2‐(6‐Hydroxyhexylthio)‐5,8‐dimethoxy‐1,4‐naphthoquinone Induces Apoptosis through ROS‐Mediated MAPK, STAT3, and NF‐κB Signalling Pathways in Lung Cancer A549 Cells
Guinan ShenCheng WangYing‐Hua LuoJiaru WangRui WangWanting XuYi ZhangYu ZhangDongjie ZhangCheng‐Hao Jin
7
Citation
39
Reference
10
Related Paper
Citation Trend
Abstract:
Two novel compounds, 2‐(2‐hydroxyethylthio)‐5,8‐dimethoxy‐1,4‐naphthoquinone (HEDMNQ) and 2‐(6‐hydroxyhexylthio)‐5,8‐dimethoxy‐1,4‐naphthoquinone (HHDMNQ), were synthesized to investigate the kill effects and mechanism of 1,4‐naphthoquinone derivatives in lung cancer cells. The results of the CCK‐8 assay showed that HEDMNQ and HHDMNQ had significant cytotoxic effects on A549, NCI‐H23, and NCI‐H460 NSCLC cells. Flow cytometry and western blot results indicated that HHDMNQ induced A549 cell cycle arrest at the G2/M phase by decreasing the expression levels of cyclin‐dependent kinase 1/2 and cyclin B1. Fluorescence microscopy and flow cytometry results indicated that HHDMNQ could induce A549 cell apoptosis, and western blot analysis showed that HHDMNQ induced apoptosis through regulating the mitochondria pathway, as well as the MAPK, STAT3, and NF‐ κ B signalling pathways. Flow cytometry results showed that intracellular reactive oxygen species (ROS) levels were increased after HHDMNQ treatment, and western blot showed that ROS could modulate the intrinsic pathway and MAPK, STAT3, and NF‐ κ B signalling pathways. These effects were blocked by the ROS inhibitor N‐acetyl‐L‐cysteine in A549 cells. Our findings suggest that compared with HEDMNQ, HHDMNQ had the stronger ability to inhibit the cell viability of lung cancer cells and induce apoptosis by regulating the ROS‐mediated intrinsic pathway and MAPK/STAT3/NF‐ κ B signalling pathways. Thus, HHDMNQ might be a potential antitumour compound for treating lung cancer.Keywords:
Signalling pathways
OBJECTIVE:To investigate the effect and the molecular mechanism of Gab2 on migration and invasion of human lung adenocarcinoma cell line A549.METHODS:The plasmid pGCsilencerU6/GFP/Neo-RNAi-Gab2#1,pGCsilencerU6/GFP/Neo-RNAi-Gab2#2 and the empty vector were transfected into A549 cells respectively to establish Gab2-deficient A549 cells(siGab2/A549#1,siGab2/A549#2 cells) and the SCR/A549 cells(as the control cells).RT-PCR and Western blot analyse were used to test the mRNA and protein expression levels of Gab2.The abilities of migration and invasion were determined by chemotaxis assay and invasion assay.The IGF-1 induced expression of MMP-2,MMP-9 and the activation of pAkt,and pmTOR in Gab2-deficient A549 cells and SCR/A549 cells were detected by Western blot.The Gab2 mRNA expression of A549 cell,Scr/A549 cell,siGab2#1/A549 cell and siGab2#2/A549 cell were 1.340±0.009,1.201±0.074,0.315±0.008 and 0.289±0.007 respectively.The protein expression of A549 cell,Scr/A549 cell,siGab2#1/A549 cell and siGab2#2/A549 cell were 0.205±0.003,0.241±0.004,0.128±0.002 and 0.066±0.001,respectively.RESULTS:The Gab2 mRNA expression level of Gab2 silencing siGab2#2/A549 cells was obviously lower than that of A549 cells(t=168.032,P0.001);Gab2 protein expression in siGab2#2/A549 cells was also significantly down-regulated than that of A549 cells(t=64.352,P0.001).The abilities of migration and invasion of Gab2 silencing A549 cells were obviously decreased as compared with those of the SCR/A549 cells(t=5.101,P=0.029;t=10.812,P=0.002).In addition,our research showed that IGF-1-induced expression of MMP-2,MMP-9(t=-2.051,P=0.054;t=-2.652,P=0.064) and the phosphorylation of pAkt,pmTOR were obviously suppressed in Gab2 silencing A549 cells as compared with those in the SCR/A549 cells.CONCLUSIONS:This study identifies that Gab2 is a critical factor in A549 cell invasion.It seems that Gab2 promotes invasive potential via activating the Akt/mTOR pathway.
Cite
Citations (0)
One of the hallmarks of cAMP is its ability to inhibit proliferation in many cell types, but stimulate proliferation in others. Clearly cAMP has cell type specific effects and the outcome on proliferation is largely attributed to crosstalk from cAMP to the RAS/RAF/mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase (ERK) kinase (MEK)/ERK pathway. We review the crosstalk between these two ancient and conserved pathways, describing the molecular mechanisms underlying the interactions between these pathways and discussing their possible biological importance.
