Co-reporter:Bo Zhu, Jia-yu Wang, Jun-jie Zhou, Feng Zhou, Wei Cheng, Ying-ting Liu, Jie Wang, Xiao Chen, Dian-hua Chen, Lan Luo, Zi-Chun Hua
Journal of Inorganic Biochemistry 2017 Volume 175(Volume 175) pp:
Publication Date(Web):1 October 2017
DOI:10.1016/j.jinorgbio.2017.07.007
•Zinc stabilized promyelocytic leukemia protein-retinoic acid receptor alpha (PML-RARα).•Zinc depletion triggered PML-RARα degradation via the proteasome pathway.•Autophagy inhibited PML-RARα degradation triggered by zinc depletion.•Crosstalk between zinc and nitric oxide signaling stabilized PML-RARα.•Zinc depletion induced apoptosis in NB4 cells.Acute promyelocytic leukemia (APL) is characterized and driven by the promyelocytic leukemia protein-retinoic acid receptor alpha (PML-RARα) fusion gene. Previous studies have highlighted the importance of PML-RARα degradation in the treatment against APL. Considering the presence of two zinc fingers in the PML-RARα fusion protein, we explored the function of zinc homeostasis in maintaining PML-RARα stability. We demonstrated for the first time that zinc depletion by its chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) triggered PML-RARα degradation in NB4 APL cells via the proteasome pathway rather than the autophagy-lysosomal pathway. In contrast, autophagy protected TPEN-mediated PML-RARα degradation in NB4 APL cells. We further demonstrated that crosstalk between zinc homeostasis and nitric oxide pathway played a key role in maintaining PML-RARα stability in NB4 APL cells. These results demonstrate that zinc homeostasis is vital for maintaining PML-RARα stability, and zinc depletion by TPEN may be useful as a potential strategy to trigger PML-RARα degradation in APL cells. We also found that TPEN triggered apoptosis of NB4 APL cells in a time-dependent manner. The relationship between PML-RARα degradation and apoptosis triggered by TPEN deserves further study.Zinc depletion triggers promyelocytic leukemia protein-retinoic acid receptor alpha (PML-RARα) degradation via the proteasome pathway and induces apoptosis in NB4 cells. Autophagy may play a protective role in PML-RARα degradation triggered by zinc depletion. The crosstalk between zinc and nitric oxide pathway is important for PML-RARα stability.Download high-res image (178KB)Download full-size image
Co-reporter:Kaizong Huang;Lingli Zhu;Yunke Wang;Ran Mo
Journal of Materials Chemistry B 2017 vol. 5(Issue 31) pp:6356-6365
Publication Date(Web):2017/08/09
DOI:10.1039/C7TB00190H
Developing a smart cancer drug delivery carrier with enhanced cancer-targeting and effective drug release in tumors is critical for efficient cancer chemotherapy. Herein, we designed a pH-responsive copolymer (PEG-ELP[VH4-70]) that entraps DOX into a hydrophobic core and self-assembles into smart DOX-loaded nanoparticles. The DOX-loaded nanoparticles were stabilized by Zn2+ and disrupted as the pH drops from 7.4 to 5.6. An in vitro study demonstrated that the DOX-loaded nanoparticle system exhibited efficient internalization, triggered the release of DOX into the cytoplasm and enabled the inhibition of tumors effectively. When used to deliver chemotherapeutics to a murine cancer model, PEG-ELP[VH4-70]/DOX accomplished a 4.8-fold suppressed effect relative to the free drug after intravenous injection. This simple strategy can promote preeminent stability for targeting hydrophobic drugs to tumor tissues.
