Anti-Cancer Effect of Betulin on a Human Lung Cancer Cell Line: A Pharmacoproteomic Approach Using 2 D SDS PAGE Coupled with Nano-HPLC Tandem Mass Spectrometry

Abstract

Betulin is a representative compound of Betula platyphylla, a tree species belonging to the Betulaceae family. In this investigation, we revealed that betulin showed anticancer activity on human lung cancer A549 cells by inducing apoptosis and changes in protein expression profiles were observed. Upon flow cytometry analysis, the surface of betulin-treated cells was found to be annexin-V positive and propidium iodide (PI) negative, which indicated that the cells were apoptotic. In order to identify the molecular players involved in betulin-induced apoptosis, cellular proteins were applied to two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2 D SDS PAGE) for differential proteomic analysis. As a result, four downregulated proteins and three upregulated proteins were identified by nano-HPLC MS/MS. The four downregulated proteins were poly(rC)-binding protein 1, isoform 1 of 3-hydroxyacyl-CoA dehydrogenase type 2, heat shock protein 90-alpha 2, and enoyl-CoA hydratase; the three upregulated proteins were aconitate hydratase, malate dehydrogenase, and splicing factor arginine/serine-rich 1. These differentially expressed proteins explained the cytotoxicity of betulin against human lung cancer A549 cells, and the proteomic approach was thus shown to be a potential tool for understanding the pharmacological activities of pharmacophores.

Abbreviations

ACN:acetonitrile

DMSO:dimethyl sulfoxide

FACS:fluorescence activated cell sorter

HEPES:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

MTT:methylthiazolelyldiphenyl-tetrazolium bromide

PI:prodidium iodide

TFA:trifluoroacetic acid

Key words

Betulaceae - Betula platyphylla - betulin - A549 cell line - proteomics - nano-HPLC MS/MS

References

• 1 Ulrich Merzenich G, Zeitler H, Jobst D, Panek D, Vetter H, Wagner H. Application of the ”-Omic-” technologies in phytomedicine.  Phytomedicine. 2007;  14 70-82
• 2 Matsuda H, Ishikado A, Nishida N, Ninomiya K, Fujiwara H, Kobayashi Y. et al . Hepatoprotective, superoxide scavenging, and antioxidative activities of aromatic constituents from the bark of Betula platyphylla var. japonica. .  Bioorg Med Chem Lett. 1998;  8 2939-44
• 3 Ju E M, Lee S E, Hwang H J, Kim J H. Antioxidant and anticancer activity of extract from Betula platyphylla var. japonica. .  Life Sci. 2004;  74 1013-26
• 4 Manez S, Recio M C, Giner R M, Rios J L. Effect of selected triterpenoids on chronic dermal inflammation.  Eur J Pharmacol. 1997;  334 103-5
• 5 Gong Y, Raj K M, Luscombe C A, Gadawski I, Tam T, Chu J. et al . The synergistic effects of betulin with acyclovir against Herpes simplex viruses.  Antiviral Res. 2004;  64 127-30
• 6 Hwang B Y, Chai H B, Kardono L B, Riswan S, Farnsworth N R, Cordell G A. et al . Cytotoxic triterpenes from the twigs of Celtis philippinensis. .  Phytochemistry. 2003;  62 197-201
• 7 Mayer B, Oberbauer R. Mitochondrial regulation of apoptosis.  News Physiol Sci. 2003;  18 89-94
• 8 Li X, Wang J, Manley J L. Loss of splicing factor ASF/SF2 induces G2 cell cycle arrest and apoptosis, but inhibits internucleosomal DNA fragmentation.  Genes Dev. 2005;  19 2705-14
• 9 Townsend P A, Cutress R I, Sharp A, Brimmell M, Packham G. BAG-1: a multifunctional regulator of cell growth and survival.  Biochim Biophys Acta. 2003;  1603 83-98
• 10 Pickering B M, Mitchell S A, Spriggs K A, Stoneley M, Willis A E. Bag-1 internal ribosome entry segment activity is promoted by structural changes mediated by poly(rC) binding protein 1 and recruitment of polypyrimidine tract binding protein 1.  Mol Cell Biol. 2004;  24 5595-605
• 11 Lanneau D, Brunet M, Frisan E, Solary E, Fontenay M, Garrido C. Heat shock proteins: essential proteins for apoptosis regulation.  J Cell Mol Med. 2008;  12 743-61
• 12 Kooijman R. Regulation of apoptosis by insulin-like growth factor (IGF)-I.  Cytokine Growth Factor Rev. 2006;  17 305-23
• 13 Nielsen T O, Andrews H N, Cheang M, Kucab J E, Hsu F D, Ragaz J. et al . Expression of the insulin-like growth factor I receptor and urokinase plasminogen activator in breast cancer is associated with poor survival: potential for intervention with 17-allylamino geldanamycin.  Cancer Res. 2004;  64 286-91
• 14 Xu W, Neckers L. Targeting the molecular chaperone heat shock protein 90 provides a multifaceted effect on diverse cell signaling pathways of cancer cells.  Clin Cancer Res. 2007;  13 1625-9