Le cause of HIF-1a induction by triptolide, we used semi-quantitative
Le cause of HIF-1a induction by triptolide, we used semi-quantitative RT-PCR and quantitative real-time PCR assays for the levels of HIF-1a mRNA in the triptolide-treated SKOV-3 cells. The results showed that triptolide enhanced the levels of HIF-1a mRNA in a concentration-dependent mannerZhou et al. Molecular Cancer 2010, 9:268 http://www.molecular-cancer.com/content/9/1/Page 5 ofAorder BAY1217389 proteasome activity 1.Bproteasome activity 1.0 0.8 0.6 0.4 0.2 0 0 100 1000 10000 MG132 triptolide (nM),1 h, *0.8 0.6 0.4 0.2 0.Con50 100 500 1000 MG132 triptolide (nM), hypoxia,12 h, D triptolide (nM),12 h,hypoxia HIF-1 mRNA level 8 6 4 2 0C0 HIF-* * *-actin50 100 500 1000 triptolide (nM),12 h,hypoxiaFigure 3 Triptolide did not affect the 26 S proteasome activity but enhanced the levels of HIF-1a mRNA. A. SKOV-3 cells were treated with triptolide for 12 h under hypoxia condition and then harvested for the detection of 26 S proteasome activity as described in the Materials and Methods. B. The lysates of SKOV-3 cells were incubated in the presence or absence of triptolide and then assayed for the 26 S proteasome activity. C and D. The levels of HIF-1a mRNA from reverse transcription -PCR (C) and real-time PCR (D) analyses in the hypoxic SKOV-3 cells treated with triptolide for 12 h. The levels of HIF-1a mRNA were normalized with b-actin mRNA expression; columns, mean of three independent experiments; bars, SD. The significant difference between triptolide-treated groups and hypoxia-control groups was analyzed by Student t test. * P < 0.05.(Fig. 3C and 3D), which may be responsible for the HIF-1a accumulation.Triptolide reduces the transcriptional activity of HIF-1a proteinHIF-1a protein functions as a critical transcription factor in adaptive response to hypoxia [1]. To determine whether triptolide also increases the transcriptional activity of HIF-1a protein when enhancing its accumulation, we examined the expression levels of its several target genes including vascular endothelial growth factor (VEGF), BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and carbonic anhydrase IX (CAIX) [30,31] in the triptolide-treated SKOV-3 cells. Unexpectedly, however, the results revealed that the mRNA levels of the three genes did not increase but decreased typically in a concentration-dependent manner (Fig. 4A). Moreover, the secretion of VEGF protein, a critical angiogenesis factor, also reduced (Fig. 4B). Triptolide was further revealed to obviouslyinhibit the sprout outgrowth from the rat aorta rings (Fig. 4C), indicating its antiangiogenesis capability as previously reported [10,11,21]. To confirm whether triptolide reduces the transcriptional activity of HIF-1a protein, we used the hypoxia responsive element (HRE)-driven luciferase reporter gene assays. After failure with SKOV-3 or A549 cells due to the low transfection efficiency, we co-transfected the HRE luciferase reporter plasmid and the renilla luciferase reporter vector pGL-3 into MCF-7 cells for 24 h. Then the cells were treated with triptolide for additional 12 h. Triptolide reduced the luciferase-elicited fluorescence in a concentration-dependent fashion, and at 1000 nM of triptolide, the fluorescence almost lowered to the basal level (Fig. 4D). Collectively, the above evidence arising from all the levels of the transcription of the target genes, the reporter gene and the subsequent biological effects indicates that triptolide, though increasing the cellular accumulation of HIF-1a PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28300835 protein, reduc.