Quantitative analysis of the level of p53 and p21WAF1 mRNA in human colon cancer HT-29 cells treated with inositol hexaphosphate
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The aim of this study was to analyze the molecular mechanism of inositol hexaphosphate (InsP6) action through which it may inhibit proliferation of colon cancer cells and cell cycle progression. A kinetic study of p53 and p21WAF1 mRNA increase was performed on human colon cancer HT-29 cells after treatment with 1, 5 and 10 mM InsP6 for 6, 12, 24 and 48 h. Real-time-QPCR based on TaqMan methodology was applied to analyze quantitatively the transcript levels of these genes. The transcription of β-actin and GAPDH genes was assessed in parallel to select the control gene with least variability. The 2-ΔΔCt method was used to analyze the relative changes in gene transcription. InsP6 stimulated p53 and p21WAF1 expression at the mRNA level, with the highest increase in p21WAF1 mRNA occurring at 24 h, i.e., following the highest increase in p53 mRNA observed at 12 h. Based on these studies it may be concluded that the ability of InsP6 to arrest the cell cycle may be mediated by the transcriptional up-regulation of the p53-responsive p21WAF1 gene.
- Bas A, Forsberg G, Hammarström S, Hammarström M-L (2004) Utility of the housekeeping genes 18S rRNA, β-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol 59: 566-573.
- Blanquicett C, Johnson MR, Heslin M, Diasio RB (2002) Housekeeping gene variability in normal and carcinomatous colorectal and liver tissues: applications in pharmacogenomic gene expression studies. Anal Biochem 303: 209-214.
- Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25: 169-193.
- Chai E, Evdokiou A, Young GP, Zalewski PD (2000) Involvement of p21 WAF1/CIP1 and its cleavage by DEVD-caspase during apoptosis of colorectal cancer cells induced by butyrate. Carcinogenesis 21: 7-14.
- Chen Y-C, Shen S-C, Chow J-M, Ko CH, Tseng S-W (2004) Flavone inhibition of tumor growth via apoptosis in vitro and in vivo. Int J Oncol 25: 661-670.
- Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Elledge SJ, Reed SI (1994) p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76: 1013-1023.
- El-Sherbiny Y, Cox MC, Ismail ZA, Shamsuddin AM, Vucenik I (2001) G0/G1 arrest and S phase inhibition of human cancer cell lines by inositol hexaphosphate (IP6). Anticancer Res 21: 2393-2404.
- Fang JY, Chen YX, Lu J, Lu R, Yang L, Zhu HY, Gu WQ, Lu LG (2004) Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Col-320 and SW1116. Cell Res 14: 217-226.
- Ferry S, Matsuda M, Yoshida H, Hirata M (2002) Inositol hexakisphosphate blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting the Akt/NFκB-mediated cell survival pathway. Carcinogenesis 23: 2031-2041.
- Graf E, Eaton JW (1985) Dietary suppression of colonic cancer. Fiber or phytate? Cancer 56: 717-718.
- Grases F, Costa-Bauza A (1999) Phytate (IP6) is a powerful agent for preventing calcification in biological fluids: usefulness in renal lithiasis treatment. Anticancer Res 19: 3717-3722.
- Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25: 386-401.
- Hainaut P, Hernandez T, Robinson A, Rodriques-Thome P, Flores T, Hollstein M, Harris CC, Montesano R (1998) IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualization tools. Nucleic Acids Res 26: 205-213.
- Hsu IC, Tokiwa T, Bennet W, Melealf RA, Welsh JA, Sun T, Harris CC (1993) p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines. Carcinogenesis 14: 987-992.
- Huang C, Ma WY, Hecht S, Dong Z (1997) Inositol hexaphosphate inhibits cell transformation and activator protein 1 activation by targeting phosphatidylinositol-3'-kinase. Cancer Res 57: 2873-2878.
- Ishikawa T, Nakatsuru Y, Zarkovic M, Shamsuddin AM (1999) Inhibition of skin cancer by IP6 in vitro: initiation-promotion model. Anticancer Res 19: 3749-3752.
- Jenab M, Thompson LU (2000) Phytic acid in wheat bran affects colon morphology, cell differentiation and apoptosis. Carcinogenesis 21: 1547-1552.
