17-丙烯胺基-17-去甲氧基格尔德霉素研究进展

17-丙烯胺基-17-去甲氧基格尔德霉素（17-AAG）是一种热休克蛋白90（HSP90）抑制剂,能够靶向性抑制肿瘤源性的HSP90,使其效应蛋白即多条细胞信号转导通路的关键调节蛋白降解,从而使细胞生长、分化受抑制,引起细胞凋亡,抑制肿瘤生长。大量试验证明,17-AAG是一种独特的靶向性治疗肿瘤的药物,能有效抑制多条维持肿瘤增殖和生存的细胞信号转导途径。临床Ⅰ-Ⅱ期试验显示该药具有良好的药理作用特征。此外,它还能和放疗、传统化疗药物联合应用协同发挥抗肿瘤作用。     

17-AAG及其衍生物治疗肺癌研究进展

17-丙烯胺基-17-去甲氧基格尔德霉素(17-AAG)及衍生物17-AAGH12、17-DMAG是热休克蛋白90竞争性抑制剂,能够降解多条信号传导通路关键蛋白,抑制肿瘤细胞生长,促使凋亡.临床Ⅰ～Ⅱ期研究证明,17-AAG及衍生物能够抑制T790M表达类型肺癌细胞,抑制肺癌细胞表皮生长因子受体,协同化疗药物细胞毒性、放射增敏作用.     

HSP90抑制剂17-AAG经下调HIF-1α抑制甲状腺未分化癌侵袭

目的：研究热休克蛋白90（heat shock protein 90, HSP90）抑制剂17-丙烯氨基-去甲氧基-格尔德霉素（17-AAG）对甲状腺未分化癌（ATC）侵袭的影响，并探究HSP90与低氧诱导因子-1α（HIF-1α）及其下游侵袭相关蛋白在ATC细胞中的分子机制。方法：本实验通过CCK8明确17-AAG对ATC细胞系8505c的药物IC50并确定进一步实验的药物浓度。通过Transwell实验检测17-AAG对ATC细胞侵袭能力的影响，并通过Western blot和蛋白质免疫共沉淀（CO-IP）探索潜在的作用机制。结果：CCK8提示经过不同浓度的17-AAG处理ATC细胞系后，17-AAG表现出对ATC生长有抑制作用，且有浓度依赖性。根据CCK8试验结果，选取浓度0.5μM和1μM作用24h用于后续试验。Transwell实验结果显示17-AAG对ATC细胞系8505c侵袭能力具有抑制作用，并且具有浓度依赖性。Western blot结果表明17-AAG使ATC细胞系HIF-1α与侵袭相关蛋白MMP2和VEGF的表达降低。CO-IP结果提示HIF-1α与HSP90在ATC细胞中形成蛋白质复合物。进一步通过挽救实验证明过表达HIF-1α后，MMP2和VEGF的表达水平升高，从而减弱了17-AAG对ATC细胞侵袭能力的抑制。结论：17-AAG可能通过调控HSP90与HIF-1α形成的复合物，使HIF-1α下调，导致侵袭相关蛋白MMP2和VEGF表达降低，来抑制ATC细胞的侵袭能力。     

卡马西平促进乳腺癌SKBR-3细胞中人表皮生长因子受体-2的降解

目的探讨卡马西平对乳腺癌细胞SKBR-3中人表皮生长因子受体-2(HER-2)表达的影响及其机制。方法分别用卡马西平、赫赛汀、17烯丙胺-17脱甲氧格尔德霉素(17-AAG)处理乳腺癌细胞SKBR-3,通过Western blot检测HER-2蛋白的表达;通过RT-PCR检测HER-2 mRNA的表达;通过免疫沉淀检测热休克蛋白90(HSP90)的分子伴侣功能及乙酰化水平;通过四甲基偶氮唑蓝(MTT)法检测细胞增殖活性。结果10μmol／L卡马西平处理即可降低乳腺癌细胞SKBR-3中HER-2蛋白水平,100μmoL／L卡马西平则在蛋白和mRNA水平均抑制HER-2的表达。卡马西平处理可以使HSP90乙酰化水平升高,并且破坏HSP90的分子伴侣功能。卡马西平对赫赛汀的促HER-2降解作用有协同作用。100μmol/L卡马西平+290μg/ml赫赛汀处理组、100μmol／L卡马西平+1μmol／L 17-AAG处理组的细胞增殖率分别为27．8％±3．0％和26．9％±2．8％,分别明显低于单独赫赛汀或17-AAG处理组(P均<0．01)。结论卡马西平可能通过破坏HSP90的分子伴侣功能、提高HSP90的乙酰化水平,降低了乳腺癌细胞SKBR-3中HER-2的表达水平,并且与赫赛汀和17-AAG有协同作用。卡马西平在肿瘤治疗中有潜在的应用价值。     

