NF-κB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death

BMC Complementary and Alternative Medicine

Yan Zhao²  Mohamed Sabry Hamza⁴  Kandhadayar Gopalan Srinivasan⁵  Jinming Li²  XiaoLing Feng¹  Hong Shuang Zhu²  Nung Ky²  Pan You Fu³  Chuan Bian Lim² 

[1] HeiLongJiang University of Chinese Medicine, Harbin, People’s Republic of China;Division of Chemical Biology and Biotechnology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore;National Cancer Centre of Singapore, NCCS-VARI Translational Research Laboratory, #501, Level 5, 11 Hospital Drive, Singapore 169610, Singapore;MSD, Translational Medicine Research Center, 8 Biomedical Grove, #04-01/-05 & #05-01/05, Neuros Building, Singapore 138665, Singapore;1stBASE Pte Ltd., 41 Singapore Science Park II, The Gemini, Singapore 117610, Singapore

关键词: Atg12 and LC3-B;    NF-κB;    Caspase;    Autophagy;    Helenalin or Hele(Helenalin);   

Others  :  1232122
DOI  :  10.1186/1472-6882-12-93

 received in 2012-01-20, accepted in 2012-07-11,  发布年份 2012

【 摘 要 】

Background

Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin’s multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained.

Methods

To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers.

Results

We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death.

Conclusions

Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.

