hGCN5在Burkkit淋巴瘤细胞株Daudi中的表达及TSA对细胞增殖凋亡的影响

目的:研究hGCN5(human general control of amino acid synthesis protein 5)在Burkkit淋巴瘤细胞株Daudi中的表达及曲古柳菌素A(TSA)对细胞增殖、凋亡的影响,探讨其抗肿瘤的分子机制.方法:采用MTT法检测细胞增殖,流式细胞仪检测细胞周期和凋亡率,采用免疫细胞化学法和Western blot观察Daudi细胞hGCN5蛋白的表达.结果:TSA可明显抑制Daudi细胞增殖;TSA(50、100μg/L)使细胞积聚于G0/G1期;TSA(200μg/L)则使细胞周期受抑于S期,而对G2/M期的作用不明显;TSA(200、400μg/L)处理24h可以诱导细胞凋亡;免疫细胞化学和Westernblot结果均显示处理组hGCN5的吸光度比未处理组明显增加(P＜0.05).结论:TSA通过上调HATs家族成员中hGCN5的表达,实现对Daudi细胞的抗增殖作用.     

抑制人RAD51基因表达对人骨肉瘤细胞体内外增殖能力的影响

目的:通过抑制人RAD51修复基因(hRAD51)表达,研究hRAD51对骨肉瘤细胞和裸鼠移植瘤生长的影响.方法:构建hRAD51基因沉默的骨肉瘤细胞株HOS-shA2,利用MTT和克隆形成实验从体外观察hRAD51基因沉默对HOS细胞增殖的影响,FCM法检测抑制hRAD51基因表达后细胞周期的变化情况,裸鼠成瘤实验观察降低hRAD51表达对骨肉瘤移植瘤生长情况的影响.结果:成功构建了hRAD51基因沉默细胞株HOS-shA2;体外实验显示,HOS-shA2细胞的增殖能力较亲本及阴性对照细胞明显降低(P<0.01);FCM法检测结果显示,hRAD51基因沉默后G2/M期细胞比例增多(P<0.01);HOS-shA2细胞的裸鼠移植瘤生长速度缓慢,剥离瘤体质量明显轻于亲本HOS组和阴性对照组,差异有统计学意义(P<0.01).结论:hRAD51在骨肉瘤细胞增殖中发挥重要作用,有望成为肿瘤治疗的新靶点.     

雷帕霉素对人淋巴瘤Raji细胞株增殖影响及其机制的探讨

目的:探讨雷帕霉素(宜欣可)对人淋巴瘤细胞株Raji细胞体外增殖及细胞周期的影响,并分析其分子作用机制.方法:采用MTT法检测不同浓度(0、1、5、10、20、40、50和100 nmol/L)雷帕霉素作用不同时间(24、48和72 h)对Raji细胞增殖的影响.用流式细胞仪测定雷帕霉素对Raji细胞周期分布和凋亡的影响.应用蛋白质印迹法检测雷帕霉素对Raji细胞周期蛋白Cyclin D1、Cyclin E、Cyclin A和p27及凋亡蛋白抑制因子Survivin蛋白表达的影响.结果:雷帕霉素浓度＞5 nmol/L对Raji细胞增殖有明显的抑制作用(P＜0.05).且呈现明显的剂量-效应和时间-效应依赖关系.雷帕霉素处理后,可明显抑制Raji细胞周期发展(P＜0.05),随着药物浓度的加大、作用时间的延长,处于G0/G1期细胞逐渐增多,S期和G2/M期细胞则逐渐减少.但Raji细胞没有发生明显的凋亡现象(P＞0.05).50 nmol/L雷帕霉素处理后,Raji细胞的Cyclin D1、Cyclin E、p27表达水平没有明显变化(P＞0.05),但Cyclin A和Survivin表达水平被明显抑制(P＜0.05).结论:雷帕霉素通过阻滞细胞周期发展抑制Raji细胞增殖,其作用机制可能与下调细胞Cyclin A和Survivin表达有关.     

