西乐葆和雷帕霉素联合作用对人乳腺癌MDA-MB-231细胞的抗肿瘤效应

目的 观察西乐葆和雷帕霉素联合作用对于人乳腺癌MDA-MB-231细胞的抗肿瘤效应.方法 不同浓度的西乐葆(20、40、60、80 μmol/L)、雷帕霉素(1、10、100、1000 nmoVL)单独及60μmol/L西乐葆和100 nmol/L雷帕霉素两者联合作用对MDA-MB-231细胞生长的影响通过噻唑蓝(MTT)比色法检测.对细胞周期和凋亡的影响使用流式细胞仪进行分析.蛋白印迹实验检测HER2和HER3的表达情况及磷酸化Akt(473)水平.结果 西乐葆、雷帕霉素对于MDA-MB-231细胞生长抑制具有浓度和时间依赖性.60 μmol/L西乐葆和100 nmol/L雷帕霉素联合应用细胞生长抑制率及凋亡率为[(88.0±8.0)%,(32.5±3.0)%],与两者单独应用细胞生长抑制率及凋亡率[(52.0±5.0)%,(12.6±2.0)%;(54.0±6.0)%,(7.2±2.0)%]比较差异有统计学意义(P<0.01).联合应用对MDA-MB-231细胞抗肿瘤效应与降低细胞HER2和HER3的表达及磷酸化Akt(473)水平有关.结论 西乐葆和雷帕霉素联合能增强对人乳腺癌DA-MB-231细胞的抗肿瘤效应,对于HER2、HER3阳性乳腺癌具有潜在的临床应用价值.     

白藜芦醇对胰腺癌细胞的抑制作用

目的检测白藜芦醇(resveratrol,Res)体外单独及联合化疗药物对胰腺癌MIAPaCa-2细胞的抑制作用。方法在体外培养条件下观察Res,5-氟脲嘧啶(5-flurouracil,5-FU)和吉西他滨(Gemcitabine,Gem)分别对胰腺癌MIAPaCa-2细胞增殖的影响,然后根据以上抑制结果,选择对MIAPaCa-2细胞抑制率在15%～30%的5-FU或Gem药物浓度,再与不同浓度的Res联合用药。在联合药物处理MIAPaCa-2细胞48 h后,同样用噻唑蓝(methyl thia-zolyl tetrazolium,MTT)法检测联合用药,检测其对细胞增殖的抑制作用。结果Res,5-FU及Gem体外单独用药均能显著抑制MIAPaCa-2细胞的生长增殖,Res联合384.4μmol/L 5-FU或5.0μmol/L Gem后,对MIAPaCa-2细胞增殖的抑制作用显著提高,与单独用药组比较,差异有统计学意义(P<0.05)。结论体外Res能显著抑制人胰腺癌MIAPaCa-2细胞的增殖,Res联合5-FU或Gem能显著提高对MIAPaCa-2细胞增殖的抑制作用。     

雷帕霉素和紫杉醇对激素非依赖性前列腺癌细胞株影响的体外实验研究

目的 通过前列腺癌细胞株的体外实验对照研究初步探讨雷帕霉素、紫杉醇及两者联合应用对激素非依赖性前列腺癌细胞株的影响.方法 分别采用噻唑蓝(MTT)比色法和流式细胞技术观察雷帕霉素、紫杉醇、雷帕霉素+紫杉醇对不同前列腺癌细胞株(LNCaP-C4、LNCaP-C4-2、PC-3)细胞增生及细胞凋亡的影响.结果 对于LNCaP-C4细胞株,同对照组相比,当雷帕霉素浓度为0.01 μmol/L时,其抑制肿瘤细胞的作用开始出现显著性差异;而对于LNCaP-C4-2、PC-3细胞株,雷帕霉素浓度为0.001 μmol/L时,其抑制肿瘤细胞的作用同对照组相比具有显著性差异.当紫杉醇浓度为0.2 ng/mL时,同对照组相比,紫杉醇表现对3种前列腺癌细胞株有显著的抑制作用(P<0.05),随着紫杉醇浓度的提高,其抑制肿瘤细胞的作用也越强.10 nmol/L的雷帕霉素、1 ng/mL的紫杉醇对前列腺癌细胞株的抑制作用同对照组相比已经有了显著性差异(P<0.05),而5 nmol/L的雷帕霉素+0.5 ng/mL的紫杉醇组对所有3种前列腺癌细胞株的抑制作用均显著高于10 nmol/L的雷帕霉素组和1 ng/mL的紫杉醇组.结论 雷帕霉素和紫杉醇对3种前列腺癌细胞株(LNCaP-C4、LNCaP-C4-2、PC-3)均有一定抑制作用,且表现为剂量依赖关系.小剂量的雷帕霉素和紫杉醇联合应用可以起到更好的抑制肿瘤细胞的作用.     

