .Imperatorin down-regulated c-met expression , sugges-ting the c-met was the target of imperatorin in the PC9 CD133 +cell subsets.The results of MTT assay, Western blot analy-sis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI 3K/AKT protein levels by down-regulating the expression of c-met, which subsequently induced the cleavage of caspases and apoptosis in the PC 9 CD133 +cell subsets.CONCLUSION:Imperatorin increases the sensitivity of lung cancer CD 133 +cell subsets to gefitinib by down-regulating the expression of c-met, and the synergistic anti-tumor effect exists between imperatorin and gefitinib .

Inhibitory Effect of β-asarone on Proliferation of Three Kinds of Tumor Cells / 广州中医药大学学报

Objective To observe the effect of β-asarone on the proliferation, cycle, apoptosis and migration of tumor cells A549, PC3, and PC9-R, thus to provide experimental basis for the application of β-asarone to the treatment of cancer. Methods After A549, PC3, and PC9-R were cultured with different concentrations ofβ-asarone for 24 h, 48 h, and 72 h respectively, CCK-8 was used to detect the optical density (D) of cell proliferation, and then the cell proliferation rate was calculated. The flow cytometry was used to measure the cell DNA cycle and cell apoptosis, and Transwell Chambers were used to detect the cell migration. Results After treatment with different concentrations of β-asarone for 24 h, 48 h, and 72 h respectively, the growth of A549, PC3, and PC9-R showed declining trend in concentration- and time-dependent manner. The proportion of A549, PC3, and PC9-R at G0/G1 phase was increased, the proportion of the three kinds of cells at S phase and the proliferation indexes were declined, the apoptotic rate of A549, PC3, and PC9-R was increased, and the migration of A549, PC3, and PC9-R was reduced (P<0.05 or P<0.01 compared with those of the normal control group). Conclusion β-asarone has certain effects on suppressing proliferation, blocking G0/G1 phase developing into S phrase, inhibiting DNA synthesis, promoting the apoptosis, and inhibiting the migration of A549, PC3 and PC9-R.

Berberine exerts pro-apoptotic effects on PC-9 cells via activation of JNK/FOXO3 signaling / 中国肿瘤临床

Objective:To investigate the pro-apoptotic effect of berberine (Ber) on human lung adenocarcinoma PC-9 cell line, and to detect the role of c-Jun N-terminal kinase (JNK)/forkhead box protein O3 (FOXO3) signaling in this process. Methods:The PC-9 cells were randomly divided into the control group and the Ber group, which was treated with 30 and 60μM Ber. The survival rate, apoptot-ic rate, ROS generation, caspase-3 activity, and mitochondrial membrane potential of cells were detected. Western blot was per-formed to detect the expression of JNK/FOXO3 signaling and apoptosis-related proteins. A JNK-specific activation inhibitor, SP600125, was used to block the phosphorylation of FOXO3 in PC-9 cells, and then treated with Ber (30 and 60μM) for further detection after 24 h. Results:Ber treatment resulted in an obvious reduction in cell viability, promotion of cell apoptosis, downregulation of mitochondri-al membrane potential, and an increase of ROS and caspase-3 in a dose-dependent manner. Western blot analysis demonstrated that Ber treatment resulted in a significant upregulation of p-JNK, FOXO3, and Bax expression, and a downregulation of p-FOXO3 and Bcl2 levels. Moreover, the inhibition of JNK activation by SP600125 antagonized the anti-FOXO3 phosphorylation role and the pro-apoptotic role of Ber on PC-9 cells. Conclusion:Ber treatment effectively inhibits the viability of PC-9 cells and enhances apoptosis and oxidative stress injury, which may be related to the upregulation of p-JNK and FOXO3 and the downregulation of p-FOXO3.

Berberine exerts pro-apoptotic effects on PC-9 cells via activation of JNK/FOXO3 signaling / 中国肿瘤临床

Objective:To investigate the pro-apoptotic effect of berberine (Ber) on human lung adenocarcinoma PC-9 cell line, and to detect the role of c-Jun N-terminal kinase (JNK)/forkhead box protein O3 (FOXO3) signaling in this process. Methods:The PC-9 cells were randomly divided into the control group and the Ber group, which was treated with 30 and 60μM Ber. The survival rate, apoptot-ic rate, ROS generation, caspase-3 activity, and mitochondrial membrane potential of cells were detected. Western blot was per-formed to detect the expression of JNK/FOXO3 signaling and apoptosis-related proteins. A JNK-specific activation inhibitor, SP600125, was used to block the phosphorylation of FOXO3 in PC-9 cells, and then treated with Ber (30 and 60μM) for further detection after 24 h. Results:Ber treatment resulted in an obvious reduction in cell viability, promotion of cell apoptosis, downregulation of mitochondri-al membrane potential, and an increase of ROS and caspase-3 in a dose-dependent manner. Western blot analysis demonstrated that Ber treatment resulted in a significant upregulation of p-JNK, FOXO3, and Bax expression, and a downregulation of p-FOXO3 and Bcl2 levels. Moreover, the inhibition of JNK activation by SP600125 antagonized the anti-FOXO3 phosphorylation role and the pro-apoptotic role of Ber on PC-9 cells. Conclusion:Ber treatment effectively inhibits the viability of PC-9 cells and enhances apoptosis and oxidative stress injury, which may be related to the upregulation of p-JNK and FOXO3 and the downregulation of p-FOXO3.

The inhibitory effects and mechanisms of oridonin on invasion of human lung cancer A549 and PC9 cells / 天津医药

Objective To investigate the inhibitory effects and mechanisms of a nature product derivate oridonin on in?vasion of human lung cancer. Methods Human lung cancer A549 and PC-9 cell lines were treated with oridonin. MTS as?say was used to determine cell proliferation. Transwell assay was used to determine the cell invasion, and adhesion assay to determine the cell adhesion. Flow cytometry was used to determine cell cycle. Western blotting and realtime-PCR were used to detect expression levels of CDK1, mTOR, p53, p21, E-cadherin, CD44,β-catenin, uPA, MMP-2/9, p-AKT and p-Src. The luciferase reporter assay was used to detect the NF-κB promoter activity. Results In vitro proliferation, invasion and adhesion of A549 and PC-9 cells were significantly inhibited by oridonin. The cell cycle was halted by G2/M phase, and ex?pressions of E-cadherin, p53 and p21 were promoted, while expressions of CDK1, mTOR, CD44,β-catenin, uPA, MMP-2/9, p-AKT and p-Src and promoter activity of NF-κB were down-regulated. Conclusion Oridonin is able to inhibit the in vitro invasion of human lung cancer A549 and PC-9 cell lines, which might be correlated with its abilities to regulate the ty?rosine kinase activity.