曲古抑菌素A对人肝癌细胞增殖凋亡影响及其机制的探讨

目的:观察曲古抑菌素A(TSA)对人肝癌SMMC-7721细胞的增殖凋亡及细胞周期的影响,探讨其作用机制。方法:设TSA处理组和对照组。光镜观察细胞形态;噻唑蓝(MTT)比色法检测增殖抑制;流式细胞术(FCM)检测细胞凋亡及细胞周期变化;RT-PCR法检测HDAC1mRNA的表达。结果:TSA处理组细胞增殖减慢,形态改变,增殖抑制作用呈明显剂量和时间依赖关系,同一浓度下,24h的抑制效果最佳;FCM检测结果显示,不同浓度TSA作用24h后,细胞凋亡率明显升高,早期凋亡率由8.72%升至18.22%(P=0.026),TSA处理后,G0/G1期细胞明显增加,由28.97%升至52.31%(P=0.043),S期细胞由26.01%升至34.28%(P=0.051),G2/M期细胞由45.02%降至13.41%(P=0.036),细胞被阻滞于G0/G1期;RT-PCR法检测结果显示,HDAC1mRNA的表达明显减少,由0.34±0.02降至(0.11±0.01),P=0.020。结论:TSA对SMMC-7721有显著的抑制增殖和诱导凋亡作用,其机制可能与抑制HDAC1的活性,下调HDAC1mRNA表达及阻滞细胞周期有关。     

曲古抑菌素A诱导HL-60细胞凋亡机制的研究

目的:探讨曲古抑菌素A(TSA)在体外诱导急性早幼粒白血病HL-60细胞凋亡的机制.方法:采用MTT方法检测TSA对HL-60细胞增殖的影响.流式细胞术检测细胞周期和细胞凋亡,RT-PCR检测凋亡相关基因Bax、Caspase-9和Caspase-3的表达.结果:0.1μmol/L的TSA可明显抑制细胞增殖,P<0.01;0.05 μmol/L的TSA可使细胞周期阻滞在G0/G1期(P<0.05),但0.1 μmol/L的TSA才能引起细胞凋亡(P<0.01);经0.1 μmol/L的TSA作用12 h后,Bax、Caspase-9和Caspase-3基因表达明显升高,P<0.01.结论:TSA诱导HL-60细胞凋亡的机制在于引起细胞周期阻滞,上调Bax、Caspase-9和Caspase-3的表达.     

舒林酸对胃癌细胞株SGC7901细胞周期调控及凋亡的影响

目的观察舒林酸影响胃癌细胞株SGC7901细胞周期分布,诱导细胞凋亡,探讨其作用机制方法采用MTT比色法观察舒林酸对胃癌SGC7901细胞增殖的影响,采用流式细胞仪检测舒林酸对细胞周期分布的影响,同时结合透射电子显微镜观察舒林酸诱导SGC901细胞凋亡的作用;免疫细胞化学方法观察舒林酸对胃癌细胞周期调控蛋白cyvclinE及P21WAF/CIPI的影响结果舒林酸可抑制胃癌SGC7901细胞的增殖,改变细胞周期分布,使G-0/G1期细胞比例增高,S期比例降低,并对SGC7901细胞有促凋亡作用.上述作用具有剂量和时间依赖性(P＜0.05).舒林酸还可上调P21WAF/CIPI蛋白表达.下调cyolinE蛋白表达.结论舒林酸可改变细胞周期分布,影响细胞周期调控蛋白表达,并可诱导SGC7901细胞凋亡,从而抑制细胞增殖.     

