Apoptosis Induced by Low-concentration Ethanol in Hepatocellular Carcinoma Cell Strains and Down-regulated AFP and Survivin Analysis by Proteomic Technology
Ethanol is generally used as a therapeutic reagent against Hepatocellular carcinoma (HCC or hepatoma) worldwide, as it can induce Hepatocellular carcinoma cell apoptosis at low concentration through a multifactorial process regulated by several unknown proteins. This paper provides a simple and available proteomic strategy for exploring differentially expressed proteins in the apoptotic pathway. The appropriate concentrations of ethanol required to induce HepG2 cell apoptosis were first assessed by MTT assay, Gisma and fluorescence staining. Next, the central proteins involved in the apoptosis pathway processs were determined using 2D-PAGE, SDS-PAGE, and bio-software analysis. Finally the downregulation of two proteins, AFP and survivin, were determined by immunocytochemistry and reverse transcriptase PCR (RT-PCR) technology. The simple, useful method demonstrated here provides a new approach to proteomic analysis in key bio-regulating process including proliferation, differentiation, apoptosis, immunity and metastasis.
[1] Llovet, J. M., Burroughs, A., and Bruix, J. (2003). Hepatocellular
carcinoma. Lancet, 362, 1907-1917.
[2] Llovet, J. M., and Beaugrand, M. (2003). Hepatocellular
carcinoma:present status and future prospects. J Hepatol, 38, 136-149.
[3] McGlynn, K. A., Tsao, L., Hsing, A. W., Devesa, S. S., and Fraumeni, J.
F. J. (2001). International trends and patterns of primary liver cancer. Int
J Cancer, 94, 290-296.
[4] Seow, T. K., Liang, R. C., Leow, C. K., and Chung, M. C. (2001).
Hepatocellular carcinoma,From bedside to proteomics. Proteomics, 1,
1249-1263.
[5] Thorgeirsson, S. S., and Grisham, J. W. (2002). Molecular pathogenesis
of human hepatocellular carcinoma. Nat Genet, 31, 339-346.
[6] Leung, T. W., Patt, Y. Z., Lau, W. Y., Ho, S. K., Yu, S. C., et al. (1999).
Complete pathological remission is possible with systemic combination
chemotherapy for inoperable hepatocellular carcinoma. Clin Cancer Res,
5, 1676-1681.
[7] Poon, R. T., Fan, S. T., Lo, C. M., Liu, C. L., and Wong, J. (1999).
Intrahepatic recurrence after curative resection of hepatocellular
carcinoma, long term results of treatment and prognostic factors. Ann
Surg, 229, 216 -222.
[8] Lowe, S. W., and Lin, A. W. (2000). Apoptosis in cancer.
Carcinogenesis, 21, 485- 495.
[9] Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature, 407,
770-776.
[10] Ghobrial, I. M., Witzig, T. E., and Adjei, A. A. (2005) Targeting
apoptosis pathways in cancer therapy. CA Cancer J Clin, 55, 178-194.
[11] Denicourt, C., and Dowdy, S. F. (2004). Targeting apoptotic pathway in
cancer cells. Science, 305, 1411-1413.
[12] Yim, E. K., Lee, K. H., Namkoong, S. E., Um, S. J., and Park, J. S.
(2006). Proteomic analysis of ursolic acid-induced apoptosis in cervical
carcinoma cell. Cancer lett, 235, 209-220.
[13] Neo, J. C., Rose, P., Ong, C. N., and Chung, M. C. (2005). beta-
Phenylethyl isothiocyanate mediated apoptosis, A proteomic
investigation of early apoptotic protein changes. Proteomics, 5, 1075-
1082.
[14] Dong, H., Ying, T., Li, T., Cao, T., Wang, J., et al. (2006). Comparative
Proteomic Analysis of Apoptosis Induced by Sodium Selenite in Human
Acute Promyelocytic Leukemia NB4 Cells. Journal of Cellular
Biochemistry, 98, 1495-1506.
[15] Monge, M., Vilaseca, M., Soto-Cerrato, V., Montaner, B., Giralt, E., and
Perez-Tomas, R. (2007). Proteomic analysis of prodigiosin-induced
apoptosis in a breast cancer mitoxantrone-resistant MCF-7 MR cell line.
