Kaempferol Alleviates Mitochondrial Damage by Reducing Mitochondrial Reactive Oxygen Species Production in Lipopolysaccharide-Induced Prostate Organoids
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemical Reagents
2.2. Organoid Formation
2.3. LPS Treatment in Organoids
2.4. Immunofluorescence
2.5. Western Blot Analysis
2.6. Total RNA Extraction and RT-qPCR
2.7. Measurement of Intracellular ROS Production Using DCF-DA
2.8. Detection of Mitochondria and Mitochondrial ROS
2.9. Measurement of Oxygen Consumption Rate through Mitochondrial Metabolism
2.10. Statistical Analysis
3. Results
3.1. Establishment of Mouse Prostate Organoid Expressing Basal and Luminal Epithelial Layers
3.2. Kaempferol Inhibits iNOS, COX-2, p-IκB, and Inflammatory Cytokines in LPS-Stimulated Prostate Organoids
3.3. Kaempferol Is Effective in Inhibiting Intracellular ROS Produced by LPS
3.4. Kaempferol Induces an Antioxidant Response by Activating the Nrf2 Pathway
3.5. Kaempferol Can Reduce the Generation of Mitochondrial ROS (mtROS)
3.6. Reduced mtROS in Prostate Organoids Can Be Due to Enhanced Autophagy and an Increase in Mitochondrial Function by Kaempferol
3.7. ROS Increases Prostate Organoid Oxygen Consumption Rate (OCR), and Co-Treatment of Kaempferol Restores Mitochondrial Dysfunction via Mitophagy
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
- Collins, M.M.; Stafford, R.S.; O’leary, M.P.; Barry, M.J. How common is prostatitis? A national survey of physician visits. J. Urol. 1998, 159, 1224–1228. [Google Scholar] [CrossRef]
- Kirby, R.S.; Lowe, D.; Bultitude, M.I.; Shuttleworth, K.E. Intra-prostatic urinary reflux: An aetiological factor in abacterial prostatitis. Br. J. Urol. 1982, 54, 729–731. [Google Scholar] [CrossRef]
- Cho, S.Y.; Bae, W.J.; Cho, Y.-H.; Lee, S.-J. Clinical characteristics and treatment results of acute bacterial prostatitis. Infect. Chemother. 2009, 41, 36–41. [Google Scholar] [CrossRef]
- Vignozzi, L.; Rastrelli, G.; Corona, G.; Gacci, M.; Forti, G.; Maggi, M. Benign prostatic hyperplasia: A new metabolic disease? J. Endocrinol. Investig. 2014, 37, 313–322. [Google Scholar] [CrossRef]
- De Nunzio, C.; Kramer, G.; Marberger, M.; Montironi, R.; Nelson, W.; Schröder, F.; Sciarra, A.; Tubaro, A. The controversial relationship between benign prostatic hyperplasia and prostate cancer: The role of inflammation. Eur. Urol. 2011, 60, 106–117. [Google Scholar] [CrossRef] [PubMed]
- Kramer, G.; Steiner, G.E.; Handisurya, A.; Stix, U.; Haitel, A.; Knerer, B.; Gessl, A.; Lee, C.; Marberger, M. Increased expression of lymphocyte-derived cytokines in benign hyperplastic prostate tissue, identification of the producing cell types, and effect of differentially expressed cytokines on stromal cell proliferation. Prostate 2002, 52, 43–58. [Google Scholar] [CrossRef]
- Khandrika, L.; Kumar, B.; Koul, S.; Maroni, P.; Koul, H.K. Oxidative stress in prostate cancer. Cancer Lett. 2009, 282, 125–136. [Google Scholar] [CrossRef] [PubMed]
