Taurolidine: a novel anti-neoplastic agent induces apoptosis of osteosarcoma cell lines

Taurolidine, the active agent of Taurolin, is a broad spectrum anti-biotic that has been used for over 15 years for the treatment of severe surgical infections. Recently, taurolidine has been shown to possess anti-neoplastic properties in vitro and in vivo against a variety of cancers including ovarian, colon and prostate. In this study we assessed the cytotoxic activity of taurolidine against human osteosarcoma (OS) cell lines and normal human bone cells. Treatment with taurolidine inhibited the growth of all ten osteosarcoma cell lines tested and taurolidine was equally potent against cell lines with and without distinct genetic defects (i.e. p53, Rb). Moreover, taurolidine-induced growth inhibition was found to be associated with a dose dependent increase in the number of apoptotic cells and apoptosis was shown to be caspase-dependent. Taurolidine treatment was also found to inhibit adhesion of OS cell lines. Compared to OS cell lines, normal bone cells in primary culture were found to be less sensitive to the cytotoxic and anti-adhesive effects of taurolidine. These data indicate that taurolidine possesses potent anti-neoplastic activity against osteosarcoma cell lines and may have potential as a novel OS chemotherapeutic agent.     

利多卡因调控微小RNA-195对骨肉瘤MG63细胞增殖、凋亡及化疗敏感性的影响

目的 探讨利多卡因(lidocaine)对骨肉瘤MG63细胞增殖、凋亡及化疗敏感性的影响及其可能的作用机制.方法 研究于2018年5―12月,体外培养骨肉瘤MG63细胞,不同浓度的利多卡因干预MG63细胞,采用甲基噻唑基四唑(MTT)法检测MG63细胞存活率及顺铂半抑制浓度(IC50值);流式细胞术检测细胞凋亡率;实时...     

微小RNA-301b-3p靶向第10号染色体缺失的磷酸酶及张力蛋白同源物对骨肉瘤细胞增殖、凋亡的影响

目的 探讨微小RNA-301b-3p(miR-301b-3p)对骨肉瘤细胞增殖、凋亡的影响及其作用机制.方法 本研究起止时间为2019年3—9月.人成骨细胞(hFOB)和骨肉瘤细胞U2OS、MG63购自美国菌种保藏中心.U2OS细胞分为miRNA抑制物阴性对照(anti-miR-NC)组、miR-301b-3p抑制物(...     

Pharmacogenomics of genes involved in antifolate drug response and toxicity in osteosarcoma

Introduction: Antifolates are structural analogs of folates, which have been used as antitumor drugs for more than 60 years. The antifolate drug most commonly used for treating human tumors is methotrexate (MTX), which is utilized widely in first-line treatment protocols of high-grade osteosarcoma (HGOS). In addition to MTX, two other antifolates, trimetrexate and pemetrexed, have been tested in clinical settings for second-line treatment of recurrent HGOS with patients unfortunately showing modest activity.

Areas covered: There is clinical evidence which suggsest that, like other chemotherapeutic agents, not all HGOS patients are equally responsive to antifolates and do not have the same susceptibility to experience adverse drug-related toxicities. Here, we summarize the pharmacogenomic information reported so far for genes involved in antifolate metabolism and transport and in MTX-related toxicity in HGOS patients.

Expert opinion: Identification and validation of genetic biomarkers that significantly impact clinical antifolate treatment response and related toxicity may provide the basis for a future treatment modulation based on the pharmacogenetic and pharmacogenomic features of HGOS patients.     

长链非编码前列腺癌相关转录因子6靶向微小RNA-139-3p对骨肉瘤细胞增殖和凋亡的影响

目的 探讨长链非编码RNA(LncRNA)前列腺癌相关转录因子6(PCAT6)对骨肉瘤细胞增殖和凋亡的影响和分子机制.方法 实时荧光定量PCR(RT-qPCR)检测20例骨肉瘤组织和与其对应的癌旁组织中PCAT6和微小RNA-139-3p(miR-139-3p)的表达水平.双荧光素酶报告基因实验和RT-qPCR验证PC...     

