Effects of Ophiopogon D combined with cyclooxygenase-2 silencing on proliferation, migration and invasion of human pancreatic cancer BxPC-3 cells

doi:10.3760/cma.j.cn371439-20201208-00115

Abstract

Abstract:

Objective To explore the effects of Ophiopogon D combined with cyclooxygenase-2 (COX-2) gene silencing on the proliferation, migration and invasion of human pancreatic cancer BxPC-3 cells. Methods BxPC-3 cells were divided into blank control group, Ophiopogonin D high-dose group (40 μmol/L), medium-dose group (20 μmol/L) and low-dose group (10 μmol/L). The COX-2-slienced cells were divided into control group, COX-2 inhibited group (50 pmol/ml siRNA-COX-2), Ophiopogonin D group (20 μmol/L) and combination treatment group (Ophiopogonin D 20 μmol/L+50 pmol/ml siRNA-COX-2). The proliferation activity of BxPC-3 cells was detected by CCK-8, and the migration distance of BxPC-3 cells was detected by scratched assay. The invasion degree of BxPC-3 cells was detected by Transwell, the relative expression level of COX-2 gene in BxPC-3 cells was detected by real-time quantitative PCR (RT-qPCR), and the relative expressions of COX-2,hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) proteins in BxPC-3 cells were detected by Western blotting. Results The cell proliferation rates of blank control group, Ophiopogonin D high-dose, medium-dose and low-dose groups were (100.0±4.9)%, (71.8±5.4)%, (80.5±5.8)% and (89.7±5.7)%, respectively. The migration distances were (279.8±24.0) μm, (141.9±21.2) μm, (168.8±37.1) μm and (224.6±19.9) μm, respectively. The absorbance (A) values of invasion number were 1.107±0.095, 0.390±0.030, 0.596±0.017 and 0.826±0.034, respectively.There were statistically significant differences (F=19.770, P<0.001; F=48.270, P<0.001; F=198.400, P<0.001).The above indexes of the Ophiopogonin D high-, medium- and low-dose groups were significantly lower than those in the blank control group (all P<0.05).The relative expression levels of COX-2 gene were 1.007±0.178, 0.387±0.169, 0.567±0.142 and 0.740±0.030, respectively,and the relative protein expression levels were 1.000±0.033, 0.654±0.085, 0.762±0.110 and 0.881±0.049, respectively, with statistically significant differences (F=10.280, P=0.004;F=11.780, P=0.003). The above indexes of the Ophiopogonin D high- and medium-dose groups were significantly lower than those in the blank control group (all P<0.05), and there was no statistically significant difference between the Ophiopogonin D low-dose group and blank control group (both P>0.05). The medium-dose of Ophiopogonin D (20 μmol/L) was selected as the subsequent concentration.After COX-2 silencing,the proliferation rates of the control group, COX-2 inhibited group, Ophiopogonin D group and combination treatment group were (100.0±2.8)%, (68.4±6.7)%, (67.7±5.9)% and (57.0±8.5)%, respectively,the migration distances were (274.4±23.8) μm, (217.0±18.8) μm, (186.2±18.6) μm and (115.7±15.8) μm, respectively,and the A values of invasion number were 1.143±0.092, 0.791±0.058, 0.715±0.026 and 0.424±0.058, respectively, with statistically significant differences (F=34.430, P<0.001; F=103.400, P<0.001; F=131.100, P<0.001). The proliferation rates, migration distances and invasion numbers in each treatment group were significantly lower than those in the control group (all P<0.001). Compared with the COX-2 inhibited group and Ophiopogonin D group, the cell proliferation, migration and invasion were significantly inhibited in the combination treatment group (all P<0.05). Compared with the Ophiopogonin D group, only the migration distance of the COX-2 inhibited group was significantly different (P<0.05).The relative expression levels of COX-2 protein in the above groups were 0.995±0.037, 0.779±0.060, 0.806±0.076 and 0.645±0.079, respectively,the relative expression levels of HIF-1α were 1.083±0.104, 0.749±0.070, 0.736±0.070 and 0.394±0.016, respectively,and the relative expression levels of VEGF protein were 1.016±0.103, 0.757±0.090, 0.745±0.021 and 0.603±0.023, respectively,with statistically significant differences (F=14.650, P=0.001; F=45.220, P<0.001; F=18.180, P<0.001).The expression levels of the three proteins in each treatment group were significantly lower than those in the control group (all P<0.05). Compared with the COX-2 inhibited group and Ophiopogonin D group, the relative protein expression levels of COX-2, HIF-1α and VEGF in the combination treatment group were significantly decreased (all P<0.05). Compared with the Ophiopogonin D group, there were no significant differences in the expression of the three proteins in the COX-2 inhibited group (all P>0.05). Conclusion Ophiopogon D combined with COX-2 gene silencing can inhibit the proliferation, migration and invasion of pancreatic cancer cells, and the mechanism may be related to the inhibition of COX-2 pathway and the decrease of HIF-1α and VEGF protein expression levels.

