The anthraquinones emodin and aloe-emodin are abundant in rhubarb. Several lines of evidence indicate that emodin and aloe-emodin have estrogenic activity as phytoestrogens. However, their effects on estrogen receptor α (ERα) activation and breast cancer cell growth remain controversial. The goal of this study is to investigate the effects and molecular mechanisms of emodin and aloe-emodin on breast cancer cell proliferation. Our results indicate that both emodin and aloe-emodin are capable of inhibiting breast cancer cell proliferation by downregulating ERα protein levels, thereby suppressing ERα transcriptional activation. Furthermore, aloe-emodin treatment led to the dissociation of heat shock protein 90 (HSP90) and ERα and increased ERα ubiquitination. Although emodin had similar effects to aloe-emodin, it was not capable of promoting HSP90/ERα dissociation and ERα ubiquitination. Protein fractionation results suggest that aloe-emodin tended to induce cytosolic ERα degradation. Although emodin might induce cytosolic ERα degradation, it primarily affected nuclear ERα distribution similar to the action of estrogen when protein degradation was blocked. In conclusion, our data demonstrate that emodin and aloe-emodin specifically suppress breast cancer cell proliferation by targeting ERα protein stability through distinct mechanisms. These findings suggest a possible application of anthraquinones in preventing or treating breast cancer in the future.

Recent reports demonstrate that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) correlates with decreased survival outcomes. However, off-target effects of targeting PKCα and limited understanding of the signaling mechanisms upstream of PKCα have hampered previous efforts to manipulate this ubiquitous gene. This study shows that the expression of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) correlates with PKCα expression in TNBC. We found that the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 facilitate the heterodimeric interaction between the two genes before the complex formation binds to the PKCα promoter. Blocking the formation of the heterodimer by transfection of MZF-160–72 or Elk-1145–157 peptide fragments at the MZF-1 / Elk-1 interface decreases DNA-binding activity of the MZF-1 / Elk-1 complex at the PKCα promoter. Subsequently, PKCα expression, migration, tumorigenicity, and the epithelial–mesenchymal transition potential of TNBC cells decrease. These subsequent effects are reversed by transfection with full-length PKCα, confirming that the MZF-1/Elk-1 heterodimer is a mediator of PKCα in TNBC cells. These data suggest that the next therapeutic strategy in treating PKCα-related cancer will be developed from blocking MZF-1/Elk-1 interaction through their binding domain.

In this study, the molecular mechanism of protein kinase C alpha (PKCα) gene regulation in hepatocellular carcinoma (HCC) involving Ets-like protein-1 (Elk-1) and myeloid zinc finger-1 (MZF-1) was investigated. The luciferase reporter assay results revealed that the presence of both MZF-1 and Elk-1 significantly contributed to the upregulation of PKCα gene transcription activity, and the transcriptional activity decreased when the transfection included a DNA-binding-deficient (∆DBD) gene vector of either MZF-1 or Elk-1 DNA-binding deficiency (MZF-1∆DBD or Elk-1∆DBD), thereby indicating that the enhanced expression of PKCα was caused by the binding of MZF-1 and/or Elk-1 with the PKCα promoter. We investigated MZF-1 and Elk-1 to determine whether they bind to each other. The results of immunoprecipitation (IP), Co-IP, chromatin IP (ChIP), and Re-ChIP analyses indicated that Elk-1 can directly bind to the N-terminal region of MZF-1 and MZF-1 can directly bind to the C-terminal region of Elk-1 to form a complex before attaching to the PKCα promoter. Furthermore, when MZF-1∆DBD or Elk-1∆DBD was added to the cells, PKCα expression decreased, and cell proliferation, migration, invasion, and tumorigenicity also decreased. These findings suggest that PKCα expression in HCC could be stimulated by the formation of MZF-1/Elk-1 complex, which directly binds to the PKCα promoter.