Effect of forsythigenin on the malignant progression of lung cancer cells by regulating the cAMP/EPAC1/RAP1 signal pathway

doi:10.11958/20250063

Abstract

Abstract:

Objective To investigate the effect of forsythigenin on the malignant progression of lung cancer cells by regulating the cyclic adenosine monophosphate/exchange protein directly activated by cAMP1/Ras-associated protein 1 (cAMP/EPAC1/RAP1) signaling pathway. Methods Lung cancer cell line A549 was cultured in vitro and grouped into the control group, the low dose forsythigenin group (25 mg/L), the medium dose forsythigenin group (50 mg/L), the high dose forsythigenin group (100 mg/L), the high dose forsythigenin+specific increase in intracellular cAMP content (pertussis toxin PTX) group (100 mg/L forsythigenin+5 μmol/L PTX) and high dose forsythigenin+EPAC1 antagonist (ESI-09) group (100 mg/L forsythigenin+1.5 μmol/L ESI-09). CCK-8 experiment was applied to detect cell proliferation. Scratch test was applied to detect cell migration. Flow cytometry was applied to detect cell apoptosis. Transwell was applied to detect cell invasion. ELISA method was applied to detect cAMP level in cell supernatant. Western blot assay was applied to detect expression levels of cAMP/EPAC1/RAP1 signaling pathway proteins and apoptotic proteins [B lymphoblastoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax)]. Results Compared with the control group, the OD450 value of A549 cells, number of cell invasions, scratch healing rate, level of cAMP, expression levels of Bcl-2, EPAC1 and RAP1 proteins were significantly reduced in the low dose, medium dose and high dose forsythigenin groups, and the expression of Bax protein and the rate of cell apoptosis were significantly increased in a dose-dependent manner (P<0.05). Compared with the high-dose forsythigenin group, the OD450 value of A549 cells, scratch healing rate, number of cell invasions, level of cAMP, expression levels of Bcl-2, EPAC1 and RAP1 proteins were obviously increased in the high-dose forsythigenin+PTX group, the expression of Bax protein and the apoptosis rate were obviously reduced (P<0.05). Levels of all indexes in the high dose forsythigenin+ESI-09 group were opposite. Conclusion Forsythigenin inhibits proliferation, migration, and invasion of A549 cells and promotes apoptosis by down-regulating the cAMP/EPAC1/RAP1 signaling pathway.

Key words: lung neoplasms, forsythiaside, cell proliferation, apoptosis, cAMP/EPAC1/RAP1 signaling pathway

CLC Number:

