Qing-Bing Zha

Jinan University (Guangzhou, China), Shengcheng, Guangdong, China

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Publications (13)23.25 Total impact

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    ABSTRACT: Cucurbitacins, the natural triterpenoids possessing many biological activities, have been reported to suppress the mTORC1/p70S6K pathway and to induce autophagy. However, the correlation between such activities is largely unknown. In this study, we addressed this issue in human cancer cells in response to cucurbitacin E (CuE) treatment. Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells. However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5. Consistent with autophagy induction, mTORC1 activity (as reflected by p70S6K and ULK1S758 phosphorylation) was inhibited by CuE treatment. The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1. In contrast, CuE rapidly activated AMPK leading to increased phosphorylation of its substrates. AMPK activation contributed to CuE-induced suppression of mTORC1/p70S6K signaling and autophagy induction, since AMPK knockdown diminished these effects. Collectively, our data suggested that CuE induced autophagy in human cancer cells at least partly via downregulation of mTORC1 signaling and upregulation of AMPK activity.
    PLoS ONE 05/2015; 10(5):e0124355. DOI:10.1371/journal.pone.0124355 · 3.53 Impact Factor
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    ABSTRACT: The anti-inflammatory effect of piperine has been largely investigated in macrophages, but its activity on epithelial cells in inflammatory settings is unclear. The present study aimed to investigate the effect of piperine on the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated human epithelial-like SW480 and HT-29 cells. Our data showed that although piperine inhibited the proliferation of SW480 and HT-29 cells in a dose-dependent manner, it had low cytotoxicity on these cell lines with 50 % inhibiting concentration (IC50) values greater than 100 μM. As epithelial-like cells, SW480 and HT-29 cells secreted high levels of the chemokine CXCL8 upon LPS stimulation. Importantly, piperine dose-dependently suppressed LPS-induced secretion of CXCL8 and the expression of CXCL8 messenger RNA (mRNA). Although piperine failed to affect the critical inflammatory nuclear factor-κB pathway, it attenuated the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling. Consistent with previous reports, p38 signaling seemed to play a more pronounced role on the CXCL8 expression than JNK signaling since inhibition of p38, instead of JNK, greatly suppressed LPS-induced CXCL8 expression. Collectively, our results indicated that piperine could attenuate the inflammatory response in epithelial cells via downregulating the MAPK signaling and thus the expression of CXCL8, suggesting its potential application in anti-inflammation therapy.
    Inflammation 12/2014; 38(3). DOI:10.1007/s10753-014-0075-z · 1.92 Impact Factor
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    ABSTRACT: Accumulating evidence indicates that cucurbitacin B (CuB), as well as other cucurbitacins, damages the actin cytoskeleton in a variety of cell types. However, the underlying mechanism of such an effect is not well understood. In this study, we showed that CuB rapidly induced actin aggregation followed by actin rod formation in melanoma cells. Cofilin, a critical regulator of actin dynamics, was dramatically dephosphorylated (i.e. activated) upon CuB treatment. Notably, the activated cofilin subsequently formed rod-like aggregates which were highly colocalized with actin rods, indicating the formation of cofilin-actin rods. Cofilin knockdown significantly suppressed rod formation but did not prevent actin aggregation. Furthermore, knockdown of the cofilin phosphatase slingshot homolog 1 (SSH1), but not chronophin (CIN), alleviated CuB-induced cofilin hyperactivation and cofilin-actin rod formation. The activity of Rho kinase and LIM kinase, two upstream regulators of cofilin activation, was downregulated after cofilin hyperactivation. Pretreatment with a thiol-containing reactive oxygen species (ROS) scavenger N-acetyl cysteine, but not other ROS inhibitors without thiol groups, suppressed CuB-induced actin aggregation, cofilin hyperactivation and cofilin-actin rod formation, suggesting that thiol oxidation might be involved in these processes. Taken together, our results demonstrated that CuB-induced formation of cofilin-actin rods was mediated by SSH1-dependent but CIN-independent cofilin hyperactivation. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
    Journal of Cellular Biochemistry 10/2013; 114(10). DOI:10.1002/jcb.24587 · 3.37 Impact Factor