霉酚酸、雷帕霉素及FTY720在体外对成人胰岛功能的影响

目的探讨霉酚酸、雷帕霉素及FTY720对体外培养的成人胰岛功能的影响。方法将分离、纯化后获得的成人胰岛细胞分别与不同浓度的霉酚酸、雷帕霉素和FTY720混合培养24h,然后测定经各药物作用后的胰岛细胞在低糖(2.8mmol/L)和高糖(16.7mmol/L)刺激下胰岛素的释放量,并计算刺激指数。结果对照组(未添加药物)的胰岛细胞在低糖和高糖刺激下胰岛素的释放量分别为(7.37±1.74)ng/ml和(15.15±5.39)ng/ml,刺激指数为2.06±0.46。与对照组相比,添加霉酚酸的胰岛细胞在低糖和高糖刺激下胰岛素的释放量均明显下降(P<0.01),刺激指数亦明显下降(P<0.05);添加雷帕霉素组,低糖和高糖刺激时胰岛素的释放量均有下降(P<0.05),但刺激指数仅在雷帕霉素浓度为1.0ng/ml时明显下降(P<0.05);添加FTY720组,低糖刺激时胰岛素的释放量明显低于对照组(P<0.05),高糖刺激时,胰岛素释放量仅在FTY720高浓度组有明显下降(P<0.05),刺激指数也有明显下降。结论相对来说,在体外雷帕霉素和FTY720对成人胰岛细胞的毒性作用较小;胰岛移植后,选择免疫抑制方案应遵循低剂量原则。     

雷帕霉素对前列腺癌细胞株22RV1增殖及S6K1活性的影响

目的:探讨雷帕霉素对前列腺癌细胞22RV1增殖及对S6 Kinase 1(S6K1)活性的影响。方法:用不同浓度(0、50、100、200、400 nmol/L)雷帕霉素作用于体外培养的前列腺癌细胞22RV1,MTT法检测细胞生长抑制率;应用液闪激酶活性测定法检测不同浓度雷帕霉素对S6K1活性的影响。结果:雷帕霉素能显著抑制22RV1细胞的生长,呈现明显的量-效关系,随着雷帕霉素剂量的增加,细胞生长抑制率逐渐升高,400 nmol/L的雷帕霉素对22RV1细胞的抑制率最高(P<0.01);同时雷帕霉素还能使S6K1蛋白活性下降,随剂量增高降低越明显,400 nmol/L的雷帕霉素使S6K1蛋白活性下降最明显(P<0.01)。结论:雷帕霉素可通过调控mammal Target ofrapamycin(mTOR)下游蛋白S6K1表达来有效地抑制前列腺癌细胞22RV1的增殖。     

沙利霉素逆转P-gp介导的肾癌ACHN细胞多药耐药的实验研究

目的 探讨沙利霉素对肾癌ACHN细胞多药耐药的逆转及其机制.方法 实验分为对照组、阿霉素组、沙利霉素组及阿霉素和沙利霉素联合用药组,药物作用24 h后,CCK-8方法 检测肾癌细胞的生长活性,免疫细胞化学法检测肾癌细胞P-糖蛋白(P-gp)的表达情况.结果 10 μg/ml 阿霉素对肾癌ACHN细胞的生长抑制率仅为4.758%.沙利霉素组对肾癌ACHN细胞生长抑制率呈剂量依赖性,并高于阿霉素组(P<0.05),其中以10 μmol/L组抑制率最高(达17.555%).联合用药组的生长抑制率高于沙利霉素组及阿霉素组(P<0.05),以10 μmol/L 沙利霉素 +10 μg/ml 阿霉素组的抑制率最高(达45.447%).与阿霉素组比较,经沙利霉素处理过的肾癌ACHN细胞中的P-gp蛋白表达下降(P<0.05).结论 沙利霉素增强了肾癌ACHN细胞对阿霉素的敏感性,耐药性逆转的机制之一可能与沙利霉素下调癌细胞中P-gp的表达有关.     