Sirolimus induces apoptosis and reverses multidrug resistance in human osteosarcoma cells in vitro via increasing microRNA-34b expression

Aim:

Multi-drug resistance poses a critical bottleneck in chemotherapy. Given the up-regulation of mTOR pathway in many chemoresistant cancers, we examined whether sirolimus (rapamycin), a first generation mTOR inhibitor, might induce human osteosarcoma (OS) cell apoptosis and increase the sensitivity of OS cells to anticancer drugs in vitro.

Methods:

Human OS cell line MG63/ADM was treated with sirolimus alone or in combination with doxorubicin (ADM), gemcitabine (GEM) or methotrexate (MTX). Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. MiRNAs in the cells were analyzed with miRNA microarray. The targets of miR-34b were determined based on TargetScan analysis and luciferase reporter assays. The expression of relevant mRNA and proteins was measured using qRT-PCR and Western blotting. MiR-34, PAK1 and ABCB1 levels in 40 tissue samples of OS patients were analyzed using qRT-PCR and in situ hybridization assays.

Results:

Sirolimus (1–100 nmol/L) dose-dependently suppressed the cell proliferation (IC₅₀=23.97 nmol/L) and induced apoptosis. Sirolimus (10 nmol/L) significantly sensitized the cells to anticancer drugs, leading to decreased IC₅₀ values of ADM, GEM and MTX (from 25.48, 621.41 and 21.72 μmol/L to 4.93, 73.92 and 6.77 μmol/L, respectively). Treatment of with sirolimus increased miR-34b levels by a factor of 7.5 in the cells. Upregulation of miR-34b also induced apoptosis and increased the sensitivity of the cells to the anticancer drugs, whereas transfection with miR-34b-AMO, an inhibitor of miR-34b, reversed the anti-proliferation effect of sirolimus. Two key regulators of cell cycle, apoptosis and multiple drug resistance, PAK1 and ABCB1, were demonstrated to be the direct targets of miR-34b. In 40 tissue samples of OS patients, significantly higher miR-34 ISH score and lower PAK5 and ABCB1 scores were detected in the chemo-sensitive group.

Conclusion:

Sirolimus increases the sensitivity of human OS cells to anticancer drugs in vitro by up-regulating miR-34b interacting with PAK1 and ABCB1. A low miR-34 level is an indicator of poor prognosis in OS patients.     

The multifunctional protein PEA-15 is involved in the control of apoptosis and cell cycle in astrocytes

PEA-15 is a small protein (15 kDa) that was first identified as an abundant phosphoprotein in brain astrocytes [Araujo et al., J Biol Chem 1993;268(8):5911-20], and subsequently shown to be widely expressed in different tissues and highly conserved among mammals [Estelles et al., J Biol Chem 1996;271(25):14800-6; Danziger et al., J Neurochem 1995;64(3):1016-25]. It is composed of a N-terminal death effector domain and a C-terminal tail of irregular structure. PEA-15 is regulated by multiple calcium-dependent phosphorylation pathways that account for its different forms: a non-phosphorylated form in equilibrium with a mono and a biphosphorylated variety. This already suggested that PEA-15 may play a major role in signal integration. Accordingly, it has been demonstrated to modulate signaling pathways that control apoptosis and cell proliferation. In particular, PEA-15 diverts astrocytes from TNFalpha-triggered apoptosis and regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been modelized and recently determined using NMR spectroscopy, and may help to understand the various functions played by the protein through its molecular interactions.     