探讨鸦胆子素D对骨肉瘤细胞增殖、凋亡与自噬的作用

目的探讨鸦胆子素D对骨肉瘤细胞(143B HOS)增殖,骨肉瘤细胞凋亡与自噬的作用。方法用CCK8实验观察鸦胆子素D对143B HOS增殖的影响;用平板克隆实验观察鸦胆子素D对143B HOS细胞集落形成的影响;用流式细胞仪检测鸦胆子素D对143B HOS周期阻滞以及诱导凋亡的作用情况;用Western Blot检测143B HOS基础凋亡与自噬以及经鸦胆子素D诱导之后的凋亡与自噬情况;用荧光显微镜检测143B细胞基础LC3荧光鼬合蛋白表达以及经鸦胆子素D诱导之后的LC3荧光鼬合蛋白表达情况。结果(1)CCK8实验显示鸦胆子素D作用后143B HOS细胞的存活率下降,同时,也减少了143B HOS细胞克隆数的形成;(2)流式细胞仪检测细胞周期显示鸦胆子素D使143B HOS的G0/G1期细胞比例增高;(3)流式细胞仪检测细胞凋亡实验显示,鸦胆子素D使143B HOS的细胞凋亡比例增加;Western Blot检测143B HOS细胞Cleaved PARP的表达,结果显示,鸦胆子素D诱导Cleaved PARP表达增加;(4)Western Blot检测143B HOS细胞LC3B的表达,结果显示,鸦胆子素D诱导LC3B-II的表达增加;荧光显微镜检测143B细胞的LC3荧光鼬合蛋白实验,结果显示,鸦胆子素D诱导143B细胞绿色荧光斑点生成增多。结论 (1)鸦胆子素D抑制骨肉瘤细胞增殖;(2)鸦胆子素D诱导骨肉瘤细胞的凋亡;(3)鸦胆子素D诱导骨肉瘤细胞自噬。     

紫杉醇对p53突变型肺癌细胞株H322的生长抑制作用

目的:通过体外实验评价紫杉醇对p53突变型肺癌细胞株H322的生长抑制作用. 方法: 紫杉醇不同作用浓度不同作用时间处理H322细胞后,应用MTT法检测紫杉醇对肺癌细胞生长抑制作用,通过流式细胞术分析细胞周期及凋亡的变化,荧光显微镜观察细胞核形态的变化. 结果: 紫杉醇对H322的细胞毒性呈时间依赖性(P<0.05),(10-1000)nmol/L的浓度对H322生长抑制差异不显著(P>0.05).浓度≤100nmol/L时,G2-M期比率随浓度增加而增高(P<0.05);凋亡比例在低浓度范围内(0.1-10)nmol/L呈浓度依赖性,浓度进一步增加,反而降低.时间依赖性实验中,10nmol/L作用时,凋亡发生呈时间效应;G2-M期所占比例在12h达最高.1000nmol/L作用时,G2-M期阻滞程度和持续时间均高于10nmol/L,但不如低浓度诱导凋亡出现得早.典型凋亡细胞出现浓缩、成片的染色质.紫杉醇诱导的凋亡和G2-M期阻滞无相关性(P>0.05).结论: 体外实验中,紫杉醇对H322细胞有明显生长抑制作用,并且这种作用呈时间依赖性,但在一定浓度范围内剂量效应不明显;低浓度紫杉醇虽然对G2-M期阻滞影响较小,却能比高浓度更早诱发凋亡,延长作用时间亦可达到有效抑制肿瘤细胞增殖的目的.     

孕激素对人宫颈癌细胞株Hela细胞体外增殖、凋亡的影响

[目的]探讨孕激素对人宫颈癌细胞株Hela细胞体外增殖及凋亡的影响.[方法]体外培养Hela细胞,采用四甲基偶氮唑蓝(MTT)比色法观察不同浓度孕酮对细胞增殖的作用,流式细胞仪及流式细胞仪间接免疫荧光技术检测其细胞周期、细胞凋亡率及细胞内bcl-2蛋白的表达,光、电镜观察其形态学和超微结构变化.[结果]当孕酮浓度为0.10μmol/L～10.00umol/L时,对细胞生长有明显的抑制作用(P＜0.01),并具有剂量依赖性.流式细胞仪分析,实验组G0/G1期上升,S期下降,凋亡率上升,细胞内bcl-2蛋白表达率明显降低(P＜0.01),并呈现剂量依赖性.倒置显微镜、光学显微镜和透射电子显微镜可观察到细胞增殖情况及凋亡的形态学改变.[结论]孕酮抑制宫颈癌Hela细胞增殖,并诱导其凋亡,同时可使抗凋亡基因bcl-2蛋白的表达下降.     

