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DC-81-enediyne induces apoptosis of human melanoma A375 cells: involvement of the ROS, p38 MAPK, and AP-1 signaling pathways

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Abstract

Melanoma is one of the most chemoresistant cancers in patient care. The remission rate of current therapy remains low. DC-81, an antitumor antibiotic produced by Streptomyces species, belongs to pyrrolo[2,1-c][1,4]benzodiazepine (PBD), which is a potent inhibitor of nucleic acid synthesis. An enediyne contains either DNA intercalating groups or DNA minor groove binding functions and these are potent DNA-damaging agents due to their ability to generate benzenoid diradicals. We have previously reported an efficient synthesis and antitumor activity of a series of novel PBD hybrids linked with enediyne. The purpose of this study was to examine the mechanism of the antiproliferative effect of DC-81-enediyne agent on human melanoma A375 cells. DC-81-enediyne induced an increase in Ca2+ level and reactive oxygen species (ROS) generation as detected by flow cytometric assay. Western blot analysis showed that DC-81-enediyne induced the phosphorylation of p38 and activating transcription factor 2 (ATF-2). By using the luciferase reporter assay, activating protein-1 (AP-1) activity was further enhanced after A375 cells were treated with graded concentrations of DC-81-enediyne. DC-81-enediyne treatment-induced A375 cell apoptosis was significantly abrogated by the addition of Ca2+, ROS, and p38 inhibitors. Collectively, our studies indicate that DC-81-enediyne induces A375 cell apoptosis through an increased Ca2+ and ROS generation, which involves p38 phosphorylation and enhanced ATF-2/AP-1 expressions, leading to caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 CytoDeath staining, and subsequent apoptotic cell death.

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Abbreviations

AP-1:

Activating protein-1

ATF-2:

Activating transcription factor 2

DCFH-DA:

2′,7′-Dichlorofluorescein diacetate

DMEM:

Dulbecco’s minimal essential medium

ERK:

Extracellular signal-regulated kinase

JNK:

c-Jun N-terminal kinase

MAPK:

Mitogen-activated protein kinase

PARP:

Poly(ADP-ribose)polymerase

PBD:

Pyrrolo[2,1-c][1,4]benzodiazepine

ROS:

Reactive oxygen species

References

  • Aggeli IK, Gaitanaki C, Beis I. Involvement of JNKs and p38-MAPK/MSK1 pathways in H2O2-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells. Cell Signal. 2006;18:1801–12.

    Article  PubMed  CAS  Google Scholar 

  • Ameyar M, Wisniewska M, Weitzman JB. A role for AP-1 in apoptosis: the case for and against. Biochimie. 2003;85:747–52.

    Article  PubMed  CAS  Google Scholar 

  • Bacus SS, Gudkov AV, Lowe M, Lyass L, Yung Y, Komarov AP, Keyomarsi K, Yarden Y, Seger R. Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene. 2001;202:147–55.

    Article  Google Scholar 

  • Barzilai A, Yamamoto K. DNA damage responses to oxidative stress. DNA Repair. 2004;3:1109–15.

    Article  PubMed  CAS  Google Scholar 

  • Brantley-Finley C, Lyle CS, Du L. The JNK, ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine, doxorubicin, and etoposide. Biochem Pharmacol. 2003;66:459–69.

    Article  PubMed  CAS  Google Scholar 

  • Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis. Immunol Today. 1994;15:7–10.

    Article  PubMed  CAS  Google Scholar 

  • Choi EM. Regulation of intracellular Ca(2+) by reactive oxygen species in osteoblasts treated with antimycin A. J Appl Toxicol. 2012;32:118–25.

    Article  PubMed  CAS  Google Scholar 

  • Cross PE, Dickinson RP, Parry MJ, Randall MJ. Selective thromboxane synthetase inhibitors. 3. 1H-imidazol-1-yl-substituted benzo[b]furan-, benzo[b]thiophene-, and indole-2- and -3-carboxylic acids. J Med Chem. 1986;29:1637–43.