S100A4蛋白在骨肉瘤中表达及其与肿瘤侵袭转移的关系

目的探讨S100A4蛋白的表达与骨肉瘤的侵袭转移的关系。方法应用免疫组织化学方法检测61例骨肉瘤患者和15例骨软骨瘤患者的S100A4蛋白表达情况,分析其与骨肉瘤临床病理特征的关系。结果 S100A4蛋白在骨肉瘤中的表达阳性率75.4%,明显高于骨软骨瘤患者的13.3%(P<0.05)。S100A4的表达与病理分型和肺转移显著相关,随着肺转移的发生,S100A4表达量增高(P<0.05);但与患者的性别、年龄、肿瘤部位和临床分期无关。结论 S100A4表达增强与骨肉瘤侵袭转移有关,可能成为判断骨肉瘤恶性程度及预后指标。     

miR-301b在调节脂肪细胞分化中的作用

摘要： 目的 研究 miR-301b 在调节间充质干细胞向脂肪细胞分化过程中的作用。方法 对小鼠骨髓基质细胞 ST2 进行脂肪细胞诱导分化, 并利用 qRT-PCR 检测细胞分化过程中成脂分化诱导组相对于阴性对照组的 miR-301b 表达变化。对 ST2 细胞转染 miR-301b mimics 并进行成脂诱导分化, 利用 qRT-PCR 和 Western blot 技术检测 miR- 301b mimics 转染组和阴性对照片段 （NC） 转染组细胞的脂肪特异性基因和蛋白表达水平的变化。结果 成脂分化诱导组 miR-301b 相对表达水平 （0.219±0.021） 较对照组 （1.000±0.425） 减少 （P＜0.05）。miR-301b mimics 转染组的脂肪细胞特异性转录因子过氧化物酶体增殖物激活受体 （PPARγ）、 CCAAT 增强子结合蛋白 （C/EBPα） 和脂肪型脂肪酸结合蛋白 （aP2） 的相对表达量均较 NC 转染组降低 （P＜0.05）。miR-310b mimics 转染组与 NC 转染组相比, 标志基因aP2、 转录因子PPARγ 和C/EBPα 蛋白的表达量均减少 （P＜0.05）。结论 miR-301b可抑制脂肪细胞分化。     

IkappaB kinase activation is involved in regulation of paclitaxel-induced apoptosis in human tumor cell lines.

Paclitaxel (Taxol), a naturally occurring antimitotic agent, has shown significant cell-killing activity against human solid tumor cells through induction of apoptosis. The molecular mechanism underlying paclitaxel-induced apoptosis is not entirely clear. Using the unique inhibitory effect of glucocorticoids on paclitaxel-induced apoptosis, we recently discovered that paclitaxel-induced inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor-kappaB (NF-kappaB) activation might contribute to the mediation of paclitaxel-induced apoptosis. In this study, using a novel IkappaBalpha phosphorylation inhibitor, we demonstrated that the blockage of paclitaxel-induced IkappaBalpha degradation inhibited apoptotic cell death in human breast cancer BCap37 and ovarian cancer OV2008 cell lines. Furthermore, in vitro kinase assays showed that the activity of IkappaB kinase (IKK), which is responsible for the phosphorylation and degradation of IkappaB proteins, was significantly activated by paclitaxel in these paclitaxel-sensitive tumor cells. Stable transfection of a mutant IkappaBalpha lacking Ser(32) and Ser(36) that was insensitive to IKK-mediated phosphorylation and degradation resulted in reduced sensitivity of tumor cells to paclitaxel-induced apoptosis. Moreover, we also found that the expression of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, an upstream regulator of IKK, was up-regulated by paclitaxel. These findings suggest that the activation of IKK might play a critical role in the regulation of paclitaxel-induced NF-kappaB activation that subsequently mediates paclitaxel-induced apoptotic cell death in solid tumor cells.     

