Berberine Inhibits Cell Growth and Mediates Caspase-Independent Cell Death in Human Pancreatic Cancer Cells

Abstract

Pancreatic cancer is one of the most aggressive human malignancies with an increasing incidence worldwide. In addition to the poor survival rates, combinations using gemcitabine as a backbone have failed to show any benefit beyond monotherapy. These facts underscore an urgent need for novel therapeutic options and motivated us to study the effect of berberine on pancreatic cancer cells. Here, we undertook an mRNA-based gene expression profiling study in order to get deeper insight into the molecular targets mediating the growth inhibitory effects of berberine on pancreatic cancer cells compared to normal ones. Twenty-four hours after treatment, berberine showed preferential selectivity toward pancreatic cancer cells compared to normal ones. Moreover, expression profiling and Ingenuity pathway analysis results showed that the cytotoxicity of berberine was accompanied with an activation of BRCA1-mediated DNA damage response, G1/S and G2/M cell cycle checkpoint regulation, and P53 signalling pathways. The activation of these signalling pathways might be explained by the fact that berberine intercalates DNA and induces DNA strand break through inhibition of topoisomerases and induction of DNA lesions.

Key words

berberine - pancreatic cancer - microarray - cell death - photodynamic therapy

References

• 1 Bezakova L, Misik V, Malekova L, Svajdlenka E, Kostalova D. Lipoxygenase inhibition and antioxidant properties of bisbenzylisoqunoline alkaloids isolated from Mahonia aquifolium.  Pharmazie. 1996;  51 758-761
• 2 Misik V, Bezakova L, Malekova L, Kostalova D. Lipoxygenase inhibition and antioxidant properties of protoberberine and aporphine alkaloids isolated from Mahonia aquifolium.  Planta Med. 1995;  61 372-373
• 3 Kraft K, Hobbs H. Pocket guide to herbal medicine. Stuttgart; Georg Thieme Verlag 2004
• 4 Lans C, Turner N, Khan T, Brauer G, Boepple W. Ethnoveterinary medicines used for ruminants in British Columbia, Canada.  J Ethnobiol Ethnomed. 2007;  3 11
• 5 Zhu X Z, Li X Y, Liu J. Recent pharmacological studies on natural products in China.  Eur J Pharmacol. 2004;  500 221-230
• 6 Imanshahidi M, Hosseinzadeh H. Pharmacological and therapeutic effects of Berberis vulgaris and its active constituent, berberine.  Phytother Res. 2008;  22 999-1012
• 7 Zha W, Liang G, Xiao J, Studer E J, Hylemon P B, Pandak W M, Wang G, Li X, Zhou H. Berberine inhibits HIV protease inhibitor-induced inflammatory response by modulating ER stress signaling pathways in murine macrophages.  PLoS One. 2010;  5 e9069
• 8 Pasqual M S, Lauer C P, Moyna P, Henriques J A. Genotoxicity of the isoquinoline alkaloid berberine in prokaryotic and eukaryotic organisms.  Mutat Res. 1993;  286 243-252
• 9 Jantova S, Letasiova S, Brezova V, Cipak L, Labaj J. Photochemical and phototoxic activity of berberine on murine fibroblast NIH-3T3 and Ehrlich ascites carcinoma cells.  J Photochem Photobiol B. 2006;  85 163-176
• 10 Lin C C, Yang J S, Chen J T, Fan S, Yu F S, Yang J L, Lu C C, Kao M C, Huang A C, Lu H F, Chung J G. Berberine induces apoptosis in human HSC-3 oral cancer cells via simultaneous activation of the death receptor-mediated and mitochondrial pathway.  Anticancer Res. 2007;  27 3371-3378
• 11 Lin J P, Yang J S, Lee J H, Hsieh W T, Chung J G. Berberine induces cell cycle arrest and apoptosis in human gastric carcinoma SNU-5 cell line.  World J Gastroenterol. 2006;  12 21-28
• 12 Mantena S K, Sharma S D, Katiyar S K. Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells.  Mol Cancer Ther. 2006;  5 296-308
