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Pawlus M.R.,Aurora University | Wang L.,Aurora University | Murakami A.,Aurora University | Dai G.,Zhejiang Academy of Traditional Chinese Medicine | Hu C.-J.,Aurora University
PLoS ONE | Year: 2013

The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein. © 2013 Pawlus et al. Source


Ji L.,Zhejiang University | Yuan Y.,Zhejiang University | Luo L.,Zhejiang University | Chen Z.,Chinese Traditional Medicine Hospital of Zhejiang Province | And 3 more authors.
Steroids | Year: 2012

Michael reaction acceptors (MRAs) are a class of active molecules that are directly or indirectly involved in various cellular processes, including the regulation of many signaling pathways. In this study, the inducible nitric oxide synthase (iNOS) assay was used to demonstrate that the dichloromethane extract of Physalis alkekengi var. franchetii (DCEP) possesses anti-inflammatory activity that might be attributed to the modification of key cysteine residues in IKKβ by the MRAs in DCEP. To isolate these MRAs, glutathione (GSH) was employed, and a simple ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) screening method was developed to investigate the GSH conjugates with potential MRAs. Five physalins, including one new compound isophysalin A (2), together with four known steroidal compounds, physalin A (1), physalin O (3), physalin L (4) and physalin G (5), were isolated to evaluate the GSH conjugating abilities, and it was indicated that compounds 1, 2 and 3, which had a common α,β-unsaturated ketone moiety, exhibited conjugating abilities with GSH and also showed significant nitric oxide (NO) production inhibiting activities. The anti-inflammatory activities of compounds 1, 2 and 3 might be attributed to their targeting multiple cysteine residues on IKKβ; therefore, the alkylation of IKKβ by compound 1 was further studied by micrOTOF-MS. The result showed that six cysteine residues (C 59, C 179, C 299, C 370, C 412, and C 618) were alkylated, which indicated that IKKβ is a potential target for the anti-inflammatory activity of physalin A. © 2011 Elsevier Inc. All rights reserved. Source


Hu Y.,Zhejiang University | Hu Y.,Zhejiang Pharmaceutical College | Xu B.,Zhejiang Pharmaceutical College | Ji Q.,Zhejiang Pharmaceutical College | And 6 more authors.
Biomaterials | Year: 2014

Polyethylenimine (PEI) is widely applied in non-viral gene delivery vectors. PEI with high molecular weight is highly effective in gene transfection but is high cytotoxic. Conversely, PEI with low molecular weight displays lower cytotoxicity but less delivering efficiency. To overcome this issue, a novel copolymer with mannosylated, a cell-penetrating peptide (CPP), grafting into PEI with molecular weight of 1800 (Man-PEI1800-CPP) were prepared in this study to target antigen-presenting cells (APCs) with mannose receptors and enhance transfection efficiency with grafting CPP. The copolymer was characterized by 1H NMR and FTIR. Spherical nanoparticles were formed with diameters of about 80-250nm by mixing the copolymer and DNA at various charge ratios of copolymer/DNA(N/P). Gel retardation assays indicated that Man-PEI1800-CPP polymers efficiently condensed DNA at low N/P ratios. Cytotoxicity studies showed that Man-PEI1800-CPP/DNA complexes maintained in a high percentage of cell viability compared to the PEI with molecular weight of 25k (PEI25k). Laser scan confocal microscopy and flow cytometry confirmed that Man-PEI1800-CPP/DNA complexes resulted in higher cell uptake efficiency on DC2.4 cells than on Hela cells line. The transfection efficiency of Man-PEI1800-CPP was significantly higher than that of PEI25k on DC2.4 cells. More importantly, the complexes were mainly distributed in the epidermis and dermis of skin and targeted on splenocytes after percutaneous coating based on microneedles invivo. These results indicated that Man-PEI1800-CPP was a potential APCs targeted of non-virus vector for gene therapy. © 2014 . Source


Zhu H.,Zhejiang University | Yang W.,Zhejiang University | He L.-j.,Zhejiang University | Ding W.-j.,Zhejiang University | And 6 more authors.
PLoS ONE | Year: 2012

The human hepatocellular carcinoma (HCC) represents biologically aggressive and chemo-resistant cancers. Owing to the low affinity with the apoptotic factor Mcl-1, the BH3 mimetic drug ABT-737 failed to exert potent cancer-killing activities in variety of cancer models including HCC. The current study demonstrated that combining ABT-737 and Celastrol synergistically suppressed HCC cell proliferation, and induced apoptosis which was accompanied with the activation of caspase cascade and release of cytochrome c from mitochondria. Further study revealed that the enhanced Noxa caused by Celastrol was the key factor for the synergy, since small interfering RNA-mediated knockdown of Noxa expression in HCC cells resulted in decreased apoptosis and attenuated anti-proliferative effects of the combination. In addition, our study unraveled that, upon Celastrol exposure, the activation of endoplasmic reticulum (ER) stress, specifically, the eIF2α-ATF4 pathway played indispensable roles in the activation of Noxa, which was validated by the observation that depletion of ATF4 significantly abrogated the Noxa elevation by Celastrol. Our findings highlight a novel signaling pathway through which Celastrol increase Noxa expression, and suggest the potential use of ATF4-mediated regulation of Noxa as a promising strategy to improve the anti-cancer activities of ABT-737. © 2012 Zhu et al. Source


Zhong L.-R.,Zhejiang Chinese Medical University | Chen X.,ZheJiang HuaYi Pharmaceutical Co. | Wei K.-M.,Zhejiang Academy of Traditional Chinese Medicine
Asian Pacific Journal of Cancer Prevention | Year: 2013

Radix Tetrastigma Hemsleyani Flavone (RTHF) is widely used as a traditional herb for its detoxification and anti-inflammation activity. Recently, several studies have shown that RTHF can inhibit growth and induce apoptosis in human cancer cell lines. However, the mechanisms are not completely understood yet. In this study we investigated the potential effects of RTHF on growth and apoptosis in human lung adenocarcinoma A549 cells as well as its mechanisms. A549 cells were treated with RTHF at various concentrations for different times. In vitro the MTT assay showed that RTHF had obvious anti-proliferation effects on A549 cells in a dose- and timedependent manner. Cell morphological changes observed by inverted microscope and Hoechst33258 methods were compared with apoptotic changes observed by fluorescence microscope. Cell apoptosis inspected by flow cytometry showed significant increase in the treatment group over the control group (P<0.01). Expression of apoptosis related Bax/Bcl-2, caspases and MAPK pathway proteins were detected by Western blotting. The results showed that RTHF up-regulated the Bax/Bcl-2 ratio and cle-caspase3/9, cle-PARP expression in a dosedependent manner. Expression of p-p38 increased, p-ERK decreased significantly and that of p-JNK was little changed in the RTHF group when compared with the control group. These results suggest that RTHF might exert anti-growth and apoptosis activity against lung cancer A549 cells through activation of caspases and Bcl-2 family proteins and the MAPK pathway, therefore presenting as a promising therapeutic agent for the treatment of lung cancer. Source

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