光活化的金丝桃素诱导胶质瘤细胞调亡的实验研究

目的 探讨光活化的金丝桃素对体外培养胶质瘤细胞凋亡的诱导作用。方法 体外培养的Ｗisar大鼠C6胶质瘤细胞株,加入金丝桃素后进行光诱导作用。然后应用ＭＴＴ法、^３Ｈ－ＴdＲ掺入实验、ＤＮＡ片断化分析等方法,测试金丝桃素对Ｃ６细胞的细胞毒作用及诱导细胞凋亡作用。结果 金丝桃素在低剂量２μg/ml时,７５Ｗ钨灯光照１０min,即可对Ｃ６细胞株具有细胞毒作用;抽提其ＤＮＡ做琼脂糖凝胶电泳可显示凋亡特有的ＤＮＡ“阶梯”。结论 金丝桃素在光诱导下具有抗胶质瘤作用。     

不同剂量γ射线照射对体外培养胶质瘤细胞系的影响及对神经功能的保护作用

目的：比较不同剂量γ射线对胶质瘤细胞系C6和SHG-44的影响，建立γ射线诱导胶质瘤细胞系C6和SHG-44凋亡研究的生物模型，探索γ射线照射对胶质瘤细胞周期的影响。方法：采用对数生长期的C6和SHG-44胶质瘤细胞经4，8，16，32和64Gy的γ射线照射后培养48h，用流式细胞仪进行细胞凋亡检测并分析细胞周期的改变。结果：不同剂量γ射线照射均可诱导C6和SHG-44胶质瘤细胞凋亡。C6细胞对照组、4，8，16，32和64Gy组照射后48h的凋亡百分数分别为(3．1&;#177;0.5)，(16．7&;#177;0.6)，(27．9&;#177;0.8)，(27．3&;#177;0.4)，(33．8&;#177;0.7)和(36．7&;#177;0.7)，照射组凋亡率均高于对照组(t=30．16～67．65，P&;lt;0.01)。SHG-44细胞对照组，4，8，16，32和64Gy组照射后48h的凋亡率均高于对照组(t=35．51～103．79，P&;lt;0.01)，但两组细胞的坏死比例均无明显增加(P&;gt;0.05)。SHG-44细胞对电离辐射更加敏感。两种细胞G0／G1期的细胞比例与γ射线照射剂量呈正相关；S期的细胞比例与照射剂量呈负相关。结论：以γ射线照射C6和SHG-44胶质瘤细胞可建立理想的射线诱导的细胞凋亡模型，γ射线照射可引起两种胶质瘤细胞系G1期阻滞和S期延迟。     

多西紫杉醇对鼠血管肉瘤细胞株的体外抑制作用

目的 探讨多西紫杉醇（Docetaxel，TXT）对鼠血管肉瘤细胞株的增殖抑制作用。方法 采用Alamar Blue法测定，TXT对ISOS-1细胞增殖抑制的影响，并与其它抗肿瘤药物进行比较；用流式细胞仪测定细胞周期的变化。结果 TXT能显著抑制ISOS-1细胞的增殖，IC50为15．8ng／ml，明显低于其它抗肿瘤药物；TXT作用后细胞周期发生明显变化，高浓度TXT对ISOS-1细胞株有G2-M期阻滞作用，低浓度TXT主要将ISOS-1细胞阻断于C0-G1期并诱导细胞凋亡。结论 TXT能强烈抑制鼠血管肉瘤细胞株ISOS-1的增殖，这种抑制作用可能通过作用G2-M期及诱导细胞凋亡来实现的。     

pEgr-hPTEN体外稳定转染对人脑胶质瘤SHG-44 细胞周期及增殖的影响

目的　探讨外源野生型PTEN基因对人脑胶质瘤SHG 4 4细胞周期及增殖的影响 ,阐明PTEN基因抑制肿瘤细胞增殖的机制。方法　脂质体包裹重组质粒pEgr hPTEN体外转染内源性PTEN基因突变的SHG 4 4胶质瘤细胞 ;G4 18筛选稳定表达细胞株并扩增培养 ;采用细胞计数法检测肿瘤细胞增殖速度 ;流式细胞术检测肿瘤细胞周期的变化。结果　经 10到 2 0dG4 18筛选得到稳定表达细胞株 ;重组质粒pEgr hPTEN稳定转染可明显抑制SHG 4 4细胞的体外增殖 ,细胞周期明显改变 ,细胞从G1期到S期发生阻滞。结论　提示转染外源野生型PTEN基因可使SHG 4 4细胞周期阻滞于G1期 ,并可抑制肿瘤细胞增殖。     