Co-reporter:Qing Zhang, Weijie Lu, Lina Ji, Zi-Chun Hua
Protein Expression and Purification 2017 Volume 135(Volume 135) pp:
Publication Date(Web):1 July 2017
DOI:10.1016/j.pep.2017.04.016
•Recombinant 8 kDa fragment of gelsolin was overexpressed in fusion with intein to increase its stability and solubility.•Released by intein-mediated protein cleavage, 8 kDa fragment of gelsolin is obtained with a yield up to 4.25 mg/L.•Purified 8 kDa fragment of gelsolin is validated to be amyloidogenic.A mutation (D187N/Y) in human plasma gelsolin (GSN) leads to the generation of an 8 kDa GSN fragment (8 kDa-GSN), and consequently causes the familial amyloidosis of Finnish type. Because of its faster kinetics of amyloid formation under physiologically relevant conditions, 8 kDa-GSN is used to explore gelsolin amyloidosis and screen small molecules that can disaggregate amyloids. However, the synthetic 8 kDa-GSN is expensive, and substantial quantities of 8 kDa-GSN are needed for the screen. Here we report a study to obtain recombinant 8 kDa-GSN with high yield from Escherichia coli. Firstly, 8 kDa-GSN in fusion with Mxe GyrA intein was purified by Ni-affinity chromatography. Then 8 kDa-GSN was released by intein-mediated protein cleavage, and separated from intein by ion-exchange chromatography. The yield of 8 kDa-GSN was only 1.5 mg/L from bacterial culture in the previous report, while it was improved to 4.25 mg/L in our study. Finally, the amyloidogenic property of 8 kDa-GSN was validated by circular dichroism spectrometry and dynamic light scattering.
Co-reporter:Kun-Zhi Jia, Shu-Lei Jin, Chun Yao, Rong Rong, ... Zi-Chun Hua
Biochimie 2017 Volume 138(Volume 138) pp:
Publication Date(Web):1 July 2017
DOI:10.1016/j.biochi.2017.04.005
•Pthrp KI mice display abnormal development of T cells.•Pthrp KI mice show impaired T-cell proliferation.•Pthrp KI mice show increased apoptosis of T cells.Parathyroid hormone-related protein (PTHrP), a ubiquitously expressed protein, is composed of four functional domains including N-terminus, mid region, nuclear localization signal (NLS) and C-terminus. Under the direction of NLS, PTHrP can enter cell nucleus from cytoplasm and stimulate mitogenesis. Although PTHrP is considered to have important developmental roles, the role of PTHrP NLS and C-terminus in developmental process remains unknown, especially in T-cell development. Here, we used a knock-in mouse model, which expresses a truncated form of PTHrP missing the NLS (87–107) and C-terminus (108–139) of the protein, to examine the role of PTHrP NLS and C-terminus in T-cell development. Our results showed that the truncated PTHrP (1-84) led to abnormal subpopulations, impaired proliferation and increased apoptosis in the thymus, indicating that PTHrP is involved in the development of T cells, and the NLS and C-terminus part is necessary for the normal role of PTHrP in T-cell development.
Co-reporter:Xiufeng Liu, Kai Wang, Ningjun Duan, Yan Lan, Pengcheng Ma, Heng Zheng, Weijuan Zheng, Jiahuang Li and Zi-chun Hua
RSC Advances 2015 vol. 5(Issue 44) pp:34572-34579
Publication Date(Web):09 Apr 2015
DOI:10.1039/C4RA17009A
Triptolide, triptonide and triptriolide are active ingredients of traditional Chinese herbal medicine Tripterygium wilfordii Hook.f. Although these compounds are found to have significant anti-inflammatory, immune-suppressive or anti-tumour effects, the molecular mechanisms of actions, especially their binding to proteins remain unclear. Since the chemical structures of triptolide, triptonide and triptriolide are similar to steroid hormones, we try to identify potential target proteins from the steroid hormone receptors (or “nuclear receptors”). In this study, using the reverse docking strategy, 12 nuclear receptors are reversely docked to triptolide and ranked by the binding energy scores. Based on this, human estrogen receptor alpha (ERα) was selected as a potential interaction protein for triptolide and the binding mode of three compounds to ERα-LBD (ligand binding domain) was further assessed by Docking and Molecular Dynamics (MD) simulation. To further analyze the docking results, Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) and Reporter Gene assays were used to validate the interactions of ERα-LBD with the three compounds. SPR studies together with ITC measurements indicated that the three compounds could bind to ERα-LBD with weak affinity. Triptonide showed the highest affinity and triptriolide exhibited the weakest affinity. Furthermore, the binding of triptonide or triptolide to ERα significantly increased the reporter gene activity in human cervical cancer cell lines HeLa. This study not only further defines the binding proteins of triptolide and its analogues but also provides useful information for application of these compounds.
