罗格列酮对人结肠癌Lovo细胞增殖的抑制作用及其机制

目的探讨罗格列酮对人结肠癌Lovo细胞增殖的抑制作用及其可能的作用机制。方法人结肠癌Lovo细胞分别经不同浓度(10-6、10-5、10-4和10-3 mol/L)的罗格列酮和美洛昔康作用6、12和24 h后,采用MTT法检测细胞的增殖活性,RT-PCR法检测罗格列酮处理24 h的细胞COX-2基因mRNA的转录水平,Western blot法检测细胞COX-2蛋白的表达水平。结果罗格列酮浓度大于10-5 mol/L,美洛昔康浓度大于10-4 mol/L均可明显抑制Lovo细胞增殖,且呈浓度和时间依赖性;罗格列酮可明显降低Lovo细胞COX-2基因mRNA和蛋白的表达水平,且均呈浓度依赖性。结论罗格列酮能抑制人结肠癌Lovo细胞增殖,其作用机制与抑制细胞COX-2的表达有关。     

小球藻类金属硫蛋白抗紫外辐射的作用研究

建立了小鼠胚胎成纤维(NIH/3T3)细胞紫外辐射损伤的实验模型,考察小球藻类金属硫蛋白(Zn-MT-like)对NIH/3T3细胞紫外辐射的保护作用。结果表明,小球藻Zn-MT-like对NIH/3T3细胞的生长无抑制作用,且具有较强的抗紫外辐射能力。质量浓度为10 g/L的小球藻Zn-MT-like可显著减少NIH/3T3细胞空泡、降低细胞裂解的程度,可使经紫外辐射的NIH/3T3细胞保持较好的活性。     

Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines

TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). β-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte^® system, cell cycle/apoptosis, and clonogenic potential. COX-2, β-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, β-catenin, MGMT, and SOX-2 up-regulation.     

Artesunate Induces Apoptosis of Bladder Cancer Cells by miR-16 Regulation of COX-2 Expression

Bladder cancer is the most common malignant tumor of the urinary tract and remains one of the major causes of cancer death worldwide. In this study, we investigated the effect and mechanism of Artesunate (ART), a traditional Chinese medicine, on inducing apoptosis of human bladder cancer cells. In vivo antitumor activity was investigated in bladder cancer in rat by subcutaneous injection of different concentration of ART. The effect of ART on growth inhibition and apoptosis of bladder cancer cells was evaluated using dimethylthiazoly-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. Cyclooxygenase-2 (COX-2) and miR-16 expression levels were determined with real-time PCR. The concentrations of prostaglandin E2 (PGE2) in the supernatants of bladder cancer cells were measured with an ELISA kit. The miR-16 inhibitor or mimic were transfected into cells to up- or down-regulate miR-16 expression. ART efficiently inhibited orthotopic tumor growth in the bladder cancer rat, which is accompanied with an increase of miR-16 expression and a decrease of COX-2 expression. In vitro, ART could induce cytotoxicity and apoptosis in bladder cancer cells, but presented a much lighter toxicity effect against normal human urothelial cells. ART significantly increased miR-16 expression and decreased the expression of COX-2 and the production of PGE2. More importantly, down-regulation of miR-16 expression could reverse the effect of ART on apoptosis and COX-2 expression in bladder cells. Moreover, exogenous PGE2 could inhibit apoptosis of bladder cancer cells treated with ART. In conclusion, ART can elicit an anti-tumor effect against bladder cancer by up-regulation of miR-16 expression, which resulted in the decrease of COX-2 expression and PGE2 production. Hence, ART might be an effective drug for the treatment of bladder cancer.     

