PANC-1 cancer stem-like cell death with silybin encapsulated in polymersomes and deregulation of stemness-related miRNAs and their potential targets

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): Cancer stem cells (CSCs) have powerful self-renewal ability and tumor recurrence. Pancreatic ductal adenocarcinoma is a malignancy with high mortality rate and ˃5% survival. Silybin has anticancer and hepatoprotective properties. We loaded silybin in PEG400-OA (SPNs) and evaluated its cytotoxic effects on PANC-1 cells and PANC-1 CSCs.  
Materials and Methods: Spheroids from PANC-1 cells were obtained by the hanging drop method. Anti-proliferative and apoptotic functions of SPNs were evaluated in spheroids and non-spheroids with MTT, DNA fragmentation, PI and PI/AnnexinV assays. The expression of CD markers was assessed with flow cytometry. QRT-PCR was used to evaluate the expression of some miRNAs and targets.   
Results: IC50 of SPNs was identified to be 50 µg/ml, 45 µg/ml, and 42µg/ml, respectively after 24 hr, 48 hr, and 72 hr in PANC-1 treated cells. PI staining and PI/AnnexinV assay showed that ~20%, ~60%, and ~80%, of cells treated with 30, 50, and 60 µg/ml of SPNs were in sub-G1 and apoptosis phase, respectively. DNA degradation was confirmed after SPNS stimulation. CD24, CD44, and CD133 expression decreased after SPNs treatment both in PANC-1 spheroid cells and PANC-1 cancer cell line. Under-expression of onco-miRs (miR-21, miR-155, and miR-221), over-expression of several apoptotic potential targets of oncomiRs (Bax, Casp-9, and P53), over-expression of tumor suppressive-miRs (let-7b, miR-34a, and miR-126), and under-expression of Bcl-2 was found in SPNs-treated cells.  
Conclusion: We suggest that silybin encapsulated in polymersomes (SPNs) may be useful as a complementary agent for destroying both pancreatic cancer cells and pancreatic CSCs  along with chemotherapeutic agents.

