Cytotoxic and proapoptotic effects of alizarin in mice with Ehrlich solid tumor: novel insights into ERα-mediated MDM2/p-Rb/E2F1 signaling pathway

Abstract

The role of estrogen receptor (ER) and its related effects is crucial for growth of breast cancer cells. This study aimed to assess the cytotoxic impact of alizarin in models of breast cancer in vivo and investigate its antiestrogenic and apoptotic properties. MTT assay was used to evaluate alizarin cytotoxic effect against MCF7 and MDA-MB-231. In vivo, 30 mice were insulted with Ehrlich tumor cells. They were randomly allocated into 3 groups, 10 mice each. Alizarin was orally administered to two treatment groups (50 mg/kg and 100 mg/kg), respectively. The third group was considered as a positive control group. Western blot was used to evaluate the expression of mouse double minute 2 (MDM2), phosphorylated retinoblastoma (pRb), and E2F1. Caspase threefold change was measured by RT-PCR. ERα, Bax, and p53 expressions were investigated by immunohistochemistry. Molecular docking study of alizarin effect on ER was performed. Alizarin demonstrated dose dependent cytotoxicity against MCF7 and MDA-MB cell lines. Alizarin decreased tumor weight in mice, prompted cell cycle arrest, and stimulated cell apoptosis by impeding ERα-mediated tumorigenic effects, and inactivating its related MDM2/p-Rb/E2F1 signaling cascade with upregulation of target genes involved in cell apoptosis (Bax, caspase 3, and p53). Molecular docking proposed that alizarin is a very promising inhibitor to ER. Alizarin represented a promising approach to inhibit cell proliferation of breast cancer by modulating estrogen receptor mediated effects on MDM2/p-Rb/E2F1 axis concomitantly with activating apoptosis of cancer cells.

Keywords: Alizarin; Breast cancer; ERα; Ehrlich solid tumor; MDM2.

Fig. 1

A ¹H-NMR spectrum of Alizarin in (DMSO-d₆, 400 MHz). B ¹³C-NMR spectrum of Alizarin in (DMSO-d₆, 100 MHz). C APT spectrum of Alizarin in (DMSO-d₆, 100 MHz)

Fig. 2

Effect of alizarin, tamoxifen, and doxorubicin on the viability of A MCF-7 and B MDA-MB- 231

Fig. 3

Effect of alizarin (50 mg/kg and 100 mg/kg) on tumor weight compared to Ehrlich control group (A). Effect of alizarin (50 mg/kg and 100 mg/kg) on tumor volume compared to Ehrlich control group (B). Representative photograph for Ehrlich control group (C). Representative photograph for group treated with (50 mg/kg) alizarin (D). Representative photograph for group treated with (100 mg/kg) alizarin (E). All data expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. (a) Significant compared with the control group at p < 0.05. (b) Significant compared with the small dose group at p < 0.05

Fig. 4

Effect of alizarin (50 mg/kg and 100 mg/kg) on ERα expression in different experimental groups. A Representative photomicrographs of ERα. B The percentage of positive immunohistochemical reaction (brown stained area) analyzed by ImageJ software. All data expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. (a) Significantly different compared with the control group at p < 0.05. (b) Significantly different compared with the small dose group at p < 0.05

Fig. 5

Effect of alizarin (50 mg/kg and 100 mg/kg) on MDM2/p-Rb/E2F1 axis. A Western blot analysis showing protein expression of MDM2, p-Rb, E2 F1 and β-actin in tumor tissues. B Quantified data of MDM2 normalized to β-actin. C Quantified data of E2F1 normalized to β-actin. D Quantified data of p-Rb normalized to β-actin. Results are expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. (a) Significantly different compared with the control group at p < 0.05. (b) Significantly different compared with the small dose group at p < 0.05

Fig. 6

Effect of alizarin (50 and 100 mg/kg) on p53 & BAX expression in different experimental groups. A, C Representative photomicrographs of p53 & BAX respectively. B, D The percentage of positive immunohistochemical reaction of p53 & BAX respectively (brown stained area) analyzed by Image J software. All data expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. (a) Significantly different compared with the control group at p < 0.05. (b) Significantly different compared with the small dose group at p < 0.05

Fig. 7

Effect of alizarin (50 and 100 mg/kg) on caspase 3 expression in different experimental groups. All data expressed as mean fold change ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. (a) Significantly different compared with the control group at p < 0.05. (b) Significantly different compared with the small dose group at p < 0.05

Fig. 8

3D binding interactions, and 3D positioning of alizarin inside both the core binding pocket (LBD) of estrogen receptor (A) and the second site on its surface (B). Alizarin alone in the LBD (C); Superimposition of alizarin and the co-crystallized HT antagonist in the LBD (D); Binding interactions of alizarin inside the LBD (E); Alizarin alone in the surface pocket (F); Superimposition of alizarin and the co-crystallized HT antagonist in the surface receptor (G); Binding interactions of alizarin inside the surface receptor (H)

Schematic representation illustrates the micro-environment in the EST with overexpression of MDM2 and activation of E2F1 and hyperphosphorylation of RB because of estrogen receptor signaling; at the same time, the apoptotic function of p53 and its target genes is prohibited due to the overexpression of MDM2. The right side of the graphical abstract represents the mechanistic pathway by which alizarin worked on antagonizing ERα and consequently, MDM2/E2F1/p-Rb axis was downregulated, synchronously; the p53 tumor suppressor gene is liberated and reactivated once again to alleviate its target genes Bax and caspase 3 restoring the apoptotic function