Smac和survivin基因在骨肉瘤中的表达及其临床意义

目的探讨人骨肉瘤组织中Smac和survivin基因表达状态,及其对骨肉瘤生物学行为的影响。方法采用免疫组织化学技术(SABC法)检测46例骨肉瘤、10例骨软骨瘤组织中Smac和survivin蛋白表达,比较二者表达与骨肉瘤主要临床病理参数的关系。结果Smac和survivin基因在骨软骨瘤组织表达分别为3例、2例,骨肉瘤中分别为29例(63%)和31例(67%);Smac表达率与骨肉瘤组织学分级、WHO分型无关,与转移有关(P<0.05),Survivin表达率与骨肉瘤组织学分级无关,与WHO分型及转移有关(P<0.05);骨肉瘤中Smac和survivin基因表达呈正相关(r=0.506,P<0.01)。结论Smac和survivin基因高表达于骨肉瘤组织中,与骨肉瘤生物学行为密切相关,二者可能共同参与骨肉瘤的发生发展。     

白细胞介素-1β对大鼠软骨细胞MMP-13 表达的影响及 miR-27b 的调控作用

目的观察白细胞介素（IL）-1β对大鼠软骨细胞基质金属蛋白酶（MMP）-13 表达的影响及miR-27b 的调控作用。方法雄性Wistar 大鼠7 只提取软骨细胞。Western blot 检测IL-1β刺激软骨细胞0 h、24 h、48 h 各时间点 MMP-13 表达变化;miRNAs 微阵列分析48 h 内软骨细胞差异表达的miRNAs;Real-time PCR 定量分析筛选出下调最为明显的miRNAs;荧光素酶报告基因实验验证miR-27b 与MMP-13 的靶定调控关系。结果IL-1β刺激软骨细胞后,MMP-13 蛋白在0 h、24 h、48 h 各时间点表达逐渐增加（P < 0.05）;miRNAs 微阵列分析发现48 h 内软骨细胞有36 个miRNAs 出现表达变化,变化最为明显的有6 个,分别为miR-27b、miR-31、miR-26a、miR-26b、miR-23、 miR-204;Real-time PCR 显示miR-27b 下调最为明显;miR-27b 拟似物和荧光素酶表达质粒共转染软骨细胞后,荧光素酶活性受到明显抑制（P < 0.05）。结论IL-1β刺激软骨细胞后,出现miR-27b 的表达下调和MMP-13 蛋白的表达上调,miR-27b 与MMP-13 存在靶定调控关系。     

Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line

To support the argument that bisphenol-A (BPA) poses a risk for ovarian cancer, OVCAR-3 cell line was exposed to environmentally relevant concentration of BPA. Expression of selected genes involved in cell cycle and apoptosis were evaluated by real-time PCR. In a dose-dependent manner, BPA increased OVCAR-3 cell proliferation and decreased caspase-3 activity, but it had no effect on DNA fragmentation. We noted 1.2-1.5-fold induction of genes responsible for inducing cell proliferation and 1.2-46-fold suppression of genes responsible for inhibition of proliferation. Moreover, 1.6-8-fold suppression of genes involved in the extrinsic apoptotic pathway was observed. In parallel, 1.3-2.5-fold suppression pro-apoptotic genes and 1.6-51-fold induction of pro-survival genes involved in the intrinsic apoptotic pathway were observed. Additionally, 1.7-fold induction of p53 and 5-fold induction of endonuclease G genes involved in CAD-independent DNA fragmentation were noted under the influence of BPA.In conclusion, we hypothesize that induction of p53 and suppression of caspase-3 and 7 gene expression observed in this study activate the DNA repair process. Therefore, despite the observed induction of endo G gene expression, the action of BPA on DNA fragmentation was not observed.     

