Human MCAF Gene Transfer Enhances the Metastatic Capacity of a Mouse Cachectic Adenocarcinoma Cell Line in Vivo

Purpose. To evaluate the effect of monocyte chemotactic and activating factor (MCAF/MCP-1/JE) on tumor progression and metastasis. Methods. Cachexia-inducing adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid or MCAF expression vector. Spontaneous lung metastases were determined in mouse. Results. The production of MCAF reached 0.4 ng/ml in vitro when transfectant cells were cultured at a cell density of 5 × 10⁴ cells/ml for 3 days. Transfection of MCAF expression vector did not affect the growth rate in vitro. Also, after MCAF-transfection, the size of tumors after intra-footpad inoculation was similar to that of the parental cells. When the primary tumors were resected on the 10th day after inoculation, the incidence of spontaneous lung metastasis was less than 20% in both cells. The number of endothelial cells in the primary tumor rapidly increased from the 10th to the 14th day after inoculation, as revealed by immunohistochemical staining. In accordance with enhanced angiogenesis, the incidence rates of spontaneous metastasis increased when the primary tumors were resected on the 14th day after inoculation. Moreover, the spontaneous lung metastases were augmented in the animals injected with MCAF-transfectants compared to those injected with parental cells with a concomitant increase of angiogenesis. Conclusions. These results suggest that MCAF may augment the metastatic potential by modulating tumor associated angiogenesis.     

A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice

Purpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein l (hu-MIP-l), murine-macrophage inflammatory protein l (mu-MIP-l), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-l, mu-MIP-l, or hu-IL-8 expression vector. The production of hu-MIP-1 reached >1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 10⁵ cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-l, mu-MIP-l, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-l and mu-MIP-l. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-l showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-l gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-l gene might be useful as an effective therapy for the treatment of certain tumors.     

Interaction Between Polyalkylcyanoacrylate Nanoparticles and Peritoneal Macrophages: MTT Metabolism,NET Reduction,and NO Production

Purpose. The nature of interactions between macrophages and drug carriers is of primordial importance either in the design of more effective therapeutic strategies for macrophage-associated pathogenesis or in establishing new approaches for pharmacological action avoiding macrophages. Methods. Polyalkylcyanoacrylate nanoparticles (PMCA, PECA, PBCA and PIBCA nanoparticles) were assayed for their toxicity on peritoneal resident and thioglycolate-elicited macrophages. Cellular viability was assessed by MTT tetrazolium salt assay, oxidative burst by NBT reduction and NO production by nitrite evaluation. Results. The nanoparticles tested led to cellular morphological modifications and induced toxicity in both types of macrophages in culture. The polyalkylcyanoacrylate nanoparticles uptake by peritoneal macrophages caused an increase in respiratory burst, as assessed by the NBT reduction assay, and induced the release of soluble toxic factors to the culture medium. The association of LPS with the PMCA nanoparticles significantly stimulated the production of nitric oxide (NO) by resident macrophages. In contrast, the association of PBCA nanoparticles with LPS does not increase the nitrite production as compared with LPS alone, which may be due to a different physico-chemical interaction between LPS and the two types of polymers. Conclusions. In cultured mice peritoneal macrophages, nanoparticles of PACA induce the production of oxygen reactive products, which cause changes in the cell metabolism of both resident and elicited macrophages. PMCA nanoparticles in association with LPS significantly increase the expression of the inducible isoform of nitric oxide synthase, leading to the release of large amount of NO, which may be highly cytotoxic to the cultured cells in the presence of peroxide generated from the oxidative burst.     

