Skip to main content
Log in

JNK inhibition blocks piperlongumine-induced cell death and transcriptional activation of heme oxygenase-1 in pancreatic cancer cells

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Piperlongumine (PL) is an alkaloid that inhibits glutathione S-transferase pi 1 (GSTP1) activity, resulting in elevated reactive oxygen species (ROS) levels and cancer-selective cell death. We aimed to identify stress-associated molecular responses to PL treatment in pancreatic ductal adenocarcinoma (PDAC) cells. GSTP1 directly interacts with JNK, which is activated by oxidative stress and can lead to decreased cancer cell proliferation and cell death. Therefore, we hypothesized that JNK pathways are activated in response to PL treatment. Our results show PL causes dissociation of GSTP1 from JNK; robust JNK, c-Jun, and early ERK activation followed by suppression; increased expression of cleaved caspase-3 and cleaved PARP; and nuclear translocation of Nrf2 and c-Myc in PDAC cells. Gene expression analysis revealed PL caused a > 20-fold induction of heme oxygenase-1 (HO-1), which we hypothesized was a survival mechanism for PDAC cells under enhanced oxidative stress. HO-1 knockout resulted in enhanced PL-induced PDAC cell death under hypoxic conditions. Similarly, high concentrations of the HO-1 inhibitor, ZnPP (10 µM), sensitized PDAC cells to PL; however, lower concentrations ZnPP (10 nM) and high or low concentrations of SnPP both protected PDAC cells from PL-induced cell death. Interestingly, the JNK inhibitor significantly blocked PL-induced PDAC cell death, Nrf-2 nuclear translocation, and HMOX-1 mRNA expression. Collectively, the results demonstrate JNK signaling contributes to PL-induced PDAC cell death, and at the same time, activates Nrf-2 transcription of HMOX-1 as a compensatory survival mechanism. These results suggest that elevating oxidative stress (using PL) while at the same time impairing antioxidant capacity (inhibiting HO-1) may be an effective therapeutic approach for PDAC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Schaeffer HJ, Weber MJ (1999) Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 19(4):2435–2444

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH (2001) Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22(2):153–183. https://doi.org/10.1210/edrv.22.2.0428

    Article  CAS  PubMed  Google Scholar 

  3. Davis RJ (2000) Signal transduction by the JNK group of MAP kinases. Cell 103(2):239–252

    Article  CAS  PubMed  Google Scholar 

  4. Zeke A, Misheva M, Remenyi A, Bogoyevitch MA (2016) JNK signaling: regulation and functions based on complex protein-protein partnerships. Microbiol Mol Biol Rev 80(3):793–835. https://doi.org/10.1128/MMBR.00043-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ventura JJ, Hubner A, Zhang C, Flavell RA, Shokat KM, Davis RJ (2006) Chemical genetic analysis of the time course of signal transduction by JNK. Mol Cell 21(5):701–710. https://doi.org/10.1016/j.molcel.2006.01.018

    Article  CAS  PubMed  Google Scholar 

  6. Mansouri A, Ridgway LD, Korapati AL, Zhang Q, Tian L, Wang Y, Siddik ZH, Mills GB, Claret FX (2003) Sustained activation of JNK/p38 MAPK pathways in response to cisplatin leads to Fas ligand induction and cell death in ovarian carcinoma cells. J Biol Chem 278(21):19245–19256. https://doi.org/10.1074/jbc.M208134200

    Article  CAS  PubMed  Google Scholar 

  7. Cobb MH, Goldsmith EJ (1995) How map kinases are regulated. J Biol Chem 270(25):14843–14846. https://doi.org/10.1074/jbc.270.25.14843

    Article  CAS  PubMed  Google Scholar 

  8. Garg TK, Chang JY (2003) Oxidative stress causes ERK phosphorylation and cell death in cultured retinal pigment epithelium: prevention of cell death by AG126 and 15-deoxy-delta 12, 14-PGJ2. BMC Ophthalmol 3:5

    Article  PubMed  PubMed Central  Google Scholar 

  9. Kim J, Wong PKY (2009) Oxidative stress is linked to ERK1/2-p16 signaling-mediated growth defect in ATM-deficient astrocytes. J Biol Chem 284(21):14396–14404. https://doi.org/10.1074/jbc.M808116200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mebratu Y, Tesfaigzi Y (2009) How ERK1/2 activation controls cell proliferation and cell death is subcellular localization the answer? Cell Cycle 8(8):1168–1175. https://doi.org/10.4161/Cc.8.8.8147