Invest New Drugs, 25, 21-29.
[16] Prince, P., and Mcmillan, T. J. (1990). Use of the tetrazolium assay in
measuring the response of human tumor cells to ionizing radiation.
Cancer Res, 50, 1392-1396.
[17] Yu, L. R., Zeng, R., Shao, X. X., Wang, N., Xu, Y. H., and Xia, Q. C.
(2000). Identification of differentially expressed proteins between
human hepatoma and normal liver cell lines by two-dimensional
electrophoresis and liquid chromatography-ion trap mass spectrometry.
Electrophoresis, 21, 3058-3068.
[18] Ding, S. J., Li, Y., Shao, X. X., Zhou, H., Zeng, R., et al. (2004).
Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H
and MHCC97-L, with different metastasis potentials. Proteomics, 4,
982-994.
[19] Yokoo, H., Kondo, T., Fujii, K., Yamada, T., Todo, S., and Hirohashi, S.
(2004). Proteomic signature corresponding to alpha fetoprotein
expression in liver Cancer cells. Hepatology, 40, 609-617.
[20] Ding, S. J., Li, Y., Tan, Y. X., Jiang, M. R., Tian, B., et al. (2004). From
proteomic analysis to clinical significance, overexpression of cytokeratin
19 correlates with hepatocellular carcinoma metastasis. Mol Cell
Proteomics, 3, 73-81.
[21] Ramagli, L. S. (1999). 2-D Proteome Analysis Protocols, Humana Press,
Totowa, NJ, USA 99-103.
[22] Liang, R. C., Neo, J. C., Lo, S. L., Tan, G. S., Seow, T. K., and Chung,
M. C. (2002). Proteome database of hepatocellular carcinoma. J
Chromatogr B Analyt Technol biomed Life Sci, 771, 202-228.
[23] Neuhooff, V., Arold, N., Taube, D., and Ehrhardt, W. (1988). Improved
staining of proteins in polyacrylamide gels including isoelectric focusing
gels with clear background at nanogram sensitivity using Coomassie
Brilliant Blue G-250 and R-250. Electrophoresis, 9, 255-62.
[24] Oertel, J., and Huhn, J. (2000). Immunocytochemical methods in
haematology and oncology. J Cancer Res Clin Oncol, 126, 425-440.
[25] Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., et al.
(2001). The sequence of human genome. Science, 291, 1304-1351.
[26] Strausberg, R. L., Feingold, E. A., Grouse, L. H., Derge, J. G., Klausner,
R. D., et al. (2002). Generation and initial analysis of more than 15000
full-length human and mouse cDNA sequences. Proc Natl Acad Sci, 99,
16899-16903.
[27] Ambrosini, G., Adida, C., and Altieri, D. C. (1997). A noval antiapoptosis
gene, survivin, expressed in cancer and lymphoma. Nat Med,
3, 917-921.
[28] Vilana, R., Bruix, J., Bru, C., Ayuso, C., Sole, M., and Rodes, J. (1992).
Tumor size determines the efficacy of percutaneous ethanol injection for
the treatment of small hepatocellular carcinoma. Hepatology, 16, 353-
357.
[29] Shiina, S., Tagawa, K., Niwa, Y., Unuma, T., Komatsu, Y., et al. (1993).
Percutaneous ethanol injection therapy for hepatocellular carcinoma,
results in 136 patients. Am J Roentgenol, 160, 1023-1028.
[30] Redvanly, R. D., Chezmar, J. L., Strauss, R. M., Galloway, J. R., Boyer,
T. D., and Bernardino, M. E. (1993). Malignant hepatic tumors, safety of
high-dose percutaneous ethanol ablation therapy. Radiology, 188, 283-
285.
[31] Castaneda, F., and Kinne, R. H. K. (2000). Cytotoxicity of milliolar
concentrations of ethanol on tumor cell line compared to normal rat
hepatocytes in vitro. J Cancer Res Clin Oncol, 126, 505-510.
[32] Castaneda, F., and Kinne, R. H. K. (2001). Apoptosis induced in HepG2
cells by short exposure to millimolar concentrations of ethanol involves
the Fas-receptor pathway. J Cancer Res Clin Oncol, 127, 418-424.
