The effect of CTB on P53 protein acetylation and consequence apoptosis on MCF-7 and MRC-5 cell lines

Authors

Department of Anatomical Sciences and Molecular Biology, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: P300 is a member of the mammalian histone acetyl transferase (HAT) family, an enzyme that acetylates histones and several non-histone proteins including P53 (the most important tumor suppressor gene) during stress, which plays an important role in the apoptosis of tumor cells. Hereby, this study describes the potency of CTB (Cholera Toxin B subunit) as a P300 activator to induce apoptosis in a breast cancer cell line (MCF-7) and a lung fibroblast cell line (MRC-5) as a non-tumorigenic control sample.
Materials and Methods: MCF-7 and MRC-5 were cultured in RPMI-1640 and treated with or without CTB at a concentration of 85.43 μmol/L, based on half-maximal inhibitory concentration (IC50) index at different times (24, 48 and 72 h). The percentage of apoptotic cells were measured by flow cytometry. Real-time quantitative RT-PCR was performed to estimate the mRNA expression of P300 in MCF-7 and MRC-5 with CTB at different times. ELISA and Bradford protein techniques were used to detect levels of total and acetylated P53 protein generated in MCF-7 and MRC-5.
Results: Our findings indicated that CTB could effectively induce apoptosis in MCF-7 significantly higher than MRC-5. We showed that expression of P300 was up-regulated by increasing time of CTB treatment in MCF-7 but not in MRC-5 and the acetylated and total P53 protein levels were increased more in MCF-7 cells than MRC-5.
Conclusion: CTB could induce acetylation of P53 protein through increasing expression of P300 and consequently induce the significant cell death in MCF-7 but it could be well tolerated in MRC-5. Therefore, CTB could be used as an anti-cancer agent.

Keywords

 
1. Zou XM, Li YL, Wang H, Cui W, Li XL, Fu SB, et al. Gastric cancer cell lines induced by Trichostatin A. World J Gastroenterol 2008;14:4810-5.  Back to cited text no. 1
    
2. Giahuai T, Shundong C, Yuehua M, Richard LP, Delong L. Novel histone deacetylase inhibitors in clinical trials as anti-cancer agents. Hematol Oncol 2010;3:15-28.  Back to cited text no. 2
    
3. Lara E, Mai A, Calvanese V, Altucci L, Lopez P, Chantar ML, et al. Salermide, a sirtuin inhibitor with a strong cancer-specific proapoptic effect. Oncogene 2009;28:781-91.  Back to cited text no. 3
    
4. Dworkin AM, Hung TH, Toland AE. Epigenetic alteration in breast: Implication for breast cancer detection, prognosis and treatment. Semin Cancer Biol 2009;19:165-71.  Back to cited text no. 4
    
5. Isharwal S, Miller MC, Marlow C, Makarov M, Partin AW, Veltri RW. P300 (histone acetyl transferase) biomarker predicts prostate cancer biochemical recurrence and correlates with changes in epithelia nuclear size and shape. Prostate 2008;68:1097-104.  Back to cited text no. 5
    
6. Vempati RK, Jayani RS, Notani D, Sengupta A, Galande S, Haldar D. P300-mediated acetylation of histone H3 lysine 56 functions in DNA damage response in mammals. J Biol Chem 2010;285:28553-64.  Back to cited text no. 6
    
7. Doran D, Shimuizo H, Perkins N, Hupp T. DNA-dependent acetylation of P53 by the transcription coactivators P300. J Biol Chem 2003;278:13431-41.  Back to cited text no. 7
    
8. Emanuele S, Lauricella M, Tesoriere GN. Histone deacetylase inhibitors: Apoptotic effects and clinical implications. Int J Oncol 2008;33:637-64.  Back to cited text no. 8
    
9. Cazzalini O, Perucca P, Savio M, Necchi D, Bianchi L, Stivala LA, et al. Interaction of P21 with PCANA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair. Nucleic Acids Res 2008;36:1713-22.  Back to cited text no. 9
    
10. Huanh W, Chen C. Akt phosphorylation of P300 at Ser-1834 is essential for its histone acetyltransferases and transcriptional activity. Mol Cell Biol 2005;25:6592-602.  Back to cited text no. 10
    
