Isolation,extraction and identification of exosomes from non-small cell lung cancer A549 cell culture supernatant / 中国生物制品学杂志

@#Objective To isolate,extract and identify exosomes from the culture supernatant of non-small cell lung cancer A549 cells. Methods Exosomes from supernatant of non-small cell lung cancer A549 cells were isolated by ultracentrifugation(Ult)and modified method[Ult combined with Exo Quick~(TM)(Ult-Exo Quick~(TM),U-EQ)],which were determined for the concentration by BCA,measured for the particle size distribution by nanoparticle tracking analysis(NTA),observed for the morphology by fluorescence staining and transmission electron microscope(TEM),and detected for the expression of CD63 and HSP70 proteins on the surface of exosomes by Western blot. Results The concentrations of exosomes isolated by U-EQ and Ult were 1. 355 and 0. 909 mg/mL,respectively;The total number of particles was 4. 65 × 10~9 and 4. 06 × 10~9 particles/mL,the peak sizes ranged from 81. 5 to 165. 5 and 59. 5 to 176. 5 nm,and the average size was(152. 3 ± 8. 8)and(94. 3 ±9. 8)nm,respectively;The exosomes were all 30～150 nm in diameter,while the morphology of exosomes extracted by U-EQ method was more uniform;CD63 and HSP70 proteins were expressed in all of them,while the exosomes extracted by U-EQ method showed higher expression. Conclusion Both U-EQ and Ult methods can isolate exosomes from the supernatant of non-small cell lung cancer A549 cells,while the concentration of exosomes extracted by U-EQ method is higher.

Effects of lncRNA CDKN2B-AS1 targeting miR-98-5p on proliferation and invasion of lung cancer A549 cells / 中华内分泌外科杂志

Objective:To investigate the effects of long non-coding RNA (lncRNA) CDKN2B-AS1 targeting miR-98-5p on proliferation and invasion of lung cancer A549 cells.Methods:A549 cells cultured in vitro were divided into control group, pcDNA group (transfected with pcDNA) , CDKN2B-AS1 group (transfected with pcDNA CDKN2B-AS1) and double transfection group (transfected with pcDNA CDKN2B-AS1 and pcDNA miR-98-5p) . The expression of lncRNA CDKN2B-AS1, miR-98-5p and the protein expression of PCNA, MMP-9 in A549 cells were detected. The activity, clone number, cloning efficiency, and the number of invasive cells of A549 cells were detected.Results:Compared with pcDNA group, the expression level of lncRNA CDKN2B-AS1 [ (2.14±0.14) vs (1.03±0.10) ], OD value in each time points, clone number [ (314.60±18.13) vs (220.08±12.46) ], cloning efficiency [ (85.81±3.06) % vs (60.03±2.85) %], invasive cell number [ (233.30±18.98) vs (140.84±12.30) ], expression levels of PCNA [ (0.78±0.08) vs (0.48±0.07) ] and MMP-9 [ (0.75±0.06) vs (0.38±0.06) ] proteins in A549 cells in CDKN2B-AS1 group were significantly increased ( P<0.05) ; the expression level of miR-98-5p [ (0.23±0.03) vs (0.99±0.09) ] was significantly decreased ( P<0.05) ; compared with CDKN2B-AS1 group, there was no significant difference in the expression level of lncRNA CDKN2B-AS1 in A549 cells in double transfection group ( P>0.05) , while the expression level of miR-98-5p in A549 cells was significantly increased ( P<0.05) . The OD value in each time points, clone number, cloning efficiency, invasive cell number, expression levels of PCNA and MMP-9 proteins were significantly decreased ( P<0.05) . Conclusion:LncRNA CDKN2B-AS1 can promote the proliferation and invasion of lung cancer A549 cells by targetingly inhibiting the expression of miR-98-5p.