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    ABSTRACT: The cucurbitacins are a class of triterpenoid molecules that possess cytotoxic characteristics for plant defense against herbivore feeding. 23,24-dihydrocucurbitacin F (DHCF), a derivative of the cucurbitacin family, has been isolated as an active component from the root of Hemsleya amabilis (Cucurbitaceae), an ancient Chinese remedy for bacillary dysentery, gastroenteritis, and cancers. While the toxicity of other cucurbitacins has been explored in several cancers, little data exist on the effect of DHCF on human cancers, including prostate cancer (PCa). In this study, we explore the level and mechanisms of DHCF toxicity on human PCa cell lines. Human PCa DU145, PC3, and LNCaP cells were treated with graded doses of DHCF in vitro, and anti-proliferative, cytotoxic, and proteomic effects were determined using MTS assay, cell cycle analysis, immunofluorescent staining, and western blotting. DHCF inhibited cell growth and induced cell cycle arrest at G(2)/M phase, formation of binucleated cells, and increased levels of apoptosis in all PCa cell lines tested. G-actin depletion, actin aggregation, and rod-like actin fibers, with little effect on microtubule structure, were observed after DHCF treatment. Actin aggregation and cofilin-actin rod formation were highly correlated with rapid and persistent dephosphorylation of cofilin-1 (cofilin). DHCF treatment resulted in upregulation of p21(Cip1) and downregulation of cyclin A in all three PCa cell lines. The anti-proliferative activity of DHCF on human PCa cells may be brought about by inducing actin aggregation and cofilin-actin rod formation, leading to cell cycle arrest, cytokinesis failure, and apoptosis.
    Cancer Chemotherapy and Pharmacology 07/2012; 70(3):415-24. DOI:10.1007/s00280-012-1921-z · 2.57 Impact Factor
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    ABSTRACT: This research intends to amplify the entire coding region sequences of SLC25A13 mRNA which encodes citrin, and to investigate sequence features of the transcripts for this gene in cultured human amniocytes. This study will provide laboratory evidence for prenatal diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) at mRNA level. One amniocyte sample was collected from a pregnant woman who underwent prenatal diagnosis of citrin deficiency and whose fetus has proven a carrier of 851del4 mutation by genomic DNA analysis. Another amniocyte sample, as a control, was from a fetus without family history of citrin deficiency. Total RNA was extracted from cultured amniocytes, cDNA was synthesized, and then nested-PCR was performed to amplify the entire coding region sequences of SLC25A13. The PCR products were cloned and analyzed by sequencing. The entire coding region of SLC25A13 gene was successful amplified from two cultured human amniocytes. The splice variant of SLC25A13, SLCA (normal mRNA), was identified in the two samples. SLCB (CAG insertion between exon 9-10) was identified in the control. SLCC (exon 5-11 skipping), but not transcriptional product from the allele with 851del4 mutation, was identified in the 851del4 mutation carrier. This study demonstrated that the entire coding region of SLC25A13 cDNA can be successfully amplified from two cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA predominated in the transcripts in normal control and 851del4 mutation carrier, suggesting that the two fetuses were not at risk for NICCD. These SLC25A13 transcription features provided laboratory evidence for prenatal diagnosis of NICCD.
    Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics 03/2012; 14(3):221-5.
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    ABSTRACT: Citrin deficiency (CD) is an autosomal recessive disorder with SLC25A13 as causative gene that encodes citrin, the liver-type aspartate/glutamate carrier isoform 2 (AGC2). Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), the major CD phenotype at pediatric age, has been previously reported as a self-limiting condition with clinical presentations resolving between 6 months and 1 year of life. We report the prenatal diagnosis of CD in a family with a fatal NICCD proband. The proband was a 10-month-old male presenting cough for 8 days and jaundiced skin 1 day. Physical examination revealed fever, dark jaundiced sclera and skin, hoarse breathing sounds, and hepatosplenomegaly. Laboratory tests uncovered elevated cholestatic indices, increased ammonia, and prolonged activated partial thromboplastin time and prothrombin time, and reduced fibrinogen. Sonography showed the features of liver cirrhosis. Metabolome analysis uncovered large quantity of 4-hydroxyphenyllactate and dicarboxylates in urine and increased citrulline and methionine in blood. The patient passed away due to liver failure at his age of 13.5 months. Mutation analysis revealed him a homozygote of 851del4, a four-base deletion in exon 9 of SLC25A13 gene. On request of the parents who had a second fetus, prenatal diagnosis of CD was performed by PCR-electrophoresis following amniocentesis and amniocyte culture, and demonstrated the fetus a carrier of the same mutation. The fatal proband in the present report has provided clinical evidence challenging the traditional concept on NICCD prognosis. Moreover, as the first trial on CD prenatal diagnosis, this study might open a novel area for clinical management of CD.