无血清摇动悬浮培养富集乳腺癌干细胞

目的探讨不同培养条件下乳腺癌MCF-7细胞的分裂特点,建立快速、有效的乳腺癌干细胞富集方法。方法将MCF-7细胞分别采用完全培养基静置培养(A组)、完全培养基摇动培养(B组)、细胞因子静置培养(C组)和细胞因子摇动培养(D组)12、24、36 h,倒置相差显微镜下观察各组细胞分裂情况,计算克隆形成率,采用流式细胞术检测CD44+/CD24-/low细胞亚群的比例变化。结果 B组和C组培养12 h,棒状分裂细胞约占30%;24 h后分别约占50%和60%;36 h后形成大的细胞克隆。D组培养12 h,棒状分裂细胞约占50%;24 h后约占80%,可见多个细胞克隆;36 h后克隆细胞数减少。D组培养12 h,CD44+/CD24-/low细胞亚群比例(8.05%)为B组(0.99%)的8倍,C组(3.80%)的2.1倍;培养24 h,D组(15.24%)为B组(4.83%)的3倍,C组(2.30%)的6倍;培养36 h,D组CD44+/CD24-/low细胞亚群比例降至9.68%,约为B组(0.95%)和C组(1.03%)的9倍。结论在细胞因子摇动培养条件下,MCF-7细胞分裂加快,分裂象增多,细胞系中CD44+/CD24-/low亚群比例增加较快,干细胞池增加明显,表明无血清摇动悬浮培养为富集乳腺癌干细胞的快速、有效的方法。     

A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells

Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions.     

Accumulation and Toxicity of Superparamagnetic Iron Oxide Nanoparticles in Cells and Experimental Animals

The uptake and distribution of negatively charged superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs) in mouse embryonic fibroblasts NIH3T3, and magnetic resonance imaging (MRI) signal influenced by SPIONs injected into experimental animals, were visualized and investigated. Cellular uptake and distribution of the SPIONs in NIH3T3 after staining with Prussian Blue were investigated by a bright-field microscope equipped with digital color camera. SPIONs were localized in vesicles, mostly placed near the nucleus. Toxicity of SPION nanoparticles tested with cell viability assay (XTT) was estimated. The viability of NIH3T3 cells remains approximately 95% within 3–24 h of incubation, and only a slight decrease of viability was observed after 48 h of incubation. MRI studies on Wistar rats using a clinical 1.5 T MRI scanner were showing that SPIONs give a negative contrast in the MRI. The dynamic MRI measurements of the SPION clearance from the injection site shows that SPIONs slowly disappear from injection sites and only a low concentration of nanoparticles was completely eliminated within three weeks. No functionalized SPIONs accumulate in cells by endocytic mechanism, none accumulate in the nucleus, and none are toxic at a desirable concentration. Therefore, they could be used as a dual imaging agent: as contrast agents for MRI and for traditional optical biopsy by using Prussian Blue staining.     

CLA Reduces Inflammatory Mediators from A427 Human Lung Cancer Cells and A427 Conditioned Medium Promotes Differentiation of C2C12 Murine Muscle Cells

Conjugated linoleic acid (CLA) is thought to have anti-proliferative and anti-inflammatory properties, but its effect on cancer cachexia is unknown. Two effects were here investigated: that of CLA on inflammatory mediator production in human lung cancer cells, and that of reduced mediators on the myogenic differentiation of murine muscle C2C12 cells. The latter cells were grown in medium conditioned by human lung cancer A427 cells, with or without CLA, to mimic only the effect of molecules released from the tumor “in vivo”, excluding the effect of host-produced cachectic factors. The results obtained show that CLA was found to reduce the production of tumor necrosis factor-α, interleukin (IL)-1β and prostaglandin E2 (PGE2), but had no effect on IL-6 production. The mechanisms underlying the effect of CLA on cytokine or PGE2 release in A427 cells are probably mediated by activation of peroxisome proliferator-activated receptor (PPAR)α, which increased at 24 h CLA treatment. In turn, the reduced content of inflammatory mediators in medium conditioned by A427 cells, in the presence of CLA, allowed muscle cells to proliferate, again by inducing PPAR. The involvement of PPARα was demonstrated by treatment with the antagonist MK-886. The findings demonstrate the anti-inflammatory and myogenic action of CLA and point to its possible application as a novel dietary supplement and therapeutic agent in inflammatory disease states, such as cachexia.     

