The effect of Crocetin on cholesterol depletion-mediated lipid raft disruption-induced apoptosis in breast cancer cells

Year 2025, Volume: 12 Issue: 2, 407 - 419

Serife Buket Bozkurt Polat , Esma Özmen

Abstract

The purpose of this study was to determine the effect of lipid raft function loss due to depletion of cholesterol in the lipid raft structure of cell membrane by crocetin and Methyl β cyclodextrin (MβCD) on cell viability and lipid raft-associated gene and apoptotic gene expressions of breast cancer cell (MCF-7). For this purpose, MCF-7 cells were treated with different concentrations of MβCD and crocetin. Cell viability was evaluated by WST-1 at 24 and 48 hours. The mRNA expressions of caveolin 1, LRP 6, survivin, Bcl2, Bax, and Caspase3 were assessed in the MβCD-treated group; crocetin-treated group; mixed-treated group MβCD+ crocetin MCF-7 cells by reverse transcription polymerase chain reaction at 24 h exposure. Cell viability indicated that all concentrations of MβCD decreased the viability of MCF-7 cells compared with control; reduction in cell viability was greatest with 1 mM. Additionally, exposure to all crocetin concentrations significantly reduced the cell viability of MCF-7 in a time-dependent manner. There was statistically significant down-regulation of caveolin 1, LRP-6, survivin, Bcl2 in response to MβCD, and crocetin at 24 h but Bax ve caspase 3 expressions were increased compared to control at 24h. These results indicated that crocetin application to MCF-7 in addition to MβCD regulated mRNA expression of lipid raft-associated genes and apoptotic genes. These findings suggest that crocetin affects MCF-7 function via cholesterol depletion-related deterioration in the lipid raft structure, which is critical for the induction of apoptosis in MCF-7 cells.

Keywords

Crocetin, Lipid raft, Methyl β cyclodextrin, MCF-7, Apoptosis

Ethical Statement

The study was approved by the Ethics Committee of the Faculty of Medicine (2021/112) of Niğde Ömer Halisdemir University.

