24-Hydroxyursolic Acid from the Leaves of the Diospyros kaki (Persimmon) Induces Apoptosis by Activation of AMP-activated Protein Kinase

Abstract

There are multiple lines of evidence that persimmon extract and its constituents have potent antitumor activity against human cancer cells. However, the molecular mechanisms of 24-hydroxyursolic acid, a triterpenoid found in persimmon, on antitumor activities are not yet understood. Here, we demonstrate that 24-hydroxyursolic acid inhibited cell proliferation, strongly activated AMP-activated protein kinase (AMPK) and mediated critical anticancer effects by inhibition of cyclooxygenase (COX-2) expression in HT-29 cells. In addition, 24-hydroxyursolic acid induced cellular apoptosis by activation of poly(ADP-ribose) polymerase (PARP), caspase-3, and phosphorylation of p53 at Ser15. It also strongly induced DNA fragmentation in HT-29 cells and thereby significantly inhibited colony formation of HT-29 cells in soft agar. In addition, 24-hydroxyursolic acid blocked the EGF-induced ERKs phosphorylation and led to the inhibition of AP-1 activity and cell transformation in JB6 CL41 cells. Collectively, these findings are the first to reveal a molecular basis for the anticarcinogenic action of 24-hydroxyursolic acid and might account for the reported chemopreventive and chemotherapic effects of persimmon extracts.