Liposomal Induction of a Heat-stable Macrophage Priming Factor to Induce Nitric Oxide in Response to LPS

Purpose. The effects of liposomes on nitric oxide (NO) production from mouse peritoneal macrophages following intraperitoneal injection of liposomes were investigated. Methods. Mouse peritoneal macrophages were collected following intraperitoneal injection of liposomes and cultured with and without lipopolysaccharide (LPS). Peritoneal washing fluid was also collected from the mice injected with liposomes. NO production was evaluated by measuring the concentration of nitrite in the macrophage culture supernatant by Griess reagent. Results. NO production stimulated by LPS was observed in peritoneal macrophages obtained from the liposome-treated mice, but liposomes did not activate macrophages directly to induce NO in response to LPS. NO production was higher in the liposomes composed of phospha-tidylcholine than that of negatively charged liposomes composed of phosphatidylserine. Peritoneal washing fluid obtained from mice injected with liposomes has a capacity to induce NO production in the macrophages from naive mice. This capacity was not diminished by heat-treatment at 100°C for 5 min. Conclusions. Peritoneal macrophages were activated to produce NO in response to LPS following intraperitoneal injection of liposomes. They did not activate macrophages directly, and the induction of heat-stable macrophage priming factor, but not cytokines, is suggested.     

Transfected Rat cMOAT Is Functionally Expressed on the Apical Membrane in Madin-Darby Canine Kidney (MDCK) Cells

Purpose. The purpose of the present study is to investigate the expression of canalicular multispecific organic anion transporter (cMOAT) by its cDNA transfection in polarized Madin-Darby canine kidney cells (MDCK). Methods. MDCK cells were transfected with an expression vector (pCXN2) containing the rat cMOAT cDNA with lipofectamine to obtain the stable transfectant under G418. Cells from a single colony whose cMOAT expression was the highest were seeded to form a tight epithelial monolayer on microporous membrane filters. Export of glutathione S-bimane (GS-B) from monolayers was determined after preloading its precursor, monochloro bimane (MCB). Results. A comparable amount of GS-B was excreted to the apical and basal compartments in the vector-transfected cells. In contrast, in cMOAT-transfected cells, the amount apically excreted was approximately twice that excreted into the basal compartment. Cyclosporin A (CsA) (30M), an inhibitor of cMOAT at higher concentrations, inhibited the preferential apical export of GS-B from cMOAT-transfected cells. Conclusions. Rat cMOAT is functionally expressed on the apical membrane of MDCK cells after transfection.     

ZCH-2B8a,an antibody targeting actin-binding protein coronin-1a,is a potential therapeutic agent for B-lineage malignancies

Abstract

Background: Monoclonal antibody (mAb)-based targeted therapy is one of the most promising strategies to cure cancers. MAb ZCH-2B8a (2B8a) was a novel antibody generated in our laboratory, which presented potential to be a therapeutic agent for hematologic malignancies.

Methods: We investigated the reactivity profile of 2B8a mAb, identified the targeting antigen by proteomic and genetic approaches and evaluated its potential to exert tumor cell killing.

Results: 2B8a antigen was strictly expressed on lymph tissues and hematopoietic cells (mainly leukocytes), and was highly expressed on B-lineage leukemia cell lines and acute lymphoblastic leukemia (ALL) cells from patients. 2B8a antibody was quickly internalized into the target cells once binding to the antigen, but was capable of killing tumor cells through complement dependent cytotoxicity. To identify the 2B8a antigen, the proteins of Raji cells were immunoprecipitated with 2B8a antibody and analyzed by mass spectrometry, which indicated that coronin-1a was a potential candidate. Then, coronin-1a gene was cloned from Raji cells, inserted into plasmid pcDNA3.1 (+), and transfected into CHO cells. The intracellular 2B8a antigen level was significantly increased in the coronin-1a transfectant cell line.

Conclusion: 2B8a mAb is a novel antibody targeting coronin-1a, which has the potential to be a therapeutic agent for B-lineage malignancies.     

BICC1 as a novel prognostic biomarker in gastric cancer correlating with immune infiltrates