2-Chlorohexadecanal and 2-Chlorohexadecanoic Acid Induce COX-2 Expression in Human Coronary Artery Endothelial Cells

2-Chlorohexadecanal (2-ClHDA), a 16-carbon chain chlorinated fatty aldehyde that is produced by reactive chlorinating species attack of plasmalogens, is elevated in atherosclerotic plaques, infarcted myocardium, and activated leukocytes. We tested the hypothesis that 2-ClHDA and its metabolites, 2-chlorohexadecanoic acid (2-ClHA) and 2-chlorohexadecanol (2-ClHOH), induce COX-2 expression in human coronary artery endothelial cells (HCAEC). COX-2 protein expression increased in response to 2-ClHDA treatments at 8 and 20 h. 2-ClHA also increased COX-2 expression following an 8 h treatment. Quantitative PCR showed that 2-ClHDA treatment increased COX-2 mRNA over 8 h, while 2-ClHA treatment led to a modest increase by 1 h and those levels remained constant over 8 h. 2-ClHDA led to a significant increase in 6-keto-PGF(1alpha) release (a measure of PGI(2) release) by HCAEC. These data suggest that 2-ClHDA and its metabolite 2-ClHA, which are produced during leukocyte activation, may alter vascular endothelial cell function by upregulation of COX-2 expression.     

Self-Targeting of Carbon Dots into the Cell Nucleus: Diverse Mechanisms of Toxicity in NIH/3T3 and L929 Cells

It is important to understand the nanomaterials intracellular trafficking and distribution and investigate their targeting into the nuclear area in the living cells. In our previous study, we firstly observed penetration of nonmodified positively charged carbon dots decorated with quaternary ammonium groups (QCDs) into the nucleus of mouse NIH/3T3 fibroblasts. Thus, in this work, we focused on deeper study of QCDs distribution inside two healthy mouse NIH/3T3 and L929 cell lines by fluorescence microspectroscopy and performed a comprehensive cytotoxic and DNA damage measurements. Real-time penetration of QCDs across the plasma cell membrane was recorded, concentration dependent uptake was determined and endocytic pathways were characterized. We found out that the QCDs concentration of 200 µg/mL is close to saturation and subsequently, NIH/3T3 had a different cell cycle profile, however, no significant changes in viability (not even in the case with QCDs in the nuclei) and DNA damage. In the case of L929, the presence of QCDs in the nucleus evoked a cellular death. Intranuclear environment of NIH/3T3 cells affected fluorescent properties of QCDs and evoked fluorescence blue shifts. Studying the intracellular interactions with CDs is essential for development of future applications such as DNA sensing, because CDs as DNA probes have not yet been developed.     

特异性抗原致敏的DC-CIK细胞对肿瘤细胞的杀伤效应

目的检测特异性抗原致敏的DC-CIK细胞对恶性肿瘤细胞的杀伤效应。方法采用肿瘤抗原致敏的DC与CIK细胞共培养,分析其免疫表型,并用MTT染色法检测其对肿瘤细胞的杀伤力。结果所获得DC-CIK细胞的CD3异质性T细胞群,对肾透明细胞癌786-0和前列腺癌PC-3细胞的杀伤率分别为70.64%和65.65%。结论DC-CIK细胞对肾透明细胞癌786-0细胞和前列腺癌PC-3细胞具有较强的杀伤效应。     

Inhibitory Effect of N-Acyl Dopamines on IgE-Mediated Allergic Response in RBL-2H3 Cells