Key words

Diospyros kaki - Ebenaceae - persimmon - 24‐hydroxyursolic acid - AMPK - COX‐2

References

• 1 Mallavadhani U V, Panda A K, Rao Y R. Pharmacology and chemotaxonomy of Diospyros.  Phytochemistry. 1998;  49 901-951
• 2 Kameda K, Takaku T, Okuda H, Kimura Y, Okuda T, Hatano T, Agata I, Arichi S. Inhibitory effects of various flavonoids isolated from leaves of persimmon on angiotensin-converting enzyme activity.  J Nat Prod. 1987;  50 680-683
• 3 Kim S Y, Jeong S M, Kim S J, Jeon K I, Park E, Park H R, Lee S C. Effect of heat treatment on the antioxidative and antigenotoxic activity of extracts from persimmon (Diospyros kaki L.) peel.  Biosci Biotechnol Biochem. 2006;  70 999-1002
• 4 Bei W, Peng W, Zang L, Xie Z, Hu D, Xu A. Neuroprotective effects of a standardized extract of Diospyros kaki leaves on MCAO transient focal cerebral ischemic rats and cultured neurons injured by glutamate or hypoxia.  Planta Med. 2007;  73 636-643
• 5 Achiwa Y, Hibasami H, Katsuzaki H, Imai K, Komiya T. Inhibitory effects of persimmon (Diospyros kaki) extract and related polyphenol compounds on growth of human lymphoid leukemia cells.  Biosci Biotechnol Biochem. 1997;  61 1099-1101
• 6 Chakrabarty S, Roy M, Hazra B, Bhattacharya R K. Induction of apoptosis in human cancer cell lines by diospyrin, a plant-derived bisnaphthoquinonoid, and its synthetic derivatives.  Cancer Lett. 2002;  188 85-93
• 7 Das Sarma M, Ghosh R, Patra A, Hazra B. Synthesis and antiproliferative activity of some novel derivatives of diospyrin, a plant-derived naphthoquinonoid.  Bioorg Med Chem. 2007;  15 3672-3677
• 8 Corradetti M N, Inoki K, Bardeesy N, DePinho R A, Guan K L. Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome.  Genes Dev. 2004;  18 1533-1538
• 9 Rattan R, Giri S, Singh A K, Singh I. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase.  J Biol Chem. 2005;  280 39582-39593
• 10 Garcia-Gil M, Pesi R, Perna S, Allegrini S, Giannecchini M, Camici M, Tozzi M G. 5′-Aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells.  Neuroscience. 2003;  117 811-820
• 11 Kefas B A, Cai Y, Kerckhofs K, Ling Z, Martens G, Heimberg H, Pipeleers D, Van de Casteele M. Metformin-induced stimulation of AMP-activated protein kinase in beta-cells impairs their glucose responsiveness and can lead to apoptosis.  Biochem Pharmacol. 2004;  68 409-416
• 12 Saitoh M, Nagai K, Nakagawa K, Yamamura T, Yamamoto S, Nishizaki T. Adenosine induces apoptosis in the human gastric cancer cells via an intrinsic pathway relevant to activation of AMP-activated protein kinase.  Biochem Pharmacol. 2004;  67 2005-2011
• 13 Motoshima H, Goldstein B J, Igata M, Araki E. AMPK and cell proliferation–AMPK as a therapeutic target for atherosclerosis and cancer.  J Physiol. 2006;  574 63-71
• 14 Ayasolla K R, Singh A K, Singh I. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) attenuates the expression of LPS- and Abeta peptide-induced inflammatory mediators in astroglia.  J Neuroinflamm. 2005;  2 21
• 15 Thuong P T, Lee C H, Dao T T, Nguyen P H, Kim W G, Lee S J, Oh W K. Triterpenoids from the leaves of Diospyros kaki (Persimmon) and their inhibitory effects on protein tyrosine phosphatase 1B.  J Nat Prod. 2008;  71 1775-1778
• 16 Dong Z, Cmarik J L. Harvesting cells under anchorage-independent cell transformation conditions for biochemical analyses.  Sci STKE. 2002;  2002 PL7
• 17 Huang C, Ma W Y, Young M R, Colburn N, Dong Z. Shortage of mitogen-activated protein kinase is responsible for resistance to AP-1 transactivation and transformation in mouse JB6 cells.  Proc Natl Acad Sci USA. 1998;  95 156-161
• 18 Hwang J T, Kim Y M, Surh Y J, Baik H W, Lee S K, Ha J, Park O J. Selenium regulates cyclooxygenase-2 and extracellular signal-regulated kinase signaling pathways by activating AMP-activated protein kinase in colon cancer cells.  Cancer Res. 2006;  66 10057-10063
• 19 Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases.  J Biol Chem. 1995;  270 16483-16486
• 20 Dong Z, Birrer M J, Watts R G, Matrisian L M, Colburn N H. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells.  Proc Natl Acad Sci USA. 1994;  91 609-613
• 21 Sinicrope F A. Targeting cyclooxygenase-2 for prevention and therapy of colorectal cancer.  Mol Carcinogen. 2006;  45 447-454
• 22 Oshima M, Dinchuk J E, Kargman S L, Oshima H, Hancock B, Kwong E, Trzaskos J M, Evans J F, Taketo M M. Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2).  Cell. 1996;  87 803-809
• 23 Tsujii M, DuBois R N. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2.  Cell. 1995;  83 493-501
• 24 Kundu J K, Chun K S, Kim S O, Surh Y J. Resveratrol inhibits phorbol ester-induced cyclooxygenase-2 expression in mouse skin: MAPKs and AP-1 as potential molecular targets.  Biofactors. 2004;  21 33-39
• 25 Sebolt-Leopold J S, Dudley D T, Herrera R, Van Becelaere K, Wiland A, Gowan R C, Tecle H, Barrett S D, Bridges A, Przybranowski S, Leopold W R, Saltiel A R. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo.  Nat Med. 1999;  5 810-816
• 26 Hwang J T, Ha J, Park I J, Lee S K, Baik H W, Kim Y M, Park O J. Apoptotic effect of EGCG in HT-29 colon cancer cells via AMPK signal pathway.  Cancer Lett. 2007;  247 115-121
• 27 Kim M S, Park J Y, Namkoong C, Jang P G, Ryu J W, Song H S, Yun J Y, Namgoong I S, Ha J, Park I S, Lee I K, Viollet B, Youn J H, Lee H K, Lee K U. Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase.  Nat Med. 2004;  10 727-733
• 28 Hardie D G. New roles for the LKB1–>AMPK pathway.  Curr Opin Cell Biol. 2005;  17 167-173
• 29 Shaw R J, Kosmatka M, Bardeesy N, Hurley R L, Witters L A, DePinho R A, Cantley L C. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress.  Proc Natl Acad Sci USA. 2004;  101 3329-3335
• 30 Cao C, Lu S, Kivlin R, Wallin B, Card E, Bagdasarian A, Tamakloe T, Chu W M, Guan K L, Wan Y. AMP-activated protein kinase contributes to UV- and H2O2-induced apoptosis in human skin keratinocytes.  J Biol Chem. 2008;  283 28897-28908
• 31 Van Erk M J, Teuling E, Staal Y C, Huybers S, Van Bladeren P J, Aarts J M, Van Ommen B. Time- and dose-dependent effects of curcumin on gene expression in human colon cancer cells.  J Carcinogen. 2004;  3 8
• 32 Andersson D, Liu J J, Nilsson A, Duan R D. Ursolic acid inhibits proliferation and stimulates apoptosis in HT29 cells following activation of alkaline sphingomyelinase.  Anticancer Res. 2003;  23 3317-3322

1 These authors contributed equally to this work.