AimBicC family RNA-binding protein 1 (BICC1) codes an RNA-binding protein that regulates gene expression and modulates cell proliferation and apoptosis. We aim at investigating the role of BICC1 in gastric carcinogenesis.MethodsBICC1 mRNA expression in gastric cancer (GC) was examined using the Tumor Immune Estimation Resource (TIMER), The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Correlations between BICC1 expression and clinicopathological parameters were analyzed. The Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier plotter databases were used to examine the clinical prognostic significance of BICC1 in GC. Signaling pathways related to BICC1 expression were identified by gene set enrichment analysis (GSEA).TIMER and CIBERSORT were used to analyze the correlations among BICC1, BICC1-coexpressed genes and tumor-infiltrating immune cells.ResultsBICC1 was highly expressed in GC and significantly correlated with grade (P = 0.002), TNM stage (P = 0.033), invasion depth (P = 0.001) and vital status (P = 0.009) of GC patients. High BICC1 expression correlated with poor overall survival. The GSEA results showed that cell adhesion-, tumor- and immune- related pathways were significantly enriched in samples with high BICC1 expression. BICC1 and its coexpressed genes were positively related to tumor-infiltrating immune cells and were strongly correlated with tumor-infiltrating macrophages (all r ≥ 0.582, P < 0.0001). The CIBERSORT database revealed that BICC1 correlated with M2 macrophages (P < 0.0001), regulatory T cells (P < 0.0001), resting mast cells (P < 0.0001), activated memory CD4+ T cells (P = 0.002), resting NK cells (P = 0.002), activated dendritic cells (P = 0.002), and follicular helper T cells (P = 0.016). The results from TIMER database confirmed that BICC1 is closely associated with the markers of M2 macrophages and tumor-associated macrophages (all r ≥ 0.5, P < 0.0001).ConclusionBICC1 may be a potential prognostic biomarker in GC and correlates with immune infiltrates.     

ALCAM基因1沉默对胃癌细胞增殖和凋亡的影响及分子机制

目的 探讨活化白细胞黏附分子（ALCAM）基因沉默对胃癌细胞增殖和凋亡的影响,并对其分子机制进行初步研究。方法 将ALCAM 和对照 shRNA转染至胃癌细胞SGC-7901中,建立稳定细胞系,分别采用实时荧光定量（RT-PCR）技术和蛋白免疫印迹法（Western blot）检测转染后SGC-7901细胞中ALCAM mRNA和蛋白水平;采用CCK-8法检测转染后SGC-7901细胞的增殖情况;采用流式细胞术检测转染后SGC-7901细胞凋亡情况。采用胃癌肿瘤模型分析ALCAM基因沉默对肿瘤生长的影响。结果 ALCAM shRNA稳定细胞系ALCAM mRNA（1.01±0.26）和蛋白质的表达水平（1.66±0.23）低于对照 shRNA组细胞ALCAM mRNA（0.12±0.06）和蛋白质水平（0.23±0.09）,差异有统计学意义（P<0.05）。与对照shRNA稳定细胞系比较,ALCAM shRNA 稳定细胞系细胞增殖活性下降（P<0.05）、凋亡水平升高（P<0.05）、荷瘤小鼠肿瘤生长速度减缓（P<0.05）。ALCAM下调并不影响总蛋白激酶B（AKT）和哺乳动物雷帕霉素靶蛋白（mTOR）的表达,而降低了AKT和mTOR磷酸化水平,抑制了mTOR活性,此外,ALCAM蛋白敲低提高了Caspase-3蛋白的表达水平,激活了凋亡信号通路。结论 ALCAM基因沉默能够降低胃癌细胞中ALCAM的表达水平,抑制胃癌细胞的增殖,增加细胞凋亡。     

LncRNA-5657 silencing alleviates sepsis-induced lung injury by suppressing the expression of spinster homology protein 2

