Pholidonone, an active stilbene derivative from Pholidota cantonensis, exhibits pro-apoptotic effect via induction of endoplasmic reticulum stress in human gastric cancer
Backgroud: Gastric cancer (GC) has become the second leading cause of death due to the worldwide incidence, mortality and prevalence. Therefore, it is urgent to find new drugs with low toxicity and high efficacy for the treatment of gastric cancer. Natural products as well as functional foods have always been the rich source of potential antitumor agents. Pholidota cantonensis Rolfe, a well-known functional food and a folk medicine, has been used for a long time in China for inflammatory diseases. Previously, we have evaluated its possible antitumor potentials by screening different solvent extracts, and found that the ethyl acetate (EtOAc) extract showed much higher cytotoxicity on human GC cell line AGS with IC50 value of 33.68 ± 1.68 μg/mL. In view of the poor knowledge concerning the phytochemical and pharmacological study of P. cantonensis, it is urgent to characterize the active compounds from EtOAc extract and the mechanisms of action underlying the anti-tumor effect of the herb.
Objective: This study aimed to identify the primary compounds in EtOAc extract of P. cantonensis involved in the antitumor activity of the plant by evaluating the cytotoxicity in two human GC cell lines including AGS and BGC-823 cells. Since endoplasmic reticulum (ER) stress-induced cell apoptosis represents attractive targets for cancer therapy recently, we focused on the underlying mechanisms associated with ER stress-induced cell apoptosis and related signaling pathways.
Methods: Various chromatographic techniques including silica gel, sephadex LH-20 and ODS C18 were used to separate the main active compound from EtOAc extract of P. cantonensis. The cell viability of AGS and BGC-823 cells upon purified compound treatment was determined by MTT assay. The alteration of cell morphology was observed using an inverted microscope. Cell apoptosis was determined by FITC-labeled annexin-V/PI double staining and flow cytometry analysis. Western blot analyses were performed to examine the levels of intracellular signaling molecules involved in ER stress-induced apoptosis.
Results: A rare stilbene derivative pholidonone was isolated and identified. The results showed that pholidonone displayed potent cytotoxicity on human gastric cancer cells. The IC50 values for 24 and 48 h in AGS cells were 26.54 ± 0.32 and 25.20 ± 3.67 μM, and the IC50 values for 24 and 48 h in BGC-823 cells were 32.41 ± 3.83 and 17.28 ± 2.30 μM, respectively). Additionally, pholidonone had pro-apoptotic effect on AGS and BGC-823 cells, and it dose-dependently up-regulated the levels of proteins involved in ER stress, including BiP, PDI, Calnexin, Ero1-Lα, IRE1α, PERK, CHOP and cleaved-caspase-3 in AGS and BGC-823 cells.
Conclusion: Pholidonone can trigger ER stress-induced apoptosis through PERK and IRE1α signaling pathway. Pholidonone might be a potential naturally occurred antitumor agent.
- Hamashima C. Current issues and future perspectives of gastric cancer screening. World J Gastroenterol 2014; 20(38): 13767–74. doi: 10.3748/wjg.v20.i38.13767
- Dong HM, Wang Q, Wang WL,Wang G, Li XK, Li GD, et al. A clinical analysis of systemic chemotherapy combined with radiotherapy for advanced gastric cancer. Medicine 2018; 97(23): e10786. doi: 10.1097/MD.0000000000010786
- Editorial Committee of the Administration Bureau of Traditional Chinese Medicine, Chinese Materia Medica (Zhong Hua Ben Cao). Shanghai: Shanghai Science and Technology Press; 1999, 8: 748.
- Chen XB, Hung LY, Xu J, Deng DS, Ling ZJ. Isolation of flavonoids from Pholidota cantonensis and their anti-tumour activities. J Clin Med 2017; 4(55): 10822–3. doi: 10.16281/j.cnki.jocml.2017.55.105
- Li B, Ali Z, Chan M, Li J, Wang M, Abe N, et al. Chemical constituents of Pholidota cantonensis. Phytochemistry 2017; 137: 132–8. doi: 10.1016/j.phytochem.2017.02.005
- Li JC, Feng L, Nohara T. Chemical constituents from herb of Pholidota cantonensis. Chin J Chin Meter Med 2008; 33(14): 1691–3.