- Kim JS, Lee S, Lee T, Lee YW, Trepel JB (2001) Transcriptional activation of p21 (WAF1/CIP1) by apicidin, a novel histone deacetylase inhibitor. Biochem Biophys Res Commun 281: 866-871.
- Kubbutat MHG, Vousden KH (1998) Keeping an old friend under control: regulation of p53 stability. Mol Med Today 4: 250-256.
- Litvak DA, Evers B, Hwang KO, Hellmich MR, Ko TC, Townsed CM (1998) Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1. Surgery 124: 161-170.
- Maltzman W, Czyzyk L (1984) UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol Cell Biol 4: 1689-1694.
- Pretlow TP, O'Riordan MA, Somich GA, Amini SB, Pretlow TG (1992) Aberrant crypts correlate with tumor incidence in F344 rats treated with azoxymethane and phytate. Carcinogenesis 13: 1509-1512.
- Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene for selection for quantitative real-time PCR. Biochem Biophys Res Commun 313: 856-862.
- Regunathan S, Reis DJ, Wahlestedt C (1992) Specific binding of inositol hexakisphosphate (phytic acid) to adrenal chromaffin cell membrane and effects on calcium-dependent catecholamine release. Biochem Pharmacol 43: 1331-1336.
- Saied IT, Shamsuddin AM (1998) Up-regulation of the tumor suppressor gene p53 and WAF1 gene expression by IP6 in HT-29 human colon carcinoma cell line. Anticancer Res 18: 1479-1484.
- Sakamoto K, Venkatraman G, Shamsuddin AM (1993) Growth inhibition and differentiation of HT-29 cells in vitro by inositol hexaphosphate (phytic acid). Carcinogenesis 14: 1815-1819.
- Shamsuddin AM, Yang GY (1995) Inositol hexaphosphate inhibits growth and induces differentiation of PC-3 human prostate cancer cells. Carcinogenesis 16: 1975-1979.
- Shamsuddin AM, Vucenik I (1999) Mammary tumor inhibition by IP6: a review. Anticancer Res 19: 3671-3674.
- Shamsuddin AM, Yang G, Vucenik I (1996) Novel anticancer function of IP6: growth inhibition and differentiation of human mammary cancer cell lines in vitro. Anticancer Res 16: 3287-3292.
- Shamsuddin AM, Vucenik I, Cole KE (1997) IP6: a novel anticancer agent. Life Sci 61: 343-354.
- Siavoshian S, Segain JP, Kornprobst M, Bonnet C, Cherbut C, Galmiche JP, Blottiere HM (2000) Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression. Gut 46: 507-514.
- Singh RP, Agarwal R (2005) Prostate cancer and inositol hexaphosphate: efficacy and mechanisms. Anticancer Res 25: 2891-2903.
- Singh RP, Agarwal C, Agarwal R (2003) Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU 145 cells: modulation of CDK1-CDK-cyclin and pRb-related protein-E2F complexes. Carcinogenesis 24: 555-562.
- Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75: 291-295.
- Vucenik I, Tantivejkul K, Zhang ZS, Cole KE, Saied I, Shamsuddin AM (1998) IP6 treatment of liver cancer. I. IP6 inhibits growth and reverses transformed phenotype in HepG2 human liver cancer cell line. Anticancer Res 18: 4083-4090.
- Vucenik I, Podczasy JJ, Shamsuddin AM (1999) Antiplatelet activity of inositol hexaphosphate (IP6). Anticancer Res 19: 3689-3693.
- Vucenik I, Ramakrishna G, Tantivejkul K, Anderson LM, Ramljak D (2005) Inositol hexaphosphate (IP6) blocks proliferation of human breast cancer cells through a PKCδ-dependent increase in p27Kip1 and decrease in retinoblastoma protein (pRb) phosphorylation. Breast Cancer Res Treat 91: 35-45.
- Wattenberg LW (1999) Chemoprevention of pulmonary carcinogenesis by myo-inositol. Anticancer Res 19: 3659-3661.
- Yang G, Shamsuddin AM (1995) IP6 induced growth inhibition and differentiation of HT-29 human colon cancer cells: Involvement of intracellular inositol phosphates. Anticancer Res 15: 2479-2488.
- Zi X, Singh RP, Agarwal R (2000) Impairment of erbB1 receptor and fluid-phase endocytosis and associated mitogenic signalling by inositol hexaphosphate in human prostate carcinoma DU 145 cells. Carcinogenesis 21: 2225-2235.
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