Nobiletin, a hexamethoxyflavonoid from citrus pomace, attenuates G1 cell cycle arrest and apoptosis in hypoxia-induced human trophoblast cells of JEG-3 and BeWo via regulating the p53 signaling pathway
Background: Hypoxia is associated with abnormal cell apoptosis in trophoblast cells, which causes fetal growth restriction and related placental pathologies. Few effective methods for the prevention and treatment of placenta-related diseases exist. Natural products and functional foods have always been a rich source of potential anti-apoptotic drugs. Nobiletin (NOB), a hexamethoxyflavonoid derived from the citrus pomace, shows an anti-apoptotic activity, which is a non-toxic constituent of dietary phytochemicals approved by the Food and Drug Administration. However, their effects on hypoxia-induced human trophoblast cells have not been fully studied.
Objective: The aim of this study was to investigate the protective effects of NOB on hypoxia-induced apoptosis of human trophoblast JEG-3 and BeWo cells, and their underlying mechanisms.
Design: First, the protective effect of NOB on hypoxia-induced apoptosis of JEG-3 and BeWo cells was studied. Cell viability and membrane integrity were determined by CCK-8 assay and lactate dehydrogenase activity, respectively. Real Time Quantitative PCR (RT-qPCR) and Western blot analysis were used to detect the mRNA and protein levels of HIF1α. Propidium iodide (PI)-labeled flow cytometry was used to detect cell cycle distribution. Cell apoptosis was detected by flow cytometry with Annexin V-FITC and PI double staining, and the expression of apoptosis marker protein cl-PARP was detected by Western blot analysis. Then, the molecular mechanism of NOB against apoptosis was investigated. Computer molecular docking and dynamics were used to simulate the interaction between NOB and p53 protein, and this interaction was verified in vitro by Ultraviolet and visible spectrum (UV-visible spectroscopy), fluorescence spectroscopy and circular dichroism. Furthermore, the changes in the expression of p53 signaling pathway genes and proteins were detected by RT-qPCR and Western blot analysis, respectively.
Results: Hypoxia treatment resulted in a decreased cell viability and cell membrane integrity in JEG-3 and BeWo cell lines, and an increased expression of HIF1α, cell cycle arrest in the G1 phase, and massive cell apoptosis, which were alleviated after NOB treatment. Molecular docking and dynamics simulations found that NOB spontaneously bonded to human p53 protein, leading to the change of protein conformation. The intermolecular interaction between NOB and human p53 protein was further confirmed by UV-visible spectroscopy, fluorescence spectroscopy and circular dichroism. After the treatment of 100 μM NOB, a down-regulation of mRNA and protein levels of p53 and p21 and an up-regulation of BCL2/BAX mRNA and protein ratio were observed in JEG-3 cells; however, there was also a down-regulation of mRNA and protein levels observed for p53 and p21 in BeWo cells after the treatment of NOB. The BCL2/BAX ratio of BeWo cells did not change after the treatment of 100 μM NOB.
Conclusion: NOB attenuated hypoxia-induced apoptosis in JEG-3 and BeWo cell lines and might be a potential functional ingredient to prevent pregnancy-related diseases caused by hypoxia-induced apoptosis. These findings would also suggest the exploration and utilization of citrus resources, and the development of citrus industry.
