GC-MS Analysis of Volatile Oil from Chaihu Guizhi Decoction and Study on Its Inhibitory Effects on in vitro Proliferation of Human Lung Adenocarcinoma A 549 Cells / 中国药房

OBJECTIVE：To study the compositi on of the volatile oil from Compound chaihu guizhi decoction ，and to evaluate its in vitro anti-proliferative activity on human lung adenocarcinoma A 549 cells. METHODS ：The volatile oil from Chaihu guizhi decoction was extracted according to the steam distillation method of general rules 2004 in the 2015 edition of Chinese Pharmacopoeia（part Ⅳ）. The volatile oil components were analyzed by GC-MS combined with Kováts index ，and the relative content of each component was calculated by peak area normalization method. Using different concentrations of cisplatin （4，8， 16，32，64 mg/L）as positive control ，MTT assay was used to detect the inhibitory effects of different concentrations of volatile oil from Chaihu guizhi decoction （25，50，100，200，400 mg/L）on in vitro proliferation of A 549 cell after 48 h of treatment. Negative control group （with cells but without drugs ）was set up. RESULTS ：A total of 71 chemical components were isolated from the volatile oil ，among which there were 59 compounds identified ，sum of peak areas accounting for 84.99％ of the total peak area. The compounds with relatively high content included ar-curcumene （17.65％），β-bisabolene（9.57％），β-ocimene（7.05％）， α-curcumene（5.35％），2，5-dimethylbenzaldehyde（4.24％），linalyl isobutyrate （2.70％），α-cedrene（2.48％），δ-cadinene （2.07％）. Compared with negative control group ，the proliferation rate of cells were decreased significantly in 4-64 mg/L cisplatin groups and 25-400 mg/L volatile oil from Chaihu guizhi decoction groups （P＜0.05）. IC 50 of cisplatin and volatile oil from Chaihu guizhi decoction to in vitro proliferation of A 549 cells were 10.150 and 73.526 mg/L. CONCLUSIONS ：The volatile oil from Chaihu guizhi decoction mainly includes ar-curcumene ，β-bisabolene，β-ocimene，α-curcumene，which shows certain inhibitory effect on in vitro proliferation of A 549 cells.

Mechanism of Scutellariae Radix-Hedyotidis Herba Against Proliferation of Lung Adenocarcinoma A549 Cells / 中国实验方剂学杂志

