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Pharmacological inhibition of p38 potentiates antimicrobial peptide TP4-induced cell death in glioblastoma cells

  • Bor-Chyuan Su
  • Jyh-Yih ChenEmail author
Article
  • 46 Downloads

Abstract

Glioblastoma is the most common and deadly type of brain cancer. The poor prognosis may be largely attributed to inadequate disease response to current chemotherapeutic agents. Activation of p38 is associated with deleterious outcomes in glioblastoma patients, as its signaling mediates chemoresistance mechanisms. Antimicrobial peptide tilapia piscidin (TP) 4 was identified from Nile tilapia (Oreochromis niloticus) and exhibits strong bactericidal effects on Gram-positive and Gram-negative bacteria. TP4 also has anticancer activity toward human triple-negative breast cancer cells and glioblastoma cells. In the present study, we tested the cytotoxic effects of combined TP4 and p38 inhibitors on glioblastoma U251 cells. We found that the combination of TP4 and p38 inhibitors (SB202190 and VX-745) enhanced cytotoxicity in U251 glioblastoma cells but not noncancerous neural cells. Cytotoxicity from the combination treatments proceeded via necrosis and not apoptosis. Mechanistically, SB202190 potentiated TP4-induced mitochondrial dysfunction, reactive oxygen species generation and unbalanced antioxidant status, which resulted in necrotic cell death. Thus, we demonstrated for the first time that combinations of TP4 and p38 inhibitors have the potential to preferentially target glioblastoma cells, while sparing noncancerous neural cells.

Keywords

Antimicrobial peptide, TP4 p38 inhibitors Mitochondrial dysfunction Necrosis 

Abbreviations

TP4

Tilapia piscidin 4

TMZ

Temozolomide

BCNU

Bis-chloroethylnitrosourea

Nrf2

Nuclear factor erythroid 2-related factor 2

VEGF

Vascular endothelial growth factor

DHE

Dihydroethidium

PI

Propidium iodide

TMRE

Tetramethylrhodamine, ethyl ester

DCF-DA

2′,7′-Dichlorodihydrofluorescein diacetate

NAC

N-Acetyl-l-cysteine

UCP

Uncoupling protein

SOD

Superoxide dismutase

DMEM

Dulbecco’s modified Eagle’s medium

RIP3

Receptor-interacting protein kinase 3

Notes

Acknowledgements

This research was supported by intramural funding from the Marine Research Station (Jiaushi, Ilan), Institute of Cellular and Organismic Biology, Academia Sinica to Dr. Jyh-Yih Chen. We thank Dr. Pei-Jung Lu, National Cheng Kung University (Taiwan), for kindly providing the U251 human glioblastoma cell line.

Author contributions

B-CS performed the experiments; B-CS analyzed the data; B-CS contributed reagents/materials/analysis tools; B-CS and J-YC wrote the paper.

Funding

This research was supported by intramural funding from the Marine Research Station (Jiaushi, Ilan), Institute of Cellular and Organismic Biology, Academia Sinica to Jyh-Yih Chen (Research Fellow).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