Mechanism of Qiyu Sanlong Prescription in Inhibiting "Addiction" of Lung Cancer A549 Cells to miRNA21 / 中国实验方剂学杂志

Objective:To investigate the molecular mechanism of Qiyu Sanlong prescription （QYSL） in inhibiting the "addiction" of lung cancer A549 cells to miRNA21. Method:The human lung cancer A549 cells were routinely passaged and divided into the blank group， blank serum group， QYSL-containing serum group， and siRNA group. The prepared QYSL-containing serum was used for intervention， with the optimal concentration and action time determined in previous studies. The protein and mRNA expression levels of miRNA21 and related molecules in its target phosphatase and tensin homolog deleted on chromosome 10 （PTEN）/phosphatidylinositol 3-kinase （PI3K） signaling pathway were detected by real-time polymerase chain reaction （Real-time PCR） and Western blot assay. Result:The comparison with the blank serum group revealed that the mRNA expression levels of miRNA21 in the QYSL-containing serum group and the siRNA group were decreased， while the PTEN mRNA expression in the QYSL-containing serum group was increased， showing significant differences （<italic>P</italic><0.01）. Compared with the blank serum， the QYSL-containing serum and siRNA significantly down-regulated PI3K and mammalian target of rapamycin （mTOR） mRNA expression （<italic>P</italic><0.01）， whereas the QYSL-containing serum did not change the mRNA expression of protein kinase B （Akt）. The protein expression levels of PTEN in the QYSL-containing serum group and the siRNA group were obviously elevated in contrast to that in the blank serum group （<italic>P</italic><0.05）. Meanwhile， the protein expression levels of phosphorylated Akt （p-Akt） and phosphorylated mTOR （p-mTOR） evidently declined in the QYSL-containing serum group （<italic>P</italic><0.05）， but there was no significant reduction in total Akt and mTOR protein expression. The PI3K protein expression was slightly down-regulated， with no statistical significance. Conclusion:QYSL inhibits the transcription of miRNA21， increases the expression of PTEN， and reduces the expression of key molecules in PI3K/Akt/mTOR signaling pathway， thus mildly inhibiting the "addiction" of lung cancer cells to oncogenes and blocking their proliferation.

Lotus leaf flavonoids induce apoptosis of human lung cancer A549 cells through the ROS/p38 MAPK pathway

BACKGROUND: Leaves of the natural plant lotus are used in traditional Chinese medicine and tea production. They are rich in flavonoids. METHODS: In this study, lotus leaf flavonoids (LLF) were applied to human lung cancer A549 cells and human small cell lung cancer cells H446 in vitro to verify the effect of LLF on apoptosis in these cells through the ROS/p38 MAPK pathway. RESULTS: LLF had no toxic effect on normal cells at concentrations up to 500 µg/mL, but could significantly inhibit the proliferation of A549 cells and H446 cells. Flow cytometry showed that LLF could induce growth in A549 cells. We also found that LLF could increase ROS and MDA levels, and decrease SOD activity in A549 cells. Furthermore, qRT-PCR and western blot analyses showed that LLF could upregulate the expression of p38 MAPK (p-p38 MAPK), caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and Bax and downregulate the expression of Cu/Zn SOD, CAT, Nrf2, NQO1, HO-1, and Bcl-2 in A549 cells. Results of HPLC showed that LLF mainly contain five active substances: kaemp-feritrin, hyperoside, astragalin, phloridzin, and quercetin. The apoptosis-inducing effect of LLF on A549 cells came from these naturally active compounds. CONCLUSIONS: We have shown in this study that LLF is a bioactive substance that can induce apoptosis in A549 cells in vitro, and merits further research and development.

