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Study on the Effects of Calpeptin on Estrogen-induced Transformation and Stemness Markers Expression of Mammary Epithelial Cells MCF- 10A Based on Calpain-ERK Signaling Pathway / 中国药房
China Pharmacy ; (12): 1549-1556, 2020.
Article in Chinese | WPRIM | ID: wpr-822618
Responsible library: WPRO
ABSTRACT
OBJECTIVE:To study the effects of Calpeptin inhibitor Calpeptin on the transformation and stemness markers expression induced by estradiol(E2),and to investigate its mechanism. METHODS :Taking human mammary epithelial cells MCF-10A as research object ,transformed cells were induced by E 2 treatment. Cells were divided into control group (0.1%DMSO), E2-transformed group (50 nmol/L),E2-transformed+Calpeptin group (50 nmol/L E 2+1 μmol/L Calpeptin),then continuously treated with corresponding drug-containing culture medium for 15 generations. Then ,MTT assay was used to determine the proliferation rate of cells (24,48 h);plate colony test was used to detect the Clone formation rate of cells ;the number of sphere-forming cells was measured by suspension spheroidization test ;mRNA expressions of stemness marker (CD44,Nanog,OCT4)and extracellular sigal-regulated kinase (ERK)were detected by RT-qPCR ,and protein expressions of CD 44,Nanog,OCT4 ,ERK and p-ERK were detected by Western blotting assay. Another E 2-transformed cells were divided into control group (0.1%DMSO)and U0126 (ERK inhibitor )group(10 μmol/L). Clone formation rate ,the number of sphere-forming ,protein expressions of CD 44,Nanog, OCT4,ERK and p-ERK were determined with above methods ,and to validate the relationship of ERK inhibition with transformed cell behavior and the expression of stemness markers. RESULTS :Compared with control group ,proliferation rate and clone formation rate of E 2 transformed group were increased significantly (P<0.01),and the number of sphere-forming was increased significantly(P<0.01);mRNA expression levels of CD 44,Nanog,OCT4,ERK and protein expression levels of CD 44,Nanog, OCT4 and p-ERK in cells were increased significantly (P<0.01). Compared with E 2-transformed group ,proliferation rate (24,48 h)and clone formation rate of E 2-transformed + Calpeptin group were decreased significantly (P<0.01),and the number of sphere-forming was decreased significantly (P<0.05);mRNA expression levels of CD 44,Nanog,OCT4 ,ERK and protein expression levels of CD 44,Nanog,OCT4,p-ERK in cells were decreased significantly (P<0.05 or P<0.01). After treated with ERK inhibitor U 0126,clone formation rate of E 2-transformed cells ,the number of sphere-forming ,protein expression levels of CD44,Nanog,OCT4 and p-ERK were increased significantly (P<0.05 or P<0.01). CONCLUSIONS :Calpeptin can inhibit the transformation and the expression of stemness markers of human mammary epithelial cells MCF- 10A,and the mechanism of it may be associated with inhibiting the activation of Calpain-ERK signaling pathway.

Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: China Pharmacy Year: 2020 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: China Pharmacy Year: 2020 Type: Article