Korean J Physiol Pharmacol.  2020 Sep;24(5):413-422. 10.4196/kjpp.2020.24.5.413.

Delphinidin enhances radio-therapeutic effects via autophagyinduction and JNK/MAPK pathway activation in non-small celllung cancer

• Kang SH
• Bak DH
• Chung BY
• Bai HW
• Kang BS

• KMID: 2505776
• DOI: http://doi.org/10.4196/kjpp.2020.24.5.413

Abstract

Delphinidin is a major anthocyanidin compound found in various vegetablesand fruits. It has anti-oxidant, anti-inflammatory, and various other biologicalactivities. In this study we demonstrated the anti-cancer activity of delphinidin,which was related to autophagy, in radiation-exposed non-small cell lung cancer(NSCLC). Radiosensitising effects were assessed in vitro by treating cells with a subcytotoxicdose of delphinidin (5 M) before exposure to -ionising radiation (IR). Wefound that treatment with delphinidin or IR induced NSCLC cell death in vitro; howeverthe combination of delphinidin pre-treatment and IR was more effective thaneither agent alone, yielding a radiation enhancement ratio of 1.54 at the 50% lethaldose. Moreover, combined treatment with delphinidin and IR, enhanced apoptoticcell death, suppressed the mTOR pathway, and activated the JNK/MAPK pathway.Delphinidin inhibited the phosphorylation of PI3K, AKT, and mTOR, and increasedthe expression of autophagy-induced cell death associated-protein in radiation-exposedNSCLC cells. In addition, JNK phosphorylation was upregulated by delphinidinpre-treatment in radiation-exposed NSCLC cells. Collectively, these results show thatdelphinidin acts as a radiation-sensitizing agent through autophagy induction andJNK/MAPK pathway activation, thus enhancing apoptotic cell death in NSCLC cells.

Keyword

Autophagy; Delphinidin; Non-small-cell lung carcinoma; Radiation-sensitizing agent

Figure

• 

  Fig. 1 Cytotoxicity of delphinidin on A549 cells after 24 h of incubation. (A) MTT assay to evaluate A549 cell viability; (B) Statistical graph of Annexin V-positive A549 cells; (C) Annexin V-positive A549 cells. The results revealed no statistically significant cytotoxicity for media with delphinidin concentrations lower than 5 µM. Statistical differences were compared between the control group and the treated groups. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. **p < 0.01, ***p < 0.001.

• 

  Fig. 2 Delphinidin increased apoptosis in gamma ray-exposed NSCLC cells. (A) Cell viability detection by the CCK-8 assay in A549, NCI-H460, and HCC827 cells. (B) Clonogenic assay to evaluate A549 cell viability. (C) Representatieve images of Annexin V-positive reaction of A549 cells. (D) Statistical graph of the muse analysis. (E) Expression level of cleaved caspase-3 protein in A549 cells. (F) Statistical analysis of the cleaved caspase-3/caspase 3 ratio. IR, ionising radiation. *p < 0.05, **p < 0.01 vs. the IR group.

• 

  Fig. 3 Delphinidin induced autophagy and suppressed the PI3K/AKT/mTOR signalling pathway in gamma ray-exposed A549 cells. (A) Western blotting for LC3 and GAPDH after treatment of cells with the indicated concentration of delphinidin for 24 h, (B) bar graph, quantitative evaluation of densitometric analysis. (D) Western blotting for LC3 and GAPDH after IR exposure to cells, (C) bar graph, quantitative evaluation of densitometric analysis. (G) Western blotting for LC3 and GAPDH after IR exposure with 5 μM delphinidin-pretreated A549 cells, (E) bar graph, quantitative evaluation of densitometric analysis. The relative expression of (F) ATG5 and (H) ATG12 mRNA was determined by qRT-PCR. (J) LC3 immunofluorescence-positive dots in A549 cells (×400). Quantification of the LC3 puncta average number from the image in (I) performed using the Image J quantification tool. (K) Suppression of upstream proteins from the mTOR signalling pathway, including PI3K, phospho-PI3K, AKT, phospho-AKT, mTOR and phospho-mTOR after IR. (L) The phospho-PI3K/PI3K, (M) the phospho-AKT/AKT, (N) the phospho-mTOR/mTOR ratio in the mTOR signalling pathway. Statistical analysis was performed for results from three independent experiments ± standard deviation. IR, ionising radiation. *p < 0.05, **p < 0.01, ***p < 0.001 compared with IR.