【 授权许可】

   
2012 Lim et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Paterson I, Anderson EA: Chemistry. The renaissance of natural products as drug candidates. Science 2005, 310(5747):451-453.
• [2]Efferth T, Benakis A, Romero MR, Tomicic M, Rauh R, Steinbach D, Hafer R, Stamminger T, Oesch F, Kaina B, et al.: Enhancement of cytotoxicity of artemisinins toward cancer cells by ferrous iron. Free Radic Biol Med 2004, 37(7):998-1009.
• [3]Efferth T, Rucker G, Falkenberg M, Manns D, Olbrich A, Fabry U, Osieka R: Detection of apoptosis in KG-1a leukemic cells treated with investigational drugs. Arzneimittelforschung 1996, 46(2):196-200.
• [4]Efferth T, Sauerbrey A, Olbrich A, Gebhart E, Rauch P, Weber HO, Hengstler JG, Halatsch ME, Volm M, Tew KD, et al.: Molecular modes of action of artesunate in tumor cell lines. Mol Pharmacol 2003, 64(2):382-394.
• [5]Huang PR, Yeh YM, Wang TC: Potent inhibition of human telomerase by helenalin. Cancer Lett 2005, 227(2):169-174.
• [6]Berges C, Fuchs D, Opelz G, Daniel V, Naujokat C: Helenalin suppresses essential immune functions of activated CD4+ T cells by multiple mechanisms. Mol Immunol 2009, 46(15):2892-2901.
• [7]Lyss G, Knorre A, Schmidt TJ, Pahl HL, Merfort I: The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65. J Biol Chem 1998, 273(50):33508-33516.
• [8]Zhao Y, Hamza MS, Leong HS, Lim CB, Pan YF, Cheung E, Soo KC, Iyer NG: Kruppel-like factor 5 modulates p53-independent apoptosis through Pim1 survival kinase in cancer cells. Oncogene 2008, 27(1):1-8.
• [9]Traganos F, Darzynkiewicz Z: Lysosomal proton pump activity: supravital cell staining with acridine orange differentiates leukocyte subpopulations. Methods Cell Biol 1994, 41:185-194.
• [10]Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C: A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991, 139(2):271-279.
• [11]Telford WG, King LE, Fraker PJ: Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 1992, 13(2):137-143.
• [12]Hoffmann R, von Schwarzenberg K, Lopez-Anton N, Rudy A, Wanner G, Dirsch VM, Vollmar AM: Helenalin bypasses Bcl-2-mediated cell death resistance by inhibiting NF-kappaB and promoting reactive oxygen species generation. Biochem Pharmacol 2011, 82(5):453-463.
• [13]Kim HP, Wang X, Chen ZH, Lee SJ, Huang MH, Wang Y, Ryter SW, Choi AM: Autophagic proteins regulate cigarette smoke-induced apoptosis: protective role of heme oxygenase-1. Autophagy 2008, 4(7):887-895.
• [14]Telleria CM, Goyeneche AA, Stocco CO, Gibori G: Involvement of nuclear factor kappa B in the regulation of rat luteal function: potential roles as survival factor and inhibitor of 20alpha-hydroxysteroid dehydrogenase. J Mol Endocrinol 2004, 32(2):365-383.
• [15]Copetti T, Bertoli C, Dalla E, Demarchi F, Schneider C: p65/RelA modulates BECN1 transcription and autophagy. Mol Cell Biol 2009, 29(10):2594-2608.
• [16]Newman DJ, Cragg GM: Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007, 70(3):461-477.
• [17]Normile D: Asian medicine. The new face of traditional Chinese medicine. Science 2003, 299(5604):188-190.
• [18]Wiseman N: Traditional Chinese medicine: a brief outline. J Chem Inf Comput Sci 2002, 42(3):445-455.
• [19]Yuan R, Lin Y: Traditional Chinese medicine: an approach to scientific proof and clinical validation. Pharmacol Ther 2000, 86(2):191-198.
• [20]Powis G, Gallegos A, Abraham RT, Ashendel CL, Zalkow LH, Grindey GB, Bonjouklian R: Increased intracellular Ca2+ signaling caused by the antitumor agent helenalin and its analogues. Cancer Chemother Pharmacol 1994, 34(4):344-350.
• [21]Boulanger D, Brouillette E, Jaspar F, Malouin F, Mainil J, Bureau F, Lekeux P: Helenalin reduces Staphylococcus aureus infection in vitro and in vivo. Vet Microbiol 2007, 119(2–4):330-338.
• [22]Francois G, Passreiter CM: Pseudoguaianolide sesquiterpene lactones with high activities against the human malaria parasite Plasmodium falciparum. Phytother Res 2004, 18(2):184-186.
• [23]Shacka JJ, Klocke BJ, Roth KA: Autophagy, bafilomycin and cell death: the “a-B-cs” of plecomacrolide-induced neuroprotection. Autophagy 2006, 2(3):228-230.
• [24]Shacka JJ, Klocke BJ, Shibata M, Uchiyama Y, Datta G, Schmidt RE, Roth KA: Bafilomycin A1 inhibits chloroquine-induced death of cerebellar granule neurons. Mol Pharmacol 2006, 69(4):1125-1136.
• [25]Wu YC, Wu WK, Li Y, Yu L, Li ZJ, Wong CC, Li HT, Sung JJ, Cho CH: Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells. Biochem Biophys Res Commun 2009, 382(2):451-456.
• [26]Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y: Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 1998, 23(1):33-42.
• [27]Jin F, Liu X, Zhou Z, Yue P, Lotan R, Khuri FR, Chung LW, Sun SY: Activation of nuclear factor-kappaB contributes to induction of death receptors and apoptosis by the synthetic retinoid CD437 in DU145 human prostate cancer cells. Cancer Res 2005, 65(14):6354-6363.
• [28]Buchele B, Zugmaier W, Lunov O, Syrovets T, Merfort I, Simmet T: Surface plasmon resonance analysis of nuclear factor-kappaB protein interactions with the sesquiterpene lactone helenalin. Anal Biochem 2010, 401(1):30-7.
• [29]Reikvam H, Olsnes AM, Gjertsen BT, Ersvar E, Bruserud O: Nuclear Factor-kappaB Signaling: A Contributor in Leukemogenesis and a Target for Pharmacological Intervention in Human Acute Myelogenous Leukemia. Crit Rev Oncog 2009, 15(1):1-36.
• [30]Fabre C, Carvalho G, Tasdemir E, Braun T, Ades L, Grosjean J, Boehrer S, Metivier D, Souquere S, Pierron G, et al.: NF-kappaB inhibition sensitizes to starvation-induced cell death in high-risk myelodysplastic syndrome and acute myeloid leukemia. Oncogene 2007, 26(28):4071-4083.
• [31]Djavaheri-Mergny M, Amelotti M, Mathieu J, Besancon F, Bauvy C, Codogno P: Regulation of autophagy by NFkappaB transcription factor and reactives oxygen species. Autophagy 2007, 3(4):390-392.
• [32]Nivon M, Richet E, Codogno P, Arrigo AP, Kretz-Remy C: Autophagy activation by NFkappaB is essential for cell survival after heat shock. Autophagy 2009, 5(6):766-783.
• [33]Xiao G: Autophagy and NF-kappaB: fight for fate. Cytokine Growth Factor Rev 2007, 18(3–4):233-243.
• [34]Fan Y, Mao R, Zhao Y, Yu Y, Sun W, Song P, Shi Z, Zhang D, Yvon E, Zhang H, et al.: Tumor necrosis factor-alpha induces RelA degradation via ubiquitination at lysine 195 to prevent excessive nuclear factor-kappaB activation. J Biol Chem 2009, 284(43):29290-29297.
• [35]Baumgartner B, Weber M, Quirling M, Fischer C, Page S, Adam M, Von Schilling C, Waterhouse C, Schmid C, Neumeier D, et al.: Increased IkappaB kinase activity is associated with activated NF-kappaB in acute myeloid blasts. Leukemia 2002, 16(10):2062-2071.
• [36]Braun T, Carvalho G, Coquelle A, Vozenin MC, Lepelley P, Hirsch F, Kiladjian JJ, Ribrag V, Fenaux P, Kroemer G: NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome. Blood 2006, 107(3):1156-1165.
• [37]Cilloni D, Messa F, Rosso V, Arruga F, Defilippi I, Carturan S, Catalano R, Pautasso M, Panuzzo C, Nicoli P, et al.: Increase sensitivity to chemotherapeutical agents and cytoplasmatic interaction between NPM leukemic mutant and NF-kappaB in AML carrying NPM1 mutations. Leukemia 2008, 22(6):1234-1240.
• [38]Frelin C, Imbert V, Griessinger E, Peyron AC, Rochet N, Philip P, Dageville C, Sirvent A, Hummelsberger M, Berard E, et al.: Targeting NF-kappaB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells. Blood 2005, 105(2):804-811.
• [39]Itoh M, Fu L, Tohda S: NF-kappaB activation induced by Notch ligand stimulation in acute myeloid leukemia cells. Oncol Rep 2009, 22(3):631-634.