References

• Badana, A.K., Chintala, M., Gavara, M.M., Naik, S., Kumari, S., Kappala, V.R., Iska, B.R., & Malla, R.R. (2018). Lipid rafts disruption induces apoptosis by attenuating expression of LRP6 and survivin in triple negative breast cancer. Biomedicine & Pharmacotherapy, 97, 359–368. https://doi.org/10.1016/j.biopha.2017.10.045
• Bathaie, S., Farajzade, A., & Hoshyar, R. (2014). A review of the chemistry and uses of crocins and crocetin, the carotenoid natural dyes in saffron, with particular emphasis on applications as colorants including their use as biological stains. Biotechnic & Histochemistry, 89(6), 401–411. https://doi.org/10.3109/10520295.2014.890741
• Bolhassani, A., Khavari, A., & Bathaie, S.Z. (2014). Saffron and natural carotenoids: Biochemical activities and anti-tumor effects. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 1845(1), 20–30. https://doi.org/10.1016/j.bbcan.2013.11.001
• Burger, K., Gimpl, G., & Fahrenholz, F. (2000). Regulation of receptor function by cholesterol. Cell Mol Life Sci CMLS, 57,1577–1592. https://doi.org/10.1007/pl00000643
• Chen, X., Duan, N., Zhang, C., & Zhang, W. (2016). Survivin and tumorigenesis: molecular mechanisms and therapeutic strategies. Journal of Cancer, 7(3), 314. https://doi.org/10.7150/jca.13332
• Chryssanthi, D.G., Lamari, F.N., Iatrou, G., Pylara, A., Karamanos, N.K., Cordopatis, P. (2007). Inhibition of breast cancer cell proliferation by style constituents of different Crocus species. Anticancer Research, 27(1A), 357–362.
• Elsheikh, S.E., Green, A.R., Rakha, E.A., Samaka, R.M., Ammar, A.A., Powe, D., Reis-Filho, J.S., & Ellis, I.O. (2008). Caveolin 1 and Caveolin 2 are associated with breast cancer basal-like and triple-negative immunophenotype. British Journal of Cancer, 99(2), 327–334. https://doi.org/10.1038/sj.bjc.6604463
• Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R., & Mathieu, C. (2001). An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods, 25(4), 386–401. https://doi.org/10.1006/meth.2001.1261
• Gutheil, G.W., Reed, G., Ray, A., Anant, S., & Dhar, A. (2012). Crocetin: an agent derived from saffron for prevention and therapy for cancer. Current Pharmaceutical Biotechnology, 13(1), 173–179. https://doi.org/10.2174/138920112798868566
• Hanahan, D., & Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell, 144(5), 646–674. https://doi.org/10.1016/j.cell.2011.02.013
• Hirsch, H.A., Iliopoulos, D., Joshi, A., Zhang, Y., Jaeger, S.A., Bulyk, M., Tsichlis, P.N., Liu, X.S., & Struhl, K. (2010). A transcriptional signature and common gene networks link cancer with lipid metabolism and diverse human diseases. Cancer Cell, 17(4), 348–361. https://doi.org/10.1016/j.ccr.2010.01.022
• Li, Y., Shan, F., & Chen, J. (2017). Lipid raft-mediated miR-3908 inhibition of migration of breast cancer cell line MCF-7 by regulating the interactions between AdipoR1 and Flotillin-1. World Journal of Surgical Oncology, 15(1), 69. https://doi.org/10.1186/s12957-017-1120-9
• Li, S., Shen, X.Y., Ouyang, T., Qu, Y., Luo, T., & Wang, H.Q. (2017). Synergistic anticancer effect of combined crocetin and cisplatin on KYSE-150 cells via p53/p21 pathway. Cancer Cell International, 17(1), 98. https://doi.org/10.1186/s12935-017-0468-9
• Livak, K.J., & Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2- ΔΔCT method. Methods, 25(4), 402 408. https://doi.org/10.1006/meth.2001.1262
• Luo, X., Cheng, C., Tan, Z., Li, N., Tang, M., Yang, L., & Cao, Y. (2017). Emerging roles of lipid metabolism in cancer metastasis. Molecular Cancer, 16(1), 76. https://doi.org/10.1186/s12943-017-0646-3
• Maja, M., Mohammed, D., Dumitru, A.C., Verstraeten, S., Lingurski, M., Mingeot-Leclercq, M-P., Alsteens, D., & Tyteca, D. (2022). Surface cholesterol-enriched domains specifically promote invasion of breast cancer cell lines by controlling invadopodia and extracellular matrix degradation. Cell Mol Life Sci, 79(8), 417. https://doi.org/10.1007/s00018-022-04426-8
• Mathupala, S.P., Ko, Y.H., & Pedersen, P.L. (2006). Hexokinase II: cancer’s double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria. Oncogene, 25(34), 4777–4786. https://doi.org/10.1038/sj.onc.1209603