• 

  Fig. 4 Delphinidin activated the autophagic cell death pathway and JNK/MAPK signalling pathway in gamma-ray exposed A549 cells. (A) The activity of up/downstream proteins from the autophagic cell death signalling pathway including p53 and DRAM after IR. (B) Statistical analysis of the p53/GAPDH ratio. (C) Statistical analysis of the DRAM/GAPDH ratio. (D) The relative expression of DRAM mRNA was determined by qRT-PCR. (E) The suppression and activation of MAPK signalling pathway, including ERK, phospho-ERK, p38, phospho-p38, JNK, and phospho-JNK after IR. (F) Statistical analysis of phospho-ERK/ERK. (G) Statistical analysis of phospho-p38/p38. (H) Statistical analysis of phospho-JNK/JNK. Statistical analysis was performed for results from three independent experiments ± standard deviation. DRAM, damage-regulator autophagy modulator; IR, ionising radiation; MAPK, mitogen‐activated protein kinase. **p < 0.01 compared with IR.

Reference

1. Tsuyuki S, Fukui S, Watanabe A, Akune S, Tanabe M, Yoshida K. 2012; Delphinidin induces autolysosome as well as autophagosome formation and delphinidin-induced autophagy exerts a cell protective role. J Biochem Mol Toxicol. 26:445–453. DOI: 10.1002/jbt.21443. PMID: 23129091.

2. Wang H, Nair MG, Strasburg GM, Chang YC, Booren AM, Gray JI, DeWitt DL. 1999; Antioxidant and antiinflammatory activities of anthocyanins and their aglycon, cyanidin, from tart cherries. J Nat Prod. 62:294–296. DOI: 10.1021/np980501m. PMID: 10075763.

3. Kim B, Lee SG, Park YK, Ku CS, Pham TX, Wegner CJ, Yang Y, Koo SI, Chun OK, Lee JY. 2016; Blueberry, blackberry, and blackcurrant differentially affect plasma lipids and pro-inflammatory markers in diet-induced obesity mice. Nutr Res Pract. 10:494–500. DOI: 10.4162/nrp.2016.10.5.494. PMID: 27698956. PMCID: PMC5037066.

4. Kasten-Pisula U, Windhorst S, Dahm-Daphi J, Mayr G, Dikomey E. 2007; Radiosensitization of tumour cell lines by the polyphenol Gossypol results from depressed double-strand break repair and not from enhanced apoptosis. Radiother Oncol. 83:296–303. DOI: 10.1016/j.radonc.2007.04.024. PMID: 17521756.

5. Bump EA, Hoffman SJ, Foye WO. Abraham DJ, editor. 2003. Radiosensitizers and radioprotective agents. Burger's medicinal chemistry and drug discovery. 6th ed. Wiley;Hoboken (NJ): p. 151–214. DOI: 10.1002/0471266949.bmc077.

6. Nambiar D, Rajamani P, Singh RP. 2011; Effects of phytochemicals on ionization radiation-mediated carcinogenesis and cancer therapy. Mutat Res. 728:139–157. DOI: 10.1016/j.mrrev.2011.07.005. PMID: 22030216.

7. Kim MH, Jeong YJ, Cho HJ, Hoe HS, Park KK, Park YY, Choi YH, Kim CH, Chang HW, Park YJ, Chung IK, Chang YC. 2017; Delphinidin inhibits angiogenesis through the suppression of HIF-1α and VEGF expression in A549 lung cancer cells. Oncol Rep. 37:777–784. DOI: 10.3892/or.2016.5296. PMID: 27959445.