    Article  PubMed  CAS  Google Scholar 

  • Engesæter B, Engebraaten O, Flørenes VA, Mælandsmo GM. Dacarbazine and the agonistic TRAIL receptor-2 antibody lexatumumab induce synergistic anticancer effects in melanoma. PLoS One. 2012;7:e45492. doi:10.1371/journal.pone.0045492.

    Article  PubMed  Google Scholar 

  • Garbe C, Eigentler TK. Diagnosis and treatment of cutaneous melanoma: state of the art 2006. Melanoma Res. 2007;17:117–27.

    Article  PubMed  Google Scholar 

  • Gius D, Botero A, Shah S, Curry HA. Intracellular oxidation/reduction status in the regulation of transcription factors NF-kappaB and AP-1. Toxicol Lett. 1999;106:93–106.

    Article  PubMed  CAS  Google Scholar 

  • Gupta A, Rosenberger SF, Bowden GT. Increased ROS levels contribute to elevated transcription factor and MAP kinase activities in malignantly progressed mouse keratinocyte cell lines. Carcinogenesis. 1999;20:2063–73.

    Article  PubMed  CAS  Google Scholar 

  • Hess J, Angel P, Schorpp-Kistner M. AP-1 subunits: quarrel and harmony among siblings. J Cell Sci. 2004;117:5965–73.

    Google Scholar 

  • Hsieh MC, Hu WP, Yu HS, Wu WC, Chang LS, Kao YH, Wang JJ. A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1α-mediated signaling. Toxicol Appl Pharmacol. 2011;255:150–9.

    Article  PubMed  CAS  Google Scholar 

  • Hu WP, Wang JJ, Lin FL, Lin YC, Lin SR, Hsu MH. An efficient synthesis of pyrrolo[2,1-c]-[1,4]benzodiazepine. Synthesis of the antibiotic DC-81. J Org Chem. 2001;66:2881–3.

    Article  PubMed  CAS  Google Scholar 

  • Hu WP, Yu HS, Sung PJ, Tsai FY, Shen YK, Chang LS, Wang JJ. DC-81-indole conjugate agent induces mitochondria mediated apoptosis in human melanoma A375 cells. Chem Res Toxicol. 2007;20:905–12.

    Article  PubMed  CAS  Google Scholar 

  • Hu WP, Tsai FY, Yu HS, Sung PJ, Chang LS, Wang JJ. Induction of apoptosis by DC-81-indole conjugate agent through NF-kappaB and JNK/AP-1 pathway. Chem Res Toxicol. 2008;21:1330–6.

    Article  PubMed  CAS  Google Scholar 

  • Hu WP, Liang JJ, Kao CL, Chen YC, Chen CY, Tsai FY, Wu MJ, Chang LS, Chen YL, Wang JJ. Synthesis and antitumor activity of novel enediyne-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids. Bioorgan Med Chem. 2009;17:1172–80.

    Article  CAS  Google Scholar 

  • Jemal A, Devesa SS, Hartge P, Tucker MA. Recent trends in cutaneous melanoma incidence among whites in the United States. J Natl Cancer Inst. 2001;93:678–83.

    Article  PubMed  CAS  Google Scholar 

  • Karin M, Liu ZG, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol. 1997;9:240–6.

    Article  PubMed  CAS  Google Scholar 

  • Konishi M, Ohkuma H, Tsuno T, Oki T, VanDuyne GD, Clardy J. Crystal and molecular structure of dynemicin A: a novel 1,5-diyn-3-ene antitumor antibiotic. J Am Chem Soc. 1990;112:3715–6.

    Article  CAS  Google Scholar 

  • Krol J, Mengele K, Ottl-Mantchenko I, Welk A, Wasilewitsch I, von Steinburg SP, Schneider KT, Schmitt M. Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitope. Int J Mol Med. 2005;16:415–20.

