Indian toad (Bufo melanostictus, Schneider) skin extract induces apoptosis and shows cytotoxic effect on Ehrlich ascites carcinoma (EAC) cells

  • Biplab Giri
  • Sananda Dey Department of Physiology, University of Gour Banga, Mokdumpur, Malda 732103, India
  • Antony Gomes Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92 APC Road, Kolkata 700 009, India;

Abstract

Indian toad (Bufo melanostictus) skin extract (TSE) was evaluated for cytotoxic and apoptogenic activities on Ehrlich ascites carcinoma (EAC) cells of EAC bearing Swiss albino male mice. LD50 of TSE was found to be 400 mg kg-1, (i.v.) and 750 mg kg-1, (i.p.) in mice. EAC cells (1-2 ´ 105 / mouse) were inoculated (i.p.) into mice (20.6 ± 0.14 gm) and the status of EAC cell proliferation and viability were studied in control and TSE treated groups. TSE (10, 20, 30 mg kg-1 d-1, i.p. for 10 d) significantly inhibit EAC cell growth in dose dependent manner. TSE (50, 100 mg kg-1 d-1, i.p. for 1 d) significantly reduced the viability of EAC cells and decreased the MTT values compared to control cells. TSE induced DNA breakage was reflected in DNA ladder and single cell gel electrophoresis (comet assay). TSE significantly (P<0.001) increased the number tailed cells and length-width ratio of DNA mass in EAC cells as compared to control. Fluorescent microscopy of TSE treated cells showed significant increase in number of early and late apoptotic cells compared to the control EAC cells. Apoptotic index of TSE treated cells was significantly (P<0.001) higher than that of the control cells. Changes in serum LDH, β2-microglobulin in TSE treated EAC mice and our earlier studies indicated the involvement of immunomodulation which may indirectly be associated with the anticancer activity of TSE.


Keywords: Toad skin extract; Bufo melanostictus; Ehrlich ascites carcinoma; Cytotoxic agent;     

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Author Biographies

Biplab Giri
Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology,  University of Calcutta,  92 APC Road,  Kolkata 700 009, India;
Sananda Dey, Department of Physiology, University of Gour Banga, Mokdumpur, Malda 732103, India

Department of Physiology, University of Gour Banga, Mokdumpur, Malda 732103, India

Antony Gomes, Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92 APC Road, Kolkata 700 009, India;
Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology,  University of Calcutta,  92 APC Road,  Kolkata 700 009, India;