References

  1. 1.
    Davis ME (2016) Glioblastoma: overview of disease and treatment. Clin J Oncol Nurs 20(5):S2–8.  https://doi.org/10.1188/16.CJON.S1.2-8 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Nizamutdinov D, Stock EM, Dandashi JA, Vasquez EA, Mao Y, Dayawansa S, Zhang J, Wu E, Fonkem E, Huang JH (2018) Prognostication of survival outcomes in patients diagnosed with glioblastoma. World Neurosurg 109:e67–e74.  https://doi.org/10.1016/j.wneu.2017.09.104 CrossRefPubMedGoogle Scholar
  3. 3.
    Ma L, Liu J, Zhang X, Qi J, Yu W, Gu Y (2015) p38 MAPK-dependent Nrf2 induction enhances the resistance of glioma cells against TMZ. Med Oncol 32(3):69.  https://doi.org/10.1007/s12032-015-0517-y CrossRefPubMedGoogle Scholar
  4. 4.
    Reithmeier T, Graf E, Piroth T, Trippel M, Pinsker MO, Nikkhah G (2010) BCNU for recurrent glioblastoma multiforme: efficacy, toxicity and prognostic factors. BMC Cancer 10:30.  https://doi.org/10.1186/1471-2407-10-30 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Demuth T, Reavie LB, Rennert JL, Nakada M, Nakada S, Hoelzinger DB, Beaudry CE, Henrichs AN, Anderson EM, Berens ME (2007) MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival. Mol Cancer Ther 6(4):1212–1222.  https://doi.org/10.1158/1535-7163.MCT-06-0711 CrossRefPubMedGoogle Scholar
  6. 6.
    Yoshino Y, Aoyagi M, Tamaki M, Duan L, Morimoto T, Ohno K (2006) Activation of p38 MAPK and/or JNK contributes to increased levels of VEGF secretion in human malignant glioma cells. Int J Oncol 29(4):981–987PubMedGoogle Scholar
  7. 7.
    Munoz L, Yeung YT, Grewal T (2016) Oncogenic Ras modulates p38 MAPK-mediated inflammatory cytokine production in glioblastoma cells. Cancer Biol Ther 17(4):355–363.  https://doi.org/10.1080/15384047.2016.1139249 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Duffy JP, Harrington EM, Salituro FG, Cochran JE, Green J, Gao H, Bemis GW, Evindar G, Galullo VP, Ford PJ, Germann UA, Wilson KP, Bellon SF, Chen G, Taslimi P, Jones P, Huang C, Pazhanisamy S, Wang YM, Murcko MA, Su MS (2011) The discovery of VX-745: a Novel and selective p38alpha kinase inhibitor. ACS Med Chem Lett 2(10):758–763.  https://doi.org/10.1021/ml2001455 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Melloni C, Sprecher DL, Sarov-Blat L, Patel MR, Heitner JF, Hamm CW, Aylward P, Tanguay JF, DeWinter RJ, Marber MS, Lerman A, Hasselblad V, Granger CB, Newby LK (2012) The study of LoSmapimod treatment on inflammation and InfarCtSizE (SOLSTICE): design and rationale. Am Heart J 164(5):646–653.  https://doi.org/10.1016/j.ahj.2012.07.030 CrossRefPubMedGoogle Scholar
  10. 10.
    Patnaik A, Haluska P, Tolcher AW, Erlichman C, Papadopoulos KP, Lensing JL, Beeram M, Molina JR, Rasco DW, Arcos RR, Kelly CS, Wijayawardana SR, Zhang X, Stancato LF, Bell R, Shi P, Kulanthaivel P, Pitou C, Mulle LB, Farrington DL, Chan EM, Goetz MP (2016) A First-in-human phase i study of the oral p38 MAPK inhibitor, ralimetinib (LY2228820 Dimesylate), in patients with advanced cancer. Clin Cancer Res 22(5):1095–1102.  https://doi.org/10.1158/1078-0432.CCR-15-1718 CrossRefPubMedGoogle Scholar
  11. 11.
    Pan CY, Tsai TY, Su BC, Hui CF, Chen JY (2017) Study of the antimicrobial activity of tilapia piscidin 3 (TP3) and TP4 and their effects on immune functions in hybrid tilapia (Oreochromis spp.). PLoS ONE 12(1):e0169678.  https://doi.org/10.1371/journal.pone.0169678 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ting CH, Chen JY (2018) Nile Tilapia derived TP4 shows broad cytotoxicity toward to non-small-cell lung cancer cells. Mar Drugs.  https://doi.org/10.3390/md16120506 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ting CH, Chen YC, Wu CJ, Chen JY (2016) Targeting FOSB with a cationic antimicrobial peptide, TP4, for treatment of triple-negative breast cancer. Oncotarget 7(26):40329–40347.  https://doi.org/10.18632/oncotarget.9612 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Su BC, Pan CY, Chen JY (2019) Antimicrobial peptide TP4 induces ROS-mediated necrosis by triggering mitochondrial dysfunction in wild-type and mutant p53 glioblastoma cells. Cancers (Basel).  https://doi.org/10.3390/cancers11020171 CrossRefPubMedCentralGoogle Scholar
