Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
권호 표지
DOI QR코드

DOI QR Code

Doxorubicin Attenuates Free Fatty Acid-Induced Lipid Accumulation via Stimulation of p53 in HepG2 Cells

  • Chawon Yun (Department of Pharmacy, College of Pharmacy, Research Institute for Drug Development, Pusan National University) ;
  • Sou Hyun Kim (Department of Pharmacy, College of Pharmacy, Research Institute for Drug Development, Pusan National University) ;
  • Doyoung Kwon (College of Pharmacy, Jeju Research Institute of Pharmaceutical Sciences, Jeju National University) ;
  • Mi Ran Byun (College of Pharmacy, Daegu Catholic University) ;
  • Ki Wung Chung (Department of Pharmacy, College of Pharmacy, Research Institute for Drug Development, Pusan National University) ;
  • Jaewon Lee (Department of Pharmacy, College of Pharmacy, Research Institute for Drug Development, Pusan National University) ;
  • Young-Suk Jung (Department of Pharmacy, College of Pharmacy, Research Institute for Drug Development, Pusan National University)
  • Received : 2023.11.09
  • Accepted : 2023.11.14
  • Published : 2024.01.01
Download PDF
⟨ Previous Next ⟩

Abstract

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of fat in the liver, and there is a global increase in its incidence owing to changes in lifestyle and diet. Recent findings suggest that p53 is involved in the development of non-alcoholic fatty liver disease; however, the association between p53 expression and the disease remains unclear. Doxorubicin, an anticancer agent, increases the expression of p53. Therefore, this study aimed to investigate the role of doxorubicin-induced p53 upregulation in free fatty acid (FFA)-induced intracellular lipid accumulation. HepG2 cells were pretreated with 0.5 ㎍/mL of doxorubicin for 12 h, followed by treatment with FFA (0.5 mM) for 24 h to induce steatosis. Doxorubicin pretreatment upregulated p53 expression and downregulated the expression of endoplasmic reticulum stress- and lipid synthesis-associated genes in the FFA -treated HepG2 cells. Additionally, doxorubicin treatment upregulated the expression of AMP-activated protein kinase, a key modulator of lipid metabolism. Notably, siRNA-targeted p53 knockdown reversed the effects of doxorubicin in HepG2 cells. Moreover, doxorubicin treatment suppressed FFA -induced lipid accumulation in HepG2 spheroids. Conclusively, these results suggest that doxorubicin possesses potential application for the regulation of lipid metabolism by enhance the expression of p53 an in vitro NAFLD model.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (NRF-2019R1I1A3A01058584) and the Commercializations Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korea government (MSIT) (No. 2021N400).

References

  1. Adamovich, Y., Adler, J., Meltser, V., Reuven, N. and Shaul, Y. (2014) AMPK couples p73 with p53 in cell fate decision. Cell Death Differ. 21, 1451-1459.  https://doi.org/10.1038/cdd.2014.60
  2. Almanza, A., Carlesso, A., Chintha, C., Creedican, S., Doultsinos, D., Leuzzi, B., Luis, A., McCarthy, N., Montibeller, L., More, S., Papaioannou, A., Puschel, F., Sassano, M. L., Skoko, J., Agostinis, P., de Belleroche, J., Eriksson, L. A., Fulda, S., Gorman, A. M., Healy, S., Kozlov, A., Munoz-Pinedo, C., Rehm, M., Chevet, E. and Samali, A. (2019) Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications. FEBS J. 286, 241-278.  https://doi.org/10.1111/febs.14608
  3. Asensio-Lopez, M. C., Soler, F., Pascual-Figal, D., Fernandez-Belda, F. and Lax, A. (2017) Doxorubicin-induced oxidative stress: the protective effect of nicorandil on HL-1 cardiomyocytes. PLoS One 12, e0172803.  https://doi.org/10.1371/journal.pone.0172803
  4. Bravo, R., Parra, V., Gatica, D., Rodriguez, A. E., Torrealba, N., Paredes, F., Wang, Z. V., Zorzano, A., Hill, J. A., Jaimovich, E., Quest, A. F. and Lavandero, S. (2013) Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration. Int. Rev. Cell Mol. Biol. 301, 215-290.  https://doi.org/10.1016/B978-0-12-407704-1.00005-1