雷帕霉素对人骨肉瘤细胞增殖和凋亡的影响

目的 观察雷帕霉素(RAPA)对人骨肉瘤细胞U-2OS和Saos-2增殖和凋亡的影响并探讨其机制.方法 体外培养骨肉瘤细胞株U-2OS和Saos-2;噻唑蓝(MTT)比色法和流式细胞仪检测不同浓度雷帕霉素对骨肉瘤细胞增殖和凋亡的影响;Western blot检测雷帕霉素作用后骨肉瘤细胞Fas蛋白表达的变化.结果 MTT检测显示雷帕霉素可抑制入骨肉瘤细胞增殖,在雷帕霉素浓度达到20 nmol/L时,抑制作用显著提高(P＜0.05),增殖抑制率分别达到31.2％和28.7％.流式细胞检测显示在雷帕霉素浓度达到20 nmol/L时,其诱导骨肉瘤细胞凋亡的作用显著增强(P＜0.05).Western blot检测显示经雷帕霉素作用48 h后的骨肉瘤细胞Fas蛋白的表达明显增强.结论 雷帕霉素对入骨肉瘤细胞株U-2OS和Saos-2的增殖有抑制作用并可诱导其发生凋亡,其可能是通过上调Fas蛋白的表达而发挥作用的.     

茶多酚通过调节Akt通路诱导胰腺癌细胞凋亡

目的 观察茶多酚主要成分没石子儿茶素没石子酸酯( EGCG)诱导人胰腺癌Panc-1细胞凋亡过程中对Akt信号途径的调节作用.方法 以不同浓度EGCG (6.3、12.5、25.0、50.0 μmol/L)处理Panc-1细胞,用噻唑蓝(MTT)比色法检测其生长抑制作用,用琼脂糖凝胶电泳法检测细胞凋亡,用Western blot技术检测细胞中Akt分子(60 x103)的变化及Bad分子(23×103)的变化.结果 EGCG以时间及剂量依赖的方式抑制Panc-1细胞增殖并诱导Panc-1细胞凋亡(P<0.05).同时ECCG剂量依赖性降低了p-Akt( ser473)和p-Bad(ser136)蛋白的含量,而Akt总蛋白和Bad总蛋白无明显改变(P<0.05).结论 EGCG可通过抑制Akt信号通路并减轻该通路对Bad蛋白的阻断作用而诱导胰腺癌细胞凋亡.     

索拉菲尼联合树突状细胞与细胞因子诱导杀伤细胞抗肝细胞癌生长作用的实验研究

目的观察索拉菲尼联合树突状细胞与细胞因子诱导杀伤细胞抗肝细胞癌生长作用。方法体外培养细胞因子诱导杀伤细胞与树突状细胞,采用不同剂量索拉菲尼6.9μmol/L、13.8μmol/L、20.8μmol/L联合树突状细胞与细胞因子诱导杀伤细胞作用于小鼠H22肝癌细胞,应用酶联免疫分析技术测定二者联合对肝癌细胞增殖抑制率。结果随着索拉非尼剂量增加,对H22肝癌细胞增殖抑制作用增强,与索拉非尼相比,抑制率差异具有统计学意义(F=1.236,P0.05;4.4 mg/ml剂量除外);索拉非尼在13.25 mg/ml时联合DC-CIK细胞对肝癌细胞增殖抑制作用最强,与索拉非尼4.4 mg/ml剂量联合DC-CIK细胞作用相比,抑制率差异具有统计学意义(F=2.175,P0.05)。结论索拉非尼联合树突状细胞与细胞因子诱导杀伤细胞共同作用于肝癌细胞,能够提高索拉非尼杀伤肝癌细胞作用,且对肝癌细胞增殖抑制作用呈现剂量依赖性,为进一步提高分子靶向药物临床治疗肝细胞癌的疗效提供了理论依据。     