R734.2

Figures/Tables 6

References 22

[1]	YIN X, LIAO H, YUN H, et al. Artificial intelligence-based prediction of clinical outcome in immunotherapy and targeted therapy of lung cancer[J]. Semin Cancer Biol, 2022, 86(Pt 2):146-159. doi:10.1016/j.semcancer.2022.08.002.
[2]	LI R J, QIN C, HUANG G R, et al. Phillygenin inhibits helicobacter pylori by preventing biofilm formation and inducing ATP leakage[J]. Front Microbiol, 2022,13:863624. doi:10.3389/fmicb.2022.863624.
[3]	LI H, CHEN M, YANG Z, et al. Phillygenin,a MELK inhibitor,inhibits cell survival and epithelial-mesenchymal transition in pancreatic cancer cells[J]. Onco Targets Ther, 2020, 13:2833-2842. doi:10.2147/OTT.S238958.
[4]	吴林斌, 吴元肇, 李晓丹, 等. 连翘苷经PI3K/Akt信号通路干预肾细胞癌的机制研究[J]. 中草药, 2019, 50(10):2377-2382.
	WU L B, WU Y Z, LI X D, et al. Effect of forsythin in inhibiting renal cell carcinoma via PI3K/Akt signaling pathway[J]. Chinese Traditional and Herbal Drugs, 2019, 50(10):2377-2382. doi:10.7501/j.issn.0253-2670.2019.10.018.
[5]	吴华英, 邓凯, 李静, 等. 益气活血方调控cAMP/Epac1/Rap1信号改善冠心病气虚血瘀证大鼠的验证[J]. 中国实验方剂学杂志, 2024, 30(18):107-116.
	WU H Y, DENG K, LI J, et al. Effect of Yiqi Huoxue prescription on cAMP/Epac1/Rap1 signaling pathway in coronary heart disease rats with Qi deficiency and blood stasis syndrome[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2024, 30(18):107-116. doi:10.13422/j.cnki.syfjx.20231819.
[6]	MOON E Y, LEE G H, LEE M S, et al. Phosphodiesterase inhibitors control A172 human glioblastoma cell death through cAMP-mediated activation of protein kinase A and Epac1/Rap1 pathways[J]. Life Sci, 2012, 90(9-10):373-380. doi:10.1016/j.lfs.2011.12.010.
[7]	邓英, 赵兴桃, 周梦婷, 等. 连翘脂素通过调控P2X7R/NF-KB/NLRP3炎性小体信号通路缓解LPS/ATP诱导的L02细胞炎症[J]. 中国实验方剂学杂志, 2022, 28(14):61-69.
	DENG Y, ZHAO X T, ZHOU M T, et al. Phillygenin mitigates lps/atp-induced l02 cell inflammation by regulating P2X7R/NF-KB/NLRP3 inflammasome signaling pathway[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2022, 28(14):61-69. doi:10.13422/j.cnki.syfjx.20220808.
[8]	杨婷婷, 王慧, 石鹏, 等. cAMP/Epac/Rap1信号通路调控NG2细胞分泌IL-1β、TNF-α、BDNF及酸枣仁皂苷A的作用研究[J]. 安徽医科大学学报, 2023, 58(2):265-270.
	YANG T T, WANG H, SHI P, et al. The effects of cAMP/Epac/Rap1 signaling pathway on the secretion of IL-1β,TNF-α,BDNF and Jujube saponin A in NG2 cells[J]. Acta Universitatis Medicinalis Anhui, 2023, 58(2):265-270. doi:10.19405/j.cnki.issn1000-1492.2023.02.016.
[9]	THAI A A, SOLOMON B J, SEQUIST L V, et al. Lung cancer[J]. Lancet, 2021, 398(10299):535-554. doi:10.1016/S0140-6736(21)00312-3.
[10]	QI C, SUN S W, XIONG X Z. From COPD to lung cancer:Mechanisms linking,diagnosis,treatment,and prognosis[J]. Int J Chron Obstruct Pulmon Dis, 2022, 17:2603-2621. doi:10.2147/COPD.S380732.
[11]	ACQUAVIVA R, MALFA G A, LOIZZO M R, et al. Advances on natural Abietane,Labdane and Clerodane diterpenes as anti-cancer agents:Sources and mechanisms of action[J]. Molecules, 2022, 27(15):4791. doi:10.3390/molecules27154791.
[12]	郝建玲, 信婧婧, 王靖, 等. 连翘苷调节NLRP3炎性通路对急性胸膜炎大鼠肺损伤的影响[J]. 天津医药, 2024, 52(2):161-166.
	HAO J L, XIN J J, WANG J, et al. Effect of phillyrin regulating NLRP3 inflammatory pathway on exudates and lung injury in rats with acute pleurisy[J]. Tianjin Med J, 2024, 52(2):161-166. doi:10.11958/20230399.
[13]	WU S, ZHANG Y, ZHANG Y, et al. Phillygenin regulates proliferation and apoptosis of non-small cell lung cancer through by AMPK/ERK/NF-κB axis[J]. Pharmazie, 2020, 75(10):512-515. doi:10.1691/ph.2020.0558.
[14]	DING X, LU D, FAN J. A natural product phillygenin suppresses osteosarcoma growth and metastasis by regulating the SHP-1/JAK2/STAT3 signaling[J]. Biosci Biotechnol Biochem, 2021, 85(2):307-314. doi:10.1093/bbb/zbaa007.
[15]	CHU T, NING Y, MA M, et al. Phillygenin regulates the colorectal cancer tumor microenvironment by inhibiting hypoxia-inducible factor 1 alpha[J]. Cytotechnology, 2025, 77(1):17. doi:10.1007/s10616-024-00679-2.
[16]	申红梅, 王丽霞, 冯绪强, 等. 连翘脂素调节HIF-1α/VEGF信号通路对宫颈癌细胞恶性生物学行为的影响[J]. 中国优生与遗传杂志, 2024, 32(9):1820-1825.
	SHEN H M, WANG L X, FENG X Q, et al. Effect of phillygenin on the malignant biological behavior of cervical cancer cells by regulating the HIF-1α/VEGF signaling pathway[J]. Chinese Journal of Birth Health & Heredity, 2024, 32(9):1820-1825. doi:10.13404/j.cnki.cjbhh.2024.09.005.
[17]	王亮, 李强, 刘晶, 等. 连翘脂素调节JAK2/STAT3信号通路对前列腺癌增殖、凋亡和上皮间质转化的影响[J]. 中国性科学, 2024, 33(3):40-44.
	WANG L, LI Q, LIU J, et al. Impacts of phillygenin on proliferation, apoptosis, and epithelial mesenchymal transformation in prostate cancer by regulating Janus kinase 2/signal transduction and transcription activating factor 3 signaling pathway[J]. Chinese Journal of Human Sexuality, 2024, 33(3):40-44. doi:10.3969/j.issn.1672-1993.2024.03.010.
[18]	ZACCOLO M, ZERIO A, LOBO M J. Subcellular organization of the cAMP signaling pathway[J]. Pharmacol Rev, 2021, 73(1):278-309. doi:10.1124/pharmrev.120.000086.
[19]	HERNÁNDEZ C, GÓMEZ-PERALTA F, SIMÓ-SERVAT O, et al. Usefulness of circulating EPAC1 as biomarkers of therapeutic response to GLP-1 receptor agonists[J]. Acta Diabetol, 2022, 59(11):1437-1442. doi:10.1007/s00592-022-01928-6.
[20]	SARI-AK D, TORRES-GOMEZ A, YAZICIOGLU Y F, et al. Structural,biochemical,and functional properties of the Rap1-interacting adaptor molecule(RIAM)[J]. Biomed J, 2022, 45(2):289-298. doi:10.1016/j.bj.2021.09.005.
[21]	沈利聪, 板得莹, 张瑜. RAP1A和EPAC1在卵巢子宫内膜异位症组织中的表达及意义[J]. 中国现代医学杂志, 2020, 30(21):12-17.
	SHEN L C, BAN D Y, ZHANG Y. Expression and significance of RAP1A and EPAC1 in ovarian endometriosis[J]. China Journal of Modern Medicine, 2020, 30(21):12-17. doi:10.3969/j.issn.1005-8982.2020.21.003.
[22]	KUMAR N, GUPTA S, DABRAL S, et al. Role of exchange protein directly activated by cAMP(EPAC1)in breast cancer cell migration and apoptosis[J]. Mol Cell Biochem, 2017, 430(1/2):115-125. doi:10.1007/s11010-017-2959-3.