- Wang, Y.; Hayward, S.W.; Cao, M.; Thayer, K.A.; Cunha, G.R. Cell differentiation lineage in the prostate. Differentiation 2001, 68, 270–279. [Google Scholar] [CrossRef] [PubMed]
- Karthaus, W.R.; Iaquinta, P.J.; Drost, J.; Gracanin, A.; Van Boxtel, R.; Wongvipat, J.; Dowling, C.M.; Gao, D.; Begthel, H.; Sachs, N. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell 2014, 159, 163–175. [Google Scholar] [CrossRef]
- Mehta, V.; Abler, L.L.; Keil, K.P.; Schmitz, C.T.; Joshi, P.S.; Vezina, C.M. Atlas of Wnt and R-spondin gene expression in the developing male mouse lower urogenital tract. Dev. Dyn. 2011, 240, 2548–2560. [Google Scholar] [CrossRef]
- Carmon, K.S.; Gong, X.; Lin, Q.; Thomas, A.; Liu, Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc. Natl. Acad. Sci. USA 2011, 108, 11452–11457. [Google Scholar] [CrossRef]
- De Lau, W.; Barker, N.; Low, T.Y.; Koo, B.-K.; Li, V.S.; Teunissen, H.; Kujala, P.; Haegebarth, A.; Peters, P.J.; Van De Wetering, M. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 2011, 476, 293–297. [Google Scholar] [CrossRef]
- Gonzalez, R.; Ballester, I.; Lopez-Posadas, R.; Suarez, M.D.; Zarzuelo, A.; Martinez-Augustin, O.; Sanchez de Medina, F. Effects of flavonoids and other polyphenols on inflammation. Crit. Rev. Food Sci. Nutr. 2011, 51, 331–362. [Google Scholar] [CrossRef]
- Pan, M.H.; Lai, C.S.; Ho, C.T. Anti-inflammatory activity of natural dietary flavonoids. Food Funct. 2010, 1, 15–31. [Google Scholar] [CrossRef]
- Crespo, I.; Garcia-Mediavilla, M.V.; Gutierrez, B.; Sanchez-Campos, S.; Tunon, M.J.; Gonzalez-Gallego, J. A comparison of the effects of kaempferol and quercetin on cytokine-induced pro-inflammatory status of cultured human endothelial cells. Br. J. Nutr. 2008, 100, 968–976. [Google Scholar] [CrossRef] [PubMed]
- Halliwell, B.; Gutteridge, J.M. Free Radicals in Biology and Medicine; Oxford University Press: Oxford, UK, 2015. [Google Scholar]
- Saw, C.L.; Guo, Y.; Yang, A.Y.; Paredes-Gonzalez, X.; Ramirez, C.; Pung, D.; Kong, A.N. The berry constituents quercetin, kaempferol, and pterostilbene synergistically attenuate reactive oxygen species: Involvement of the Nrf2-ARE signaling pathway. Food Chem. Toxicol. 2014, 72, 303–311. [Google Scholar] [CrossRef]
- Lin, C.W.; Chen, P.N.; Chen, M.K.; Yang, W.E.; Tang, C.H.; Yang, S.F.; Hsieh, Y.S. Kaempferol reduces matrix metalloproteinase-2 expression by down-regulating ERK1/2 and the activator protein-1 signaling pathways in oral cancer cells. PLoS ONE 2013, 8, e80883. [Google Scholar] [CrossRef] [PubMed]
- Cairns, G.; Thumiah-Mootoo, M.; Burelle, Y.; Khacho, M. Mitophagy: A new player in stem cell biology. Biology 2020, 9, 481. [Google Scholar] [CrossRef] [PubMed]
- Lin, Q.; Chen, J.; Gu, L.; Dan, X.; Zhang, C.; Yang, Y. New insights into mitophagy and stem cells. Stem Cell Res. Ther. 2021, 12, 452. [Google Scholar] [CrossRef]
