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      抑制Src酪氨酸激酶活性对人肺癌A549/DDP细胞耐药性及MDR1和LRP表达的影响 Translated title: Effect of Src Tyrosine Kinase Inhibition on the Drug-resistance as well as MDR1 and LRP Expression of the Human Cis-platinum-resistant Lung Cancer Cell Line A549/DDP

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          Abstract

          背景与目的

          本研究旨在探讨抑制Src酪氨酸激酶活性对人肺癌A549/DDP细胞耐药性及多药耐药蛋白(muti-drug resistance 1, MDR1)和肺耐药相关蛋白(lung resistance-related protein, LRP)表达的影响。

          方法

          以Src酪氨酸激酶抑制剂作用于A549/DDP细胞,应用Western blot法检测肿瘤细胞Src酪氨酸激活性的变化,CellTiter-Glo发光法检测肿瘤细胞药物敏感性的变化,流式细胞仪检测肿瘤细胞Rh-123含量变化,Western blot法和RT-PCR检测肿瘤细胞MDR1和LRP表达变化。

          结果

          Src酪氨酸激酶抑制剂可下调A549/DDP细胞中Src酪氨酸激活性,2.5 μM和10 μM Src酪氨酸激酶抑制剂作用肿瘤细胞后,肿瘤细胞药物敏感性提高,逆转倍数(reversal fold, RF)分别为1.59倍和2.10倍,肿瘤细胞中Rh-123含量分别提高了1.21倍和1.59倍,MDR1 mRNA表达分别是对照组的53.8%和27.5%,LRP mRNA表达分别是对照组的59.3%和21.4%,MDR1和LRP蛋白表达水平明显下降。

          结论

          抑制A549/DDP细胞中Src酪氨酸激酶活性可逆转肿瘤细胞多药耐药性,提高肿瘤细胞药物敏感性,其机制可能与降低细胞MDR1和LRP表达有关。

          Translated abstract

          Background and objective

          The aim of this study is to investigate the effect of Src tyrosine kinase inhibition on the drug-resistance as well as the expression of multidrug resistance 1 (MDR1) and lung resistance-related protein (LRP) of the human cis-platinum-resistant lung cancer cell line A549/DDP.

          Methods

          4-Anilinoquirazoline was used to inhibit Src tyrosine kinase activity in A549/DDP. Western blot analysis was used to detect the Src tyrosine kinase activity. CellTiter-Glo assay was used to detect the drug sensitivity of tumor cells. Flow cytometry was used to detect the intracellular Rh-123 content. Western blot and real-time PCR assay were used to detect the expression of tumor MDR1 and LRP.

          Results

          4-Anilinoquirazoline can down-regulate the cellular Src tyrosine kinase activity in A549/DDP. After treatment with 2.5 μM and 10 μM of 4-anilinoquirazoline, the cells became more sensitive to the drug and the reversal folds (RFs) of tumor cell sensitivity to the drug were 1.59- and 2.10-fold, respectively. The intracellular content of Rh-123 improved by 1.21- and 1.59-fold, respectively. The mRNA levels of MDR1 were 53.8% and 27.5% of the control, respectively. The mRNA level of LRP was 59.3% and 21.4% of the control, respectively. The expression of MDR1 and LRP protein significantly decreased.

          Conclusion

          The inhibition of Src tyrosine kinase activity in A549/DDP cells can reverse multi-drug resistance and increase the sensitivity of the cells to the drug. The mechanism may be related to the down-regulation of cellular MDR1 and LRP.

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          Most cited references15

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          Role of intratumoural heterogeneity in cancer drug resistance: molecular and clinical perspectives

          Drug resistance continues to be a major barrier to the delivery of curative therapies in cancer. Historically, drug resistance has been associated with over-expression of drug transporters, changes in drug kinetics or amplification of drug targets. However, the emergence of resistance in patients treated with new-targeted therapies has provided new insight into the complexities underlying cancer drug resistance. Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance. Single cell sequencing studies that identified multiple genetically distinct variants within human tumours clearly demonstrate the heterogeneous nature of human tumours. The major contributors to intratumoural heterogeneity are (i) genetic variation, (ii) stochastic processes, (iii) the microenvironment and (iv) cell and tissue plasticity. Each of these factors impacts on drug sensitivity. To deliver curative therapies to patients, modification of current therapeutic strategies to include methods that estimate intratumoural heterogeneity and plasticity will be essential.
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            Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling.

            Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src-LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity.
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              Blocking the PI3K/AKT and MEK/ERK signaling pathways can overcome gefitinib-resistance in non-small cell lung cancer cell lines.

              To investigate the effects of gefitinib (EGFR-TKI), LY294002 (PI3K inhibitor) and U0126 (MEK inhibitor) on proliferation and apoptosis in five non-small cell lung cancer (NSCLC) cell lines (PC9, PC9/AB2, H1975, H1299 and A549). The inhibitory rates of cells were tested by MTT and apoptosis was detected through flow cytometry when treated with gefitinib, LY294002 and U0126. The sensitivity to gefitinib was different in different cell lines, which was associated with EGFR mutation type. The cells with EGFR mutation were more sensitive than those with EGFR wild-type, except PC9/AB2 cells. LY294002 and U0126 can inhibit cell proliferation and promote apoptosis in all five cell lines. The sensitivity to gefitinib was restored partially in the resistant cell lines by combining gefitinib with LY294002 or U0126. The effects on cell proliferation and apoptosis were stronger in cells with EGFR mutation when PI3K/AKT pathway was blocked, however, for cells with EGFR wild-type, the effects were stronger when the MEK pathway was blocked. When the PI3K and MEK pathways were blocked together, proliferation inhibition and apoptosis level in NSCLC cells was similar to that in cells treated with EGFR TKI. There were some differences according to EGFR mutation type, suggesting that EGFR mutations may result in alterations of downstream signaling pathways. The sensitivity of gefitinib resistant cell lines can be restored partially when the two downstream signaling pathways are blocked. However, these cells were still drug resistant, suggesting that the activation of PI3K and MEK pathways is not the only mechanism of EGFR-resistance.
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                Author and article information

                Contributors
                Journal
                Zhongguo Fei Ai Za Zhi
                Zhongguo Fei Ai Za Zhi
                ZGFAZZ
                Chinese Journal of Lung Cancer
                中国肺癌杂志编辑部 (天津市和平区南京路228号300020 )
                1009-3419
                1999-6187
                20 September 2012
                : 15
                : 9
                : 501-506
                Affiliations
                [ ] 276826 日照,山东省日照市人民医院检验科 Department of Laboratory Medicine, Rizhao People's Hospital, Rizhao 276826, China
                Author notes
                律洁, Jie LV, E-mail: lujie_rz@ 123456yahoo.com.cn
                Article
                zgfazz-15-9-501 R734.2
                10.3779/j.issn.1009-3419.2012.09.01
                5999856
                22989452
                2b1881cf-7f67-4077-9182-d4621ade546c
                版权所有©《中国肺癌杂志》编辑部2012Copyright ©2012 Chinese Journal of Lung Cancer. All rights reserved.

                This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 3.0) License. See: https://creativecommons.org/licenses/by/3.0/

                History
                : 30 June 2012
                : 26 August 2012
                Categories
                基础研究
                Basic Research

                src酪氨酸激酶,a549/ddp,多药耐药,mdr1,lrp,src tyrosine kinase,multi-drug resistance

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