BackgroundLncRNAs are closely associated with many human major diseases, however, the roles of lncRNAs in sepsis-induced lung injury remain unclear.MethodsHigh-throughput sequencing was used to detect the changes in lncRNA expression profile in alveolar macrophages after LPS stimulation. The BALF of patients with sepsis-induced lung injury was collected. Rats with CLP-induced septic lung injury were treated with sh-lncRNA-5657 via intravenous injection. NR8383 cells were transfected with lentiviral vectors expressing lncRNA-5657, lncRNA-5657 smart silencer, or si-spns2. The BALF cells expression levels of lncRNA-5657 and proinflammatory cytokines in BALF of patients and rats as well as in rat macrophages were measured. Changes in histopathologic score, lung wet/dry weight ratio, and spns2 expression in macrophages were examined. The relationship between lncRNA-5657 and its potential target gene spns2 was validated using a dual-luciferase reporter assay.ResultsThe lncRNA expression profile of LPS-stimulated macrophages demonstrated that lncRNA-5657 showed the greatest fold-change. The BALF cells of patients with sepsis-induced ARDS and the lung tissue of rats with CLP-induced sepsis had significantly increased lncRNA-5657 levels. LncRNA-5657 silencing alleviated CLP-induced lung inflammation in rats. In NR8383 cells, lncRNA-5657 overexpression enhanced, whereas lncRNA-5657 silencing attenuated the expression of proinflammatory cytokines and spns2. A dual-luciferase reporter assay showed that lncRNA-5657 interacted with the promoter of the spns2 gene. Spns2 silencing alleviated LPS-induced inflammatory response and blocked the proinflammatory function of lncRNA-5657 in alveolar macrophages.ConclusionLncRNA-5657 is closely associated with sepsis-induce lung injury. In vitro and in vivo data demonstrated that LncRNA-5657 silencing alleviates sepsis-induced lung injury by inhibiting lung inflammatory response via suppressing spns2 expression.     

Vaccination with MCP-1 cDNA transfectant on human malignant glioma in nude mice induces migration of monocytes and NK cells to the tumor

Recently, studies on vaccination with tumor cells genetically engineered to produce monocyte chemoattractant protein-1 (MCP-1) have provided some encouragement. These studies have shown infiltration of certain types of lymphocytes at the tumor site. However, natural killer (NK) cells have not yet been assessed. We obtained a human malignant glioma cell line producing human MCP-1 constitutively by transfection of MCP-1 cDNA. We then test the effect of vaccination with the MCP-1 transfectant on nude mice. Although vaccination with MCP-1 transfectant did not reduce the tumor in our study, it was associated with the infiltration of large numbers of NK cells and monocytes at the tumor site. The site of vaccination also showed large numbers of monocytes. NK cells were detected with anti-asialo GM1 antibody, and monocytes were detected immunohistochemically with F4/80. We assumed that infiltrating monocytes at the site of vaccination could promote the infiltration of monocytes and NK cells into the tumor site without T-cell mediated transduction because the host lacked T-cell function.     

Non-histone nuclear factor HMGB1 as a therapeutic target in colorectal cancer

Introduction: High-motility group box (HMGB)-1 is the focus of recent cancer research. HMGB1 plays a critical role in cancer development, progression, and metastasis by activation of cancer cells, enhancement of tumor angiogenesis, and suppression of host anti-cancer immunity. HMGB1 is a relevant target for cancer treatment.

Areas covered: This review aims to overview the biological feature and diverses role in cancer of HMGB1. HMGB1 is a non-histone chromosomal protein, a secretory protein binding to the receptor for advanced glycation end products in cancer cells and monocyte-lineage immune cells, and a DNA presenting chaperon for toll-like receptors. HMGB1 enhances proliferation, motility, invasion and survival of cancer cells. In contrast, HMGB1 induces apoptosis in monocyte-lineage immune cells and inhibits tumor-infiltrating macrophages and dendritic cells, lymph node sinus macrophages and liver Kupffer cells to attenuate anti-cancer immune responses and anti-metastatic organ defense. Then the novel techniques for inhibiting HMGB1 are reviewed.

Expert opinion: Various techniques targeting HMGB1 are subjected to trial. HMGB1 targeting is a potential therapeutic techniqueagainst cancer development, progression, and especially metastasis. Technical breakthroughs in application of HMGB1 targeting to human diseases are now urgently required.     