热休克蛋白90抑制剂对LoVo细胞生长及细胞周期的影响

背景与目的:热休克蛋白90(heat shock protein90,Hsp90)是体内重要的伴侣分子,体内许多重要的蛋白质是其底物蛋白质,但其本身不能降解底物蛋白,只能与底物相互作用,起到分子伴侣的作用.Hsp90抑制剂能抑制肿瘤细胞增殖,诱导肿瘤细胞凋亡,导致周期阻滞,破坏tfsp90与底物的结合及促进Hsp90底物蛋白质降解.为探讨Hsp90与生存素(Survivin)同时作为治疗靶点的效应及可能机制,本实验观察Hsp90抑制剂17-烯丙胺-17-脱甲氧格尔得霉素(17-AAG)对人结肠癌LoVo细胞增殖、细胞周期以及对周期相关蛋白Survivin水平的影响.方法:用四氮唑盐还原法(MTT法)检测17-AAG对LoVo细胞增殖影响;Propidium Iodide(PI)染色检测细胞周期变化;Western blot检测Hsp90底物蛋白Survivin的变化.结果:17-AAG呈时间-剂量-依赖性抑制LoVo细胞增殖.100、500、和800 ng/ml 17-AAG作用于LoVo细胞24 h后,细胞增殖抑制率分别为21.00%、40.81%、60.34%,作用48 h后,细胞增殖抑制率分别为27.29%、48.17%、80.97%,作用72 h,细胞增殖抑制率分别为34.45%、67.81%和88.42%.17-AAG导致LoVo细胞周期改变,100和500 ng/ml 17-AAG作用72 h,G0/G1期分别占(61±3)%及(74±3)%,LoVo细胞阻滞于G0/G1期,未加药处理的LoVo细胞G0/G1期比例仅为(48.2±0.8)%(P＜0.001).500 ng/ml 17-AAG作用于LoVo细胞72 h可部分清除Hsp90底物蛋白Survivin(P ＜0.001).结论:17-AAG呈时间-剂量依赖性抑制LoVo细胞增殖,阻滞LoVo细胞于G0/G1期,并能部分清除Survivin.     

药物17-AAG对人肺癌细胞系的放射敏感性影响

目的 研究17-AAG对人NSCLC细胞系A549的放射增敏作用并简析其可能机制。方法 MTT实验检测17-AAG对A549细胞的生长抑制作用。多靶单击拟合克隆存活曲线分析药物对细胞放射增敏作用。β-半乳糖苷酶染色实验观察药物对细胞衰老产生的影响。γ-H2AX免疫荧光染色分析药物对DNA损伤修复的影响。组间比较行成组t检验或方差分析。结果 17-AAG对A549细胞增殖抑制作用呈时间剂量依赖性(P=0.01~0.05)。与单纯照射组相比加药照射组克隆形成率降低,50 nmol/L时的放射增敏比为1.79(D₀值比)、2.30(D_q值比)。药物+4 GyX射线照射后72 h引发细胞衰老比例为30.48%,高于单纯用药组(9.18%,P=0.00)与单纯照射组(12.30%,P=0.00)。免疫荧光染色观察到17-AAG使DNA损伤修复延迟。结论 17-AAG对人肺癌A549细胞有生长抑制及放射增敏作用,其增敏机制可能为与引发细胞衰老和延迟DNA损伤修复有关。     

法尼基转移酶抑制剂在抗肿瘤和逆转肿瘤耐药中的作用

法尼基转移酶抑制剂(FTI)是一类靶向作用于肿瘤细胞增殖、细胞信号转导途径的新药.它通过抑制细胞内包括Ras在内的蛋白法尼基化修饰,诱导细胞凋亡及抑制血管生成从而发挥抗肿瘤作用;通过抑制P糖蛋白(Pgp)的功能来逆转肿瘤的多药耐药(MDR).     

