The Anti-Promyelocytic Leukemia Mode of Action of Two Endophytic Secondary Metabolites Unveiled by a Proteomic Approach

Abstract

As a result of a program to find antitumor compounds of endophytes from medicinal Asteraceae, the steroid (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (a) and the diterpene aphidicolin (b) were isolated from the filamentous fungi Papulaspora immersa and Nigrospora sphaerica, respectively, and exhibited strong cytotoxicity against HL-60 cells. A proteomic approach was used in an attempt to identify the drugsʼ molecular targets and their respective antiproliferative mode of action. Results suggested that the (a) growth inhibition effect occurs by G2/M cell cycle arrest via reduction of tubulin alpha and beta isomers and 14–3–3 protein gamma expression, followed by a decrease of apoptotic and inflammatory proteins, culminating in mitochondrial oxidative damage that triggered autophagy-associated cell death. Moreover, the decrease observed in the expression levels of several types of histones indicated that (a) might be disarming oncogenic pathways via direct modulation of the epigenetic machinery. Effects on cell cycle progression and induction of apoptosis caused by (b) were confirmed. In addition, protein expression profiles also revealed that aphidicolin is able to influence microtubule dynamics, modulate proteasome activator complex expression, and control the inflammatory cascade through overexpression of thymosin beta 4, RhoGDI2, and 14–3–3 proteins. Transmission electron micrographs of (b)-treated cells unveiled dose-dependent morphological characteristics of autophagy- or oncosis-like cell death.

Key words

Chaetomiaceae - Papulaspora immersa - Trichosphaeriaceae - Nigrospora sphaerica - aphidicolin - epoxyergostane-type steroid