光活化的金丝桃素对大鼠C6胶质瘤抑制作用及其对VEGF、Flt-1和MVD的影响

目的　研究金丝桃素被光激活后对C6胶质瘤的抑制作用及其对VEGF、Flt 1和MVD的影响。方法　将接种C6胶质瘤细胞后的 4 0只Wistar大鼠随机分为 5组 ,空白对照组、高和低剂量金丝桃素作用组、高和低剂量金丝桃素 +光照组 ,16天后切取肿瘤组织 ,称重计算抑瘤率 ,标本用SP法免疫组化染色 ,检测瘤组织中VEGF、Flt 1和MVD。结果　 (1)光照下 ,当金丝桃素为 0 .8μg ml时 ,抑瘤率为 6 7.2 % ,金丝桃素为 2 .0 μg ml时抑瘤率为 82 .4 % ,非光照金丝桃素组对肿瘤无抑制作用。 (2 )VEGF在各组大鼠C6胶质瘤组织中均有表达 ,表达值较高 ,各组间无显著性差异。空白对照组中Flt 1表达较高 ,且微血管计数最多。金丝桃素 +光照组中Flt 1蛋白表达和MVD明显低于非光照组及对照组 (P <0 .0 5 ) ,呈剂量依赖关系。Flt 1与肿瘤MVD呈正相关 ,rFlt 1=0 .78。结论　光激活金丝桃素对Flt 1有抑制作用 ,对VEGF表达没有影响。光激活的金丝桃素能抑制大鼠C6胶质瘤生长的机制 ,可能是抑制肿瘤组织中Flt 1后 ,阻止了VEGF发挥效应 ,抑制了肿瘤间质血管生成 ,而抑制肿瘤的生长。     

苯乙酸钠调控人前列腺癌细胞凋亡的研究

目的　为了观察苯乙酸钠 (NaPA)对人前列腺癌细胞株 (PC - 3)的分化诱导作用。方法　应用流式细胞术(FCM)、台盼蓝染色计数法和3 H -TdR掺入法观察NaPA对人前列腺癌细胞株的细胞周期动力学变化的影响。结果　苯乙酸钠对人前列腺癌细胞株的细胞呈剂量依赖性抑制 ,细胞周期的G0 /G1期升高 ,S期、G2 /M期相对下降 ,表明NaPA可抑制人前列腺癌细胞株增殖 ,主要是S期合成降低。结论　提示NaPA通过诱导前列腺癌细胞凋亡治疗前列腺癌具有可期待前景。     

槲皮素诱导人胰癌细胞株eanpan-2凋亡的实验研究

目的探讨中草药樨皮素诱导人胰腺癌细胞株canpan-2凋亡的作用。方法运用MTT法检测樨皮素对eanpan-2细胞凋亡的抑制率，流式细胞仪技术分析樨皮素对canpan-2细胞增殖周期的影响。结果10—320μmol／L浓度的樨皮素均能抑制eanpan-2细胞的生长，且呈明显的时间，剂量依赖性关系，细胞周期分析显示樨皮素组G1期细胞比例明显增多，呈量效关系。结论樨皮素抑制eanpan-2细胞增殖，可能通过使canpan-2细胞停滞在G1期而诱导其凋亡。     

姜黄素诱导Hep-2细胞凋亡及其对细胞周期各时相的影响

目的探讨姜黄素对Hep-2（人喉癌细胞株）细胞诱导分化机制，为喉癌治疗提供理论依据。方法应用MTT（塞唑兰）比色法和流式细胞仪检测Hep-2细胞增殖抑制率及细胞周期各时相的变化和凋亡率，并通过电子显微镜观察经姜黄素诱导分化后的亚细胞结构改变。结果姜黄素对Hep-2细胞增殖有明显的抑制作用，且呈剂量依赖性，流式细胞仪分析，G1期细胞增多，S期细胞减少，C2／M期细胞相对增多，亚细胞结构、细胞空化，染色质趋边凝集。结论姜黄素能够抑制Hep-2细胞增殖，阻止G1期细胞向S期的进程，促进Hep-2细胞凋亡。     

