Role and molecular mechanism of the Wnt/β-catenin signaling pathway in the regulation of NSCLC A549 cell proliferation and apoptosis by oleanolic acid

doi:10.3760/cma.j.cn371439-20251128-00053

Abstract

Abstract:

Objective To investigate the role of the Wnt/β-catenin signaling pathway in regulating the proliferation and apoptosis of non-small cell lung cancer（NSCLC）A549 cells by oleanolic acid（OA）and its potential molecular mechanism. Methods A549 cells were cultured in vitro， and the effect of different concentrations（0， 0.625， 1.25， 2.5， 5， 10， 20 μmol/L）of OA on cell viability was detected by CCK-8 assay. The cells were divided into the negative control group， the HLY78 group（10 μmol/L HLY78）， the OA group（5 μmol/L OA）， the HLY78+OA group（10 μmol/L HLY78+5 μmol/L OA）， and the IWR-1 group（8 μmol/L IWR-1）. Western blotting was used to detect the expression levels of proteins related to the Wnt/β-catenin signaling pathway， cell cycle， and apoptosis. Real-time cell analysis was employed to detect cell proliferation dynamics. Flow cytometry was performed to analyze the cell cycle and apoptosis. Results OA inhibited the viability of NSCLC A549 cells in a generally significant dose- and time-dependent manner. The expression levels of Wnt5a/b protein in NSCLC A549 cells of the negative control group， the HLY78 group， the OA group， the HLY78+OA group， and the IWR-1 group were 1.03±0.06， 1.42±0.02， 0.98±0.07， 1.17±0.05， and 0.34±0.10， respectively， and the expression levels of β-catenin protein were 0.99±0.02， 1.22±0.08， 0.69±0.16， 0.85±0.12， and 0.39±0.19， respectively， with statistically significant differences（F=116.70， P<0.001； F=19.20， P<0.001）. Compared with the negative control group， the protein expressions of Wnt5a/b and β-catenin in the OA group were both significantly decreased（both P<0.05）， while the protein expressions of Wnt5a/b and β-catenin in the HLY78 group were significantly increased（both P<0.05）. Compared with the OA group， the protein expressions of Wnt5a/b and β-catenin in the HLY78+OA group were both significantly increased（both P<0.05）. The 72 h normalized cell indexes of the 5 groups were 7.20±0.04， 6.75±0.05， 4.74±0.03， 5.22±0.04， and 4.87±0.03， respectively， with a statistically significant difference（F=8 745.26， P<0.001）. Compared with the negative control group， the normalized cell indexes of the OA group and the IWR-1 group were both significantly decreased（both P<0.05）； compared with the OA group， the normalized cell index of the HLY78+OA group was significantly increased（P<0.05）. The proportions of Sub G₁ phase in the 5 groups were（5.45±0.21）%，（3.30±0.20）%，（9.69±1.91）%，（8.29±1.16）%， and（8.21±1.26）%， respectively； the proportions of G₀/G₁ phase were（55.46±0.37）%，（45.29±3.34）%，（45.07±0.42）%，（46.15±2.61）%， and（52.15±4.93）%， respectively； the proportions of S phase were（10.81±1.24）%，（18.53±0.92）%，（13.80±2.09）%，（15.17±1.53）%， and（11.73±2.76）%， respectively， and with statistically significant differences（F=15.16， P<0.001； F=7.86， P=0.004； F=8.36， P=0.003）. Compared with the negative control group， the OA group showed statistically significant differences. Additionally， compared with both the OA group and the negative control group， the HLY78+OA group also demonstrated statistically significant differences（all P<0.05）. The apoptosis rates of the 5 groups were（0.61±0.05）%，（0.90±0.24）%，（32.96±2.39）%，（13.02±3.91）%， and（30.65±0.94）%， respectively， with a statistically significant difference（F=166.60， P<0.001）. The apoptosis rate of A549 cells in the OA group was significantly increased compared with the negative control group（P<0.05）； the apoptosis rate in the HLY78+OA group was significantly decreased compared with the OA group（P<0.05）. There were statistically significant differences in the expression levels of cell cycle-related proteins（PCNA， Cyclin D1， Cyclin D3， CDK2， CDK6， P18）and apoptosis-related proteins（XIAP， Survivin， Bcl-2， Bax）among the 5 groups（F=20.23， P<0.001； F=17.19， P<0.001； F=63.77， P<0.001； F=24.62， P<0.001； F=127.25， P<0.001； F=11.89， P<0.001； F=18.10， P<0.001； F=24.95， P<0.001； F=41.82， P<0.001； F=23.96， P<0.001）. Compared with the negative control group， the protein expressions of PCNA， Cyclin D1， Cyclin D3， CDK2， CDK6， XIAP， Survivin， Bcl-2 in the OA group were all significantly lower， while the protein expressions of P18 and Bax were higher（all P<0.05）. Compared with the OA group， the protein expressions of PCNA， Cyclin D1， Cyclin D3， CDK2， CDK6， XIAP， Survivin， Bcl-2 in the HLY78+OA group were all higher， while the expressions of P18 and Bax were lower（all P<0.05）. Conclusions The Wnt/β-catenin signaling pathway critically mediates the anti-tumor effects of OA in NSCLC A549 cells. By targeting and inhibiting this pathway， OA downregulates cell cycle drivers and anti-apoptotic proteins while upregulating pro-apoptotic proteins， thereby effectively inhibiting proliferation， arresting the cell cycle， and promoting apoptosis.