References

1. Schweizer F, Cationic amphiphilic peptides with cancerselective toxicity. Eur J Pharmacol 2009; 625:190-194.
2. Maciel NM, Schwartz CA, Junior OR, Sebben A, Castro MS, Sousa MV, et al. Composition of indole alkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids with phylogenetic implications. Comp Biochem Physiol Part B: Biochem Mol Biol 2003; 134:641-649.
3. Lin L, Ren L, Wen L, Wang Y, Qi J. Effect of evodiamine on the proliferation and apoptosis of A549 human lung cancer cells. Molecular medicine reports. 2016; 14(3):2832-2838.
4. Liu C, Cao W, Chen Y, Qu D, Zhou J. Comparison of toad skins Bufo bufo gargarizans Cantor from different regions for their active constituents content and cytotoxic activity on lung carcinoma cell lines. Pharmacognosy magazine. 2014; 10(39):207.
5. Ferreira PM, Lima DJ, Debiasi BW, Soares BM, da Conceição Machado K, da Costa Noronha J, de Jesus Rodrigues D, Sinhorin AP, Pessoa C, Júnior GM. Antiproliferative activity of Rhinella marina and Rhaebo guttatus venom extracts from Southern Amazon. Toxicon. 2013; 72:43-51.
6. Dahham SS, Hew CS, Jaafar IB, Gam LH. The Protein Profiling of Asian Giant Toad Skin Secretions And Their Antimicrobial Activity. International Journal of Pharmacy and Pharmaceutical Sciences. 2016; 8:88-95.
7. Clarke BT. The natural history of amphibian skin secretions, there normal functioning and potential medical applications. Biol. Rev. 1997; 72: 365-379.
8. Lu CX, Nan KJ, Lei Y. Agents from amphibians with anticancer properties. Anti-cancer drugs. 2008; 19(10):931-939.
9. Oelkrug C, Hartke M, Schubert A. Mode of action of anticancer peptides (ACPs) from amphibian origin. Anticancer research. 2015; 35(2):635-643.
10. Nakata M, Kawaguchi S, Oikawa A, Inamura A, Nomoto S, Miyai H, Nonaka T, Ichimi S, Fujita-Yamaguchi Y, Luo C, Gao B. An aqueous extract from toad skin prevents gelatinase activities derived from fetal serum albumin and serum-free culture medium of human breast carcinoma MDA-MB-231 cells. Drug discoveries & therapeutics. 2015; 9(6):417-421.
11. Nakata M, Mori S, Kamoshida Y, Kawaguchi S, Fujita-Yamaguchi Y, Gao B, Tang W. Toad skin extract cinobufatini inhibits migration of human breast carcinoma MDA-MB-231 cells into a model stromal tissue. Bioscience trends. 2015; 9(4):266-269.
12. Ko WS, Park TY, Park C, Kim YH, Yoon HJ, Lee SY, Hong SH, Choi BT, Lee YT, Choi YH. Induction of apoptosis by Chan Su, a traditional Chinese medicine, in human bladder carcinoma T24 cells. Oncol. Rep. 2005; 14(2):475-480.
13. Fushimi R, Koh T, Iyama S, Yasuhara M, Tachi J, Kohda K, Amino N, Miyai K. Digoxin-like immunoreactivity in Chinese medicine. Ther Drug Monit. 1990; 12(3):242-245.
14. Craig SB, Tara LP, John HB, Michael JT. Host Defence Peptides from the Skin Glands of Australian Amphibians. Caerulein Neuropeptides and Antimicrobial, Anticancer, and nNOS Inhibiting Citropins from the Glandular Frog Litoria subglandulosa. Australian J Chem. 2004; 57(7):693-701.
15. Nogawa T, Kamano Y, Yamashita A, Pettit GR. Isolation and structure of five new cancer cell growth inhibitory bufadienolides from the Chinese traditional drug Ch'an Su. J Nat Prod. 2001; 64(9):1148-1152.
16. Das M, Dasgupta SC, Gomes A. Immunomodulatory and antineoplastic activity of common Indian toad (Bufo melanostictus, Schneider) skin extract. Ind J Phamcol. 1998; 30:311-317.
17. Giri B, Gomes A, Debnath A, Saha A, Biswas AK, Dasgupta SC. Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract on U937 and K562 cells. Toxicon. 2006; 48(4):388-400.
18. Gomes A, Giri B, Kole L, Saha A, Debnath A, Gomes A. A crystalline compound (BM-ANF1) from the Indian toad (Bufo melanostictus, Schneider) skin extract, induced antiproliferation and apoptosis in leukemic and hepatoma cell line involving cell cycle proteins. Toxicon. 2007; 50(6):835-849.
19. Giri B, Gomes A, Sengupta R, Banerjee S, Nautiyal J, Sarkar FH, Majumdar AP. Curcumin synergizes the growth inhibitory properties of Indian toad (Bufo melanostictus Schneider) skin-derived factor (BM-ANF1) in HCT-116 colon cancer cells. Anticancer Res. 2009; 29(1):395-401.
20. Bhattacharjee P, Giri B, Gomes A. Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin. Toxicon. 2011; 57(2):225-236
21. Gomes A, Giri B, Alam A, Mukherjee S, Bhattacharjee P, Gomes A. Anticancer activity of a low immunogenic protein toxin (BMP1) from Indian toad (Bufo melanostictus, Schneider) skin extract. Toxicon. 2011; 58(1):85-92.
22. Fernandes D.J. and Klubes, P., 1979. A biochemical and pharmacological study of therapeutic system with 5- fluorouracil plus cyclophosphamide in murine L1210 leukemia. Cancer Research. 39; 1396-1404.
