Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Picropodophyllotoxin Induces G1 Cell Cycle Arrest and Apoptosis in Human Colorectal Cancer Cells via ROS Generation and Activation of p38 MAPK Signaling Pathway

  • Lee, Seung-On (Department of Biomedicine, Health and Life Convergence Sciences, BK21 Four, Biomedical and Healthcare Research Institute, Mokpo National University) ;
  • Kwak, Ah-Won (Department of Pharmacy, College of Pharmacy, Mokpo National University) ;
  • Lee, Mee-Hyun (College of Korean Medicine, Dongshin University) ;
  • Seo, Ji-Hye (Department of Dental Pharmacology, School of Dentistry, Jeonbuk National University) ;
  • Cho, Seung-Sik (Department of Biomedicine, Health and Life Convergence Sciences, BK21 Four, Biomedical and Healthcare Research Institute, Mokpo National University) ;
  • Yoon, Goo (Department of Pharmacy, College of Pharmacy, Mokpo National University) ;
  • Chae, Jung-Il (Department of Dental Pharmacology, School of Dentistry, Jeonbuk National University) ;
  • Joo, Sang Hoon (College of Pharmacy, Daegu Catholic University) ;
  • Shim, Jung-Hyun (Department of Biomedicine, Health and Life Convergence Sciences, BK21 Four, Biomedical and Healthcare Research Institute, Mokpo National University)
  • Received : 2021.09.07
  • Accepted : 2021.09.13
  • Published : 2021.12.28
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Abstract

Picropodophyllotoxin (PPT), an epimer of podophyllotoxin, is derived from the roots of Podophyllum hexandrum and exerts various biological effects, including anti-proliferation activity. However, the effect of PPT on colorectal cancer cells and the associated cellular mechanisms have not been studied. In the present study, we explored the anticancer activity of PPT and its underlying mechanisms in HCT116 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to monitor cell viability. Flow cytometry was used to evaluate cell cycle distribution, the induction of apoptosis, the level of reactive oxygen species (ROS), assess the mitochondrial membrane potential (Δψm), and multi-caspase activity. Western blot assays were performed to detect the expression of cell cycle regulatory proteins, apoptosis-related proteins, and p38 MAPK (mitogen-activated protein kinase). We found that PPT induced apoptosis, cell cycle arrest at the G1 phase, and ROS in the HCT116 cell line. In addition, PPT enhanced the phosphorylation of p38 MAPK, which regulates apoptosis and PPT-induced apoptosis. The phosphorylation of p38 MAPK was inhibited by an antioxidant agent (N-acetyl-L-cysteine, NAC) and a p38 inhibitor (SB203580). PPT induced depolarization of the mitochondrial inner membrane and caspase-dependent apoptosis, which was attenuated by exposure to Z-VAD-FMK. Overall, these data indicate that PPT induced G1 arrest and apoptosis via ROS generation and activation of the p38 MAPK signaling pathway.

Keywords

Acknowledgement

We greatly appreciated using the Convergence Research Laboratory (established by the MNU Innovation Support Project in 2019) to conduct this research. This research was funded by the Basic Science Research Program of National Research Foundation Korea, grant number 2019R1A2C1005899.

