Advertisement

Journal of Cell Communication and Signaling

, Volume 12, Issue 2, pp 467–478 | Cite as

Novel combination of 2-methoxyestradiol and cyclophosphamide enhances the antineoplastic and pro-apoptotic effects on S-180 ascitic tumour cells

  • Srabantika Mallick
  • Atish Barua
  • Goutam Paul
  • Samarendra Nath BanerjeeEmail author
Research Article
  • 153 Downloads

Abstract

Sarcoma 180 (S-180) tumour cell line is a stable murine tumour cell line with 98–99% stumour takes capacity in Swiss albino mouse - Mus musculus. 2 Methoxyestradiol (2ME) - a promising anti-neoplastic and anti-angiogenic agent, showed toxicity to host body in higher concentration. Cyclophosphamide (CP), the anti-neoplastic agent has long been used as a chemotherapeutic drug for treatment of different cancers. Our studies have shown that the combination effect of 2ME and CP on S-180 tumour cell line is anti-proliferative and less toxic. The treatment with lower concentrations of 2ME and CP (6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) antagonistically increased the life span of tumour bearing mice and synergistically inhibited the viable cell population. 2ME or CP treatment individually induces G2/M arrest. The combination treatment of 2ME + CP (6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) produced a significant increase of cells in the G0 which is the indication of cell arrest or apoptosis. Reduction of cell viability by 2ME + CP treatments is due to apoptotic cell death. This combination therapy produced a significant inhibitory effect of cell proliferation and augmentation of cell accumulation in the G0 phase (i.e. apoptosis). Apoptosis is validated by Fluorescence staining of control and treated S-180 tumour cells with Acridine Orange and EtBr dye. Moreover, a steady increase in the frequency of complex chromosomal aberrations (i.e. tri-, qudri-radial translocations) in tumour cells was noted in that particular concentration of combination therapy treated series along with the increase in dead cell frequency and tumour regression pattern. It is assumed that, these chromosomal abnormalities or damages recorded in higher frequency prevent the affected metaphases to enter into the next cell cycle through apoptosis or necrosis. This study introduces a novel combination, where this particular concentration of 2ME + CP (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) not only enhanced the life span of tumour bearing mouse and decreased the tumour volume antagonistically but also inhibited the viable cell population synergistically, which could serve as a potential effective regimen for cancer treatment.

Keywords

Apoptosis Chromosome Chou-Talalay method Combination effect Combination index Cyclophosphamide 2 Methoxyestradiol S-180 tumour cell line 

Abbreviations

AO

Acridine orange

CP

Cyclophosphamide

EtBr

Ethidium bromide

2ME

2Methoxyestradiol

S-180

Sarcoma 180

CI

Combination Index

Notes

Acknowledgements

Acknowledgement is due to UGC, New Delhi (Ref.F.42-602/2013 (SR) dated 22.03.2013 MRP) for financial support. The paper is dedicated to late Professor Samar Chakrabarti, Cancer Cytogenetic Unit, Department of Zoology, Burdwan University. Authors are grateful to Dr. R.N. Boral and Dr. C.K. Panda, CNCI, for help. Authors are thankful to Dr. Snigdha Banerjee and Prof. Sushanta K. Banerjee, Kansas University Medical Center, Kansas, U.S.A. for encouragement. Authors are also grateful to Dr. Samiran Mondal and Dr. Ashesh Garai, Department of Chemistry, Rammohan College, Kolkata for help. Authors are thankful to the Principal Dr. Saswati Sanyal of Rammohan College, Kolkata for support and encouragement.

