Eupatorin Suppressed Tumor Progression and Enhanced Immunity in a 4T1 Murine Breast Cancer Model

Abstract

Eupatorin is a polymethoxy flavone extracted from Orthosiphon stamineus and was reported to exhibit cytotoxic effects on several cancer cell lines. However, its effect as an anti-breast cancer agent in vivo has yet to be determined. This study aims to elucidate the potential of eupatorin as an anti-breast cancer agent in vivo using 4T1 challenged BALB/c mice model. In this article, BALB/c mice (20-22 g) challenged with 4T1 cells were treated with 5 mg/kg or 20 mg/kg eupatorin, while the untreated and healthy mice were fed with olive oil (vehicle) via oral gavage. After 28 days of experiment, the mice were sacrificed and blood was collected for serum cytokine assay, while tumors were harvested to extract RNA and protein for gene expression assay and hematoxylin-eosin staining. Organs such as spleen and lung were harvested for immune suppression and clonogenic assay, respectively. Eupatorin (20 mg/kg) was effective in delaying the tumor development and reducing metastasis to the lung compared with the untreated mice. Eupatorin (20 mg/kg) also enhanced the immunity as the population of NK1.1+ and CD8+ in the splenocytes and the serum interferon-γ were increased. Concurrently, eupatorin treatment also has downregulated the expression of pro-inflammatory and metastatic related genes (IL-1β. MMP9, TNF-α, and NF-κB). Thus, this study demonstrated that eupatorin at the highest dosage of 20 mg/kg body weight was effective in delaying the 4T1-induced breast tumor growth in the animal model.

Keywords: 4T1; CD8+; MMP-9; NF-κB; NK1.1; clonogenic; eupatorin.

Figure 1.

Overview of the in vivo experimental design.

Figure 2.

Effect of eupatorin on 4T1 cell cytotoxicity for 24, 48, and 72 hours using MTT assay. The IC₅₀ value of eupatorin after 24-, 48-, and 72-hour incubation time on 4T1 cells. Values are expressed as mean ± SD for 3 independent observations.

Figure 3.

(A) Tumor weight after 28 days of the experiment. The images of harvested tumor represent the untreated tumor, tumor mice treated with 5 mg/kg eupatorin, and tumor mice treated with 20 mg/kg eupatorin. Excised tissue sections were stained with hematoxylin and eosin (H&E) staining and viewed under the microscope (Nikon) at 20× magnification. The representative images of tumor tissue section stained with H&E in (B) untreated tumor, (C) tumor mice treated with 5 mg/kg eupatorin, and (D) tumor mice treated with 20 mg/kg eupatorin. Statistical analysis was performed using unpaired t test. Data are presented as mean values ± SD of n = 5 independent experiments (*Statistical significance [P < .05] between untreated and treated groups).

Figure 4.

Apoptotic cells quantification of the harvested tumor section stained with TUNEL apoptosis detection kit (FITC-labelled) and viewed under the microscope (Nikon) at 40× magnification. The number of apoptotic cells in the tumor section from 5 hematoxylin and eosin (H&E)-stained slides in each group is represented in (A). The representative images of TUNEL labeled tumor tissue from (B) untreated mice, (C) tumor mice treated with 5 mg/kg eupatorin, and (D) tumor mice treated with 20 mg/kg eupatorin. Statistical analysis was performed using unpaired t test. Data are presented as mean values ± SD of n = 5 independent experiments (*statistical significance [P < .05] between untreated and treated groups).

Figure 5.

Lung metastases were investigated on day 28 via clonogenic assay from untreated mouse (fed with olive oil), 5 mg/kg and 20 mg/kg body weight (BW) eupatorin-treated mouse. Blue colony represented the breast cancer cells that invaded into the lung and survived. The number of cancer cell colonies that survived in lung harvested from breast cancer mice was enumerated. Statistical analysis was performed using the unpaired t test. Data are presented as mean values ± SD of n = 3 independent experiments (*statistical significance [P < .05] between untreated and treated group).

Figure 6.

The weight of harvested spleen was presented in (A) where representative images of harvested spleen presented healthy mice (fed with olive oil), untreated tumor mice (fed with olive oil), mice fed with 5 mg/kg eupatorin in olive oil, and mice fed with 20 mg/kg eupatorin in olive oil. (B-E) Representative images of the spleen tissue section harvested from (B) healthy mice, (C) untreated 4T1 tumor mice (fed with olive oil), (D) 4T1 tumor mice fed with 5 mg/kg body weight (BW) eupatorin in olive oil, and (E) breast cancer mice fed with 20 mg/kg BW eupatorin in olive oil. Excised tissue sections were stained with hematoxylin and eosin staining and viewed under the microscope (Nikon) at 400× magnification. Scale bar: 50 µm. (B) Normal distribution of white pulp (WP) and red pulp (RP) in the healthy spleen. (C) Diffuse WP and RP distribution with the presence of giant cells (black arrow) in the untreated mouse. Statistical analysis was performed using the unpaired t test. Data are presented as mean values ± SD of n = 3 independent experiments (*statistical significance [P < .05] between healthy mice and tumor mice).

Figure 7.

Immune cells expression by the percentage of (A) NK1.1⁺CD3⁺ and NK1.1⁺CD3⁻ expression and (B) CD3⁺CD4⁺ and CD3⁺CD8⁺ expression as assessed by flow cytometry analysis. Data are presented as mean values ± SD of n = 3 independent experiments. *Statistical significance (P < .05) compared with the mice in the untreated group.

Figure 8.

(A) Cytokine value of IL-1β and IFN-γ in blood serum collected from healthy mice, untreated 4T1-induced mice, and treated group of 4T1-induced mice fed with 5 mg/kg eupatorin and 20 mg/kg eupatorin daily for 28 days. (B) Expression of targeted genes MMP9, TNF-α, IL-1β, and NF-κB in tumor harvested from untreated mice and treated mice with 5 mg/kg and 20 mg/kg eupatorin. Data were normalized with the reference gene of GAPDH and transformed in fold change number. (C) The representative of stained-free gel. Immunofluorescence imaging of protein lysate of the putative housekeeping proteins of β-actin and MMP9, extracted from the tumor. The sample sequences are as follows: untreated tumor mice (right), treated tumor mice fed with 5 mg/kg eupatorin (left), and treated tumor mice fed with 20 mg/kg eupatorin (center) daily for 28 days. Statistical analysis was performed using the unpaired t test. Data are presented as mean values ± SD of n = 3 independent experiments. *Statistical significance (P < .05) compared with the mice in the untreated group.

Figure 9.

Mode of action for the eupatorin in vivo antitumor and antimetastatic effects.