miR-200b 靶向DNMT3A 抑制非小细胞肺癌细胞增殖与诱导凋亡

摘要： 目的 探讨miR-200b是否通过靶向调控DNMT3A抑制人非小细胞肺癌A549细胞增殖与诱导凋亡。方法 运用qRT-PCR检测miR-200b在不同非小细胞肺癌细胞株中的表达; 将miR-200b mimics、 scramble、 DNMT3A-siRNA、 control-siRNA分别转染于A549细胞, 其中scramble与control-siRNA分别作为miR-200b mimics与DNMT3A-siRNA 的阴性对照组。采用 Western blot 检测 A549 细胞中 DNMT3A 蛋白表达; 采用 MTT 与 AnnexinV-FITC/PI 染色法分别检测 A549 细胞的增殖与凋亡, 比较 miR-200b mimics 与 DNMT3A-siRNA 对 A549 细胞增殖与凋亡的影响。结果 qRT-PCR 结果显示, miR-200b 在非小细胞肺癌 A549、 H1299、 L78、 H460 细胞中的表达均明显低于正常人支气管上皮 16HBE 细胞, 其中以 A549 细胞下调最为明显 （P < 0.05）。Western blot 结果显示, 外源过表达 miR-200b 或沉默 DNMT3A 能明显下调 A549 细胞中 DNMT3A 蛋白的水平。MTT 结果显示, 转染 miR-200b mimics 或沉默 DN⁃ MT3A 48 h、 72 h、 96 h 后, 反映细胞增殖的光密度 （OD） 值与各自阴性对照组比较明显减小 （P < 0.05）。AnnexinV- FITC/PI 染色结果显示, 转染 miR-200b mimics 或沉默 DNMT3A 后, A549 细胞的凋亡率分别为 （23.33%±0.90%、 20.41%±0.70%） 均高于各自阴性对照组 （5.28%±0.55%、 5.68%±0.47%, P < 0.05）。结论 miR-200b通过下调DNMT3A 抑制人非小细胞肺癌细胞增殖与诱导凋亡。     

微小 RNA-490-3p靶向 FK506相关蛋白 14表达抑制骨肉瘤 MG63细胞侵袭和迁移的实验研究

目的探讨微小 RNA-490-3p(miR-490-3p)对骨肉瘤 MG63细胞侵袭、迁移的影响及其机制。方法 2019年 9月至 2020年 4月,从中科院上海细胞库购买人骨肉瘤细胞株 MG63进行体外培养,分为对照组(未转染)miR-NC组(转染 miR-NC)、 miR-490-3p组(转染 miR-490-3p mimics),miR-490-3p+pcDNA组(共转染 miR-490-3p mimics与空载体、)和 miR-490-3p+FKBP14组(共转染 miR-490-3p mimics与 FKBP14过表达载体),采用 RT-PCR检测 miR-490-3p表达, Transwell小室法检测细胞侵袭、迁移,蛋白质印迹法检测钙黏蛋白 E(E-cadherin)、基质金属蛋白酶 -2(MMP-2)、上皮间质转化因子 Twist转录因子( Twist)和 FK506相关蛋白 14(FKBP14)蛋白表达,双荧光素酶报告基因实验检测 miR-490-3p和 FKBP14的靶向关系。结果与对照组比较, miR-490-3p组细胞中 miR-490-3p表达水平明显升高( 3.68±0.37比 1.02±0.10,P<0.05),而侵袭细胞数( 33.25±2.02比     

Synergistic apoptotic effect of crocin and cisplatin on osteosarcoma cells via caspase induced apoptosis

Crocin is well-known traditional Chinese medicine which is extracted from saffron. However, its role in osteosarcoma has not been well understood. Therefore, we used crocin and cisplatin individually or jointly on MG63 and OS732 cells so as to explore whether crocin could induce cellular apoptosis and suppress the ability of invasion of osteosarcoma cells. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di- phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope and fluorescence microscope, ?ow cytometry, and Transwell invasion chamber methods. The expressions of caspase-3 and caspase-8 were detected by Western blot. The survival rate of combined application was significantly lower than that of the individual application. Apoptosis-inducing effect of combined application was much stronger than that of individual application. The invasion ability of MG63 and OS732 cells was restrained significantly in the combined group compared with the individual group and control group. Combined group has the effect of up-regulating the expressions of cleaved-caspase-3 and caspase-8. The results suggested that combination of crocin and cisplatin has a strong killing effect on osteosarcoma cells and suppresses the ability of invasion of MG63 and OS732 cells which might be related to up-regulate the expression of caspase-3 and caspase-8.     

