Doxorubicin (DOX) is a cytostatic agent belonging to anthracycline group. Important role in mechanism associated with negative effects of DOX plays an oxidative stress. Heat shock proteins (HSPs) are part of mechanisms initiated in response to stressful stimuli and play an important role in cellular responses to oxidative stress through interaction with components of redox signaling. The present work was aimed to study the role of HSPs and autophagy in mechanisms underlying effects of sulforaphane (SFN), a potential activator of Nrf-2, on doxorubicin-induced toxicity in human kidney HEK293 cells. We investigated effects of SFN and DOX on proteins associated with regulation of heat shock response, redox signaling, and autophagy. Results show that SFN significantly reduced cytotoxic effects of DOX. The positive effects of SFN on DOX-induced changes were associated with up-regulation of Nrf-2 and HSP60 protein levels. In the case of another heat shock protein HSP40, SFN increased its levels when was administered alone but not in conditions when cells were exposed to the effects of DOX. Sulforaphane also reversed negative effects of DOX on activities of superoxide dismutases (SODs) and up-regulation of autophagy markers (LC3A/B-II, Atg5, and Atg12). In conclusion, the changes observed in HSP60 are of particular importance in terms of protecting cells from the effects of DOX. Finding that under conditions where SFN reduced cytotoxic effects of DOX were significantly increased protein levels of both Nrf-2 and HSP60 point to the role of HSP60 in mechanisms of redox signaling underlying effects of SFN on DOX-induced toxicity in HEK293 cells. Moreover, data confirmed an important role of autophagy in effects of SFN on DOX-induced toxicity.

Centre of Experimental Medicine Institute for Heart Research Slovak Academy of Sciences Bratislava Slovak Republic

Zobrazit více v PubMed

Pommier Y, Leo E, Zhang H, Marchand C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol. 2010;17:421–433. doi: 10.1016/j.chembiol.2010.04.012. PubMed DOI PMC

da Costa RM, Fais RS, Dechandt CRP, Louzada-Junior P, Alberici LC, Lobato NS, Tostes RC. Increased mitochondrial ROS generation mediates the loss of the anti-contractile effects of perivascular adipose tissue in high-fat diet obese mice. Br J Pharmacol. 2017;174:3527–3541. doi: 10.1111/bph.13687. PubMed DOI PMC

Yi G, Din Jud, Zhao F, Liu X. Effect of soybean peptides against hydrogen peroxide induced oxidative stress in HepG2 cells via Nrf2 signaling. Food Funct. 2020;11:2725–737. doi: 10.1039/C9FO01466G. PubMed DOI

Xia M-H, Yan X-Y, Zhou L, Xu L, Zhang L-C, Yi H-W, Su J. p62 suppressed VK3-induced oxidative damage through Keap1/Nrf2 pathway in human ovarian cancer cells. J Cancer. 2020;11:1299–1307. doi: 10.7150/jca.34423. PubMed DOI PMC

Wu J, Pan X, Fu H, Zheng Y, Dai Y, Yin Y, Chen Q, Hao Q, Bao D, Hou D. Effect of curcumin on glycerol-induced acute kidney injury in rats. Sci Rep. 2017;7:10114. doi: 10.1038/s41598-017-10693-4. PubMed DOI PMC

Xin Y, Bai Y, Jiang X, Zhou S, Wang Y, Wintergerst KA, Cui T, Ji H, Tan Y, Cai L. Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3β/Fyn pathway. Redox Biol. 2018;15:405–417. doi: 10.1016/j.redox.2017.12.016. PubMed DOI PMC

Ebrahimpour S, Shahidi SB, Abbasi M, Tavakoli Z, Esmaeili A. Quercetin-conjugated superparamagnetic iron oxide nanoparticles (QCSPIONs) increases Nrf2 expression via miR-27a mediation to prevent memory dysfunction in diabetic rats. Sci Rep. 2020;10:15957. doi: 10.1038/s41598-020-71971-2. PubMed DOI PMC

Tanito M, Masutani H, Kim YC, Nishikawa M, Ohira A, Yodoi J. Sulforaphane induces thioredoxin through the antioxidant-responsive element and attenuates retinal light damage in mice. Invest Opthalmol Vis Sci. 2005;46:979–987. doi: 10.1167/iovs.04-1120. PubMed DOI