References

  1. Adler AD (1982) Cytogenetic Assays of Environmental mutagens. In: Hsu TC (eds) Oxford IBH, New Delhi, pp 249–276Google Scholar
  2. Ahmed AR, Hombal SM (1984) Cyclophosphamide (Cytoxan): A review on relevant pharmacology and clinical uses. J Am Acad Dermatol 11:1115–1126CrossRefPubMedGoogle Scholar
  3. Anton E (1987) Delayed toxicity of cyclophosphamide in normal mice. Br J Exp pathol 68:237–249PubMedPubMedCentralGoogle Scholar
  4. Banerjee S, Kambhampati S, Banerjee SK, Haque I (2011) Pomegranate sensitizes Tamoxifen action in ER-α positive breast cancer cells. J Cell Commun Signal 5:317–324CrossRefPubMedPubMedCentralGoogle Scholar
  5. Banerjee SN (2017) Tumour angiogenesis and anti-angiogenic therapy. Lambert Academic Publishing, GermanyGoogle Scholar
  6. Banerjee SN, Banerjee SK (2005) 2-ME induced tumour angiogenesis inhibition – A new strategy for Cancer treatment. Int J Mol Med 16:S42Google Scholar
  7. Banerjee SN, Banerjee SK (2008) Antiangiogenic therapy – new avenue for cancer treatment. In: Director (eds) Zoological Research in Human Welfare, Zoological Survey of India, Kolkata, pp 267–272Google Scholar
  8. Banerjee SN, Mallick S (2013) Anti-angiogenic therapy on in vivo tumor bearing mouse model system. 3rd International Cancer Research Symposium Kolkata, India December 18-21 2012. J Cell Commun Signal 7(1). doi: 10.1007/s12079-013-0191-9
  9. Banerjee SN, Sengupta K, Banerjee S, Saxena N, Banerjee SK (2003) 2-methoxyestradiol exhibits a biphasic effect on VEGF-A in tumor cells and upregulation is mediated through ER-α: A possible signaling pathway associated with the impact of 2-ME2 on proliferative cells. Neoplasia 5:417–426CrossRefPubMedPubMedCentralGoogle Scholar
  10. Bello V, Goding JW, Greengrass V, Sali A, Dubljevic V, Lenoir C, Trugnan G, Maurice M (2001) Characterization of a di-leucine-based signal in the cytoplasmic tail of the nucleotide-pyrophosphatase NPP1 that mediates basolateral targeting but not endocytosis. Mol Biol Cell 12:3004–3015CrossRefPubMedPubMedCentralGoogle Scholar
  11. Browder T, Butterfield CE, Kräling BM, Shi B, Marshall B, O'Reilly MS, Folkman J (2000) Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60:1878–1886PubMedGoogle Scholar
  12. Brueggemeier RW, Singh U (1989) Inhibition of rat liver microsomal estrogen 2-hydroxylase by 2-methoxyestrogens. J Steroid Biochem 33:589–593CrossRefPubMedGoogle Scholar
  13. Chakrabarti S, Banerjee SN, Ray Choudhuri S (1985) Similar clastogenic sensitivity of mouse and rat somatic chromosomes exposed in vivo to the leaf extract of Lathyrus sativus. Indian J Exp Biol 23:138PubMedGoogle Scholar
  14. Chakrabarti A, Chakrabarti S (1987) High yield of micronuclei and micronuclei premature chromosome condensation in a mouse tumor cell line cultured in vivo with prearrested mitotic metaphases. Neoplasma 34:5Google Scholar
  15. Chakrabarti S, Huda R, Biswas T (1998) Cytogenetic Alterations Associated with the Acquisition of Drug Resistance in a Murine Tumour Cell line. Perspect Cytol Genet 9:165–171Google Scholar
  16. Chou TC (1991) The median-effect principle and the combination index for quantification of synergism and antagonism. In: Chou TC, Rideout DC (eds) Synergism and antagonism in chemotherapy. Academic Press, New York, pp 61–89Google Scholar
  17. Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzym Regul 22:27–55CrossRefGoogle Scholar
  18. Colleoni M, Rocca A, Sandri MT, Zorzino L, Masci G, Nole F, Peruzzotti G, Robertson C, Orlando L, Cinieri S, de Braud F, Viale G, Goldhirsch A (2002) Low dose oral methotrexate and cyclophosphamide in metastatic breast cancer: antitumor activity and correlation with vascular endothelial growth factor levels. Ann Oncol 13:73–80CrossRefPubMedGoogle Scholar
