海绵来源的smenospongine通过抑制非小细胞肺癌细胞中的EGFR-Akt-ABCG2信号通路抑制顺铂耐药

doi:10.3969/j.issn.1674-8115.2022.08.004

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (8): 997-1007.doi: 10.3969/j.issn.1674-8115.2022.08.004

海绵来源的smenospongine通过抑制非小细胞肺癌细胞中的EGFR-Akt-ABCG2信号通路抑制顺铂耐药

廖雅慧¹^,²^,³(), 刘丽云², 朱泓睿², 林厚文², 严继舟¹(), 孙凡²()

^1.上海海洋大学海洋生物与组织再生实验室，上海 201306
^2.上海交通大学医学院附属仁济医院海洋药物实验室，上海 200127
^3.上海交通大学医学院附属第九人民医院黄浦分院药剂科，上海 200011

收稿日期:2022-03-11 接受日期:2022-05-13 出版日期:2022-08-28 发布日期:2022-10-08
通讯作者: 严继舟,孙凡 E-mail:15800578349@163.com;jyan2@shou.edu.cn;sunfan2017@163.com
作者简介:廖雅慧（1994—），女，硕士生；电子信箱：15800578349@163.com。
基金资助:
国家重点研发计划(2018YFC0310900);国家自然科学基金(82073713);上海市高水平地方高校创新团队(SSMU-ZLCX20180702)

Marine sponge-derived smenospongine overcomes resistance of cisplatin via inhibiting EGFR-Akt-ABCG2 pathway in NSCLC cells

LIAO Yahui¹^,²^,³(), LIU Liyun², ZHU Hongrui², LIN Houwen², YAN Jizhou¹(), SUN Fan²()

^1.Institute for Marine Biosystem and Neurosciences, Shanghai Ocean University, Shanghai 201306, China
^2.Research Center for Marine Drugs, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^3.Department of Pharmacy, Shanghai Ninth People's Hospital Huangpu Branch, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

Received:2022-03-11 Accepted:2022-05-13 Online:2022-08-28 Published:2022-10-08
Contact: YAN Jizhou,SUN Fan E-mail:15800578349@163.com;jyan2@shou.edu.cn;sunfan2017@163.com
Supported by:
National Key Research and Development Program of China(2018YFC0310900);National Natural Science Foundation of China(82073713);Innovative Research Team of High-level Local Universities in Shanghai(SSMU-ZLCX20180702)

摘要/Abstract

摘要：

目的·研究海绵来源的倍半萜胺醌类化合物smenospongine（SME）对非小细胞肺癌（non-small cell lung cancer，NSCLC）细胞A549及其顺铂耐药株A549/DDP的抗肿瘤活性及作用机制。方法·利用2种细胞（母本细胞A549和顺铂耐药细胞A549/DDP）作为研究模型，采用CCK-8实验检测2株细胞对一线化学治疗药物的半抑制浓度（half maximum inhibitory concentration，IC₅₀）以验证A549/DDP多药耐药的情况；通过克隆形成实验检测SME对母本及耐药细胞增殖的影响，同时采用Transwell侵袭实验以及蛋白质印迹法（Western blotting）检测SME对A549/DDP上皮间质转化（epithelial to mesenchymal transition，EMT）的影响；应用Western blotting和实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测2株细胞中受到SME调节的多药耐药基因的蛋白和mRNA表达水平，探讨其耐药分子机制；进一步采用Western blotting检测SME对多药耐药蛋白ATP结合盒蛋白G超家族成员2（ATP-binding cassette superfamily G member 2，ABCG2）上游蛋白的影响；通过流式细胞技术检测SME对母本细胞和顺铂耐药株细胞周期的影响，同时应用TUNEL（TdT-mediated dUTP Nick-end labeling）染色以及Western blotting检测SME对2株细胞凋亡的影响。结果·与母本细胞相比，顺铂耐药株A549/DDP细胞对顺铂表现出显著耐药性，同时对多种临床一线肺癌化学治疗药物表现出多药耐药的特征；SME显著抑制A549和A549/DDP的增殖、克隆形成，同时抑制耐药株的EMT；通过筛选多药耐药蛋白发现，SME显著下调ABCG2的蛋白以及mRNA表达；SME可抑制ABCG2上游的表皮生长因子受体（epidermal growth factor receptor，EGFR）/丝氨酸苏氨酸蛋白激酶（serine/threonine kinase，Akt）信号通路，进而下调ABCG2，并上调FoxO1；SME诱导母本细胞和耐药株细胞周期阻滞在G0/G1期，同时诱导2株细胞凋亡。结论·SME作为抑制NSCLC耐药的新小分子化合物通过抑制EGFR-Akt信号通路，下调ABCG2蛋白、上调FoxO1蛋白，进而抑制A549和A549/DDP的细胞活力，抑制耐药株的EMT，最终促进细胞凋亡。