17-AAG联合紫杉醇对人未分化甲状腺癌FRO细胞增殖和凋亡影响的研究

目的探讨热休克蛋白90(HSP90)抑制剂17-丙烯胺基-17-去甲氧格尔德霉素(17-AAG)联合紫杉醇对人未分化甲状腺癌FRO细胞增殖和凋亡的影响。方法 1采用四甲基偶氮唑盐微量酶反应比色法(MTT比色法)测定不同浓度(17-AAG:0.312 5、0.625 0、1.250 0、2.500 0及5.000 0μmol/L;紫杉醇:0.001 0、0.010 0、0.100 0及1.000 0μmol/L)、不同时间(24、48及72 h)17-AAG、紫杉醇单药和联合处理(17-AAG:0.625 0μmol/L,紫杉醇:0.001 0、0.010 0、0.100 0及1.000 0μmol/L)后FRO细胞的增殖抑制率。2采用流式细胞仪检测17-AAG、紫杉醇单药及联合处理24 h后(17-AAG:0.625 0μmol/L、紫杉醇:0.100 0μmol/L;联合用药:17-AAG的浓度为0.625 0μmol/L,紫杉醇的浓度为0.100 0μmol/L)FRO细胞的细胞周期变化及凋亡率。3采用胱天蛋白酶-3(Caspase-3)和Caspase-9检测试剂盒检测17-AAG、紫杉醇单药及联合处理24 h后(17-AAG:0.625 0μmol/L、紫杉醇:0.100 0μmol/L;联合用药:17-AAG的浓度为0.625 0μmol/L,紫杉醇的浓度为0.100 0μmol/L)后FRO细胞中的Caspase-3和Caspase-9活性。空白对照组均不加任何药物,只加培养液。结果 1同时点空白对照组、各剂量17-AAG组/紫杉醇组/17-AAG联合紫杉醇组的增殖抑制率随浓度升高而逐渐升高,任2组比较差异均有统计学意义(P0.05);各剂量17-AAG组/紫杉醇组/17-AAG联合紫杉醇组的增殖抑制率在24、28及72 h逐渐增高,任2组比较差异均有统计学意义(P0.05);同时点同浓度情况下,17-AAG联合紫杉醇组的增殖抑制率均高于单独用药组(P0.05)。各时点17-AAG与紫杉醇联合的q值均大于1.15,两者之间呈协同作用。2 17-AAG组、紫杉醇组及17-AAG联合紫杉醇组FRO细胞的凋亡率均明显高于空白对照组(P0.05),且17-AAG联合紫杉醇组FRO细胞的凋亡率高于17-AAG组和紫杉醇组(P0.05)。3 17-AAG组、紫杉醇组及17-AAG联合紫杉醇组FRO细胞的Caspase-3和Caspase-9活性均高于空白对照组(P0.05);且17-AAG联合紫杉醇组细胞的Caspase-3和Caspase-9活性均高于17-AAG组和紫杉醇组相应指标(P0.05)。结论 17-AAG和紫杉醇均可明显抑制FRO细胞的增殖并诱导细胞凋亡,联合用药有一定的协同效应,呈剂量依赖关系。     

Antiproliferative and overadditive effects of rapamycin and FTY720 in pancreatic cancer cells in vitro