- Wang W, Yan X, Yin QM, Ling T, Wang RK, Liu L. Chemical constituents from Pholidota cantonensis herbs. Chin Med Mat 2017; 40(8): 1861–3. doi: 10.13863/j.issn1001-4454.2017.08.025
- Liu L, Wang W, Yin QM, Yan X, Huang YX, Fan YY, et al. The separation and identification of the chemical constituents from ethyl acetate extract of Pholidota cantonensis. J Yangzhou Univ Nat Sci Ed 2015; 18(4): 53–6. doi: 10.19411/j.1007-824x.2015.04.014
- Brower V. Back to nature: extinction of medicinal plants threatens drug discovery. J Natl Cancer Inst 2008; 100(12): 838–9. doi: 10.1093/jnci/djn199
- Kaufman RJ. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Gene Dev 1999; 13(10): 1211–33. doi: 10.1101/gad.13.10.1211
- Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer 2004; 4: 966–77. doi: 10.1038/nrc1505
- Pahl HL. Signal transduction from the endoplasmic reticulum to the cell nucleus. Physiol Rev 1999; 79(3): 683–701. doi: 10.1152/physrev.1922.214.171.1243
- Breckenridge DG, Germain M, Mathai JP, Nguyen M, Shore GC. Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene 2003; 22(53): 8608–18. doi: 10.1038/sj.onc.1207108
- Lemasters JJ. Dying a thousand deaths: redundant pathways from different organelles to apoptosis and necrosis. Gastroenterology 2005; 129(1): 351–60. doi: 10.1053/j.gastro.2005.06.006
- Ellgaard L, Helenius A. Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 2003; 4(3): 181–91. doi: 10.1038/nrm1052
- Anelli T, Alessio M, Bachi A, Bergamelli L, Bertoli G, Camerini S, et al. Thiol-mediated protein retention in the endoplasmic reticulum: the role of ERp44. Embo J 2003; 22(19): 5015–22. doi: 10.1093/emboj/cdg491
- Freedman RB. Protein disulfide isomerase: multiple roles in the modification of nascent secretory proteins. Cell 1989; 57(7): 1069–72. doi: 10.1016/0092-8674(89)90043-3
- Tien AC, Rajan A, Schulze KL, Ryoo HD, Acar M, Steller H, et al. Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster. J Cell Biol 2008; 182(6): 1113–25. doi: 10.1083/jcb.200805001
- Kaufman RJ, Scheuner D, Schroder M, Shen X, Lee K, Liu CY, et al. The unfolded protein response in nutrient sensing and differentiation. Nat Rev Mol Cell Bio 2002; 3(6): 411–21. doi: 10.1038/nrm829
- Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Bio 2000; 2(6): 326–32. doi: 10.1038/35014014
- Okamura K, Kimata Y, Higashio H, Tsuru A, Kohno K. Dissociation of Kar2p/BiP from an ER sensory molecule, Ire1p, triggers the unfolded protein response in yeast. Biochem Bioph Res Co 2000; 279(2): 445–50. doi: 10.1006/bbrc.2000.3987
- Kimata Y, Oikawa D, Shimizu Y, Ishiwata-Kimata Y, Kohno K. A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1. J Cell Biol 2004; 167(3): 445–56. doi: 10.1083/jcb.200405153
- Oikawa D, Kimata Y, Kohno K. Self-association and BiP dissociation are not sufficient for activation of the ER stress sensor Ire1. J Cell Sci 2007; 120(9): 1681–8. doi: 10.1242/jcs.002808
- Nishitoh H, Matsuzawa A, Tobiume K, Saegusa K, Takeda K, Inoue K, et al. ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Gene Dev 2002; 16(11): 1345–55. doi: 10.1101/gad.992302
- Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 2000; 287(5453): 664–6. doi: 10.1126/science.287.5453.664
- Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 1999; 397(6716): 271–4. doi: 10.1038/16729
- Shi Y, An J, Liang J, Hayes SE, Sandusky GE, Stramm LE, et al. Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells. J Biol Chem 1999; 274(9): 5723–30. doi: 10.1074/jbc.274.9.5723
- Shi Y, Vattem KM, Sood R, An J, Liang J, Stramm L, et al. Identification and characterization of pancreatic eukaryotic initiation factor 2 α-subunit kinase, PEK, involved in translational control. Mol Cell Biol 1998; 18(12): 7499–509. doi: 10.1128/MCB.18.12.7499
- Marciniak SJ, Garcia-Bonilla L, Hu J, Harding HP, Ron D. Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK. J Cell Biol 2006; 172(2): 201–9. doi: 10.1083/jcb.200508099
- Barone MV, Crozat A, Tabaee A, Philipson L, Ron D. CHOP (GADD153) and its oncogenic variant, TLS-CHOP, have opposing effects on the induction of G1/S arrest. Gene Dev 1994; 8(4): 453–64. doi: 10.1101/gad.8.4.453
- Zhan Q, Lord KA, Alamo IJ, Hollander MC, Carrier F, Ron D, et al. The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mol Cell Biol 1994; 14(4): 2361–71. doi 10.1128/MCB.14.4.2361
- Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 2000; 6(5): 1099–108. doi: 10.1016/s1097-2765(00)00108-8
- Okada T, Yoshida H, Akazawa R, Negishi M, Mori K. Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response. Biochem J 2002; 366(2): 585–94. doi: 10.1042/BJ20020391
- Scheuner D, Song B, Mcewen E, Liu C, Laybutt R, Gillespie P, et al. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol Cell 2001; 7(6): 1165–76. doi: 10.1016/S1097-2765(01)00265-9
- Wang DD, Wu QX, Pan WJ, Hussain S, Mehmood S, Chen Y. A novel polysaccharide from the Sarcodon aspratus triggers apoptosis in Hela cells via induction of mitochondrial dysfunction. Food Nutr Res 2018; 62: 1285–94. doi: 10.29219/fnr.v62.1285
- Song S, Tan J, Miao Y, Li M, Zhang Q. Crosstalk of autophagy and apoptosis: involvement of the dual role of autophagy under ER stress. J Cell Physiol 2017; 232(11): 2977–84. doi: 10.1002/jcp.25785
- Senft D, Ronai ZA. UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci 2015; 40(3): 141–8. doi: 10.1016/j.tibs.2015.01.002
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