Antisurvivin oligonucleotides inhibit growth and induce apoptosis in human medullary thyroid carcinoma cells

Survivin is a novel member of the inhibitor of apoptosis protein (IAP) family, which is known to be over-expressed in various carcinomas and associated with their biologically aggressive characteristics. The aim of this study was to investigate survivin expression in human medullary thyroid carcinoma (MTC) and a MTC cell line TT, correlate survivin expression with clinicopathologic features of MTC, and test effects of antisurvivin oligonucleotides (ASODNs) on growth and apoptosis of TT cells. Survivin expression was immunohistochemically determined in formalin-fixed and paraffin-embedded specimens obtained from 10 cases of normal thyroid (NT) and 10 cases of MTC, and in TT cells. In TT cells, we confirmed survivin expression and its down-regulation by ASODNs using RT-PCR and Western blot analyses, and investigated effects of ASODNs on viability and growth by MTT assay and apoptosis by apoptotic analyses including DNA laddering assay, acridine orange/ethidium bromide staining and flow cytometric cell cycle analysis. Immunohistochemical analysis showed high survivin expression in MTC and TT cells, whereas no immunoreactivity was detectable in NT. Statistical analyses revealed no significant correlation of survivin expression with the clinicopathologic features of MTC. In TT cells, survivin expression at both mRNA and protein levels was confirmed and could be down-regulated by ASODNs concomitant with decrease in viability and growth, and increase in apoptosis. Our results suggest that survivin plays an important role in MTC independent of the conventional clinicopathologic factors, and ASODNs is a promising survivin-targeted gene therapy for MTC.     

Down-regulation of survivin suppresses uro-plasminogen activator through transcription factor JunB

Survivin, a member of the inhibitors of apoptosis protein family, is expressed during development and in various human cancers. However, the clinical relevance of survivin in cancer is still a matter of debate. Genes induced by hepatocyte growth factor (HGF) were screened using cDNA microarray technology in the stomach cancer cell lines, NUGC3 and MKN28. The levels of JunB, survivin, and uro-plasminogen activator (uPA) were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced up regulation of JunB, survivin, and uPA was inhibited by pre-treatment with a MEK inhibitor (PD 98059). HGF-induced up-regulation of uPA was repressed by survivin knockdown. HGF enhanced the binding activity of JunB to the survivin promoter in control cells, but not in the JunB-shRNA cells. Transfection with survivin- shRNA resulted in a decrement of cell proliferation, as determined with MTT assays. In an in vitro invasion assay, significantly fewer cells transfected with survivin shRNA than control cells were able to invade across a Matrigel membrane barrier. In conclusion, survivin appeared to play an important role in the up-regulation of uPA induced by HGF via JunB and might contribute to HGF-mediated tumor invasion and metastasis, which may serve as a promising target for gastric cancer therapy.     

Optimization of photodynamic therapy response by survivin gene knockdown in human metastatic breast cancer T47D cells

Photodynamic therapy (PDT) leads to the generation of cytotoxic oxygen species that appears to stimulate several different signaling pathways, some of which lead to cell death, whereas others mediate cell survival. In this context, we observed that PDT mediated by methyl-5-aminolevulinic acid as the photosensitizer resulted in over-expression of survivin, a member of the inhibitor of apoptosis (IAP) family that correlates inversely with patient prognosis. The role of survivin in resistance to anti-cancer therapies has become an area of intensive investigation. In this study, we demonstrate a specific role for survivin in modulating PDT-mediated apoptotic response. In our experimental system, we use a DNA vector-based siRNA, which targets exon-1 of the human survivin mRNA (pSil_1) to silence survivin expression. Metastatic T47D cells treated with both pSil_1 and PDT exhibited increased apoptotic indexes and cytotoxicity when compared to single-agent treated cells. The treatment resulted in increased PARP and caspase-3 cleavage, a decrease in the Bcl-2/Bak ratio and no participation of heat shock proteins. In contrast, the overexpression of survivin by a survivin-expressed vector increased cell viability and reduced cell death in breast cancer cells treated with PDT. Therefore, our data suggest that combining PDT with a survivin inhibitor may attribute to a more favorable clinical outcome than the use of single-modality PDT.     