    PubMed  CAS  Google Scholar 

  • Lee CH, Hu WP, Hong CH, Yu HS, Liao WT, Chen CY, Chen YL, Chen BH, Chen GS, Wang JJ. Pyrrolo[2,1-c][1,4]benzodiazepine and indole conjugate (IN6CPBD) has better efficacy and superior safety than the mother compound DC-81 in suppressing the growth of established melanoma in vivo. Chem Biol Interact. 2009;180:360–7.

    Article  PubMed  CAS  Google Scholar 

  • Lee MD, Dunne TS, Siegel MM, Chang CC, Morton GO, Borders DB. Calichemicins, a novel family of antitumor antibiotics. 1. Chemistry and partial structure of calichemicin.gamma.1I. J Am Chem Soc. 1987;109:3464–6.

    Article  CAS  Google Scholar 

  • Lipton SA, Nicotera P. Calcium, free radicals and excitotoxins in neuronal apoptosis. Cell Calcium. 1998;23:165–71.

    Article  PubMed  CAS  Google Scholar 

  • Mahboobi S, Pongratz H, Hufsky H, Hockemeyer J, Frieser M, Lyssenko A, Paper DH, Bürgermeister J, Böhmer FD, Fiebig HH, Burger AM, Baasner S, Beckers T. Synthetic 2-aroylindole derivatives as a new class of potent tubulin-inhibitory, antimitotic agents. J Med Chem. 2001;44:4535–53.

    Article  PubMed  CAS  Google Scholar 

  • Merighi S, Simioni C, Gessi S, Varani K, Mirandola P, Tabrizi MA, Baraldi PG, Borea PA. A2B and A3 adenosine receptors modulate vascular endothelial growth factor and interleukin-8 expression in human melanoma cells treated with etoposide and doxorubicin. Neoplasia. 2009;11:1064–73.

    PubMed  CAS  Google Scholar 

  • Mingo-Sion AM, Marietta PM, Koller E, Wolf DM, Van Den Berg CL. Inhibition of JNK reduces G2/M transit independent of p53, leading to endoreduplication, decreased proliferation, and apoptosis in breast cancer cells. Oncogene. 2004;23:596–604.

    Article  PubMed  CAS  Google Scholar 

  • Morton S, Davis RJ, Cohen P. Signalling pathways involved in multisite phosphorylation of the transcription factor ATF-2. FEBS Lett. 2004;572:177–83.

    Article  PubMed  CAS  Google Scholar 

  • Nagata Y, Todokoro K. Requirement of activation of JNK and p38 for environmental stress-induced erythroid differentiation and apoptosis and of inhibition of ERK for apoptosis. Blood. 1999;94:853–63.

    PubMed  CAS  Google Scholar 

  • Nicolaou KC, Dai WM. Chemistry and biology of the enediyne anticancer antibiotics. Angew Chem Int Ed Engl. 1991;30:1387–416.

    Article  Google Scholar 

  • Nicotera P, Orrenius S. The role of calcium in apoptosis. Cell Calcium. 1998;23:173–80.

    Article  PubMed  CAS  Google Scholar 

  • Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium–apoptosis link. Nat Rev Mol Cell Biol. 2003;4:552–65.

    Article  PubMed  CAS  Google Scholar 

  • Qian Y, Guan T, Huang M, Cao L, Li Y, Cheng H, Jin H, Yu D. Neuroprotection by the soy isoflavone, genistein, via inhibition of mitochondria-dependent apoptosis pathways and reactive oxygen induced-NF-κB activation in a cerebral ischemia mouse model. Neurochem Int. 2012;60:759–67.

    Article  PubMed  CAS  Google Scholar 

  • Roulston A, Reinhard C, Amiri P, Williams LT. Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor alpha. J Biol Chem. 1998;273:10232–9.

    Article  PubMed  CAS  Google Scholar 

  • Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteolysis. Cell. 1997;91:443–6.

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Russell DW. Assay for β-galactosidase in extracts of mammalian cells. In: Irwin N, editor. Molecular cloning, vol. 17. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2001a. p. 48–51.

