Abstract
Zerumbone (ZER) is a phytochemical isolated from plants of the Zingiberaceae family. Numerous studies have demonstrated its diverse pharmacological properties, particularly its potent antitumorigenic activity. This study aimed to assess the antiproliferative effects of ZER on HT-29 cells cultivated in both two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture systems. The evaluation of growth (size), cell death, and cell cycle arrest in 3D spheroid HT-29 cells was correlated with mRNA expression data. Treatment of 2D cells revealed that ZER exhibited cytotoxicity at concentrations above 30 µM, and an IC50 of 83.54 µM (24-h post-ZER treatment) effectively suppressed cell migration. In the 3D model, ZER induced an increase in spheroid volume over a 72-h period attributed to disaggregation and reconfiguration of characteristic zones. Analysis of cell death demonstrated a significant rise in apoptotic cells after 24 h of ZER treatment, along with cell cycle arrest in the G1 phase. Furthermore, ZER treatment resulted in alterations in mRNA expression, affecting key signaling pathways involved in cell death (BCL2 and BBC3), endoplasmic reticulum stress (ERN1), DNA damage (GADD45A), cell cycle regulation (CDKN1A, NFKB1, MYC, and TP53), and autophagy (BECN1 and SQSTM1). These findings suggested that ZER holds promise as a potential candidate for the development of novel anticancer agents that can modulate crucial cell signaling pathways. Additionally, the use of the 3D culture system proved to be a valuable tool in our investigation.
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References
Abdelwahab SI, Abdul AB, Zain ZN, Hadi AH (2012) Zerumbone inhibits interleukin-6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells. Int Immunopharmacol 2(4):594–602. https://doi.org/10.1016/j.intimp.2012.01.014
Bhat P, Kriel J, Shubha PB, Basappa SNS, Loos B (2018) Modulating autophagy in cancer therapy: advancements and challenges for cancer cell death sensitization. Biochem Pharmacol 147:170–182. https://doi.org/10.1016/j.bcp.2017.11.021
Bossi G, Lapi E, Strano S, Rinaldo C, Blandino G, Sacchi A (2006) Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression. Oncogene 25(2):304–309. https://doi.org/10.1038/sj.onc.1209026
Bracci L, Fabbri A, Del Cornò M, Conti L (2021) Dietary polyphenols: promising adjuvants for colorectal cancer therapies. Cancers 13(18):4499. https://doi.org/10.3390/cancers13184499
Brüningk SC, Rivens I, Box C, Oelfke U, Ter Haar G (2020) 3D tumour spheroids for the prediction of the effects of radiation and hyperthermia treatments. Sci Rep 10(1):1653. https://doi.org/10.1038/s41598-020-58569-4
Choudhari AS, Mandave PC, Deshpande M, Ranjekar P, Prakash O (2020) Phytochemicals in cancer treatment: from preclinical studies to clinical practice. Front Pharmacol 10:1614. https://doi.org/10.3389/fphar.2019.01614
De Simone U, Roccio M, Gribaldo L, Spinillo A, Caloni F, Coccini T (2018) Human 3D cultures as models for evaluating magnetic nanoparticle CNS cytotoxicity after short- and repeated long-term exposure. Int J Mol Sci 19(7):1993. https://doi.org/10.3390/ijms19071993
Dehghan R, Bahreini F, Najafi R, Saidijam M, Amini R (2021) The combination of zerumbone and 5-FU: a significant therapeutic strategy in sensitizing colorectal cancer cells to treatment. BioMed Res Int 2021:1–18. https://doi.org/10.1155/2021/6635874