平瘤康方剂对C6胶质瘤细胞增殖的抑制作用

目的探讨中药方剂平瘤康含药血清对体外培养的C6胶质瘤细胞增殖及细胞周期的影响。方法2.5%、5%、10%和20%平瘤康含药血清处理体外培养的C6胶质瘤细胞24h、48h和72h后,应用MTT比色法检测C6胶质瘤细胞增殖;PI染色后,应用流式细胞仪观测细胞周期时相。结果10%和20%的含药血清能抑制C6胶质瘤细胞的增殖;10%和20%的含药血清处理C6细胞48h、72h后,S期细胞百分率下降,呈剂量依赖性。结论平瘤康含药血清可能通过阻滞细胞周期而抑制胶质瘤细胞增殖。     

不同剂量γ射线照射对体外培养胶质瘤细胞系的影响及对神经功能的保护作用

目的:比较不同剂量γ射线对胶质瘤细胞系C6和SHG-44的影响,建立γ射线诱导胶质瘤细胞系C6和SHG-44凋亡研究的生物模型,探索γ射线照射对胶质瘤细胞周期的影响。方法:采用对数生长期的C6和SHG-44胶质瘤细胞经4,8,16,32和64Gy的γ射线照射后培养48h,用流式细胞仪进行细胞凋亡检测并分析细胞周期的改变。结果:不同剂量γ射线照射均可诱导C6和SHG-44胶质瘤细胞凋亡。C6细胞对照组、4,8,16,32和64Gy组照射后48h的凋亡百分数分别为(3.1±0.5),(16.7±0.6),(27.9±0.8),(27.3±0.4),(33.8±0.7)和(36.7±0.7),照射组凋亡率均高于对照组(t=30.16～67.65,P<0.01)。SHG-44细胞对照组,4,8,16,32和64Gy组照射后48h的凋亡率均高于对照组(t=35.51～103.79,P<0.01),但两组细胞的坏死比例均无明显增加(P>0.05)。SHG-44细胞对电离辐射更加敏感。两种细胞G0/G1期的细胞比例与γ射线照射剂量呈正相关;S期的细胞比例与照射剂量呈负相关。结论:以γ射线照射C6和SHG-44胶质瘤细胞可建立理想的射线诱导的细胞凋亡模型,γ射线照射可引起两种胶质瘤细胞系G1期阻滞和S期延迟。     

5-ALA介导的PDT对大鼠体内外C6胶质瘤的杀伤作用研究

目的探讨5-ALA介导的光动力对大鼠C6胶质瘤细胞体内外杀伤作用。方法MTT法探讨培育时间和5-ALA浓度对光动力效应的影响,为动物试验提供时间和剂最参考。取近皮层大鼠胶质瘤模型39只,其中PDT组12只,单纯开骨窗组9只,单用5-ALA＋开骨窗组9只,开骨窗＋激光组9只,各组处理后观察生存状态和生存期,部分大鼠行病理及透射电镜检查。结果5-ALA与C6细胞共同培育5h其介导的PDT作用达到最强;5-ALA浓度为0．8mmol／L以上时对细胞的抑制率可超过50％;电镜示PDT组瘤组织可见较多量凋亡,细胞间连接松散。结论该实验设计的大鼠近皮层模型可以很好地满足脑肿瘤动物实验的要求。5-ALA介导的PDT对体外及颅内种植C6细胞都有杀伤作用。     

Erythropoietin inhibits apoptosis induced by photodynamic therapy in ovarian cancer cells

Recombinant human erythropoietin is widely used to treat anemia associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Erythropoietin is the principal regulator of erythroid cell proliferation, differentiation, and apoptosis. Recently, the antiapoptotic and proliferative effects of erythropoietin on nonhematopoietic cells were also established. We now show the effect of erythropoietin treatment on the response of A2780 and SKOV3 ovarian carcinoma cell lines to photodynamic therapy (PDT) using hypericin. SKOV3 exhibited an increased resistance to hypericin when cells were treated with erythropoietin. This resistance was reversed by treatment of SKOV3 cells with the specific Janus kinase 2 kinase inhibitor AG490 or the tyrosine kinase inhibitor genistein. These results support a role for the specific erythropoietin-induced Janus kinase 2/STAT signal transduction pathway in PDT resistance. Evidence of erythropoietin signaling was obtained by the demonstration of Akt phosphorylation in both A2780 and SKOV3 cells. Erythropoietin-treated SKOV3 cells exhibited decreased apoptosis induced by hypericin, an effect that was blocked by the phosphoinositide 3-kinase/Akt inhibitor wortmannin. These results may have important implications for ovarian cancer patients undergoing PDT and receiving erythropoietin.     

