Then, 10 l of the CCK-8 solution was added to each well and the cells were cultured at 37 C for another hour

Then, 10 l of the CCK-8 solution was added to each well and the cells were cultured at 37 C for another hour. also has been used in anti-tumor therapy for many years10,11,12. Interestingly, recent research has shown that one of the main active ingredients, physcion 8-O–glucopyranoside (PG), causes apoptosis and blocks cell cycle progression in the human lung cancer cell line A54913. However, little is known about the mechanism by which PG induces apoptosis in cancer cells. In present study, the OSCC cell line KB was used as model to examine whether PG induces apoptosis and to determine the underlying mechanism. In addition to showing the pro-apoptotic effect of PG in the KB cell line, data from this study demonstrated that survivin plays a key role in the apoptosis-inducing effect of PG, and PG modulates survivin through miR-21/PTEN/Akt/GSK3 signaling. Materials and methods Cell culture The human OSCC-derived cell line KB (ATCC, Shanghai. China) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Chemical Co, St Louis, MO, USA) containing 10% heat-inactivated FBS (fetal bovine serum), 50 U/ml penicillin and streptomycin. The cell cultures were maintained at 37 C in a humidified atmosphere of 5% CO2. Cell viability test Cell viability was determined via a commercial kit (WST-8 Cell Counting Kit-8, Beyotime, Nantong, China). According the manufacturer’s instructions, cells at a density of 3104 were placed in 96-well culture plates and cultured for the indicated time. Then, 10 l of the CCK-8 solution was added to each well and the cells were cultured at 37 C for another hour. Cell viability was assessed by measuring absorbance at 450 nm (ELX-800, Bio-Tek Instruments, Winooski, USA). Cell apoptosis assay The proapoptotic effect of PG was determined by beta-Interleukin I (163-171), human flow cytometry (FITC Annexin V apoptosis kit, BD Pharmingen, NJ, USA). Briefly, the cells were rinsed with ice-cold PBS buffer before being resuspended in binding buffer at a final density of 1106 cells/ml. The cells were then stained with annexin V-FITC and propidium iodide (PI) for 15 min in the dark, and the apoptosis rate was analyzed (Beckman Coulter Inc, FL, USA). Annexin V-FITC positive cells were regarded as undergoing apoptosis, and those negative for FITC were regarded as living cells. Determination of miRNA and mRNA expression Gene expression was determined by quantitative real time PCR (qPCR) using gene-specific primers as described previously14. In brief, total RNA was extracted using a commercial kit (RNeasy Mini kit, Qiagen, Dusseldorf, Germany). For miRNA expression, 40 ng of cDNA, which was obtained by reverse-transcription, beta-Interleukin I (163-171), human was used as a template for the PCR reaction14. mRNA expression beta-Interleukin I (163-171), human was detected using a master mix that included a SYBR Rabbit Polyclonal to KLF11 GREEN master mix (Solarbio Co, Beijing, China), a forward primer, a reverse primer, and template cDNA (10 beta-Interleukin I (163-171), human ng), on a BioRad iCycler. Gene expression was analyzed by using U6 or GAPDH as an internal standard. Construction of plasmids and cell transfection To investigate the role of survivin in PG-induced apoptosis in KB cells, survivin was overexpressed as previously described15. Briefly, a full-length cDNA fragment, encoding human survivin, was obtained by reverse transcription and PCR with the survivin primers15 and was inserted into the pEGFP-N1 vector (Takara Biomedical Technology Co, Ltd, Beijing, China). The resulting plasmid was named pEGFP-N1-survivin. Then, the pEGFP-N1-survivin vector was cloned into KB cells to produce survivin overexpression. KB cells were transfected with an empty pEGFP-N1 vector that used as a control. Forty-eight hours after transfection, a G418 solution was used to select the stable clones. Knockdown of survivin in.

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