Effects of ligustilide regulating RhoA/ROCK signaling pathway on biological behavior of esophageal cancer cells

doi:10.11958/20240758

Abstract

Abstract:

Objective To investigate effects of ligustilide (LIG) on proliferation, apoptosis, angiogenic mimicry and Ras homolog gene family member A (RhoA)/Rho associated coiled coil containing protein kinase 1 (ROCK) signaling pathway in esophageal cancer cells. Methods Esophageal cancer cell line EC-109 was treated with LIG at concentrations of 0, 12.5, 25, 50, 100, and 200 μmol/L to detect cell activity, and the suitable concentration was selected for subsequent experiments. EC-109 cells were grouped into the control group, the LIG low, medium and high concentration groups (LIG-L, LIG-M and LIG-H groups), and the LIG-H+RhoA activator Naciclassine group (LIG-H+Naciclassine group). Edu was applied to detect cell proliferation, and flow cytometry was applied to detect cell apoptosis. Angiogenetic mimicry was observed. Western blot assay was applied to detect expression levels of proteins related to cell proliferation and apoptosis, and RhoA, ROCK proteins. Nude mouse tumor transplantation experiment was applied to verify the effect of LIG on the growth of esophageal cancer tumors. Immunohistochemistry was applied to detect expression levels of angiogenesis related factors (VEGF), RhoA and ROCK proteins in transplanted tumors. Results Compared with the control group, the vascular mimicry tubular structure of EC-109 cells decreased sequentially in the LIG-L group, the LIG-M group and the LIG-H group. The positive rate of Edu, the expression levels of Cyclin D1, Ki67, Bcl-2, RhoA and ROCK reduced in turn. P21, cell apoptosis rate, the expression of Bax and Caspase-3 increased in sequence (P<0.05). Naciclasine, RhoA activator, partially reversed the effect of LIG on cell proliferation, apoptosis and vasculogenic mimicry of esophageal cancer cells. Nude mouse transplantation tumor experiment showed that compared with the control group, the growth rate of transplanted tumor showed down, tumor volume decreased and the expression levels of RhoA, ROCK and VEGF decreased in the LIG group (P<0.05). Conclusion Ligustilide inhibits the proliferation and angiogenic mimicry of esophageal cancer cells by inhibiting RhoA/ROCK signaling pathway, and promotes the apoptosis of esophageal cancer cells.

Key words: ligustilide, esophageal neoplasms, rhoA GTP-binding protein, rho-associated kinases, cell proliferation, apoptosis, neoplasm transplantation

CLC Number:

R735.1

Figures/Tables 13

References 22

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组别	Edu阳性率/%	Cyclin D1
Control组	42.36±2.14	1.03±0.08
LIG-L组	36.79±1.85^a	0.71±0.07^a
LIG-M组	28.52±1.43^ab	0.48±0.06^ab
LIG-H组	19.48±0.97^abc	0.24±0.04^abc
LIG-H+Naciclasine组	26.13±1.32^d	0.45±0.06^d
F	191.213^＊＊	135.030^＊＊
组别	P21	Ki67
Control组	0.25±0.03	1.21±0.09
LIG-L组	0.37±0.04^a	0.89±0.08^a
LIG-M组	0.69±0.05^ab	0.47±0.07^ab
LIG-H组	0.95±0.07^abc	0.31±0.05^abc
LIG-H+Naciclasine组	0.72±0.08^d	0.48±0.06^d
F	147.387^＊＊	160.494^＊＊

组别	Edu阳性率/%	Cyclin D1
Control组	42.36±2.14	1.03±0.08
LIG-L组	36.79±1.85^a	0.71±0.07^a
LIG-M组	28.52±1.43^ab	0.48±0.06^ab
LIG-H组	19.48±0.97^abc	0.24±0.04^abc
LIG-H+Naciclasine组	26.13±1.32^d	0.45±0.06^d
F	191.213^＊＊	135.030^＊＊
组别	P21	Ki67
Control组	0.25±0.03	1.21±0.09
LIG-L组	0.37±0.04^a	0.89±0.08^a
LIG-M组	0.69±0.05^ab	0.47±0.07^ab
LIG-H组	0.95±0.07^abc	0.31±0.05^abc
LIG-H+Naciclasine组	0.72±0.08^d	0.48±0.06^d
F	147.387^＊＊	160.494^＊＊

组别	细胞凋亡率/%	Bax	Bcl-2	Caspase-3
Control组	3.16±0.25	0.26±0.04	0.76±0.08	0.34±0.04
LIG-L组	20.73±1.82^a	0.48±0.05^a	0.54±0.06^a	0.64±0.05^a
LIG-M组	27.15±2.53^ab	0.80±0.06^ab	0.32±0.05^ab	0.97±0.07^ab
LIG-H组	36.74±3.24^abc	1.15±0.08^abc	0.18±0.04^abc	1.22±0.11^abc
LIG-H+Naciclasine组	25.38±2.36^d	0.83±0.06^d	0.39±0.05^d	0.99±0.06^d
F	176.966^＊＊	199.712^＊＊	88.952^＊＊	143.769^＊＊

组别	细胞凋亡率/%	Bax	Bcl-2	Caspase-3
Control组	3.16±0.25	0.26±0.04	0.76±0.08	0.34±0.04
LIG-L组	20.73±1.82^a	0.48±0.05^a	0.54±0.06^a	0.64±0.05^a
LIG-M组	27.15±2.53^ab	0.80±0.06^ab	0.32±0.05^ab	0.97±0.07^ab
LIG-H组	36.74±3.24^abc	1.15±0.08^abc	0.18±0.04^abc	1.22±0.11^abc
LIG-H+Naciclasine组	25.38±2.36^d	0.83±0.06^d	0.39±0.05^d	0.99±0.06^d
F	176.966^＊＊	199.712^＊＊	88.952^＊＊	143.769^＊＊

组别	管状结构数	RhoA	ROCK
Control组	34.50±3.27	1.03±0.09	1.14±0.12
LIG-L组	27.16±2.54^a	0.80±0.08^a	0.91±0.07^a
LIG-M组	20.33±1.83^ab	0.47±0.07^ab	0.58±0.06^ab
LIG-H组	11.83±0.96^abc	0.32±0.05^abc	0.23±0.04^abc
LIG-H+Naciclasine组	18.17±1.62^d	0.54±0.06^d	0.55±0.06^d
F	94.523^＊＊	93.729^＊＊	131.819^＊＊