Keywords


1. Shao Y, Zhang L, Cui L, Lou W, Wang D, Lu W, et al. LIN28B suppresses microRNA let-7b expression to promote CD44+/LIN28B+ human pancreatic cancer stem cell proliferation and invasion. Am J Cancer Res 2015;5:2643-2659.
2. Yang Z, Zhang Y, Tang T, Zhu Q, Shi W, Yin X, et al. Transcriptome profiling of panc-1 spheroid cells with pancreatic cancer stem cells properties cultured by a novel 3d semi-solid system. Cell Physiol Biochem 2018;47:2109-2125.
3. Ning X, Du Y, Ben Q, Huang L, He X, Gong Y, et al. Bulk pancreatic cancer cells can convert into cancer stem cells(CSCs) in vitro and 2 compounds can target these CSCs. Cell Cycle. 2016;15:403-412.
4. Peng Y, Croce CM. The role of microRNAs in human cancer. Signal Transduct Target Ther 2016;1:15004.
5. 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.
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:nep164.
7. Sharma A, Houshyar R, Bhosale P, Choi J-I, Gulati R, Lall C. Chemotherapy induced liver abnormalities: an imaging perspective. Clin Mol Hepatol. 2014;20:317-326.
8. Dilova V, Stoianova Y. Increasing the solubility of a poor soluble api-milk thistle dry extract (silymarin). Pharmacia. 2014;61:18-21.
9. Rahbar Takrami S, Ranji N, Sadeghizadeh M. Antibacterial effects of curcumin encapsulated in nanoparticles on clinical isolates of Pseudomonas aeruginosa through downregulation of efflux pumps. Mol Biol Rep 2019;46:2395-404. 410.    
10. Duran N, Marcato P, Teixeira Z, Durán M, Costa F, Brocchi M. State of the art of nanobiotechnology applications in neglected diseases. Curr Nanosci 2009;5:396408.
11.    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 Cell Cycle Res 2015;5:143-158.
12.    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.
13.    Zhang L, Liao Y, Tang L. MicroRNA-34 family: A potential tumor suppressor and therapeutic candidate in cancer. J Exp Clin Cancer Res 2019;38:53-82.
14.    Ranji N, Sadeghizadeh M, Karimipoor M, Shokrgozar MA, Nakhaei Sistani R, Paylakhi SH. MicroRNAs signature in IL-2-induced CD4+ T cells and their potential targets. Biochem Genet. 2015;53:169-183.
15.    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.
16.    Peng Y, Croce CM. The role of MicroRNAs in human cancer. Signal transduction and targeted therapy. 2016;1:15004.
17.    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:e6816.
18.    Khan AQ, Ahmed EI, Elareer NR, Junejo K, Steinhoff M, Uddin S. Role of miRNA-regulated cancer stem cells in the pathogenesis of human malignancies. Cells. 2019;8.
19.    Xu Y-F, Hannafon BN, Ding W-Q. microRNA regulation of human pancreatic cancer stem cells. Stem Cell Investig. 2017;4:5-18.
20.    Pang Y, Liu J, Li X, Zhang Y, Zhang B, Zhang J, et al. Nano Let-7b sensitization of eliminating esophageal cancer stem‑like cells is dependent on blockade of Wnt activation of symmetric division. Int J Oncol 2017;51:1077-1088.
21.    Dorrance AM, Neviani P, Ferenchak GJ, Huang X, Nicolet D, Maharry KS, et al. Targeting leukemia stem cells in vivo with antagomiR-126 nanoparticles in acute myeloid leukemia. Leukemia. 2015;29:2143-2153.
22.    de Leeuw DC, Denkers F, Olthof MC, Rutten AP, Pouwels W, Jan Schuurhuis G, et al. Attenuation of microRNA-126 expression that drives CD34+38− stem/progenitor cells in acute myeloid leukemia leads to tumor eradication. Cancer Res 2014;74:2094-2105.
23.    Zhao Y, Zhao L, Ischenko I, Bao Q, Schwarz-Mörtl B, Niess H, et al. Antisense inhibition of microRNA-21 and microRNA-221 in tumor-initiating stem-like cells modulates tumorigenesis, metastasis, and chemotherapy resistance in pancreatic cancer. Target Oncol 2015;10:535-548.
24.    Chung W-M, Chang W-C, Lumin C, Chang Y-Y, Shyr C-R, Hung Y-C, et al. MicroRNA-21 promotes the ovarian teratocarcinoma PA1 cell line by sustaining cancer stem/progenitor populations in vitro. Stem Cell Res Ther 2013;4:88-103.
25.    Słotwiński R, Lech G, Słotwińska S. MicroRNAs in pancreatic cancer diagnosis and therapy. Cent Eur J Immunol 2018;43:314-324.
26.    Poustchi F, Amani H, Ahmadian Z, Niknezhad SV, Mehrabi S, Santos HA, et al. Combination therapy of killing diseases by injectable hydrogels: from concept to medical applications. Adv Healthcare Mater 2020; 2001571.
27.    Delmas D, Xiao J, Vejux A, Aires V. Silymarin and Cancer: A Dual Strategy in Both in Chemoprevention and Chemosensitivity. Molecules. 2020;25:2009-2038.
28.    Deep G, Agarwal R. Antimetastatic efficacy of silibinin: molecular mechanisms and therapeutic potential against cancer. Cancer Metastasis Rev. 2010;29:447-463.
29.    Wang J-Y, Chang C-C, Chiang C, Chen W-M, Hung S-C. Silibinin suppresses the maintenance of colorectal cancer stem-like cells by inhibiting PP2A/AKT/mTOR pathways. J Cell Biochem 2011;113:1733-1743.
30.    Patel S, Waghela B, Shah K, Vaidya F, Mirza S, Pathak C, et al. Silibinin, A natural blend in polytherapy formulation for targeting cd44v6 expressing colon cancer stem cells. Sci Rep 2018;8:16985.
31.    Wang L, Li P, Hu W, Xia Y, Hu C, Liu L, et al. CD44(+)CD24(+) subset of PANC-1 cells exhibits radiation resistance via decreased levels of reactive oxygen species. Oncol Lett. 2017;14:1341-1346.
32.    Abdollahi P, Ebrahimi M, Motamed N, Samani FS. Silibinin affects tumor cell growth because of reduction of stemness properties and induction of apoptosis in 2D and 3D models of MDA-MB-468. Anticancer Drugs 2015;26:487-497.
33.    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.
34.    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.
35.    Liu C, Kelnar K, Liu B, Chen X, Calhoun-Davis T, Li H, et al. The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nat Med 2011;17:211-215.
36.    Soengas MS, Alarcon RM, Yoshida H, Giaccia AJ, Hakem R, Mak TW, et al. Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition. Science. 1999;284:156-159.
37.    Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ 2018;25:486-541.