Evaluation of Firefly Luciferase Bioluminescence Mediated Photodynamic Toxicity in Cancer Cells

Purpose This work investigated whether fLuc-catalyzed oxidation of d-luciferin generates sufficient light to induce photodynamic toxicity in cancer cells.Procedures Light emission was assessed via cooled CCD (charge-coupled device) camera. Parental and fLuc expressing cancer cells were exposed to subtoxic concentrations of photosensitizers (Rose Bengal or hypericin) and d-luciferin, sunlight, or lamplight. Toxicity was assessed by MTT assay.Results fLuc expressing cells emitted up to 500-fold higher levels of photons than parental cell lines. Although exposure to photosensitizer and sunlight reduced survival of various cell lines, survival of fLuc expressing cells incubated with photosensitizer and d-luciferin, or photosensitizer and lamplight, did not differ significantly from parental or untreated cells.Conclusions Contesting recent reports, fLuc bioluminescence does not generate sufficient photons to induce Rose Bengal or hypericin photodynamic toxicity in a range of malignant and nonmalignant cell lines, and is not suitable as a generalizable approach to antineoplastic therapy.     

Highly efficient green synthesis and photodynamic therapeutic study of hypericin and its derivatives

A highly efficient synthetic pathway for hypericin (7a) was achieved under mild conditions with an overall yield over two steps of 92% using emodinanthrone as a starting material, where protohypericin, a key precursor of hypericin, was synthesized in water with microwave assistance, which was then photocyclized to hypericin with a high yield via 1 h irradiation in a visible light reactor equipped with 575 nm monochromatic lamps. In addition, the method could be used to synthesize hypericin derivatives (7b–d) with similar overall yields. Furthermore, their effects of photodynamic therapy (PDT) were evaluated on A431, HepG-2, and MCF-7 cell lines. The PDT of 7b was better than that of 7a, whereas 7c and 7d were worse. Unlike other cell lines, MCF-7 was not sensitive to any of 7a–d at the same concentrations.

A highly efficient synthetic pathway for hypericin as well as its derivatives was achieved under mild and green conditions with high yields.     

On hypericin application in fluorescence diagnosis and cancer treatment: Pharmacokinetics and photosensitizing efficiency in nude mice bearing WiDr carcinoma

Hypericin is a potential photosensitizer for photodynamic diagnosis and therapy of cancer. Using a non-invasive fluorescence method we have shown that it is selectively accumulated in WiDr colon carcinoma tumours grown in nude mice after intraperitoneal (i.p.) administration (2–5 mg/kg). The maximal concentration of hypericin in the tumour was recorded 24 h after injection. The fluorescence of the drug recorded from the surface of the tumour was 6.5, 17, 34 or 6 times higher than that measured in skin, brain, liver or muscles, respectively. Photodynamic therapy with high drug doses and light exposures (5 mg/kg hypericin, i.p., 120 J/cm²) at 6 or 24 h after drug administration delayed tumour growth. Hypericin appears to be a suitable drug, mainly for fluorescence diagnosis.     

Influence of epidermal growth factor on photodynamic therapy of glioblastoma cells in vitro

Photodynamic therapy (PDT) could be a useful adjuvant in glioblastoma treatment. The fact that epidermal growth factor (EGF) and its receptor are involved in glioblastoma growth control led us to investigate the relationships between EGF and PDT with respect to three different glioma cell lines (C6, T98 G, U87 MG) responsive to growth stimulation by EGF. Flow cytometric analysis revealed that each cell line expressed EGF receptors. PDT was then applied to the cells using haematoporphyrin derivative (HPD) as photosensitizer and argon laser irradiation. When cells were incubated for 2 h with HPD (0.1–10 μg/ml) and then laser-irradiated (λ = 514 nm; energy density 25 J/cm²), all three cell lines showed photosensitivity. The median lethal dose was respectively 3, 4.5 and 2.7 μg/ml for C6, T98 G and U87 MG. EGF (2–50 ng/ml) had no effect on HPD- and laser-induced toxicity when added to cells before PDT, whereas toxicity decreased for all three cell lines when EGF was added after PDT. HPD (1–2 μg/ml, incubation times 30–180 min) also induced an increase in EGF receptor expression for the C6 line.     