  5. Cappetta, D., De Angelis, A., Sapio, L., Prezioso, L., Illiano, M., Quaini, F., Rossi, F., Berrino, L., Naviglio, S. and Urbanek, K. (2017) Oxidative stress and cellular response to doxorubicin: a common factor in the complex milieu of anthracycline cardiotoxicity. Oxid. Med. Cell. Longev. 2017, 1521020.  https://doi.org/10.1155/2017/1521020
  6. Carrara, M., Prischi, F., Nowak, P. R., Kopp, M. C. and Ali, M. M. (2015) Noncanonical binding of BiP ATPase domain to Ire1 and Perk is dissociated by unfolded protein CH1 to initiate ER stress signaling. Elife 4, e03522.  https://doi.org/10.7554/eLife.03522
  7. Chen, L. L. and Wang, W. J. (2021) p53 regulates lipid metabolism in cancer. Int. J. Biol. Macromol. 192, 45-54.  https://doi.org/10.1016/j.ijbiomac.2021.09.188
  8. Dauer, P., Sharma, N. S., Gupta, V. K., Durden, B., Hadad, R., Banerjee, S., Dudeja, V., Saluja, A. and Banerjee, S. (2019) ER stress sensor, glucose regulatory protein 78 (GRP78) regulates redox status in pancreatic cancer thereby maintaining "stemness". Cell Death Dis. 10, 132.  https://doi.org/10.1038/s41419-019-1408-5
  9. Derdak, Z., Villegas, K. A., Harb, R., Wu, A. M., Sousa, A. and Wands, J. R. (2013) Inhibition of p53 attenuates steatosis and liver injury in a mouse model of non-alcoholic fatty liver disease. J. Hepatol. 58, 785-791.  https://doi.org/10.1016/j.jhep.2012.11.042
  10. Estes, C., Razavi, H., Loomba, R., Younossi, Z. and Sanyal, A. J. (2018) Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease. Hepatology 67, 123-133.  https://doi.org/10.1002/hep.29466
  11. Flowers, M. T. and Ntambi, J. M. (2008) Role of stearoyl-coenzyme A desaturase in regulating lipid metabolism. Curr. Opin. Lipidol. 19, 248-256.  https://doi.org/10.1097/MOL.0b013e3282f9b54d
  12. Galic, S., Loh, K., Murray-Segal, L., Steinberg, G. R., Andrews, Z. B. and Kemp, B. E. (2018) AMPK signaling to acetyl-CoA carboxylase is required for fasting- and cold-induced appetite but not thermogenesis. Elife 7, e32656.  https://doi.org/10.7554/eLife.32656
  13. Ge, R. and Kao, C. (2019) Cell surface GRP78 as a death receptor and an anticancer drug target. Cancers (Basel) 11, 1787.  https://doi.org/10.3390/cancers11111787
  14. Goldstein, I., Ezra, O., Rivlin, N., Molchadsky, A., Madar, S., Goldfinger, N. and Rotter, V. (2012) p53, a novel regulator of lipid metabolism pathways. J. Hepatol. 56, 656-662.  https://doi.org/10.1016/j.jhep.2011.08.022
  15. Han, J. and Kaufman, R. J. (2016) The role of ER stress in lipid metabolism and lipotoxicity. J. Lipid Res. 57, 1329-1338.  https://doi.org/10.1194/jlr.R067595
  16. Herzig, S. and Shaw, R. J. (2018) AMPK: guardian of metabolism and mitochondrial homeostasis. Nat. Rev. Mol. Cell Biol. 19, 121-135.  https://doi.org/10.1038/nrm.2017.95
  17. Huh, Y., Cho, Y. J. and Nam, G. E. (2022) Recent epidemiology and risk factors of nonalcoholic fatty liver disease. J. Obes. Metab. Syndr. 31, 17-27.  https://doi.org/10.7570/jomes22021
  18. Jensen-Urstad, A. P. and Semenkovich, C. F. (2012) Fatty acid synthase and liver triglyceride metabolism: housekeeper or messenger? Biochim. Biophys. Acta 1821, 747-753.  https://doi.org/10.1016/j.bbalip.2011.09.017
  19. Jiang, P., Du, W. and Yang, X. (2013) p53 and regulation of tumor metabolism. J. Carcinog. 12, 21.  https://doi.org/10.4103/1477-3163.122760
  20. Kciuk, M., Gielecinska, A., Mujwar, S., Kolat, D., Kaluzinska-Kolat, Z., Celik, I. and Kontek, R. (2023) Doxorubicin-an agent with multiple mechanisms of anticancer activity. Cells 12, 659.  https://doi.org/10.3390/cells12040659
  21. Kim, S. H., Yun, C., Kwon, D., Lee, Y. H., Kwak, J. H. and Jung, Y. S. (2023) Effect of isoquercitrin on free fatty acid-induced lipid accumulation in HepG2 cells. Molecules 28, 1476.  https://doi.org/10.3390/molecules28031476
  22. Konar, S., Hedges, C. P., Callon, K. E., Bolam, S., Leung, S., Cornish, J., Naot, D. and Musson, D. S. (2023) Palmitic acid reduces viability and increases production of reactive oxygen species and respiration in rat tendon-derived cells. bioRxiv doi: 10.1101/2023.02.08.527761 [Preprint]. 