自噬蛋白LC3B参与伏立诺他联合顺铂对骨肉瘤细胞增殖抑制及凋亡的研究

摘 要：［目的］ 探讨伏立诺他( suberoylanilide hydroxamic acid,SAHA)联合顺铂(cisplatin,CDDP)对骨肉瘤细胞的增殖抑制及其可能机制。［方法］ 以骨肉瘤细胞系HOS为研究对象,通过MTS测定伏立诺他联合顺铂对骨肉瘤细胞增殖抑制的效果,运用流式细胞术（flow cytometry,FCM）,透射电子显微技术（transmission electron microscope,TEM）,蛋白质印迹技术（Western Blot）检测对细胞周期阻滞,自噬小体产生及凋亡与自噬相关蛋白的表达情况。［结果］ 伏立诺他联合顺铂能够明显抑制骨肉瘤细胞HOS的增殖（P<0.05）。经伏立诺他联合顺铂（SAHA 1μmol/L+CDDP 4μmol/L）处理HOS细胞后,细胞周期阻滞于S期（P<0.01）。TEM结果表明联合处理组诱导了自噬小体的产生。Western Blot结果显示联合处理组使得HOS细胞LC3B蛋白的表达增加,与对照组相比差异有统计学意义（P<0.05）。在加入自噬抑制剂3-MA后,不仅降低LC3B的表达,而且Caspase3、PARP表达也降低,与对照组相比差异有统计学意义（均P<0.05）。 ［结论］ 伏立诺他联合顺铂通过细胞周期阻滞及诱导自噬的发生,从而发挥对骨肉瘤细胞增殖抑制及诱导凋亡的作用。     

Hypoxia increases the expression of stem cell markers in human osteosarcoma cells

Osteosarcoma (OS) is the most common primary malignant tumor of bone. It is a common phenomenon that osteosarcoma cells have a hypoxic microenvironment. Hypoxia can dedifferentiate cells of several malignant tumor types into stem cell-like phenotypes. However, the role of hypoxia in stemness induction and the expression of cancer stem cell (CSC) markers in human osteosarcoma cells has not been reported. The present study examined the effects of hypoxia on stem-like cells in the human osteosarcoma MNNG/HOS cells. Under the incubation with 1% oxygen, the expression of CSCs markers (Oct-4, Nanog and CD133) in MNNG/HOS cells were increased. Moreover, MNNG/HOS cells cultured under hypoxic conditions were more likely to proliferate into spheres and resulted in larger xenograft tumor. Hypoxia also increased the mRNA and protein levels of hypoxia-inducible factor (HIF)-1α. Then rapamycin was used, which has been shown to lower HIF-1α protein level, to inhibit the hypoxic response. Rapamycin suppressed the expression of HIF-1α protein and CSCs markers (Oct4, Nanog and CD133) in MNNG/HOS cells. In addition, pretreatment with rapamycin reduced the efficiency of MNNG/HOS cells in forming spheres and xenograft tumors. The results demonstrated that hypoxia (1% oxygen) can dedifferentiate some of the MNNG/HOS cells into stem cell-like phenotypes, and that the mTOR signaling pathway participates in this process via regulating the expression of HIF-1α protein.     