Involvement of Nrf2 and JNK1 in the Activation of Antioxidant Responsive Element (ARE) by Chemopreventive Agent Phenethyl Isothiocyanate (PEITC)

Purpose. Phenethyl isothiocyanate (PEITC) has been of great interest as a promising cancer chemopreventive agent. To better understand its chemopreventive activity, we examined the effect of PEITC on the antioxidant responsive element (ARE), which is an important gene regulatory element of many phase II drug-metabolizing/detoxification enzymes as well as cellular defensive enzymes. Methods. HeLa cells were transiently transfected with different cDNA plasmids using calcium phosphate precipitation. Subsequently, the cells were maintained in fresh media, and various concentrations of PEITC were added to the transfected cells. After harvesting and lysing of the cells, ARE-luciferase reporter gene activity was measured and normalized against -galactosidase activity. Results. Treatments of HeLa cells with PEITC transiently stimulated ARE-reporter gene expressions in a dose-dependent manner. Overexpression of wild-type NF-E2 related factor-2 (Nrf2) dramatically increased ARE-reporter gene expression in a dose-dependent manner. Similar effects were seen when wild-type c-Jun N-terminal kinase 1 (JNK1) was transfected, although the transactivating potential of JNK1 was much less than that of Nrf2. Cotransfection of Nrf2 and JNK1 showed additional enhancement of ARE reporter gene expression, implying that JNK1 might be an upstream activator of Nrf2. To support this, overexpression of dominant-negative JNK1 suppressed Nrf2-induced ARE reporter gene expression in a dose-dependent manner. When PEITC was added, slight enhancement of ARE reporter gene expression was observed in either Nrf2- or JNK1-transfected cells. Finally, ARE reporter activity induced by PEITC was substantially attenuated by transfection of either dominant-negative mutant of Nrf2 or dominant-negative mutant of JNK1. Conclusion. Taken together, these data suggest that JNK1 acts as an upstream activator of Nrf2 and that PEITC activates ARE-mediated phase II drug metabolism gene expressions via the JNK1- and Nrf2-dependent pathways.     

Ability of Doxorubicin-Loaded Nanoparticles to Overcome Multidrug Resistance of Tumor Cells After Their Capture by Macrophages

Purpose. Investigation of the ability of doxorubicin-loaded nanoparticles (NP/Dox) to overcome multidrug resistance (MDR) when they have first been taken up by macrophages. Methods. The growth inhibition of P388 sensitive (P388) and resistant (P388/ADR) tumor cells was evaluated in a coculture system consisting of wells with two compartments. The tumor cells were seeded into the lower compartment, the macrophages were introduced into the upper part in which the drug preparations were also added. Results. Doxorubicin exerted lower cytotoxicity on tumor cells in coculture compared with direct contact. In P388/ADR, NP/Dox cytotoxicity was far higher than that of free doxorubicin (Dox). Three different formulations of cyclosporin A (either free (CyA), loaded to nanoparticles (NP/CyA) or in a combined formulation with doxorubicin (NP/Dox-CyA)), were added to modulate doxorubicin efficacy. The addition of cyclosporin A to Dox increased drug cytotoxicity. Both CyA added to NP/Dox and NP/Dox-CyA were able to bypass drug resistance. Conclusions. Despite the barrier role of macrophages, NP/Dox remained far more cytotoxic than Dox against P388/ADR. Both NP/ Dox + CyA and NP/Dox-CyA allowed to overcome MDR, but the last one should present greater advantagein vivo by confining both drugs in the same compartment, hence reducing the adverse effects.     

Effects of Transfection with the Cu,Zn-Superoxide Dismutase Gene on Xanthine/Xanthine Oxidase-Induced Cytotoxicity in Fibroblasts from Rat Skin

Purpose. The effects of transfection with the human Cu, Zn-superoxide dismutase (hSOD)⁴ gene on active oxygen-induced cytotoxicity in rat skin fibroblasts (FR) were studied for the purpose of developing the novel delivery system of hSOD using hSOD gene. Methods. An expression plasmid for hSOD, pRc/RSV-SOD, was constructed and used to transfect FR cells. Xanthine (X)/xanthine oxidase (XO) system were used to generate active oxygen species. The effects of transfection with the hSOD gene on active oxygen-induced cytotoxicity were assessed by comparing the number of surviving cells and the level of lipid peroxidation in host and transformants after exposure to X/XO system. Results. The cellular SOD activity in RSV-SOD cells transfected with pRc/RSV-SOD was significantly increased in comparison with host or RSV cells transfected with the pRc/RSV plasmid containing no hSOD gene as a control. Furthermore, Western blot analysis using an anti-hSOD antibody indicated the production of hSOD in RSV-SOD cells. On the other hand, although the numbers of surviving cells in both host and RSV-SOD cultures after exposure to X/XO system decreased in a time-dependent manner, the decrease in number of surviving RSV-SOD cells was less than that in host cells. In the presence of catalase, the decreases in number of surviving cells in both host and RSV-SOD cultures after exposure to the X/XO system were also less than those in the absence of catalase. However, the decreases in cell survival in RSV-SOD cultures were significantly less than those in host cells in the presence of catalase. Furthermore, the levels of lipid peroxidation in RSV-SOD cells exposed to the X/XO system in the presence or absence of catalase were lower than those in host cells. These results indicated that the increase in cellular SOD activity by transfection with the hSOD gene protects cells from oxidative stress. Conclusions. Human SOD gene therapy may be useful for treatment of diseases in which oxidative tissue damage is produced.     