    Article  CAS  PubMed  Google Scholar 

  11. Tang DM, Wu DC, Hirao A, Lahti JM, Liu LQ, Mazza B, Kidd VJ, Mak TW, Ingram AJ (2002) ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. J Biol Chem 277(23):21110

    CAS  Google Scholar 

  12. Burotto M, Chiou VL, Lee JM, Kohn EC (2014) The MAPK pathway across different malignancies: a new perspective. Cancer 120(22):3446–3456. https://doi.org/10.1002/cncr.28864

    Article  CAS  PubMed  Google Scholar 

  13. Roh JL, Kim EH, Park JY, Kim JW, Kwon M, Lee BH (2014) Piperlongumine selectively kills cancer cells and increases cisplatin antitumor activity in head and neck cancer. Oncotarget 5(19):9227–9238. https://doi.org/10.18632/oncotarget.2402

    Article  PubMed  PubMed Central  Google Scholar 

  14. Bezerra DP, Castro FO, Alves AP, Pessoa C, Moraes MO, Silveira ER, Lima MA, Elmiro FJ, Costa-Lotufo LV (2006) In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper. Braz J Med Biol Res 39(6):801–807. https://doi.org/10.1590/s0100-879X2006000600014

    Article  CAS  PubMed  Google Scholar 

  15. Li W, Wen CY, Bai HY, Wang XY, Zhang XL, Huang LL, Yang XL, Iwamoto A, Liu HL (2015) JNK signaling pathway is involved in piperlongumine-mediated apoptosis in human colorectal cancer HCT116 cells. Oncol Lett 10(2):709–715. https://doi.org/10.3892/ol.2015.3371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li JH, Sharkey CC, King MR (2015) Piperlongumine and immune cytokine TRAIL synergize to promote tumor death. Sci Rep. 2:89. https://doi.org/10.1038/srep09987

    Article  CAS  Google Scholar 

  17. Thongsom S, Suginta W, Lee KJ, Choe H, Talabnin C (2017) Piperlongumine induces G2/M phase arrest and apoptosis in cholangiocarcinoma cells through the ROS-JNK-ERK signaling pathway. Apoptosis 22(11):1473–1484. https://doi.org/10.1007/s10495-017-1422-y

    Article  CAS  PubMed  Google Scholar 

  18. Randhawa H, Kibble K, Zeng H, Moyer MP, Reindl KM (2013) Activation of ERK signaling and induction of colon cancer cell death by piperlongumine. Toxicol In Vitro 27(6):1626–1633. https://doi.org/10.1016/j.tiv.2013.04.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Chen Y, Liu JM, Xiong XX, Qiu XY, Pan F, Liu D, Lan SJ, Jin S, Yu SB, Chen XQ (2015) Piperlongumine selectively kills hepatocellular carcinoma cells and preferentially inhibits their invasion via ROS-ER-MAPKs-CHOP. Oncotarget 6(8):6406–6421. https://doi.org/10.18632/oncotarget.3444

    Article  PubMed  PubMed Central  Google Scholar 

  20. Dhillon H, Chikara S, Reindl KM (2014) Piperlongumine induces pancreatic cancer cell death by enhancing reactive oxygen species and DNA damage. Toxicol Rep 1:309–318. https://doi.org/10.1016/j.toxrep.2014.05.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang T, Arifoglu P, Ronai Z, Tew KD (2001) Glutathione S-transferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus. J Biol Chem 276(24):20999–21003. https://doi.org/10.1074/jbc.M101355200

    Article  CAS  PubMed  Google Scholar 

  22. Adler V, Yin Z, Fuchs SY, Benezra M, Rosario L, Tew KD, Pincus MR, Sardana M, Henderson CJ, Wolf CR, Davis RJ, Ronai Z (1999) Regulation of JNK signaling by GSTp. EMBO J 18(5):1321–1334. https://doi.org/10.1093/emboj/18.5.1321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yin Z, Ivanov VN, Habelhah H, Tew K, Ronai Z (2000) Glutathione S-transferase p elicits protection against H2O2-induced cell death via coordinated regulation of stress kinases. Cancer Res 60(15):4053–4057

    CAS  PubMed  Google Scholar 

  24. Ascione A, Cianfriglia M, Dupuis ML, Mallano A, Sau A, Tregno F, Pezzola S, Caccuri AM (2009) The glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol overcomes the MDR1-P-glycoprotein and MRP1-mediated multidrug resistance in acute myeloid leukemia cells. Cancer Chemother Pharm 64(2):419–424. https://doi.org/10.1007/s00280-009-0960-6