Inhibition of Survivin Restores the Sensitivity of Breast Cancer Cells to Docetaxel and Vinblastine

Combination therapy is considered a viable strategy to overcome the resistance to chemotherapeutics. Survivin as a member of the inhibitor of apoptosis protein (IAP) family, which is involved in resistance to various drugs. We investigated the role of combination therapy in downregulating survivin and increasing drug’s efficacy in MDA-MB-231 cells. MTT assay and DAPI staining were applied to study the anti-proliferative activity and apoptosis response of the agents. Real-time RT-PCR and Western blot analysis were applied to study survivin mRNA and protein. Our findings showed that combined treatment of cells with docetaxel and vinblastine reduces survivin expression and consequently decreases the IC₅₀ value of docetaxel from 70 to 5 nM (p?p?相似文献   

Ultraviolet-B radiation causes an upregulation of survivin in human keratinocytes and mouse skin

Understanding of the mechanism of ultraviolet (UV)-mediated cutaneous damages is far from complete. The cancer-specific expression of Survivin, a member of the inhibitor of apoptosis family of proteins, coupled with its importance in inhibiting cell death and in regulating cell division, makes it a target for cancer treatment. This study was designed to investigate the modulation of Survivin during UV response, both in vitro and in vivo. We used UV-B-mediated damages in normal human epidermal keratinocytes (NHEK) cells as an in vitro model and SKH-1 hairless mouse model for the in vivo studies. For in vitro studies, NHEK were treated with UV-B and samples were processed at 5, 15, 30 min, 1, 3, 6, 12 and 24 h after treatment. Our data demonstrated that UV-B exposure (50 mJ/cm2) to NHEK resulted in a significant upregulation in Survivin messenger RNA (mRNA) and protein levels. We also observed that UV-B exposure to NHEK resulted in significant (1) decrease in Smac/DIABLO and (2) increase in p53. For in vivo studies, the SKH-1 hairless mice were subjected to a single exposure of UV-B (180 mJ/cm2), and samples were processed at 3, 6, 12 and 24 h after UV-B exposure. UV-B treatment resulted in a significant increase in protein or mRNA levels (or both) of Survivin, phospho-Survivin and p53 and a concomitant decrease in Smac/DIABLO in mouse skin. This study demonstrated, for the first time, the involvement of Survivin (and the associated events) in UV-B response in vitro and in vivo in experimental models regarded to have relevance to human situations.     

Tunicamycin enhances TRAIL-induced apoptosis by inhibition of cyclin D1 and the subsequent downregulation of survivin

TNF-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising cancer therapy that preferentially induces apoptosis in cancer cells, but not most normal tissues. However, many cancers are resistant to TRAIL by mechanisms that are poorly understood. In this study, we showed that tunicamycin, a naturally occurring antibiotic, was a potent enhancer of TRAIL-induced apoptosis through downregulation of survivin. The tunicamycin-mediated sensitization to TRAIL was efficiently reduced by forced expression of survivin, suggesting that the sensitization was mediated at least in part through inhibition of survivin expression. Tunicamycin also repressed expression of cyclin D1, a cell cycle regulator commonly overexpressed in thyroid carcinoma. Furthermore, silencing cyclin D1 by RNA interference reduced survivin expression and sensitized thyroid cancer cells to TRAIL; in contrast, forced expression of cyclin D1 attenuated tunicamycin-potentiated TRAIL-induced apoptosis via over-riding downregulation of survivin. Collectively, our results demonstrated that tunicamycin promoted TRAIL-induced apoptosis, at least in part, by inhibiting the expression of cyclin D1 and subsequent survivin. Of note, tunicamycin did not sensitize the differentiated thyroid epithelial cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may offer an attractive strategy for safely treating resistant thyroid cancers.     