Co-reporter:Jie Wang;Liangqiang He;Dianhua Chen;Yazhou Pi
European Biophysics Journal 2015 Volume 44( Issue 5) pp:325-336
Publication Date(Web):2015/07/01
DOI:10.1007/s00249-015-1026-9
We constructed a green fluorescent phosphatidylserine (PS)-binding probe, which was generated by fusing enhanced green fluorescent protein (EGFP) to the C terminus of human annexin V (anxV). With this probe, we investigated anxV–membrane interaction under different calcium and anxV-EGFP concentrations through flow cytometry (FCM). A mathematical description of the binding characteristics is proposed and validated to quantify the relationship concerning the relative concentration of membrane-bound anxV (B), calcium concentration ([C]), and protein concentration ([P]). Further analyses reveal that \(\frac{1}{B}\) is linear with \(\frac{1}{{[\text{C}]^{2} }}\) or \(\frac{1}{[\text{P}]}\) when [P] and [C] are fixed, respectively, which indicates that the anxV–membrane binding reaction may involve sequential multiple steps. Our study provides a reference for application of anxV in apoptosis detection. The mathematical expression facilitates exploration of the possible interactions between calcium, anxV, and membrane. The corresponding mathematical analysis strengthens the interpretation of the interaction data.
Co-reporter:Jiahuang Li;Yuan Chen;Jie Yang
Biopolymers 2015 Volume 103( Issue 5) pp:247-259
Publication Date(Web):
DOI:10.1002/bip.22589
ABSTRACT
The Schistosoma juponicum 26 kDa glutathione S-transferase (sj26GST) consists of the N-terminal domain (N-domain), containing three alpha-helices (named H1-H3) and four anti-parallel beta-strands (S1-S4), and the C-terminal domain (C-domain), comprising five alpha-helices (named H4-H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N-domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N-domain could not fold independent, suggesting that correct folding of N-domain depended on its interactions with C-domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C-domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C-domain and N-domain. The paper can offer insights into the molecular mechanism of GSTs unfolding. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 247–259, 2015.
Co-reporter:Xiufeng Liu;Fan Qiu;Zhipeng Liu;Yan Lan;Kai Wang;Ping-Kun Zhou;Yao Wang
Apoptosis 2014 Volume 19( Issue 10) pp:1532-1544
Publication Date(Web):2014 October
DOI:10.1007/s10495-014-1025-9
The urokinase-type plasminogen activator receptor (uPAR) serves not only as an anchor for urokinase-type plasminogen activator but also participates in intracellular signal transduction events. In this study, we investigated whether uPAR could modulate TRAIL-induced apoptosis in human colon cancer cells HCT116. Using an antisense strategy, we established a stable HCT116 cell line with down-regulated uPAR. The sensitivity to TRAIL-induced apoptosis was evaluated by FACS analysis. Our results show that the inhibition of uPAR could sensitize HCT116 to TRAIL-induced apoptosis. uPAR inhibition changed the expression of mitochondrial apoptotic pathway proteins, including Bcl-2, Bax, Bid and p53, in a pro-apoptotic manner. We also found that the inhibition of uPAR down-regulated the phosphorylation of FAK, ERK and JNK. The inhibition of p53 by RNA interference rescued cells from enhanced apoptosis, thus indicating that p53 is critical for enhancing TRAIL-induced apoptosis. Furthermore, JNK, but not ERK, inhibition involved in the up-regulation of p53. JNK negatively regulated p53 protein level. Overall, our results show that uPAR inhibition can sensitize colon cancer cells HCT116 to TRAIL-induced apoptosis via active p53 and mitochondrial apoptotic pathways that JNK inhibition is involved.
Co-reporter:Yan Lan;Xiufeng Liu;Rong Zhang;Kai Wang;Yao Wang;Zi-Chun Hua
BioMetals 2013 Volume 26( Issue 2) pp:241-254
Publication Date(Web):2013 April
DOI:10.1007/s10534-012-9607-x
Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.