Objective:To observe the effects of Scutellariae Radix-Hedyotidis Herba on the proliferation of human lung adenocarcinoma A549 cells and the expression of interleukin-6（IL-6）， phosphatidylinositol 3-kinase （PI3K），protein kinase B （Akt）， p-protein kinase B （p-Akt）， mechanistic target of rapamycin （mTOR）， hypoxia-inducible factor-1<italic>α </italic>（HIF-1<italic>α</italic>）， and Cyclin D₁ at the cellular level， and to explore their molecular mechanism. Method:Following the set-up of the blank group （complete medium）， low-， moderate-，and high-dose （20， 40， and 60 mg·L^-1） Scutellariae Radix-Hedyotidis Herba groups， and low-， moderate-， and high-dose （5， 10， and 20 mg·L^-1） cisplatin groups， the cell were treated with the corresponding drugs for 24， 48， and 72 h for detecting their viability by tetrazolium bromide （MTT） colorimetry. A549 cells were then divided into the blank group， Scutellariae Radix-Hedyotidis Herba group， cisplatin group， and combined medication group and intervened with thecomplete medium， 40 mg·L^-1Scutellariae Radix-Hedyotidis Herba， 10 mg·L^-1 cisplatin， and 40 mg·L^-1Scutellariae Radix-Hedyotidis Herba + 10 mg·L^-1cisplatin， respectively， for 24， 48 and 72 h， followed by the measurement of inhibitory effects against the proliferation of A549 cells in each experimental group. The level of IL-6 in cell culture supernatant was determined by enzyme-linked immunosorbent assay （ELISA） after 72 h. The mRNA expression levels of HIF-1<italic>α</italic> and Cyclin D₁ in each group were assayed by real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of PI3K， Akt， p-Akt， mTOR， HIF-1<italic>α</italic>， and Cyclin D₁ by Western blot. Result:After 24 h intervention， Scutellariae Radix-Hedyotidis Herba did not significantly inhibit the proliferation of A549 cells. However， 48 h later， the inhibitory effect in Scutellariae Radix-Hedyotidis Herba groups were significantly enhanced in comparison with that in the blank group （<italic>P</italic><0.05）， exhibiting a time-dependent response. After 72 h of action， no significant change was present in the inhibitory effect of each Scutellariae Radix-Hedyotidis Herba group， so the optimal concentration of Scutellariae Radix-Hedyotidis Herba was set at 40 mg·L^-1 for follow-up experiments. As demonstrated by the comparison with the blank group， cisplatin at each concentration inhibited the cell proliferation in a time-dependent manner （<italic>P<</italic>0.05）. Considering the cell survival rate， the best concentration of cisplatin was set at 10 mg·L^-1. Compared with the blank group， Scutellariae Radix-Hedyotidis Herba combined with cisplatin remarkably inhibited the proliferation of A549 cells in a time-dependent manner （<italic>P<</italic>0.05）， and the differences between the combined medication group and the other two groups became more significant after 72 h of medication （<italic>P<</italic>0.01）. The IL-6 level in each experimental group， especially in the combined medication group， significantly declined in contrast to that in the blank group （<italic>P<</italic>0.01）. The mRNA expression levels of HIF-1<italic>α</italic> and Cyclin D₁ in all experimental groups were obviously lower than those in the blank group， with the most significant changes observed in the combined medication group （<italic>P</italic><0.05，<italic>P</italic><0.01）. The protein expression levels of PI3K， p-Akt， mTOR， HIF-1<italic>α</italic>， and Cyclin D₁ in each experimental group was significantly down-regulated（<italic>P</italic><0.05，<italic>P</italic><0.01）， and the levels in the combined medication group were even lower than those in the cisplatin group （<italic>P<</italic>0.01）. Conclusion:Scutellariae Radix-Hedyotidis Herba has an inhibitory effect on the proliferation of A549 cells， which may be related to its inhibition against the expression and secretion of IL-6/PI3K/Akt/mTOR-HIF-1<italic>α</italic> axis.

Effect of cereblon on inhibition of human lung cancer A549 cell migration by thalidomide / 国际药学研究杂志

Objective:To investigate the influence of cereblon(CRBN)on the inhibitory effect of thalidomide on the migration of human lung adenocarcinoma A549 cells and related mechanism. Methods: The effect of thalidomide on the A549 cell proliferation was assayed by the MTS method. CRBN in A549 cells was knocked down(A549CRBN cells) with RNA interference and lentivirus infection. Transwell assay was performed to detect the effect of thalidomide on the cell migration of A549 and A549CRBN cells. RT-qPCR assay was performed to detect the gene expression of N-cadherin and vimentin. Results: The thalidomide at the 1, 10, 50 and 100 μmol/L concentration did not affect the proliferation of A549 cells, while the 100 μmol/L thalidomide could significantly inhibit the migration of A549 cells. After the CRBN gene knockdown the migration ability of A549 cells was significantly decreased(P<0.01), and the gene expression of N-cadherin and vimentin was also significantly reduced in the A549CRBN cells. The inhibitory effect of thalidomide on the cell migration in the A549 cells disappeared after CRBN gene knockdown. Thalidomide could inhibit the gene expression of N-cadherin and vimentin in a CRBN-dependent manner. Conclusion: Thalidomide could inhibit the migration of A549 cells and the expression of the migration-related genes via CRBN.

