Inhibition of Cancer Stem Cells Growth with Silibinin Encapsulated in Nanoparticles with Deregulation of miR-34a, miR-221, and miR-222


Cancer Stem Cells, miR-34a, miR-221/222, Silibinin, SPNs


How to Cite

Hossainzadeh, S. ., Nouhi Kararoudi, A. ., Mousavi Eshkelani, S. M. ., Pakizehkar, S. ., Naderi Sohi, A. ., Najafi, F. ., & Ranji, N. (2023). Inhibition of Cancer Stem Cells Growth with Silibinin Encapsulated in Nanoparticles with Deregulation of miR-34a, miR-221, and miR-222. Iranian Red Crescent Medical Journal, 25(4).


Background: Colorectal Cancer (CRC) is the most common malignant gastrointestinal cancer. Cancer stem cells (CSCs) are the major cause of cancer recurrence and cancer drug resistance. Silibinin, as an herbal compound, has anticancer properties.

Objectives: The present study aimed to evaluate the antiproliferative effects of silibinin on HT29 stem-like cells (spheroids).

Methods: In this study, antiproliferative and apoptotic properties of Silibinin encapsulated in Polymersome Nanoparticles (SPNs) were evaluated by MTT assay, propidium iodide (PI) /AnnexinV assay, cell cycle analysis, and DAPI (4',6-diamidino-2-phenylindole) staining. The expression of some miRNAs and their potential targets was evaluated by real-time reverse transcription-polymerase chain reaction (qRT-PCR).

Results: IC50 of SPNs was determined at 28.13±0.78µg/ml after 24 h. SPNs (28µg/ml) induced apoptosis by 32.36% in HT29 cells after 24 h. DAPI staining indicated a decrease in stained nuclei after SPNs induction. SPNs treatment increased the expression of miR-34a, as well as P53, BAX, CASP9, CASP3, and CASP8. The downregulation of miR-221 and miR-222 was observed in SPNs treated cells. Moreover, SPNs decrease the expression level of CD markers in HT29 spheroids (cancer stem cells) compared to untreated spheroids. Spheroids were completely destroyed after 72 h treatment with SPNs (28µg/ml). 

Conclusion: As evidenced by the obtained results, SPNs can be used as an effective anticancer agent in multi-layer (cancer stem cells) and mono-layer cancerous cells with the upregulation of tumor suppressive miRs and genes, as well as downregulation of oncomiRs and oncogenes.


Akbari F, Peymani M, Salehzadeh A, Ghaedi K. Integrative in silico and in vitro transcriptomics analysis revealed new lncRNAs related to intrinsic apoptotic genes in colorectal cancer. Cancer Cell Int. 2020;20(1):546. doi: 10.1186/s12935-020-01633-w. [PubMed:33292233]

Granados-Romero JJ, Valderrama-Treviño AI, Contreras-Flores EH, Barrera-Mera B, Herrera Enríquez M, Uriarte-Ruíz K, et al. Colorectal cancer: a review. Int J Res Med. 2017;5(11):4667. doi:10.18203/2320-6012.ijrms20174914

Akbari F, Peymani M, Salehzadeh A, Ghaedi K. Identification of modules based on integrative analysis for drug

prediction in colorectal cancer. Gene Rep. 2021;25:101403. doi:10.1016/j.genrep.2021.101403

Orang AV, Barzegari A. MicroRNAs in colorectal cancer: from diagnosis to targeted therapy. Asian Pac J Cancer Prev. 2014;15(17):6989-99. doi:10.7314/apjcp.2014.15.17.6989. [PubMed:25227782]

Tehrani FK, Ranji N, Kouhkan F, Hosseinzadeh S. PANC-1 cancer stem-like cell death with silybin encapsulated in polymersomes and deregulation of stemness-related miRNAs and their potential targets. Iran J Basic Med Sci. 2021;24(4):514-23. doi:10.22038/ijbms.2021.54001.12136. [PubMed:34094034]

Pakizehkar S, Ranji N, Naderi Sohi A, Sadeghizadeh M. Curcumin loaded PEG400-OA nanoparticles: A suitable system to increase apoptosis, decrease migration, and deregulate miR-125b/miR182 in MDA-MB-231 human breast cancer cells. Polym Adv Technol. 2020;31(8):1793-804. doi:10.1002/pat.4906.

