Asian Pacific Journal of Cancer Prevention

Asian Pacific Journal of Cancer Prevention (아시아태평양암예방학회)
권호 표지
DOI QR코드

DOI QR Code

Molecular Mechanism of Crocin Induced Caspase Mediated MCF-7 Cell Death: In Vivo Toxicity Profiling and Ex Vivo Macrophage Activation

  • Bakshi, Hamid A (Department of Food Science and Human Nutrition, College of Applied science, A'Sharqiyah University) ;
  • Hakkim, Faruck Lukmanul (Biology Division, Department of Basic science, College of Applied science, A'Sharqiyah University) ;
  • Sam, Smitha (Department of Research Jawaharlal Nehru Cancer hospital and Research Center)
  • Published : 2016.04.11
Download PDF
⟨ Previous Next ⟩

Abstract

Background: Crocus sativus and its major constituent crocin are well established to have anti-cancer properties in breast cancer cells (MCF-7). However the role of C. sativus extract (CSE) and crocin on caspase signaling mediated MCF-7 cell death at molecular level is remains unclear. In this study, we tried to unravel role of CSE and crocin on caspase mediated MCF-7 cells death and their in vivo preclinical toxicity profiling and immune stimulatory effect. Materials and Methods: CSE extract was fractionated by HPLC and crocin was isolated and characterized by NMR, IR, and MS. MCF-7 cells were treated with both CSE and crocin and expression of Bcl-2 and Bax was assessed after 24 and 36 hours. Furthermore, caspase 3, caspase 8 and caspase 9 expression was determined by Western blotting after 24 hours of treatment. DNA fragmentation analysis was performed for genotoxicity of CSE and crocin in MCF-7 cells. The in vivo toxicity profile of CSE (300 mg/kg of b.wt) was investigated in normal Swiss albino mice. In addition, peritoneal macrophages were collected from crocin (1, 1.5 and 2 mg/kg body weight) treated mice and analyzed for ex vivo yeast phagocytosis. Results: Immunoblot analysis revealed that there was time dependent decline in anti-apoptotic Bcl-2 with simultaneous upregulation of Bax in CSE and crocin treated MCF-7 cells. Further CSE and crocin treatment downregulated caspase 8 and 9 and cleaved the caspase 3 after 24 hours. Both CSE and crocin elicited considerable DNA damage in MCF-7 cells at each concentration tested. In vivo toxicity profile by histological studies revealed no observable histopathologic differences in the liver, kidney, spleen, lungs and heart in CSE treated and untreated groups. Crocin treatment elicited significant dose and time dependent ex vivo yeast phagocytosis by peritoneal macrophages. Conclusions: Our study delineated involvement of pro-apoptotic and caspase mediated MCF-7 cell death by CSE and crocin at the molecular level accompanied with extensive DNA damage. Further we found that normal swiss albino mice can tolerate the maximum dose of CSE. Crocin enhanced ex vivo macrophage yeast phagocytic ability.