Rapamycin inhibits the growth of several tumors including pancreatic carcinoma both in vitro and in vivo. The antitumor effects of FTY720 were also shown recently. The present study was performed to investigate the in vitro antiproliferative capacity of combined treatment with rapamycin and FTY720 on pancreatic cacinoma cell lines. MATERIALS AND METHODS: The Panc-1 and AsPc-1 cell lines were employed as the pancreatic carcinoma model in vitro. For monotreatment experiments, rapamycin was added in increasing doses from 0.002 micromol/L to 200 micromol/L; FTY720 was used from 1 micromol/L to 15 micromol/L. For combined treatment, two concentrations of rapamycin were combined with seven concentrations of FTY720; or two concentrations of FTY720 with five concentrations of rapamycin. The antiproliferative capacity was assessed by the MTT assay. RESULTS: Rapamycin and FTY720 inhibited MTT incorporation into Panc-1 and AsPc-1 in dose-dependent fashion with or without serum stimulation. In coincubation experiments, great susceptibility to rapamycin was seen when combined with 10 micromol/L FTY720. An effective combination for AsPc-1 was 10 micromol/L FTY720 with 0.002 micromol/L rapamycin, resulting in more than 50% inhibition of MTT incorporation, and for Panc-1, 10 micromol/L FTY720 with 0.002 micromol/L rapamycin and 10 micromol/L FTY720 with 20 micromol/L rapamycin; the corresponding inhibition levels reached about 40% and 60%, respectively. CONCLUSION: Rapamycin and FTY720 showed dose-dependent antiproliferative effects on pancreatic carcinoma cell lines in vitro both alone and in combination. The combined use of rapamycin and FTY720 showed additive and supra-additive antiproliferative effects on pancreatic carcinoma cell lines. The susceptibility of pancreatic carcinoma cells to rapamycin was significantly enhanced when combined with FTY720.     

Antiproliferative and overadditive effects of everolimus and mycophenolate mofetil in pancreas and lung cancer cells in vitro

BACKGROUND: Everolimus inhibits the growth of several tumor cell lines in vitro as well as tumor growth in a rat model. Mycophenolate mofetil (MMF) inhibits growth of a Walker sarcoma in a rat model in vivo. Herein we tested the in vitro antiproliferative capacity of everolimus and MMF on a pancreatic tumor cell line Panc-1 and on a small cell lung cancer cell line ScLc. MATERIALS AND METHODS: Cells were cultured under standardized conditions. Everolimus was added in increasing doses from 0.005 to 500 microg/mL; MMF was used from 0.05 to 5000 microg/mL. For co-incubation experiments, we combined everolimus (0.005 microg/mL and 0.05 microg/mL) with five concentrations of MMF; and MMF (0.5 microg/mL and 5 microg/mL) with five concentrations of everolimus. The antiproliferative capacity was assessed by a BrdU incorporation assay. RESULTS: Everolimus and MMF inhibited BrdU incorporation into Panc-1 and ScLc in a dose-dependent fashion. A 50% inhibition was seen in Panc-1 only at 50 microg/mL everolimus, but in ScLc at 5 microg/mL everolimus. MMF was clearly more potent in Panc-1: 50% inhibition was observed at 5 microg/L. In ScLc, 40% inhibition of BrdU incorporation was seen only at 50 microg/L MMF. In co-incubation, an effective combination for both Panc-1 and ScLc was 5 microg/mL MMF with 0.005 microg/mL everolimus resulting in 50% inhibition of BrdU incorporation (P < .001). CONCLUSIONS: Everolimus and MMF showed dose-dependent antiproliferative effects in tumor cell lines in vitro both alone and in combination. The combined use of everolimus and MMF showed supra-additive effects at concentrations used for therapeutic immunosuppression in patients.     

Defining the role of the epidermal growth factor receptor in pancreatic cancer grown in vitro

BACKGROUND: The purpose of this study was to determine the effect of a novel epidermal growth factor (EGF) receptor tyrosine kinase inhibitor, Erlotinib, on pancreatic cancer cell lines of varying differentiation in vitro. METHODS: Six pancreatic cancer cell lines (AsPc-1, CAPAN-1, HPAC, HPAF-II, Mia PaCa-2, PANC-1) were grown in the presence of 50 microM or 100 microM of Erlotinib or recombinant EGF. Cell proliferation was determined using the MTT assay over 72 hours. The EGF receptor gene and protein expression were determined by polymerase chain reaction and immunohistochemistry respectively. RESULTS: All cell lines demonstrated the presence of the EGF receptor gene and its gene product. Five of six cell lines showed significant growth inhibition at 72 hours compared with controls (P <0.05). The EGF augmentation increased proliferation of each cell line but this increase was only significant in AsPc-1. CONCLUSIONS: Inhibition of EGF receptor is a valid therapeutic strategy in pancreatic cancer.     