热休克蛋白 90--癌症治疗的新靶点

热休克蛋白90(heat shock protein90,Hsp90)作为分子伴侣参与调控、维持细胞内多种蛋白的构象和功能,以帮助细胞在应激环境刺激下正常生长。近年来研究表明很多癌基因蛋白均为Hsp90的作用靶点,因此抑制Hsp90的功能将促进这些癌基因蛋白的降解,有助于癌症治疗。体内外实验也证实了Hsp90抑制剂的抗肿瘤活性,其中17-烯丙胺-17-脱甲氧格尔德霉素(17-allylamio-17-desmethoxygeldanamycin,17-AAG)正在进行临床试验。     

热休克蛋白90抑制剂研究进展

热休克蛋白90(HSP90)抑制剂能够抑制HSP90,促使在肿瘤生长信号通路中起重要作用的HSP90效应蛋白通过泛素化降解,从而阻断肿瘤增殖生长信号通路的多个靶点,有效阻止肿瘤的生长.现综述近年来有关HSP90抑制剂在肿瘤治疗方面的研究进展.     

Comparison of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) and 17-allylamino-17-demethoxygeldanamycin (17AAG) in vitro: effects on Hsp90 and client proteins in melanoma models

The heat shock protein Hsp90 is a potential target for drug discovery of novel anticancer agents. By affecting this protein, several cell signaling pathways may be simultaneously modulated. The geldanamycin analog 17AAG has been shown to inhibit Hsp90 and associated proteins. Its clinical use, however, is hampered by poor solubility and thus, difficulties in formulation. Therefore, a water-soluble derivative was desirable and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) is such a derivative. Studies were carried out in order to evaluate the activity and molecular mechanism(s) of 17DMAG in comparison with those of 17-allylamino-demethoxygeldanamycin (17AAG). 17DMAG was found to be more potent than 17AAG in a panel of 64 different patient-derived tumor explants studied in vitro in the clonogenic assay. The tumor types that responded best included mammary cancers (six of eight), head and neck cancers (two of two), sarcomas (four of four), pancreas carcinoma (two of three), colon tumors (four of eight for 17AAG and six of eight for 17DMAG), and melanoma (two of seven). Bioinformatic comparisons suggested that, while 17AAG and 17DMAG are likely to share the same mode(s) of action, there was very little similarity with standard anticancer agents. Using three permanent human melanoma cell lines with differing sensitivities to 17AAG and 17DMAG (MEXF 276L, MEXF 462NL and MEXF 514L), we found that Hsp90 protein was reduced following treatment at a concentration associated with total growth inhibition. The latter occurred in MEXF 276L cells only, which are most sensitive to both compounds. The depletion of Hsp90 was more pronounced in cells exposed to 17DMAG than in those treated with 17AAG. The reduction in Hsp90 was associated with the expression of erbB2 and erbB3 in MEXF 276L, while erbB2 and erbB3 were absent in the more resistant MEXF 462NL and MEXF 514L cells. Levels of known Hsp90 client proteins such as phosphorylated AKT followed by AKT, cyclin D1 preceding cdk4, and craf-1 declined as a result of drug treatment in all three melanoma cell lines. However, the duration of drug exposure needed to achieve these effects was variable. All cell lines showed increased expression of Hsp70 and activated cleavage of PARP. No change in PI3K expression was observed and all melanoma cells were found to harbor the activating V599E BRAF kinase mutation. The results of our in vitro studies are consistent with both 17AAG and 17DMAG acting via the same molecular mechanism, i.e. by modulating Hsp90 function. Since 17DMAG can be formulated in physiological aqueous solutions, the data reported here strongly support the development of 17DMAG as a more pharmaceutically practicable congener of 17AAG.     

