Silibinin-Induced Apoptosis and Downregulation of MicroRNA-21 and MicroRNA-155 in MCF-7 Human Breast Cancer Cells / 한국유방암학회지

PURPOSE: MicroRNAs (miRNAs) have received much attention owing to their aberrant expression in various stages of cancer. In many biological processes, miRNAs negatively regulate gene expression, and may be useful in therapeutic strategies. The present study evaluated the effects of silibinin (silybin), a natural flavonoid, on miRNA expression and attempted to elucidate therapeutic targets in MCF-7 breast cancer cells. METHODS: The rates of cell proliferation and apoptosis were determined in silibinin-treated and untreated MCF-7 cells. Furthermore, the expression levels of miR-21 and miR-155 were measured in MCF-7 cells after incubation with silibinin (100 µg/mL), and the putative targets of the miRNAs within the apoptotic pathways were predicted using bioinformatic approaches. The expression levels of some of these targets were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Silibinin induced apoptosis in MCF-7 cells in a dose- and time-dependent manner. qRT-PCR analysis revealed a decrease in miR-21 and miR-155 expression levels in silibinin-treated cells relative to the levels in the untreated cells. Potential miR-21 and miR-155 targets within the apoptotic pathways, such as CASP-9, BID, APAF-1, CASP-3, CASP-8, and PDCD4, were predicted by in silico analysis. qRT-PCR analysis showed upregulation of some of these potential targets including caspase-9 (CASP-9) and BID after silibinin treatment for 48 hours. CONCLUSION: Our results suggest a correlation between the expression of miR-21 and miR-155, and MCF-7 cell proliferation. The antiproliferative activity of silibinin may partly be attributable to the downregulation of miR-21 and miR-155, and the upregulation of their apoptotic targets. Furthermore, the upregulation of CASP-9 and BID indicates that silibinin induces apoptosis through both the extrinsic and intrinsic pathways.

Isolation and in vitro characterization of mesenchymal stem cells derived from the pulp tissue of human third molar tooth

It is still controversial that the stem cells isolated from human dental pulp meets the criteria for mesenchymal stem cells [MSCs]. The aim of the present study was to examine whether or not they are MSCs, or are distinct stem cells population residing in tooth pulp. Adherent fibroblastic cells in the culture of pulp tissue from human third molars were propagated through several successive subcultures. Passaged-3 cells with a tendency to differentiate into odontoblastic cells were used to examine the key properties of MSCs including typical tripotent differentiation potential into bone, cartilage and adipose cell lineages and the expression of typical surface antigens. Moreover, they were examined for growth capacity in culture. Dental pulp stem cells successfully progressed towards differentiation among three skeletal cell lineages. More than 90% of the cell population exhibited the expression of surface antigens known to be found on mesenchymal lineages such as CD105, CD90, CD44, and CD73, while only less than 2% expressed endothelial-hematopoietic epitopes including CD56, CDllb, CD34, CD31, CD33, and CD45. The cells exhibited a relatively high proliferation capacity with population doubling time of about 21.9 hours. The dental pulp stem cells are of MSC population, and may be considered suitable for use in regenerative medicine, owing to their relatively rapid rate of in vitro propagation

Ex vivo expansion and differentiation of mesenchymal stem cells from goat bone marrow

Mesenchymal stem cells [MSCs] from large animals as goat which is genetically more closely related to human have rarely been gained attentions. The present study tried to isolate and characterize MSCs from goat bone marrow. Fibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures. Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineages to determine their MSC nature. Differentiations were determined by RT-PCR analysis of related gene expression. To identify the best culture conditions for propagation, passage-3 cells were plated either at varying cell densities or different fetal bovine serum [FBS] concentrations for a week, at the end of which the cultures were statistically compared with respect to the cell proliferation. In this study, we also determined goat MSC population doubling time [PDT] as the index of their in vitro expansion rate. Passage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in both specific staining as well as the specific gene expression profile. Moreover, there appeared to be more expansion when the cultures were initiated at 100 cells/cm[2] in a medium supplemented with 15% FBS. A relatively short PDT [24.94 +/- 2.67 hr] was a reflection of the goat MSC rapid rate of expansion. Taken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletal cell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBS concentration

Quantitative analysis of the proliferation and differentiation of rat articular chondrocytes in alginate 3D culture

While articular chondrocytes are among those appropriate candidates for cartilage regeneration, the cell dedifferentiation during monolayer culture has limited their application. Several investigations have indicated the usefulness of alginate, but the topic of proliferation and differentiation of chondrocytes in alginate culture has still remained controversial. Rat articular chondrocytes were released by enzymatic digestion, plated at 5 x 10[4] cells/cm[2] and culture-expanded. Passaged-5 cells were then cultivated in alginate as 2-mm beads for a period of two months during which the expansion rate and the level of cell differentiation were determined by [3-[A, 5- dimethylthiazolyl- 2-yl]-l, 5-diphenyl tetrazolium bromide] assay and real-time PCR analysis respectively and compared with those of chondrocytes in monolayer culture. Average population doubling time in alginate cultures [10.04 +/- 0.9 days] tended to be significantly [P<0.05] higher than that in monolayer cultures [2.94 +/- 0.3 days]. The period of alginate culture could be subdivided into expansion phase [Days 0-40]; during which proliferation appeared to be high and differentiation phase [Days 40-60] during which the expression of cartilage-specific genes including collagen II and aggrecan tended to be upregulated. During both the proliferation and differentiation phases, the expression of collagen I was low. At chondrocytes monolayer cultures, the proliferating cells appeared to have a very low expression level of cartilage-specific genes and a high expression level of collagen I gene during the entire culture period [P<0.05]. It seems that alginate provides conditions in which rat articular chondrocytes are able to undergo proliferation and differentiation in certain time point of cultivation period