Apoptosis induction and proliferation inhibition by silibinin encapsulated in nanoparticles in MIA PaCa-2 cancer cells and deregulation of some miRNAs

Document Type: Original Article

Authors

1 Department of Biology, Faculty of Sciences, Rasht Branch, Islamic Azad University, Rasht, Iran

2 Stem cell Technology Research Center, Tehran, Iran

3 Department of Tissue Engineering and Regenerative Medicine, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Abstract

Objective(s): Silibinin, as an herbal compound, has anti-cancer activity. Because of low solubility of silibinin in water and body fluids, it was encapsulated in polymersome nanoparticles and its effects were evaluated on pancreatic cancer cells and cancer stem cells.
Materials and Methods: MIA PaCa-2 pancreatic cancer cells were treated with different doses of silibinin encapsulated in polymersome nanoparticles (SPNs). Stemness of MIA PaCa-2 cells was evaluated by hanging drop technique and CD133, CD24, and CD44 staining. The effects of SPNs on cell cycle, apoptosis and the expression of several genes and miRNAs were investigated.
Results: IC50 of SPNs was determined to be 40 µg/ml after 24 hr. Our analysis showed that >98% of MIA PaCa-2 cells expressed three stem cell markers. FACS analysis showed a decrease in these markers in SPNs-treated cells. PI/AnnexinV staining revealed that 40 µg/ml and 50 µg/ml of SPNs increased apoptosis up to ~40% and >80% of treated cells, respectively. Upregulation of miR-34a, miR-126, and miR-let7b and downregulation of miR-155, miR-222 and miR-21 was observed in SPNs-treated cells. In addition, downregulation of some genes involved in proliferation or migration such as AKT3, MASPINE, and SERPINEA12, and upregulation of apoptotic genes were observed in treated cells.
Conclusion: Our results suggested that SPNs induced apoptosis and inhibited migration and proliferation in pancreatic cells and cancer stem cells through suppression of some onco-miRs and induction of some tumor suppressive miRs, as well as their targets.