In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative

Purpose To describe the preclinical basis for further development of 17-dimethyl aminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG, NSC 707545).Methods In vitro proliferation assays, and in vivo model studies in metastatic pancreatic carcinoma and subcutaneous xenograft melanoma and small-cell lung carcinoma models.Results 17-DMAG emerged from screening studies as a potent geldanamycin analog, with the average concentration inhibiting the growth of the NCI anticancer cell line drug screen by 50% being 0.053 M. Head to head comparison with 17-allylamino-17-demethoxygeldanamycin (17-AAG, NSC 330507) revealed 17-DMAG to possess potent activity against certain cell types, e.g., MDA-MB-231 breast carcinoma and HL60-TB leukemia which were relatively insensitive to 17-AAG. Evidence of oral bioavailability of 17-DMAG in a saline-based formulation prompted more detailed examination of its antitumor efficacy in vivo. 17-DMAG inhibited the growth of the AsPC-1 pancreatic carcinoma xenografts growing as intrahepatic metastases at doses of 6.7–10 mg/kg twice daily for 5 days administered orally under conditions where 17-AAG was without activity. 17-DMAG in an aqueous vehicle at 7.5–15 mg/kg per day for 3 days on days 1–3, 8–10 and 13–17, or 1–5 and 8–12 showed evidence of antitumor activity by the parenteral and oral routes in the MEXF 276 and MEXF 989 melanomas and by the parenteral route in the LXFA 629 and LXFS 650 adenocarcinoma and small-cell carcinoma models. The latter activity was comparable to the historical activity of 17-AAG.Conclusions Taken together, the in vivo activity of 17-DMAG supports the further development of this water-soluble and potentially orally administrable geldanamycin congener.     

Population pharmacokinetic analysis of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) in adult patients with solid tumors

Purpose

To identify sources of exposure variability for the tumor growth inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) using a population pharmacokinetic analysis.

Methods

A total 67 solid tumor patients at 2 centers were given 1 h infusions of 17-DMAG either as a single dose, daily for 3 days, or daily for 5 days. Blood samples were extensively collected and 17-DMAG plasma concentrations were measured by liquid chromatography/mass spectrometry. Population pharmacokinetic analysis of the 17-DMAG plasma concentration with time was performed using nonlinear mixed effect modeling to evaluate the effects of covariates, inter-individual variability, and between-occasion variability on model parameters using a stepwise forward addition then backward elimination modeling approach. The inter-individual exposure variability and the effects of between-occasion variability on exposure were assessed by simulating the 95 % prediction interval of the AUC per dose, AUC_0–24 h, using the final model and a model with no between-occasion variability, respectively, subject to the five day 17-DMAG infusion protocol with administrations of the median observed dose.

Results

A 3-compartment model with first order elimination (ADVAN11, TRANS4) and a proportional residual error, exponentiated inter-individual variability and between occasion variability on Q2 and V1 best described the 17-DMAG concentration data. No covariates were statistically significant. The simulated 95% prediction interval of the AUC_0–24 h for the median dose of 36 mg/m² was 1,059–9,007 mg/L h and the simulated 95 % prediction interval of the AUC_0–24 h considering the impact of between-occasion variability alone was 2,910–4,077 mg/L h.

Conclusions

Population pharmacokinetic analysis of 17-DMAG found no significant covariate effects and considerable inter-individual variability; this implies a wide range of exposures in the population and which may affect treatment outcome. Patients treated with 17-DMAG may require therapeutic drug monitoring which could help achieve more uniform exposure leading to safer and more effective therapy.     

Th17细胞及IL-17在肿瘤免疫中的作用

辅助性T细胞17( Th17)和白细胞介素-17(IL-17)在多种肿瘤组织中分布和表达,其在肿瘤发生发展过程中起抗肿瘤还是促肿瘤作用目前尚未定论,因而有必要进一步研究它们在肿瘤微环境中的作用,希望在肿瘤治疗过程中趋利避害,提高疗效.