Qazi A, Pal J, Maitah M, Fulciniti M, Pelluru D, Nanjappa P, Lee S, et al. Anticancer activity of a broccoli derivative, sulforaphane, in barrett adenocarcinoma: potential use in chemoprevention and as adjuvant in chemotherapy. Transl Oncol. 2010;3:389–399. doi: 10.1593/tlo.10235. PubMed DOI PMC

Silva-Palacios A, Ostolga-Chavarría M, Sánchez-Garibay C, Rojas-Morales P, Galván-Arzate S, Buelna-Chontal M, Pavón N, et al. Sulforaphane protects from myocardial ischemia-reperfusion damage through the balanced activation of Nrf2/AhR. Free Radic Biol Med. 2019;143:331–340. doi: 10.1016/j.freeradbiomed.2019.08.012. PubMed DOI

Yoon HY, Kang NI, Lee HK, Jang KY, Park JW, Park BH. Sulforaphane protects kidneys against ischemia-reperfusion injury through induction of the Nrf2-dependent phase 2 enzyme. Biochem Pharmacol. 2008;75:2214–223. doi: 10.1016/j.bcp.2008.02.029. PubMed DOI

Zhao J, Kobori N, Aronowski J, Dash PK. Sulforaphane reduces infarct volume following focal cerebral ischemia in rodents. Neurosci Lett. 2006;393:108–112. doi: 10.1016/j.neulet.2005.09.065. PubMed DOI

Kapoor S, Kala D, Svoboda J, Daněk J, Faridová A, Brnoliaková Z, Mikulecká A, Folbergrová J, Otáhal J. The effect of sulforaphane on perinatal hypoxic-ischemic brain injury in rats. Physiol Res. 2022;71:401–441. doi: 10.33549/physiolres.934878. PubMed DOI PMC

Singh P, Sharma R, McElhanon K, Allen CD, Megyesi JK, Beneš H, Singh SP. Sulforaphane protects the heart from doxorubicin-induced toxicity. Free Radic Biol Med. 2015;86:90–101. doi: 10.1016/j.freeradbiomed.2015.05.028. PubMed DOI PMC

Dovinova I, Kvandova M, Balis P, Gresova L, Majzunova M, Horakova L, Chan J, Barancik M. The role of Nrf2 and PPARγ in the improvement of oxidative stress in hypertension and cardiovascular diseases. Physiol Res. 2020;69(Suppl 4):S541–S553. doi: 10.33549/physiolres.934612. PubMed DOI PMC

Bose C, Awasthi S, Sharma R, Beneš H, Hauer-Jensen M, Boerma M, Singh SP. Sulforaphane potentiates anticancer effects of doxorubicin and attenuates its cardiotoxicity in a breast cancer model. PLoS One. 2018;13:e0193918. doi: 10.1371/journal.pone.0193918. PubMed DOI PMC

Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) Autophagy. 2016;12:1–22. doi: 10.1080/15548627.2015.1100356. PubMed DOI PMC

Yang F, Wang F, Liu Y, Wang S, Li X, Huang Y, Xia Y, Cao C. Sulforaphane induces autophagy by inhibition of HDAC6-mediated PTEN activation in triple negative breast cancer cells. Life Sci. 2018;213:149–157. doi: 10.1016/j.lfs.2018.10.034. PubMed DOI

Reeg S, Jung T, Castro JP, Davies KJA, Henze A, Grune T. The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome. Free Radic Biol Med. 2016;99:153–166. doi: 10.1016/j.freeradbiomed.2016.08.002. PubMed DOI PMC

Gomez-Pastor R, Burchfiel ET, Thiele DJ. Regulation of heat shock transcription factors and their roles in physiology and disease. Nat Rev Mol Cell Biol. 2018;19:4–19. doi: 10.1038/nrm.2017.73. PubMed DOI PMC

Klumpen E, Hoffschröer N, Zeis B, Gigengack U, Dohmen E, Paul RJ. Reactive oxygen species (ROS) and the heat stress response of Daphnia pulex: ROS-mediated activation of hypoxia-inducible factor 1 (HIF-1) and heat shock factor 1 (HSF-1) and the clustered expression of stress genes. Biol Cell. 2017;109:39–64. doi: 10.1111/boc.201600017. PubMed DOI

Kovács Sigmond, Hotzi Bohár, Fazekas Deák, Vellai Barna. HSF1Base: A Comprehensive Database of HSF1 (Heat Shock Factor 1) Target Genes. Int J Mol Sci. 2019;20:5815. doi: 10.3390/ijms20225815. PubMed DOI PMC