体外血管内皮生长因子单克隆抗体对C6胶质瘤细胞增殖、侵袭和迁移力的影响

目的:在体外观察血管内皮生长因子(VEGF)单克隆抗体对C6胶质瘤细胞增殖、侵袭和迁移力的影响。方法:采用氯化钴(Co Cl2)模拟缺氧环境,予以0、0.1、1、10μg/m L的VEGF抗体,对常氧或缺氧条件下对C6胶质瘤细胞进行处理。通过MTT实验检测细胞增殖,Transwell实验检测细胞的侵袭和迁移能力,Western blotting检测HIF-1α蛋白、FAK/Pyk2总蛋白及磷酸化FAK-Tyr397/Pyk2-Tyr402表达水平变化。结果:200μmol/L Co Cl2明显抑制C6细胞增殖(P<0.05),10μg/m L VEGF抗体可明显抑制C6细胞增殖(P<0.05)。与常氧相比,缺氧可使C6细胞的迁移、侵袭能力增强(P<0.05)。常氧和缺氧下,VEGF抗体的干预均增强C6细胞的迁移、侵袭能力(P<0.05),且相对于常氧,缺氧可进一步增强VEGF抗体促C6细胞迁移、侵袭的作用(P<0.05)。Co Cl2干预可增加C6细胞中HIF-1α的含量;在常氧和缺氧情况下,只有1μg/m L VEGF抗体可降低FAK的总蛋白量(P<0.05);在缺氧情况下,VEGF抗体可明显增高Pyk2-Tyr402位点的磷酸化水平。结论:在缺氧情况下,VEGF抗体的干预可使Pyk2-Tyr402位点的磷酸化水平增高,引起C6细胞的侵袭迁移能力进一步增强。     

The role of thrombin in gliomas

BACKGROUND: In a previous study we found that intracerebral infusion of argatroban, a specific thrombin inhibitor, reduces brain edema and neurologic deficits in a C6 glioma model. OBJECTIVES: To examine the role of thrombin in gliomas and whether systemic argatroban administration can reduce glioma mass and neurologic deficits and extend survival time in C6 and F98 gliomas. METHODS: The presence of thrombin in human glioblastoma samples and rat C6 glioma cells (in vitro and in vivo) was assessed using immunohistochemistry. The effect of thrombin on C6 cell proliferation in vitro was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The role of thrombin in vivo was assessed in rat C6 and F98 glioma cell models using argatroban, a thrombin inhibitor. The effects of argatroban on tumor mass, neurologic deficits and survival time were investigated. RESULTS: Thrombin immunoreactivity was found in cultured rat C6 glioma cells and human glioblastomas. Thrombin induced C6 cell proliferation in vitro. In C6 glioma, argatroban reduced glioma mass (P < 0.05) and neurologic deficits (P < 0.05) at day 9. In F98 glioma, argatroban prolonged survival time (P < 0.05). CONCLUSION: These results suggest that thrombin plays an important role in glioma growth. Thrombin may be a new therapeutic target for gliomas.     

Experimental therapy of malignant gliomas using the inhibitor of histone deacetylase MS-275

Inhibitors of histone deacetylases are promising compounds for the treatment of cancer but have not been systematically explored in malignant brain tumors. Here, we characterize the benzamide MS-275, a class I histone deacetylase inhibitor, as potent drug for experimental therapy of glioblastomas. Treatment of four glioma cell lines (U87MG, C6, F98, and SMA-560) with MS-275 significantly reduced cell growth in a concentration-dependent manner (IC(90), 3.75 micromol/L). Its antiproliferative effect was corroborated using a bromodeoxyuridine proliferation assay and was mediated by G(0)-G(1) cell cycle arrest (i.e., up-regulation of p21/WAF) and apoptotic cell death. Implantation of enhanced green fluorescent protein-transfected F98 glioma cells into slice cultures of rat brain confirmed the cytostatic effect of MS-275 without neurotoxic damage to the organotypic neuronal environment in a dose escalation up to 20 micromol/L. A single intratumoral injection of MS-275 7 days after orthotopic implantation of glioma cells in syngeneic rats confirmed the chemotherapeutic efficacy of MS-275 in vivo. Furthermore, its propensity to pass the blood-brain barrier and to increase the protein level of acetylated histone H3 in brain tissue identifies MS-275 as a promising candidate drug in the treatment of malignant gliomas.