- Palikaras, K.; Tavernarakis, N. Mitochondrial homeostasis: The interplay between mitophagy and mitochondrial biogenesis. Exp. Gerontol. 2014, 56, 182–188. [Google Scholar] [CrossRef]
- Filomeni, G.; Graziani, I.; De Zio, D.; Dini, L.; Centonze, D.; Rotilio, G.; Ciriolo, M.R. Neuroprotection of kaempferol by autophagy in models of rotenone-mediated acute toxicity: Possible implications for Parkinson’s disease. Neurobiol. Aging 2012, 33, 767–785. [Google Scholar] [CrossRef] [PubMed]
- Cheaito, K.; Bahmad, H.F.; Hadadeh, O.; Msheik, H.; Monzer, A.; Ballout, F.; Dagher, C.; Telvizian, T.; Saheb, N.; Tawil, A.; et al. Establishment and characterization of prostate organoids from treatment-naïve patients with prostate cancer. Oncol. Lett. 2022, 23, 6. [Google Scholar] [CrossRef] [PubMed]
- Gleave, A.M.; Ci, X.; Lin, D.; Wang, Y. A synopsis of prostate organoid methodologies, applications, and limitations. Prostate 2020, 80, 518–526. [Google Scholar] [CrossRef]
- de Lau, W.; Peng, W.C.; Gros, P.; Clevers, H. The R-spondin/Lgr5/Rnf43 module: Regulator of Wnt signal strength. Genes. Dev. 2014, 28, 305–316. [Google Scholar] [CrossRef]
- García-Mediavilla, V.; Crespo, I.; Collado, P.S.; Esteller, A.; Sánchez-Campos, S.; Tuñón, M.J.; González-Gallego, J. The anti-inflammatory flavones quercetin and kaempferol cause inhibition of inducible nitric oxide synthase, cyclooxygenase-2 and reactive C-protein, and down-regulation of the nuclear factor kappaB pathway in Chang Liver cells. Eur. J. Pharmacol. 2007, 557, 221–229. [Google Scholar] [CrossRef]
- Kim, S.H.; Park, J.G.; Lee, J.; Yang, W.S.; Park, G.W.; Kim, H.G.; Yi, Y.-S.; Baek, K.-S.; Sung, N.Y.; Hossen, M.J.; et al. The Dietary Flavonoid Kaempferol Mediates Anti-Inflammatory Responses via the Src, Syk, IRAK1, and IRAK4 Molecular Targets. Mediat. Inflamm. 2015, 2015, 904142. [Google Scholar] [CrossRef] [PubMed]
- Zhang, J.; Wang, X.; Vikash, V.; Ye, Q.; Wu, D.; Liu, Y.; Dong, W. ROS and ROS-mediated cellular signaling. Oxid. Med. Cell Longev. 2016, 2016, 4350965. [Google Scholar] [CrossRef]
- Starkov, A.A. The role of mitochondria in reactive oxygen species metabolism and signaling. Ann. N. Y. Acad. Sci. 2008, 1147, 37–52. [Google Scholar] [CrossRef]
- Cantó, C.; Gerhart-Hines, Z.; Feige, J.N.; Lagouge, M.; Noriega, L.; Milne, J.C.; Elliott, P.J.; Puigserver, P.; Auwerx, J. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 2009, 458, 1056–1060. [Google Scholar] [CrossRef]
- Miller, A.-F. Superoxide dismutases: Ancient enzymes and new insights. FEBS Lett. 2012, 586, 585–595. [Google Scholar] [CrossRef]
- Schieber, M.; Chandel, N.S. ROS function in redox signaling and oxidative stress. Curr. Biol. 2014, 24, R453–R462. [Google Scholar] [CrossRef]