Deorukhkar A, Ahuja N, Mercado AL, Diagaradjane P, Raju U, Patel N, Mohindra P, Diep N, Guha S, Krishnan S (2015) Zerumbone increases oxidative stress in a thiol-dependent ROS-independent manner to increase DNA damage and sensitize colorectal cancer cells to radiation. Cancer Med 4(2):278–292. https://doi.org/10.1002/cam4.367
Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4(3):309–324. https://doi.org/10.1038/nprot.2008.226
Galanos P, Vougas K, Walter D, Polyzos A, Maya-Mendoza A, Haagensen EJ, Kokkalis A, Roumelioti FM, Gagos S, Tzetis M, Canovas B, Igea A, Ahuja AK, Zellweger R, Havaki S, Kanavakis E, Kletsas D, Roninson IB, Garbis SD, Lopes M, Gorgoulis VG (2016) Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing. Nat Cell Biol 18(7):777–789. https://doi.org/10.1038/ncb3378
Girisa S, Shabnam B, Monisha J, Fan L, Halim CE, Arfuso F, Ahn KS, Sethi G, Kunnumakkara AB (2019) Potential of zerumbone as an anti-cancer agent. Molecules 24(4):734. https://doi.org/10.3390/molecules24040734
Green SK, Francia G, Isidoro C, Kerbel RS (2004) Antiadhesive antibodies targeting E-cadherin sensitize multicellular tumor spheroids to chemotherapy in vitro. Mol Cancer Ther 3(2):149–159. https://doi.org/10.1158/1535-7163.149.3.2
Haque MA, Jantan I, Arshad L, Bukhari SNA (2017) Exploring the immunomodulatory and anticancer properties of zerumbone. Food Funct 8(10):3410–3431. https://doi.org/10.1039/c7fo00595d
Haque A, Brazeau D, Amin AR (2021) Perspectives on natural compounds in chemoprevention and treatment of cancer: an update with new promising compounds. Eur J Cancer 149:165–183. https://doi.org/10.1016/j.ejca.2021.03.009
Hassanzadeh P (2011) Colorectal cancer and NF-κB signaling pathway. Gastroenterol Hepatol Bed Bench 4(3):127–132
Hoffman A, Spetner LM, Burke M (2001) Cessation of cell proliferation by adjustment of cell redox potential. J Theor Biol 211(4):403–407. https://doi.org/10.1006/jtbi.2001.2356
Hsieh AL, Dang CV (2016) MYC, Metabolic synthetic lethality, and cancer. In: Cramer, T., A. Schmitt, C. (eds) Metabolism in cancer. Recent Results in Cancer Research, vol 207, 73-91. Springer, Cham. https://doi.org/10.1007/978-3-319-42118-6_4
Jensen C, Teng Y (2020) Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci 7:33. https://doi.org/10.3389/fmolb.2020.00033
Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV (2014) In vitro cell migration and invasion assays. J Visualized Exp: JoVE 88:51046. https://doi.org/10.3791/51046
Kang CG, Lee HJ, Kim SH, Lee EO (2016) Zerumbone suppresses osteopontin-induced cell invasion through inhibiting the FAK/AKT/ROCK pathway in human non-small cell lung cancer A549 cells. J Nat Prod 79(1):156–160. https://doi.org/10.1021/acs.jnatprod.5b00796
Khanna S, Chauhan A, Bhatt AN, Dwarakanath BSR (2020) Chapter 13—Multicellular tumor spheroids as in vitro models for studying tumor responses to anticancer therapies. In Animal biotechnology, 2nd ed.; Verma AS, Singh A, Eds. Academic Press: Boston, MI, USA, 2020 pp 251–268. https://doi.org/10.1016/B978-0-12-811710-1.00011-2
Kirana C, McIntosh GH, Record IR, Jones GP (2003) Antitumor activity of extract of Zingiber aromaticum and its bioactive sesquiterpenoid zerumbone. Nutr Cancer 45(2):218–225. https://doi.org/10.1207/S15327914NC4502_12