Crosstalk
Signalling pathways
Cite
Citations (282)
To explore the effects of oxidative damage induced by mainstream smoke (MS) on A549 and A549-R cells of hOGG1 deficient cell.A549 cells and A549-R cells with down-regulated hOGG1 gene were treated at the concentrations of MS for 2h. The cellular sensitivities and DNA damages were measured by MTT assay and comet assay, respectively. The contents of ROS and 8-OHdG in both cells were examined by fluorescence method and HPLC-ECD.The cell viabilities decreased with increases of concentration of MS. The IC50 of hOGG1 deficient A549-R cell was more lower than that in A549 cell (P < 0.05). With dose increases of MS, the contents of ROS increased in both cell lines. When MS was more than or equal to 1.25 No. of cigarette/L, the contents of ROS in A549-R cells were much higher than those in A549 cells. The comet assay indicated that DNA damages of A549-R cells were more higher than those in A549 cells, and comet rate, tail length and OTM in A549-R cells were more higher in comparison with A549 cells (P < 0.05) at the levers of all concentrations. Furthermore, the levels of 8-OHdG in A549-R cells were higher than those of A549 cells at the doses of 2.5 and 5 No. of cigarette/L (P < 0.05).Mainstream smoke could induce oxidative damage in A549 and A549-R cells and hOGG1 deficiency cell could increase sensitivity to MS.
Comet Assay
Cite
Citations (0)
Objective Dentin matrix phosphoprotein 1(DMP1) is a non-collagenous, acidic extracellular matrix protein expressed chiefly in bone and dentin. The present study was aimed to investigate whether DMP1 can trigger MAPK signalling pathway in hMSC, MC3T3-E1 and MDPC-23 cells. Methods The activation of MAPK-ERK by rDMP1F / C and inhibition of ERK by MAPK inhibitor at different timepoints in hMSC, MC3T3-E1 and MDPC-23 cells was detected using Western blot. The inhibition of ERK cytoplasm / nucleus translocation by the MAPK inhibitor was detected using immunofluorescence microscopy. The activation of MAPK-ERK signalling pathway by DMP1 was further validated through knowdown of Ras gene expression by siRNA. Results The MAPK-ERK could be activated by both recombinant DMP1 C-terminal(rDMP1C) and full lenth(rDMP1F) from 5 min to 3 h in three cell lines. The phosphorylated-ERK increased, but not total ERK, in these cell lines treated by rDMP1. The phosphorylation of MAPK-ERK by rDMP1F in hMSC cells last longer than that in MC3T3 or MDPC-23 cells. The activation of MAPK lasted longer by rDMP1F than by rDMP1C. MAPK inhibitor could decrease the phosphorylation of MAPK-ERK by rDMP1C and rDMP1F and inhibit the ERK translocation from the cytoplasm into the nucleus. Knowdown of Ras gene expression by siRNA could significantly decrease the phosphoralated of ERK. Conclusion Both rDMP1C and rDMP1F can active the MEK-ERK, however, rDMP1F has a stronger effect in trigger MAPK signalling pathway than rDMP1C.
DMP1
Cite
Citations (0)
Objective: To investigate whether cancer-associated- fibroblasts (CAF), the key component of tumor microenvironment, regulate the chemoresistant capacity of lung cancer cell line A549 through SDF-1 secretion. Methods: Primary cell isolation techniques was used to isolate cancer-associated-fibroblasts from lung cancer patients. MTT assay was applied to determine the proliferation and chemoresistance of A549 cells. Quantative PCR was used to detect the mRNA changes of Bcl-xL. Western blotting was used to detect the protein expression of Bcl-xL. ELISA was applied to detect the SDF-1 secretion from normal fibroblasts (NF) and CAF. Results: CAF promoted the proliferation of A549 cells, while NF had no significant effect on them. After 72 hrs incubation, the absorbance value of A549+ CAF medium group was 0.814±0.006, significantly different from the 0.753±0.006 of the A549+ NF medium group (P<0.05). The Q-PCR assay indicated that mRNA