    Article  CAS  Google Scholar 

  25. Bauer M, Bauer I (2002) Heme oxygenase-1: redox regulation and role in the hepatic response to oxidative stress. Antioxid Redox Signal 4(5):749–758. https://doi.org/10.1089/152308602760598891

    Article  CAS  PubMed  Google Scholar 

  26. Dhillon H, Mamidi S, McClean P, Reindl KM (2016) Transcriptome analysis of piperlongumine-treated human pancreatic cancer cells reveals involvement of oxidative stress and endoplasmic reticulum stress pathways. J Med Food 19(6):578–585. https://doi.org/10.1089/jmf.2015.0152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Poss KD, Tonegawa S (1997) Reduced stress defense in heme oxygenase 1-deficient cells. Proc Natl Acad Sci USA 94(20):10925–10930. https://doi.org/10.1073/pnas.94.20.10925

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pileggi A, Cattan P, Molano RD, Berney T, Vizzardelli C, Fraker C, Oliver R, Ricordi C, Pastori R, Bach FH, Inverardi L (2001) Induced heme oxygenase-1 upregulation protects pancreatic beta cells from apoptosis in vitro. Sci World J 1:108. https://doi.org/10.1100/tsw.2001.125

    Article  Google Scholar 

  29. Kietzmann T, Samoylenko A, Immenschuh S (2003) Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat Hepatocytes. J Biol Chem 278(20):17927–17936. https://doi.org/10.1074/jbc.M203929200

    Article  CAS  PubMed  Google Scholar 

  30. Shan Y, Pepe J, Lu TH, Elbirt KK, Lambrecht RW, Bonkovsky HL (2000) Induction of the heme oxygenase-1 gene by metalloporphyrins. Arch Biochem Biophys 380(2):219–227. https://doi.org/10.1006/abbi.2000.1921

    Article  CAS  PubMed  Google Scholar 

  31. Gaitanaki C, Konstantina S, Chrysa S, Beis I (2003) Oxidative stress stimulates multiple MAPK signalling pathways and phosphorylation of the small HSP27 in the perfused amphibian heart. J Exp Biol 206(16):2759–2769. https://doi.org/10.1242/jeb.00483

    Article  CAS  PubMed  Google Scholar 

  32. Chikara S, Mamidi S, Sreedasyam A, Chittem K, Pietrofesa R, Zuppa A, Moorthy G, Dyer N, Christofidou-Solomidou M, Reindl KM (2018) Flaxseed consumption inhibits chemically induced lung tumorigenesis and modulates expression of phase II enzymes and inflammatory cytokines in A/J mice. Cancer Prev Res (Phila) 11(1):27–37. https://doi.org/10.1158/1940-6207.CAPR-17-0119

    Article  CAS  Google Scholar 

  33. Mohammad J, Dhillon H, Chikara S, Mamidi S, Sreedasyam A, Chittem K, Orr M, Wilkinson JC, Reindl KM (2018) Piperlongumine potentiates the effects of gemcitabine in in vitro and in vivo human pancreatic cancer models. Oncotarget 9(12):10457–10469. https://doi.org/10.18632/oncotarget.23623

    Article  PubMed  Google Scholar 

  34. Harshbarger W, Gondi S, Ficarro SB, Hunter J, Udayakumar D, Gurbani D, Singer WD, Liu Y, Li L, Marto JA, Westover KD (2017) Structural and biochemical analyses reveal the mechanism of glutathione S-transferase Pi 1 inhibition by the anti-cancer compound piperlongumine. J Biol Chem 292(1):112–120. https://doi.org/10.1074/jbc.M116.750299

    Article  CAS  PubMed  Google Scholar 

  35. Lee HN, Jin HO, Park JA, Kim JH, Kim JY, Kim B, Kim W, Hong SE, Lee YH, Chang YH, Hong SI, Hong YJ, Park IC, Surh YJ, Lee JK (2015) Heme oxygenase-1 determines the differential response of breast cancer and normal cells to piperlongumine. Mol Cells 38(4):327–335. https://doi.org/10.14348/molcells.2015.2235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Siegel RL, Miller KD (2018) Jemal A (2018) Cancer statistics. CA: A Cancer J Clin 68(1):7–30. https://doi.org/10.3322/caac.21442

    Article  Google Scholar 

  37. Xiong XX, Liu JM, Qiu XY, Pan F, Yu SB, Chen XQ (2015) Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways. Acta Pharmacol Sin 36(3):362–374. https://doi.org/10.1038/aps.2014.141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Sosa V, Moline T, Somoza R, Paciucci R, Kondoh H, LLeonart ME (2013) Oxidative stress and cancer: an overview. Ageing Res Rev 12(1):376–390. https://doi.org/10.1016/j.arr.2012.10.004

