Abstract
BackgroundEstrogen receptor beta (ERβ) was considered as a tumor-inhibiting factor in estrogen-sensitive malignant tumors. In this study, we intended to investigate whether ERβ was involved in inducing autophagy in osteosarcoma.MethodsThis is an experimental study. The associations between ERβ and autophagy were detected in osteosarcoma U2-OS cells which were treated with E2, E2 + 2,3-Bis (4-hydroxyphenyl) propionitrile (DPN, ERβ agonists), E2 + DPN + water, E2 + DPN + 3-Methyladenine (3-MA, autophagy inhibitor), respectively. Cell viability and death were detected using cell counting kit 8 assay and flow cytometry, respectively. In addition, the expression of autophagy marker LC3II/I, sequestosome 1 (P62), mammalian target of rapamycin (mTOR), and phosphorylated-mTOR (p-mTOR) was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting.ResultsCell viability was significantly decreased with DPN treatment, while was reversed with 3-MA treatment. DPN treatment decreased living cells proportion and increased cell apoptosis proportion, while 3-MA treatment reversed those changes. However, there were significant differences between the E2 group and the E2 + DPN + 3-MA group for the living cell proportion and cell apoptosis proportion, suggesting apoptosis and autophagy all were induced. In addition, DPN treatment upregulated the LC3II/I expression level and downregulated P62 and mTOR (mRNA level) and p-mTOR (protein level) expression levels.ConclusionERβ inhibited the cell viability and mediated cell death by inducing apoptosis and autophagy in osteosarcoma. ERβ-induced autophagy in osteosarcoma was associated with downregulating the P62 expression level and inhibiting mTOR activation.
Highlights
Estrogen receptor beta (ERβ) was considered as a tumor-inhibiting factor in estrogen-sensitive malignant tumors
ERβ inhibited the viability of U2-OS cells The cells viability were inhibited with DPN treatment compared with that in control (E2 vs. E2 + DPN; E2 vs. E2 + DPN + water; P < 0.001), while the inhibited cells viability were reversed with 3-MA treatment (E2 + DPN + 3-MA vs. E2 + DPN; E2 + DPN + 3-MA vs. E2 + DPN + water; P < 0.001), indicating that ERβ could inhibit the
The expression of Light Chain 3 (LC3)-II/I, P62, and mammalian target of rapamycin (mTOR) at mRNA and protein levels To demonstrate the mechanisms of ERβ-induced autophagy in U2-OS cells, the expression of LC3-II/I, P62, and mTOR were determined (Fig. 4)
Summary
Estrogen receptor beta (ERβ) was considered as a tumor-inhibiting factor in estrogen-sensitive malignant tumors. We intended to investigate whether ERβ was involved in inducing autophagy in osteosarcoma. The two subtypes, ERα and ERβ, are highly homologous in structure, while being different in tissue distribution and the biological effects under binding to ligand [9]. There are no reports focusing on whether ERβ is implicated in mediating autophagy in osteosarcoma. We found that ERβ mediated the proliferation, migration, and invasion of osteosarcoma cells by the regulation of the integrin, Bcl-2, and PI3K/Akt signal pathways [11]. We intended to investigate whether ERβ is involved in inducing autophagy in osteosarcoma. DPN and 3-MA were selected to examine the association among ERβ and autophagy on osteosarcoma cell lines U2-OS
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