- Lv Y, Lu C, Ji X, Miao Z, Long W, Ding H, et al. Roles of microRNAs in preeclampsia. J Cell Physiol 2019; 234(2): 1052–61. doi: 10.1002/jcp.27291
- Groom KM, David AL. The role of aspirin, heparin, and other interventions in the prevention and treatment of fetal growth restriction. Am J Obstet Gynecol 2018; 218(2S): S829–40. doi: 10.1016/j.ajog.2017.11.565
- Zhang Y, Zhang M, Li L, Wei B, He A, Lu L, et al. Methylation-reprogrammed Wnt/beta-catenin signalling mediated prenatal hypoxia-induced brain injury in foetal and offspring rats. J Cell Mol Med 2018; 22(8): 3866–74. doi: 10.1111/jcmm.13660
- Kohan-Ghadr HR, Kilburn BA, Kadam L, Johnson E, Kolb BL, Rodriguez-Kovacs J, et al. Rosiglitazone augments antioxidant response in the human trophoblast and prevents apoptosisdagger. Biol Reprod 2019; 100(2): 479–94. doi: 10.1093/biolre/ioy186
- Gupta SK, Malhotra SS, Malik A, Verma S, Chaudhary P. Cell signaling pathways involved during invasion and syncytialization of trophoblast cells. Am J Reprod Immunol 2016; 75(3): 361–471. doi: 10.1111/aji.12436
- Brukman NG, Uygur B, Podbilewicz B, Chernomordik LV. How cells fuse. J Cell Biol 2019; 218(5): 1436–51. doi: 10.1083/jcb.201901017
- Mahato N, Sharma K, Sinha M, Baral ER, Koteswararao R, Dhyani A, et al. Bio-sorbents, industrially important chemicals and novel materials from citrus processing waste as a sustainable and renewable bioresource: A review. J Adv Res 2020; 23: 61–82. doi: 10.1016/j.jare.2020.01.007
- Zheng Y, Bu J, Yu L, Chen J, Liu H. Nobiletin improves propofol-induced neuroprotection via regulating Akt/mTOR and TLR 4/NF-kappaB signaling in ischemic brain injury in rats. Biomed Pharmacother 2017; 91: 494–503. doi: 10.1016/j.biopha.2017.04.048
- Zhang N, Wei WY, Yang Z, Che Y, Jin YG, Liao HH, et al. Nobiletin, a polymethoxy flavonoid, protects against cardiac hypertrophy induced by pressure-overload via inhibition of NAPDH oxidases and endoplasmic reticulum stress. Cell Physiol Biochem 2017; 42(4): 1313–25. doi: 10.1159/000478960
- Wu Y, Zhang W, Li M, Cao D, Yang X, Gong J. Nobiletin ameliorates ischemia-reperfusion injury by suppressing the function of Kupffer cells after liver transplantation in rats. Biomed Pharmacother 2017; 89: 732–41. doi: 10.1016/j.biopha.2017.02.087
- Andrey N. Safety evaluation dossier supporting a generally recodnized as safe (GRAS) conclusion for orange pomace. Available from: https://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/default.htm [cited 10 February 2021].
- Zhang M, Zhang R, Liu J, Wang H, Wang Z, Liu J, et al. The effects of 5,6,7,8,3’,4’-hexamethoxyflavone on apoptosis of cultured human choriocarcinoma trophoblast cells. Molecules 2020; 25(4): 946. doi: 10.3390/molecules25040946
- Abou-Kheir W, Barrak J, Hadadeh O, Daoud G. HTR-8/SVneo cell line contains a mixed population of cells. Placenta 2017; 50: 1–7. doi: 10.1016/j.placenta.2016.12.007
- Costa MA. Scrutinising the regulators of syncytialization and their expression in pregnancy-related conditions. Mol Cell Endocrinol 2016; 420: 180–93. doi: 10.1016/j.mce.2015.11.010
- Chen PY, Chen YT, Gao WY, Wu MJ, Yen JH. Nobiletin down-regulates c-KIT gene expression and exerts antileukemic effects on human acute myeloid leukemia cells. J Agric Food Chem 2018; 66(51): 13423–34. doi: 10.1021/acs.jafc.8b05680
- Zhang R, Chen J, Mao L, Guo Y, Hao Y, Deng Y, et al. Nobiletin triggers reactive oxygen species-mediated pyroptosis through regulating autophagy in ovarian cancer cells. J Agric Food Chem 2020; 68(5): 1326–36. doi: 10.1021/acs.jafc.9b07908
- Xu M, Liu G, Li M, Huo M, Zong W, Liu R. Probing the cell apoptosis pathway induced by perfluorooctanoic acid and perfluorooctane sulfonate at the subcellular and molecular levels. J Agric Food Chem 2020; 68(2): 633–41. doi: 10.1021/acs.jafc.9b07072