  15. 15.
    Cho KJ, Seo JM, Kim JH (2011) Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species. Mol Cells 32(1):1–5.  https://doi.org/10.1007/s10059-011-1021-7 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Dan Dunn J, Alvarez LA, Zhang X, Soldati T (2015) Reactive oxygen species and mitochondria: a nexus of cellular homeostasis. Redox Biol 6:472–485.  https://doi.org/10.1016/j.redox.2015.09.005 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Mittler R (2017) ROS are good. Trends Plant Sci 22(1):11–19.  https://doi.org/10.1016/j.tplants.2016.08.002 CrossRefPubMedGoogle Scholar
  18. 18.
    D’Arcy MS (2019) Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biol Int 43(6):582–592.  https://doi.org/10.1002/cbin.11137 CrossRefPubMedGoogle Scholar
  19. 19.
    Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N (2013) Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta 1833(12):3448–3459.  https://doi.org/10.1016/j.bbamcr.2013.06.001 CrossRefPubMedGoogle Scholar
  20. 20.
    Gu WJ, Liu HL (2013) Induction of pancreatic cancer cell apoptosis, invasion, migration, and enhancement of chemotherapy sensitivity of gemcitabine, 5-FU, and oxaliplatin by hnRNP A2/B1 siRNA. Anticancer Drugs 24(6):566–576.  https://doi.org/10.1097/CAD.0b013e3283608bc5 CrossRefPubMedGoogle Scholar
  21. 21.
    Kaufmann SH, Earnshaw WC (2000) Induction of apoptosis by cancer chemotherapy. Exp Cell Res 256(1):42–49.  https://doi.org/10.1006/excr.2000.4838 CrossRefPubMedGoogle Scholar
  22. 22.
    Fernald K, Kurokawa M (2013) Evading apoptosis in cancer. Trends Cell Biol 23(12):620–633.  https://doi.org/10.1016/j.tcb.2013.07.006 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Trejo-Solis C, Serrano-Garcia N, Escamilla-Ramirez A, Castillo-Rodriguez RA, Jimenez-Farfan D, Palencia G, Calvillo M, Alvarez-Lemus MA, Flores-Najera A, Cruz-Salgado A, Sotelo J (2018) Autophagic and apoptotic pathways as targets for chemotherapy in glioblastoma. Int J Mol Sci.  https://doi.org/10.3390/ijms19123773 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Su Z, Yang Z, Xie L, DeWitt JP, Chen Y (2016) Cancer therapy in the necroptosis era. Cell Death Differ 23(5):748–756.  https://doi.org/10.1038/cdd.2016.8 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Lehar J, Krueger AS, Avery W, Heilbut AM, Johansen LM, Price ER, Rickles RJ, Short GF 3rd, Staunton JE, Jin X, Lee MS, Zimmermann GR, Borisy AA (2009) Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nat Biotechnol 27(7):659–666.  https://doi.org/10.1038/nbt.1549 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Masgras I, Carrera S, de Verdier PJ, Brennan P, Majid A, Makhtar W, Tulchinsky E, Jones GD, Roninson IB, Macip S (2012) Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21. J Biol Chem 287(13):9845–9854.  https://doi.org/10.1074/jbc.M111.250357 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA (2011) Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques 50(2):98–115.  https://doi.org/10.2144/000113610 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Wang X, Ottosson A, Ji C, Feng X, Nordenskjold M, Henter JI, Fadeel B, Zheng C (2009) Proteasome inhibition induces apoptosis in primary human natural killer cells and suppresses NKp46-mediated cytotoxicity. Haematologica 94(4):470–478.  https://doi.org/10.3324/haematol.13783 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Johannessen TC, Bjerkvig R (2012) Molecular mechanisms of temozolomide resistance in glioblastoma multiforme. Expert Rev Anticancer Ther 12(5):635–642.  https://doi.org/10.1586/era.12.37 CrossRefPubMedGoogle Scholar
  30. 30.
