Efficacy of punarnavine in restraining organ-specific tumour progression in 4T1-induced murine breast tumour model

  • Gilcy George Kallivalappil
  • Girija Kuttan
Original Article


Most of the breast cancer deaths occur when cancer cells depart from their tumour of origin and spread systemically and colonise distant organs. The present study was to find out whether punarnavine, the quinolizidine alkaloid, with already proven antimetastatic effect on spontaneous B16F10 pulmonary metastasis has got any effect on a drastic organ-specific breast cancer spread. For the study, we selected a syngenic mouse 4T1 breast tumour model that mimics stage four of human breast cancer. The metastatic progression of 4T1 to lymph nodes, lungs, and liver was reduced by punarnavine (40 mg/kg body weight) administration in BALB/c mice. This was evident from the histopathology of these organs as well as from the reduction in the metastatic cell density of cultured 6-thioguanine-resistant 4T1 cells in the punarnavine-treated group compared to the control group. There was also a significant (p < 0.0001) inhibition of the primary breast tumour growth in the orthotopic site of induction with a simultaneous increase (p < 0.0001) in the life span of treated animals. The assessment of biochemical parameters such as hydroxyproline, hexosamine, uronic acid, sialic acid and γ-glutamyl transferase and the analysis of various cytokines VEGF, IL-1β, TNF-α and GM-CSF showed a similar pattern of reduction in punarnavine (p < 0.0001) treated group compared to the control group. The gene expression study revealed the inhibitory effect of punarnavine on the major genes MMP-2, MMP-9, TIMP-1, TIMP-2 and VEGF involved in the metastatic process. These findings undeniably proved the potential of this quinolizidine alkaloid in combating breast tumour development and its progression in the studied murine model.


Breast cancer 4T1 mouse tumour model Punarnavine Organ-specific tumour progression Cytokines Matrix metalloproteinase 



Dulbecco’s modified Eagle’s medium


Foetal bovine serum


Hanks balanced salt solution


Enzyme-linked immunosorbent assay




Tumour necrosis factor-α


Granulocyte monocyte colony-stimulating factor


Vascular endothelial growth factor


Matrix metalloproteinase


Tissue inhibitor of matrix metalloproteinase


Glyceraldehyde 3-phosphate dehydrogenase


Triple-negative breast cancer



The authors express gratitude to the Council of Scientific and Industrial Research (CSIR), Government of India for the senior research fellowship provided to Ms. Gilcy George K.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