    Google Scholar 

  • Sambrook J, Russell DW. Assay for luciferase in extracts of mammalian cells. In: Irwin N, editor. Molecular cloning, vol. 17. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2001b. p. 42–7.

    Google Scholar 

  • Scott B, Eaton CJ. Role of reactive oxygen species in fungal cellular differentiations. Curr Opin Microbiol. 2008;11:488–93.

    Article  PubMed  CAS  Google Scholar 

  • Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat Cell Biol. 2002;4:131–6.

    Google Scholar 

  • Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell. 2002;9:459–70.

    Google Scholar 

  • Simon HU, Haj-Yehia A, Levi-Schaffer F. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis. 2000;5:415–8.

    Google Scholar 

  • Stoian I, Oros A, Moldoveanu E. Apoptosis and free radicals. Biochem Mol Med. 1996;59:93–7.

    Article  PubMed  CAS  Google Scholar 

  • Susin SA, Zamzami N, Kroemer G. Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta. 1998;1366:151–65.

    Article  PubMed  CAS  Google Scholar 

  • Tobiume K, Matsuzawa A, Takahashi T, Nishitoh H, Morita K, Takeda K, Minowa O, Miyazono K, Noda T, Ichijo H. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep. 2001;2:222–8.

    Article  PubMed  CAS  Google Scholar 

  • Wang JJ, Shen YK, Hu WP, Hsieh MC, Lin FL, Hsu MK, Hsu MH. Design, synthesis, and biological evaluation of pyrrolo[2,1-c][1,4]benzodiazepine and indole conjugates as anticancer agents. J Med Chem. 2006;49:1442–9.

    Article  PubMed  CAS  Google Scholar 

  • Wu J, Suzuki H, Akhand AA, Zhou YW, Hossain K, Nakashima I. Modes of activation of mitogen-activated protein kinases and their roles in cepharanthine-induced apoptosis in human leukemia cells. Cell Signal. 2002;14:509–15.

    Article  PubMed  CAS  Google Scholar 

  • Xiao D, Singh SV. Phenethyl isothiocyanate-induced apoptosis in p53-deficient PC-3 human prostate cancer cell line is mediated by extracellular signal-regulated kinases. Cancer Res. 2002;62:3615–9.

    PubMed  CAS  Google Scholar 

  • Xu C, Shen G, Yuan X. ERK and JNK signaling pathways are involved in the regulation of activator protein 1 and protein 1 and cell death elicited by three isothiocyanates in human prostate cancer PC-3 cells. Carcinogenesis. 2006;27:437–45.

    Article  PubMed  Google Scholar 

  • Zhu F, Zhang Y, Bode AM, Dong Z. Involvement of ERKs and mitogen- and stress-activated protein kinase in UVC-induced phosphorylation of ATF2 in JB6 cells. Carcinogenesis. 2004;25:1847–52.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank the National Science Council of the Republic of China and the Kaohsiung Medical University Research Foundation for the financial support.

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Authors and Affiliations

  1. Graduate Institute of Pharmacy, Faculty of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan

    Chung-Yu Chen & Jeh-Jeng Wang

  2. Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan

    Yin-Kai Chen, Chia-Chen Hsu & Wan-Ping Hu

  3. Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan

    Jeh-Jeng Wang & Hsien-Chang Lin

  4. Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli, Taiwan

    Feng-Yuan Tsai

  5. National Museum of Marine Biology and Aquarium, Pingtung, Taiwan

    Ping-Jyun Sung

  6. Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan

    Long-Sen Chang

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  1. Chung-Yu Chen
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Correspondence to Wan-Ping Hu.

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Chen, CY., Chen, YK., Wang, JJ. et al. DC-81-enediyne induces apoptosis of human melanoma A375 cells: involvement of the ROS, p38 MAPK, and AP-1 signaling pathways. Cell Biol Toxicol 29, 85–99 (2013). https://doi.org/10.1007/s10565-012-9238-6

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