8. Jeong MH, Ko H, Jeon H, Sung GJ, Park SY, Jun WJ, Lee YH, Lee J, Lee SW, Yoon HG, Choi KC. 2016; Delphinidin induces apoptosis via cleaved HDAC3-mediated p53 acetylation and oligomerization in prostate cancer cells. Oncotarget. 7:56767–56780. DOI: 10.18632/oncotarget.10790. PMID: 27462923. PMCID: PMC5302952.

9. Chen J, Zhou J, Li F, Zhu Y, Zhang W, Yu X. 2017; [Delphinidin induces autophagy in HER-2+ breast cancer cells via inhibition of AKT/mTOR pathway]. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 42:264–270. Chinese. DOI: 10.11817/j.issn.1672-7347.2017.03.005. PMID: 28364098.

10. Levine B, Kroemer G. 2008; Autophagy in the pathogenesis of disease. Cell. 132:27–42. DOI: 10.1016/j.cell.2007.12.018. PMID: 18191218. PMCID: PMC2696814.

11. Jin S, White E. 2007; Role of autophagy in cancer: management of metabolic stress. Autophagy. 3:28–31. DOI: 10.4161/auto.3269. PMID: 16969128. PMCID: PMC2770734.

12. Kondo Y, Kondo S. 2006; Autophagy and cancer therapy. Autophagy. 2:85–90. DOI: 10.4161/auto.2.2.2463. PMID: 16874083.

13. Bialik S, Dasari SK, Kimchi A. 2018; Autophagy-dependent cell death - where, how and why a cell eats itself to death. J Cell Sci. 131:jcs215152. DOI: 10.1242/jcs.215152. PMID: 30237248.

14. Gozuacik D, Kimchi A. 2004; Autophagy as a cell death and tumor suppressor mechanism. Oncogene. 23:2891–2906. DOI: 10.1038/sj.onc.1207521. PMID: 15077152.

15. Kim TH, Kim HS, Kang YJ, Yoon S, Lee J, Choi WS, Jung JH, Kim HS. 2015; Psammaplin A induces Sirtuin 1-dependent autophagic cell death in doxorubicin-resistant MCF-7/adr human breast cancer cells and xenografts. Biochim Biophys Acta. 1850:401–410. DOI: 10.1016/j.bbagen.2014.11.007. PMID: 25445714.

16. Mohammadpour R, Safarian S, Ejeian F, Sheikholya-Lavasani Z, Abdolmohammadi MH, Sheinabi N. 2014; Acetazolamide triggers death inducing autophagy in T-47D breast cancer cells. Cell Biol Int. 38:228–238. DOI: 10.1002/cbin.10197. PMID: 24155029.

17. Kulkarni YM, Kaushik V, Azad N, Wright C, Rojanasakul Y, O'Doherty G, Iyer AK. 2016; Autophagy-induced apoptosis in lung cancer cells by a novel digitoxin analog. J Cell Physiol. 231:817–828. DOI: 10.1002/jcp.25129. PMID: 26264876. PMCID: PMC4751068.

18. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, Mukherjee C, Shi Y, Gélinas C, Fan Y, Nelson DA, Jin S, White E. 2006; Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 10:51–64. DOI: 10.1016/j.ccr.2006.06.001. PMID: 16843265. PMCID: PMC2857533.

19. Liu L, Liao JZ, He XX, Li PY. 2017; The role of autophagy in hepatocellular carcinoma: friend or foe. Oncotarget. 8:57707–57722. DOI: 10.18632/oncotarget.17202. PMID: 28915706. PMCID: PMC5593678.

20. Feng R, Wang SY, Shi YH, Fan J, Yin XM. 2010; Delphinidin induces necrosis in hepatocellular carcinoma cells in the presence of 3-methyladenine, an autophagy inhibitor. J Agric Food Chem. 58:3957–3964. DOI: 10.1021/jf9025458. PMID: 20025272.

