Dichloroacetate enhances the antitumor effect of pirarubicin via regulating the ROS-JNK signaling pathway in liver cancer cells.

Aim: Liver cancer is one of the most common malignancies and has a high recurrence rate. However, current treatment strategies do not achieve satisfactory outcomes in the clinic. To explore a new strategy to enhance the effectiveness of chemotherapy in liver cancer, we investigated whether dichloroacetate (DCA) could enhance the sensitivity of liver cancer cells to pirarubicin (THP). Methods: Liver cancer cells were treated with DCA alone, THP alone, or DCA and THP combined. Cell viability was determined by the CCK-8 assay. Cell apoptosis was analyzed by flow cytometer. Reactive oxygen species (ROS) were detected using a CM-H2DCFDA fluorescence probe. Protein levels were identified by immunoblotting. Results: The results revealed that DCA significantly enhanced the antitumor effect of THP in liver cancer cells. Changes in morphology and adherence ability were observed, as well as decreased cell viability. The results of flow cytometry showed that the combination of THP and DCA significantly increased apoptosis of liver cancer cells. Moreover, compared with THP alone, combination treatment with DCA significantly increased THP-triggered ROS generation in liver cancer cells. The antioxidant N-acetyl-L-cysteine reversed the synergistic effect of DCA and THP on ROS generation, cell viability and apoptosis. Furthermore, phosphorylation of c-Jun N-terminal kinase (JNK) was significantly increased in the DCA and THP combination group. The effects of DCA and THP on cell viability and apoptosis were inhibited by the JNK inhibitor SP600125. Conclusion: The results obtained in the present study indicated that DCA enhanced the antitumor effect of THP in liver cancer cells via regulating the ROS-JNK signaling pathway.

A feedback loop in PPARγ-adenosine A2A receptor signaling inhibits inflammation and attenuates lung damages in a mouse model of LPS-induced acute lung injury.

Although peroxisome proliferator-activated receptor-γ (PPARγ) and adenosine A2A receptor (A2AR) are reported to be anti-inflammatory factors in acute lung injury (ALI), their internal link and synergic or antagonistic effect after activation are poorly understood. Here, we found that PPARγ and A2AR could upregulate the mRNA and protein expressions of each other in lung tissues of LPS-induced mouse ALI model and murine macrophages. Further investigation demonstrated that PPARγ upregulated A2AR expression by directly binding to a DR10 response element (-218 to -197) within A2AR gene promoter region. Instead of directly interacting with PPARγ, A2AR stimulated PPARγ expression via protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling by provoking the binding of CREB to a cAMP responsive element (CRE)-like site in PPARγ gene promoter region. In addition, combination of PPARγ and A2AR agonists was found to exert obviously better effect on suppressing neutrophil infiltration and inflammatory cytokine expressions, attenuating lung edema, pathological changes and improving lung function of blood gas exchange than their single application. These findings reveal a novel functional positive feedback loop between PPARγ and A2AR signaling to potentialize their effect on inhibiting inflammation and attenuating lung damages in ALI. It suggests that targeting this PPARγ-A2AR signaling rather than PPARγ or A2AR alone may be a more attractive and efficient potential therapeutic strategy for ALI.

MicroRNA-613 represses lipogenesis in HepG2 cells by downregulating LXRα.

BACKGROUND: MicroRNAs (miRNAs) emerge as new important regulators of lipid homeostasis by regulating corresponding genes. MiR-613 is a newly discovered microRNA, of which the biological function is unknown. A recent report has shown that miR-613 downregulates liver X receptor α (LXRα), a ligand-activated nuclear receptor playing an important role in the regulation of lipid metabolism. The purpose of this study is to explore the effect and the molecular basis of miR-613 on lipogenesis in HepG2 cells. METHODS: HepG2 cells were transiently transfected with miR-613 mimic or control microRNA. Real time PCR, Western blot, Luciferase reporter assay and Oil Red O staining were employed to examine the expression of LXRα and its target genes involved in lipogenesis, binding site for miR-613 in 3'-untranslated region (3'-UTR) of LXRα mRNA and lipid droplet accumulation in the cells. RESULTS: MiR-613 dramatically suppressed the expression of LXRα and its target genes including sterol-regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), carbohydrate responsive element-binding protein (ChREBP) and acetyl-CoA carboxylase (ACC). Reporter assay showed that miR-613 directly bound to 3'-UTR of LXRα mRNA. Moreover, miR-613 significantly repressed LXRα-induced lipid droplet accumulation in HepG2 cells. Ectopic expression of LXRα without 3'-UTR markedly attenuated the miR-613-mediated downregulation of LXRα's target genes and LXRα-induced lipid droplet accumulation. CONCLUSIONS: MiR-613 suppresses lipogenesis by directly targeting LXRα in HepG2 cells, suggesting that miR-613 may serve as a novel target for regulating lipid homeostasis.