Delivery of Survivin siRNA Using Cationic Diphenylalanine Vesicles

Small interfering RNA(siRNA) has been proved to be a powerful tool for silencing target gene in cells, raising the possibility that siRNA can be employed as a therapy for treating cancers and other genetic diseases. However, siRNA transfection has the limitation due to the difficulty in the delivery of siRNA to target cells and tissues. To explore an efficient biocompatible siRNA delivery system, cationic diphenylalanine vesicles(CDPVs) were constructed to transfer survivin siRNA to human ovarian cancer cells. The morphology of CDPVs was characterized by scanning electron microscopy(SEM) and the distribution of survivin siRNA was characterized by confocal laser scanning microscopy, which reveal that diphenylalanine and the survivin siRNA were successfully co-delivered. After co-incubation for 48 h, the CDPVs/siRNA exhibited enhanced tumor cell growth inhabitation and apoptosis inducted in human SK-OV-3 ovarian carcinoma cells. Overall, CDPVs is an efficient siRNA delivery system and has a promising prospect for cancer therapy.     

Paxilline enhances TRAIL-mediated apoptosis of glioma cells via modulation of c-FLIP, survivin and DR5

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. Here, we report that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Paxilline treatment markedly upregulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly downregulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPs) and the caspase inhibitor, survivin, through proteasome-mediated degradation. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5 or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.     

钛/活细胞界面原位EIS研究

设计了原位电解池用于生物材料/活细胞界面电化学交流阻抗谱(EIS)的测量, 原位观测了钛/MG63细胞界面EIS行为及其随时间的变化. 原位电解池中引入Ag/AgCl作为参比电极, 解决了EIS测量过程中高频部分信号失真的问题. 钛/MG63细胞界面EIS研究初步结果表明: 活细胞在钛电极表面附着, 可改变界面双电层结构和电极的表面状态, 进而影响界面的电化学性质. 实验发现, 由于钛电极表面氧化层的阻抗信号处于中低频段, 而细胞膜层自身的电化学阻抗响应位于高频范围, 由此, 可分别分析不同的电极过程, 解析其相互关系, 研究活细胞在生物材料表面的作用机理.     

Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.     

Cellular toxicity induced by different pH levels on the R3230AC rat mammary tumour cell line. An in vitro model for investigation of the tumour destructive properties of electrochemical treatment of tumours

INTRODUCTION: The aim of this study was to evaluate the cellular toxicity of different pH levels on the R3230AC mammary tumour cell line (clone-D) in vitro and to determine in what way the pH affects the tumour cells. The results could be used to interpret the cell damaging effects seen in electrochemical treatment of tumours (EChT), where pH alteration in tissue is the major event. METHODS: Tumour cells were treated with pH 3.5, 5, 7, 9, 10 and 11 for 10, 20 or 30 min, respectively, followed by studies with the viability assay 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (methyltetrazolium (MTT)), morphological observation in phase contrast microscope (PCM) and light microscope, nucleotide analogue incorporation (BrdU; 5-Brdmo-2'-deoxyuridine), Caspase-3 activity measurement and detection of DNA fragmentation by an agarose gel electrophoresis. RESULTS: In the viability assay, it was found that different pH levels had cytotoxic effects; these effects were dependent on the pH value and on the time of exposure at a given pH. Morphologically, cells in pH 3.5 and 5 had shrunk, were rounded and had condensed chromatin, whereas prominent cell swelling and nuclear expansion were seen in the pH 9- and 10-treated cells. Gross cytolysis was found in pH 11. A BrdU incorporation assay indicated that proliferation rate is inhibited markedly both with decreasing and increasing pH. Significant Caspase-3 activity was found in pH 3.5 and 5 groups. Caspase-3 levels for the alkaline exposure were equal or below the normal control. DNA ladder formation, a characteristic of apoptosis, was only visualised in the treatment of pH 3.5 for 30 min. CONCLUSIONS: pH changes inhibit cell proliferation and decrease cell viability. The pathway of killing tumour cell in low pH probably has at least two directions: apoptosis and cell necrosis, whereas high pH results in only cell necrosis. The study suggests that low pH environment can induce apoptosis in unphysiological condition comparable with tissue pH at EChT. In addition, it seems that R3230AC mammary tumour cells are more tolerant to high pH than to acidic changes. This supports the theory that anodic EChT should be more efficient than cathodic.     