Co-reporter:Wenjing Zhang;Yan Lan;Qilai Huang
Cytotechnology 2013 Volume 65( Issue 3) pp:447-455
Publication Date(Web):2013 May
DOI:10.1007/s10616-012-9499-1
Galangin, an active flavonoid present at high concentration in Alpinia officinarum Hance and propolis, shows cytotoxicity towards several cancer cell lines, including melanoma. However, the specific cellular targets of galangin-induced cytotoxicity in melanoma are still unknown. Here, we investigated the effects of galangin in B16F10 melanoma cells and explored the possible molecular mechanisms. Galangin significantly decreased cell viability of B16F10 cells, and also induced cell apoptosis shown by Hoechst 33342 staining and Annexin V-PI double staining flow cytometric assay. Furthermore, upon galangin treatment, disruption of mitochondrial membrane potential was observed by JC-1 staining. Western blotting analysis indicated that galangin activated apoptosis signaling cascades by cleavage of procaspase-9, procaspase-3 and PARP in B16F10 cells. Moreover, galangin significantly induced activation of phosphor-p38 MAPK in a time and dose dependent manner. SB203580, an inhibitor of p38, partially attenuated galangin-induced apoptosis in B16F10 cells. Taken together, this work suggests that galangin has the potential to be a promising agent for melanoma treatment and may be further evaluated as a chemotherapeutic agent.
Co-reporter:Guo Chen;Bo Tang;Bing-Ya Yang;Jian-Xiang Chen
Applied Microbiology and Biotechnology 2013 Volume 97( Issue 10) pp:4393-4401
Publication Date(Web):2013 May
DOI:10.1007/s00253-012-4321-8
The PNP/6-methylpurine 2′-deoxyriboside (6MePdR) system is an efficient gene-directed enzyme prodrug therapy system with significant antitumor activities. In this system, Escherichia coli purine nucleoside phosphorylase (ePNP) activates nontoxic 6MePdR into potent antitumor drug 6-methylpurine (6MeP). The Salmonella typhimurium PNP (sPNP) gene has a 96-% sequence homology in comparison with ePNP and also has the ability to convert 6MePdR to 6MeP. In this study, we used tumor-targeting S. typhimurium VNP20009 expressing endogenous PNP gene constitutively to activate 6MePdR and a combination treatment of bacteria and prodrug in B16F10 melanoma model. The conversion of 6MePdR to 6MeP by S. typhimurium was analyzed by HPLC and the enzyme activity of sPNP was confirmed by in vitro (tetrazolium-based colorimetric assay) MTT cytotoxicity assay. After systemic administration of VNP20009 to mice, the bacteria largely accumulated and specifically delivered endogenous sPNP in the tumor. In comparison with VNP20009 or 6MePdR treatment alone, combined administration of VNP20009 followed by 6MePdR treatment significantly delayed the growth of B16F10 tumor and increased the CD8+ T-cell infiltration. In summary, our results demonstrated that the combination therapy of S. typhimurium and prodrug 6MePdR is a promising strategy for cancer therapy.
Co-reporter:Su Feng;Wei Chen;Dan Cao;Jinjun Bian
Cellular and Molecular Life Sciences 2011 Volume 68( Issue 1) pp:109-124
Publication Date(Web):2011 January
DOI:10.1007/s00018-010-0444-1
Increasing evidence demonstrates that Na+, K+-ATPase plays an important role in pulmonary inflammation, but the mechanism remains largely unknown. In this study, we used cardiotonic steroids as Na+, K+-ATPase inhibitors to explore the possible involvement of Na+, K+-ATPase in pulmonary epithelial inflammation. The results demonstrated that mice after ouabain inhalation developed cyclooxygenase-2-dependent acute lung inflammation. The in vitro experiments further confirmed that Na+, K+-ATPase inhibitors significantly stimulated cyclooxygenase-2 expression in lung epithelial cells of human or murine origin, the process of which was participated by multiple cis-elements and trans-acting factors. Most importantly, we first described here that Na+, K+-ATPase inhibitors could evoke a significant Hu antigen R nuclear export in lung epithelial cells, which stabilized cyclooxygenase-2 mRNA by binding with a proximal AU-rich element within its 3′-untranslated region. In conclusion, HuR-mediated mRNA stabilization opens new avenues in understanding the importance of Na+, K+-ATPase, as well as its inhibitors in inflammation.
