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

Abu Hanifah Ramadhani , Ahmad Hafidul Ahkam, Aditya Ragil Suharto, Yoga Dwi Jatmiko, Hideo Tsuboi, Muhaimin Rifa’i


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.


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

Full Text:



Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer. 2004;4(11):891-899.

DOI: 10.1038/nrc1478.

Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell. 2011;146(6):873-887.

DOI: 10.1016/j.cell.2011.08.039.

Vaupel P, Höckel M, Mayer A. Detection and characterization of tumor hypoxia using pO2 histography. Antioxid Redox Signal. 2007;9(8):1221-1235.

DOI: 10.1089/ars.2007.1628.

Murugaiyan G, Saha B. Protumor vs antitumor functions of IL-17. J Immunol. 2009;183(7):4169-4175.

DOI: 10.4049/jimmunol.0901017.

Sun X, Cheng G, Hao M, Zheng J, Zhou X, Zhang J, et al. CXCL12/CXCR4/CXCR7 chemokine axis and cancer progression. Cancer Metastasis Rev. 2010;29(4):709-722.

DOI: 10.1007/s10555-010-9256-x.

Burchiel SW, De Ann PD, Gomez MP, Montano RM, Barton SL, Seamer LC. Inhibition of lymphocyte activation in splenic and gut-associated lymphoid tissues following oral exposure of mice to 7,12-dimethylbenz[a]anthracene. Toxicol Appl Pharmacol. 1990;105:434-442.

DOI: 10.1016/0041-008X(90)90147-M.

Buters J, Quintanilla‐Martinez L, Schober W, Soballa VJ, Hintermair J, Wolff T, et al. CYP1B1 determines susceptibility to low doses of 7,12-dimethylbenz[a]anthracene‐induced ovarian cancers in mice: correlation of CYP1B1‐mediated DNA adducts with carcinogenicity. Carcinogenesis. 2003;24(2):327-334.

DOI: 10.1093/carcin/24.2.327.

Ramadhani AH, Nafisah W, Isnanto H, Sholeha TK, Jatmiko YD, Tsuboi H, et al. Immunomodulatory effects of Cyperus rotundus extract on 7,12-dimethylbenz[a]anthracene (DMBA) exposed BALB/c mice. Pharm Sci. 2020;27(1):46-55.

DOI: 10.34172/PS.2020.61.

Balmain A, Harris CC. Carcinogenesis in mouse and human cells: parallels and paradoxes. Carcinogenesis. 2000;21(3):371-377.

DOI: 10.1093/carcin/21.3.371.

Currier N, Solomon SE, Demicco EG, Chang DLF, Farago M, Ying H, et al. Oncogenic signaling pathways activated in DMBA-induced mouse mammary tumors. Toxicol Pathol. 2005;33(6):726-737.

DOI: 10.1080/01926230500352226.

Arulkumaran S, Ramprasath VR, Shanthi P, Sachdanandam P. Restorative effect of Kalpaamruthaa, an indigenous preparation, on oxidative damage in mammary gland mitochondrial fraction in experimental mammary carcinoma. Mol Cell Biochem. 2006;291(1-2):77-82.

DOI: 10.1007/s11010-006-9199-2.

Kuczynski EA, Vermeulen PB, Pezzella F, Kerbel RS, Reynolds AR. Vessel co-option in cancer. Nat Rev Clin Oncol. 2019;16(8):469-493.

DOI: 10.1038/s41571-019-0181-9.

Kaur N, Kaur B, Sirhindi G. Phytochemistry and pharmacology of Phyllanthus niruri L.: a review. Phytother Res. 2017;31(7):980-1004.

DOI: 10.1002/ptr.5825.

Tang YQ, Jaganath IB, Sekaran SD. Phyllanthus spp. induces selective growth inhibition of PC-3 and MeWo human cancer cells through modulation of cell cycle and induction of apoptosis. PLoS One. 2010;5(9):e12644,1-11.

DOI: 10.1371/journal.pone.0012644.

Lee SH, Jaganath IB, Wang SM, Sekaran SD. Antimetastatic effects of Phyllanthus on human lung (A549) and breast (MCF-7) cancer cell lines. PLoS One. 2011;6(6):e20994,1-14.