关键词: smenospongine（SME）, 非小细胞肺癌, 顺铂耐药, ATP结合盒蛋白G超家族成员2, 丝氨酸苏氨酸蛋白激酶

Abstract:

Objective ·To explore the antitumor activities and mechanisms of the sesquiterpene amine quinone compound smenospongine (SME) in non-small cell lung cancer (NSCLC) cell line A549 and cisplatin-resistant cell line A549/DDP. Methods ·Parental cell A549 and its cisplatin-resistant cell A549/DDP were used as research models. CCK-8 assay was used to detect the half maximum inhibitory concentration (IC₅₀) of the two cell lines treated with first-line chemotherapeutic drugs, in order to verify the multidrug resistance of A549/DDP. Colony formation assay was used to detect the effect of SME on the proliferation of parental and drug-resistant cells. Transwell invasion assay and Western blotting were used to detect the effect of SME on epithelial to mesenchymal transformation (EMT) of A549/DDP; Western blotting and quantitative real-time PCR (qRT-PCR) were used to detect the protein and mRNA expression levels of multidrug resistance genes regulated by SME in two cell lines and the molecular mechanism of drug resistance. Furthermore, Western blotting was used to detect the effect of SME on the upstream protein of multidrug resistance protein, ATP-binding cassette superfamily G member 2 (ABCG2), and flow cytometry was used to detect the effect of SME on the cell cycle of parental and cisplatin-resistant cells. TdT-mediated dUTP Nick-end labeling (TUNEL) and Western blotting were used to detect the effect of SME on apoptosis of the two cell lines. Results ·Compared with parental cells, cisplatin-resistant A549/DDP cells showed significant resistance to cisplatin and showed multidrug resistance to the first-line chemotherapy drugs used on lung cancer. SME markedly inhibited the proliferation and clone formation of A549 and A549/DDP as well as the EMT of drug-resistant cell. SME notably down-regulated the expression of ABCG2 protein and mRNA, and inhibited the epidermal growth factor receptor (EGFR)-serine/threonine kinase (Akt) signal pathway upstream of ABCG2, which thereby down-regulated ABCG2, and positively regulated FoxO1. SME induced G0/G1 arrest and induced apoptosis of both cells. Conclusion ·As a new small molecular compound in overcoming the drug resistance of NSCLC, SME inhibits the cell viability of A549 and A549/DDP by restraining EGFR-Akt signal pathway which thereby down-regulates ABCG2, positively regulates FoxO1 and inhibits EMT of A549/DDP, which finally leads to apoptosis.

Key words: smenospongine (SME), non-small cell lung cancer (NSCLC), cisplatin resistance, ATP-binding cassette superfamily G member 2 (ABCG2), serine/threonine kinase (Akt)

中图分类号:

廖雅慧, 刘丽云, 朱泓睿, 林厚文, 严继舟, 孙凡. 海绵来源的smenospongine通过抑制非小细胞肺癌细胞中的EGFR-Akt-ABCG2信号通路抑制顺铂耐药[J]. 上海交通大学学报（医学版）, 2022, 42(8): 997-1007.

LIAO Yahui, LIU Liyun, ZHU Hongrui, LIN Houwen, YAN Jizhou, SUN Fan. Marine sponge-derived smenospongine overcomes resistance of cisplatin via inhibiting EGFR-Akt-ABCG2 pathway in NSCLC cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(8): 997-1007.

图/表 7

图1 顺铂耐药株的鉴定Note： A549 and A549/DDP were treated with various concentrations of cisplatin for 48 h. The cell viability was determined by CCK-8 assay.

Fig 1 Authentication of cisplatin-resistant cells

表1 顺铂耐药株的MDR特性

Tab 1 MDR features of cisplatin-resistant cells

Chemical	IC₅₀/（μmol×L^-1）		RI
Chemical	A549	A549/DDP	RI
DDP	3.730±0.080	155.100±6.290	41.550
GEM	0.570±0.070	2.880±0.100	5.050
NVB	0.006±0.001	5.370±0.050	895.000
Pemetrexed	48.040±6.340	304.670±0.754	6.342
5-FU	31.630±0.930	33.710±0.100	1.070

