Suppression of hypoxia and inflammatory pathways by Phyllanthus niruri extract inhibits angiogenesis in DMBA-induced breast cancer mice

Abstract

Background and purpose: Angiogenesis has been one of the hallmarks of cancer. In recent years, Phyllanthus niruri extract (PNE) was reported to inhibit angiogenesis by decreasing the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in breast cancer. However, the experimental results were confirmed in cancer cell lines only, whereas the anti-angiogenic activity in animal models has not been demonstrated. In this study, we tried to examine the anti-angiogenic activity of PNE on BALB/c strain mice models that were induced for breast cancer using the carcinogenic substance 7,12- dimethylbenz[a]anthracene (DMBA).

Experimental approach: Experimental animals were divided into five different groups; vehicle, DMBA, PNE 500 mg/kg, PNE 1000 mg/kg; and PNE 2000 mg/kg. Mammary carcinogenesis was induced using a subcutaneous injection of 15 mg/kg of DMBA for 12 weeks. Afterward, oral PNE treatment was given for the following 5 weeks. VEGFA and HIF-1α were observed using immunohistochemistry. Endothelial cell markers CD31, CD146, and CD34 were observed using the fluorescent immunohistochemistry method. The levels of interleukin-6 (IL-6), IL-17, and C-X-C motif chemokine (CXCL12) were measured using flow cytometry.

Findings/results: The survival analysis indicated that PNE increased the survival rate of mice (P = 0.043, log-rank test) at all doses. The PNE treatment decreased the immunoreactive score of angiogenic factors (VEGF and HIF-1α), as well as the endothelial cell markers (CD31, CD146, and CD34). The PNE- treated groups also decreased the levels of inflammatory cytokines (IL-6, IL-17, and CXCL12) at all doses.

Conclusion and implications: This finding suggests that PNE may inhibit the progression of angiogenesis in breast cancer mice by targeting the hypoxia and inflammatory pathways.

Keywords: Angiogenesis; Breast cancer; DMBA; Inflammation; Phyllanthus niruri.

Fig. 1

Kaplan-Meier analysis was measured for 5 weeks after receiving PNE treatment. Mice were exposed to DMBA for 12 weeks prior to the treatment. (A) The survival analysis showed that the mice treated with PNE had a better chance of survival. (B) Similarly, the hazard function also reveals that treated mice had a lower hazard rate compared to the DMBA group. DMBA, 7,12-dimethylbenz[a]anthracene; PNE, Phyllanthus niruri extract.

Fig. 2

H&E staining reveals blood vessels (arrowheads) that grow in mammary tissue. The expression of HIF-1α and VEGFA were observed using immunohistochemistry staining. DMBA, 7,12-Dimethylbenz[a]anthracene; H&E, hematoxylin and eosin; HIF, hypoxia-inducible factor; PNE, Phyllanthus niruri extract; VEGFA, vascular endothelial growth factor A.

Fig. 3

Immunoreactive score of (A) angiogenic factors and (B) endothelial cell markers. Scores range from 0-12. ###P < 0.001 indicates significant difference compared to the vehicle group; *P < 0.05, **P < 0.01, ***P < 0.001 versus DMBA group. CD, Cluster of differentiation; DMBA, 7,12-dimethylbenz[a]anthracene; HIF, hypoxia inducible factor; PNE, Phyllanthus niruri extract; VEGFA, vascular endothelial growth factor A.

Fig. 4

Fluorescent immunohistochemical staining of endothelial cell markers. The expression of CD31, CD146, and CD34 were marked by blue, green, and red, respectively. CD, Cluster of differentiation; DMBA, 7,12-dimethylbenz[a]anthracene; PNE, Phyllanthus niruri extract.

Fig. 5

Flow cytometry analysis of (A) IL-6, (B) IL-17, and (C) CXCL12. All data represent mean ± SD. ^###P < 0.001 indicates significant difference compared to the vehicle group; *P < 0.05, **P < 0.01, ***P < 0.001 in contrast to DMBA group. CD, Cluster of differentiation; DMBA, 7,12-dimethylbenz[a]anthracene; IL, interleukin; PNE, Phyllanthus niruri extract.