Formation of plasmid-based transfection complexes with an acid-labile cationic lipid: characterization of in vitro and in vivo gene transfer

Purpose. This study tests the hypothesis that gene transfer efficiency may be improved through the use of transiently stable transfection complexes that degrade within endosomal compartments and promote plasmid escape into the cytosol. Method. An acid labile cationic lipid, O-(2R-1,2-di-O-(1`Z, 9`Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), was designed, synthesized, and tested for enhanced gene transfer activity relative to non-labile controls. Results. The O-alkenyl chains of BCAT were completely hydrolyzed after 4 h incubation in pH 4.5 buffer at 25°C. Addition of BCAT to plasmid DNA in 40%ethanol followed by ethanol evaporation yielded transfection complexes that transfected several cell types in the presence of fetal calf serum and without the need of a helper lipid. Transfection complexes prepared from BCAT displayed higher luciferase expression than the corresponding DCAT complexes (an acid-insensitive derivative of BCAT) for all cell types tested. Uptake studies showed that this increase was not due to a difference in the amount of DNA being delivered. FACS analysis for GFP expression showed that BCAT transfection complexes yielded 1.6 more transfected cells and 20%higher log mean fluorescence than DCAT transfection complexes. In vivo gene transfer was demonstrated in subcutaneous tumor-bearing mice by systemic administration of a 60 g plasmid dose. Expression was observed in the lungs and in the tumor, with the highest activity being observed in the lungs. Conclusions. Our results show that increased transfection can be obtained by coupling the cationic headgroup to the hydrophobic amphiphilic tails via acid-labile bonds. Acid-catalyzed release of the alkyl chains should facilitate dissociation of the cationic lipid headgroup from the plasmid, thus accelerating one of the rate-limiting steps in cationic lipid mediated transfection.     

Human Dipeptide Transporter,hPEPTl, Stably Transfected into Chinese Hamster Ovary Cells

Purpose. A cDNA encoding the H⁺-coupled peptide transporter, hPEPTl, has previously been cloned from human ileum (8). The objective of this study was to establish a stably transfected cell line expressing hPEPTl in mammalian cell culture. Methods. The hPEPTl cDNA was subcloned into an expression vector carrying the CMV promoter and a neomycin resistance gene. This vector, pCDNA3-PEPT1, was transiently transfected into several cell lines to identify those capable of expressing PEPT1 transport function. CHO cells were selected and stably transfected with PEPT1 (CHO-PEPT1). Dipeptide transport activity was measured with ³H-Gly-Sar, in the presence and absence of inhibitors. Results. The clonal cell line, CHO-PEPT1, displayed high transport activity. Dipeptide transport was sensitive to pH and specific for dipeptides and other small peptides. Peptidomimetic antibiotics, such as cephalexin, were competitors for peptide transport. Conclusions. The stably transfected cell line, CHO-PEPT1 exhibits enhanced transport over that of cell lines with native expression of PEPT1, and therefore, represents a useful tool for rapid screening of drugs that utilize the peptide transporter in the human intestine for absorption.     