    The Tohoku Journal of Experimental Medicine 12/2011; 225(4):273-6. DOI:10.1620/tjem.225.273 · 1.28 Impact Factor
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    ABSTRACT: Gossypol (GOS), a BH3 mimetic, has been investigated as a sensitizing co-therapy to radiation and chemotherapy in treatment of metastatic prostate cancer. In this study, we found that valproic acid (VPA), a histone deacetylase inhibitor (HDACI), counteracted the suppressive effect of GOS on histone H3 acetylation and enhanced the cytotoxicity of GOS to DU145 prostate cancer cells. Significant synergistic effects were observed in combined GOS and VPA treatment, culminating in more DNA damage and cell death. The iTRAQ-based quantitative proteomic analysis revealed differential proteomic profiles in cells treated with VPA, GOS or their combination. In GOS-treated cells, oxidative phosphorylation-related proteins were depressed and endoplasmic reticulum stress markers were upregulated. In the presence of VPA, the GOS-induced mitochondrial stress was further enhanced since glycolysis- and hypoxia-associated proteins were upregulated, suggesting a disruption of energy metabolism in these cells. Furthermore, the DNA damage repair ability of cells co-treated with GOS and VPA was also decreased, as evidenced by the downregulation of DNA damage repair proteins and the enhancement of DNA fragmentation and cell death. These findings suggest that GOS in combination with an HDACI has the potential to increase its clinical efficacy in the treatment of prostate cancer.
    Journal of proteomics 06/2011; 74(10):2180-93. DOI:10.1016/j.jprot.2011.06.016 · 3.93 Impact Factor
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    ABSTRACT: Anti-viral CD8(+) T cell responses involve an initial expansion and effector phase, followed by contraction phase and formation of CD8(+) memory T cells. During this contraction phase, increased surface expression of the negative regulator PD-1 is associated with functional exhaustion of CD8(+) T cells. Although its role in T cell suppression has been established, the importance of PD-1 in the differentiation of CD8(+) T cells remains unclear. In this study, we examine PD-1 expression in relation to viral specificity of CD8(+) T cells against persistent or non-persistent viruses, and further define differentiation phenotypes of CD8(+) T cells by CD27 and CD28 expression. Surprisingly, the inhibitory receptor PD-1 was expressed by Flu-specific CD8(+) T cells in a level comparable to HCMV-and EBV-specific cells. Moreover, in virus-specific CD8(+) T cells, CD127(+)/CD127(-) and CD62L(+)/CD62L(-) cells expressed similar levels of PD-1 molecules. These results suggest that the PD-1/PD-L1 pathway may play a regulatory role in memory T cell subsets in addition to its association with T-cell exhaustion.
    Clinical Immunology 03/2008; 126(2):222-34. DOI:10.1016/j.clim.2007.08.021 · 3.99 Impact Factor
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    ABSTRACT: HLA-A* 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8+ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A* 2402 tetramer loaded with HCMV pp65(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A* 2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A* 2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of HLA-A* 2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A* 2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A* 2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A* 2402-QYD monomers with streptavidin-PE at a molar ratio of 4:1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24+ donors and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8+ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A* 2402 individuals.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 04/2007; 23(2):284-91. DOI:10.1016/S1872-2075(07)60025-9
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    ABSTRACT: To optimize expression condition of HLA-A*0203 heavy chain ectodomain fused with a BirA substrate peptide (BSP) (HLA-A*0203-BSP) for E.coli BL21(DE3) transformant and to prepare a functional HLA-A*0203 tetramer loaded with an antigenic peptide derived from EBNA3(596-604) of Epstein-Barr virus (EBV). The temperature, IPTG concentration and inductive duration of HLA-A*0203-BSP fusion protein expressed for E.coli BL21(DE3) transformant were optimized. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. The monomer of soluble HLA-A*0203-peptide was generated from the fusion protein by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin (beta2m) and HLA-A*0203 restricted EBV EBNA3(596-604) peptide (SVRDRLARL, SVR). Refolded and purified monomer was then biotinylated with BirA. Following the purification of the obtained biotinylated monomer, the tetramer was formed by incubation with streptavidin-PE at a ratio of 4:1. Flow cytometry (FCM) analysis was performed to determine its binding activity with specific cytotoxic T lymphocytes (CTL). SDS-PAGE and Western blot showed that the optimized expression condition was overnight induction at 37 degrees C with 0.4 mmol/L IPTG. The expressed protein of about 34 kDa in the form of inclusion bodies accumulated up to about 30% of total bacterial protein under the optimized expression condition. The monomer of soluble HLA-A*0203/SVR was successfully generated and purified. Non-reducing SDS-PAGE analysis showed that the biotinylation was above 85%. HLA-A*0203/SVR tetramer was constructed by mixing the monomer with streptavidin-PE at a ratio of 4:1. FCM analysis indicated that this tetramer could bind specific CTL from HLA-A2+ donors. HLA-A*0203-BSP fusion protein was overexpressed in E.coli under the optimized condition. The tetramers of HLA-A*0203/SVR were prepared from this fusion protein and it possessed binding activity with specific CTL, which provided a powerful tool for direct visualization and quantification of specific CTL from HLA-A*0203 donors.
    Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 03/2007; 23(2):97-101.
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    ABSTRACT: This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 02/2007; 23(1):106-11. DOI:10.1016/S1872-2075(07)60008-9
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    ABSTRACT: Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus which persists lifelong after primary infection and can lead to a significant disease in the immunocompromised individuals. CD8(+) T cells are believed to play a crucial role in both the elimination of active infection and maintenance of HCMV latency. Large expansions of CD8(+) T cells specific for a single epitope of HCMV have been well documented in Caucasoid population. To date, no similar study has been performed in Chinese populations. Here we report the characteristics of HCMV-specific CD8(+) T cells in healthy young and elderly Chinese donors using pp65(495-503)-loaded HLA-A*0201 tetramers. Cells were stained with a combination of the tetramers and antibodies for CD28 and CD57 or a panel of TCR Vbeta and analyzed by three-color flow cytometry. The frequencies of pp65(495-503)-specific T cells within total CD8(+) T cell population were between 0.14 and 6.84% (mean 2.45%) in the young donors and were from 0.33 to 6.89% (mean 1.95%) in the elderly donors, respectively. There was no significant difference between the two groups. The expression of CD28 was decreased whereas CD57 expression was increased in tetramer-negative CD8(+) T cells in the elderly when compared with the young group. However, neither of these changes was found within tetramer-positive cell populations. Moreover, TCR Vbeta usage within tetramer-positive population was predominated by certain TCR Vbeta subsets. These results demonstrate that large expansions of HCMV-specific CD8(+) T cells with certain subsets TCR Vbeta exist both in the healthy young and in the elderly Chinese individuals, which may play a role in the maintenance of virus latency but have potential detrimental influence on the immune responses to other pathogens or vaccinations.
    Journal of Clinical Immunology 10/2006; 26(5):417-29. DOI:10.1007/s10875-006-9035-1 · 2.65 Impact Factor
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    ABSTRACT: To optimize tetramer staining condition using HLA-A*0201 tetramer (A2-NLV tetramer) loaded with NLV peptide (pp65(495-503)) derived from structural protein pp65 of human cytomegalovirus and to investigate its application in phenotyping of specific cytotoxic T lymphocytes (CTL). Peripheral blood from HLA-A2(+) donors was first stained with A2-NLV tetramer/PE under different conditions and then labeled with anti-CD3-FITC and anti-CD8-APC. The stained samples were analyzed with flow cytometry to find out the optimized staining condition. Meanwhile, the phenotype and activation antigen expression were determined. Tetramer staining with whole blood was superior to peripheral blood mononuclear cells. The optimized condition for tetramer staining was incubating 100 muL of whole blood with 0.3 mug of A2-NLV tetramer for 1 h at 4 degrees Celsius. Under this condition the specific staining was strong while unspecific staining of CD8(-) T cells was quite weak. Phenotypic analysis under this condition showed that the ratio of CD28 positive A2-NLV tetramer specific CTL was lower than that of nonspecific CTL, whereas the ratio of CD57 positive specific CTL was higher than that of nonspecific CTL. CD25 molecules were only expressed on the activated specific CTL. The optimized tetramer staining condition can increase the specificity of tetramer staining and decrease unspecific binding, therefore it is applicable for phenotyping and functional analysis of antigen-specific CTL.
    Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2006; 22(2):247-51.