[33] Nobuaki, N., Eichhorst, S. T., Muller, M., and Krammer, P. H. (2001).
Ethanol-induced apoptosis in hepatoma cells proceeds via intracellular
Ca+; elevation activation of TLCK-sensitive proteases, and cytochrome
c release. Exp Cell Res, 269, 202-213.
[34] Kurose, I., Higuchi, H., Miura, S., Saito, H., Watanabe, N., et al. (1997).
Oxidative stress mediated apoptosis of hepatocytes exposed to acute
ethanol intoxication. Hepatology, 25, 368-378.
[35] Santamaria, E., Munoz, J., Fernandez-Irigoyen, J., Prieto, J., and
Corrales, F. J. (2007). Toward the discovery of new biomarkers of
hepatocellular carcinoma by proteomics. Liver Int, 27, 163-173.
[36] Feng, J. T., Shang, S., and Beretta, L. (2006). Proteomics for the early
detection and treatment of hepatocellular carcinoma. Oncogene, 25,
2810-2817.
[37] Chignard, N., and Beretta, L. (2004). Proteomics for hepatocellular
carcinoma marker discovery. Gastroenterology, 127, 120-125.
[38] Guo, L., Eisenman, J. R., Mahomkar, R. M., Peschon, J. J., Paxton, R. J.,
et al. (2002). A proteomic approach for the identification of cell-surface
proteins shed by metalloproteases. Mol cell proteomics, 1, 30-36.
[39] Falini, B., and Mason, D. Y. (2002). Protein encoded by genes involved
in chromosomal alterations in lymphoma and leukemia, clinical value
their detection by immunocytochemistry. Blood, 99, 409-426.
[40] Tanke, H. J., Dirks, R. W., and Raap, T. (2005). FISH and
immunocytochemistry, towards visualising single target molecules in
living cells. Curr Opin Biotechnol, 16, 49-54.
[41] Mizejewski, G. J. (2001). Alpha-fetoprotein structure and function,
relevance to isoforms, epitopes, and conformational variants. Exp Biol
Med, 226, 377-408.
[42] Dudich, E., Semenkova, L., Gorbatova, E., Dudich, I., Khromykh, L., et
al. (1998). Growth-regulative activity of human alpha-fetoprotein for
different types of tumor and normal cells. Tumour Biol, 19, 30-40.
[43] Li, M. S., Ma, Q. L., Chen, Q., Liu, X. H., Li, P. F., et al. (2005). Alphafetoprotein
triggers hepatoma cells escaping from immune surveillance
through altering the expression of Fas/FasL and tumor necrosis factor
related apoptosis-inducing ligand and its receptor of lymphocytes and
liver cancer cells. World J Gastroenterol, 11, 2564-2569.
[44] Walczak, H., and Krammer, P. H. (2000). The CD95 (APO-1/Fas) and
the TRAIL(APO-2L) Apoptosis systems. Exp Cell Res, 256, 58-66.
[45] Altierti, D. C. (2003). Survivin, versatile modulation of cell division and
apoptosis in cancer. Oncogene, 22, 8581-8589.
[46] Duffy, M. J., O-Donovan, N., Brennan, D. J., Gallagher, W. M., and
Ryan, B. M. (2007). Survivin, a promising tumor biomarker. Cancer lett,
249, 49-60.
[47] Chious, S. K., Jones, M. K., and Tarnawski, A. S. (2003). Survivin - an
anti-apoptosis protein, its biological roles and implications for cancer
and beyond. Med Sci Monit, 9, 125-129.
[48] Conway, E. M., Pollefeyt, S., Steiner-Mosonyi, M., Luo, W., Devriese,
A., et al. (2002). Deficiency of survivin in transgenic mice exacerbates
Fas-induced apoptosis via mitochondrial pathway. Gastroenterology,
123, 619-631.
[49] Sah, N. K., Khan, Z., Khan, G. J., and Bisen, P. S. (2006). Structural,
functional and therapeutic biology of survivin. Cancer Lett, 244, 166-
171.
[1] Llovet, J. M., Burroughs, A., and Bruix, J. (2003). Hepatocellular
carcinoma. Lancet, 362, 1907-1917.