McDuffee AT, Senisterra G, Huntley S, Lepock JR, Sekhar KR, Meredith MJ, Borrelli MJ, et al. Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response. J Cell Physiol. 1997;171:143–151. doi: 10.1002/(SICI)1097-4652(199705)171:2<143::AID-JCP4>3.0.CO;2-O. PubMed DOI

McMahon M, Itoh K, Yamamoto M, Hayes JD. Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J Biol Chem. 2003;278:21592–1600. doi: 10.1074/jbc.M300931200. PubMed DOI

Sajadimajd S, Khazaei M. Oxidative stress and cancer: the role of Nrf2. Curr Cancer Drug Targets. 2018;18:538–557. doi: 10.2174/1568009617666171002144228. PubMed DOI

Akagi R, Kubo T, Hatori Y, Miyamoto T, Inouye S. Heme oxygenase-1 induction by heat shock in rat hepatoma cell line is regulated by the coordinated function of HSF1, NRF2 and BACH1. J Biochem. 2021;170:501–510. doi: 10.1093/jb/mvab065. PubMed DOI

Almeida DV, da Silva Nornberg BF, Geracitano LA, Barros DM, Monserrat JM, Marins LF. Induction of phase II enzymes and hsp70 genes by copper sulfate through the electrophile-responsive element (EpRE): insights obtained from a transgenic zebrafish model carrying an orthologous EpRE sequence of mammalian origin. Fish Physiol Biochem. 2010;36:347–353. doi: 10.1007/s10695-008-9299-x. PubMed DOI

Samarasinghe B, Wales CTK, Taylor FR, Jacobs AT. Heat shock factor 1 confers resistance to Hsp90 inhibitors through p62/SQSTM1 expression and promotion of autophagic flux. Biochem Pharmacol. 2014;87:445–455. doi: 10.1016/j.bcp.2013.11.014. PubMed DOI PMC

Dou J, Su P, Xu C, Wen Z, Mao Z, Li W. Targeting Hsc70-based autophagy to eliminate amyloid β oligomers. Biochem Biophys Res Commun. 2020;524:923–928. doi: 10.1016/j.bbrc.2020.02.016. PubMed DOI PMC

Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, Sou YS, et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nat Cell Biol. 2010;12:213–23. doi: 10.1038/ncb2021. PubMed DOI

Sun B, Xu Y, Liu ZY, Meng WX, Yang H. Autophagy assuages myocardial infarction through Nrf2 signaling activation-mediated reactive oxygen species clear. Eur Rev Med Pharmacol Sci. 2020;24:7381–7390. doi: 10.26355/eurrev_202007_21906. PubMed DOI

Zang H, Wu W, Qi L, Tan W, Nagarkatti P, Nagarkatti M, Wang X, Cui T. Autophagy inhibition enables Nrf2 to exaggerate the progression of diabetic cardiomyopathy in mice. Diabetes. 2020;69:2720–734. doi: 10.2337/db19-1176. PubMed DOI PMC

Hou K, Shen J, Yan J, Zhai C, Zhang J, Pan JA, Zhang Y, et al. Loss of TRIM21 alleviates cardiotoxicity by suppressing ferroptosis induced by the chemotherapeutic agent doxorubicin. EBioMedicine. 2021;69:103456. doi: 10.1016/j.ebiom.2021.103456. PubMed DOI PMC

Dayalan Naidu S, Dikovskaya D, Gaurilcikaite E, Knatko EV, Healy ZR, Mohan H, Koh G, et al. Transcription factors NRF2 and HSF1 have opposing functions in autophagy. Sci Rep. 2017;7:11023. doi: 10.1038/s41598-017-11262-5. PubMed DOI PMC

Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36:59–74. doi: 10.1099/0022-1317-36-1-59. PubMed DOI

Li W, Trieu J, Blazev R, Parker BL, Murphy KT, Swiderski K, Lynch GS. Sulforaphane attenuates cancer cell-induced atrophy of C2C12 myotubes. Am J Physiol Cell Physiol. 2023;324:C205–C221. doi: 10.1152/ajpcell.00025.2022. PubMed DOI

Bai Y, Chen Q, Sun YP, Wang X, Lv L, Zhang LP, Liu JS, Zhao S, Wang XL. Sulforaphane protection against the development of doxorubicin-induced chronic heart failure is associated with Nrf2 upregulation. Cardiovasc Ther. 2017;35:e12277. doi: 10.1111/1755-5922.12277. PubMed DOI