  19. Culo F, Allegretti N, Maruwrc M (1977) Lymphotoxic effect of cyclophosphamide in therapy of Ehrlich ascites carcinoma in mice. J Natl Cancer Inst 58:1759–1764CrossRefPubMedGoogle Scholar
  20. Curtis RE, Boice JD Jr, Stovall M, Bernstein L, Greenberg RS, Flannery JT, Schwartz AG, Weyer P, Moloney WC, Hoover RN (1992) Risk of leukemia after chemotherapy and radiation treatment for breast cancer. N Engl J Med 326:1745–1751CrossRefPubMedGoogle Scholar
  21. D’ Amato RJ, Lin CM, Flynn E, Folkman J, Hamel E (1994) 2-methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site. Proc Natl Acad Sci U S A 91:3964–3968CrossRefPubMedPubMedCentralGoogle Scholar
  22. Das SB, Chakrabarti S (1989) Cytotoxic potential of a drug combination on S-180 tumour model. Perspect Cytol Genet 6:601–606Google Scholar
  23. Dolai N, Karmakar I, Kumar RBS, Bala A, Mazumder UK, Haldar PK (2012) Antitumour potential of Castanopsis indica (Roxb. ex Lindl.) A.DC. leaf extract against Ehrlich’s ascites carcinoma cell. Indian J Exp Biol 50:359–365PubMedGoogle Scholar
  24. Duncan GS, Brenner D, Tusche MW, Brustle A, Knobbe CB, Elia AJ, Mock T, Bray MR, Krammer PH, Mak TW (2012) 2-Methoxyestradiol inhibits experimental autoimmune encephalomyelitis through suppression of immune cell activation. Proc Natl Acad Sci U S A 109:21034–21039CrossRefPubMedPubMedCentralGoogle Scholar
  25. El Naga RN, El-Demerdash E, Youssef SS, Abdel-Naim AB, El-Merzabani M (2009) Cytotoxic effects of 2-methoxyestradiol in the hepatocellular carcinoma cell line HepG2. Pharmacology 84:9–16CrossRefPubMedGoogle Scholar
  26. Folkman J (2008) Angiostatin and Endostatin: Angiogenesis Inhibitors in Blood and Stroma. In: Fiqq WD, Judah F (eds) Angiogenesis An integrative approach from Science to Medicine. Springer, BerlinGoogle Scholar
  27. Friedman OM, Myles A, Colvin M (1979) Cyclophosphamide and related phosphoramide mustards-current status and future prospects. Adv Cancer Chemother 1:143–204Google Scholar
  28. Huang Z, Roychowdhury MK, Waxman DJ (2000) Impact of liver P450 reductase suppression on cyclophosphamide activation, pharmacokinetics and anti-tumoral activity in cytochrome P450 – based cancer gene therapy model. Cancer Gene Ther 7:1034–1042CrossRefPubMedGoogle Scholar
  29. Imreh G, Norberg HV, Imreh S, Zhivotovsky B (2011) Chromosomal breaks during mitotic catastrophe trigger cH2AX–ATM–p53-mediated apoptosis. J Cell Sci 124:2951–2963CrossRefPubMedGoogle Scholar
  30. Kasibhatla S, Amarante-Mendes GP, Finucane D, Brunner T, Bossy-Wetzel E, Green DR (2006) Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis. Cold Spring Harb Protoc. doi: 10.1101/pdb.prot4493
  31. Khan TS, Sundin A, Juhlin C, Wilander E, Oberg K, Eriksson B (2004) Vincristine, cisplatin, teniposide, and cyclophosphamide combination in the treatment of recurrent or metastatic adrenocortical cancer. Med Oncol 21:167–170CrossRefPubMedGoogle Scholar
  32. Kimura M, Tomita Y, Morishita H, Takahashi K (1998) Presence of mucosal change in the urinary bladder in nonhematuric patients with long-term exposure and/or accumulating high-dose Cyclophosphamide, Possible significance of follow-up cystoscopy on preventing development of Cyclophosphamide-induced hemorrhagic cystitis. Urol Int 61:8–11CrossRefPubMedGoogle Scholar
  33. Klauber N, Parangi S, Flynn E, Hamel E, D’Amato RJ (1997) Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and Taxol. Cancer Res 57:81–86PubMedGoogle Scholar
  34. Krishnamoorthy D, Mirunalini S (2016) Antiproliferative and apoptotic effect of Pleurotus ostreatus on human mammary carcinoma cell line (michigan cancer foundation-7). Cancer Transl Med 2:95–104CrossRefGoogle Scholar
  35. Kuan L, Peng-cheng L, Run L, Xing W (2015) Dual AO/EB Staining to Detect Apoptosis in Osteosarcoma Cells Compared with Flow Cytometry. Med Sci Monit Basic Res 21:15–20CrossRefGoogle Scholar