Silencing carbonic anhydrase 1 inhibits the proliferation, invasion and migration of A549 cells in lung cancer / 中国临床药理学与治疗学

AIM: To investigate the effects of silencing carbonic anhydrase 1 (CA1) on proliferation, apoptosis, invasion and migration of human lung cancer A549 cells. METHODS: CA1-specific siRNA (si-CA1 group) and negative control (si-NC group) were transfected into lung cancer A549 cells by lipofection. The A549 cells transfected with empty liposome were used as blank control group. Real-time quantitative PCR (qPCR) and Western blot (Western blot) were used to detect the expression of CA1 mRNA and protein. Cell counting kit method (CCK-8), flow cytometry and Transwell assay were used to detect proliferation and apoptosis of A549 cells, invasion and migration capabilities. RESULTS:qPCR and Western blot showed that the expression levels of CA1 mRNA and protein in A549 cells transfected with CA1 siRNA were significantly down-regulated (P0.05). CONCLUSION: Silencing CA1 can inhibit the proliferation, invasion and migration of lung cancer A549 cells and promote cell apoptosis.

Sodium selenite induced human lung cancer A549 cells apoptosis through Keap1/Nrf2/ARE signaling pathway / 中国药理学通报

Aim: To study the induction of apoptotic effect of sodium selenite on human lung cancer A549 cells and its mechanisms. Methods: A549 cells were exposed to different concentrations of sodium selenite for 24 h. MTT assay was applied to determine A549 cell proliferation. Inverted fluorescence microscope was used to investigate the morphological changes in A549 cells. Flow cytometry analysis was applied to assess the apoptotic rates of A549 cells. Laser confocal microscope was employed to measure the reactive oxygen species (ROS) fluorescence intensity. A multi-detection reader was used to determine the antioxidant parameter. Western blot was utilized to detect the expression of Keapl, Nrf2, HO-1 and Nrf2 in cytoplasm and nucleus. Results: MTT results showed that sodium selenite inhibited the proliferation of A549 cells in a concentration-dependent manner. After treatment with sodium selenite for 24 h, the apoptotic rate of A549 cells was markedly increased through Hoechst 33342 staining and flow cytometry measurement. Sodium selenite significantly up-regulated ROS and malondialdehyde (MDA) content and down-regulated the levels of superoxide dismutase (SOD) and glutathione (GSH). Meanwhile, sodium selenite treatment also reduced the expressions of Keapl, Nrf2 and HO-1 at protein levels and inhibited Nrf2 protein nuclear translocation in A549 cells. Conclusions: Treatment with sodium selenite induces A549 cells apoptosis, which may contribute to the anti-proliferation activity, induction of apoptosis and regulation of oxidative stress reaction and Keapl/Nrf2/ARE antioxidative signaling pathway expression.

Atractylenolide I regulates proliferation and invasion of lung cancer A549 cells through the TLR4/MyD88 pathway / 实用肿瘤学杂志

Objective The objective of this study was to investigate the role and molecular mechanism of AtractylenolideⅠin the progression of lung cancer. Methods qRT-qPCR and immunohistochemistry were used to detect the expression of TLR4 and MyD88 at levels of mRNA and protein in lung cancer and adjacent tissues. Transwell and MTT assays were used to detect effects of at-ractylenolide I(100 μM)on the invasion,migration and proliferation of A549 cells. Western blot was also used to detect the effect of atractylenolide I on the expression of TLR4 and MyD88 proteins. Results The expression of TLR4 and MyD88 at levels of mRNA and protein was highly expressed in lung cancer tissues when compared to adjacent tissues(P<0. 05). Compared with the control group,the invasion ability of A549 cells in the Atractylenolide I group was significantly decreased,and the proliferative activity was in-hibited(P<0. 05). Atractylenolide I inhibited the expression of TLR4 and MyD88 protein in A549 cells( P<0. 05). Conclusion

Study on Inhibitory Mechanism of Timosaponin B-Ⅱ on the Proliferation and Migration of Human Lung Cancer A549 Cells / 中国药房

