Study On Crocin Inhibiting The Proliferation Of Breast Cancer MCF-7 Cells Under 2D/3D Conditions

Crocin is one of the main active ingredients extracted from the dry stigma of the Crocus sativus L.Recently,studies have shown that saffron,a traditional Chinese medicine,has obvious anti-cancer activity on a variety of cancer cells,and has almost no toxic side effects on normal cells.It is expected to become a novel natural anti-cancer drug.The purpose of this paper is to investigate its efficacy and influencing factors from in vitro models.The traditional in vitro 2D?Two dimensional,2D?cell culture model was selected to analyzed the inhibitory effect of Crocin on human breast cancer cell line?MCF-7?.Crocin can significantly inhibit the growth of MCF-7 cells in the 2D cell model,and the inhibitory effect was significantly enhanced with the increase of the action time and concentration following CCK-8 assays.And MCF-7 cells treated with Crocin shrunk from polygon into a ball,the number of living cells decreasd significantly following multiple assays including compound fluorescent staining,hematoxylin-Eosin staining?HE?and scanning electron microscope?SEM?.With the concentration of Crocin increasing,nucleus were denatured and fragmented and most of the cells were apoptotic.In addition,we evaluated in vitro the effect of Crocin on the activity of mouse precranial osteoblast cell line?MC3T3-E1?and human adipose stem cells?ADSCs?.The results indicated that low concentration of Crocin has no obvious cytotoxic effect on the above two normal cells.The inhibitory effect of Crocin on MCF-7 cells in 3D cell culture model was further evaluated since 3D culture model can better mimic the microenvironment of in vivo cell growth and improve the efficiency and accuracy of anticancer drugs.In this study,we prepared microbeads using calcium alginate?Alginate,Alg?,gelatin?Gelatin,Gel?,calcium chloride?CaCl₂?and further fabricated the in vitro 3D culture model.Calcium alginate beads?CA?and calcium alginate/gelatin?CAG?were prepared by the droplet method,and characterized and analyzed their microstructure,immersion resistance,swelling,permeability,shrinkage and cell viability.The results showed that the diameter of the gel bead material was between 2.0 mm and 3.0 mm,and its internal structure was porous and interconnected,which can provide a suitable growth environment for cell proliferation and differentiation.And the mechanical properties of immersion are sufficient to support the long-term culture of isolated cells inside the beads.Compared with CA beads,CAG beads had better permeability to glucose and bovine serum albumin.And the shrinkage of CAG beads was smaller,which can reduce the damage to embedded cells.The cell culture further showed that proliferation of cells in CAG beads was significant and the cells were tightly connected to form a large number of colonies by embedding MCF-7 cells in CAG and CA beads.Afterwards,CCK-8,compound fluorescent staining and fluorescent staining of harvested cells were used to determine the embedded density of MCF-7 cells in the beads?embedded cell density:2×103cells/bead,5×10³ cells/bead,1×10⁴ cells/bead,2×10⁴ cells/bead?.The results indicated that2×10³MCF-7 cells embedded in bead were growing and proliferating,and a large number of cell colonies were generated by the tight connection between the cells.In summary,CAG microbeads embedded with 2×10⁴ MCF-7 cells were prepared to fabricate the in vitro 3D cell culture model,which was used for subsequent evaluation of the inhibitory effect of Crocin on 3D cultured MCF-7 cells.Compared with 2D culture,IC50values of 3D cultured cells increased 2.77,2.70 and 4.34 times,indicating that the 3D microbead culture environment enhanced the drug resistance of the cells.Compound fluorescent staining showed that MCF-7 cells without Crocin's treatment continued to grow and proliferate in the microbeads,and the cells were interconnected to form cell colonies.With the concentration of Crocin increasing,the cell viability rate decreased obviously,the cell colonies decreased and the colony size further decreased.In the study,the inhibitory effect of Crocin on MCF-7 cells was studied from traditional2D cell culture and 3D microbead culture by the CCK assay,composite fluorescence staining,HE staining and SEM.The study found that Crocin has a time-dependent and concentration-dependent inhibitory effect on the growth of cells in 2D cultured cells and 3D microbead culture,and IC₅₀0 values were obtained by fitting the inhibition rate-concentration curve.Through compound fluorescence staining and HE staining,it was found that Crocin also had a certain influence on the cell morphology,which resulted in shrinkage and rupture of cell in 2D culture and reduction of cell colony size in the 3D microbeads culture.In addition,it was confirmed that Crocin at low concentration had almost no effect on the activity of two normal cells?MC3T3-E1 cells and ADSCs cells?.