Co-reporter:Yuan Chen, Dingyuan Ma, Qi-Lai Huang, Weijuan Zheng, Jing Zhang, Yi Shen, Jiahuang Li, Wei Dong, Min Lu, Jin Wang and Zi-Chun Hua
Cellular & Molecular Immunology 2009 6(4) pp:295-301
Publication Date(Web):2009-08-01
DOI:10.1038/cmi.2009.39
FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecular structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain an FADD variant that containing C-terminal region. In this study, mouse FADD (80-205) containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni2+ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical -helix spectrum in CD (circular dichroism) assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK I-binding activity which was important for its non-apoptotic function.
Co-reporter:Faiz MMT Marikar, Dingyuan Ma, Jianqiang Ye, Bo Tang, Weijuan Zheng, Jing Zhang, Min Lu and Zichun Hua
Cellular & Molecular Immunology 2008 5(6) pp:471-474
Publication Date(Web):2008-12-01
DOI:10.1038/cmi.2008.59
The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichia coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refolded and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD protein was allowed the production of high titre polyclonal antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.
Co-reporter:Xiangbai Dong;Jie Li;Shufeng Li;Jing Zhang;Zi-chun Hua
Molecular Biotechnology 2008 Volume 38( Issue 2) pp:129-135
Publication Date(Web):2008 February
DOI:10.1007/s12033-007-9002-y
The apoptotic adapter protein FADD has been shown to play diverse roles in cell survival and proliferation. FADD knockout embryos died of heart defects, rendering Cre/loxP-mediated conditional FADD knockout mice a unique tool for investigating FADD-dependent nonapoptotic mechanism. Previously, these genetically engineered mice were identified by time-consuming Southern blot or controversial real-time PCR. In this article, we report a novel genotyping strategy based on allele-specific inverse PCR (ASI-PCR) for rapid and reliable identification of conditional FADD knockout mice. In this strategy, the knockout nature of FADD was simply identified by screening the absence of the wild type FADD-specific ASI-PCR product. Using this method, we accurately identified CD4-Cre-mediated T cell specific FADD knockout mice. The whole process can be accomplished in any normal biological laboratory within 12 h using genomic DNA from tail biopsy. The proposed ASI-PCR-based approach is simple, rapid, sensitive, reproducible, and especially suitable for genotyping small amount of spatiotemporally restricted biopsies and large animal population. We believe that the strategy described in this article may be of general utility in genotyping other conditional gene knockout mice.
Co-reporter:Dingyuan Ma, Yuan Chen, Lei Fang, Guanghui Jin, Bin Zhou, Lin Cao, Jianqiang Ye, Zichun Hua
Journal of Chromatography B 2007 Volume 857(Issue 2) pp:231-239
Publication Date(Web):1 October 2007
DOI:10.1016/j.jchromb.2007.07.022
A number of approaches have been investigated to enhance the selective toxicity of tumor necrosis factor α (TNFα) to permit its systemic use in cancer therapy. Because vascular targeting has been proven to be a valid strategy for improving the therapeutic index of TNFα, we prepared RGD-hTNF consisting of human TNF fused with the ACDCRGDCFCG peptide, a ligand of αvβ3 and αvβ5 integrins. Recombinant RGD-hTNF was produced in Escherichia coli as a polyhistidine fusion protein. Between polyhistidine tag and RGD-hTNF, a tobacco etch virus (TEV) protease cleavage site (ENLYFQG) was introduced to ensure the release of intact RGD-hTNF. The purification strategy consisted of the target protein capture step by immobilized metal affinity chromatography (IMAC), TEV protease cleavage of fusion protein, the subtractive depletion of removed His-tag by IMAC and the final gel filtration step. As a result, about 18 mg of intact RGD-hTNF was obtained from 1 l of bacteria culture. The purified RGD-hTNF was characterized by SDS-PAGE, Western blot, mass spectroscopy and gel filtration. Since the RGD-hTNF molecule retained the cytotoxic activity of the TNF moiety and the integrin binding ability of the RGD moiety, the purification method provided material for assessing its anti-tumor activity in animal model.