Lactate promotes vascular smooth muscle cell switch to a synthetic phenotype by inhibiting miR-23b expression

Recent research indicates that lactate promotes the switching of vascular smooth muscle cells (VSMCs) to a synthetic phenotype, which has been implicated in various vascular diseases. This study aimed to investigate the effects of lactate on the VSMC phenotype switch and the underlying mechanism. The CCK-8 method was used to assess cell viability. The microRNAs and mRNAs levels were evaluated using quantitative PCR. Targets of microRNA were predicted using online tools and confirmed using a luciferase reporter assay. We found that lactate promoted the switch of VSMCs to a synthetic phenotype, as evidenced by an increase in VSMC proliferation, mitochondrial activity, migration, and synthesis but a decrease in VSMC apoptosis. Lactate inhibited miR-23b expression in VSMCs, and miR-23b inhibited VSMC''s switch to the synthetic phenotype. Lactate modulated the VSMC phenotype through downregulation of miR-23b expression, suggesting that overexpression of miR-23b using a miR-23b mimic attenuated the effects of lactate on VSMC phenotype modulation. Moreover, we discovered that SMAD family member 3 (SMAD3) was the target of miR-23b in regulating VSMC phenotype. Further findings suggested that lactate promotes VSMC switch to synthetic phenotype by targeting SMAD3 and downregulating miR-23b. These findings suggest that correcting the dysregulation of miR-23b/SMAD3 or lactate metabolism is a potential treatment for vascular diseases.     

177Lu‐DOTMP induces G2/M cell cycle arrest and apoptosis in MG63 cell line

Bone pain is the major manifestation of skeletal metastases. Although various treatment modalities are available for bone pain palliation, use of radiolabeled phosphonates is documented to be more effective. Among radionuclides available for this purpose, lutetium‐177 is gaining popularity due to its moderate beta energy, theranostic capability, favorable half‐life and convenient production logistics. ¹⁷⁷Lu‐DOTMP has shown considerable promise as a metastatic bone pain palliating agent in preliminary evaluations and recent clinical studies. Therefore, an attempt was made to elucidate the possible mechanism of in vitro cell death induced by ¹⁷⁷Lu‐DOTMP in MG63 cells. ¹⁷⁷Lu‐DOTMP binding studies were carried out in mineralized bone of MG63 cells and around 50% binding was observed. Skeletons of Wistar rats showed 1.78 ± 0.5% IA/g at a 3 h time period which was almost constant up to 7 days. MG63 cells were incubated with 3.7 and 37 MBq of ¹⁷⁷Lu‐DOTMP for 48 h prior to perform assays. An increase in the magnitude of cell toxicity and apoptotic DNA fragmentation was observed. Enhancement of G2/M phase cell cycle arrest and apoptosis were documented which were dose‐dependent. Thus, ¹⁷⁷Lu‐DOTMP induced apoptotic cell death in MG63 cells, which might be one of the primary causes of pain relief in osseous metastases.     

Up-regulation of miR-135b expression induced by oxidative stress promotes the apoptosis of renal tubular epithelial cells under high glucose condition

This study aimed to investigate the role and underlying mechanism of miR-135b in high glucose-induced oxidative stress of renal tubular epithelial cells. Here, in vivo experiments found that compared to the control group, miR-135b expression was significantly up-regulated in the diabetes group, whereas BMP7 mRNA and protein levels were down-regulated. In high glucose-treated renal tubular epithelial cells (HK-2) in vitro, oxidative stress was induced, which up-regulated miR-135b expression. In addition, the regulation of miR-135b on BMP7 expression was confirmed in HK-2 cells. Under high glucose conditions, oxidative stress promoted the apoptosis of HK-2 cells through the up-regulation of miR-135b expression. In vivo experiments indicated that interference with miR-135b improved renal function in mice with diabetic nephropathy. In conclusion, these results indicated that the up-regulation of miR-135b expression induced by oxidative stress promotes the apoptosis of HK-2 cells under high glucose conditions.