    Sangpairoj K, Vivithanaporn P, Apisawetakan S, Chongthammakun S, Sobhon P, Chaithirayanon K (2017) RUNX1 regulates migration, invasion, and angiogenesis via p38 MAPK pathway in human glioblastoma. Cell Mol Neurobiol 37(7):1243–1255.  https://doi.org/10.1007/s10571-016-0456-y CrossRefPubMedGoogle Scholar
  31. 31.
    Goldsmith CS, Kim SM, Karunarathna N, Neuendorff N, Gerard Toussaint L, Earnest DJ, Bell-Pedersen D (2018) Inhibition of p38 MAPK activity leads to cell type-specific effects on the molecular circadian clock and time-dependent reduction of glioma cell invasiveness. BMC Cancer 18(1):43.  https://doi.org/10.1186/s12885-017-3896-y CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Sooman L, Lennartsson J, Gullbo J, Bergqvist M, Tsakonas G, Johansson F, Edqvist PH, Ponten F, Jaiswal A, Navani S, Alafuzoff I, Popova S, Blomquist E, Ekman S (2013) Vandetanib combined with a p38 MAPK inhibitor synergistically reduces glioblastoma cell survival. Med Oncol 30(3):638.  https://doi.org/10.1007/s12032-013-0638-0 CrossRefPubMedGoogle Scholar
  33. 33.
    Campbell RM, Anderson BD, Brooks NA, Brooks HB, Chan EM, De Dios A, Gilmour R, Graff JR, Jambrina E, Mader M, McCann D, Na S, Parsons SH, Pratt SE, Shih C, Stancato LF, Starling JJ, Tate C, Velasco JA, Wang Y, Ye XS (2014) Characterization of LY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK with antitumor activity. Mol Cancer Ther 13(2):364–374.  https://doi.org/10.1158/1535-7163.MCT-13-0513 CrossRefPubMedGoogle Scholar
  34. 34.
    Weinberg SE, Chandel NS (2015) Targeting mitochondria metabolism for cancer therapy. Nat Chem Biol 11(1):9–15.  https://doi.org/10.1038/nchembio.1712 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Kishikawa JI, Inoue Y, Fujikawa M, Nishimura K, Nakanishi A, Tanabe T, Imamura H, Yokoyama K (2018) General anesthetics cause mitochondrial dysfunction and reduction of intracellular ATP levels. PLoS ONE 13(1):e0190213.  https://doi.org/10.1371/journal.pone.0190213 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Liou GY, Storz P (2010) Reactive oxygen species in cancer. Free Radic Res 44(5):479–496.  https://doi.org/10.3109/10715761003667554 CrossRefPubMedGoogle Scholar
  37. 37.
    Gao Z, Sarsour EH, Kalen AL, Li L, Kumar MG, Goswami PC (2008) Late ROS accumulation and radiosensitivity in SOD1-overexpressing human glioma cells. Free Radic Biol Med 45(11):1501–1509.  https://doi.org/10.1016/j.freeradbiomed.2008.08.009 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Yoshii Y, Saito A, Zhao DW, Nose T (1999) Copper/zinc superoxide dismutase, nuclear DNA content, and progression in human gliomas. J Neurooncol 42(2):103–108CrossRefGoogle Scholar
  39. 39.
    Smith PS, Zhao W, Spitz DR, Robbins ME (2007) Inhibiting catalase activity sensitizes 36B10 rat glioma cells to oxidative stress. Free Radic Biol Med 42(6):787–797.  https://doi.org/10.1016/j.freeradbiomed.2006.11.032 CrossRefPubMedGoogle Scholar
  40. 40.
    Dalla Pozza E, Fiorini C, Dando I, Menegazzi M, Sgarbossa A, Costanzo C, Palmieri M, Donadelli M (2012) Role of mitochondrial uncoupling protein 2 in cancer cell resistance to gemcitabine. Biochim Biophys Acta 1823(10):1856–1863.  https://doi.org/10.1016/j.bbamcr.2012.06.007 CrossRefPubMedGoogle Scholar
  41. 41.
    Alam JJ (2015) Selective brain-targeted antagonism of p38 MAPKalpha reduces hippocampal IL-1beta levels and improves morris water maze performance in aged rats. J Alzheimers Dis 48(1):219–227.  https://doi.org/10.3233/JAD-150277 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Alam J, Blackburn K, Patrick D (2017) Neflamapimod: clinical phase 2b-ready oral small molecule inhibitor of p38alpha to reverse synaptic dysfunction in early Alzheimer’s disease. J Prev Alzheimers Dis 4(4):273–278.  https://doi.org/10.14283/jpad.2017.41 CrossRefPubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Department of Anatomy and Cell Biology, School of Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  2. 2.Marine Research Station, Institute of Cellular and Organismic BiologyAcademia SinicaJiaushiTaiwan

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