  23. Krstic, J., Galhuber, M., Schulz, T. J., Schupp, M. and Prokesch, A. (2018) p53 as a dichotomous regulator of liver disease: the dose makes the medicine. Int. J. Mol. Sci. 19, 921.  https://doi.org/10.3390/ijms19030921
  24. Kruse, J. P. and Gu, W. (2009) Modes of p53 regulation. Cell 137, 609-622.  https://doi.org/10.1016/j.cell.2009.04.050
  25. Lacroix, M., Linares, L. K., Rueda-Rincon, N., Bloch, K., Di Michele, M., De Blasio, C., Fau, C., Gayte, L., Blanchet, E., Mairal, A., Derua, R., Cardona, F., Beuzelin, D., Annicotte, J. S., Pirot, N., Torro, A., Tinahones, F. J., Bernex, F., Bertrand-Michel, J., Langin, D., Fajas, L., Swinnen, J. and Le Cam, L. (2021) The multifunctional protein E4F1 links P53 to lipid metabolism in adipocytes. Nat. Commun. 12, 7037.  https://doi.org/10.1038/s41467-021-27307-3
  26. Li, Y., Xu, S., Mihaylova, M. M., Zheng, B., Hou, X., Jiang, B., Park, O., Luo, Z., Lefai, E., Shyy, J. Y., Gao, B., Wierzbicki, M., Verbeuren, T. J., Shaw, R. J., Cohen, R. A. and Zang, M. (2011) AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice. Cell Metab. 13, 376-388.  https://doi.org/10.1016/j.cmet.2011.03.009
  27. Li, Y., Zhang, Y., Li, R., Chen, W., Howell, M., Zhang, R. and Chen, G. (2012) The hepatic Raldh1 expression is elevated in Zucker fatty rats and its over-expression introduced the retinal-induced Srebp1c expression in INS-1 cells. PLoS One 7, e45210.  https://doi.org/10.1371/journal.pone.0045210
  28. Liu, Y. and Gu, W. (2022) The complexity of p53-mediated metabolic regulation in tumor suppression. Semin. Cancer Biol. 85, 4-32.  https://doi.org/10.1016/j.semcancer.2021.03.010
  29. Livak, K. J. and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408.  https://doi.org/10.1006/meth.2001.1262
  30. Ma, Y., Lee, G., Heo, S. Y. and Roh, Y. S. (2021) Oxidative stress is a key modulator in the development of nonalcoholic fatty liver disease. Antioxidants (Basel) 11, 91.  https://doi.org/10.3390/antiox11010091
  31. Matsui, H., Yokoyama, T., Sekiguchi, K., Iijima, D., Sunaga, H., Maniwa, M., Ueno, M., Iso, T., Arai, M. and Kurabayashi, M. (2012) Stearoyl-CoA desaturase-1 (SCD1) augments saturated fatty acidinduced lipid accumulation and inhibits apoptosis in cardiac myocytes. PLoS One 7, e33283.  https://doi.org/10.1371/journal.pone.0033283
  32. McGlinchey, A. J., Govaere, O., Geng, D., Ratziu, V., Allison, M., Bousier, J., Petta, S., de Oliviera, C., Bugianesi, E., Schattenberg, J. M., Daly, A. K., Hyotylainen, T., Anstee, Q. M. and Oresic, M. (2022) Metabolic signatures across the full spectrum of non-alcoholic fatty liver disease. JHEP Rep. 4, 100477.  https://doi.org/10.1016/j.jhepr.2022.100477
  33. Moulder, D. E., Hatoum, D., Tay, E., Lin, Y. and McGowan, E. M. (2018) The roles of p53 in mitochondrial dynamics and cancer metabolism: the pendulum between survival and death in breast cancer? Cancers (Basel) 10, 189.  https://doi.org/10.3390/cancers10060189
  34. Namba, T., Chu, K., Kodama, R., Byun, S., Yoon, K. W., Hiraki, M., Mandinova, A. and Lee, S. W. (2015) Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway. Oncotarget 6, 19990-20001.  https://doi.org/10.18632/oncotarget.4598