- Ma Y, Wu Y, Xia Z, Li J, Li X, Xu P, et al. Anti-hypoxic molecular mechanisms of rhodiola crenulata extract in zebrafish as revealed by metabonomics. Front Pharmacol 2019; 10: 1356. doi: 10.3389/fphar.2019.01356
- Elks PM, Renshaw SA, Meijer AH, Walmsley SR, van Eeden FJ. Exploring the HIFs, buts and maybes of hypoxia signalling in disease: lessons from zebrafish models. Dis Model Mech 2015; 8(11): 1349–60. doi: 10.1242/dmm.021865
- Lopez-Garcia G, Cilla A, Barbera R, Alegria A. Anti-inflammatory and cytoprotective effect of plant sterol and galactooligosaccharides-enriched beverages in Caco-2 cells. J Agric Food Chem 2020; 68(7): 1862–70. doi: 10.1021/acs.jafc.9b03025
- Chen J, Chen AY, Huang H, Ye X, Rollyson WD, Perry HE, et al. The flavonoid nobiletin inhibits tumor growth and angiogenesis of ovarian cancers via the Akt pathway. Int J Oncol 2015; 46(6): 2629–38. doi: 10.3892/ijo.2015.2946
- Fischer M. Census and evaluation of p53 target genes. Oncogene 2017; 36(28): 3943–56. doi: 10.1038/onc.2016.502
- Hu M, Yang J, Qu L, Deng X, Duan Z, Fu R, et al. Ginsenoside Rk1 induces apoptosis and downregulates the expression of PD-L1 by targeting the NF-kappaB pathway in lung adenocarcinoma. Food Funct 2020; 11(1): 456–71. doi: 10.1039/c9fo02166c
- Parveen T, Kamran M, Fatmi MQ. Structural and dynamical thermostability of psychrophilic enzyme at various temperatures: Molecular dynamics simulations of tryptophan synthase. Arch Biochem Biophys 2019; 663: 297–305. doi: 10.1016/j.abb.2019.01.022
- Geng H, Chen F, Ye J, Jiang F. Applications of molecular dynamics simulation in structure prediction of peptides and proteins. Comput Struct Biotechnol J 2019; 17: 1162–70. doi: 10.1016/j.csbj.2019.07.010
- Sahihi M, Ghayeb Y. An investigation of molecular dynamics simulation and molecular docking: interaction of citrus flavonoids and bovine beta-lactoglobulin in focus. Comput Biol Med 2014; 51: 44–50. doi: 10.1016/j.compbiomed.2014.04.022
- Wang J, Zheng X, Zhang H. Exploring the conformational changes in fibrinogen by forming protein corona with CdTe quantum dots and the related cytotoxicity. Spectrochim Acta A Mol Biomol Spectrosc 2019; 220: 117143. doi: 10.1016/j.saa.2019.117143
- Chen R, Jiang H, Pu H. Interaction of artemisinin and its derivatives with human serum albumin studied using spectroscopies and molecular modeling methods. Mol Biol Rep 2013; 40(8): 4791–804. doi: 10.1007/s11033-013-2575-6
- Zohoorian-Abootorabi T, Sanee H, Iranfar H, Saberi MR, Chamani J. Separate and simultaneous binding effects through a non-cooperative behavior between cyclophosphamide hydrochloride and fluoxymesterone upon interaction with human serum albumIn: multi-spectroscopic and molecular modeling approaches. Spectrochim Acta A Mol Biomol Spectrosc 2012; 88: 177–91. doi: 10.1016/j.saa.2011.12.026
- Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 2006; 1(6): 2876–90. doi: 10.1038/nprot.2006.202
- Wang J, Zhang D, Du J, Zhou C, Li Z, Liu X, et al. Tet1 facilitates hypoxia tolerance by stabilizing the HIF-alpha proteins independent of its methylcytosine dioxygenase activity. Nucleic Acids Res 2017; 45(22): 12700–14. doi: 10.1093/nar/gkx869
- Chen B, Longtine MS, Nelson DM. Punicalagin, a polyphenol in pomegranate juice, downregulates p53 and attenuates hypoxia-induced apoptosis in cultured human placental syncytiotrophoblasts. Am J Physiol Endocrinol Metab 2013; 305(10): E1274–80. doi: 10.1152/ajpendo.00218.2013
- Sagrillo-Fagundes L, Assuncao Salustiano EM, Ruano R, Markus RP, Vaillancourt C. Melatonin modulates autophagy and inflammation protecting human placental trophoblast from hypoxia/reoxygenation. J Pineal Res 2018; 65(4): e12520. doi: 10.1111/jpi.12520