Langhans SA (2018) Three-dimensional in vitro cell culture models in drug discovery and drug repositioning. Front Pharmacol 9:6. https://doi.org/10.3389/fphar.2018.00006
Lay MM, Karsani SA, Malek SN (2014) 1-(2,6-Dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) ethanone-induced cell cycle arrest in G1/G0 in HT-29 cells human colon adenocarcinoma cells. Int J Mol Sci 15(1):468–483. https://doi.org/10.3390/ijms15010468
Liu J, Jiang G, Mao P, Zhang J, Zhang L, Liu L, Wang J, Owusu L, Ren B, Tang Y, Li W (2018) Down-regulation of GADD45A enhances chemosensitivity in melanoma. Sci Rep 8(1):4111. https://doi.org/10.1038/s41598-018-22484-6
Logue SE, McGrath EP, Cleary P, Greene S, Mnich K, Almanza A, Chevet E, Dwyer RM, Oommen A, Legembre P, Godey F, Madden EC, Leuzzi B, Obacz J, Zeng Q, Patterson JB, Jäger R, Gorman AM, Samali A (2018) Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy. Nat Commun 9(1):3267. https://doi.org/10.1038/s41467-018-05763-8
Lv D, Hu Z, Lu L, Lu H, Xu X (2017) Three-dimensional cell culture: a powerful tool in tumor research and drug discovery. Oncol Lett 14(6):6999–7010. https://doi.org/10.3892/ol.2017.7134
Lv T, Zhang W, Han X (2018) Zerumbone suppresses the potential of growth and metastasis in hepatoma HepG2 cells via the MAPK signaling pathway. Oncol Lett 15(5):7603–7610. https://doi.org/10.3892/ol.2018.8335
Miller DM, Thomas SD, Islam A, Muench D, Sedoris K (2012) c-Myc and cancer metabolism. Clin Cancer Res 18(20):5546–53. https://doi.org/10.1158/1078-0432.CCR-12-0977
Murakami A, Takahashi D, Kinoshita T, Koshimizu K, Kim HW, Yoshihiro A, Nakamura Y, Jiwajinda S, Terao J, Ohigashi H (2002) Zerumbone, a Southeast Asian ginger sesquiterpene, markedly suppresses free radical generation, proinflammatory protein production, and cancer cell proliferation accompanied by apoptosis: the α, β-unsaturated carbonyl group is a prerequisite. Carcinogenesis 23(5):795–802
Park JM, Huang S, Wu TT, Foster NR, Sinicrope FA (2013) Prognostic impact of Beclin 1, p62/sequestosome 1 and LC3 protein expression in colon carcinomas from patients receiving 5-fluorouracil as adjuvant chemotherapy. Cancer Biol Ther 14(2):100–107. https://doi.org/10.4161/cbt.22954
Park JH, Park GM, Kim JK (2015) Zerumbone, sesquiterpene photochemical from ginger, inhibits angiogenesis. Korean J Physiol Pharmacol 19(4):335–340. https://doi.org/10.4196/kjpp.2015.19.4.335
Pashirzad M, Johnston TP, Sahebkar A (2021) Therapeutic effects of polyphenols on the treatment of colorectal cancer by regulating Wnt β-catenin signaling pathway. J Oncol 2021:3619510. https://doi.org/10.1155/2021/3619510
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36. https://doi.org/10.1093/nar/30.9.e36
Piccinini F (2015) AnaSP: a software suite for automatic image analysis of multicellular spheroids. Comput Methods Programs Biomed 119(1):43–52. https://doi.org/10.1016/j.cmpb.2015.02.006
Piccinini F, Tesei A, Arienti C, Bevilacqua A (2015) Cancer multicellular spheroids: volume assessment from a single 2D projection. Comput Methods Programs Biomed 118(2):95–106. https://doi.org/10.1016/j.cmpb.2014.12.003