• 13 Mantena S K, Sharma S D, Katiyar S K. Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP.  Carcinogenesis. 2006;  27 2018-2027
• 14 Piyanuch R, Sukhthankar M, Wandee G, Baek S J. Berberine, a natural isoquinoline alkaloid, induces NAG-1 and ATF3 expression in human colorectal cancer cells.  Cancer Lett. 2007;  258 230-240
• 15 Serafim T L, Oliveira P J, Sardao V A, Perkins E, Parke D, Holy J. Different concentrations of berberine result in distinct cellular localization patterns and cell cycle effects in a melanoma cell line.  Cancer Chemother Pharmacol. 2008;  61 1007-1018
• 16 Katiyar S K, Meeran S M, Katiyar N, Akhtar S. p 53 Cooperates berberine-induced growth inhibition and apoptosis of non-small cell human lung cancer cells in vitro and tumor xenograft growth in vivo.  Mol Carcinogen. 2009;  48 24-37
• 17 Liu Z, Liu Q, Xu B, Wu J, Guo C, Zhu F, Yang Q, Gao G, Gong Y, Shao C. Berberine induces p 53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage.  Mutat Res. 2009;  662 75-83
• 18 Lin H L, Liu T Y, Lui W Y, Chi C W. Up-regulation of multidrug resistance transporter expression by berberine in human and murine hepatoma cells.  Cancer. 1999;  85 1937-1942
• 19 Kim J B, Ko E, Han W, Shin I, Park S Y, Noh D Y. Berberine diminishes the side population and ABCG2 transporter expression in MCF-7 breast cancer cells.  Planta Med. 2008;  74 1693-1700
• 20 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun M J. Cancer statistics, 2009.  CA Cancer J Clin. 2009;  59 225-249
• 21 Li J, Saif M W. Any progress in the management of advanced pancreatic cancer? Highlights from the 45th ASCO annual meeting, Orlando, FL; 2009.  JOP. 2009;  10 361-365
• 22 Burris 3rd H A, Moore M J, Andersen J, Green M R, Rothenberg M L, Modiano M R, Cripps M C, Portenoy R K, Storniolo A M, Tarassoff P, Nelson R, Dorr F A, Stephens C D, Von Hoff D D. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial.  J Clin Oncol. 1997;  15 2403-2413
• 23 Rivera F, Lopez-Tarruella S, Vega-Villegas M E, Salcedo M. Treatment of advanced pancreatic cancer: from gemcitabine single agent to combinations and targeted therapy.  Cancer Treat Rev. 2009;  35 335-339
• 24 Youns M, Efferth T, Reichling J, Fellenberg K, Bauer A, Hoheisel J D. Gene expression profiling identifies novel key players involved in the cytotoxic effect of artesunate on pancreatic cancer cells.  Biochem Pharmacol. 2009;  78 273-283
• 25 Esposito I, Bauer A, Hoheisel J D, Kleeff J, Friess H, Bergmann F, Rieker R J, Otto H F, Kloppel G, Penzel R. Microcystic tubulopapillary carcinoma of the pancreas: a new tumor entity?.  Virchows Arch. 2004;  444 447-453
• 26 Boujrad H, Gubkina O, Robert N, Krantic S, Susin S A. AIF-mediated programmed necrosis: a highly regulated way to die.  Cell Cycle. 2007;  6 2612-2619
• 27 Agnello M, Morici G, Rinaldi A M. A method for measuring mitochondrial mass and activity.  Cytotechnology. 2008;  56 145-149
• 28 Hara A, Iizuka N, Hamamoto Y, Uchimura S, Miyamoto T, Tsunedomi R, Miyamoto K, Hazama S, Okita K, Oka M. Molecular dissection of a medicinal herb with anti-tumor activity by oligonucleotide microarray.  Life Sci. 2005;  77 991-1002
• 29 Iizuka N, Oka M, Yamamoto K, Tangoku A, Miyamoto K, Miyamoto T, Uchimura S, Hamamoto Y, Okita K. Identification of common or distinct genes related to antitumor activities of a medicinal herb and its major component by oligonucleotide microarray.  Int J Cancer. 2003;  107 666-672