  35. Panasiuk, A., Dzieciol, J., Panasiuk, B. and Prokopowicz, D. (2006) Expression of p53, Bax and Bcl-2 proteins in hepatocytes in non-alcoholic fatty liver disease. World J. Gastroenterol. 12, 6198-6202.  https://doi.org/10.3748/wjg.v12.i38.6198
  36. Pawlak, M., Lefebvre, P. and Staels, B. (2015) Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver disease. J. Hepatol. 62, 720-733.  https://doi.org/10.1016/j.jhep.2014.10.039
  37. Porteiro, B., Fondevila, M. F., Buque, X., Gonzalez-Rellan, M. J., Fernandez, U., Mora, A., Beiroa, D., Senra, A., Gallego, R., Ferno, J., Lopez, M., Sabio, G., Dieguez, C., Aspichueta, P. and Nogueiras, R. (2018) Pharmacological stimulation of p53 with low-dose doxorubicin ameliorates diet-induced nonalcoholic steatosis and steatohepatitis. Mol. Metab. 8, 132-143.  https://doi.org/10.1016/j.molmet.2017.12.005
  38. Primeau, A. J., Rendon, A., Hedley, D., Lilge, L. and Tannock, I. F. (2005) The distribution of the anticancer drug Doxorubicin in relation to blood vessels in solid tumors. Clin. Cancer Res. 11, 8782-8788.  https://doi.org/10.1158/1078-0432.CCR-05-1664
  39. Prokesch, A., Graef, F. A., Madl, T., Kahlhofer, J., Heidenreich, S., Schumann, A., Moyschewitz, E., Pristoynik, P., Blaschitz, A., Knauer, M., Muenzner, M., Bogner-Strauss, J. G., Dohr, G., Schulz, T. J. and Schupp, M. (2017) Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis. FASEB J. 31, 732-742.  https://doi.org/10.1096/fj.201600845R
  40. Qu, L., Huang, S., Baltzis, D., Rivas-Estilla, A. M., Pluquet, O., Hatzoglou, M., Koumenis, C., Taya, Y., Yoshimura, A. and Koromilas, A. E. (2004) Endoplasmic reticulum stress induces p53 cytoplasmic localization and prevents p53-dependent apoptosis by a pathway involving glycogen synthase kinase-3beta. Genes Dev. 18, 261-277.  https://doi.org/10.1101/gad.1165804
  41. Schonfeld, P. and Wojtczak, L. (2008) Fatty acids as modulators of the cellular production of reactive oxygen species. Free Radic. Biol. Med. 45, 231-241.  https://doi.org/10.1016/j.freeradbiomed.2008.04.029
  42. Sozio, M. S., Lu, C., Zeng, Y., Liangpunsakul, S. and Crabb, D. W. (2011) Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells. Am. J. Physiol. Gastrointest. Liver Physiol. 301, G739-G747.  https://doi.org/10.1152/ajpgi.00432.2010
  43. Tang, C. H., Ranatunga, S., Kriss, C. L., Cubitt, C. L., Tao, J., PinillaIbarz, J. A., Del Valle, J. R. and Hu, C. C. (2014) Inhibition of ER stress-associated IRE-1/XBP-1 pathway reduces leukemic cell survival. J. Clin. Invest. 124, 2585-2598.  https://doi.org/10.1172/JCI73448
  44. Tsang, W. P., Chau, S. P., Kong, S. K., Fung, K. P. and Kwok, T. T. (2003) Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis. Life Sci. 73, 2047-2058.  https://doi.org/10.1016/S0024-3205(03)00566-6
  45. Tsuru, A., Imai, Y., Saito, M. and Kohno, K. (2016) Novel mechanism of enhancing IRE1α-XBP1 signalling via the PERK-ATF4 pathway. Sci. Rep. 6, 24217.  https://doi.org/10.1038/srep24217