Keywords


1. Maleki Zadeh M, 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: 45-52.
2. Ilic M, Ilic I. Epidemiology of pancreatic cancer. World J Gastroenterol 2016; 22 :9694-9705.
3. Hussain SP. Pancreatic Cancer: Current Progress and Future Challenges. Int J Biol Sci 2016;12:270–272.
4. Chen K, Huang Y, Chen J. Understanding and targeting cancer stem cells: therapeutic implications and challenges. Acta Pharmacol Sin 2013 ;34:732-740.
5. Rao CV, Mohammed A. New insights into pancreatic cancer stem cells. World J Stem Cells 2015;7:547–555.
6. Haddad Y, Vallerand D, Brault A, Haddad PS. Antioxidant and hepatoprotective effects of silibinin in a rat model of nonalcoholic steatohepatitis. Evid Based Complement Alternat Med 2011;2011:1-10.
7.Ge Y, Zhang Y, Chen Y, Li Q, Chen J, Dong Y, et al. Silibinin causes apoptosis and cell cycle arrest in some human pancreatic cancer cells. Int J Mol Sci 2011;12:4861–4871.
8. Ibrahim S, Tagami T, Kishi T, Ozeki T. Curcumin marinosomes as promising nano-drug delivery system for lung cancer. Int J Pharm 2018;540:40–49.
9. Natrajan D, Srinivasan S, Sundar K, Ravindran A. Formulation of essential oil-loaded chitosan–alginate nanocapsules. J Food Drug Anal 2015;23:560–568.
10. Soppimath KSK, Aminabhavi TMTM, Kulkarni ARAR, Rudzinski WE. Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release 2001;70:1–20.
11. Kumari A, Singla R, Guliani A, Yadav SK. Nanoencapsulation for drug delivery. Excli J 2014;13: 265–86.
12. Scarpa E, Bailey JL, Janeczek AA, Stumpf PS, Johnston AH, Oreffo ROC, et al. Quantification of intracellular payload release from polymersome nanoparticles. Sci Rep 2016;6:1–13.
13. Tahmasebi Birgani M, Erfani-Moghadam V, Babaei E, Najafi F, Zamani M, Shariati M, et al. Dendrosomal nano-curcumin; The novel formulation to improve the anticancer properties of curcumin. Prog Biol Sci 2015;5:143–158.
14. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834–838.
15. Gao Y, Schug J, McKenna LB, Le Lay J, Kaestner KH, Greenbaum LE. Tissue-specific regulation of mouse microRNA genes in endoderm-derived tissues. Nucleic Acids Res 2011;39:454–463.
16. Rachagani S, Kumar S, Batra SK. MicroRNA in pancreatic cancer: Pathological, diagnostic and therapeutic implications. Cancer Lett 2010;292:8–16.
17. Liang Y, Ridzon D, Wong L, Chen C. Characterization of microRNA expression profiles in normal human tissues. BMC Genomics 2007;8:1–20.
18. He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, et al. The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci 2005;102:19075–19080.
19. Ranji N, Mapar M,Sadat SM. miR-17 and miR-20a expression in IL-2 signaling pathway in Jurkat T cells. Mol Genet Microbiol Virol 2017;32:224–229.
20. Jusufović E, Rijavec M, Keser D, Korošec P, Sodja E, Iljazović E, et al. Let-7b and miR-126 are down-regulated in tumor tissue and correlate with microvessel density and survival outcomes in non-small-cell lung cancer. PLoS One 2012;7: 1–10.
21. Zhang DG, Zheng JN, Pei DS. P53/microRNA-34-induced metabolic regulation: new opportunities in anticancer therapy. Mol Cancer 2014;13:1–7.
22. Luu HN, Lin H-Y, Sørensen KD, Ogunwobi OO, Kumar N, Chornokur G, et al. miRNAs associated with prostate cancer risk and progression. BMC Urol 2017;17:1–18.
23. Feng SD, Mao Z, Liu C, Nie YS, Sun B, Guo M, et al. Simultaneous overexpression of miR-126 and miR-34a induces a superior antitumor efficacy in pancreatic adenocarcinoma. Onco Targets Ther  2017;10:5591–5604.
24. Ranji N, Sadeghizadeh M, Shokrgozar MA, Bakhshandeh B, Karimipour M, Amanzadeh A, et al. miR-17-92 cluster: an apoptosis inducer or proliferation enhancer. Mol Cell Biochem  2013;380:229–238.
25. Feng YH, Tsao CJ. Emerging role of microRNA-21 in cancer. Biomed reports 2016;5:395–402.
26. Pfeffer SR, Yang CH, Pfeffer LM. The Role of miR-21 in cancer. Drug Dev Res 2015;76:270–277.
27. Song J, Ouyang Y, Che J, Li X, Zhao Y, Yang K, et al. Potential value of miR-221/222 as diagnostic, prognostic, and therapeutic biomarkers for diseases. Front Immunol 2017; 8: 1–9.
28. Yu H, Xu W, Gong F, Chi B, Chen J, Zhou L. microRNA‑155 regulates the proliferation, cell cycle, apoptosis and migration of colon cancer cells and targets CBL. Exp Ther Med 2017;14:4053–4060.
29. Gu S, Lai Y, Chen H, Liu Y, Zhang Z. miR-155 mediates arsenic trioxide resistance by activating Nrf2 and suppressing apoptosis in lung cancer cells. Sci Rep 2017;7:1–13.