expressions of Bcl-xL in the A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.11, 1.10±0.09 and 3.50±0.30, respectively, showing a significant difference between the A549+ NF medium group and A549+ CAF medium group (P<0.05). The Western blot showed that protein expressions of Bcl-xL in the A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.08, 1.10±0.12 and 3.10±0.25, respectively, with a significant difference between the A549+ NF medium group and A549+ CAF medium group (P<0.05). The ELISA results showed that the SDF-1 concentrations in the A549+ NF medium group and A549+ CAF medium group were 3.23±0.02 and 9.53±0.10, respectively, significantly different from each other (P<0.05). The MTT assay indicated that the absorbance values of OD of A549 group, A549+ AMD3100 group, A549+ NF medium group, A549+ NF medium+ AMD3100 group, A549+ CAF medium and A549+ CA Fmedium+ AMD3100 group were 0.43±0.03, 0.25±0.02, 0.48±0.03, 0.31±0.03, 0.72±0.06 and 0.45±0.03, respectively. The data of A549+ NF medium group was significantly different from that of A549+ CAF medium group (P<0.05). Conclusions: Cancer-associated-fibroblasts enhance the drug resistance of A549 cells through SDF-1 secretion, upregulating the expression level of Bcl-xL through interaction with CXCR4. Our study not only illustrates that tumor microenvironment is able to enhance drug resistance of tumor, but also provides experimental evidence for the cancer-associated-fibroblasts as a potential therapeutic target for the treatment of lung cancer.目的： 探讨肿瘤相关成纤维细胞(CAF)是否通过分泌基质细胞衍生因子1(SDF-1)调节肺癌A549细胞的化疗耐药性。 方法： 采用原代细胞分离方法获得肺癌患者的CAF，采用四甲基偶氮唑蓝(MTT)法检测A549细胞的增殖能力和对化疗药物的耐药性，荧光定量PCR检测Bcl-xL mRNA水平，Western blot检测Bcl-xL蛋白水平，酶联免疫吸附试验(ELISA)检测CAF分泌SDF-1的情况。 结果： CAF可以促进肺癌A549细胞的增殖，而正常纤维细胞对A549细胞增殖的影响不明显。培养72 h后，A549＋CAF上清组和A549＋NF上清组的吸光度(A)值分别为0.814±0.006和0.753±0.006，差异有统计学意义(P<0.05)。荧光定量PCR检测结果显示，A549组、A549＋NF上清组和A549＋CAF上清组的Bcl-xL mRNA表达水平分别为1.00±0.11、1.10±0.09和3.50±0.30，A549＋CAF上清组与A549＋NF上清组比较，差异有统计学意义(P<0.05)。Western blot检测显示，A549组、A549＋NF上清组和A549＋CAF上清组的Bcl-xL蛋白表达水平分别为1.00±0.08、1.10±0.12和3.10±0.25，A549＋CAF上清组与A549＋NF上清组比较，差异有统计学意义(P<0.05)。ELISA检测显示，A549＋NF上清组和A549＋CAF上清组中SDF-1含量分别为3.23±0.02和9.53±0.10，差异有统计学意义(P<0.05)。MTT检测显示，A549组、A549＋AMD3100组、A549＋NF上清组、A549＋NF上清＋AMD3100组、A549＋CAF上清组和A549＋CAF上清＋AMD3100组的A值分别为0.43±0.03、0.25±0.02、0.48±0.03、0.31±0.03、0.72±0.06和0.45±0.03，A549＋CAF上清和A549＋NF上清组比较，差异有统计学意义(P<0.05)。 结论： CAF通过分泌SDF-1激活肺癌A549细胞中的趋化因子受体4(CXCR4)，提高Bcl-xL的表达，增强肺癌A549细胞对顺铂的化疗耐药性，这为CAF作为肺癌潜在的治疗靶点提供了实验依据。.
Cite
Citations (5)
Signalling
Signalling pathways
Cite
Citations (0)
Objective To investigate different gene expressions of EGFR and HER-3 in human lung adenocarcinoma cells A549 and pemetrexed disodium(PEM)-resistant cells A549/PEM.Methods A549/PEM cell line was established with large dose of PEM.Growth inhibitions of A549 and A549/PEM cells were determined by CCK-8 assay.Cell cycle analysis was performed by flow cytometry(FCM).RT-PCR was used to analyse mRNA expressions of EGFR and HER-3 in A549 and A549/PEM cells.Results CCK-8 assay showed that the IC50 of PEM for A549 cells was 3.43×10-6mol/L.PEM-resistant cell line A549/PEM was established after being exposed to PEM repeatedly for 4 months,and the resistance index(RI) was 5.15.Flow cytometry analysis showed that proportions in G1/G0 phase of A549 and A549/PEM cells were(56.25±0.10)% and(65.12±0.40)%(P0.05).The proportions in S phase were(40.87±0.30) % and(31.74±0.40)%(P0.05).The mRNA expressions of EGFR in A549 and A549/PEM were 0.55±0.08 and 0.64±0.07,and mRNA expressions of HER-3 in A549 and A549/PEM were 1.29±0.03 and 1.56±0.12.The differences were statistically significant(P0.05).Conclusion Gene expressions of EGFR and HER-3 increase in A549/PEM cells,compared with that in A549 cells.It suggestes that changes of the two genes expressions appear to be associated with resistance to PEM.
Cite
Citations (0)