Co-reporter:W Yin, W Cheng, W Shen, L Shu, J Zhao, J Zhang and Z-C Hua
Leukemia 2007 21(8) pp:1669-1678
Publication Date(Web):June 7, 2007
DOI:10.1038/sj.leu.2404791
Human T-cell leukemia is a malignant disease that needs various regimens of cytotoxic chemotherapy to overcome drug resistance. Recently, Na+,K+-ATPase has emerged as a potential target for cancer therapy. However, its exact signaling pathway in human T-cell leukemia cell death has not been well defined. In the current study, we found CD95(APO-1) was able to trigger the internalization of plasma membrane Na+,K+-ATPase in Jurkat cells or primary T cells as a mechanism to suppress its activity. This internalization was closely relevant to intracellular glutathione (GSH) depletion in Jurkat cells downstream of Fas-associated death domain protein (FADD) and caspase 8. GSH depletion in Fas L-treated Jurkat cells induced the generation of hydrogen peroxide (H2O2), which subsequently increased the serine phosphorylation of Na+,K+-ATPase 1 subunit. Exogenous H2O2 even mimicked the effect of Fas L to upregulate the serine phosphorylation of Na+,K+-ATPase 1 subunit and suppress Na+,K+-ATPase activity. Overall, our results indicate that CD95(APO-1) induces the FADD- and caspase 8-dependent internalization of Na+,K+-ATPase through intracellular GSH loss, and the subsequent generation of H2O2-mediated serine phosphorylation of Na+,K+-ATPase 1 subunit. Taken together, this study presents a novel regulatory mechanism of Na+,K+-ATPase in CD95(APO-1)-mediated human T-leukemia cell apoptosis.
Co-reporter:Qi-Ming SUN;Faiz M. M. T. MARIKAR;Zi-Chun HUA
Acta Biochimica et Biophysica Sinica 2006 Volume 38(Issue 5) pp:305-309
Publication Date(Web):18 MAY 2006
DOI:10.1111/j.1745-7270.2006.00167.x
Abstract Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the cDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli. Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.
Edited by Ming-Hua XU
Co-reporter:Guang-Hui JIN;Wei DONG;Shu-Feng LI;Qi-Ming SUN;Ding-Yuan MA;Zi-Chun HUA
Acta Biochimica et Biophysica Sinica 2006 Volume 38(Issue 11) pp:780-787
Publication Date(Web):15 NOV 2006
DOI:10.1111/j.1745-7270.2006.00220.x
Abstract Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. To enhance its gene delivery efficiency and minimize cytotoxicity, we have synthesized small cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro. In this study, branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate [1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2–6 h. The efficiencies of the cross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein (EGFP) reporter gene were assessed in melanoma B16F10 cell line and other cell lines. Flow cytometry was used to quantify the cellular entry efficiency of plasmid and the transgene expression level. The cytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay. EGDMA-PEI 800–4h, a typical cross-linked PEI reported here, mediated a more efficient expression of reporter gene than the commercially available 25-kDa branched PEI control, and resulted in a 9-fold increase in gene delivery in B16F10 cells and a 16-fold increase in 293T cells, while no cytotoxicity was found at the optimized condition for gene delivery. Furthermore, the transfection activity of polyplexes was preserved in the presence of serum proteins.