  36. LaVallee TM, Zhan XH, Johnson MS, Herbstritt CJ, Swartz G, Williams MS, Hembrough WA, Green SJ, Pribluda VS (2003) 2-methoxyestradiol up-regulates death receptor 5 and induces apoptosis through activation of the extrinsic pathway. Cancer Res 63:468–475PubMedGoogle Scholar
  37. Mallick S, Paul G, Banerjee SN (2015a) Effect of 2-Methoxyestradiol and Cyclophosphamide on S-180 Mouse Tumour Model System ‘15 34th Annual Convention of Indian Association for Cancer Research, Jaipur, India, February 19–21, 178Google Scholar
  38. Mallick S, Paul G, Banerjee SN (2015b) Effect of 2-Methoxyestradiol (2ME) an anti-angiogenic agent on in vivo tumour bearing mouse. Issue Biol Sci Pharm Res 3:63–70Google Scholar
  39. Nowell PC, Hungerford DA, Cole LJ (1964) Chromosome Changes Following Irradiation In Mammals. Ann N Y Acad Sci 114:252–258CrossRefPubMedGoogle Scholar
  40. Pal AK, Neogi LN, Chakrabarti A, Chakrabarti S (1984) C-band-like effect produced by mitomycin C on mouse ascites tumour chromosomes in vivo. Indian J Exp Biol 22:61–62PubMedGoogle Scholar
  41. Panse VG, Sukhatme PV (1985) Statistical methods for agricultural workers. Indian Council of Agricultural Research, New Delhi, pp 1–359Google Scholar
  42. Patel JM (1987) Stimulation of cyclophosphamide-induced pulmonary microsomal lipid peroxidation by oxygen. Toxicology 45:79–91CrossRefPubMedGoogle Scholar
  43. Prasad B, Giri A (1994) Antitumor effect of cisplatin against murine ascites Dalton’s lymphoma. Indian J Exp Biol 32:155–162PubMedGoogle Scholar
  44. Pribluda VS, Gubish ER, LaVallee TM Jr, Treston A, Swartz GM, Green SJ (2000) 2-methoxyestradiol: An endogenous Antiangiogenic and antiproliferative drug candidate. Cancer Metastasis Rev 19:173–179CrossRefPubMedGoogle Scholar
  45. Ray G, Banerjee S, Saxena NK, Campbell DR, Veldhuizen PV, Banerjee SK (2005) Stimulation of MCF- 7 tumour progression in athymic nude mice by 17β-estradiol induces WISP-2/CCN5 expression in xenografts: A novel signaling molecule in hormonal carcinogenesis. Oncol Rep 13:445–448PubMedGoogle Scholar
  46. Sattler M, Salgia R (2003) Molecular and cellular biology of small cell lung cancer. Semin Oncol 30:57–71CrossRefPubMedGoogle Scholar
  47. Shand FL (1979) The immunopharmacology of cyclophosphamide. Int J Immunopharmacol 1:165–171CrossRefPubMedGoogle Scholar
  48. Shokrzadeh M, Ahmadi A, Naghshvar F, Chabra A, Jafarinejhad M (2014) Prophylactic Efficacy of Melatonin on Cyclophosphamide-Induced Liver Toxicity in Mice. Bio Med Research International, Hindawi Publishing Corporation Article ID 470425:1-6Google Scholar
  49. Yamamoto M, Maehara Y, Oda S, Ichiyoshi Y, Kusumoto T, Sugimachi K (1999) The p53 tumour suppressor gene in anticancer agent induced apoptosis and chemosensitivity of human gastrointestinal cancer cell lines. Cancer Chemother Pharmacol 43:43–49CrossRefPubMedGoogle Scholar
  50. Zhu BT, Conney AH (1998a) Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis? Cancer Res 58:2269–2277PubMedGoogle Scholar
  51. Zhu BT, Conney AH (1998b) Functional role of estrogen metabolism in target cells: review and prospectives. Carcinogenesis 19:1–27CrossRefPubMedGoogle Scholar
  52. Zoubine MN, Weston AP, Johnson DC, Campbell DR, Banerjee SK (1999) 2-Methoxyestradiol-induced growth suppression and lethality in estrogen-responsive MCF-7 cells may be mediated by down regulation of p34cdc2 and cyclin B1 expression. Int J Oncol 15:639–646PubMedGoogle Scholar

Copyright information

© The International CCN Society 2017

Authors and Affiliations

  • Srabantika Mallick
    • 1
  • Atish Barua
    • 2
  • Goutam Paul
    • 3
  • Samarendra Nath Banerjee
    • 1
    Email author
  1. 1.Department of ZoologyRammohan CollegeKolkataIndia
  2. 2.Chittaranjan National Cancer InstituteKolkataIndia
  3. 3.Department of PhysiologyUniversity of KalyaniKalyaniIndia

Personalised recommendations