Prasannan R, Kalesh KA, Shanmugam MK, Nachiyappan A, Ramachandran L, Nguyen AH, Kumar AP, Lakshmanan M, Ahn KS, Sethi G (2012) Key cell signaling pathways modulated by zerumbone: role in the prevention and treatment of cancer. Biochem Pharmacol 84(10):1268–1276. https://doi.org/10.1016/j.bcp.2012.07.015
Rahman HS, Rasedee A, Yeap SK, Othman HH, Chartrand MS, Namvar F, Abdul AB, How CW (2014) Biomedical properties of a natural dietary plant metabolite, zerumbone, in cancer therapy and chemoprevention trials. BioMed Res Int 2014:920742. https://doi.org/10.1155/2014/920742
Rajedadram A, Pin KY, Ling SK, Yan SW, Looi ML (2021) Hydroxychavicol, a polyphenol from Piper betle leaf extract, induces cell cycle arrest and apoptosis in TP53-resistant HT-29 colon cancer cells. J Zhejiang Univ Sci B 22(2):112–122. https://doi.org/10.1631/jzus.B2000446
Raymundo DP, Doultsinos D, Guillory X, Carlesso A, Eriksson LA, Chevet E (2020) Pharmacological targeting of IRE1 in cancer. Trends Cancer 6(12):1018–1030. https://doi.org/10.1016/j.trecan.2020.07.006
Rejhová A, Opattová A, Čumová A, Slíva D, Vodička P (2018) Natural compounds and combination therapy in colorectal cancer treatment. Eur J Med Chem 144:582–594. https://doi.org/10.1016/j.ejmech.2017.12.039
Ren F, Shu G, Liu G, Liu D, Zhou J, Yuan L, Zhou J (2014) Knockdown of p62/sequestosome 1 attenuates autophagy and inhibits colorectal cancer cell growth. Mol Cell Biochem 385(1–2):95–102. https://doi.org/10.1007/s11010-013-1818-0
Riaz TA, Junjappa RP, Handigund M, Ferdous J, Kim HR, Chae HJ (2020) Role of endoplasmic reticulum stress sensor IRE1α in cellular physiology, calcium, ROS signaling, and metaflammation. Cells 9(5):1160. https://doi.org/10.3390/cells9051160
Sehrawat A, Arlotti JA, Murakami A, Singh SV (2012) Zerumbone causes Bax- and Bak-mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo. Breast Cancer Res Treat 136(2):429–441. https://doi.org/10.1007/s10549-012-2280-5
Seidlitz T, Stange DE (2021) Gastrointestinal cancer organoids-applications in basic and translational cancer research. Exp Mol Med 53(10):1459–1470. https://doi.org/10.1038/s12276-021-00654-3
Sheng X, Nenseth HZ, Qu S, Kuzu OF, Frahnow T, Simon L, Greene S, Zeng Q, Fazli L, Rennie PS, Mills IG, Danielsen H, Theis F, Patterson JB, Jin Y, Saatcioglu F (2019) IRE1α-XBP1s pathway promotes prostate cancer by activating c-MYC signaling. Nat Commun 10(1):323. https://doi.org/10.1038/s41467-018-08152-3
Shieh YH, Huang HM, Wang CC, Lee CC, Fan CK, Lee YL (2015) Zerumbone enhances the Th1 response and ameliorates ovalbumin-induced Th2 responses and airway inflammation in mice. Int Immunopharmacol 24(2):383–391. https://doi.org/10.1016/j.intimp.2014.12.027
Sies H, Jones DP (2020) Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21:363–383. https://doi.org/10.1038/s41580-020-0230-3
Silva TM, Pinheiro CD, Orlandi PP, Pinheiro CC, Pontes GS (2018) Zerumbone from Zingiber zerumbet (L.) Smith: a potential prophylactic and therapeutic agent against the cariogenic bacterium Streptococcus mutans. BMC Complement Altern Med 18(1):301. https://doi.org/10.1186/s12906-018-2360-0
Sithara T, Dhanya BP, Arun KB, Sini S, Dan M, Kokkuvayil Vasu R, Nisha P (2018) Zerumbone, a cyclic sesquiterpene from Zingiber zerumbet induces apoptosis, cell cycle arrest, and antimigratory effects in SW480 colorectal cancer cells. J Agric Food Chem 66(3):602–612. https://doi.org/10.1021/acs.jafc.7b04472
