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가설 설정
- Analysis of cytotoxicity and determination of IC50 values by MTT assay in A-549, MDA-MB-231, U87-MG, MCF-7, PANC-1, MRC-5 and DPSCs cell lines treated with PGG. A: Inhibition curves by MTT assay in each cell line. B: Mean±SEM of IC50 values) of each cell line in triplicate.
제안 방법
- 1. Analysis of cytotoxicity and determination of IC50 values by MTT assay in A-549, MDA-MB-231, U87-MG, MCF-7, PANC-1, MRC-5 and DPSCs cell lines treated with PGG. A: Inhibition curves by MTT assay in each cell line.
- Both cancer and normal cell lines were seeded at 1×104 cells/well into a 6-well plate and cultured in complete A-DMEM media containing 0 (control) and 10 μM PGG for 7 days at 37.5˚C in a humidified atmosphere of 5% CO2 in air by changing culture medium for every 3 days.
- Changes of telomerase activiy analyzed by RQ-TRAP assay in A-549, MDA-MB-231, U87-MG, MCF-7, PANC1, MRC-5 and DPSCs cell lines treated with 10 μM PGG for 7 days.
- The RQ-TRAP was performed using the PCR reagent LightCycler FastStart DNA Master SYBR Green 1 (Roche, Germany), containing lysed samples, 2.5 mM MgCl2, 0.02 μg of primer TS (5’-AAT CCG TCG GAG CAG AGT T-3’), 0.04 μg of primer ACX (5’-GCG CGG CTT ACC CTT ACC CTT ACC CTA ACC-3’), according to the manufacturer’s protocol and final volume was adjusted to 20 μl with RNase-free water.
- The cells were then incubated with 1 ml senescence-associated ß-galactosidase staining solution at 37o C for overnight. The cells stained with blue color were examined under an inverted microscope (Nikon, Tokyo, Japan) equipped with CCD camera and image program.
- The cytotoxicity test with IC50 values assay was determined by MTT in the A-549, MDA-MB-231, U87-MG, MCF-7 and PANC-1 human cancer cell lines, and DPSCs derived from dental papilla tissues and MRC-5 fetal fibroblasts treated with PGG, as shown in Fig. 1. The IC50 value (mean±SEM) in three replicate was 6.
대상 데이터
- 4 and 280 mOsm/kg, respectively. A-549 lung adenocarcinoma, MDA-MB-231 breast Adenocarcinoma, MCF-7 breast Adenocarcinoma, U87-MG brain glioblastoma astrocytoma, PANC1 pancreatic carcinoma cancer cells and MRC-5 normal fetal lung fibroblasts were purchased from the American Type Culture Collection (Manassas, VA, USA). The DPSCs used for this experiments were derived from extracted third molar tooth and the basic characterizations of stem cell in the isolated DPSCs were demonstrated in the previous study [15].
데이터처리
- Differences among the cell groups were analyzed by using one-way analysis of variance (ANOVA). Differences in the IC50 value, population doubling time and relative telomerase activity were analyzed using a Student’s t-test.
- Differences in the IC50 value, population doubling time and relative telomerase activity were analyzed using a Student’s t-test.
- The PDT value was calculated using the formula DT = t (log 2)/(log Nt-log No), where t represents the culture time, and No and Nt are the initial and final cell numbers respectively. Furthermore, when IC50 values and PDT were determined, the correlation coefficient analysis was performed between two values using Exel program (Microsoft, Redmond, WA, USA).
성능/효과
- 2B. A considerably high correlation coefficient degree (r2=0.9496) was exhibited between IC50 values and PDT, and the IC50 values were proportionally increased along with the increase of PDT in each cell lines.
- All samples were quantified using the LightCycler Quantification Software’s (Roche, Mannheim, Germany) second derivative method of crossing point (Cp) determination, and the relative telomerase activity of untreated control MRC-5 fibroblasts was considered as 100% for comparison with the treatment groups in five replicates.
- Our results demonstrated that PGG treatment has induced the cellular cytotoxicity, including inhibition of cell proliferation, high level of senescence-associated ß-glucosidase activity and downregulation of telomerase activity in the human cell lines of various origins.
- Our results have demonstrated that PGG induces the cytotoxic effects, including the inhibition of proliferation rate, high senescence-associated-ß-galactosidase activity and down-regulation of telomerase activity.
- Values indicated the mean telomease activity (mean±SEM) of five replicates and the telomerase activity in untreated control MRC-5 fibroblasts was considered as 100% for comparison with other cell lines.
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