• Mir, M. A., Ganai, S. A., Mansoor, S., Jan, S., Mani, P., Masoodi, K. Z., Amin, H., Rehman, M.U., Ahmad, P. (2020). Isolation, purification and characterization of naturally derived Crocetin beta-d-glucosyl ester from Crocus sativus L. against breast cancer and its binding chemistry with ER-alpha/HDAC2. Saudi Journal of Biological Sciences, 27(3), 975-984. https://doi.org/10.1016/j.sjbs.2020.01.018
• Murai, T. (2015). Cholesterol lowering: role in cancer prevention and treatment. Biological Chemistry, 396(1), 1–11. https://doi.org/10.1515/hsz-2014-0194
• Naseri, M.H., Mahdavi, M., Davoodi, J., Tackallou, S.H., Goudarzvand, M., & Neishabouri, S.H. (2015). Up regulation of Bax and down regulation of Bcl2 during 3-NC mediated apoptosis in human cancer cells. Cancer Cell International, 15(1), 55. https://doi.org/10.1186/s12935-015-0204-2
• Onodera, R., Motoyama, K., Okamatsu, A., Higashi, T., Kariya, R., Okada, S., & Arima, H. (2013). Involvement of cholesterol depletion from lipid rafts in apoptosis induced by methyl-β-cyclodextrin. International Journal of Pharmaceutics, 452(1–2), 116 123. https://doi.org/10.1016/j.ijpharm.2013.04.071
• Patel, S., Sarwat, M., & Khan, T.H. (2017). Mechanism behind the anti-tumour potential of saffron (Crocus sativus L.): The molecular perspective. Critical Reviews in Oncology/Hematology, 115, 27–35. https://doi.org/10.1016/j.critrevonc.2017.04.010
• Pfaffl, M.W. (2001). A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Research, 29(9), e45–e45. https://doi.org/10.1093/nar/29.9.e45
• Pike, L.J. (2006). Rafts defined: A report on the keystone symposium on lipid rafts and cell function. Journal of Lipid Research, 47(7), 1597–1598. https://doi.org/10.1194/jlr.e600002-jlr200
• Raghu, H., Sodadasu, P.K., Malla, R.R., Gondi, C.S., Estes, N., & Rao, J.S. (2010). Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells. BMC Cancer, 10(1), 647. https://doi.org/10.1186/1471-2407-10-647
• Sarı, C., Kolaylı, S., & Eyüpoğlu, F. C. (2021). A comparative study of MTT and WST-1 assays in cytotoxicity analysis. Haydarpaşa Numune Medical Journal, 61(3), 281. http://dx.doi.org/10.14744/hnhj.2019.16443
• Shah, A.D., Inder, K.L., Shah, A.K., Cristino, A.S., McKie, A.B., Gabra, H., Davis, M.J., & Hill, M.M. (2016). Integrative analysis of subcellular quantitative proteomics studies reveals functional cytoskeleton membrane–lipid raft interactions in cancer. Journal of Proteome Research, 15(10), 3451–3462. https://doi.org/10.1021/acs.jproteome.5b01035
• Simons, K., & Ikonen, E. (2000). How cells handle cholesterol. Science, 290(5497), 1721-1726. https://doi.org/10.1126/science.290.5497.1721
• Sajjadi, M., & Bathaie, Z. (2017). Comparative study on the preventive effect of saffron carotenoids, crocin and crocetin, in NMU-induced breast cancer in rats. Cell Journal (Yakhteh), 19(1), 94. https://doi.org/10.22074/cellj.2016.3901
• Sezgin, E., Levental, I., Mayor, S., & Eggeling, C. (2017). The mystery of membrane organization: composition, regulation and roles of lipid rafts. Nature Reviews Molecular Cell Biology, 18(6), 361–374. https://doi.org/10.1038/nrm.2017.16
• Umigai, N., Murakami, K., Ulit, M.V., Antonio, L.S., Shirotori, M., Morikawa, H., & Nakano, T. (2011). The pharmacokinetic profile of crocetin in healthy adult human volunteers after a single oral administration. Phytomedicine, 18(7), 575 578. https://doi.org/10.1016/j.phymed.2010.10.019
• Varshney, P., Yadav, V., & Saini, N. (2016). Lipid rafts in immune signalling: current progress and future perspective. Immunology, 149(1), 13–24. https://doi.org/10.1111/imm.12617
• Yeo, S.K., & Guan, J.L. (2017). Breast cancer: multiple subtypes within a tumor? Trends Cancer, 3(11),753–760. https://doi.org/10.1016/j.trecan.2017.09.001
• Zhang, Z., Wang, C.Z., Wen, X.D., Shoyama, Y., & Yuan, C.S. (2013). Role of saffron and its constituents on cancer chemoprevention. Pharmaceutical Biology, 51(7), 920–924. https://doi.org/10.3109/13880209.2013.771190
• Zheng, J., Zhou, Y., Li, Y., Xu, D-P., Li, S., & Li, H-B. (2016). Spices for prevention and treatment of cancers. Nutrients, 8(8), 495. https://doi.org/10.3390/nu8080495
• Zidovetzki, R., & Levitan, I. (2007). Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochimica et Biophysica Acta (BBA) – Biomembranes, 1768(6), 1311 1324. https://doi.org/10.1016/j.bbamem.2007.03.026