Chu CY, Rana TM. Small RNAs: regulators and guardians

of the genome. J Cell Physiol. 2007;213(2):412-9. doi:10.1002/jcp.21230. [PubMed:17674365]

Sahlberg SH, Spiegelberg D, Glimelius B, Stenerlow B, Nestor M. Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells. PloS one. 2014;9(4):e94621. doi:10.1371/journal.pone.0094621. [PubMed:24760019]

Jaggupilli A, Elkord E. Significance of CD44 and CD24 as

cancer stem cell markers: an enduring ambiguity. Clin Dev Immunol. 2012;2012:708036. doi: 10.1155/2012/708036. [PubMed:22693526]

Chabner BA, Roberts TG. Chemotherapy and the war on cancer. Nat Rev Cancer. 2005;5(1):65-72. doi:10.1038/nrc1529

Priya ML, Subbaiah Kotakadi V. Herbal and medicinal plants molecules towards treatment of cancer: a mini review. Am J Ethnomed. 2014.

Yin SY, Wei WC, Jian FY, Yang NS. Therapeutic applications of herbal medicines for cancer patients. Evid Based Complement Alternat Med. 2013;2013:302426. doi:10.1155/2013/302426. [PubMed:23956768]

Desai AG, Qazi GN, Ganju RK, El-Tamer M, Singh J, Saxena AK, et al. Medicinal plants and cancer chemoprevention. Curr Drug Metab. 2008;9(7):581-91. doi:10.2174/138920008785821657. [PubMed:18781909]

Chang YC, Jan CI, Peng CY, Lai YC, Hu FW, Yu CC. Activation of microRNA-494-targeting Bmi1 and ADAM10 by

silibinin ablates cancer stemness and predicts favourable prognostic value in head and neck squamous cell carcinomas. Oncotarget. 2015;6(27):24002-16. doi: 10.18632/oncotarget.4365. [PubMed:26090866]

Lim R, Morwood CJ, Barker G, Lappas M. Effect of silibinin in reducing inflammatory pathways in in vitro and in vivo models of infection-induced preterm birth. PloS one. 2014;9(3):e92505. doi: 10.1371/journal.pone.0092505. [PMID:24647589]

Kidd P, Head K. A review of the bioavailability and clinical efficacy of milk thistle phytosome: a silybin-phosphatidylcholine complex (Siliphos). Altern Med Rev. 2005;10(3):193-203. [PubMed:16164374]

Deshpande PP, Biswas S, Torchilin VP. Current trends in the use of liposomes for tumor targeting. Nanomedicine. 2013;8(9):1509-28. doi:10.2217/nnm.13.118. [PubMed:23914966]

Baker JR Jr. Dendrimer-based nanoparticles for cancer therapy. Hematology Am Soc Hematol Educ Program. 2009;2009(1):708-19. doi:10.1182/asheducation-2009.1.708. [PubMed:20008257]

Keskin D, Tezcaner A. Micelles as delivery system for

cancer treatment. Curr Pharm Des. 2017;23(35):5230-41. doi:10.2174/1381612823666170526102757. [PubMed:28552065]

Amiji MM. Nanotechnology for cancer therapy. CRC press; 2006.

Zhang XY, Zhang PY. Polymersomes in

nanomedicine-A review. Curr Med Chem. 2017;13(2):124-9. doi:10.2174/1573413712666161018144519.

Discher DE, Eisenberg A. Polymer vesicles. Science. 2002;297(5583):967-73. doi:10.1126/science.1074972. [PubMed:12169723]

Ma Y, Bao-Han W, Lv X, Su Y, Zhao X, Yin Y, et al. MicroRNA-34a mediates the autocrine signaling of PAR2-activating

proteinase and its role in colonic cancer cell proliferation. PloS one. 2013;8(8):e72383. doi:10.1371/journal.pone.0072383. [PubMed:23991105]

Pakizehkar S, Ranji N, Sohi AN, Sadeghizadeh M. Polymersome-assisted delivery of curcumin: A suitable approach to decrease cancer stemness markers and regulate miRNAs expression in HT29 colorectal cancer cells. Polym Adv Technol. 2020;31(1):160-77. doi:10.1002/pat.4759.