[2] Llovet, J. M., and Beaugrand, M. (2003). Hepatocellular
carcinoma:present status and future prospects. J Hepatol, 38, 136-149.
[3] McGlynn, K. A., Tsao, L., Hsing, A. W., Devesa, S. S., and Fraumeni, J.
F. J. (2001). International trends and patterns of primary liver cancer. Int
J Cancer, 94, 290-296.
[4] Seow, T. K., Liang, R. C., Leow, C. K., and Chung, M. C. (2001).
Hepatocellular carcinoma,From bedside to proteomics. Proteomics, 1,
1249-1263.
[5] Thorgeirsson, S. S., and Grisham, J. W. (2002). Molecular pathogenesis
of human hepatocellular carcinoma. Nat Genet, 31, 339-346.
[6] Leung, T. W., Patt, Y. Z., Lau, W. Y., Ho, S. K., Yu, S. C., et al. (1999).
Complete pathological remission is possible with systemic combination
chemotherapy for inoperable hepatocellular carcinoma. Clin Cancer Res,
5, 1676-1681.
[7] Poon, R. T., Fan, S. T., Lo, C. M., Liu, C. L., and Wong, J. (1999).
Intrahepatic recurrence after curative resection of hepatocellular
carcinoma, long term results of treatment and prognostic factors. Ann
Surg, 229, 216 -222.
[8] Lowe, S. W., and Lin, A. W. (2000). Apoptosis in cancer.
Carcinogenesis, 21, 485- 495.
[9] Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature, 407,
770-776.
[10] Ghobrial, I. M., Witzig, T. E., and Adjei, A. A. (2005) Targeting
apoptosis pathways in cancer therapy. CA Cancer J Clin, 55, 178-194.
[11] Denicourt, C., and Dowdy, S. F. (2004). Targeting apoptotic pathway in
cancer cells. Science, 305, 1411-1413.
[12] Yim, E. K., Lee, K. H., Namkoong, S. E., Um, S. J., and Park, J. S.
(2006). Proteomic analysis of ursolic acid-induced apoptosis in cervical
carcinoma cell. Cancer lett, 235, 209-220.
[13] Neo, J. C., Rose, P., Ong, C. N., and Chung, M. C. (2005). beta-
Phenylethyl isothiocyanate mediated apoptosis, A proteomic
investigation of early apoptotic protein changes. Proteomics, 5, 1075-
1082.
[14] Dong, H., Ying, T., Li, T., Cao, T., Wang, J., et al. (2006). Comparative
Proteomic Analysis of Apoptosis Induced by Sodium Selenite in Human
Acute Promyelocytic Leukemia NB4 Cells. Journal of Cellular
Biochemistry, 98, 1495-1506.
[15] Monge, M., Vilaseca, M., Soto-Cerrato, V., Montaner, B., Giralt, E., and
Perez-Tomas, R. (2007). Proteomic analysis of prodigiosin-induced
apoptosis in a breast cancer mitoxantrone-resistant MCF-7 MR cell line.
Invest New Drugs, 25, 21-29.
[16] Prince, P., and Mcmillan, T. J. (1990). Use of the tetrazolium assay in
measuring the response of human tumor cells to ionizing radiation.
Cancer Res, 50, 1392-1396.
[17] Yu, L. R., Zeng, R., Shao, X. X., Wang, N., Xu, Y. H., and Xia, Q. C.
(2000). Identification of differentially expressed proteins between
human hepatoma and normal liver cell lines by two-dimensional
electrophoresis and liquid chromatography-ion trap mass spectrometry.
Electrophoresis, 21, 3058-3068.
[18] Ding, S. J., Li, Y., Shao, X. X., Zhou, H., Zeng, R., et al. (2004).
Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H
and MHCC97-L, with different metastasis potentials. Proteomics, 4,
982-994.
[19] Yokoo, H., Kondo, T., Fujii, K., Yamada, T., Todo, S., and Hirohashi, S.
(2004). Proteomic signature corresponding to alpha fetoprotein
expression in liver Cancer cells. Hepatology, 40, 609-617.