Upregulation of ski in fibroblast is implicated in the peroxisome proliferator--activated receptor δ-mediated wound healing.

BACKGROUND/AIM: Both peroxisome proliferator-activated receptor (PPAR) δ and Ski are investigate the interaction of PPARδ and Ski and this interaction-associated effect in wound healing. METHODS: Effect of PPARδ activation on Ski expression was detected in rat skin fibroblasts by real-time PCR and western blot. Luciferase assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay were performed to identify the binding site of PPARδ in the promoter region of rat Ski gene. And the functional activity of PPARδ regulation to Ski was detected in fibroblast proliferation and rat skin wound healing model. RESULTS: PPARδ agonist GW501516 upregulated Ski expression in a dose-dependent manner. Direct repeat-1 (DR1) response element locating at -865∼-853 in Ski promoter region was identified to mediate PPARδ binding to Ski and associated induction of Ski. Furthermore, PPARδ upregulated Ski to promote fibroblasts proliferation and rat skin wound repair, which could be largely blocked by pre-treated with Ski RNA interference. CONCLUSION: This study demonstrates that Ski is a novel target gene for PPARδ and upregulation of Ski to promote fibroblast proliferation is implicated in the PPARδ-mediated wound healing.

Panax notoginseng saponins attenuate atherosclerosis via reciprocal regulation of lipid metabolism and inflammation by inducing liver X receptor alpha expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng (Burk.) F.H. Chen has been used as a health product and natural remedy in traditional medicine for cardiovascular diseases for more than 1000 years in Asia, including China, Japan, and Korea. Panax notoginseng saponins (PNS) are the major effective ingredients extracted from Panax notoginseng. AIM OF THE STUDY: The purpose of this study was to investigate whether Panax notoginseng saponins (PNS) attenuated atherosclerosis by inducing liver X receptor alpha (LXRα) expression and to elucidate the mechanisms responsible for the effects. MATERIALS AND METHODS: The AS rats were treated once daily with PNS (100 mg/kg, i.p.), and pathological changes in the aorta were observed using Sudan IV staining. The expression of LXRα in the aortic wall was measured by Western blot analysis. THP-1 macrophages were cultured with PNS in the presence or absence of geranylgeranyl pyrophosphate ammonium salt (GGPP), an LXRα antagonist. The expression of LXRα and its target genes ATP-binding cassette A1 and G1 (ABCA1, ABCG1) were determined by qRT-PCR. The transcriptional activation of the LXRα gene promoter was analyzed by a reporter assay. The NF-κB DNA binding activity and the expression of interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1) was evaluated respectively by Trans-AM NF-κB ELISA and ELISA in THP-1 macrophages that were stimulated with LPS after treatment with PNS and GGPP. RESULTS: PNS treatment alleviated the typical pathological changes associated with atherosclerosis in rats. The expression of LXRα was increased in rat aortas after treatment with PNS. In vitro, PNS increased LXRα mRNA levels in THP-1 macrophages. The reporter assays showed that PNS enhanced transcriptional activation of the LXRα gene promoter and led to the upregulation of ABCA1 and ABCG1 expression. This upregulation could be reversed by treatment with GGPP. Additionally, PNS inhibited NF-κB DNA binding activity and reduced secretion of IL-6 and MCP-1 in LPS-stimulated THP-1 macrophages. These effects could be reversed by GGPP. CONCLUSIONS: The results indicated that the PNS-mediated attenuation of AS may, at least partly, due to LXRα uprergulation. The mechanisms of action included enhancement transcriptional activation of the LXRα gene promoter by PNS and subsequent upregulation of ABCA1 and ABCG1 and inhibition of NF-κB DNA binding activity.