OBJECTIVE:To study the inhibitory mechanism of timosaponin B-Ⅱ(TB-Ⅱ) on the proliferation and migration of human lung cancer A549 cells. METHODS:A549 cells were treated with TB-Ⅱ [0(blank control),1,10 and 100 μg/ml] for 48 h,and total RNA and total protein were extracted respectively. Real time fluorescence quantitative-PCR and Western blot were used to detect mRNA and protein levels of IL-18. IL-18 in A549 cells was silenced by transfection;the expression of IL-18 mRNA and protein were compared among untransfection group,negative control group and transfection group;and then human lung can-cer A549 cells with silenced gene were treated with 10 μg/ml TB-Ⅱ for 24,48 and 72 h. The activity of cell proliferation was de-tected with CCK-8,and the change of cell migration ability was observed by streak method. RESULTS:Compared with blank con-trol,the expression of IL-18 mRNA and protein in A549 cells all increased after treated with TB-Ⅱ(P<0.05 or P<0.01),and were positively correlated with concentration. Compared with untransfection group,the expression of IL-18 mRNA and protein de-creased in transfection group(P<0.01). Compared with untransfected cell treated with TB-Ⅱ,the viability and migration ability of A549 cells with transfection gene increased after treated with TB-Ⅱ for 72 h(P<0.01). CONCLUSIONS:TB-Ⅱ can inhibit the proliferation and migration of A549 cells by up-regulating IL-18 gene expression.

Effects of Thymalfasin for Injection on the Apoptosis of Humun Lung Cancer A549 Cells / 中国药房

OBJECTIVE:To study the effects of Thymalfasin for injection on the apoptosis of human lung cancer A549 cells. METHODS:After treated with 0(blank control),25,50,100,200 and 400 mg/L Thymalfasin for injection for 24,48 and 72 h, the cell proliferation inhibitory rate was analyzed with MTT and calculated. After treated with 0(blank control),50 and 100 mg/L Thymalfasin for injection for 48 h,cell apoptosis was detected by flow cytometry,and the expression of Caspase-3,Bcl-2 and Bax and the phosphorylation level of Akt were deteced by Western blot. RESULTS:Compared with blank control group,proliferation in-hibitory rate of A549 cells increased after treated with Thymalfasin for injection,in concentration and time-dependent manner(P<0.05). The apoptotic rate of A549 cells increased after treated with Thymalfasin for injection 50,100 mg/L for 48 h (P<0.05). The expression of Caspase-3 increased while the Bcl-2/Bax and phosphorylation level of Akt decreased in A549 cells after treated with Thymalfasin for injection 100 mg/L (P<0.05). CONCLUSIONS:Thymalfasin for injection can inhibit the proliferation of A549 cells by activating Caspase-3,decreasing Bcl-2/Bax ratio,inhibiting Akt signal pathway and induce the apoptosis of A549 cells.

Preparation of Folic Acid-loaded Vincristine Nano Liposome and Its Effects on Human Liver and Lung Cancer Cells / 中国药房

OBJECTIVE:To prepare folic acid(FA)-loaded vincristine(VCR)nano liposome(VCR-nLip-FA)and to study its effects on human liver and lung cancer cells. METHODS:VCR-nLip-FA was prepared by ammonium sulfate gradient method,and particle size,Zeta-potential,encapsulation rate and release rate were investigated. Taking human liver cancer HepG2 cells and lung cancer A549 cells as example,uptake rate and inhibitory effect in vitro (5-80 μg/ml) were compared between VCR-nLip-FA and VCR-nLip. RESULTS:The particle size distribution,average particle size,average Zeta-potential,average encapsulation rate and 24 h accumulative release rate of VCR-nLip-FA were 98.1-159.0 nm,132.2 nm,-40.1 mV,(86.6±3.5)%(n=4)and(42.2± 2.6)%. Compared with VCR-nLip,there was no statistical significance in uptake rate of A549 cells to VCR-nLip-FA and inhibitory effect of VCR-nLip-FA on A549 cell viability (P>0.05);uptake rate of HepG2 cells to VCR-nLip-FA and inhibitory effect of VCR-nLip-FA on HepG2 cell viability increased significantly (P<0.01),in dose-dependent manner. CONCLUSIONS:Prepared VCR-nLip-FA can target anti-tumor drug to HepG2 cells efficiently,and highly inhibit the growth of HepG2 cells. But it has no higher effects on A549 cells.