Keywords

References

  1. Abdullaev FI (2002). Cancer chemopreventive and tumoricidal properties of saffron (Crocus sativus L.). Exp Biol Med, 227, 20-25. https://doi.org/10.1177/153537020222700104
  2. Abdullaev FI, Espinosa-Aguirre JJ (2004). Biomedical properties of saffron and its potential use in cancer therapy and chemoprevention trials. Cancer Detec Prev, 28, 426-432. https://doi.org/10.1016/j.cdp.2004.09.002
  3. Abe K, Saito H (2000). Effects of saffron extract and its constituent crocin on learning behaviour and long-term potentiation. Phytother Res, 14, 149-52. https://doi.org/10.1002/(SICI)1099-1573(200005)14:3<149::AID-PTR665>3.0.CO;2-5
  4. Aung HH, Wang CZ, Ni M, et al (2007). Crocin from Crocus sativus possesses significant anti-proliferation effects on human colorectal cancer cells. Exp Oncol, 29, 175-80.
  5. Bakshi H, Touseef T, Fassal G, et al (2012). Crocus sativus L. prevents progression of cell growth and enhances cell toxicity in human breast cancer and lung cancer cell lines. Int J pharma Life Sci, 2, 120-4.
  6. Bakshi H, Sam S, Feroz A, et al (2009). Crocin from Kashmiri saffron (Crocus sativus) induces in vitro and In vivo xenograft growth inhibition of dalton's lymphoma in mice. Asian Pac J Cancer Prev, 10, 887-90.
  7. Bakshi H, Sam S, Rozati R, et al (2010). DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from khashmiri saffron in human pancreatic cancer cell line. Asian Pac J Cancer Prev, 11, 675-9.
  8. Bakshi H, Sam S, Sharma M, et al (2008). Assessment of cytotoxic and apoptogenic activity of Crocus sativus on Human cancer cell lines. Ind J Applied Life Sci, 4, 34-38.
  9. Carol ED, Chun CL, Angela BM, et al (2014). Cancer Treatment and Survivorship Statistics, 2014. CA Cancer J Clin, 64, 252-71. https://doi.org/10.3322/caac.21235
  10. Chen Y, Zhang H, Tian X, et al (2008). Antioxidant potential of crocins and ethanol extracts of Gardenia jasminoides ELLIS and Crocus sativus L.: a relationship investigation between antioxidant activity and crocin contents. Food Chem, 109, 484-92. https://doi.org/10.1016/j.foodchem.2007.09.080
  11. Cohen GM, Sun XM, Fearnhead H, et al (1994). Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes. J Immunol, 153, 507-16.
  12. De Groote MA, Fang FC (1995). NO inhibitions: antimicrobial properties of nitric oxide. Clin Infect Dis, 21, 162-5. https://doi.org/10.1093/clinids/21.1.162
  13. Deng Y, Guo ZG, Zeng ZL, et al (2002). Studies on the pharmacological effects of saffron (Crocus sativus L.) - A review. Zhongguo Zhong Yao Za Zhi, 27, 565-8.
  14. Dong X, Yang X, Xie K, et al (1995). Activation of inducible nitric oxide synthase gene in murine macrophages requires protein phosphatase 1 and 2A activities. J Leuko Biol, 58, 725-32. https://doi.org/10.1002/jlb.58.6.725
  15. Escribano J, Diaz-Guerra MJ, Riese HH, et al (1999). In vitro activation of macrophages from corms of Crocus sativus L. Cancer Lett, 144, 107-14. https://doi.org/10.1016/S0304-3835(99)00211-6
  16. He SY, Qian ZY, Tang FT, et al (2005). Effect of crocin on experimental atherosclerosis in quails and its mechanisms. Life Sci, 77, 907-21. https://doi.org/10.1016/j.lfs.2005.02.006
  17. Hosseinzadeh H, Ziaee T, Sadeghi A (2008). The effect of saffron, Crocus sativus stigma, extract and its constituents, safranal and crocin on sexual behaviors in normal male rats. Phytomedicine, 15, 491-95. https://doi.org/10.1016/j.phymed.2007.09.020
  18. Janeway CA, Medzhitov R (2002). Innate immune recognition. Ann Rev Immunol, 20, 197-216. https://doi.org/10.1146/annurev.immunol.20.083001.084359
  19. Jorgensen LV, Andersen HJ, Skibsted, LH (1997). Kinetics of reduction of hypervalent iron in myoglobin by crocin in aqueous solution. Free Rad Res, 27, 73-87. https://doi.org/10.3109/10715769709097840
  20. Jun CD, Yoon HJ, Park YC, et al (1996). Synergistic cooperation between thapsigargin and phorbol ester for induction of nitric oxide synthesis in murine peritoneal macrophages. Free Rad Biol Med, 20, 769-76. https://doi.org/10.1016/0891-5849(95)02157-4
  21. Kim T, Jung U, Cho DY, et al (2001). Se-methylselenocysteine induces apoptosis through caspase activation in HL-60 cells. Carcinogenesis, 22, 559-65. https://doi.org/10.1093/carcin/22.4.559
  22. Kumar S, Ikram AB, Adel Al-Ajmi, et al (2011). Changing trends of breast cancer survival in sultanate of oman. J Oncol, 1-7.
  23. Landen JW, Lang R, McMahon SJ, et al (2002). Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma. Cancer Res, 64, 4109-14.
  24. Liakopoulou-Kyriakides M, Skubas AI (1990). Characterization of the platelet aggregation inducer and inhibitor isolated from Crocus sativus. Biochem Int, 22, 103-10.