Analysis of UVB-modulated Gene Expression in Human Keratinocytes by mRNA Differential Display Polymerase Chain Reaction

Abstract— EUltraviolet (UV) light is the most important environmental insult to skin. Even a single exposure to UVB radiation can result in inflammation and may also lead to DNA damage and apoptosis in the acute response of the cutaneous tissue. To elucidate the complex alterations of gene expression in human keratinocytes underlying these UV responses we took advantage of differential display polymerase chain reaction (DD-PCR) technology's ability to detect qualitative and quantitative changes in gene expression in more than two cell populations simultaneously. We demonstrate that low-dose UVB (100 Jm_-2) leads to both induction and down regulation of different genes during the 24 h after irradiation in a time-dependent manner. In addition to the identification of known genes as possible effectors or targets in the UV response of human keratinocytes, we here identify a new sequence that is negatively regulated by UVB irradiation and was termed HUR 7 (HaCaT UV repressed). In general our results showed that DD-PCR is a useful tool in the analysis of quantitative changes of mRNA levels in human keratinocytes after UV irradiation. The identification of new UVB-repressed genes offers the opportunity to identify unrecognized molecular mechanisms in the UV response of human cells.     

Optical sensor for the detection of caspase-9 activity in a single cell

We demonstrate for the first time, the application and utility of a unique optical sensor having a nanoprobe for monitoring the onset of the mitochondrial pathway of apoptosis in a single living cell by detecting enzymatic activities of caspase-9. Minimally invasive analysis of single live MCF-7 cells for caspase-9 activity is demonstrated using the optical sensor which employs a modification of an immunochemical assay format for the immobilization of nonfluorescent enzyme substrate, Leucine-GlutamicAcid-Histidine-AsparticAcid-7-amino-4-methylcoumarin (LEHD-AMC). LEHD-AMC covalently attached on the nanoprobe tip of an optical sensor is cleaved during apoptosis by caspase-9 generating free AMC. An evanescent field is used to excite cleaved AMC and the resulting fluorescence signal is detected. By quantitatively monitoring the changes in fluorescence signals, caspase-9 activity within a single living MCF-7 cell was detected. By comparing of the fluorescence signals from apoptotic cells induced by photodynamic treatment and nonapoptotic cells, we successfully detected caspase-9 activity, which indicates the onset of apoptosis in the cells.     

Ultrasmall Nanopipette: Toward Continuous Monitoring of Redox Metabolism at Subcellular Level

Ionic current rectification (ICR) based nanopipettes allow accurate monitoring of cellular behavior in single living cells. Herein, we proposed a 30 nm nanopipette functionalized with G‐quadruplex DNAzyme as an efficient biomimetic recognizer for ROS generation at subcellular level via the changes of current–voltage relationship. Taking advantages of the ultra‐small tip, the nanopipette could penetrate into a single living cell repeatedly or keep measuring for a long time without compromising the cellular functions. Coupled with precision nanopositioning system, generation of ROS in mitochondria in response to cell inflammation was determined with high spatial resolution. Meanwhile, the changes of aerobic metabolism in different cell lines under drug‐induced oxidative stress were monitored continuously. We believe that the ICR‐nanopipette could be developed as a powerful approach for the study of cellular activities via electrochemical imaging in living cells.     

