Data Availability StatementThe data used to support the findings of this
June 3, 2019
Data Availability StatementThe data used to support the findings of this study are available from the corresponding author upon request. hST3Gal Torisel cost V promoter: (forward) 5-GCCCCGGGTGCGTCCCTG-3 and (reverse) 5-AGCGCCGCTCTCGCGCC-3. 3. Results 3.1. Effect of Curcumin around the Proliferation of HCT116 Cells The cytotoxicity of curcumin in HCT116 cells was investigated using MTT assay. HCT116 cells were treated with curcumin in various concentrations for 12 and 24 h. As shown Physique 1, curcumin inhibited dose- and time-dependently HCT116 cell proliferation. Cell viability in cells treated with curcumin for 24 h was significantly decreased compared to those for 12 h. Open in a separate window Physique 1 Effect of curcumin on cell viability of HCT116 cells. HCT 116 cells were exposed to various concentrations of curcumin (0, 20, 40, 60, 80 hST3Gal V gene expressionis transcriptionally downregulated by curcumin in HCT116 cells. Open in a separate window Physique 3 Effect of curcumin on hST3Gal V mRNA levels in HCT116 cells. HCT116 cells were treated for 24 h at Rabbit Polyclonal to TF2H1 different concentrations (0, 10, 20, 30, 40, 50 0.001versusthe control. 3.4. Effect of Curcumin on Ganglioside GM3 Expression in HCT116 Cells To investigate whether or not the decrease of hST3Gal V gene expression by curcumin treatment causes the reduction of ganglioside GM3 level synthesized by hST3Gal Torisel cost V in HCT116 cells, the level of cellular expression of ganglioside GM3 was determined by immunofluorescence confocal microscopy using anti-GM3 mAb and FITC-conjugated anti-mouse IgG/M/A mixture as secondary. As shown in Physique 4, ganglioside GM3 expression was significantly decreased in HCT116 cells treated with 30 Renillaluciferase activity derived from pRL-TK. Data are presented as the means SD of three impartial experiments with triplicate measurements. (b) ChIP assay performed in curcumin-treated HCT116 cells, or nontreated cells with insight control (without Torisel cost antibody) and non-specific immunoglobulin (IgG), CREB, and ATF antibodies. The precipitated chromatin was amplified by PCR with primers particular for the CREB/ATF consensus binding site on hST3Gal V promoter. Our prior studies have confirmed that Torisel cost a putative CREB/ATF consensus binding site (5-TGACGTCA-3) located at placement -143 in your community -177/-83 is vital for the appearance of hST3Gal V gene in a variety of kind of cells [13, 14, 16C18]. Hence, we next looked into whether CREB/ATF was mixed up in appearance from the hST3Gal V gene induced by curcumin. The pGL3-177 CREB/ATF Mut build using a mutation at CREB/ATF site was transfected into HCT116 cells and promoter assay was completed. As proven in Body 5(a), the promoter activity attained with pGL3-177 CREB/ATF Mut was less than that using the pGL3-Simple vector irrespective of curcumin treatment, indicating that destruction from the CREB/ATF site affected significantly the promoter activity. This result shows that this CREB/ATF site is vital for the appearance of hST3Gal V gene in HCT116 cells. To confirm that CREB and ATF interact with the hST3Gal V promoter in vivo, we performed ChIP experiments with CREB and ATF antibodies and nuclear extracts from HCT116 cells treated with curcumin. After formaldehyde cross-linking, sonication, and precipitation of the chromatin with CREB, ATF, or IgG antibodies, the precipitated DNA was subjected to PCR amplification using primers flanking the CREB/ATF binding site around the hST3Gal V Torisel cost promoter. As shown in Physique 5(b), the amplified products were observed in CREB and ATF immunoprecipitations, whereas no amplified product was detected in IgG immunoprecipitation, indicating that CREB/ATF expressed in HCT116 cells experienced the ability to bind specifically to the CRE/ATF site of hST3Gal V promoter. On the other hand, the amplified transmission in CREB and ATF immunoprecipitations in curcumin-treated cells was decreased to about 45% and 58% of that of untreated cells, respectively. This result indicated that curcumin suppressed the transcription of hST3Gal V gene, at least in part, by inhibiting CREB/ATF-mediated transcriptional activity. 3.6. Transcriptional Activation of hST3Gal V Gene via AMPK Pathway in HCT116 Cells We next investigated the transmission transduction pathway responsible for transcriptional activation of hST3Gal V gene expression in HCT116 cells. As shown in Figure.