Recently, endogenous N-acyl dopamines have been found to show anti-inflammatory and immunomodulatory activities. However, the effect of the N-acyl dopamines on allergic responses was not reported. In this study, we investigated whether N-acyl dopamines might inhibit immunoglobulin E-mediated degranulation in RBL-2H3 cells. When RBL-2H3 cells were exposed to palmitoyl dopamine (NP-DA), oleoyl dopamine (NO-DA) or arachidonoyl dopamine (NA-DA) at micromolar levels, all these compounds significantly inhibited the release of β-hexosaminidase, a marker of degranulation, as well as tumor necrosis factor (TNF)-α. In comparison, NP-DA, potently suppressing the release of β-hexosaminidase (IC₅₀, 3.5 μM) and TNF-α (IC₅₀, 2.2 μM), was more potent than NO-DA or NA-DA. Additionally, NP-DA markedly suppressed the formation of prostaglandin E₂, prostaglandin D₂ and leukotriene C₄, corresponding to pro-inflammatory lipid mediators in asthma. In the mechanistic analyses, where the effect of NP-DA on the FcεRI cascade was examined, NP-DA significantly inhibited the phosphorylation and expression of Syk, but not Lyn. And, NP-DA also suppressed phosphorylation of ERK1/2 and Akt. Further, NP-DA decreased the phosphorylation of cPLA₂ and 5-lipoxygenase (5-LO), but not cyclooxygenase-2 (COX-2). Based on these results, it is suggested that NP-DA exert anti-allergic effect on allergic response through suppressing the activation of Syk, ERK1/2, Akt, cPLA₂ and 5-LO. Besides, a strong inhibition of COX-2 activity by NP-DA may be additional mechanism for its anti-allergic action. Such an anti-allergic action of N-acyl dopamines may contribute to further information about biological functions of N-acyl dopamines.     

Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on ovarian carcinoma in vivo

The present study was designed to investigate the combined effects of cyclooxygenase (COX)-1 and COX-2 selective inhibitors on human ovarian SKOV-3 carcinoma cells xenograft-bearing mice. The animals were treated with 3 mg/kg SC-560 (a COX-1 selective inhibitor) alone, 25 mg/kg celecoxib (a COX-2 selective inhibitor) alone, or SC-560/celecoxib by gavage, twice a day for three weeks. To test the mechanism of inhibition of tumor growth by COX selective inhibitors, the index of proliferating cells in tumor tissues was determined by immunostaining and the index of apoptotic cells by the terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. The inhibitory rate on tumor growth in the combination group was 35.54% which is significant statistically compared with that of the control group (P < 0.05). In the combination group, the index of cell proliferation and apoptosis were 12.40% and 51.03% respectively, which are significant statistically compared with those of the control group (22.56%, 19.07%, all P < 0.05). These studies indicate that synergism between two COX inhibitors and inhibitor combination treatment has particular potential for chemoprevention of ovarian cancer growth.     

Elevated STAT3 Signaling-Mediated Upregulation of MMP-2/9 Confers Enhanced Invasion Ability in Multidrug-Resistant Breast Cancer Cells

The development of multidrug resistance greatly impedes effective cancer therapy. Recent advances in cancer research have demonstrated that acquisition of multidrug resistance by cancer cells is usually accompanied by enhanced cell invasiveness. Several lines of evidence indicated that cross activation of other signaling pathways during development of drug resistance may increase invasive potential of multidrug-resistant (MDR) cancer cells. However, the accurate mechanism of this process is largely undefined. In this study, to better understand the associated molecular pathways responsible for cancer progression induced by drug resistance, a MDR human breast cancer cell line SK-BR-3/EPR with P-glycoprotein overexpression was established using stepwise long-term exposure to increasing concentration of epirubicin. The SK-BR-3/EPR cell line exhibited decreased cell proliferative activity, but enhanced cell invasive capacity. We showed that the expression of metastasis-related matrix metalloproteinase (MMP)-2/9 was elevated in SK-BR-3/EPR cells. Moreover, SK-BR-3/EPR cells showed elevated activation of STAT3. Activation of STAT3 signaling is responsible for enhanced invasiveness of SK-BR-3/EPR cells through upregulation of MMP-2/9. STAT3 is a well-known oncogene and is frequently implicated in tumorigenesis and chemotherapeutic resistance. Our findings augment insight into the mechanism underlying the functional association between MDR and cancer invasiveness.     