Regulation of Wnt5a in apoptosis of human lung adenocarcinoma A549 cells and its mechanism / 吉林大学学报(医学版)

Objective:To investigate the regulation effect of Wnt5a on the apoptosis of lung adenocarcinoma A549 cells,and to clarify its mechanism.Methods:The human lung adenocarcinoma cells were selected.The A549 cells treated with Wnt5a were used as treatment group,and the A549 cells treated with C culture solution were used as control group.The apoptotic body induced by Wnt5a was assessed with TUNEL assay;the apoptotic rates of the A549 cells in various groups were detected by Annexin V-FITC/PI double staining;the reactive oxygen species (ROS)levels in the A549 cells in various groups were determined with DCFH-DA fluorescence probe,and the mitochondria membrane potential was assessed with JC-1 staining method.Western blotting was used to analyze the expression levels of apoptosis-related proteins in the A549 cells in various groups.Results:Compared with control group,the apoptotic rates of the A549 cells in treatment group 12,24,and 48 h after treatment were significantly increased(P<0.01);the ROS levels were increased(P<0.05);the mitochondria membrane potentials were decreased(P<0.05),the expressing amount of BAX was up-regulated and the expression amount of AIF was down-regulated.Conclusion:Wnt5a has regulation on the apoptosis of human lung adenocarcinoma cells and can promote the apoptosis of A549 cells through mitochondrial pathway.

Regulation of Wnt5a in apoptosis of human lung adenocarcinoma A549 cells and its mechanism / 吉林大学学报(医学版)

Objective: To investigate the regulation effect of Wnt5a on the apoptosis of lung adenocarcinoma A549 cells, and to clarify its mechanism. Methods, The human lung adenocarcinoma cells were selected. The A549 cells treated with Wnt5a were used as treatment group, and the A549 cells treated with C culture solution were used as control group. The apoptotic body induced by Wnt5a was assessed with TUNEL assay; the apoptotic rates of the A549 cells in various groups were detected by Annexin V-FITC/PI double staining; the reactive oxygen species (ROS) levels in the A549 cells in various groups were determined with DCFH-DA fluorescence probe, and the mitochondria membrane potential was assessed with JC-1 staining method. Western blotting was used to analyze the expression levels of apoptosis-related proteins in the A549 cells in various groups. Results; Compared with control group, the apoptotic rates of the A549 cells in treatment group 12, 24, and 48 h after treatment were significantly increased (P<0.01); the ROS levels were increased (P<0.05); the mitochondria membrane potentials were decreased (P<0.05), the expressing amount of BAX was up-regulated and the expression amount of AIF was down-regulated. Conclusion: Wnt5a has regulation on the apoptosis of human lung adenocarcinoma cells and can promote the apoptosis of A549 cells through mitochondrial pathway.

Effects of Akt Inhibitor MK-2206 on Proliferation and Apoptosis of Lung Adenocarcinoma A549 Cells / 中国药房

OBJECTIVE:To study the effects of Akt inhibitor MK-2206 on the proliferation and apoptosis of lung adenocarcino-ma A549 cells. METHODS:The optical density of A549 cells was detected by MTT assay after treated with 0(blank control),0.5, 1,2.5,5,10,20 and 30 μmol/L MK-2206 for 24 h;after pretreatment with 0(blank control),5,10 and 20 μmol/L MK-2206 for 24 h,morphological changes of A549 cells were observed with inverted microscope. Cell cycle and apoptosis rate were analyzed by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins Cyclin D1,p21 and p27 and apopto-sis-related protein PARP(cf-PARP),cf-caspase-3,Bcl-2 and Bax. RESULTS:Compared with blank control,the optical density of A549 cells decreased,cells shrank and presented vesicular state after treatment of MK-2206;A549 cells arrested in G0/G1 stage, the protein expression of p21 and p27 strengthened while that of Cyclin D1 decreased;the apoptotic rate of cells increased,the ex-pression of cf-PARP,cf-caspase-3 and Bax in cells increased while that of Bcl-2 decreased. All reponse were in concentration-de-pendant manner (P<0.05 or P<0.01). CONCLUSIONS:MK-2206 can inhibit the proliferation of A549 cells,and induce the apoptosis of A549 cells by adjusting the expression of activating caspase-3,down-regulating Bcl-2 and up-regulating Bax.