  1. Agarwal RR, Dutt SS (1935) Chemical examination of punarnava or Boerhavia diffusa Linn II, isolation of an alkaloid punarnavine. Proc Acad Sci United Prov Agra Audh India 5:240–242Google Scholar
  2. Aher V, Chattopadhyay P, Goyary D, Veer V (2013) Evaluation of the genotoxic and antigenotoxic potential of the alkaloid punarnavine from Boerhavia diffusa. Planta Med 79:939–945CrossRefPubMedGoogle Scholar
  3. Bergman I, Loxley R (1970) The determination of hydroxyproline in urine hydrolysates. Clin Chim Acta 27:347–349CrossRefPubMedGoogle Scholar
  4. Bibby MC (2004) Orthotopic models of cancer for preclinical drug evaluation. Eur J Cancer 40:852–857CrossRefPubMedGoogle Scholar
  5. Bitter T, Muir HM (1962) A modified uronic acid carbazole reaction. Anal Biochem 4:330–334CrossRefPubMedGoogle Scholar
  6. Braithwaite D, Demb J, Henderson ML (2016) Optimal breast cancer screening strategies for older women: current perspectives. Clin Interv Aging 11:111–125CrossRefPubMedPubMedCentralGoogle Scholar
  7. Brew K, Dinakarpandian D, Nagase H (2000) Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 1477:267–283CrossRefPubMedGoogle Scholar
  8. Büll C, Stoel MA, den Brok MH, Adema GJ (2014) Sialic acids sweeten a tumor’s life. Cancer Res 74:3199–3204CrossRefPubMedGoogle Scholar
  9. Carlson RW, Allred DC, Anderson BO et al (2009) Breast cancer. Clinical practice guidelines in oncology. J Natl Compr Canc Netw 7:122–192CrossRefPubMedGoogle Scholar
  10. Chiang AC, Massagué J (2008) Molecular basis of metastasis. N Engl J Med 359:2814–2823CrossRefPubMedPubMedCentralGoogle Scholar
  11. Coffelt SB, Kersten K, Doornebal CW et al (2015) IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis. Nature 522:345–348CrossRefPubMedPubMedCentralGoogle Scholar
  12. Dawson J, Smith GD, Boak J, Peters TJ (1979) gamma-Glutamyltransferase in human and mouse breast tumours. Clin Chim Acta 96:37–42CrossRefPubMedGoogle Scholar
  13. Dhingra D, Valecha R (2014) Evidence for involvement of the monoaminergic system in antidepressant-like activity of an ethanol extract of Boerhavia diffusa and its isolated constituent, punarnavine, in mice. Pharm Biol 52:767–774CrossRefPubMedGoogle Scholar
  14. DuPré SA, Redelman D, Hunter KW (2007) The mouse mammary carcinoma 4T1: characterization of the cellular landscape of primary tumours and metastatic tumour foci. Int J Exp Pathol 88:351–360CrossRefPubMedPubMedCentralGoogle Scholar
  15. Eccles SA, Box G, Court W et al (1994) Preclinical models for the evaluation of targeted therapies of metastatic disease. Cell Biophys 24:279–291CrossRefPubMedGoogle Scholar
  16. Elson LA, Morgan WT (1933) A colorimetric method for the determination of glucosamine and chondrosamine. Biochem J 27:1824–1828CrossRefPubMedPubMedCentralGoogle Scholar
  17. Engebraaten O, Vollan HKM, Børresen-Dale A-L (2013) Triple-negative breast cancer and the need for new therapeutic targets. Am J Pathol 183:1064–1074CrossRefPubMedGoogle Scholar
  18. Greenwell M, Rahman PKSM (2015) Medicinal plants: their use in anticancer treatment. Int J Pharm Sci Res 6:4103–4112PubMedPubMedCentralGoogle Scholar
  19. Gupta GP, Nguyen DX, Chiang AC et al (2007) Mediators of vascular remodelling co-opted for sequential steps in lung metastasis. Nature 446:765–770CrossRefPubMedGoogle Scholar
  20. Ho B-Y, Lin C-H, Apaya MK et al (2012) Silibinin and paclitaxel cotreatment significantly suppress the activity and lung metastasis of triple negative 4T1 mammary tumor cell in mice. J Tradit Complement Med 2:301–311CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hoffman RM (1999) Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 17:343–359CrossRefPubMedGoogle Scholar
  22. Hogan-Ryan A, Fennelly JJ, Jones M et al (1980) Serum sialic acid and CEA concentrations in human breast cancer. Br J Cancer 41:587–592CrossRefPubMedPubMedCentralGoogle Scholar
  23. Hussaini SAB, Andola SK, Mahanta A (2015) Study of metastasis in lymphnode biopsies with special reference to immunohistochemistry (IHC) in metastatic breast carcinoma. J Clin Diagn Res 9:13–16Google Scholar
  24. Kalluri R, Weinberg RA (2009) The basics of epithelial–mesenchymal transition. J Clin Invest 119:1420–1428CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kessenbrock K, Plaks V, Werb Z (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141:52–67CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kim S, Takahashi H, Lin W-W et al (2009) Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis. Nature 457:102–106CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lamouille S, Xu J, Derynck R (2014) Molecular mechanisms of epithelial–mesenchymal transition. Nat Rev Mol Cell Biol 15:178–196CrossRefPubMedPubMedCentralGoogle Scholar