Key words: Carcinoma, non-small-cell lung, Oleanolic acid, Cell proliferation, Apoptosis

Wang Junqi, Li Kairui, Yuan Rong, Lu Ying, Xu Zhaojun, Song Lan. Role and molecular mechanism of the Wnt/β-catenin signaling pathway in the regulation of NSCLC A549 cell proliferation and apoptosis by oleanolic acid[J]. Journal of International Oncology, 2026, 53(6): 321-330.

Figures/Tables 16

References 14

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浓度（μmol/L）	24 h	48 h
0（阴性对照）	100.00±0.00	100.00±0.00
0.625	88.63±11.39^a	82.80±3.87^ac
1.25	81.91±11.92^ab	78.92±4.47^abc
2.5	70.14±9.19^ab	63.68±1.65^abc
5	71.37±8.78^a	62.10±5.81^ab
10	13.60±3.12^ab	11.03±1.32^abc
20	6.05±0.16^ab	1.16±0.38^abc
F值	107.57	656.96
P值	<0.001	<0.001

组别	Wnt5a/b	β-catenin
0 μmol/L OA	0.99±0.02	0.99±0.02
1.25 μmol/L OA	0.78±0.03^a	0.80±0.07^a
5 μmol/L OA	0.67±0.03^ab	0.65±0.03^ab
0.004 g/L顺铂	0.68±0.03^ac	0.82±0.05^ac
F值	80.55	26.37
P值	<0.001	<0.001

组别	Wnt5a/b	β-catenin
阴性对照组	1.03±0.06	0.99±0.02
HLY78组	1.42±0.02^a	1.22±0.08^a
OA组	0.98±0.07^ab	0.69±0.16^ab
HLY78+OA组	1.17±0.05^bc	0.85±0.12^abc
IWR-1组	0.34±0.10^abcd	0.39±0.19^abcd
F值	116.70	19.20
P值	<0.001	<0.001

组别	Sub G₁期	G₀/G₁期	S期	G₂/M期
阴性对照组	5.45±0.21	55.46±0.37	10.81±1.24	26.64±0.15
HLY78组	3.30±0.20^a	45.29±3.34^a	18.53±0.92^a	30.38±2.38
OA组	9.69±1.91^ab	45.07±0.42^ab	13.80±2.09^ab	27.89±0.87
HLY78+OA组	8.29±1.16^abc	46.15±2.61^abc	15.17±1.53^abc	28.37±3.73
IWR-1组	8.21±1.26^d	52.15±4.93^d	11.73±2.76^d	25.94±2.85
F值	15.16	7.86	8.36	1.56
P值	<0.001	0.004	0.003	0.260

组别	PCNA	Cyclin D1	Cyclin D3	CDK2	CDK6	P18
阴性对照组	0.99±0.01	0.98±0.02	0.98±0.01	0.99±0.02	0.99±0.02	0.99±0.01
HLY78组	1.30±0.14^a	1.31±0.18^a	1.15±0.02^a	1.28±0.13^a	1.52±0.13^a	0.80±0.12^a
OA组	0.81±0.07^ab	0.72±0.08^ab	0.81±0.02^ab	0.74±0.09^ab	0.43±0.09^ab	1.42±0.14^ab
HLY78+OA组	1.02±0.09^abc	0.78±0.14^abc	0.93±0.05^abc	0.95±0.21^abc	1.00±0.03^abc	1.17±0.10^abc
IWR-1组	0.75±0.04^d	0.61±0.09^d	0.83±0.03^d	0.38±0.03^d	0.33±0.04^d	1.26±0.06^d
F值	20.23	17.19	63.77	24.62	127.25	11.89
P值	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001