- Alagal, R.I.; AlFaris, N.A.; Alshammari, G.M.; ALTamimi, J.Z.; AlMousa, L.A.; Yahya, M.A. Kaempferol attenuates doxorubicin-mediated nephropathy in rats by activating SIRT1 signaling. J. Funct. Foods 2022, 89, 104918. [Google Scholar] [CrossRef]
- Malpass, K. Defective mitochondrial dynamics in the hot seat—A therapeutic target common to many neurological disorders? Nat. Rev. Neurol. 2013, 9, 417. [Google Scholar] [CrossRef] [PubMed]
- Lemasters, J.J.; Nieminen, A.L.; Qian, T.; Trost, L.C.; Elmore, S.P.; Nishimura, Y.; Crowe, R.A.; Cascio, W.E.; Bradham, C.A.; Brenner, D.A.; et al. The mitochondrial permeability transition in cell death: A common mechanism in necrosis, apoptosis and autophagy. Biochim. Biophys. Acta 1998, 1366, 177–196. [Google Scholar] [CrossRef]
- Tang, C.; Han, H.; Yan, M.; Zhu, S.; Liu, J.; Liu, Z.; He, L.; Tan, J.; Liu, Y.; Liu, H. PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury. Autophagy 2018, 14, 880–897. [Google Scholar] [CrossRef] [PubMed]
- Angajala, A.; Lim, S.; Phillips, J.B.; Kim, J.-H.; Yates, C.; You, Z.; Tan, M. Diverse roles of mitochondria in immune responses: Novel insights into immuno-metabolism. Front. Immunol. 2018, 9, 1605. [Google Scholar] [CrossRef]
- Viscomi, C.; Bottani, E.; Civiletto, G.; Cerutti, R.; Moggio, M.; Fagiolari, G.; Schon, E.A.; Lamperti, C.; Zeviani, M. In vivo correction of COX deficiency by activation of the AMPK/PGC-1α axis. Cell Metab. 2011, 14, 80–90. [Google Scholar] [CrossRef]
- Narendra, D.P.; Jin, S.M.; Tanaka, A.; Suen, D.-F.; Gautier, C.A.; Shen, J.; Cookson, M.R.; Youle, R.J. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 2010, 8, e1000298. [Google Scholar] [CrossRef] [PubMed]
- Huang, K.; Gao, X.; Wei, W. The crosstalk between Sirt1 and Keap1/Nrf2/ARE anti-oxidative pathway forms a positive feedback loop to inhibit FN and TGF-β1 expressions in rat glomerular mesangial cells. Exp. Cell Res. 2017, 361, 63–72. [Google Scholar] [CrossRef]
- Suliman, H.B.; Carraway, M.S.; Welty-Wolf, K.E.; Whorton, A.R.; Piantadosi, C.A. Lipopolysaccharide stimulates mitochondrial biogenesis via activation of nuclear respiratory factor-1. J. Biol. Chem. 2003, 278, 41510–41518. [Google Scholar] [CrossRef]
- Gasparrini, M.; Forbes-Hernandez, T.Y.; Giampieri, F.; Afrin, S.; Alvarez-Suarez, J.M.; Mazzoni, L.; Mezzetti, B.; Quiles, J.L.; Battino, M. Anti-inflammatory effect of strawberry extract against LPS-induced stress in RAW 264.7 macrophages. Food Chem. Toxicol. 2017, 102, 1–10. [Google Scholar] [CrossRef] [PubMed]
- Lee, D.; Lee, J.H.; Lee, S.H. Flavonoids baicalein and kaempferol reduced inflammation in benign prostate hyperplasia patient-derived cells through regulating mitochondrial respiration and intracellular oxygen species. Korean J. Food Sci. Technol. 2021, 53, 213–217. [Google Scholar]
- Divakaruni, A.S.; Paradyse, A.; Ferrick, D.A.; Murphy, A.N.; Jastroch, M. Analysis and interpretation of microplate-based oxygen consumption and pH data. In Meth Enzymol; Elsevier: Amsterdam, The Netherlands, 2014; Volume 547, pp. 309–354. [Google Scholar]