Establishment of new multidrug-resistant human osteosarcoma cell lines

Multidrug-resistant clones of human osteosarcoma MNNG/HOS and MG63 cells were isolated by stepwise selection on exposure to increasing doses of doxorubicin (DXR). The final clones MNNG/HOS/DXR1000 and MG63/DXR1000, established after ethylmethane sulfonate mutagenesis, showed 96-fold and 121-fold higer resistance to DXR than their parental cell lines. They were also cross-resistant to vincristine, but not to cisplatinum or methotrexate. The levels of multidrug-resistance-1 (MDR1) mRNA expression increased gradually according to the concentration of DXR in both cell lines. Although the parental MNNG/HOS cells expressed a low level of MDR1 mRNA, the parental MG63 cells showed no MDR1 expression. The IC50 values of MNNG/HOS and its resistant variant to DXR were higher than those of MG63 and its resistant clone. Multidrug-resistant associated protein (MRP) mRNA expression was detected in MNNG/HOS or MG63 parental cell lines, and in their resistant variants. MG63 and its resistant variants revealed stable expression of MRP, whereas the resistant phenotype of MNNG/HOS showed decreased MRP expression, compared to its parental cell line. No alteration in the levels of hepatocyte growth factor (HGF) or its receptor c-MET was recognized between parental lines and their resistant variants. The results indicate that our DXR-resistant variants of MNNG/HOS and MG63 reveal a classical MDR phenotype and can offer a model with which to investigate the mechanisms of multidrug resistance in osteosarcoma.     

Nuclear receptor agonists as potential differentiation therapy agents for human osteosarcoma.

PURPOSE: This study was designed to investigate whether nuclear receptor agonists can be used as potential differentiation therapy agents for human osteosarcoma. EXPERIMENTAL DESIGN: Four osteosarcoma cell lines (143B, MNNG/HOS, MG-63, and TE-85) were treated with proliferator-activated receptor (PPAR)gamma agonists, troglitazone and ciglitazone, and a retinoid X receptor (RXR) ligand, 9-cis retinoic acid. The proliferation and induction of apoptosis in the treated cells were assessed, as was the induction of alkaline phosphatase, a differentiation marker of osteoblasts. RESULTS: The expression of PPARgamma was readily detected in all tested osteosarcoma lines. On treatment with the PPARgamma and RXR ligands, all four osteosarcoma lines exhibited a significantly reduced proliferation rate and cell viability. Among the four lines, 143B and MNNG/HOS were shown to be more sensitive to ligand-induced apoptosis, as demonstrated by the Crystal Violet and Hoechst staining assays. Of the three tested ligands, troglitazone was shown to be the most effective in inducing cell death, followed by 9-cis retinoic acid. Moreover, a strong synergistic effect on the induction of cell death was observed when both troglitazone and 9-cis retinoic acid or ciglitazone and 9-cis retinoic acid were administered to osteosarcoma cells. Troglitazone was shown to effectively induce alkaline phosphatase activity, a well-characterized hallmark for osteoblastic differentiation. CONCLUSIONS: Our findings suggest that PPARgamma and/or RXR ligands may be used as efficacious adjuvant therapeutic agents for primary osteosarcoma, as well as potential chemopreventive agents for preventing the recurrence and metastasis of osteosarcoma after the surgical removal of the primary tumors.     

Oxymatrine induces mitochondria dependent apoptosis in human osteosarcoma MNNG/HOS cells through inhibition of PI3K/Akt pathway

The cytostatic drug from traditional Chinese medicinal herb has acted as a chemotherapeutic agent used in treatment of a wide variety of cancers. Oxymatrine, classified as a quinolizidine alkaloid, is a phytochemical product derived from Sophora flavescens, and has been reported to possess anticancer activities. However, the cancer growth inhibitory effects and molecular mechanisms in human osteosarcoma MNNG/HOS cell have not been well studied. In the present study, the cytotoxic effects of oxymatrine on MNNG/HOS cells were examined by MTT and bromodeoxyuridine (BrdU) incorporation assays. The percentage of apoptotic cells and the level of mitochondrial membrane potential (Δψ _m) were assayed by flow cytometry. The levels of apoptosis-related proteins were measured by Western blot analysis or enzyme assay Kit. Our results showed that treatment with oxymatrine resulted in a significant inhibition of cell proliferation and DNA synthesis in a dose-dependent manner, which has been attributed to apoptosis. Furthermore, we found that oxymatrine considerably inhibited the expression of Bcl-2 whilst increasing that of Bax. This promoted mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytoplasm, as well as the activation of caspase-9 and -3. Moreover, addition of oxymatrine to MNNG/HOS cells also attenuated phosphatidylinositol 3-kinase (PI3K) ?Akt signaling pathway cascade, evidenced by the dephosphorylation of P13K and Akt. Likewise, oxymatrine significantly suppressed tumor growth in female BALB/C nude mice bearing MNNG/HOS xenograft tumors. In addition, no evidence of drug-related toxicity was identified in the treated animals by comparing the body weight increase and mortality. Therefore, these findings should be useful for understanding the apoptotic cellular mechanism mediated by oxymatrine and might offer a therapeutic potential advantage for human osteosarcoma chemoprevention or chemotherapy.     