30. Magee P, Shi L, Garofalo M. Role of microRNAs in chemoresistance. Ann Transl Med 2015;3:1–9.
31. Santos JC, Lima N da S, Sarian LO, Matheu A, Ribeiro ML, Derchain SFM. Exosome-mediated breast cancer chemoresistance via miR-155 transfer. Sci Rep 2018;8:1–11.
32. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 2003;83:173–180.
33. Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 1995; 7:862–869
34. 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;234:22285–22298
35. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63.
36. Langdon SP. Cancer cell culture: Methods and protocols. Humana Press  2004; 360.
37. Lee PY, Costumbrado J, Hsu C-Y, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp 2012; 62. 1–5.
38. Meidhof S, Brabletz S, Lehmann W, Preca B-T, Mock K, Ruh M, et al. ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. EMBO Mol Med 2015;7:831–847.
39. Abbaszadegan MR, Bagheri V, Razavi MS, Momtazi AA, Sahebkar A, Gholamin M. Isolation, identification, and characterization of cancer stem cells: A review. J Cell Physiol 2017;232:2008–2018.
40. Fomeshi MR, Ebrahimi M, Mowla SJ, Firouzi J, Khosravani P. CD133 is not suitable marker for isolating melanoma stem cells from D10 cell line. Cell J  2016;18:21–7.
41. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al. Identification of pancreatic cancer stem cells. Cancer Res 2007;67:1030–7.
42. Salaria S, Means A, Revetta F, Idrees K, Liu E, Shi C. Expression of CD24, a stem cell marker, in pancreatic and small intestinal neuroendocrine tumors. Am J Clin Pathol 2015;144:642–648.
43. Zhan H xiang, Xu J wei, Wu D, Zhang T ping, Hu S yuan. Pancreatic cancer stem cells: New insight into a stubborn disease. Cancer Lett 2015;357:429–437.
44. Zhou P, Li B, Liu F, Zhang M, Wang Q, Liu Y, et al. The epithelial to mesenchymal transition (EMT) and cancer stem cells: Implication for treatment resistance in pancreatic cancer. Mol Cancer 2017;16:1–11.
45. Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007;1: 313–323.
46. Ramasamy K, Agarwal R. Multitargeted therapy of cancer by silymarin. Cancer Lett 2008;269:352–362.
47. Molavi O, Narimani F, Asiaee F, Sharifi S, Tarhriz V, Shayanfar A, et al. Silibinin sensitizes chemo-resistant breast cancer cells to chemotherapy. Pharm Biol 2017;55:729–739.
48. Wang S, Meng Q, Xie Q, Zhang M. Effect and mechanism of resveratrol on drug resistance in human bladder cancer cells. Mol Med Rep 2017;15:1179–1187.
49. Sun Y, Guan Z, Zhao W, Jiang Y, Li Q, Cheng Y, et al. Silibinin suppresses bladder cancer cell malignancy and chemoresistance in an NF-κB signal-dependent and signal-independent manner. Int J Oncol 2017;51:1219–1226.
50. Ghost  a, Ghost T, Jain S. Silymarin- a review on the pharmacodynamics and bioavailability enhancement approaches. J Pharm Sci Technol 2010;2:348–355.
51. Omolo CA, Kalhapure RS, Jadhav M, Rambharose S, Mocktar C, Ndesendo VMKK, et al. Pegylated oleic acid: A promising amphiphilic polymer for nano-antibiotic delivery. Eur J Pharm Biopharm 2017;112:96–108.
52. Amirsaadat S, Pilehvar-Soltanahmadi Y, Zarghami F, Alipour S, Ebrahimnezhad Z, Zarghami N. Silibinin-loaded magnetic nanoparticles inhibit hTERT gene expression and proliferation of lung cancer cells. Artif Cells, Nanomedicine, Biotechnol 2017;45:1649–1656.
53. Yazdi Rouholamini SE, Moghassemi S, Maharat Z, Hakamivala A, Kashanian S, Omidfar K. Effect of silibinin-loaded nano-niosomal coated with trimethyl chitosan on miRNAs expression in 2D and 3D models of T47D breast cancer cell line. Artif Cells Nanomed Biotechnol 2018;46:524–535.
54. Erfani-Moghadam V, Nomani A, Zamani M, Yazdani Y, Najafi F, Sadeghizadeh M. A novel diblock copolymer of (monomethoxy poly [ethylene glycol]-oleate) with a small hydrophobic fraction to make stable micelles/polymersomes for curcumin delivery to cancer cells. Int J Nanomedicine 2014;9:5541–5554.
55. Sood N, Jenkins WT, Yang X-Y, Shah NN, Katz JS, Koch CJ, et al. Biodegradable polymersomes for the delivery of gemcitabine to Panc-1 cells. J Pharm 2013;2013:1–10.
56. Maleki Zadeh M, Ranji N, Motamed N. Deregulation of miR-21 and miR-155 and their putative targets after silibinin treatment in T47D breast cancer cells. Iran J Basic Med Sci 2015;18:1209–1214.
57. Zhang X, Liu J, Zhang P, Dai L, Wu Z, Wang L, et al. Silibinin induces G1 arrest, apoptosis and JNK/SAPK upregulation in SW1990 human pancreatic cancer cells. Oncol Lett 2018 Apr 19;15:9868–9876.