线粒体在雷帕霉素诱导肝癌细胞Bel-7402凋亡中的作用

目的观察雷帕霉素（rapamycin,RAPA）体外对肝癌细胞Bel-7402的生长抑制和诱导凋亡作用,并探讨线粒体在诱导凋亡机理中的作用。方法以5、10、20、30、40和50nmol／L不同浓度的RAPA作用于体外培养的Bel-7402细胞,MTT法检测细胞生长抑制率;应用流式细胞仪检测细胞凋亡;Hoechst 33258荧光染色法观察细胞凋亡时的形态学变化;JC-1染色法检测细胞线粒体膜电位（mitochondrial membrane potential）的变化。结果RAPA可显著地抑制Bel-7402的生长,诱导细胞发生凋亡,并呈现出明显的量-效与时-效关系,50nmol／LRAPA作用72h,引起的细胞抑制率和凋亡率明显高于其他浓度药物组（P〈0．01）。RAPA作用Bel-7402细胞48h后,在Hoechst33258荧光染色图片上可见细胞核浓缩、细胞核碎裂等典型的细胞凋亡特征。凋亡过程中线粒体膜电位下降。结论RAPA能抑制Bel-7402细胞的生长,诱导细胞凋亡发生,线粒体膜电位下降在凋亡过程中可能起到重要作用。     

雷帕霉素对肾小球系膜细胞增殖及凋亡的影响

目的 观察不同浓度雷帕霉素对大鼠肾小球系膜细胞增殖及凋亡的影响,探讨其可能的作用机制。 方法 使用不同浓度雷帕霉素（1 μg/L、2 μg/L、4 μg/L、8 μg/L、16 μg/L）处理大鼠肾小球系膜细胞,并设正常对照组。MTT法测不同浓度雷帕霉素干预24 h、48 h、72 h后对细胞增殖的影响,并描记生长曲线。干预72 h后,采用RT-PCR和Western印迹法分别检测各浓度雷帕霉素组细胞周期负调蛋白p27和p53的mRNA和蛋白表达变化;用流式细胞计量术检测各浓度雷帕霉素组细胞周期及凋亡率。 结果 小剂量雷帕霉素（1 μg/L）对系膜细胞增殖即具有明显的抑制作用,停滞于G1期的细胞数明显增加,但对系膜细胞凋亡无明显影响。较大剂量雷帕霉素(8~16 μg/L)能够明显增加肾小球系膜细胞凋亡。雷帕霉素能够增加肾小球系膜细胞p27、p53 mRNA和蛋白表达,且呈剂量依赖性。 结论 雷帕霉素可能通过增加p27、p53表达抑制肾小球系膜细胞增殖和促进系膜细胞凋亡。     

Induction of apoptosis in human bladder cancer cells in vitro and in vivo caused by FTY720 treatment

PURPOSE: FTY720 is a unique immunosuppressant that induces apoptosis in activated lymphocytes but not in other hematopoietic cells. We examined whether FTY720 has anticancer effects on human bladder cancer cells by inducing apoptosis and we investigated its molecular pathway. MATERIALS AND METHODS: We used the 3 human bladder cancer cell lines T24, UMUC3 and HT1197, and the human fibroblast derived cell line CRL-2096 (American Type Tissue Collection, Rockville, Maryland) in this study. The difference in drug susceptibility to FTY720 in cancer cells and fibroblasts was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell growth assays. FTY720 induced apoptosis was determined by morphological analysis under light and electron microscopy, and DNA electrophoresis, and its molecular pathway was evaluated by Western blot analysis focusing on the p42/p44 mitogen activated protein kinase pathway. We then tested the in vivo effect of this agent using 2 mouse models of human bladder cancer xenograft. RESULTS: FTY720 treatment in vitro induced selective apoptosis in cancer cells at a concentration of less than 10 microM. Morphological analysis revealed features characteristic of apoptosis, including small cytoplasm with fragmented nuclei and condensed chromatin. DNA electrophoresis confirmed apoptosis, as evidenced by a distinct oligosomal ladder. Western blot analysis revealed that the agent significantly inhibited hepatocyte growth factor induced p42/p44 mitogen activated protein kinase activity. The in vivo anticancer effect was clearly confirmed by significantly decreased tumor growth without notable side effects in the 2 xenograft models. CONCLUSIONS: FTY720 treatment may induce selective apoptosis in vitro as well as in vivo in cancer cells. We suggest that FTY720 is a potent and clinically applicable anticancer agent for bladder cancer.     