References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 2021. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 71: 209-249. https://doi.org/10.3322/caac.21660
  2. Qian J, Fang D, Lu H, Cao Y, Zhang J, Ding R, et al. 2018. Tanshinone IIA promotes IL2-mediated SW480 colorectal cancer cell apoptosis by triggering INF2-related mitochondrial fission and activating the Mst1-Hippo pathway. Biomed. Pharmacother. 108: 1658-1669. https://doi.org/10.1016/j.biopha.2018.09.170
  3. Rijo P, Pesic M, Fernandes AS, Santos CN. 2020. Natural products: optimizing cancer treatment through modulation of redox balance. Oxid. Med. Cell Longev. 2020: 2407074.
  4. Yang SY, Sales KM, Fuller B, Seifalian AM, Winslet MC. 2009. Apoptosis and colorectal cancer: implications for therapy. Trends Mol. Med. 15: 225-233. https://doi.org/10.1016/j.molmed.2009.03.003
  5. Entezar-Almahdi E, Mohammadi-Samani S, Tayebi L, Farjadian F. 2020. Recent advances in designing 5-fluorouracil delivery systems: a stepping stone in the safe treatment of colorectal cancer. Int. J. Nanomedicine 15: 5445-5458. https://doi.org/10.2147/IJN.S257700
  6. Tian ZY, Du GJ, Xie SQ, Zhao J, Gao WY, Wang CJ. 2007. Synthesis and bioevaluation of 5-fluorouracil derivatives. Molecules 12: 2450-2457. https://doi.org/10.3390/12112450
  7. Choudhari AS, Mandave PC, Deshpande M, Ranjekar P, Prakash O. 2019. Phytochemicals in cancer treatment: from preclinical studies to clinical practice. Front. Pharmacol. 10: 1614. https://doi.org/10.3389/fphar.2019.01614
  8. Kwak AW, Yoon G, Lee MH, Cho SS, Shim JH, Chae JI. 2020. Picropodophyllotoxin, an epimer of podophyllotoxin, causes apoptosis of human esophageal squamous cell carcinoma cells through ROS-mediated JNK/P38 MAPK pathways. Int.J. Mol. Sci. 21: 4640. https://doi.org/10.3390/ijms21134640
  9. Zhao W, Cong Y, Li HM, Li S, Shen Y, Qi Q, et al. 2021. Challenges and potential for improving the druggability of podophyllotoxinderived drugs in cancer chemotherapy. Nat. Prod. Rep. 38: 470-488. https://doi.org/10.1039/D0NP00041H
  10. Wu X, Sooman L, Wickstrom M, Fryknas M, Dyrager C, Lennartsson J, et al. 2013. Alternative cytotoxic effects of the postulated IGF-IR inhibitor picropodophyllin in vitro. Mol. Cancer Ther. 12: 1526-1536. https://doi.org/10.1158/1535-7163.MCT-13-0091
  11. Schieber M, Chandel NS. 2014. ROS function in redox signaling and oxidative stress. Curr. Biol. 24: R453-462. https://doi.org/10.1016/j.cub.2014.03.034
  12. Choi BH, Kim JM, Kwak MK. 2021. The multifaceted role of NRF2 in cancer progression and cancer stem cells maintenance. Arch. Pharm. Res. 44: 263-280. https://doi.org/10.1007/s12272-021-01316-8
  13. Shi X, Zhang Y, Zheng J, Pan J. 2012. Reactive oxygen species in cancer stem cells. Antioxid. Redox Signal. 16: 1215-1228. https://doi.org/10.1089/ars.2012.4529
  14. Falone S, Lisanti MP, Domenicotti C. 2019. Oxidative stress and reprogramming of mitochondrial function and dynamics as targets to modulate cancer cell behavior and chemoresistance. Oxid. Med. Cell Longev. 2019: 4647807.
  15. Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, et al. 2020. ROS in cancer therapy: the bright side of the moon. Exp. Mol. Med. 52: 192-203. https://doi.org/10.1038/s12276-020-0384-2
  16. Cargnello M, Roux PP. 2011. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol. Mol. Biol. Rev. 75: 50-83. https://doi.org/10.1128/MMBR.00031-10
  17. Fulda S, Debatin KM. 2006. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25: 4798-4811. https://doi.org/10.1038/sj.onc.1209608
  18. Kaufmann T, Strasser A, Jost PJ. 2012. Fas death receptor signalling: roles of Bid and XIAP. Cell Death Differ. 19: 42-50. https://doi.org/10.1038/cdd.2011.121
  19. Parrish AB, Freel CD, Kornbluth S. 2013. Cellular mechanisms controlling caspase activation and function. Cold Spring Harb. Perspect. Biol. 5: a008672.
  20. De Chiara G, Marcocci ME, Torcia M, Lucibello M, Rosini P, Bonini P, et al. 2006. Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences. J. Biol. Chem. 281: 21353-21361. https://doi.org/10.1074/jbc.M511052200
  21. Ding Q, Xie XL, Wang MM, Yin J, Tian JM, Jiang XY, et al. 2019. The role of the apoptosis-related protein BCL-B in the regulation of mitophagy in hepatic stellate cells during the regression of liver fibrosis. Exp. Mol. Med. 51: 1-13.
  22. Keum N, Giovannucci E. 2019. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat. Rev. Gastroenterol. Hepatol. 16: 713-732. https://doi.org/10.1038/s41575-019-0189-8
