Study on the mechanism of resveratrol reversing temozolomide resistance in glioma cells by regulating Wnt/β-catenin pathway through BRD4

doi:10.11958/20220566

Abstract

Abstract:

Objective To explore whether the effect of resveratrol in reversing temozolomide (TMZ) resistance in glioma cells was related to the regulation of bromodomain-containing protein 4 (BRD4) and Wnt/β-catenin pathways. Methods Human glioma TMZ low-sensitivity cell line (U138), high-sensitivity cell line (U251) and drug-resistant cell line (T98G) were obtained, and Western blot assay was performed to detect the protein expressions of BRD4, Wnt3a, β-catenin and TMZ drug resistance protein (MGMT). T98G cell line was taken and divided into the control group 1 (adding 100 μmoL/L TMZ), the RES1 group (adding 50 μmoL/L RES) and RES+TMZ group (adding 100 μmoL/L TMZ and 50 μmoL/L RES). Cell proliferation and apoptosis were measured by CCK-8 method and flow cytometry. Protein expressions of BRD4, Wnt3a, β-catenin and MGMT were measured by Western blot assay. In order to analyze the effect of BRD4 overexpression on TMZ resistance, on the basis of adding 100 μmoL/L TMZ, T98G cells were transfected with BRD4 overexpression plasmid (pcDNA BRD4) or added 50 μmoL/L RES, and cells were divided into the pcDNA NC group, the pcDNA BRD4 group, the RES2 group and the RES+pcDNA BRD4 group. In order to verify the regulatory effect of BRD4 on Wnt3a/β-catenin pathway, on the basis of adding 100 μmoL/L TMZ, BRD4 inhibitor JQ1 and the Wnt3a/β-catenin pathway activator LiCl were added, and cells were divided into the control 2 groups, the JQ1 group and the JQ1+LiCl group. T98G cells were inoculated into the left scapular region of nude mice, and treated with RES, TMZ and/or JQ1. Protein expressions of Ki67, BRD4, MGMT and Wnt3a/β-catenin in tumor were measured. Results Compared with the high-susceptibility strain (U251), the expressions of BRD4, Wnt3a, β-catenin and MGMT increased in sequence in the low-susceptibility strain (U138) and the drug-resistant strain (T98G) (P<0.05). Compared with the control group 1, RES intervention could inhibit the expression and proliferation of BRD4, Wnt3a/β-catenin and MGMT proteins in T98G cells, promote apoptosis and reverse cell drug resistance (P<0.05). PcDNA BRD4 could reverse the anti-proliferative and pro-apoptotic effects of RES. BRD4 inhibitor JQ1 could inhibit the expression and proliferation of BRD4, Wnt3a/β-catenin and MGMT proteins in T98G cells, and promote apoptosis (P<0.05). LiCl could reverse the anti-proliferative and pro-apoptotic effects of JQ1. RES alone or in combination with JQ1 could activate the inhibitory effect of TMZ on Wnt3a/β-catenin pathway, MGMT expression and cell proliferation in tumors (P<0.05). Conclusion RES can reverse TMZ resistance of glioma T98G cells by downregulating BRD4 and thereby inhibiting the activation of Wnt3a/β-catenin pathway.

Key words: glioma, drug resistance, neoplasm, Wnt signaling pathway, beta catenin, resveratrol, temozolomide, bromodomain-containing protein 4

CLC Number:

R739.41

Figures/Tables 17

References 21

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细胞系	BRD4	Wnt3a	β-catenin	MGMT
U251	0.38±0.06	0.42±0.07	0.26±0.05	0.63±0.07
U138	0.91±0.11^a	1.23±0.13^a	1.52±0.14^a	1.78±0.12^a
T98G	1.85±0.21^ab	2.09±0.28^ab	2.17±0.26^ab	2.69±0.24^ab
F	166.826^＊＊	125.287^＊＊	189.237^＊＊	249.449^＊＊

细胞系	BRD4	Wnt3a	β-catenin	MGMT
U251	0.38±0.06	0.42±0.07	0.26±0.05	0.63±0.07
U138	0.91±0.11^a	1.23±0.13^a	1.52±0.14^a	1.78±0.12^a
T98G	1.85±0.21^ab	2.09±0.28^ab	2.17±0.26^ab	2.69±0.24^ab
F	166.826^＊＊	125.287^＊＊	189.237^＊＊	249.449^＊＊

组别	增殖活性（%）			凋亡率（%）		BRD4
对照1组	100.00±5.06			9.14±2.13		1.78±0.19
RES1组	51.19±6.71^a			23.19±4.08^a		1.36±0.27^a
RES+TMZ组	25.47±2.63^ab			45.36±6.52^ab		0.74±0.12^b
F	332.660^＊＊			94.239^＊＊		39.929^＊＊
组别		Wnt3a	β-catenin		MGMT
对照1组		1.32±0.18	1.86±0.33		2.19±0.48
RES1组		0.93±0.16^a	1.47±0.12^a		1.68±0.23^a
RES+TMZ组		0.42±0.11^b	0.65±0.18^b		1.04±0.13^b
F		52.305^＊＊	44.096^＊＊		19.909^＊＊

组别	增殖活性（%）			凋亡率（%）		BRD4
对照1组	100.00±5.06			9.14±2.13		1.78±0.19
RES1组	51.19±6.71^a			23.19±4.08^a		1.36±0.27^a
RES+TMZ组	25.47±2.63^ab			45.36±6.52^ab		0.74±0.12^b
F	332.660^＊＊			94.239^＊＊		39.929^＊＊
组别		Wnt3a	β-catenin		MGMT
对照1组		1.32±0.18	1.86±0.33		2.19±0.48
RES1组		0.93±0.16^a	1.47±0.12^a		1.68±0.23^a
RES+TMZ组		0.42±0.11^b	0.65±0.18^b		1.04±0.13^b
F		52.305^＊＊	44.096^＊＊		19.909^＊＊

组别	增殖活性（%）	凋亡率（%）	BRD4	Wnt3a	β-catenin	MGMT
pcDNA NC组	100.02±6.18	9.16±1.09	1.78±0.19	1.32±0.18	1.86±0.33	2.07±0.38
pcDNA BRD4组	160.73±15.02^a	2.74±0.16^a	2.53±0.26^a	2.45±0.31^a	2.84±0.37^a	2.95±0.41^a
RES2组	51.23±6.84^a	22.36±5.07^a	1.32±0.28^a	0.92±0.25^a	1.42±0.18^a	1.57±0.15^a
RES+pcDNA BRD4组	93.42±7.15^bc	13.75±1.26^bc	1.96±0.41	1.76±0.19^bc	2.15±0.37^bc	2.51±0.17^bc
F	134.977^＊＊	57.308^＊＊	17.162^＊＊	45.382^＊＊	20.542^＊＊	23.081^＊＊