    Article  CAS  PubMed  Google Scholar 

  39. Liou GY, Storz P (2010) Reactive oxygen species in cancer. Free Radic Res 44(5):479–496. https://doi.org/10.3109/10715761003667554

    Article  CAS  PubMed  Google Scholar 

  40. Alam J, Cook JL (2007) How many transcription factors does it take to turn on the heme oxygenase-1 gene? Am J Respir Cell Mol Biol 36(2):166–174. https://doi.org/10.1165/rcmb.2006-0340TR

    Article  CAS  PubMed  Google Scholar 

  41. Gozzelino R, Jeney V, Soares MP (2010) Mechanisms of cell protection by heme oxygenase-1. Annu Rev Pharmacol Toxicol 50:323–354. https://doi.org/10.1146/annurev.pharmtox.010909.105600

    Article  CAS  PubMed  Google Scholar 

  42. Tenhunen R, Marver HS, Schmid R (1968) The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA 61(2):748–755. https://doi.org/10.1073/pnas.61.2.748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Loboda A, Jazwa A, Grochot-Przeczek A, Rutkowski AJ, Cisowski J, Agarwal A, Jozkowicz A, Dulak J (2008) Heme oxygenase-1 and the vascular bed: from molecular mechanisms to therapeutic opportunities. Antioxid Redox Signal 10(10):1767–1812. https://doi.org/10.1089/ars.2008.2043

    Article  CAS  PubMed  Google Scholar 

  44. La P, Fernando AP, Wang Z, Salahudeen A, Yang G, Lin Q, Wright CJ, Dennery PA (2009) Zinc protoporphyrin regulates cyclin D1 expression independent of heme oxygenase inhibition. J Biol Chem 284(52):36302–36311. https://doi.org/10.1074/jbc.M109.031641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Podkalicka P, Mucha O, Jozkowicz A, Dulak J, Loboda A (2018) Heme oxygenase inhibition in cancers: possible tools and targets. Contemp Oncol (Pozn) 22(1A):23–32. https://doi.org/10.5114/wo.2018.73879

    Article  Google Scholar 

  46. Berberat PO, Dambrauskas Z, Gulbinas A, Giese T, Giese N, Kunzli B, Autschbach F, Meuer S, Buchler MW, Friess H (2005) Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment. Clin Cancer Res 11(10):3790–3798. https://doi.org/10.1158/1078-0432.CCR-04-2159

    Article  CAS  PubMed  Google Scholar 

  47. Zhe N, Wang J, Chen S, Lin X, Chai Q, Zhang Y, Zhao J, Fang Q (2015) Heme oxygenase-1 plays a crucial role in chemoresistance in acute myeloid leukemia. Hematology 20(7):384–391. https://doi.org/10.1179/1607845414Y.0000000212

    Article  CAS  PubMed  Google Scholar 

  48. Sass G, Leukel P, Schmitz V, Raskopf E, Ocker M, Neureiter D, Meissnitzer M, Tasika E, Tannapfel A, Tiegs G (2008) Inhibition of heme oxygenase 1 expression by small interfering RNA decreases orthotopic tumor growth in livers of mice. Int J Cancer 123(6):1269–1277. https://doi.org/10.1002/ijc.23695

    Article  CAS  PubMed  Google Scholar 

  49. Liu YS, Li HS, Qi DF, Zhang J, Jiang XC, Shi K, Zhang XJ, Zhang XH (2014) Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin. World J Gastroenterol 20(26):8572–8582. https://doi.org/10.3748/wjg.v20.i26.8572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Frank J, Lornejad-Schafer MR, Schoffl H, Flaccus A, Lambert C, Biesalski HK (2007) Inhibition of heme oxygenase-1 increases responsiveness of melanoma cells to ALA-based photodynamic therapy. Int J Oncol 31(6):1539–1545

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Jagadish Loganathan, Jeffrey Kittilson, and Megan Orr for their technical support and assistance. We would like to acknowledge the use of the Biostatistics Core Facility for statistical analysis in this manuscript. This study is funded by National Institute of General Medical Sciences of the National Institutes of Health, Award Number 1P20GM109024.

Funding

NIH Grant number 1P20GM109024 (to KMR). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Katie M. Reindl.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (CSV 37 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mohammad, J., Singh, R.R., Riggle, C. et al. JNK inhibition blocks piperlongumine-induced cell death and transcriptional activation of heme oxygenase-1 in pancreatic cancer cells. Apoptosis 24, 730–744 (2019). https://doi.org/10.1007/s10495-019-01553-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-019-01553-9

Keywords

Navigation