人参皂苷Rh2诱导人胃癌SGC-7901细胞凋亡的研究

目的研究人参皂苷Rh2(G-Rh2)对人胃癌细胞SGC-7901的影响。方法采用MTT法检测G-Rh2对SGC-7901细胞存活率的影响;流式细胞分析法检测凋亡小体的含量;免疫印迹技术及体外Caspase-3/-7活力测定方法检测Caspase的激活状态。结果G-Rh2对SGC-7901细胞生长有明显的抑制作用,且呈量-效关系,IC50为9.3μg/ml。7.5μg/mlG-Rh2作用SGC-7901细胞24h,凋亡细胞数量为6.97%。7.5μg/mlG-Rh2作用SGC-7901细胞20h,出现多聚(ADP-核糖)聚合酶[poly(ADP-ribose)polymerase,PARP]断裂,Caspase-3/-7活力开始出现,并随作用时间的延长而增强。结论G-Rh2诱导Caspase参与SGC-7901细胞凋亡。     

COX-2 Inhibition and Inhibition of Cytosolic Phospholipase A2 Increase CD36 Expression and Foam Cell Formation in THP-1 Cells

Cardiovascular safety of cyclooxygenase (COX)-2-selective inhibitors and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) is of worldwide concern. COX-2 inhibitors and NSAIDs act by inhibiting arachidonic acid metabolism to prostaglandins. They confer a cardiovascular hazard manifested as an elevated risk of myocardial infarction. Mechanisms underlying these cardiovascular effects are uncertain. Here we determine whether interference with cytosolic phospholipase A2 (cPLA-2) or COX-2 through pharmacologic blockade or silencing RNA impacts expression of scavenger receptor CD36 and scavenger receptor A, both involved in cholesterol uptake in monocytes and macrophages. THP-1 human monocytes and human peripheral blood mononuclear cells were exposed to celecoxib, a COX-2 selective inhibitor currently in clinical use, and to arachidonyl trifluoromethyl ketone (AACOCF3), an arachidonic acid analog that selectively inhibits cPLA-2. Celecoxib and AACOCF3 each upregulated expression of CD36, but not scavenger receptor A, as determined by quantitative PCR and immunoblotting. Silencing of cPLA-2 or COX-2 had comparable effects to pharmacologic treatments. Oil red O staining revealed a profound increase in foam cell transformation of THP-1 macrophages exposed to either celecoxib or AACOCF3 (both 25 μM), supporting a role for the COX pathway in maintaining macrophage cholesterol homeostasis. Demonstration of disrupted cholesterol balance by AACOCF3 and celecoxib provides further evidence of the possible mechanism by which COX inhibition may promote lipid overload leading to atheromatous lesion formation and increased cardiovascular events.     

KDRn3蛋白在人脐静脉血管内皮细胞中的表达及对其增殖的抑制作用

目的观察KDRn3蛋白在人脐静脉血管内皮细胞中的表达及对其增殖的抑制作用。方法将质粒pEGFP-N1/KDRn3扩增并鉴定后,以脂质体介导转染人脐静脉血管内皮细胞,培养一定时间后,在荧光显微镜下观察绿色荧光蛋白的表达,ELISA检测细胞培养上清中KDRn3的含量,MTT法检测KDRn3蛋白对人脐静脉血管内皮细胞增殖的影响。结果转染后48h,在荧光显微镜下可见pEGFP-N1/KDRn3转染组人脐静脉血管内皮细胞发出绿色荧光,72h发出绿色荧光的细胞增多,强度增强;转染后24、48和72h,细胞培养上清中KDRn3含量与转染前比较均提高,且差异有显著意义;转染后48和72h,pEGFP-N1/KDRn3转染组与对照组比较,细胞增殖有明显的抑制,且差异有显著意义。结论KDRn3蛋白可在人脐静脉血管内皮细胞中表达,并能抑制其增殖。     

Successful Treatment of Persistent SARS-CoV-2 Infection in a B-Cell Depleted Patient with Activated Cytotoxic T and NK Cells: A Case Report

We report a lymphoma patient with profound B-cell deficiency after chemotherapy combined with anti-CD20 antibody successfully treated with remdesivir and convalescent plasma for prolonged SARS-CoV-2 infection. Viral clearance was likely attributed to the robust expansion and activation of TCR Vβ2 CD8+ cytotoxic T cells and CD16 + CD56- NK cells. This is the first presentation of TCR-specific T cell oligoclonal response in COVID-19. Our study suggests that B-cell depleted patients may effectively respond to anti-SARS-CoV-2 treatment when NK and antigen-specific Tc cell response is induced.