Effect of receptor for activated C kinase 1 gene silencing on the sensitivity of cell line A549 to chemotherapeutic drugs / 肿瘤研究与临床

Objective To investigate the effect of shRNA-mediated down-regulation of the receptor for activated C kinase 1 (RACK1) gene on the chemotherapeutic sensitivities in human lung adenocarcinoma cell line A549.Methods The shRNA recombinant plasmid targeting to human RACK1 gene was designed and transferred into A549 cells by lipofectin technique.The protein level of RACK1 was measured by Western blot to confirm the function of shRNA plasmid.Drug sensitivities of A549 cells to cisplatin,gemcitabine,pemetrexed and paclitaxel were analyzed by MTT assay.The protein expression of LRP and MRP were detected by Western blot.Results After 24 hours transfection,the relative expression quantity of RACK1 protein in RACK1-shRNA group was 0.267± 0.470,which was significantly lower than that in vector-shRNA group (0.821±0.109) and control group (0.842±0.060) (F =54.438,P ＜ 0.05).The results of MTT showed that the growth of A549 cells in the RACK1-shRNA group was markedly inhibited.The sensitivities of A549 cells to cisplatin and paclitaxel were significantly enhanced compared with that in the vector-shRNA group and control group (P ＜ 0.05).The relative expression quantity of LRP and MRP protein in RACK1-shRNA group were 0.163±0.056 and 0.246±0.050,which were lower than that in vector-shRNA group and control group (F LRP =19.430,F MRP =61.548,both P ＜ 0.05).Conclusion Targeted gene silencing of RACK1 improves the sensitivity of A549 cells to the ascisplatin and paclitaxel medicines,which might be achieved through down-regulation of the expression of LRP and MRP.

The changes in biological characteristics of lung adenocarcinoma A549 cells under two in vitro culture systems / 解放军医学杂志

Objective To investigate the changes in cells on cell cycle,cell apoptosis and susceptibility to chemotherapeutic drugs of lung adenocarcinoma A549 cells under two dimensional (2D system) and three dimensional (3D system) culture conditions. Methods The three dimensional culture of A549 cells was carried out using rotatable culture bottle and desk-top water bath shake. Flow cytometer,in situ cell apoptosis detection kit,MTT and blood cell counter were employed to compare cell cycle,cell apoptosis and susceptibility to ADM of A549 cells under different culture conditions. Results The three dimensional culture system was confirmed to have been successful by the aid of observation under inverted and electron microscope. There were significant retardation of G_ 1 phase,lower cell apoptosis rate and decreased susceptibility to ADM in A549 cells in 3D system compared with those in 2D system. Conclusions There were remarkable differences in biological characteristics of A549 cells in two culture systems,indicating that A549 cells cultivated in 3D system simulated better solid tumor in vivo . The 3D system was very useful for further investigation of the behavior of solid tumor,so that anti-carcinoma chemotherapeutic drugs could be advantageously tested in vitro before clinical application.

Cigarette Smoke-Induced DNA Damage and Repair in Two Kinds of Human Lung Interstitial Cells / 环境与健康杂志

Objective To study the DNA damage and repair of normal lung interstitial cells and human lung adenocarcinoma cells exposed to cigarette smoke. Methods Cultured human embryo lung fibroblasts (HLF) and human lung adenocarcinoma A549 cells. Mainstream smoke was collected by using dimerhyl sulfoxide (DMSO) and phosphate buffer solution (PBS) as absorbents. MTT assay was used to test the cytotoxicity of the solutions of cigarette smoke, then selected the concentrations of the solutions with no obvious cytotoxicity to treat cells and detected DNA damage and repair by comet assay. Results As treated with original solutions or 1/2 dilution of DMSO cigarette smoke solutions only, the Viability of cells was below 80%, but it was beyond 80% when treated with PBS solutions. The results showed that a significant difference of DNA damage was seen between the treated groups and negative control groups (P0.05),but the DNA damage caused by DMSO solutions was worse than PBS solutions significantly (P