• 30 Ramachandran C, Rodriguez S, Ramachandran R, Raveendran Nair P K, Fonseca H, Khatib Z, Escalon E, Melnick S J. Expression profiles of apoptotic genes induced by curcumin in human breast cancer and mammary epithelial cell lines.  Anticancer Res. 2005;  25 3293-3302
• 31 Mathiasen I S, Jaattela M. Triggering caspase-independent cell death to combat cancer.  Trends Mol Med. 2002;  8 212-220
• 32 Maiti M, Kumar G S. Molecular aspects on the interaction of protoberberine, benzophenanthridine, and aristolochia group of alkaloids with nucleic acid structures and biological perspectives.  Med Res Rev. 2007;  27 649-695
• 33 Pereira G C, Branco A F, Matos J A, Pereira S L, Parke D, Perkins E L, Serafim T L, Sardao V A, Santos M S, Moreno A J, Holy J, Oliveira P J. Mitochondrially targeted effects of berberine [Natural Yellow 18,5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 mouse melanoma cells: comparison with direct effects on isolated mitochondrial fractions.  J Pharmacol Exp Ther. 2007;  323 636-649
• 34 Tsang C M, Lau E P, Di K, Cheung P Y, Hau P M, Ching Y P, Wong Y C, Cheung A L, Wan T S, Tong Y, Tsao S W, Feng Y. Berberine inhibits Rho GTPases and cell migration at low doses but induces G2 arrest and apoptosis at high doses in human cancer cells.  Int J Mol Med. 2009;  24 131-138
• 35 Gemin A, Sweet S, Preston T J, Singh G. Regulation of the cell cycle in response to inhibition of mitochondrial generated energy.  Biochem Biophys Res Commun. 2005;  332 1122-1132
• 36 Krishnan P, Bastow K F. The 9-position in berberine analogs is an important determinant of DNA topoisomerase II inhibition.  Anticancer Drug Des. 2000;  15 255-264
• 37 Letasiova S, Jantova S, Miko M, Ovadekova R, Horvathova M. Effect of berberine on proliferation, biosynthesis of macromolecules, cell cycle and induction of intercalation with DNA, dsDNA damage and apoptosis in Ehrlich ascites carcinoma cells.  J Pharm Pharmacol. 2006;  58 263-270
• 38 Sun Y, Xun K, Wang Y, Chen X. A systematic review of the anticancer properties of berberine, a natural product from Chinese herbs.  Anticancer Drugs. 2009;  20 757-769
• 39 Debnath D, Kumar G S, Nandi R, Maiti M. Interaction of berberine chloride with deoxyribonucleic acids: evidence for base and sequence specificity.  Indian J Biochem Biophys. 1989;  26 201-208
• 40 Letasiova S, Jantova S, Muckova M, Theiszova M. Antiproliferative activity of berberine in vitro and in vivo.  Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2005;  149 461-463
• 41 Konkimalla V B, Efferth T. Inhibition of epidermal growth factor receptor over-expressing cancer cells by the aphorphine-type isoquinoline alkaloid, dicentrine.  Biochem Pharmacol. 2010;  79 1092-1099
• 42 Efferth T, Fabry U, Osieka R. Apoptosis and resistance to daunorubicin in human leukemic cells.  Leukemia. 1997;  11 1180-1186
• 43 Johansson M, Persson J L. Cancer therapy: targeting cell cycle regulators.  Anticancer Agents Med Chem. 2008;  8 723-731
• 44 Efferth T, Fabry U, Glatte P, Osieka R. Expression of apoptosis-related oncoproteins and modulation of apoptosis by caffeine in human leukemic cells.  J Cancer Res Clin Oncol. 1995;  121 648-656
• 45 Wong A, Soo R A, Yong W P, Innocenti F. Clinical pharmacology and pharmacogenetics of gemcitabine.  Drug Metab Rev. 2009;  41 77-88
• 46 Bergman A M, Pinedo H M, Peters G J. Determinants of resistance to 2′,2′-difluorodeoxycytidine (gemcitabine).  Drug Resist Updat. 2002;  5 19-33
• 47 Kroep J R, Loves W J, van der Wilt C L, Alvarez E, Talianidis I, Boven E, Braakhuis B J, van Groeningen C J, Pinedo H M, Peters G J. Pretreatment deoxycytidine kinase levels predict in vivo gemcitabine sensitivity.  Mol Cancer Ther. 2002;  1 371-376