[PPARγ up-regulates TGFß/smad signal pathway repressor c-Ski].

TGFß/smad pathway is recognized as an important signal pathway to promote the pathogenesis of atherosclerosis (AS). Peroxisome proliferator-activated receptor γ (PPARγ) activation is considered to be important in modulating AS. Herein, we investigated the regulation of PPARγ on c-Ski, the repressor of TGFß/smad pathway, in rat AS model and cultured vascular smooth muscle cells (VSMCs). c-Ski mRNA and protein expression were detected by real-time PCR and Western blot, respectively, in vivo and in vitro with treatment of PPARγ agonist rosiglitazone and antagonist GW9662. The proliferation and collagen secretion of VSMCs after c-Ski transfection were investigated. The underlying mechanism was further investigated by online program NUBIScan and luciferase reporter gene analysis. Results showed that both mRNA and protein expressions of c-Ski in the AS lesions was down-regulated in vivo, while in cultured VSMCs, c-Ski transfection significantly suppressed the proliferation and collagen secretion of rat VSMCs. Rosiglitazone significantly up-regulated mRNA and protein levels of c-Ski in VSMCs, which could be blocked by GW9662. Online NUBIScan analysis suggested possible PPARγ binding sites in the promoter region of c-Ski. In addition, luciferase activity of c-Ski reporter gene was also increased obviously in the presence of rosiglitazone. These results indicate that c-Ski is one of the newly found target genes of PPARγ and thus involved in the anti-AS effect of PPARγ.

Chronic or high dose acute caffeine treatment protects mice against oleic acid-induced acute lung injury via an adenosine A2A receptor-independent mechanism.

The antagonism or genetic deletion of adenosine A(2A) receptors has been shown to exacerbate tissue damage in acute lung injury. Caffeine, a widely consumed behavioral drug, acts as a non-selective antagonist of A(2A) receptor and also has additional pharmacological effects. Thus, the protective vs. deleterious effects of caffeine in acute lung injury should be evaluated. In a murine oleic acid-induced model of acute lung injury, we found that chronic caffeine treatment by drinking water (0.1g/l or 0.25g/l for 2 weeks before acute lung injury) or acute caffeine treatment at high dose (i.p. 50mg/kg, injection, 30min before acute lung injury) significantly attenuated the lung edema, hemorrhage, neutrophil recruitment as well as the inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) expressions in both of the wild type (WT) and A(2A) receptor knockout (KO) mice. This profile was accompanied by increased cAMP levels and up-regulation of A2B receptor mRNAs in the lungs. In contrast, acute caffeine treatment at low dose (i.p. 5mg/kg or 15mg/kg, injection, 30min before acute lung injury) enhanced the inflammation and lung damage in WT mice with decreasing cAMP but not in A(2A) receptor KO mice. These results indicate that caffeine either enhances lung damage by antagonizing A(2A) receptor or exerts protection against lung damage via A(2A) receptor-independent mechanisms, depending on the timing of exposure (chronic vs. acute) and dose of administration (low vs. high). These findings provide new insight of caffeine in acute lung injury and highlight the potential benefit and strategy of caffeine intake or administration for preventing acute lung injury.

[Effect of androsterone on scavenger receptor class B type I in human vascular endothelial cells].

OBJECTIVE: To investigate the effect of FXR on scavenger receptor class B type I (SR-BI) expression. METHODS: Human vascular endothelium Eahy926 cells were treated with FXR agonist androsterone, and the specific target gene of FXR SHP mRNA was detected by RT-PCR. SR-BI mRNA and protein were determined using RT-PCR, real-time PCR and Western blotting. RESULTS: The level of SHP mRNA in Eahy926 cells increased after androsterone treatment at different concentrations for 24 h, demonstrating FXR activation in the cells. RT-PCR, real-time PCR and Western blotting detected increased SR-BI expression at both mRNA and protein levels after FXR activation. CONCLUSION: FXR increases the expression of SR-BI in human vascular endothelium cells.

[Expression and activity analysis of human 67kD laminin receptor in Pichia pastoris].