组别	EPAC1	RAP1	Bax	Bcl-2
对照组	1.13±0.09	1.15±0.10	0.74±0.08	1.11±0.11
连翘脂素低剂量组	0.95±0.09^a	0.93±0.08^a	0.99±0.09^a	0.92±0.08^a
连翘脂素中剂量组	0.72±0.08^ab	0.69±0.07^ab	1.26±0.10^ab	0.78±0.07^ab
连翘脂素高剂量组	0.42±0.05^abc	0.35±0.04^abc	1.57±0.18^abc	0.37±0.04^abc
连翘脂素高剂量+ PTX组	0.69±0.07^d	0.72±0.06^d	1.08±0.12^d	0.66±0.03^d
连翘脂素高剂量+ ESI-09组	0.28±0.03^d	0.21±0.02^d	1.98±0.09^d	0.23±0.05^d
F	96.544^＊＊	137.119^＊＊	74.781^＊＊	116.511^＊＊

组别	EPAC1	RAP1	Bax	Bcl-2
对照组	1.13±0.09	1.15±0.10	0.74±0.08	1.11±0.11
连翘脂素低剂量组	0.95±0.09^a	0.93±0.08^a	0.99±0.09^a	0.92±0.08^a
连翘脂素中剂量组	0.72±0.08^ab	0.69±0.07^ab	1.26±0.10^ab	0.78±0.07^ab
连翘脂素高剂量组	0.42±0.05^abc	0.35±0.04^abc	1.57±0.18^abc	0.37±0.04^abc
连翘脂素高剂量+ PTX组	0.69±0.07^d	0.72±0.06^d	1.08±0.12^d	0.66±0.03^d
连翘脂素高剂量+ ESI-09组	0.28±0.03^d	0.21±0.02^d	1.98±0.09^d	0.23±0.05^d
F	96.544^＊＊	137.119^＊＊	74.781^＊＊	116.511^＊＊