21. Lee DY, Park YJ, Hwang SC, Kim KD, Moon DK, Kim DH. 2018; Cytotoxic effects of delphinidin in human osteosarcoma cells. Acta Orthop Traumatol Turc. 52:58–64. DOI: 10.1016/j.aott.2017.11.011. PMID: 29290536. PMCID: PMC6136320.

22. Chen J, Zhu Y, Zhang W, Peng X, Zhou J, Li F, Han B, Liu X, Ou Y, Yu X. 2018; Delphinidin induced protective autophagy via mTOR pathway suppression and AMPK pathway activation in HER-2 positive breast cancer cells. BMC Cancer. 18:342. DOI: 10.1186/s12885-018-4231-y. PMID: 29587684. PMCID: PMC5870693.

23. Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E. 2005; Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy. 1:84–91. DOI: 10.4161/auto.1.2.1697. PMID: 16874052.

24. Otomo C, Metlagel Z, Takaesu G, Otomo T. 2013; Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy. Nat Struct Mol Biol. 20:59–66. DOI: 10.1038/nsmb.2431. PMID: 23202584. PMCID: PMC3540207.

25. Mizushima N. Deretic V, editor. 2006. Transgenic models of autophagy. Autophagy in immunity and infection: a novel immune effector. Wiley-VCH;Weinheim: p. 55–67. DOI: 10.1002/352760880X.ch3.

26. Hafeez BB, Siddiqui IA, Asim M, Malik A, Afaq F, Adhami VM, Saleem M, Din M, Mukhtar H. 2008; A dietary anthocyanidin delphinidin induces apoptosis of human prostate cancer PC3 cells in vitro and in vivo: involvement of nuclear factor-kappaB signaling. Cancer Res. 68:8564–8572. DOI: 10.1158/0008-5472.CAN-08-2232. PMID: 18922932. PMCID: PMC3149885.

27. Crighton D, Wilkinson S, Ryan KM. 2007; DRAM links autophagy to p53 and programmed cell death. Autophagy. 3:72–74. DOI: 10.4161/auto.3438. PMID: 17102582.

28. Crighton D, Wilkinson S, O'Prey J, Syed N, Smith P, Harrison PR, Gasco M, Garrone O, Crook T, Ryan KM. 2006; DRAM, a p53-induced modulator of autophagy, is critical for apoptosis. Cell. 126:121–134. DOI: 10.1016/j.cell.2006.05.034. PMID: 16839881.

29. Wilkinson MG, Millar JB. 2000; Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J. 14:2147–2157. DOI: 10.1096/fj.00-0102rev. PMID: 11053235.

30. Mizushima N. 2007; Autophagy: process and function. Genes Dev. 21:2861–2873. DOI: 10.1101/gad.1599207. PMID: 18006683.

31. Gewirtz DA. 2014; The four faces of autophagy: implications for cancer therapy. Cancer Res. 74:647–651. DOI: 10.1158/0008-5472.CAN-13-2966. PMID: 24459182.

32. Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, Han W, Lou F, Yang J, Zhang Q, Wang X, He C, Pan H. 2013; Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis. 4:e838. DOI: 10.1038/cddis.2013.350. PMID: 24113172. PMCID: PMC3824660.

33. Notte A, Leclere L, Michiels C. 2011; Autophagy as a mediator of chemotherapy-induced cell death in cancer. Biochem Pharmacol. 82:427–434. DOI: 10.1016/j.bcp.2011.06.015. PMID: 21704023.

34. Liu Q, Wang JJ, Pan YC, Meng LF, Zhan X, Zheng QF. 2008; [Expression of autophagy-related genes Beclin1 and MAPLC3 in non-small cell lung cancer]. Ai Zheng. 27:25–29. Chinese. PMID: 18184459.

35. Jiang ZF, Shao LJ, Wang WM, Yan XB, Liu RY. 2012; Decreased expression of Beclin-1 and LC3 in human lung cancer. Mol Biol Rep. 39:259–267. DOI: 10.1007/s11033-011-0734-1. PMID: 21556768.