11. Polley S, Guha S, Roy NS, Kar S, Sakaguchi K, Chuman Y, et al. Differential recognition of phosphorylated transactivation domains of P53 by different p300 domains. J Mol Biol 2008;376:8-12.  Back to cited text no. 11
    
12. Kim HJ, Bae SC. Histone deacetylase inhibitors: Molecular mechanisms of action and clinical trials as anti-cancer drugs. Am J Transl Res 2011;3:166-79.  Back to cited text no. 12
    
13. Cazzalini O, Perucca P, Savio M, Necchi D, Stivala LA, Ducommun B, et al. Interaction of P21CDKN1A with PCNA regulates the histone acetyltransferases activity of P300 in nucleotide excision repair. Nucleic Acids Res 2008;36:1713-22.  Back to cited text no. 13
    
14. Ozaki T, Nakagawara A. P53: The Attractive Tumor Suppressor in the cancer research field. J Biomed Biotechnol 2011;10:11-23.  Back to cited text no. 14
    
15. Leeuwen I, Lain S. Sirtuins and P53. Cancer Res 2009;9:230-55.  Back to cited text no. 15
    
16. Cang S, Ma Y, Liu D. New clinical developments in histone deacetylase inhibitors for epigenetic therapy of cancer. J Hematol Oncol 2009;2:22.  Back to cited text no. 16
    
17. Liu G, Chen X. Regulation of the P53 transcriptional activity. J Cell Biochem 2006;97:448-58.  Back to cited text no. 17
    
18. Robbins SL, Kumar V. Robbins and cotran pathologic basis of disease. 7th ed. Vol. 11. Elserier: Saunders; 2010. p. 19-54.  Back to cited text no. 18
    
19. Gregoretti IV, Lee YM, Goodson HV. Molecular evolution of the histone deacteylase family: Functional implications of phylogenetic analysis. J Mol Biol 2004;338:17-31.  Back to cited text no. 19
    
20. Olmosa Y, Brosensb JJ, Lama EW. Interplay between SIRT proteins and tumor suppressor transcription factors in chemotherapeutic resistance of cancer. Drug Resist Updat 2011;14:35-44.  Back to cited text no. 20
    
21. Chim CS, Wong AS, Kwong YL. Absence of P300 gene promoter methylation in acute leukemia. Cancer Genet Cytogenet 2004;150:164-7.  Back to cited text no. 21
    
22. Escande C, Chini CS, Nin V, Dykhouse KM. Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice. J Clin Invest 2010;120:545-58.  Back to cited text no. 22
    
23. Fermento ME, Gandini NA, Lang CA, Perez JE. Intracellular distribution of P300 and its differential recruitment to aggresomes in breast cancer. Exp Mol Pathol 2010;88:256-64.  Back to cited text no. 23
    
24. Boniol M, Gavin A, Vatten LJ. Breast cancer mortality in neighboring European countries with different levels of screening but similar access to treatment: Trend analysis of who mortality database. BMJ 2011;343:d441.  Back to cited text no. 24
    
25. Montazeri A, Vahdaninia M, Harirchi I, Harirchi AM, Sajadian A, Khaleghi F, et al. Breast cancer in Iran: Need for greater women awareness of warning signs and effective screening methods. Asia Pac Fam Med 2008;7:6.  Back to cited text no. 25
    
26. Chan HM, Thangue NB. p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J Cell Sci 2001;114:2363-73.  Back to cited text no. 26
    
27. Dworkin AM, Huang TH, Toland AE. Epigenetic alterations in the breast: Implications for breast cancer detection, prognosis, and treatment. Semin Cancer Biol 2009;19:165-71.  Back to cited text no. 27
    
28. Mantelingu K, Kishore AH, Balasubramanyam K, Pavan Kumar GV, Altaf M, Swamy SN, et al. Activation of P300 histone acetyltransferases by small molecules altering enzyme structure: Probed by surface-enhanced raman spectroscopy. J Phys Chem B 2007;111:4527-34.  Back to cited text no. 28
    