Somatostatin analog RC-160 inhibits the growth of human osteosarcomas in nude mice

We investigated the effects of the potent somatostatin analog RC-160 on the growth of human osteosarcoma cell lines SK-ES-1 and MNNG/HOS, transplanted into nude mice or cultured in vitro. Growth of SK-ES-I and MNNG/HOS tumors in nude mice was significantly inhibited after 4 weeks of treatment with daily s.c. injections of 100 μg RC-160, as measured by a reduction in tumor volume and weight. Histologically, the number of mitotic cells was decreased in the groups treated with RC-160. In mice bearing either tumor model, administration of RC-160 significantly decreased serum growth hormone and insulin-like growth factor I (IGF-I) levels. Specific high-affinity receptors for somatostatin and epidermal growth factor were found on membranes of MNNG/HOS tumors but not on SK-ES-I tumors. Receptor analyses also demonstrated high-affinity binding sites for IGF-I on membranes of both tumors. In cell cultures, the proliferation rate of MNNG/HOS cells, but not of SK-ES-I, was significantly suppressed by RC-160. Our findings demonstrate that RC-160 can significantly inhibit the growth of SK-ES-I and MNNG/HOS osteosarcomas in mice. © 1996 Wiley-Liss, Inc.     

Highly-expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/β-catenin and mTOR/HIF1α/VEGF signaling

The P2X7 receptor, an ATP-gated ion channel, is critical for cancer cell growth, invasiveness, and angiogenesis. Previous studies indicate that P2X7 regulates osteoblast proliferation and osteodeposition and that high P2X7 expression has a pro-growth effect in osteosarcoma. However, how it functions in osteosarcoma cell growth and metastasis is not clear. Thus, we elucidated molecular mechanisms of P2X7-dependent positive regulation of osteosarcoma cell proliferation, invasion, migration, epithelial to mesenchymal transition (EMT), and angiogenesis using in vitro and in vivo models. We confirm that P2X7 is highly-expressed in human osteosarcoma tumor tissues and HOS/MNNG, MG63, U2OS, SW1353 and SAOS-2 cell lines. P2X7 receptor stimulation enhanced HOS/MNNG and SAOS-2 cell proliferation, migration and invasion; but knockdown of P2X7 expression or receptor inhibition had opposite effects. P2X7 positively regulated glycogen content, epithelial to mesenchymal transition and stemness of HOS/MNNG cells. P2X7 activation promoted PI3K/Akt/GSK3β/β-catenin and mTOR/HIF1α/VEGF signaling, thereby mediating pro-tumor effects of osteosarcoma cells. Consistent with data from in vitro experiments, systemic administration of P2X7 agonist induced tumor growth, metastasis and tumor-associated bone destruction in osteosarcoma-bearing nude mice, whereas a P2X7 antagonist reversed these effects. Thus, the P2X7 receptor participates in regulation of osteosarcoma growth and metastasis and we offer evidence that P2X7 may be a promising therapeutic target for treating osteosarcoma.     

Health-related quality of life in survivors of stage I-II breast cancer: randomized trial of post-operative conventional radiotherapy and hypofractionated tomotherapy

Background

Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies.

Methods

CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis.

Results

The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs.

Conclusions

MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma.