山茱萸提取物对乳腺癌细胞MDA-MB-231侵袭和转移的抑制作用及机制研究

目的探讨山茱萸提取物对人乳腺癌细胞MDA-MB-231侵袭和转移的抑制作用及机制。方法设MDA-MB-231细胞组、顺铂组(100.0μg/mL)、山茱萸提取物低剂量组(100.0μg/mL)、高剂量组(200.0μg/mL);以上各组每孔设6个平行样,培养72h。培养结束后,MTT法测定细胞活力,结晶紫染色测定单克隆形成数目,刮擦伤口愈合试验测定细胞迁移,Transwell室测定细胞侵袭,RT-PCR法及Western blot法测定胰岛素样生长因子1受体(IGF-1R)、丝氨酸/苏氨酸激酶(Akt)基因和蛋白水平。结果与MDA-MB-231细胞组比较,顺铂组、山茱萸提取物低、高剂量组人乳腺癌MDA-MB-231细胞吸光度(OD值)[(0.34±0.02)、(0.62±0.04)、(0.18±0.04)比(0.91±0.03)]、存活率[(25.54±0.15)%、(44.65±1.35)%、(16.41±2.36)%比(93.54±0.24)%]、单克隆形成数目[(186.63±12.65)个、(325.98±17.65)个、(108.63±12.63)个比(715.63±15.63)个]、迁移率[(45.63±9.65)%、(63.25±8.99)%、(23.96±11.32)%比(85.65±10.32)%]、穿膜数[(105.63±32.54)个、(298.63±28.65)个、(54.96±16.66)个比(542.63±29.65)个]、IGF-1R、Akt mRNA [(1.62±0.16)、(3.59±0.21)、(1.11±0.18)比(6.54±0.18),(1.48±0.19)、(3.69±0.21)、(0.89±0.25)比(5.69±0.14)]和蛋白[(1.89±0.19)、(3.62±0.15)、(1.24±0.23)比(6.99±0.18),(2.65±0.23)、(3.49±0.18)、(1.05±0.24)比(6.36±0.19)]表达降低(P<0.05)。与顺铂组比较,山茱萸提取物低剂量组人乳腺癌MDA-MB-231细胞OD值[(0.62±0.04)比(0.34±0.02)]、存活率[(44.65±1.35)%比(25.54±0.15)%]、单克隆形成数目[(325.98±17.65)个比(186.63±12.65)个]、迁移率[(63.25±8.99)%比(45.63±9.65)%]、穿膜数[(298.63±28.65)个比(105.63±32.54)个]、IGF-1R、Akt mRNA [(3.59±0.21)比(1.62±0.16)、(3.69±0.21)比(1.48±0.19)]和蛋白[(3.62±0.15)比(1.89±0.19)、(3.49±0.18)比(2.65±0.23)]表达升高(P<0.05),山茱萸提取物高剂量组OD值[(0.18±0.04)比(0.34±0.02)]、存活率[(16.41±2.36)%比(25.54±0.15)%]、单克隆形成数目[(108.63±12.63)个比(186.63±12.65)个]、迁移率[(23.96±11.32)%比(45.63±9.65)%]、穿膜数[(54.96±16.66)个比(105.63±32.54)个]、IGF-1R、Akt mRNA[(1.11±0.18)比(1.62±0.16)、(0.89±0.25)比(1.48±0.19)]和蛋白[(1.24±0.23)比(1.89±0.19)、(1.05±0.24)比(2.65±0.23)]表达降低(P<0.05)。与山茱萸提取物低剂量组比较,山茱萸提取物高剂量组OD值[(0.18±0.04)比(0.62±0.04)]、存活率[(16.41±2.36)%比(44.65±1.35)%]、单克隆形成数目[(108.63±12.63)个比(325.98±17.65)个]、迁移率[(23.96±11.32)%比(63.25±8.99)%]、穿膜数[(54.96±16.66)个比(298.63±28.65)个]、IGF-1R、Akt mRNA[(1.11±0.18)比(3.59±0.21)、(0.89±0.25)比(3.69±0.21)]和蛋白[(1.24±0.23)比(3.62±0.15)、(1.05±0.24)比(3.49±0.18)]表达降低(P<0.05)。结论山茱萸提取物对人乳腺癌细胞MDA-MB-231增殖、侵袭、转移具有明显抑制作用,200.0μg/mL的山茱萸提取物效果尤为明显;其机制可能与山茱萸提取物抑制IGF-1R、Akt mRNA和蛋白的表达进而抑制IGF-1R/Akt信号通路的激活有关。

Inhibitory effect and mechanism of Cornus officinalis extract on invasion and metastasis of breast cancer cell MDA-MB-231

Objective To explore the inhibitory effect and mechanism of Cornus officinalis extract on invasion and metastasis of human breast cancer cell MDA-MB-231. Methods MDA-MB-231 cell group, cisplatin group(100.0μg/mL), Cornus officinalis extract low-dose group(100.0μg/mL), high-dose group(200.0μg/mL) were established, and six parallel samples in each set of the above groups were cultured for 72 h. After the cultivation, MTT assay was used to measure cell viability, crystal violet staining was used to determine the number of monoclonal formation, scraping wound healing test was used to check cell migration, Transwell chamber was used to measure cell invasion, RT-PCR and Western blot were used to test insulin-like growth factor 1 receptor(IGF-1 R), serine/threonine kinase(Akt) gene and protein levels. Results Compared to the MDA-MB-231 cell group, the absorbance(OD value)[(0.91±0.03) vs(0.34±0.02),(0.62±0.04) and(0.18±0.04)], survival rate [(93.54±0.24)% vs(25.54±0.15)%,(44.65±1.35)% and(16.41±2.36)%], number of monoclonal formation [(715.63±15.63) vs(186.63±12.65),(325.98±17.65) and(108.63±12.63)], migration rate[(85.65±10.32)% vs(45.63±9.65)%,(63.25±8.99)% and(23.96±11.32)%], number of membrane penetration[(542.63±29.65) vs(105.63±32.54),(298.63±28.65)and(54.96 ±16.66)], mRNA level of both IGF-1 R [(6.54 ±0.18) vs(1.62 ±0.16),(3.59 ±0.21) and(1.11 ±0.18)]and Akt [(5.69±0.14) vs(1.48±0.19),(3.69±0.21) and(0.89±0.25)], protein level of both IGF-1 R [(6.99±0.18)vs(1.89 ±0.19),(3.62 ±0.15) and(1.24 ±0.23)] and Akt [(6.36 ±0.19) vs(2.65 ±0.23),(3.49 ±0.18) and(1.05 ±0.24)] decreased significantly in cisplatin group and Cornus extract low-dose and high-dose group(P<0.05, respectively). Compared to cisplatin group, OD [(0.34±0.02) vs(0.62 ±0.04)], survival rate [(25.54 ±0.15)% vs(44.65 ±1.35)%], number of monoclonal formation [(186.63±12.65) vs(325.98±17.65)], migration rate [(45.63±9.65)% vs(63.25±8.99)%], number of membrane penetration [(105.63±32.54) vs(298.63±28.65)], mRNA level of both IGF-1 R [(1.62±0.16) vs(3.59±0.21)] and Akt [(1.48±0.19) vs(3.69±0.21)], and protein level of both IGF-1 R[(1.89±0.19) vs(3.62±0.15)] and Akt [(2.65±0.23) vs(3.49±0.18)] increased significantly in Cornus extract lowdose group(P<0.05);And these decreased significantly in thehigh-dose group, i.e., OD: [(0.34±0.02) vs(0.18±0.04)], survival rate: [(25.54±0.15)% vs(16.41±2.36)%], number of monoclonal formation: [(186.63±12.65) vs(108.63±12.63)], migration rate: [(45.63±9.65)% vs(23.96±11.32)%], number of membrane penetration: [(105.63±32.54) vs(54.96 ±16.66)], IGF-1 R mRNA: [(1.62 ±0.16) vs(1.11 ±0.18)], Akt mRNA:(1.48 ±0.19) vs(0.89 ±0.25)], IGF-1 R protein: [(1.89±0.19) vs(1.24±0.23)], Akt protein: [(2.65±0.23) vs(1.05±0.24)]. Compared to the low-dose group, these decreased significantly in thehigh-dose group, i.e., OD: [(0.62±0.04) vs(0.18±0.04)], survival rate: [(44.65±1.35)% vs(16.41±2.36)%], number of monoclonal formation: [(325.98±17.65) vs(108.63±12.63)],migration rate: [(63.25±8.99)% vs(23.96±11.32)%], number of membrane penetration: [(298.63±28.65) vs(54.96±16.66)], IGF-1 R mRNA: [(3.59±0.21) vs(1.11±0.18)], Akt mRNA: [(3.69±0.21) vs(0.89±0.25)], IGF-1 R protein: [(3.62±0.15) vs(1.24±0.23)], Akt protein: [(3.49±0.18) vs(1.05±0.24)]. Conclusion Cornus officinalis extract can inhibit the proliferation, invasion and metastasis of MDA-MB-231 cells, 200.0μg/mL dose may be especially effective. The mechanism may be related to the suppression of expression of IGF-1 R, Akt mRNA and protein, and thus activate the IGF-1 R/Akt signaling pathway.