23. Sur P, Ganguly DK. Tea plant root extract (TRE) as antineoplastic agent. Planta Medica. 1994; 60: 106-109.
24. Kawada K, Yonei T, Ueoka H, Kiura K, Tabata M, Takigawa N, Harada M, Tanimoto M. Comparison of chemosensitivity tests: clonogenic assay versus MTT assay. Acta Med. Okayama. 2002; 56(3):129-134.
25. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 1988; 175:184-191.
26. Gong J, Traganos F, Darzynkie WZ. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem. 1994; 218:314-319.
27. Leu J, Shen HM, Ong CN. Salvia miltiorrhiza inhibits cell growth and induces apoptosis in human hepatoma HepG2 cells. Cancer letters. 2000; 153(1-2):85-93.
28. Fotakis G, Timbrell JA. In vitro cytotoxicity assay: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters. 2006; 160:171-177.
29. Ko WS, Park TY, Park C, Kim YH, Yoon HJ, Lee SY, Hong SH, Choi BT, Lee YT, Choi YH. Induction of apoptosis by Chan Su, a traditional Chinese medicine, in human bladder carcinoma T24 cells. Oncol Rep. 2005; 14:475-480.
30. Yun HR, Yoo HS, Shin DY, Hong SH, Kim JH, Cho CK, Choi YH. Apoptosis induction of human lung carcinoma cells by Chan Su (Venenum Bufonis) through activation of caspases. J Acupunct Meridian Stud. 2009; 2:210-217.
31. Lu CX, Wang WY, Ma N, Cui Y, Li XY, Zhou Y. Anticancer peptide from Chinese toad (Bufo Bufo Gargarizans) skin enhanced sensitivity to 5-Fu in hepatocarcinoma cells (HepG2). Clin Oncol. Cancer Res. 2011; 8: 149-154
32. Gopalakrishna P, Khar A. Comet assay to measure DNA damage in apoptotic cells. J. Biochem. Biophys. Methods. 1995; 30:69-73.
33. Olive PL, Banath JP. Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent. Exp. Cell Res. 1995; 221: 19-26.
34. Robert SM, Chen B, Eric WA, Joseph LR. Detection of DNA damage by the alkaline comet assay after exposure to low-dose gamma radiation. Radiation Research. 1998; 149:396-400.
35. Watabe M, Kawazoe N, Masuda Y, Nakajo S, Nakaya K. Bcl-2 protein inhibits bufalin- induced apoptosis through inhibition of mitogen-activated protein kinase activation in human leukemia U937 cells. Cancer Res. 1997; 57(15): 3097-3100.
36. Lee JS, Dixon DO, Kantarjian HM, Keating MJ, Talpaz M. Prognosis of chronic lymphocytic leukemia: a multivariate regression analysis of 325 untreated patients. Blood. 1987; 69:929.
37. Molica S, Levato D, Cascavilla N, Levato L, Musto P. Clinico-prognostic implications of simultaneous increased serum levels of soluble CD23 and beta2-microglobuline in B-cell chronic lymphocytic leukemia. Eur J Haematol. 1999; 62:117.
38. Hallek M, Wanders L, Ostwald M, et al. Serum beta2-microglobulin and serum thymidine kinase are independent predictors of progression free survival in chronic lymphocytic leukemia and immunocytoma. Leuk Lymphoma. 1996; 22:439.
39. Koi M, Saito M, Ebina T, Ishida N.Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice. Microbiol Immunol. 1981; 25(6):565-574.
40. Klimp AH, De Vries EGE, Scherphof GL, Daemen T. A potential role of macrophage activation in the treatment of cancer. Critical Reviews in Oncology Hematology. 2002; 44:143-161.
41. Shukla Y, Verma AS, Mehrotra NK, Ray PK. Antitumor activity of protein A in a mouse skin model of two-stage carcinogenesis. Cancer Letters.1996; 103:41-47.
42. de Wit R, Bakker PJ, Reiss P, Hoek FJ, Lange JM, Goudsmit J, Veenhof KH. Temporary increase in serum beta 2-microglobulin during treatment with interferon-alpha for AIDS- associated Kaposi’s sarcoma. AIDS.1990; 4(5):459-462.
43. Richard AG, Thomas JK, Barbara AO. Kuby Immunology, 4th ed. New York: W.H. Freeman and Company, 2002:178-180.
44. Klimp AH, De Vries EGE, Scherphof GL, Daemen T. A potential role of macrophage activation in the treatment of cancer. Critical Reviews in Oncology Hematology. 2002; 44:143-161.
45. Bass KK, Mastrangelo MJ. Immunopotentiation with low dose cyclophosphamide in the active specific immunotherapy of cancer. Cancer Immunol Immunother. 1998; 47:1-12.
46. Lasek W, Giermasz A, Kuc K, Wankowicz A, Feleszko W, Golab J, et al. Potentiation of the anti-tumor effect of Actinomycin D by tumor necrosis factor  in mice: correlation between in vitro and in vivo results. Int J Cancer. 1996; 66:374-379.
47. Hamuro J, Kikuchi MT, Takatsuki F, Suzuki M. Cancer cell progression and chemoimmunotherapy-dual effects in the reduction of resistance to the therapy. Br J Cancer. 1996; 73:465-471.
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How to Cite
Giri, B., Dey, S., & Gomes, A. (2018). Indian toad (Bufo melanostictus, Schneider) skin extract induces apoptosis and shows cytotoxic effect on Ehrlich ascites carcinoma (EAC) cells. Journal of Drug Delivery and Therapeutics, 8(5), 303-312. https://doi.org/10.22270/jddt.v8i5.1873