FRAP-p70s6K signaling is required for pancreatic cancer cell proliferation

BACKGROUND: FRAP-p70s6K signaling regulates mitogenic responses to growth factors in eukaryotic cells. Constitutive p70s6K activation occurs in some human malignancies and may contribute to dysregulated cell growth. We examined whether inhibition of this pathway affects mitogen-induced proliferation and cell cycle progression of human pancreatic cancer cells in vitro. METHODS: Quiescent BxPC3 and Panc-1 human pancreatic cancer cells treated with or without 20 ng/mL rapamycin (FRAP inhibitor) were repleted with 10% FCS to induce cell cycle entry. Proliferation was measured with MTT assay. Cell cycle and apoptosis were determined by FACS analysis. Phosphorylation of p70s6K, Akt, and cdc2 was evaluated by Western blot. Statistical analysis was by two-tailed t test (P < 0.05). RESULTS: Rapamycin (Rapa) inhibited the phosphorylation of p70s6K while inducing G(1) cell cycle arrest (P < 0.005). In both cell lines, Rapa inhibited serum-induced proliferation (P < 0.05) without affecting apoptosis. Cdc2 phosphorylation was inhibited by 15 min with Rapa (not shown), consistent with cell cycle arrest. Akt phosphorylation was not affected, indicating FRAP specificity of Rapa. CONCLUSIONS: FRAP-p70s6K signaling appears to be necessary for G(1)-to-S phase progression and proliferation in pancreatic cancer cells. This supports earlier work demonstrating a similar regulatory role for PI-3' kinase, an upstream activator of FRAP-p70s6K.     

Oral efficacy of the new immunomodulator FTY720 in cynomolgus monkey kidney allotransplantation,given alone or in combination with cyclosporine or RAD

BACKGROUND: FTY720 is a novel immunomodulator with a unique mechanism of action, i.e. chemokine-dependent lymphocyte homing into secondary lymphoid organs associated with profound lymphocyte depletion in blood. We investigated its efficacy, either FTY720 alone or together with cyclosporine or the rapamycin derivative rapamycin derivative (RAD), in cynomolgus monkey kidney allotransplantation. METHODS: Life-supporting allotransplantation was performed in bilaterally nephrectomized hosts. Compounds were given once daily by oral gavage. Monitoring was done by serum creatinine and urea, and rejection was concluded when values exceeded 500 micromol/L and 50 mmol/L, respectively (5-6 times the upper limit of reference values). Rejection was confirmed by graft histology. The termination point was set to 100 days after transplantation. In addition, animals were monitored for 24 hr drug concentrations and thorough inspection of potential adverse side effects. RESULTS: FTY720 given alone at 3.0 mg/kg per day prolonged rejection-free survival (33-85 days, mean 24 hr concentration between 54 and 66 ng/mL [n=3]), but it was not efficacious at a 0.3 mg/kg per day dose. For cyclosporine alone, 30 mg/kg per day during maintenance was efficacious (average concentration above 100 ng/mL, historical data from our group), and for RAD alone 0.75 mg/kg per day (concentration above 10 ng/mL). Efficacious FTY720-cyclosporine-A (CsA) or FTY720-RAD combinations were established using 0.1-0.3 mg/kg per day FTY720, 10-30 mg/kg per day cyclosporine, and/or 0.25-0.50 mg/kg per day RAD. Compared with single-compound treatment, FTY720 effective doses and 24 hr trough concentrations were at least tenfold lower in combination treatment and those of cyclosporine and RAD about twofold lower, indicative of effective synergy between the compounds. Already at the lowest FTY720 dose tested (0.03 mg/kg per day), there was a profound lymphocyte depletion down to about 30% of pretransplant values, which further increased at the highest dose (3.0 mg/kg per day, to about 14% of pretransplant values). Lymphocyte depletion was reflected by a decrease in T and B subpopulations. CONCLUSION: FTY720 is an effective immunosuppressant in prevention of acute kidney allograft rejection in cynomolgus monkeys and synergizes with cyclosporine and/or RAD in yielding rejection-free allograft survival.