Meroxest improves the prognosis of immunocompetent C57BL/6 mice with allografts of E0771 mouse breast tumor cells

Abstract

Introduction: Recently, we have reported the antitumor properties of a new family of synthetic merosesquiterpenes, among which meroxest is highlighted, since it has high activity and specificity for ER⁺ breast cancer cells. In this paper, we characterize allografts of ER⁺ E0771 mouse breast tumor cells in immunocompetent C57BL/6 mice, and also analyze the effect of meroxest on the prognosis of the disease.

Material and methods: Twenty female C57BL/6 mice were injected with 10⁶ E0771 cells. Once the tumors reached the appropriate size, the mice were divided into two groups, one control and another treated orally with 15 mg/kg of meroxest. After 20 days, tumor samples were taken for histopathological study and for determination of the expression of the prognostic markers Ki67 and vascular endothelial growth factor (VEGF) by immunofluorescence.

Results: In sections stained with hematoxylin-eosin, we observed that tumors have a well-defined capsule enclosing E0771 tumor cells. The central area of tumors contains necrotic regions with leukocyte infiltration. Meroxest treatment significantly reduces tumor size (68%, p < 0.05), induces changes in its structure, decreases the degree of leukocyte infiltration, and significantly reduces the expression of Ki67 (33%, p < 0.05) and VEGF (82%, p < 0.05).

Conclusions: Meroxest improves the prognosis of mice since it reduces leukocyte infiltration, and decreases the expression of Ki67 and VEGF markers. Consequently, the merosesquiterpene could become a useful antiangiogenic drug in the treatment of breast cancer. These results encourage us to deepen the study of meroxest, in order to find more evidence that supports the convenience of its evaluation in a clinical study or trial.

Keywords: Ki67; breast cancer; in vivo; merosesquiterpene; vascular endothelial growth factor.

Figure 1

Chemical structure of meroxest

Figure 2

Allografts of E0771 cells in C57BL/6 mice. A – Mouse bearing a tumor (arrow). B – Samples of tumors: whole tumor from control (left) and from 15 mg/kg meroxest-treated animal (right) (B1). The mean volume of control tumors was 12.14 × 103 mm³, whereas that of tumors treated with meroxest was 3.89 × 103 mm³. Fragments from control tumors (B2) and from 15 mg/kg meroxest-treated tumors (B3). Arrows in B3 indicate areas with marked modifications in the structure of meroxest-treated tumors as compared to controls. Bars: 24 mm (B1) and 15 mm (B2 and B3)

Figure 3

Histological analysis of E0771 allografts. A – Micrograph of a hematoxylin-eosin-stained control tissue section showing a peripheral capsule of normal tissue (■), enclosing tumor cells (*) and necrotic centers (▲) with leukocyte infiltration (arrow). B – Micrograph with higher magnification which shows leukocyte infiltration at the edge of a necrotic center. C and D – E0771 tumor cells invading the underlying muscle tissue. E and F – Tumor sections from a 15 mg/kg meroxest-treated animal exhibiting less leukocyte infiltration than controls. Bars: 367.5 µm (A, E), 147 µm (B, C, F) and 36.75 µm (D)

Figure 4

Immunofluorescence analysis of Ki67 and VEGF expression in tumor sections from control and 15 mg/kg meroxest-treated animals. Nuclei were counterstained with DAPI (blue). Bars: 100 µm

Figure 5

Quantitative analysis of Ki67 and VEGF expression in tumor sections from control and 15 mg/kg meroxest-treated animals. Total cell fluorescence was calculated using ImageJ software. Mean values and SEM are shown *P < 0.05 compared with the control group.