医用透明质酸钠凝胶对肿瘤生长和转移影响的实验研究

目的 考察医用透明质酸钠凝胶（medical sodium hyaluronate gel,HA）在体外和裸鼠体内对腹、盆腔相关肿瘤生长和转移的影响。方法 利用Hela、CT26和HCT116等3株肿瘤细胞,通过MTT法和Transwell实验分别考察不同浓度HA体外对肿瘤细胞生长和迁移的影响;建立裸鼠结肠原位种植瘤模型,比较瘤块接种4周后不同实验组的瘤块体积和瘤重,考察HA体内对结肠癌HCT116增殖的影响;通过比较接种3周后不同实验组裸鼠的肺和肝脏的肿瘤转移率和转移病灶数,考察HA对结肠癌CT26体内转移的影响。结果 不同浓度HA在体外均未促进Hela、CT26和HCT116细胞的生长和转移,且在5 mg/ml HA浓度下,显示出一定的抑制作用。体内结果显示,HA未促进结肠癌HCT116肿瘤的生长,却显示出明显抑制CT26肿瘤的转移。结论 在本实验条件下,HA在体内、外均未促进腹、盆腔相关肿瘤细胞Hela、CT26和HCT116的生长、迁移和转移。     

Effects of schisandrin on transcriptional factors in lipopolysaccharide-pretreated macrophages

Schisandrin is the main active ingredient isolated from Schisandra chinensis Baill. Recent studies have demonstrated that schisandrin exhibits anti-inflammatory effects in vivo and in vitro. In this study, we examined whether the order of lipopolysaccharide (LPS) treatment affects the mechanism of schisandrin anti-inflammatory activity. We found that the antiinflammatory mechanisms are not the same depending on whether macrophages were treated with schisandrin before or after LPS. The main difference is that inhibitor kappaBα (IκBα) degradation was not inhibited when macrophages were pretreated by LPS before schisandrin and was weakly inhibited when macrophages were pretreated by schisandrin before LPS.     

In Vivo Gene Transfer by Intravenous Administration of Stable Cationic Lipid/DNA Complex

Purpose. A stable cationic lipid/DNA complex has been developed for in vivo gene transfer. The formulation capitalizes on a previously described procedure to obtain stable lipid/DNA complexes for in vitro gene transfer (1). Methods. Conditions for DNA/lipid complex formation were modified to yield a DNA concentration of 1 mg/ml. Heat stable alkaline phosphatase (AP) under a CMV promoter was used as a reporter gene. Results. The resulting complex was completely insensitive to serum inactivation. Tail vein injection of a 80 g DNA into Balb C mice yielded significant levels of reporter enzyme activity in the lung, heart, spleen, muscle, and liver. Less AP activity was observed in the kidney. No AP activity was observed in blood, bone marrow or brain. A titration of the lipid (DOSPA) to DNA-nucleotide ratio showed the optimal molar ratio for in vivo gene transfer to be 1/1. Using this ratio in a dose response study showed approximately 80 g of DNA/mouse yielded the highest level of gene expression. Using this dose at a 1/1 lipid to DNA nucleotide ratio, the time course for alkaline phosphatase activity was determined. Maximal AP activity was observed 24 hours after injection for all tissues. By day 5, the activity dropped approximately 10 fold for all tissues. By day 7, residual activity was detected in the lung, heart, and muscle. Histology of the lung showed both interstitial and endothelial cells to be transfected. In all other tissues, however, endothelial cells were the only transfected cell type. Conclusions. These results demonstrate that reformulation of an existing cationic lipid can result in the formation of a stable lipid/DNA complex, which is able to reproducibly transfect lung, heart, spleen, and liver upon intravenous administration.     

Two new bis-C20-diterpenoid alkaloids with anti-inflammation activity from Aconitum bulleyanum

Two new bis-C₂₀-diterpenoid alkaloids, bulleyanines A and B (1 and 2), were isolated from Aconitum bulleyanum. Their structures were elucidated by comprehensive spectroscopic analyses including UV, IR, MS, 1D and 2D NMR. Biological activity tests indicated that compound 1 exhibited significant inhibitory activity against nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW264.7 macrophages with the inhibition rate of 74.60% (40 μmol/L), while positive control dexamethasone gave 78.70% inhibition at 100 μg/ml.