- Ebegboni VJ, Dickenson JM, Sivasubramaniam SD. Antioxidative effects of flavonoids and their metabolites against hypoxia/reoxygenation-induced oxidative stress in a human first trimester trophoblast cell line. Food Chem 2019; 272: 117–25. doi: 10.1016/j.foodchem.2018.08.036
- Wang X, Simpson ER, Brown KA. P53: Protection against tumor growth beyond effects on cell cycle and apoptosis. Cancer Res 2015; 75(23): 5001–7. doi: 10.1158/0008-5472.CAN-15-0563
- Inoue T, Wu L, Stuart J, Maki CG. Control of p53 nuclear accumulation in stressed cells. FEBS Lett 2005; 579(22): 4978–84. doi: 10.1016/j.febslet.2005.08.006
- An WG, Kanekal M, Simon MC, Maltepe E, Blagosklonny MV, Neckers LM. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature 1998; 392(6674): 405–8. doi: 10.1038/32925
- Keith B, Johnson RS, Simon MC. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nat Rev Cancer 2011; 12(1): 9–22. doi: 10.1038/nrc3183
- Senturk E, Manfredi JJ. P53 and cell cycle effects after DNA damage. Methods Mol Biol 2013; 962: 49–61. doi: 10.1007/978-1-62703-236-0_4
- Mrakovcic M, Kleinheinz J, Frohlich LF. P53 at the crossroads between different types of HDAC inhibitor-mediated cancer cell death. Int J Mol Sci 2019; 20(10). doi: 10.3390/ijms20102415
- Ahmad MM, Rezk NA, Fawzy A, Sabry M. Protective effects of curcumin and silymarin against paracetamol induced hepatotoxicity in adult male albino rats. Gene 2019; 712:143966. doi: 10.1016/j.gene.2019.143966
- Li Q, Zhang Z, Li H, Pan X, Chen S, Cui Z, et al. Lycium barbarum polysaccharides protects H9c2 cells from hypoxia-induced injury by down-regulation of miR-122. Biomed Pharmacother 2019; 110: 20–8. doi: 10.1016/j.biopha.2018.11.012
- Jaafaru MS, Nordin N, Rosli R, Shaari K, Bako HY, Noor NM, et al. Prospective role of mitochondrial apoptotic pathway in mediating GMG-ITC to reduce cytotoxicity in H2O2-induced oxidative stress in differentiated SH-SY5Y cells. Biomed Pharmacother 2019; 119: 109445. doi: 10.1016/j.biopha.2019.109445
- Hammond EM, Giaccia AJ. The role of p53 in hypoxia-induced apoptosis. Biochem Biophys Res Commun 2005; 331(3): 718–25. doi: 10.1016/j.bbrc.2005.03.154
- Patra S, Panda PK, Naik PP, Panigrahi DP, Praharaj PP, Bhol CS, et al. Terminalia bellirica extract induces anticancer activity through modulation of apoptosis and autophagy in oral squamous cell carcinoma. Food Chem Toxicol 2020; 136: 111073. doi: 10.1016/j.fct.2019.111073
- He H, Feng M, Xu H, Li X, He Y, Qin H, et al. Total triterpenoids from the fruits of Chaenomeles speciosa exerted gastroprotective activities on indomethacin-induced gastric damage via modulating microRNA-423-5p-mediated TFF/NAG-1 and apoptotic pathways. Food Funct 2020; 11(1): 662–79. doi: 10.1039/c9fo02322d
- Qi Y, Cheng X, Gong G, Yan T, Du Y, Wu B, et al. Synergistic neuroprotective effect of schisandrin and nootkatone on regulating inflammation, apoptosis and autophagy via the PI3K/AKT pathway. Food Funct 2020; 11(3): 2427–38. doi: 10.1039/c9fo02927c
- Liu J, Wang Y, Hao Y, Wang Z, Yang Z, Wang Z. 5-Heptadecylresorcinol attenuates oxidative damage and mitochondria-mediated apoptosis through activation of the SIRT3/FOXO3a signaling pathway in neurocytes. Food Funct 2020; 11(3): 2535–42. doi: 10.1039/c9fo03028j
- Sharma C, Kang SC. Garcinol pacifies acrylamide induced cognitive impairments, neuroinflammation and neuronal apoptosis by modulating GSK signaling and activation of pCREB by regulating cathepsin B in the brain of zebrafish larvae. Food Chem Toxicol 2020; 138: 111246. doi: 10.1016/j.fct.2020.111246
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