  25. Llambi F, Green DR (2011). Apoptosis and oncogenesis: give and take in the Bcl-2 family. Curr Opin Genet Dev, 21, 12-20. https://doi.org/10.1016/j.gde.2010.12.001
  26. Lussignoli S, Fraccaroli M, Andrioli G, et al (1999). A microplate based colorimetric assay of the total peroxyl radical trapping capability of human plasma. Anal Biochem, 269, 38-44. https://doi.org/10.1006/abio.1999.4010
  27. Lv CF, Luo CL, Ji HY, et al (2008). Influence of crocin on gene expression profile of human bladder cancer cell lines T24. Zhongguo Zhong Yao Za Zhi, 33, 1612-7.
  28. Martini FA (2004). Fundamentals of Anatomy and Physiology. 6th ed. New York: Benjamin Cummings, 791-812.
  29. Mousavi S, Afshari JT, Brook A (2008). Study of Cytotoxic Effects of Saffron in MCF-7 Cells. Iranian J Pharm Sci, 4, 261-268.
  30. Mousavi SH, Seyed Adel Moallem, Soghra Mehri, et al (2011). Improvement of cytotoxic and apoptogenic properties of crocin in cancer cell lines by its nanoliposomal form. Pharm Biol, 49, 1039-45. https://doi.org/10.3109/13880209.2011.563315
  31. Mousavi SH, Tavakkol-Afshari J, Brook A, et al (2009a). Direct toxicity of Rose Bengal in MCF-7 cell line: Role of apoptosis. Food Chem Toxicol, 47, 855-859. https://doi.org/10.1016/j.fct.2009.01.018
  32. Mousavi SH, Tavakkol-Afshari J, Brook A, et al (2009b). Role of caspases and Bax protein in saffron-induced apoptosis in MCF-7 cells. Food Chem Toxicol, 47, 1909-13. https://doi.org/10.1016/j.fct.2009.05.017
  33. Narayanan K, Balakrishnan A, Miyamoto S (2000). NF-kappaB is essential for induction of pro-inflammatory cytokine genes by filarial parasitic sheath proteins. Mol Immunol, 37, 115-23. https://doi.org/10.1016/S0161-5890(00)00039-0
  34. Nathan CF (1987). Secretory products of macrophages. J Clin Invest, 79, 319-26. https://doi.org/10.1172/JCI112815
  35. Nicoletti I, Migliorati G, Pagliacci MC, et al (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Meth, 139, 271-9. https://doi.org/10.1016/0022-1759(91)90198-O
  36. Nikouei BM, Mousavi SH, Shahsavand S, et al (2013). Assessment of Cytotoxic Properties of Safranal and Nanoliposomal Safranal in Various Cancer Cell Lines. Phytother Res, 27, 1868-73. https://doi.org/10.1002/ptr.4945
  37. Ouyang L, Shi Z, Zhao S, et al (2012). Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Proliferat, 45, 487-98. https://doi.org/10.1111/j.1365-2184.2012.00845.x
  38. Park YC, Jun CD, Kang HS, et al (1996). Role of intracellular calcium as a priming signal for the induction of nitric oxide synthesis in murine peritoneal macrophages. Immunol, 87, 296-302. https://doi.org/10.1046/j.1365-2567.1996.456544.x
  39. Pitsikas N, Boultadakis A, Georgiadou G, et al (2008). Effects of the active constituents of Crocus sativus L., crocins, in an animal model of anxiety. Phytomedicine, 15, 1135-9. https://doi.org/10.1016/j.phymed.2008.06.005
  40. Raddassi K, Berthon B, Petit JF, et al (1994). Role of calcium in the activation of mouse peritoneal macrophage: induction of NO synthase by calcium ionophore and thapsigargin. Cell Immunol, 153, 443-55. https://doi.org/10.1006/cimm.1994.1041
  41. Rios JL, Recio MC, Giner RM, et al (1996). An update review of saffron and its active constituents. Phytother Res, 10, 189-93. https://doi.org/10.1002/(SICI)1099-1573(199605)10:3<189::AID-PTR754>3.0.CO;2-C
  42. Salomi MJ, Nair SC, Panikkar PR (1991). Inhibitory effects of Nigela sativa and saffron (Crocus sativus) on chemical carcinogenesis in mice. Nutr Cancer, 16, 67-72. https://doi.org/10.1080/01635589109514142
  43. Samarghandian S, Borji A (2014). Anticarcinogenic effect of saffron (Crocus sativus L.) and its ingredients. Pharmacognosy Res, 6, 99-107. https://doi.org/10.4103/0974-8490.128963
  44. Shamas-Din A, Brahmbhatt H, Leber B, et al (2011). BH3-only proteins: orchestrators of apoptosis. Biochim Biophys Acta, 1813, 508-20. https://doi.org/10.1016/j.bbamcr.2010.11.024
  45. Sheng L, Qian Z, Zheng S, et al (2006). Mechanism of hypolipidemic effect of crocin in rats: crocin inhibits pancreatic lipase. Eur J Pharmacol, 543, 116-22. https://doi.org/10.1016/j.ejphar.2006.05.038
  46. Tavakkol-Afshari J, Brook A, Mousavi SH (2008). Study of cytotoxic and apoptogenic properties of saffron extract in human cancer cell lines. Food Chem Toxicol, 46, 3443-7. https://doi.org/10.1016/j.fct.2008.08.018
  47. Vento S, Cainelli F (2003). Infections in patients with cancer undergoing chemotherapy: aetiology, prevention, and treatment. Lancet Oncol, 4, 595-604. https://doi.org/10.1016/S1470-2045(03)01218-X

Cited by

  1. L. Causes a Non Apoptotic Calpain Dependent Death in C6 Rat Glioma Cells, Exhibiting a Synergistic Effect with Temozolomide pp.1532-7914, 2019, https://doi.org/10.1080/01635581.2018.1506493