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. doi: 10.1016/0003-2697(76)90527-3. PubMed DOI

Li S, Yang H, Chen X. Protective effects of sulforaphane on diabetic retinopathy: activation of the Nrf2 pathway and inhibition of NLRP3 inflammasome formation. Exp Anim. 2019;68:221–31. doi: 10.1538/expanim.18-0146. PubMed DOI PMC

Miao X, Bai Y, Sun W, Cui W, Xin Y, Wang Y, Tan Y, et al. Sulforaphane prevention of diabetes-induced aortic damage was associated with the up-regulation of Nrf2 and its down-stream antioxidants. Nutr Metab (Lond) 2012;9:84. doi: 10.1186/1743-7075-9-84. PubMed DOI PMC

Bai Y, Chen Q, Sun YP, Wang X, Lv L, Zhang LP, Liu JS, Zhao S, Wang XL. Sulforaphane protection against the development of doxorubicin-induced chronic heart failure is associated with Nrf2 upregulation. Cardiovasc Ther. 2017;35:e12277. doi: 10.1111/1755-5922.12277. PubMed DOI

Gan N, Wu YC, Brunet M, Garrido C, Chung FL, Dai C, Mi L. Sulforaphane activates heat shock response and enhances proteasome activity through up-regulation of Hsp27. J Biol Chem. 2010;285:35528–35536. doi: 10.1074/jbc.M110.152686. PubMed DOI PMC

Li Y, Karagöz GE, Seo YH, Zhang T, Jiang Y, Yu Y, Duarte AMS, et al. Sulforaphane inhibits pancreatic cancer through disrupting Hsp90-p50Cdc37 complex and direct interactions with amino acids residues of Hsp90. J Nutr Biochem. 2012;23:1617–1626. doi: 10.1016/j.jnutbio.2011.11.004. PubMed DOI PMC

Gabrielson K, Bedja D, Pin S, Tsao A, Gama L, Yuan B, Muratore N. Heat shock protein 90 and ErbB2 in the cardiac response to doxorubicin injury. Cancer Res. 2007;67:1436–1441. doi: 10.1158/0008-5472.CAN-06-3721. PubMed DOI

Jing L, Jiang JR, Liu DM, Sheng JW, Zhang WF, Li ZJ, Wei LY. Structural characterization and antioxidant activity of polysaccharides from Athyrium multidentatum (Doll.) ching in D-galactose-induced aging mice via PI3K/AKT pathway. Molecules. 2019;24:3364. doi: 10.3390/molecules24183364. PubMed DOI PMC

Nordgren KKS, Wallace KB. Keap1 redox-dependent regulation of doxorubicin-induced oxidative stress response in cardiac myoblasts. Toxicol Appl Pharmacol. 2014;274:107–116. doi: 10.1016/j.taap.2013.10.023. PubMed DOI

Li B, Kim DS, Yadav RK, Kim HR, Chae HJ. Sulforaphane prevents doxorubicin-induced oxidative stress and cell death in rat H9c2 cells. Int J Mol Med. 2015;36:53–64. doi: 10.3892/ijmm.2015.2199. PubMed DOI PMC

Copple IM, Lister A, Obeng AD, Kitteringham NR, Jenkins RE, Layfield R, Foster BJ, Goldring CE, Park BK. Physical and functional interaction of sequestosome 1 with Keap1 regulates the Keap1-Nrf2 cell defense pathway. J Biol Chem. 2010;285:16782–6788. doi: 10.1074/jbc.M109.096545. PubMed DOI PMC

Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T. Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep. 2001;2:330–335. doi: 10.1093/embo-reports/kve061. PubMed DOI PMC

Zhu K, Yuan Y, Wen J, Chen D, Zhu W, Ouyang Z, Wang W. LncRNA Sox2OT-V7 promotes doxorubicin-induced autophagy and chemoresistance in osteosarcoma via tumor-suppressive miR-142/miR-22. Aging (Albany NY) 2020;12:6644–6666. doi: 10.18632/aging.103004. PubMed DOI PMC

Zhang J, Zha Y, Jiao Y, Li Y, Zhang S. Protective role of cezanne in doxorubicin-induced cardiotoxicity by inhibiting autophagy, apoptosis and oxidative stress. Toxicology. 2023;485:153426. doi: 10.1016/j.tox.2023.153426. PubMed DOI