36. Fu LL, Cheng Y, Liu B. 2013; Beclin-1: autophagic regulator and therapeutic target in cancer. Int J Biochem Cell Biol. 45:921–924. DOI: 10.1016/j.biocel.2013.02.007. PMID: 23420005.

37. Valente G, Morani F, Nicotra G, Fusco N, Peracchio C, Titone R, Alabiso O, Arisio R, Katsaros D, Benedetto C, Isidoro C. 2014; Expression and clinical significance of the autophagy proteins BECLIN 1 and LC3 in ovarian cancer. Biomed Res Int. 2014:462658. DOI: 10.1155/2014/462658. PMID: 25136588. PMCID: PMC4127242.

38. Karagounis IV, Kalamida D, Mitrakas A, Pouliliou S, Liousia MV, Giatromanolaki A, Koukourakis MI. 2016; Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy. Br J Cancer. 115:312–321. DOI: 10.1038/bjc.2016.202. PMID: 27380135. PMCID: PMC4973160.

39. Chu CT. 2006; Autophagic stress in neuronal injury and disease. J Neuropathol Exp Neurol. 65:423–432. DOI: 10.1097/01.jnen.0000229233.75253.be. PMID: 16772866. PMCID: PMC1885377.

40. Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B, Bao JK. 2012; Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 45:487–498. DOI: 10.1111/j.1365-2184.2012.00845.x. PMID: 23030059. PMCID: PMC6496669.

41. Huang P, Han J, Hui L. 2010; MAPK signaling in inflammation-associated cancer development. Protein Cell. 1:218–226. DOI: 10.1007/s13238-010-0019-9. PMID: 21203968. PMCID: PMC4875080.

42. Zhang W, Liu HT. 2002; MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res. 12:9–18. DOI: 10.1038/sj.cr.7290105. PMID: 11942415.

43. Kyriakis JM, Avruch J. 2012; Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update. Physiol Rev. 92:689–737. DOI: 10.1152/physrev.00028.2011. PMID: 22535895.

44. Reddy KB, Nabha SM, Atanaskova N. 2003; Role of MAP kinase in tumor progression and invasion. Cancer Metastasis Rev. 22:395–403. DOI: 10.1023/A:1023781114568. PMID: 12884914.

45. Burotto M, Chiou VL, Lee JM, Kohn EC. 2014; The MAPK pathway across different malignancies: a new perspective. Cancer. 120:3446–3456. DOI: 10.1002/cncr.28864. PMID: 24948110. PMCID: PMC4221543.

46. Sui X, Kong N, Ye L, Han W, Zhou J, Zhang Q, He C, Pan H. 2014; p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett. 344:174–179. DOI: 10.1016/j.canlet.2013.11.019. PMID: 24333738.

47. Wong CH, Iskandar KB, Yadav SK, Hirpara JL, Loh T, Pervaiz S. 2010; Simultaneous induction of non-canonical autophagy and apoptosis in cancer cells by ROS-dependent ERK and JNK activation. PLoS One. 5:e9996. DOI: 10.1371/journal.pone.0009996. PMID: 20368806. PMCID: PMC2848860.

48. Shajahan AN, Dobbin ZC, Hickman FE, Dakshanamurthy S, Clarke R. 2012; Tyrosine-phosphorylated caveolin-1 (Tyr-14) increases sensitivity to paclitaxel by inhibiting BCL2 and BCLxL proteins via c-Jun N-terminal kinase (JNK). J Biol Chem. 287:17682–17692. DOI: 10.1074/jbc.M111.304022. PMID: 22433870. PMCID: PMC3366801.

49. Tian PG, Jiang ZX, Li JH, Zhou Z, Zhang QH. 2015; Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1. Biochem Biophys Res Commun. 463:518–523. DOI: 10.1016/j.bbrc.2015.05.061. PMID: 26026678.