DOI: 10.1371/journal.pone.0020994.

Júnior RFDA, Soares LAL, da Costa Porto CR, de Aquino RGF, Guedes HG, Petrovick PR, et al. Growth inhibitory effects of Phyllanthus niruri extracts in combination with cisplatin on cancer cell lines. World J Gastroenterol. 2012;18(31):4162-4168.

DOI: 10.3748/wjg.v18.i31.4162.

Sharma P, Parmar J, Verma P, Sharma P, Goyal PK. Anti-tumor activity of Phyllanthus niruri (a medicinal plant) on chemical-induced skin carcinogenesis in mice. Asian Pac J Cancer Prev. 2009;10(6):1089-1094.

Tang YQ, Jaganath IB, Manikam R, Sekaran SD. Phyllanthus suppresses prostate cancer cell, PC-3, proliferation and induces apoptosis through multiple signaling pathways (MAPKs, PI3K/Akt, NF

DOI: 10.1155/2013/609581.

Tang YQ, Jaganath IB, Manikam R, Sekaran SD. Inhibition of MAPKs, Myc/Max, NFκB, and Hypoxia pathways by Phyllanthus prevent proliferation, metastasis, and angiogenesis in human melanoma (MeWo) cancer cell line. Int J Med Sci. 2014;11(6):564-577.

DOI: 10.7150/ijms.7704.

Lee SH, Jaganath IB, Atiya N, Manikam R, Sekaran SD. Suppression of ERK1/2 and hypoxia pathways by four Phyllanthus species inhibits metastasis of human breast cancer cells. J Food Drug Anal. 2016;24(4):855-865.

DOI: 10.1016/j.jfda.2016.03.010.

Rifa’i M, Widodo N. Significance of propolis administration for homeostasis of CD4+CD25+ immunoregulatory T cells controlling hyperglycemia. Springerplus. 2014;3:526-533.

DOI: 10.1186/2193-1801-3-526.

Vijayashree R, Aruthra P, Ramesh Rao K. A Comparison of manual and automated methods of quantitation of oestrogen/progesterone receptor expression in breast carcinoma. J Clin Diagn Res. 2015;9(3):EC01-EC05.

DOI: 10.7860/JCDR/2015/12432.5628.

Singh T, Singh A, Kumar R, Singh JK. Acute toxicity study of Phyllanthus niruri and its effect on the cyto-architectural structure of nephrocytes in Swiss albino mice Mus-musculus. Pharmacogn J. 2016;8(1):77-80.

DOI: 10.5530/pj.2016.1.17.

Cao G, O’Brien CD, Zhou Z, Sanders SM, Greenbaum JN, Makrigiannakis A, et al. Involvement of human PECAM-1 in angiogenesis and in vitro endothelial cell migration. Am J Physiol Cell Physiol. 2002;282(5):C1181-C1190.

DOI: 10.1152/ajpcell.00524.2001.

Stalin J, Harhouri K, Hubert L, Subrini C, Lafitte D, Lissitzky JC, et al. Soluble melanoma cell adhesion molecule (smcam/scd146) promotes angiogenic effects on endothelial progenitor cells through angiomotin. J Bio Chem. 2013;288(13):8991-9000.

DOI: 10.1074/jbc.M112.446518.

Shih LM, Hsu MY, Palazzo JP, Herlyn M. The cell-cell adhesion receptor Mel-CAM acts as a tumor suppressor in breast carcinoma. Am J Pathol. 1997;151(3):745-751.

Sidney LE, Branch MJ, Dunphy SE, Dua HS, Andrew H. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells. 2014;32(6):1380-1389.

DOI: 10.1002/stem.1661.

Zhao L, Zhang SL, Tao JY, Pang R, Jin F, Guo Y, et al. Preliminary exploration on anti-inflammatory mechanism of Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose) in vitro. Int Immunopharmacol. 2008;8(7):1059-1064.

DOI: 10.1016/j.intimp.2008.03.003.


  • There are currently no refbacks.

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.