- van der Windt, G.J.W.; Everts, B.; Chang, C.-H.; Curtis, J.D.; Freitas, T.C.; Amiel, E.; Pearce, E.J.; Pearce, E.L. Mitochondrial Respiratory Capacity Is a Critical Regulator of CD8+ T Cell Memory Development. Immunity 2012, 36, 68–78. [Google Scholar] [CrossRef] [PubMed]
- Da, J.; Xu, M.; Wang, Y.; Li, W.; Lu, M.; Wang, Z. Kaempferol Promotes Apoptosis While Inhibiting Cell Proliferation via Androgen-Dependent Pathway and Suppressing Vasculogenic Mimicry and Invasion in Prostate Cancer. Anal. Cell. Pathol. 2019, 2019, 1907698. [Google Scholar] [CrossRef]
- Wang, X.; Zhu, J.; Yan, H.; Shi, M.; Zheng, Q.; Wang, Y.; Zhu, Y.; Miao, L.; Gao, X. Kaempferol inhibits benign prostatic hyperplasia by resisting the action of androgen. Eur. J. Pharmacol. 2021, 907, 174251. [Google Scholar] [CrossRef] [PubMed]
Ingredient | Final Concentration |
---|---|
R-spondin conditioned medium | 10% |
Noggin conditioned medium | 25% |
N-acetyl-L-cysteine | 1.25 mM |
EGF | 10 μM |
DHT | 50 ng/mL |
A83-01 | 1 nM |
HEPES | 200 nM |
Glutamax | 10 mM |
B27 Supplement | 2 mM |
Glutamax | 2% |
Y-27632 | 10 μM (only 5–7 days after passage) |
Gene | Primer Sequence | |
---|---|---|
GAPDH | F | 5′-AACAGCAACTCCCACTCTTC-3′ |
R | 5′-GTGGTCCAGGGTTTCTTACTC-3′ | |
TNF-a | F | 5′-AGCCCCCAGTCTGTATCCTT-3′ |
R | 5′-GAGGCAACCTGACCACTCTC-3′ | |
IL-6 | F | 5′-GCCAGAGTCCTTCAGAGAGATA-3′ |
R | 5′-CAAACCTAGTGCGTTATGCCTA-3′ | |
IL-1B | F | 5′-CTGCTTCCAAACCTTTGACC-3′ |
R | 5′-AGCTTCTCCACAGCCACAAT-3′ | |
COX-2 | F | 5′-CCAGATGCTATCTTTGGGGA-3′ |
R | 5′-GCTCGGCTTCCAGTATTGAG-3′ | |
Nrf2 | F | 5′-TCCGCTGCCATCAGTCAGTC-3′ |
R | 5′-ATTGTGCCTTCAGCGTGCTTC-3′ | |
HO-1 | F | 5′-AACAAGCAGAACCCAGTCTATGC-3′ |
R | 5′-AGGTAGCGGGTATATGCGTGGGCC-3′ | |
NQO-1 | F | 5′-TTCTGTGGCTTCCAGGTCTT-3′ |
R | 5′-AGGCTGCTTGGAGCAAAATA-3′ | |
SOD1 | F | 5′-TGGGTTCCACGTCCATCAGTA-3′ |
R | 5′-ACCGTCCTTTCCAGCAGTCA-3′ |
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Lee, M.J.; Cho, Y.; Hwang, Y.; Jo, Y.; Kim, Y.-G.; Lee, S.H.; Lee, J.H. Kaempferol Alleviates Mitochondrial Damage by Reducing Mitochondrial Reactive Oxygen Species Production in Lipopolysaccharide-Induced Prostate Organoids. Foods 2023, 12, 3836. https://doi.org/10.3390/foods12203836
Lee MJ, Cho Y, Hwang Y, Jo Y, Kim Y-G, Lee SH, Lee JH. Kaempferol Alleviates Mitochondrial Damage by Reducing Mitochondrial Reactive Oxygen Species Production in Lipopolysaccharide-Induced Prostate Organoids. Foods. 2023; 12(20):3836. https://doi.org/10.3390/foods12203836
Chicago/Turabian StyleLee, Myeong Joon, Yeonoh Cho, Yujin Hwang, Youngheun Jo, Yeon-Gu Kim, Seung Hwan Lee, and Jong Hun Lee. 2023. "Kaempferol Alleviates Mitochondrial Damage by Reducing Mitochondrial Reactive Oxygen Species Production in Lipopolysaccharide-Induced Prostate Organoids" Foods 12, no. 20: 3836. https://doi.org/10.3390/foods12203836