To carry out the secretive expression of human 67 kD laminin receptor (67LR), recombinant expression plasmid pPIC9K-67LR was constructed by inserting of 67LR cDNA into yeast expression vector pPIC9K. The 67LR protein was expressed in Pichia pastoris after induced by methanol, and about 12.56 mg electrophoresis purity 67LR could be obtained after the purification of 1L culture using affinity chromatograph column. In vitro competitive binding assay showed that target protein has an excellent biological activity. The successful expression of 67LR has placed a solid foundation for the research on structure and functions of 67LR.

[Expression of human LNalpha4LG4-5 in Pichia yeast].

To carry out the secretive expression of human laminin alpha4 chain LG4-5 module (hLNalpha4LG4-5), recombinant expression plasmid pPICZalphaA-LG45 was constructed by inserting of hLNalpha4LG4-5 cDNA into yeast expression vector pPICZaA. The hLNalpha4LG4-5 protein was expressed in GS115 Pichia yeast strain after induced by methanol, and purified target protein can obviously promote the expansion and adhesion of 293 cells.

[Screening of antagonist peptide of BLyS from C7C phage display peptide library].

AIM: To screen an antagonist peptide of BLyS from C7C phage display peptide library. METHODS: C7C phage display peptide library was screened with BLyS. Indirect ELISA, competitive ELISA and MTT colorimetry were used to identify positive phage clones. RESULTS: After 3 rounds of screening, the gradual increase of the ratio of output to input and specific enrichment had been achieved. Two phage clones that could inhibit the BLyS activity were identified. CONCLUSION: Two phage display antagonist peptides of BLyS were obtained.

[The cloning, high level expression in Escherichia coli of human beta-defensin 3 and its antimicrobial activity analysis].

In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag.

AIM: Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pIII in order to get some information on mimotopes. METHODS: Through affinity enrichment and ELISA screening, positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced) By blocking test, specificity of the mimic phage epitopes was identified. RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced. According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-III. The percentage of blocking was from (21.0+/-1.6) % to (39.0+/-2.7) %. CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.

[Preparation of phage-displayed single chain variable fragments of monoclonal antibody MC5 recognizing colorectal carcinoma].

BACKGROUND & OBJECTIVE: MC5 is a murine monoclonal antibody with a good specificity to human colorectal carcinoma and smaller murine antibody can significantly decrease the possibility of developing human antimouse antibody response in vivo study. The aim of this study was to prepare single chain variable fragments (ScFv) of MC5. METHODS: mRNA was isolated from the hybridoma cell line producing MC5, and the DNAs encoding variable domains of heavy and light chains(VH and VL DNAs) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFvs. After two rounds of panning with cell line SW480 highly expressing MC5-binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by ELISA, and the affinity of the positive phage clones was assayed by competition ELISA. RESULTS: The VH, VL, and ScFv DNAs were about 340 bp, 320 bp, and 750 bp, respectively. Ten phage clones displayed ScFv of MC5 were selected from 25 enriched phage clones, and 3 of the 10 phage clones had higher affinity of binding to the antigen. CONCLUSION: The phage-displayed ScFv fragments of monoclonal antibody MC5 are successfully produced by phage display technique, which may provide a way for broadening the application range of the antibody.

Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells.

AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens. METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E.coli HB2151 to express soluble ScFv. RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60 %, 24 % and 30 %. MC3-ScFv had M(r) 32 000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab. CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3.

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Cloning and identification of an angiostatic molecule IP10/crg -2.

AIM:To obtain human and murine cDNAs encoding IFN-gamma inducible protein 10 (IP-10) and cytokine responsive gene-2 (Crg-2).METHODS:The encoding genes of IP-10 and Crg-2 were amplified by RT-PCR from cultured human fibroblast cells and Balb/c mouse liver treated by IFN-gamma and TNF-alpha,respectively, and cloned into plasmids of pUC19 and pGEM3Zf(+).RESULTS:The nucleotide sequences of the amplified DNA were confirmed by endonucleases digestion and sequencing.CONCLUSION:Recombinant IP-10/crg-2 gene clones with 306bp and 314bp inserts were established for further research on biological activities and ligands of hIP-10/mCrg-2.