Stine ZE, Walton ZE, Altman BJ, Hsieh AL, Dang CV (2015) MYC, metabolism, and cancer. Cancer Disc 5(10):1024–1039. https://doi.org/10.1158/2159-8290.CD-15-0507
Suarez-Arnedo A, Torres Figueroa F, Clavijo C, Arbeláez P, Cruz JC, Muñoz-Camargo C (2020) An ImageJ plugin for the high throughput image analysis of in vitro scratch wound healing assays. PloS One 15(7):e0232565. https://doi.org/10.1371/journal.pone.0232565
Takada Y, Murakami A, Aggarwal BB (2005) Zerumbone abolishes NF-kappaB and IkappaBalpha kinase activation leading to suppression of antiapoptotic and metastatic gene expression, upregulation of apoptosis, and downregulation of invasion. Oncogene 24(46):6957–6969. https://doi.org/10.1038/sj.onc.1208845
Tretter V, Hochreiter B, Zach ML, Krenn K, Klein KU (2021) Understanding cellular redox homeostasis: a challenge for precision medicine. Int J Mol Sci 23(1):106. https://doi.org/10.3390/ijms23010106
Virgone-Carlotta A, Lemasson M, Mertani HC, Diaz JJ, Monnier S, Dehoux T, Delanoë-Ayari H, Rivière C, Rieu JP (2017) In-depth phenotypic characterization of multicellular tumor spheroids: effects of 5-fluorouracil. PloS One 12(11):e0188100. https://doi.org/10.1371/journal.pone.0188100
Wang HH, Chang TY, Lin WC, Wei KC, Shin JW (2017) GADD45A plays a protective role against temozolomide treatment in glioblastoma cells. Sci Rep 7(1):8814. https://doi.org/10.1038/s41598-017-06851-3
Wani NA, Zhang B, Teng KY, Barajas JM, Motiwala T, Hu P, Yu L, Brüschweiler R, Ghoshal K, Jacob ST (2018) Reprograming of glucose metabolism by zerumbone suppresses hepatocarcinogenesis. Mol Cancer Res 16(2):256–268. https://doi.org/10.1158/1541-7786.MCR-17-0304
Xiao BD, Zhao YJ, Jia XY, Wu J, Wang YG, Huang F (2020) Multifaceted p21 in carcinogenesis, stemness of tumor and tumor therapy. World J Stem Cells 12(6):481–487. https://doi.org/10.4252/wjsc.v12.i6.481
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The authors gratefully acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/BR), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/BR), Financiadora de Projetos (FINEP/BR), and Fundação Araucária, Brazil, for their general support through infrastructure and fellows.
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Mário Sérgio Mantovani and Sandra Regina Lepri conceived and designed the research. Nayane de Oliveira Silva conducted experiments and wrote the manuscript. Luan Vitor Alves de Lima and Liana Martins de Oliveira contributed to analytical tools. Matheus Felipe da Silva and Amanda Passuello de Aguiar provided the analyzed data. Simone Cristine Semprebon and Phelipe Oliveira Favaron were responsible for the supervision. Ingrid Felicidade contributed to writing, reviewing, and editing the manuscript. All the authors read and approved the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.
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de Oliveira Silva, N., de Lima, L.V.A., de Oliveira, L.M. et al. Cellular and molecular antiproliferative effects in 2D monolayer and 3D-cultivated HT-29 cells treated with zerumbone. Naunyn-Schmiedeberg's Arch Pharmacol 397, 1561–1573 (2024). https://doi.org/10.1007/s00210-023-02701-4
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DOI: https://doi.org/10.1007/s00210-023-02701-4