  46. Vousden, K. H. and Ryan, K. M. (2009) p53 and metabolism. Nat. Rev. Cancer 9, 691-700.  https://doi.org/10.1038/nrc2715
  47. Wang, M. and Kaufman, R. J. (2014) The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat. Rev. Cancer 14, 581-597.  https://doi.org/10.1038/nrc3800
  48. Wen, S. Y., Ali, A., Huang, I. C., Liu, J. S., Chen, P. Y., Padma Viswanadha, V., Huang, C. Y. and Kuo, W. W. (2023) Doxorubicin induced ROS-dependent HIF1α activation mediates blockage of IGF1R survival signaling by IGFBP3 promotes cardiac apoptosis. Aging (Albany N.Y.) 15, 164-178.  https://doi.org/10.18632/aging.204466
  49. Winder, W. W. and Hardie, D. G. (1996) Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am. J. Physiol. 270, E299-E304.  https://doi.org/10.1152/ajpendo.1996.270.2.E299
  50. Yahagi, N., Shimano, H., Matsuzaka, T., Najima, Y., Sekiya, M., Nakagawa, Y., Ide, T., Tomita, S., Okazaki, H., Tamura, Y., Iizuka, Y., Ohashi, K., Gotoda, T., Nagai, R., Kimura, S., Ishibashi, S., Osuga, J. and Yamada, N. (2003) p53 Activation in adipocytes of obese mice. J. Biol. Chem. 278, 25395-25400.  https://doi.org/10.1074/jbc.M302364200
  51. Yahagi, N., Shimano, H., Matsuzaka, T., Sekiya, M., Najima, Y., Okazaki, S., Okazaki, H., Tamura, Y., Iizuka, Y., Inoue, N., Nakagawa, Y., Takeuchi, Y., Ohashi, K., Harada, K., Gotoda, T., Nagai, R., Kadowaki, T., Ishibashi, S., Osuga, J. I. and Yamada, N. (2004) p53 involvement in the pathogenesis of fatty liver disease. J. Biol. Chem. 279, 20571-20575.  https://doi.org/10.1074/jbc.M400884200
  52. Yang, W., Zhao, J., Zhao, Y., Li, W., Zhao, L., Ren, Y., Ou, R. and Xu, Y. (2020) Hsa_circ_0048179 attenuates free fatty acid-induced steatosis via hsa_circ_0048179/miR-188-3p/GPX4 signaling. Aging (Albany N.Y.) 12, 23996-24008.  https://doi.org/10.18632/aging.104081
  53. Yoshihara, T., Maruyama, R., Shiozaki, S., Yamamoto, K., Kato, S. I., Nakamura, Y. and Tobita, S. (2020) Visualization of lipid droplets in living cells and fatty livers of mice based on the fluorescence of π-extended coumarin using fluorescence lifetime imaging microscopy. Anal. Chem. 92, 4996-5003.  https://doi.org/10.1021/acs.analchem.9b05184
  54. Yun, C., Lee, J. H., Park, H., Jin, Y. M., Park, S., Park, K. and Cho, H. (2000) Chemotherapeutic drug, adriamycin, restores the function of p53 protein in hepatitis B virus X (HBx) protein-expressing liver cells. Oncogene 19, 5163-5172.  https://doi.org/10.1038/sj.onc.1203896
  55. Zhang, X., Lin, Y., Lin, S., Li, C., Gao, J., Feng, Z., Wang, J., Zhang, J., Zhang, H., Zhang, Y., Chen, X., Chen, S., Xu, C., Li, Y., Yu, C. and Zeng, H. (2020) Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction. Hepatol. Int. 14, 828-841.  https://doi.org/10.1007/s12072-020-10068-4
  56. Zhao, J., Zhou, X., Chen, B., Lu, M., Wang, G., Elumalai, N., Tian, C., Zhang, J., Liu, Y., Chen, Z., Zhou, X., Wu, M., Li, M., Prochownik, E. V., Tavassoli, A., Jiang, C. and Li, Y. (2023) p53 promotes peroxisomal fatty acid β-oxidation to repress purine biosynthesis and mediate tumor suppression. Cell Death Dis. 14, 87.  https://doi.org/10.1038/s41419-023-05625-2