29. Devipryia B, Paraneswari AR, Rajalakshmi G, Palvannan T, Kumaradhas P. Exploring the binding affinities of P300 enzyme activators CTBP and CTB using docking method. Indian J Biochem Biophys 2010;47:364-9.  Back to cited text no. 29
    
30. Wong P, Pickard A, Mccance DJ. P300 alters keratinocyte cell growth and differentiation through regulation of p21(Waf1/CIP1). PLoS One 2010;5:e8369.  Back to cited text no. 30
    
31. Cen Y. Sirtuins inhibitors: The approach to affinity and selectivity. Biochim Biophys Acta 2010;1804:1635-44.  Back to cited text no. 31
    
32. Calvanese V, Fraga MF. Sirt1 brings sternness closer to cancer and aging. Aging (Albany NY) 2011;3;162-7.  Back to cited text no. 32
    
33. Gallinari P, Di MS, Jones P, Pallaoro M, Steinkuhler C. HDACs, histone deacetylation and gene transcription: From molecular biology to cancer therapeutics. Cell Res 2007;17:195-211.  Back to cited text no. 33
    
34. Yang T, Fu M, Pestell R, Sauve AA. SIRT1 and endocrine signaling. Trends Endocrinol Metab 2006;17:186-91.  Back to cited text no. 34
    
35. Jones AP, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415-28.  Back to cited text no. 35
    
36. Helweg B, Gatbonton T, Schuler AD. Antitumor activity of a small - molecule inhibitor of human silent information regulator 2 enzymes. Cancer Res 2006;66:4368-77.  Back to cited text no. 36
    
37. Molversmyr AK, Saether T, Gilfillan S, Lorenzo PI, Kvaløy H, Matre V, et al. A SUMO-regulated activation function controls synergy of c-Myb through a repressor-activator switch leading to differential P300 recruitment. Nucleic Acids Res 2010;38:4970-84.  Back to cited text no. 37
    
38. Janknecht R. The versatile functions of the transcriptional coactivators p300 and CBP and their roles in disease. Histol Histopathol 2002;17:657-68.  Back to cited text no. 38
    
39. Iyer NG, Ozdag H, Caldas C. p300/CBP and cancer. Oncogene 2004;23:4225-31.  Back to cited text no. 39
    
40. Ikushima H, Miyazono K. TGFâ signalling: A complex web in cancer progression. Nat Rev Cancer 2010;10:415-24.  Back to cited text no. 40
    
41. Chen LF, Greene WC. Regulation of distinct biological activities of the NF-kappaB transcription factor complex by acetylation. J Mol Med (Berl) 2003;81:549-57.  Back to cited text no. 41
    
42. Bedford DC, Kasper LH, Fukuyama T, Brindle PK. Target gene context influences the transcriptional requirement for the KAT3 family of CBP and P300 histone acetyltransferases. Epigenetics 2010;5:9-15.  Back to cited text no. 42
    
43. Goodman RH, Smolik S. CBP/P300 in cell growth, transformation and development. Genes Dev 2000;14:1553-77.  Back to cited text no. 43
    
44. Karamouzis MV, Konstantinopoulos PA, Papavassiliou AG. Roles of CREB-binding protein (CBP)/P300 in respiratory epithelium tumorigenesis. Cell Res 2007;17:324-32.  Back to cited text no. 44
    
45. Chen S, Feng B, George B, Megan CR. Transcriptional co-activator P300 regulates glucose-induced gene expression in endothelial cells. Am J Physiol Endocrinol Metab 2010;298:E127-37.  Back to cited text no. 45
    
46. Gu W, Roeder RG. Activation of P53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997;90:595-606.  Back to cited text no. 46
    
47. Cui L, Miao J, Furuya T, Fan QI, Li X, Rathod PK, et al. Histone acetyltransferases inhibitor Anacardic acid causes changes in global gene expression during In vitro Plasmodium Falciparum development. Eukaryot Cell 2008;7:1200-10.  Back to cited text no. 47
    
48. Gauthier S, Tremblay MJ. Cholera toxin inhibits HIV-1 replication in human colorectal epithelial HT-29 cells through Adenylate cyclase activation. Antiviral Res 2010;88:207-16.  Back to cited text no. 48