Key words: Pancreatic neoplasms, Cell proliferation, Neoplasm invasiveness, Cyclooxygenase 2, Ophiopogon D

Zhong Yang, He Miao, Liu Zhi, Chen Jianyu, Zhang Guangnian, Qin Long, Li Ting, Li Jianshui. Effects of Ophiopogon D combined with cyclooxygenase-2 silencing on proliferation, migration and invasion of human pancreatic cancer BxPC-3 cells[J]. Journal of International Oncology, 2021, 48(10): 583-590.

Figures/Tables 12

References 24

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组别	增殖率 (%)	迁移距离 (μm)	侵袭能力 (A₄₅₀值)
空白对照组	100.0±4.9	279.8±24.0	1.107±0.095
麦冬皂苷D高剂量组	71.8±5.4^a	141.9±21.2^a	0.390±0.030^a
麦冬皂苷D中剂量组	80.5±5.8^a	168.8±37.1^a	0.596±0.017^a
麦冬皂苷D低剂量组	89.7±5.7^b	224.6±19.9^a	0.826±0.034^a
F值	19.770	48.270	198.400
P值	<0.001	<0.001	<0.001

组别	COX-2
组别	基因	蛋白
空白对照组	1.007±0.178	1.000±0.033
麦冬皂苷D高剂量组	0.387±0.169^a	0.654±0.085^a
麦冬皂苷D中剂量组	0.567±0.142^a	0.762±0.110^a
麦冬皂苷D低剂量组	0.740±0.030	0.881±0.049
F值	10.280	11.780
P值	0.004	0.003

组别	基因	蛋白
阴性沉默组	1.000±0.039	1.000±0.040
COX-2沉默组1^#	0.622±0.061^ab	0.392±0.016^ab
COX-2沉默组2^#	0.433±0.048^a	0.236±0.118^a
COX-2沉默组3^#	0.589±0.065^ab	0.396±0.061^ab
F值	58.960	69.830
P值	<0.001	<0.001

组别	增殖率 (%)	迁移距离 (μm)	侵袭能力 (A₄₅₀值)
空白组	100.0±2.8	274.4±23.8	1.143±0.092
COX-2抑制组	68.4±6.7^a	217.0±18.8^a	0.791±0.058^a
麦冬皂苷D组	67.7±5.9^a	186.2±18.6^ab	0.715±0.026^a
联合用药组	57.0±8.5^abc	115.7±15.8^abc	0.424±0.058^abc
F值	34.430	103.400	131.100
P值	<0.001	<0.001	<0.001

组别	COX-2	HIF-1α	VEGF
空白组	0.995±0.037	1.083±0.104	1.016±0.103
COX-2抑制组	0.779±0.060^a	0.749±0.070^a	0.757±0.090^a
麦冬皂苷D组	0.806±0.076^a	0.736±0.070^a	0.745±0.021^a
联合用药组	0.645±0.079^abc	0.394±0.016^abc	0.603±0.023^abc
F值	14.650	45.220	18.180
P值	0.001	<0.001	<0.001