58. Vinogradov S, Wei X. Cancer stem cells and drug resistance: the potential of nanomedicine. Nanomedicine 2012;7:597–615.
59. Zhou Q, Ye M, Lu Y, Zhang H, Chen Q, Huang S, et al. Curcumin improves the tumoricidal effect of mitomycin c by suppressing ABCG2 expression in stem cell-like breast cancer cells. Castresana JS, editor. PLoS One 2015;10:1-12.
60. Atashpour S, Fouladdel S, Komeili Movahhed T, Barzegar E, Ghahremani MH, Ostad SN, et al. Quercetin induces cell cycle arrest and apoptosis in CD133+ cancer stem cells of human colorectal HT29 cancer cell line and enhances anticancer effects of doxorubicin. Iran J Basic Med Sci 2015;18:635–643.
61. Lonardo E, Hermann PC, Heeschen C. Pancreatic cancer stem cells-update and future perspectives. Mol Oncol 2010;4:431–442.
62. Almanaa TN, Geusz ME, Jamasbi RJ. Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines. BMC Complement Altern Med 2012;12:1-15.
63. Nambiar D, Prajapati V, Agarwal R, Singh RP. In vitro and in vivo anticancer efficacy of silibinin against human pancreatic cancer BxPC-3 and PANC-1 cells. Cancer Lett 2013;334:109–117.
64. Ali S, Banerjee S, Logna F, Bao B, Philip PA, Korc M, et al. Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer. J Cell Physiol 2012;227:3373–3380.
65. Moriyama T, Ohuchida K, Mizumoto K, Yu J, Sato N, Nabae T, et al. MicroRNA-21 modulates biological functions of pancreatic cancer cells including their proliferation, invasion, and chemoresistance. Mol Cancer Ther 2009 ;8:1067–1074.
66. Du Rieu MC, Torrisani J, Selves J, Al Saati T, Souque A, Dufresne M, et al. MicroRNA-21 is induced early in pancreatic ductal adenocarcinoma precursor lesions. Clin Chem 2010;56:603–612.
67. Halkova T, Cuperkova R, Minarik M, Benesova L. MicroRNAs in pancreatic cancer: involvement in carcinogenesis and potential use for diagnosis and prognosis. Gastroenterol Res Pract 2015;2015:1–11.
68. Ebrahimi S, Hosseini M, Ghasemi F, Shahidsales S, Maftouh M, Akbarzade H, et al. Circulating microRNAs as potential diagnostic, prognostic and therapeutic targets in pancreatic cancer. Curr Pharm Des 2017 ;22:6444–6450.
69. Frampton AE, Krell J, Jacob J, Stebbing J, Castellano L, Jiao LR. Loss of miR-126 is crucial to pancreatic cancer progression. Expert Rev Anticancer Ther 2012 10;12:881–884.
70. Ji Q, Hao X, Zhang M, Tang W, Yang M, Li L, et al. MicroRNA mir-34 inhibits human pancreatic cancer tumor-initiating cells. PLoS One 2009;4:1-13.
71. Xu Y-F, Hannafon BN, Ding W-Q. microRNA regulation of human pancreatic cancer stem cells. Stem Cell Investig 2017;4:1-7.
72. Shi L, Chen J, Yang J, Pan T, Zhang S, Wang Z. MiR-21 protected human glioblastoma U87MG cells from chemotherapeutic drug temozolomide induced apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 activity. Brain Res 2010;1352:255–264.
73. Zhang C-Z, Zhang J-X, Zhang A-L, Shi Z-D, Han L, Jia Z-F, et al. MiR-221 and miR-222 target PUMA to induce cell survival in glioblastoma. Mol Cancer 2010;9:1-9.
74. Czochor JR, Glazer PM. microRNAs in Cancer Cell Response to Ionizing Radiation. Antioxid Redox Signal 2014;21:293–312.
75. Fu X, Wen H, Jing L, Yang Y, Wang W, Liang X, et al. MicroRNA-155-5p promotes hepatocellular carcinoma progression by suppressing PTEN through the PI3K/Akt pathway. Cancer Sci 2017;108:620–631.
76. Zou Z, Anisowicz A, Hendrix MJ, Thor A, Neveu M, Sheng S, et al. Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science 1994;263:526–529.
77. Cha HJ, Jeong MJ, Kleinman HK. Role of Thymosin 4 in Tumor Metastasis and Angiogenesis. Cancer Spectrum Knowl Environ 2003;95:1674–1680.
78. Liu J, Zhao XJ, Zhang XL, Wu XH, Zhao TP. Knock-down Akt3 inhibits ovarian cancer cell growth and migration. Int J Clin Exp Med 2017;10:8566–8573.
79. Wang L, Huang D, Jiang Z, Luo Y, Norris C, Zhang M, et al. Akt3 is responsible for the survival and proliferation of embryonic stem cells. Biol Open 2017;6:850–861.
80. Bach DH, Park HJ, Lee SK. The dual role of bone morphogenetic proteins in cancer. Mol Ther-Oncolytics 2018;8:1–13.
81. Fathy A, Abdelrahman AE. EZH2, Endothelin-1, and CD34 as biomarkers of aggressive cervical squamous cell carcinoma: An immunohistochemical study. Turk Patoloji Derg 2018;34:150–157.
82. Mandrysz M, Dybiec B. Energetics of the undamped stochastic oscillators. J Cell Sci 2018;121:3683–3692.