  23. Lopez J, Tait SW. 2015. Mitochondrial apoptosis: killing cancer using the enemy within. Br. J. Cancer 112: 957-962. https://doi.org/10.1038/bjc.2015.85
  24. Hassan M, Watari H, AbuAlmaaty A, Ohba Y, Sakuragi N. 2014. Apoptosis and molecular targeting therapy in cancer. Biomed Res. Int. 2014: 150845. https://doi.org/10.1155/2014/150845
  25. Villanueva J, Yung Y, Walker JL, Assoian RK. 2007. ERK activity and G1 phase progression: identifying dispensable versus essential activities and primary versus secondary targets. Mol. Biol. Cell 18: 1457-1463. https://doi.org/10.1091/mbc.E06-10-0908
  26. Narasimha AM, Kaulich M, Shapiro GS, Choi YJ, Sicinski P, Dowdy SF. 2014. Cyclin D activates the Rb tumor suppressor by mono-phosphorylation. Elife 3: e02872. https://doi.org/10.7554/elife.02872
  27. Cheng X, Feng H, Wu H, Jin Z, Shen X, Kuang J, et al. 2018. Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer. Cancer Lett. 431: 105-114. https://doi.org/10.1016/j.canlet.2018.05.046
  28. Ling YH, Liebes L, Zou Y, Perez-Soler R. 2003. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells. J. Biol. Chem. 278: 33714-33723. https://doi.org/10.1074/jbc.M302559200
  29. Richa S, Dey P, Park C, Yang J, Son JY, Park JH, et al. 2020. A New histone deacetylase inhibitor, MHY4381, induces apoptosis via generation of reactive oxygen species in human prostate cancer cells. Biomol. Ther. (Seoul) 28: 184-194. https://doi.org/10.4062/biomolther.2019.074
  30. Adams CJ, Kopp MC, Larburu N, Nowak PR, Ali MMU. 2019. Structure and molecular mechanism of ER stress signaling by the unfolded protein response signal activator IRE1. Front. Mol. Biosci. 6: 11. https://doi.org/10.3389/fmolb.2019.00011
  31. Lindner P, Christensen SB, Nissen P, Moller JV, Engedal N. 2020. Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B, and distinct contributions from unfolded protein response components. Cell Commun. Signal. 18: 12. https://doi.org/10.1186/s12964-019-0499-z
  32. Lee AS. 2005. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35: 373-381. https://doi.org/10.1016/j.ymeth.2004.10.010
  33. Pfaffenbach KT, Gentile CL, Nivala AM, Wang D, Wei Y, Pagliassotti MJ. 2010. Linking endoplasmic reticulum stress to cell death in hepatocytes: roles of C/EBP homologous protein and chemical chaperones in palmitate-mediated cell death. Am. J. Physiol. Endocrinol. Metab. 298: E1027-1035. https://doi.org/10.1152/ajpendo.00642.2009
  34. Hu H, Tian M, Ding C, Yu S. 2018. The C/EBP homologous protein (CHOP) transcription factor functions in endoplasmic reticulum stress-induced apoptosis and microbial infection. Front. Immunol. 9: 3083. https://doi.org/10.3389/fimmu.2018.03083
  35. Darling NJ, Cook SJ. 2014. The role of MAPK signalling pathways in the response to endoplasmic reticulum stress. Biochim. Biophys. Acta 1843: 2150-2163. https://doi.org/10.1016/j.bbamcr.2014.01.009
  36. Wada T, Penninger JM. 2004. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23: 2838-2849. https://doi.org/10.1038/sj.onc.1207556
  37. Ko YH, Kim SK, Kwon SH, Seo JY, Lee BR, Kim YJ, et al. 2019. 7,8,4'-Trihydroxyisoflavone, a metabolized product of daidzein, attenuates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells. Biomol. Ther. (Seoul) 27: 363-372. https://doi.org/10.4062/biomolther.2018.211
  38. Kulisz A, Chen N, Chandel NS, Shao Z, Schumacker PT. 2002. Mitochondrial ROS initiate phosphorylation of p38 MAP kinase during hypoxia in cardiomyocytes. Am. J. Physiol. Lung Cell Mol. Physiol. 282: L1324-1329.
  39. Zorov DB, Juhaszova M, Sollott SJ. 2014. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol. Rev. 94: 909-950. https://doi.org/10.1152/physrev.00026.2013
  40. Levine B, Sinha S, Kroemer G. 2008. Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy 4: 600-606. https://doi.org/10.4161/auto.6260
  41. Lee YJ, Kim WI, Kim SY, Cho SW, Nam HS, Lee SH, et al. 2019. Flavonoid morin inhibits proliferation and induces apoptosis of melanoma cells by regulating reactive oxygen species, Sp1 and Mcl-1. Arch. Pharm. Res. 42: 531-542. https://doi.org/10.1007/s12272-019-01158-5
  42. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479-489. https://doi.org/10.1016/S0092-8674(00)80434-1
  43. Shi Y. 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell 9: 459-470. https://doi.org/10.1016/S1097-2765(02)00482-3