Edited by Ming-Hua XU
Co-reporter:Lu Wang, Liangqiang He, Rong Zhang, Xiufeng Liu, Yongzhe Ren, Zhipeng Liu, Xiangyu Zhang, Wei Cheng, Zi-Chun Hua
Molecular Immunology (June 2014) Volume 59(Issue 2) pp:163-171
Publication Date(Web):1 June 2014
DOI:10.1016/j.molimm.2014.02.004
•MicroRNA-21 was induced immediately during early T lymphocyte activation.•MicroRNA-21 promoted ERK and JNK signaling and furthermore, enhanced AP-1 activity and interleukin-2 expression.•ERK and JNK promoted microRNA-21 expression.•MicroRNA-21 regulates TCR-activated downstream genes.MicroRNAs are small noncoding RNAs that act as posttranscriptional regulators of gene expression. To identify microRNAs involved in T cell activation, we performed a microRNA array profiling with Jurkat cells. We found that microRNA-21 (miR-21), which is upregulated in many tumors by targeting a series of tumor suppressor genes to promote tumor growth, was significantly increased in activated Jurkat cells and primary CD4+ T lymphocytes compared with that in quiescent counterparts. By using a signaling network building tool, miR-21 was predicted regulates ERK and JNK signaling in activated Jurkat cells. Indeed, miR-21 promotes ERK and JNK signaling in activated T cells. Sprouty1, a direct target of miR-21 that has been shown an inhibitor of ERK and JNK, was also inhibited by forced miR-21 expression in activated T cells. Reciprocally, miR-21 levels were induced by MEK or JNK signaling response to T cell receptor (TCR) engagement. Furthermore, transfection with miR-21 mimic promotes activator protein 1 (AP-1) activity and interleukin-2 (IL-2) expression. These results provide a missing function of miR-21 in TCR-mediated signaling transduction in T lymphocytes, suggesting that miR-21 may augment T cell immune response by a positive feedback mechanism.
Co-reporter:Lei Fang, Kun-Zhi Jia, Ya-Lan Tang, Ding-Yuan Ma, Mei Yu, Zi-Chun Hua
Protein Expression and Purification (January 2007) Volume 51(Issue 1) pp:102-109
Publication Date(Web):1 January 2007
DOI:10.1016/j.pep.2006.07.003
Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni2+ affinity chromatography, 65 mg of His6-TEV was isolated with purity over 95% from 1 L of culture. The enzyme activity of His6-TEV was generally characterized by using GST–EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties.
Co-reporter:Wenjing Zhang, Qilai Huang, Zichun Hua
Acta Pharmaceutica Sinica B (December 2012) Volume 2(Issue 6) pp:
Publication Date(Web):1 December 2012
DOI:10.1016/j.apsb.2012.10.009
Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising antitumor therapy against lung cancer which is currently undergoing a phase III clinical trial in China. Unfortunately some cancer patients in the clinical trial displayed resistance to TRAIL treatment. In investigating ways to overcome this resistance, we evaluated the inhibitory effect of galangin on TRAIL resistant A549 human lung adenocarcinoma cells. Here we report that, in comparison with the single agents, the combination of galangin and TRAIL markedly suppressed proliferation of A549 cells and induced apoptosis as shown by DAPI and JC-1 staining. The combination of galangin and TRAIL induced PARP cleavage, activation of caspase-8 and p38 MAPK (mitogen-activated protein kinases). These findings indicate that the combination of galangin and TRAIL may constitute a promising strategy for the treatment of lung cancer.Graphical abstractThe combination of galangin and TRAIL may constitute a promising strategy for the treatment of lung cancer.Download full-size image
Co-reporter:Shi-Ying LI, Xiang-Yu ZHANG, Xin ZHANG, Yan LAN, Zi-Chun HUA
Biomedical and Environmental Sciences (December 2010) Volume 23(Issue 6) pp:496-501
Publication Date(Web):December 2010
DOI:10.1016/S0895-3988(11)60013-5
Co-reporter:Luan Shu, Wu Yin, Jing Zhang, Bo Tang, Yuan-Xi Kang, Fan Ding, Zi-Chun Hua
Cell Biology International (August 2007) Volume 31(Issue 8) pp:784-789
Publication Date(Web):1 August 2007
DOI:10.1016/j.cellbi.2007.01.035
Sinomenine is an active component isolated from Sinomenium acutum and is widely used as an immunosuppressive drug for treating autoimmune diseases. CD4+ T-cell population plays a key role in adaptive immune response and is related to some autoimmune diseases. In this study, we investigated the possible immunosuppressive effect of sinomenine on CD4+ T cells and its underlying mechanism. Our data demonstrated that sinomenine remarkably suppressed the proliferation of CD4+ T cells, blocked the cell cycle progression from G0/G1 phase to S plusG2/M phases. Finally, the immunosuppressive activity elicited by sinomenine in CD4+ primary lymphocytes was found to be largely accounted for by caspase 3-dependent cells apoptosis. Sinomenine did not significantly alter the expression of bcl-2 in activated CD4+ primary T cells, suggesting that bcl-2 might not be involved in sinomenine-induced T cells apoptosis. In sum, this study proposes a novel mechanism for the immunosuppressive function of sinomenine on primary mouse CD4+ T cells.