图2 SME抑制顺铂耐药细胞增殖和转移Note：A. A549 and A549/DDP were incubated with SME for 48 h. Cell viability was determined by CCK-8 assay. B. A549 and A549/DDP were exposed to 5 μmol/L and 20 μmol/L for 48 h and subjected to colony formation assay. Colony formation assay and the quantified number of colonies of A549 and A549/DDP cells after the indicated treatment. ①P=0.000, compared with the control group; ②P=0.020, compared to A549 treated with 20 μmol/L SME group. C. Migration capacities of A549/DDP cells were determined by using transwell assay. Scale bar—100 μm. ③P=0.000, compared with the control (0 μmol/L) group. D. The protein expression of EMT-related markers of A549 and A549/DDP were examined by Western blotting. GAPDH was used as an internal standard. E. A549/DDP cells were incubated with SME for 24 h and the protein expression of EMT-related markers were detected by Western blotting. Bar graph shows the relative protein expressions of the indicated proteins. ④P=0.003, ⑤P=0.009, ⑥P=0.001, ⑦P=0.000, ⑧P=0.020, compared with the control group.

Fig 2 SME inhibits cell proliferation and EMT in cisplatin-resistant cells

图3 SME抑制顺铂耐药细胞 ABCG2 基因表达Note：A. A549/DDP was treated with SME for 24 h and the analysis of ABCC11 and ABCB1 was presented by Western blotting, with GAPDH as loading control. ①P=0.000, ②P=0.030, ③P=0.010, compared with the control group. B. The expression of ABCG2 in A549 and A549/DDP was presented by Western blotting after exposure to SME for 24 h. Bar graph shows the relative protein expressions of the indicated proteins, and GAPDH is shown as loading control. ④P=0.000, compared with the control group. C. The mRNA expression of ABCG2 was detected after A549 and A549/DDP were exposed to SME for 24 h. ⑤P=0.000, compared with the control group.

Fig 3 SME inhibits the mRNA expression of ABCG2 in cisplatin-resistant cells

图4 SME抑制顺铂耐药细胞EGFR-Akt信号通路活性Note：A. A549 and A549/DDP were exposed to various concentrations of SME for 24 h and the expression of Akt, p-Akt and FoxO1 was analyzed by Western blotting, with GAPDH as loading control. ①P=0.000, ②P=0.002, ③P=0.007, ④P=0.020, compared with the control group. B. A549 and A549/DDP were treated with deguelin and SME for 24 h and the results of the indicated protein level are shown, with GAPDH as loading control. ⑤P=0.001 ⑥P=0.000, ⑦P=0.010, ⑧P=0.006, compared with the control group without any treatment (Ctrl group). C. The expression of ABCG2 was detected by Western blotting when exposed to deguelin and SME for 24 h. The bar graphs represent the relative protein levels. ⑨P=0.000, compared with the control group. D. The EGFR and p-EGFR protein levels of both cells were detected by western blotting. GAPDH was used as loading control. The bar graphs represent the relative protein levels. ⑩P=0.001, compared with treatment of EGF alone in A549 and A549/DDP groups; ?P=0.000, compared with the control group.

Fig 4 SME inhibits the activity of EGFR-Akt pathway in cisplatin-resistant cells

图5 SME诱导顺铂耐药细胞G0/G1期阻滞和细胞凋亡Note：A/B. A549 (A) and A549/DDP (B) were both treated with SME for 24 h followed by flow cytometric assay. The histograms described the percentage of the cell cycle distributions. ①P=0.000, ②P=0.040, ③P=0.050, compared with the control group. C. TUNEL assay was used to evaluate the apoptotic status of cells. Scale bar, 500 μm. D. The bar graphs show the apoptotic rates of TUNEL assay. ④P=0.000, compared with the control group. E. The expressions of Bcl-xL and Bax were detected by Western blotting after exposure to SME for 48 h, with GAPDH as a loading control. The bar graph represents the ratio of Bax/Bcl2 at protein level. ⑤P=0.002, ⑥P=0.000, ⑦P=0.050,⑧P=0.001, compared with the control group.

Fig 5 SME induces G0/G1 arrest and apoptosis in cisplatin-resistant cells

图6 SME抑制EGFR-Akt-ABCG2信号通路模式图Note：SME inhibited the drug-resistance of A549/DDP by restraining the EGFR/Akt pathway, thereby suppressing the transcription of ABCG2. Meanwhile, SME also up-regulated FoxO1, another downstream protein of Akt, hence inducing apoptosis.

Fig 6 A schematic diagram depicting a model of the inhibition of SME on the EGFR-Akt-ABCG2 pathway

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海绵来源的smenospongine通过抑制非小细胞肺癌细胞中的EGFR-Akt-ABCG2信号通路抑制顺铂耐药

Marine sponge-derived smenospongine overcomes resistance of cisplatin via inhibiting EGFR-Akt-ABCG2 pathway in NSCLC cells

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