• References

• 1 National Cancer Institute. Leukemia. Available at http://www.cancer.gov/cancertopics/types/leukemia from National Cancer Institute. Accessed October 30, 2013
• 2 Mi J. Current treatment strategy of acute promyelocytic leukemia. Front Med 2011; 5: 341-347
• 3 Gallo MBC, Chagas FO, Almeida MO, Macedo CC, Cavalcanti BC, Barros FWA, de Moraes MO, Costa-Lotufo LV, Pessoa C, Bastos JK, Pupo MT. Endophytic fungi found in association with Smallanthus sonchifolius (Asteraceae) as resourceful producers of cytotoxic bioactive natural products. J Basic Microbiol 2009; 49: 142-151
• 4 Gallo MBC, Cavalcanti BC, Barros FWA, de Moraes MO, Costa-Lotufo LV, Pessoa C, Bastos JK, Pupo MT. Chemical constituents of Papulaspora immersa, an endophyte from Smallanthus sonchifolius (Asteraceae), and their cytotoxic activity. Chem Biodivers 2010; 7: 2941-2950
• 5 Krokan H, Wist E, Krokan RH. Aphidicolin inhibits DNA synthesis by DNA polymerase and isolated nuclei by a similar mechanism. Nucleic Acids Res 1981; 9: 4709-4719
• 6 OʼDwyer PJ, Moyer JD, Suffness M, Harrison SD, Cysyk R, Hamilton TC, Plowman J. Antitumor activity and biochemical effects of aphidicolin glycinate (NSC 303812) alone and in combination with cisplatin in vivo . Cancer Res 1994; 54: 724-729
• 7 Schimke RT, Kung A, Sherwood SS, Sheridan J, Sharma R. Life, death and genomic change in perturbed cell cycles. Trans R Soc B Biol Sciences 1994; 345: 311-317
• 8 Kurose A, Tanaka T, Huang X, Traganos F, Darzynkiewicz Z. Synchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation: an indication of DNA damage. Cell Prolif 2006; 39: 231-240
• 9 Csapó Z, Sasvári-Székely M, Spasokoukotskaja T, Talianidis I, Eriksson S, Staub M. Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes. Biochem Pharmacol 2001; 61: 191-197
• 10 Chou RH, Chervenick PA. Combined effects of aphidicolin and retinoic acid on proliferation and differentiation on human leukemic (HL-60) cells. Cell Tissue Kinet 1985; 18: 387-397
• 11 Kobayashi M, Lee NK, Son BW, Yanagi K, Kyogoku Y, Kitagawa I. Stoloniferone-A, -B, -C, and –D, four new cytotoxic steroids from the Okinawa soft coral Clavularia viridis . Tetrahedron Lett 1984; 25: 5925-5928
• 12 Samadi AK, Tong X, Mukerji R, Zhang H, Timmermann BN, Cohen MS. Withaferin A, a cytotoxic steroid from Vassobia breviflora, induces apoptosis in human head and neck squamous cell carcinoma. J Nat Prod 2010; 73: 1476-1481
• 13 de Ridder MC, Van Der Plas AJ, Erpelinck-Verschueren CAJ, Wenberg BL, Jansen JH. Dexamethasone does not counteract the response of acute promyelocytic leukaemia cells to all-trans retinoic acid. British J Haematol 1999; 106: 107-110
• 14 Distelhorst CW. Recent insights into the mechanism of glucocorticosteroid-induced apoptosis. Cell Death Differ 2002; 9: 6-19
• 15 Van Waes C. Nuclear factor-kB in development, prevention, and therapy of cancer. Clin Cancer Res 2007; 13: 1076-1082
• 16 Laane E, Tamm KP, Buentke E, Ito K, Khahariza P, Oscarsson J, Corcoran M, Björklund AC, Hultenby K, Lundin J, Heyman M, Söderhäll S, Mazur J, Porwit A, Pandolfi PP, Zhivotovsky B, Panaretakis T, Grandér D. Cell death induced by dexamethasone in lymphoid leukemia is mediated through initiation of autophagy. Cell Death Diff 2009; 16: 1018-1029
• 17 Dunn jr. WA. Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J Cell Biol 1990; 110: 1923-1933
• 18 Li L, Han W, Gu Y, Qiu S, Lu Q, Jin J, Luo J, Hu X. Honokiol induces a necrotic cell death through the mitochondrial permeability transition pore. Cancer Res 2007; 67: 4894-4903
• 19 Bera TK, Walker DA, Sherins RJ, Pastan I. POTE protein, a cancer-testis antigen, is highly expressed in spermatids in human testis and is associated with apoptotic cells. Biochem Biophys Res Communic 2012; 417: 1271-1274
• 20 Choi MR, Groot M, Drexler HCA. Functional implications of caspase-mediated RhoGDI2 processing during apoptosis of HL60 and K562 leukemia cells. Apoptosis 2007; 12: 2025-2035
• 21 Cui J, Li W, Wang J, Li A, Li H, Wang H, He K, Li W, Kang L, Yu M, Shen B, Wang G, Zhang X. Proteomics-based identification of human acute leukemia antigens that induce humoral immune response. Mol Cell Proteomics 2005; 4: 1718-1724
• 22 Arntzen MØ, Thiede B. Apoptoproteomics, an integrated database for analysis of proteomics data obtained from apoptotic cells. Mol Cell Proteomics 2012; 11: M111.010447
• 23 Jaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 2005; 21: 247-269
• 24 Lee DH, Chung K, Song JA, Kim TH, Kang H, Huh JH, Jung SG, Ko JJ, An HJ. Proteomic identification of paclitaxel-resistance associated hnRNP A2 and GDI 2 proteins in human ovarian cancer cells. J Proteome Res 2010; 9: 5668-5676
• 25 Rubin CI, Atweh GF. The role of stathmin in the regulation of the cell cycle. J Cell Biochem 2004; 93: 242-250
• 26 Bhalla KB. Microtubule-targeted anticancer agents and apoptosis. Oncogene 2003; 22: 9075-9086
• 27 Chakravarti D, Hong R. SET-ting the stage for life and death. Cell 2003; 112: 589-593
• 28 Sancho M, Diani E, Beato M, Jordan A. Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth. PLoS Genetics 2008; 4: 1-17
• 29 Fazal F, Bijli KM, Minhajuddin M, Rein T, Finkelstein JN, Rahman A. Essential role of cofilin-1 in regulating thrombin-induced RelA/p 65 nuclear translocation and intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells. J Biol Chem 2009; 284: 21047-21056
• 30 Assinder SJ, Stanton JAL, Prasad PD. Transgelin: an actin-binding protein and tumour suppressor. Int J Biochem Cell Biol 2009; 41: 482-486
• 31 Giardino AK, Yaffe MB. 14-3-3 Proteins as signaling integration points for cell cycle control and apoptosis. Semin Cell Dev Biol 2011; 22: 688-695
• 32 Shachar I, Haran M. The secret second life of an innocent chaperone: the story of CD74 and B cell/chronic lymphocytic leukemia cell survival. Leuk Lymphoma 2011; 52: 1446-1454
• 33 Stanković S, Majkić-Singh N. Myeloperoxidase: new roles for an old molecule. J Med Biochem 2011; 30: 230-236
• 34 Sarmento-Ribeiro AB, Proença MT, Sousa I, Pereira A, Guedes F, Teixeira A, Oliveira CR. A possible role for oxidation stress in lymphoid leukaemias and therapeutic failure. Leuk Res 2012; 36: 1041-1048
• 35 Rollins-Raval MA, Roth CG. The value of immunohistochemistry for CD14, CD123, CD33, myeloperoxidase and CD68R in the diagnosis of acute and chronic myelomonocytic leukaemias. Histopathol 2012; 60: 933-942
• 36 Conroy H, Mawhinney L, Donnelly SC. Inflammation and cancer: macrophage migration inhibitory factor (MIF)–the potential missing link. Q J Med 2010; 103: 831-836
• 37 Tang MC, Su Y. Thymosin β ₄ knockdown disrupts mitochondrial functions of SW480 human colon cancer cells. Cancer Sci 2011; 102: 1665-1672
• 38 Jo JO, Kang YJ, Ock MS, Kleinman HK, Chang HK, Cha H. Thymosin β ₄ expression in human tissues and in tumors using tissue microarrays. J Appl Immunohistochem Mol Morphol 2011; 19: 160-167
• 39 Moon EY, Im YS, Ryu YK, Kang JH. Actin-sequestering protein, thymosin beta-4, is a novel hypoxia responsive regulator. Clin Exp Metastasis 2010; 27: 601-609
• 40 Crockford D, Turjman N, Allan C, Angel J. Thymosin β ₄: structure, function, and biological properties supporting current and future clinical applications. Ann N Y Acad Sci 2010; 1194: 179-189
• 41 Antharavally BS, Mallia KA, Rangaraj P, Bell PA. Quantitation of proteins using a dye-metal-based colorimetric protein assay. Anal Biochem 2009; 385: 342-345
• 42 Richter L, Ruckert U, Kramer S. Learning a predictive model for growth inhibition from the NCI DTP human tumor cell line screening data: does gene expression make a difference?. Pacific Symp Biocomp 2006; 11: 596-607
• 43 McCarthy J, Hopwood F, Oxley D, Laver M, Castagna A, Righetti PG, Williams K, Herbert BJ. Carbamylation of proteins in 2-D electrophoresis myth or reality?. Proteome Res 2003; 2: 239-242