Tahmasebi Mirgani M, Isacchi B, Sadeghizadeh M, Marra F, Bilia AR, Mowla SJ, et al. Dendrosomal curcumin nanoformulation downregulates pluripotency genes via miR-145 activation in U87MG glioblastoma cells. Int J Nanomedicine. 2014;9:403-17. doi: 10.2147/IJN.S48136. [PubMed:24531649]

Zadeh MM, Motamed N, Ranji N, Majidi M, Falahi F. Silibinin-induced apoptosis and downregulation of microRNA-21 and microRNA-155 in MCF-7 human breast cancer cells. J breast cancer. 2016;19(1):45-52. doi: 10.4048/jbc.2016.19.1.45. [PubMed27066095]

Zhou Y, Xia L, Wang H, Oyang L, Su M, Liu Q, et al. Cancer

stem cells in progression of colorectal cancer. Oncotarget. 2018;9(70):33403-15. doi:10.18632/oncotarget.23607. [PubMed:30279970]

Agarwal C, Singh RP, Dhanalakshmi S, Tyagi AK, Tecklenburg M, Sclafani RA, et al. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene. 2003;22(51):8271-82. doi:10.1038/sj.onc.1207158. [PubMed:14614451]

Kauntz H. Cellular and molecular targets of silibinin, a natural flavonoid, in colorectal cancer prevention and therapy. Doctoral dissertation:Strasbourg; 2012.

Patel S, Waghela B, Shah K, Vaidya F, Mirza S, Patel S, et al. Silibinin, A natural blend in polytherapy formulation for targeting Cd44v6 expressing colon cancer stem cells. Sci Rep. 2018;8(1):16985. doi: 10.1038/s41598-018-35069-0. [PubMed:30451890]

Hossainzadeh S, Ranji N, Naderi Sohi A, Najafi F. Silibinin encapsulation in polymersome: A promising anticancer nanoparticle for inducing apoptosis and decreasing the expression level of miR-125b/miR-182 in human breast

cancer cells. J Cell Physiol. 2019;234(12):22285-98. doi:10.1002/jcp.28795. [PubMed:31073992]

Wang YX, Cai H, Jiang G, Zhou TB, Wu H. Silibinin inhibits proliferation, induces apoptosis and causes cell cycle arrest in human gastric cancer MGC803 cells via STAT3 pathway inhibition. Asian Pac J Cancer Prev. 2014;15(16):6791-8. doi:10.7314/apjcp.2014.15.16.6791. [PMID:25169527]

Shaheen S, Ahmed M, Lorenzi F, Nateri AS. Spheroid-formation (colonosphere) assay for in vitro assessment and expansion of stem cells in colon cancer. Stem Cell Rev. 2016;12(4):492-9. doi: 10.1007/s12015-016-9664-6. [PubMed:27207017]

Agarwal R, Agarwal C. Abstract B32: translational potential of a small-molecule silibinin in colorectal cancer: targeting cancer stem cells and their inflammatory niche. Clin Cancer Res. 2018;24(1):B32-B. doi:10.1158/1557-3265.TCM17-B32.

Rupaimoole R, Slack FJ. A role for miR-34 in colon cancer stem cell homeostasis. Stem Cell Investig. 2016;3(8):42. doi: 10.21037/sci.2016.08.04. [PubMed:27668249]

Li C, Wang Y, Lu S, Zhang Z, Meng H, Liang L, et al. MiR‑34a inhibits colon cancer proliferation and metastasis by inhibiting platelet‑derived growth factor receptor α. Mol Med Rep. 2015;12(5):7072-8. doi:10.3892/mmr.2015.4263. [PubMed:26324236]

Liu S, Sun X, Wang M, Hou Y, Zhan Y, Jiang Y, et al. A

microRNA 221–and 222–mediated feedback loop maintains constitutive activation of NFκB and STAT3 in colorectal

cancer cells. Gastroenterology. 2014;147(4):847-59. e11. doi:10.1053/j.gastro.2014.06.006. [PubMed:24931456]

Navarro F, Lieberman J. MiR-34 and p53: new insights into a complex functional relationship. PloS one. 2015;10(7):e0132767. doi:10.1371/journal.pone.0132767. [PMID:26177460]

Fan N, Wang J. MicroRNA 34a contributes to virus-mediated apoptosis through binding to its target gene Bax in influenza A virus infection. Biomed Pharmacother. 2016;83:1464-70. doi: 10.1016/j.biopha.2016.08.049. [PubMed:27610823]

Yacoub RA, Fawzy IO, Assal RA, Hosny KA, Zekri AN, Esmat G, et al. MiR-34a: multiple opposing targets and one destiny in hepatocellular carcinoma. J Clin Transl Hepatol. 2016;4(4):300-5. doi:10.14218/JCTH.2016.00031. [PMID:28097098]