基于DNA纳米花的细胞自噬基因沉默用于增敏抗肿瘤化疗

自噬是真核细胞降解蛋白质的重要途径之一,在细胞的更新代谢中起重要作用.肿瘤细胞借助高水平的细胞自噬能够阻断细胞凋亡途径,降低化疗药物的抗肿瘤效果.本文通过设计编码有核酸适配体序列(Aptamer)和DNA酶序列(DNAzyme)的多功能DNA纳米花,利用DNA序列可负载化疗药物阿霉素(Dox)的特性,实现了对肿瘤细胞特异靶向的药物递送,并高效沉默肿瘤细胞的自噬相关基因ATG5,达到增敏抗肿瘤化疗的效果.通过RT-PCR实验验证合成的DNA纳米花可以有效剪切肿瘤细胞中自噬相关基因ATG5的mRNA;并通过DNA纳米花的细胞毒性和细胞凋亡实验研究了其对肿瘤细胞系MCF-7的靶向治疗作用,结果显示该多功能DNA纳米花在增敏抗肿瘤化疗方面具有明显优势.     

A duplex–triplex nucleic acid nanomachine that probes pH changes inside living cells during apoptosis

A duplex–triplex switchable DNA nanomachine was fabricated and has been applied for the demonstration of intracellular acidification and apoptosis of Ramos cells, with graphene oxide (GO) not only as transporter but also as fluorescence quencher. The machine constructed with triplex-forming oligonucleotide exhibited duplex–triplex transition at different pH conditions. By virtue of the remarkable difference in affinity of GO with single-stranded DNA and triplex DNA, and the super fluorescence quenching efficiency of GO, the nanomachine functions as a pH sensor based on fluorescence resonance energy transfer. Moreover, taking advantage of the excellent transporter property of GO, the duplex–triplex/GO nanomachine was used to sense pH changes inside Ramos cells during apoptosis. Fluorescence images showed different results between living and apoptotic cells, illustrating the potential of DNA scaffolds responsive to more complex pH triggers in living systems.

Dynamic metabolic change of cancer cells induced by natural killer cells at the single-cell level studied by label-free mass cytometry

Natural killer cells (NK cells) are important immune cells which have attracted increasing attention in cancer immunotherapy. Due to the heterogeneity of cells, individual cancer cells show different resistance to NK cytotoxicity, which has been revealed by flow cytometry. Here we used label-free mass cytometry (CyESI-MS) as a new tool to analyze the metabolites in Human Hepatocellular Carcinoma (HepG2) cells at the single-cell level after the interaction with different numbers of NK92 MI cells. A large amount of chemical information from individual HepG2 cells was obtained showing the process of cell apoptosis induced by NK cells. Nineteen metabolites which consecutively change during cell apoptosis were revealed by calculating their average relative intensity. Four metabolic pathways were impacted during cell apoptosis which hit 4 metabolites including glutathione (GSH), creatine, glutamic acid and taurine. We found that the HepG2 cells could be divided into two phenotypes after co-culturing with NK cells according to the bimodal distribution of concentration of these 4 metabolites. The correlation between metabolites and different apoptotic pathways in the early apoptosis cell group was established by the 4 metabolites at the single-cell level. This is a new idea of using single-cell specific metabolites to reveal the metabolic heterogeneity in cell apoptosis which would be a powerful means for evaluating the cytotoxicity of NK cells.

Label-free mass cytometry is utilized to study the dynamic metabolic change during apoptosis in HepG2 cells induced by NK92 MI cells at the single-cell level. The metabolic heterogeneity of individual HepG2 cells during apoptosis was revealed.     

纳米TiO2-Cu2O复合材料可见光催化杀伤人宫颈肿瘤细胞研究

以人宫颈肿瘤细胞(Hela细胞)为研究对象, 研究了可见光催化(光强为50 mW/cm2)条件下, 该复合材料Fenton作用对细胞的凋亡诱导作用和细胞周期的影响, 并对抗肿瘤作用机理进行探讨. 结果表明, 该复合材料对肿瘤细胞具有明显的杀伤作用, 抑制Hela细胞增殖, 降低细胞存活率, 诱导Hela细胞产生细胞凋亡. 此外, 还能够引起细胞周期各时相改变, 使细胞生长阻滞于G2/M期. 并引发细胞氧化应激反应的发生, 最终破坏胞内抗氧化酶体系的平衡. 由此可见, 纳米TiO2-Cu2O复合材料在抗肿瘤的可见光疗应用中具有一定的应用价值.