  28. Lehembre F, Regenass U (2012) Metastatic disease: a drug discovery perspective. Semin Cancer Biol 22:261–271CrossRefPubMedGoogle Scholar
  29. Manu KA, Kuttan G (2009a) Anti-metastatic potential of Punarnavine, an alkaloid from Boerhavia diffusa Linn. Immunobiology 214:245–255CrossRefPubMedGoogle Scholar
  30. Manu KA, Kuttan G (2009b) Immunomodulatory activities of Punarnavine, an alkaloid from Boerhavia diffusa. Immunopharmacol Immunotoxicol 31:377–387CrossRefPubMedGoogle Scholar
  31. Manu KA, Kuttan G (2009c) Punarnavine induces apoptosis in B16F-10 melanoma cells by inhibiting NF-kappaB signaling. Asian Pac J Cancer Prev 10:1031–1037PubMedGoogle Scholar
  32. Nguyen DX, Bos PD, Massagué J (2009) Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 9:274–284CrossRefPubMedGoogle Scholar
  33. Oakley GJ III, Tubbs RR, Crowe J et al (2006) HER-2 amplification in tubular carcinoma of the breast. Am J Clin Pathol 126:55–58CrossRefPubMedGoogle Scholar
  34. Ouzounova M, Lee E, Piranlioglu R et al (2017) Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade. Nat Commun 8:1–13CrossRefGoogle Scholar
  35. Pulaski BA, Ostrand-Rosenberg S (2001) Mouse 4T1 Breast tumor model. In: Current protocols in immunology. Wiley, Hoboken, NJ, USA, pp 1–16Google Scholar
  36. Radisky ES, Radisky DC (2010) Matrix metalloproteinase-induced epithelial–mesenchymal transition in breast cancer. J Mammary Gland Biol Neoplasia 15:201–212CrossRefPubMedPubMedCentralGoogle Scholar
  37. Saraswati S, Alhaider AA, Agrawal SS (2013) Punarnavine, an alkaloid from Boerhaavia diffusa exhibits anti-angiogenic activity via downregulation of VEGF in vitro and in vivo. Chem Biol Interact 206:204–213CrossRefPubMedGoogle Scholar
  38. Silva VL, Ferreira D, Nobrega FL, Martins IM (2016) Selection of novel peptides homing the 4T1 CELL line: exploring alternative targets for triple negative breast cancer. PLoS One 11:1–15Google Scholar
  39. Skoza L, Mohos S (1976) Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation. Biochem J 159:457–462CrossRefPubMedPubMedCentralGoogle Scholar
  40. Su S, Liu Q, Chen J et al (2014) A positive feedback loop between mesenchymal-like cancer cells and macrophages is essential to breast cancer metastasis. Cancer Cell 25:605–620CrossRefPubMedGoogle Scholar
  41. Szasz G (1976) Reaction-rate method for gamma-glutamyltransferase activity in serum. Clin Chem 22:2051–2055PubMedGoogle Scholar
  42. Tao K, Li J, Warner J et al (2001) Multiple lysosomal trafficking phenotypes in metastatic mouse mammary tumor cell lines. Int J Oncol 19:1333–1339PubMedGoogle Scholar
  43. Vanharanta S, Massagué J (2013) Origins of metastatic traits. Cancer Cell 24:410–421CrossRefPubMedPubMedCentralGoogle Scholar
  44. Vernon AE, Bakewell SJ, Chodosh LA (2007) Deciphering the molecular basis of breast cancer metastasis with mouse models. Rev Endocr Metab Disord 8:199–213CrossRefPubMedGoogle Scholar
  45. Wajner SM, Capp C, Brasil BA et al (2014) Reduced tissue inhibitor of metalloproteinase-2 expression is associated with advanced medullary thyroid carcinoma. Oncol Lett 7:731–737CrossRefPubMedGoogle Scholar
  46. Wculek SK, Malanchi I (2015) Neutrophils support lung colonization of metastasis-initiating breast cancer cells. Nature 528:413–417CrossRefPubMedPubMedCentralGoogle Scholar
  47. Weigelt B, Peterse JL, van’t Veer LJ (2005) Breast cancer metastasis: markers and models. Nat Rev Cancer 5:591–602CrossRefPubMedGoogle Scholar
  48. West DC, Hampson IN, Arnold F, Kumar S (1985) Angiogenesis induced by degradation products of hyaluronic acid. Science 228:1324–1326CrossRefPubMedGoogle Scholar
  49. Wu Y, Deng J, Rychahou PG et al (2009) Stabilization of snail by NF-kappaB is required for inflammation-induced cell migration and invasion. Cancer Cell 15:416–428CrossRefPubMedPubMedCentralGoogle Scholar
  50. Xiong G, Deng L, Zhu J et al (2014) Prolyl-4-hydroxylase α subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition. BMC Cancer 14:1CrossRefPubMedPubMedCentralGoogle Scholar
  51. Yadav L, Puri N, Rastogi V, Satpute P (2015) Tumour angiogenesis and angiogenic inhibitors: a review. J Clin 9:1–5Google Scholar
  52. Zelenay S, van der Veen AG, Böttcher JP et al (2015) Cyclooxygenase-dependent tumor growth through evasion of immunity. Cell 162:1257–1270CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of ImmunologyAmala Cancer Research Centre (Affiliated to the University of Calicut)ThrissurIndia

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