Co-reporter:Hongqin Zhuang, Weiwei Jiang, Wei Cheng, Kui Qian, Wei Dong, Lin Cao, Qilai Huang, Shufeng Li, Fei Dou, Jen-Fu Chiu, Xue-Xun Fang, Min Lu, Zi-Chun Hua
Lung Cancer (April 2010) Volume 68(Issue 1) pp:27-38
Publication Date(Web):1 April 2010
DOI:10.1016/j.lungcan.2009.05.014
Tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it preferentially induces apoptosis in human cancer over normal cells. Most tumor cells, including lung cancer cell line A549, unfortunately, are resistant to TRAIL treatment even at high dose. Recent studies indicated that TRAIL-resistant cancer cells could be sensitized to TRAIL by combination therapy. Stress and heat shock proteins such as HSP90, HSP70 and HSP27 are induced in response to a wide variety of physiological environmental insults including heat, reactive oxygen species or anticancer drugs. Their elevated expressions facilitate cells to survive in stress circumstances. The HSP27 expression is enhanced in many tumor cells, implying that it is involved in tumor progression and the development of treatment resistance in various tumors, including lung cancer. This fact suggests a novel strategy for the treatment of cancer via inhibiting the function of HSP27. In this study, we investigated the inhibitory effect of a small interfering (si) RNA on the expression of HSP27 gene in the TRAIL-resistant human lung adenocarcinoma cell line A549, and the effect of HSP27 siRNA on drug sensitization of A549 cells to TRAIL treatment. The results showed that treatment of A549 cells with HSP27 siRNA down-regulated HSP27 expression but did not induce significant apoptosis. However, combination of HSP27 siRNA with TRAIL-induced significant apoptosis in TRAIL-resistant A549 cells. In addition to inducing caspases activation and apoptosis, combined treatment with HSP27 siRNA and TRAIL also increased JNK and p53 expression and activity. Collectively, these findings provide a conclusion that siRNA targeting of the HSP27 gene specifically down-regulated HSP27 expression in A549 cells, and sensitized the cells to TRAIL-induced apoptosis.
Co-reporter:Xiangbai Dong, Qiming Sun, Dongping Wei, Jiahuang Li, ... Zi-Chun Hua
FEBS Letters (22 December 2007) Volume 581(Issue 30) pp:5796-5802
Publication Date(Web):22 December 2007
DOI:10.1016/j.febslet.2007.11.049
A novel ferritin cDNA, SferH-5, has been cloned from 7-day-old soybean seedlings. Putative SferH-5 has 96% identity with SferH-1 reported previously. All the five amino acid variants distributed in the mature region are not involved in highly conserved residues associated with ferroxidase activity center. We speculate that SferH-5 encodes a novel 26.5-kDa subunit of soybean seed ferritin, which is designated H-5 in this study. Recombinant H-5 was able to assemble, together with co-expressed H-2, as a functional soybean seed ferritin-like complex, H-5/H-2. Our data reveal the potential heterogeneity of the 26.5-kDa subunit of soybean seed ferritin.
![[5-OXIDO-4-(THIOPHENE-2-CARBONYL)-1,2,5-OXADIAZOL-5-IUM-3-YL]-THIOPHEN-2-YLMETHANONE](http://img.cochemist.com/ccimg/7800/7733-96-2.png)
![[5-OXIDO-4-(THIOPHENE-2-CARBONYL)-1,2,5-OXADIAZOL-5-IUM-3-YL]-THIOPHEN-2-YLMETHANONE](http://img.cochemist.com/ccimg/7800/7733-96-2_b.png)