[20] Ding, S. J., Li, Y., Tan, Y. X., Jiang, M. R., Tian, B., et al. (2004). From
proteomic analysis to clinical significance, overexpression of cytokeratin
19 correlates with hepatocellular carcinoma metastasis. Mol Cell
Proteomics, 3, 73-81.
[21] Ramagli, L. S. (1999). 2-D Proteome Analysis Protocols, Humana Press,
Totowa, NJ, USA 99-103.
[22] Liang, R. C., Neo, J. C., Lo, S. L., Tan, G. S., Seow, T. K., and Chung,
M. C. (2002). Proteome database of hepatocellular carcinoma. J
Chromatogr B Analyt Technol biomed Life Sci, 771, 202-228.
[23] Neuhooff, V., Arold, N., Taube, D., and Ehrhardt, W. (1988). Improved
staining of proteins in polyacrylamide gels including isoelectric focusing
gels with clear background at nanogram sensitivity using Coomassie
Brilliant Blue G-250 and R-250. Electrophoresis, 9, 255-62.
[24] Oertel, J., and Huhn, J. (2000). Immunocytochemical methods in
haematology and oncology. J Cancer Res Clin Oncol, 126, 425-440.
[25] Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., et al.
(2001). The sequence of human genome. Science, 291, 1304-1351.
[26] Strausberg, R. L., Feingold, E. A., Grouse, L. H., Derge, J. G., Klausner,
R. D., et al. (2002). Generation and initial analysis of more than 15000
full-length human and mouse cDNA sequences. Proc Natl Acad Sci, 99,
16899-16903.
[27] Ambrosini, G., Adida, C., and Altieri, D. C. (1997). A noval antiapoptosis
gene, survivin, expressed in cancer and lymphoma. Nat Med,
3, 917-921.
[28] Vilana, R., Bruix, J., Bru, C., Ayuso, C., Sole, M., and Rodes, J. (1992).
Tumor size determines the efficacy of percutaneous ethanol injection for
the treatment of small hepatocellular carcinoma. Hepatology, 16, 353-
357.
[29] Shiina, S., Tagawa, K., Niwa, Y., Unuma, T., Komatsu, Y., et al. (1993).
Percutaneous ethanol injection therapy for hepatocellular carcinoma,
results in 136 patients. Am J Roentgenol, 160, 1023-1028.
[30] Redvanly, R. D., Chezmar, J. L., Strauss, R. M., Galloway, J. R., Boyer,
T. D., and Bernardino, M. E. (1993). Malignant hepatic tumors, safety of
high-dose percutaneous ethanol ablation therapy. Radiology, 188, 283-
285.
[31] Castaneda, F., and Kinne, R. H. K. (2000). Cytotoxicity of milliolar
concentrations of ethanol on tumor cell line compared to normal rat
hepatocytes in vitro. J Cancer Res Clin Oncol, 126, 505-510.
[32] Castaneda, F., and Kinne, R. H. K. (2001). Apoptosis induced in HepG2
cells by short exposure to millimolar concentrations of ethanol involves
the Fas-receptor pathway. J Cancer Res Clin Oncol, 127, 418-424.
[33] Nobuaki, N., Eichhorst, S. T., Muller, M., and Krammer, P. H. (2001).
Ethanol-induced apoptosis in hepatoma cells proceeds via intracellular
Ca+; elevation activation of TLCK-sensitive proteases, and cytochrome
c release. Exp Cell Res, 269, 202-213.
[34] Kurose, I., Higuchi, H., Miura, S., Saito, H., Watanabe, N., et al. (1997).
Oxidative stress mediated apoptosis of hepatocytes exposed to acute
ethanol intoxication. Hepatology, 25, 368-378.
[35] Santamaria, E., Munoz, J., Fernandez-Irigoyen, J., Prieto, J., and
Corrales, F. J. (2007). Toward the discovery of new biomarkers of
hepatocellular carcinoma by proteomics. Liver Int, 27, 163-173.
[36] Feng, J. T., Shang, S., and Beretta, L. (2006). Proteomics for the early
detection and treatment of hepatocellular carcinoma. Oncogene, 25,
2810-2817.
[37] Chignard, N., and Beretta, L. (2004). Proteomics for hepatocellular
carcinoma marker discovery. Gastroenterology, 127, 120-125.
[38] Guo, L., Eisenman, J. R., Mahomkar, R. M., Peschon, J. J., Paxton, R. J.,
et al. (2002). A proteomic approach for the identification of cell-surface
proteins shed by metalloproteases. Mol cell proteomics, 1, 30-36.
[39] Falini, B., and Mason, D. Y. (2002). Protein encoded by genes involved
in chromosomal alterations in lymphoma and leukemia, clinical value
their detection by immunocytochemistry. Blood, 99, 409-426.
[40] Tanke, H. J., Dirks, R. W., and Raap, T. (2005). FISH and
immunocytochemistry, towards visualising single target molecules in
living cells. Curr Opin Biotechnol, 16, 49-54.
[41] Mizejewski, G. J. (2001). Alpha-fetoprotein structure and function,
relevance to isoforms, epitopes, and conformational variants. Exp Biol
Med, 226, 377-408.
[42] Dudich, E., Semenkova, L., Gorbatova, E., Dudich, I., Khromykh, L., et
al. (1998). Growth-regulative activity of human alpha-fetoprotein for
different types of tumor and normal cells. Tumour Biol, 19, 30-40.
[43] Li, M. S., Ma, Q. L., Chen, Q., Liu, X. H., Li, P. F., et al. (2005). Alphafetoprotein
triggers hepatoma cells escaping from immune surveillance
through altering the expression of Fas/FasL and tumor necrosis factor
related apoptosis-inducing ligand and its receptor of lymphocytes and
liver cancer cells. World J Gastroenterol, 11, 2564-2569.
[44] Walczak, H., and Krammer, P. H. (2000). The CD95 (APO-1/Fas) and
the TRAIL(APO-2L) Apoptosis systems. Exp Cell Res, 256, 58-66.
[45] Altierti, D. C. (2003). Survivin, versatile modulation of cell division and
apoptosis in cancer. Oncogene, 22, 8581-8589.
[46] Duffy, M. J., O-Donovan, N., Brennan, D. J., Gallagher, W. M., and
Ryan, B. M. (2007). Survivin, a promising tumor biomarker. Cancer lett,
249, 49-60.
[47] Chious, S. K., Jones, M. K., and Tarnawski, A. S. (2003). Survivin - an
anti-apoptosis protein, its biological roles and implications for cancer
and beyond. Med Sci Monit, 9, 125-129.
[48] Conway, E. M., Pollefeyt, S., Steiner-Mosonyi, M., Luo, W., Devriese,
A., et al. (2002). Deficiency of survivin in transgenic mice exacerbates
Fas-induced apoptosis via mitochondrial pathway. Gastroenterology,
123, 619-631.
[49] Sah, N. K., Khan, Z., Khan, G. J., and Bisen, P. S. (2006). Structural,
functional and therapeutic biology of survivin. Cancer Lett, 244, 166-
171.
@article{"International Journal of Biological, Life and Agricultural Sciences:56268", author = "Xin Kai and Juan Li and Sexin Huang and Zengliang Bai", title = "Apoptosis Induced by Low-concentration Ethanol in Hepatocellular Carcinoma Cell Strains and Down-regulated AFP and Survivin Analysis by Proteomic Technology", abstract = "Ethanol is generally used as a therapeutic reagent against Hepatocellular carcinoma (HCC or hepatoma) worldwide, as it can induce Hepatocellular carcinoma cell apoptosis at low concentration through a multifactorial process regulated by several unknown proteins. This paper provides a simple and available proteomic strategy for exploring differentially expressed proteins in the apoptotic pathway. The appropriate concentrations of ethanol required to induce HepG2 cell apoptosis were first assessed by MTT assay, Gisma and fluorescence staining. Next, the central proteins involved in the apoptosis pathway processs were determined using 2D-PAGE, SDS-PAGE, and bio-software analysis. Finally the downregulation of two proteins, AFP and survivin, were determined by immunocytochemistry and reverse transcriptase PCR (RT-PCR) technology. The simple, useful method demonstrated here provides a new approach to proteomic analysis in key bio-regulating process including proliferation, differentiation, apoptosis, immunity and metastasis.
", keywords = "Hepatocellular carcinoma, Ethanol, Proteomics,survivin and AFP", volume = "4", number = "1", pages = "69-7", }
