17-AAG通过抑制Erk信号通路增强奥沙利铂诱导结肠癌细胞凋亡

目的 研究17-AAG与奥沙利铂是否存在协同效应,并探讨PI3K/Akt、Erk信号通路在奥沙利铂联合17-AAG诱导人结肠癌细胞凋亡过程中的作用。方法 四甲基偶氮唑盐比色实验（MTT法）检测17-AAG、奥沙利铂对细胞增殖的抑制作用;流式细胞术分析细胞凋亡情况;Western blot法检测相关蛋白的表达水平。结果 17-AAG能抑制RKO细胞的增殖;17-AAG联合奥沙利铂作用于RKO细胞24 h后,G₂/M期细胞比例及凋亡率明显增加;奥沙利铂抑制p-Akt、p-Erk的活化,上调Bax,下调Bcl-2蛋白表达,活化裂解Caspase-3,联合使用17-AAG后可进一步增强上述作用,但对PI3K/Akt信号通路影响不大。结论 本实验表明17-AAG具有增强奥沙利铂诱导结肠癌RKO细胞凋亡的作用,同时也说明17-AAG与奥沙利铂存在协同效应;17-AAG上调Bax,下调Bcl-2蛋白表达及增强奥沙利铂对Erk信号通路的抑制作用可能是其促进凋亡的重要机制之一。     

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。     

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。     

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.     

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-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。     

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阻止剂增强热诱导骨肉瘤细胞杀伤的研究

目的：探讨热休克蛋白90（heat shock protain 90,HSP90）分子伴侣复合物阻止剂17-丙烯胺基-17去甲氧基格尔德霉素（17-allylamide-17-demethoxgeldanamycin,17-AAG）,对热诱导的骨肉瘤SOSP-9607细胞杀伤作用的影响。方法：采用干细胞集落形成实验、流式细胞术（flow cytometry,FCM）和吖啶橙荧光染色等方法观察比较单纯热疗组与热疗联合17-AAG治疗组对骨肉瘤SOSP-9607细胞活力、凋亡率及形态学影响。结果：干细胞集落形成实验显示,热疗联合17-AAG治疗组较单独热疗对骨肉瘤SOSP-9607细胞的杀伤效应明显增强,P=0．036;Annexin V—FITC染色流式细胞术结果显示,热疗联合17-AAG治疗组所致凋亡率（52．2％）大于单纯热疗组（17．5％）;用吖啶橙荧光染色法观察到热疗联合17-AAG治疗组明显见骨肉瘤SOSP-9607细胞固缩着色不均而且较深,细胞核浓缩、裂解等凋亡特征。结论：HSP90分子伴侣复合物阻止剂17-AAG能增强热诱导的骨肉瘤SOSP-9607细胞的杀伤作用。     

miR-17-92基因簇反义寡核苷酸对胃癌细胞增殖与侵袭的抑制作用

目的：探讨miR-17-92基因簇反义寡核苷酸对人胃癌细胞株SGC-7901的增殖及侵袭的影响。方法：合成miR-17-92基因簇中miR-17、miR-18a、miR-20a的反义寡核苷酸,通过实时荧光定量PCR验证miR-17、miR-18a、miR-20a反义寡核苷酸对SGC-7901细胞的miR-17、miR-18a、miR-20a的抑制效果,然后利用MTT、Transwell检测抑制表达后对细胞增殖及侵袭的影响。结果：miR-17、miR-18a、miR-20a的反义寡核苷酸能够抑制对应miRNA的表达,同时抑制了胃癌细胞的增殖与侵袭。结论：miR-17-92基因簇可能在胃癌发生发展中起到了癌基因的作用。     

A limited sample model to predict area under the drug concentration curve for 17-(allylamino)-17-demethoxygeldanamycin and its active metabolite 17-(amino)-17-demethoxygeldanomycin

Purpose The Hsp90-directed anticancer agent 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) is currently undergoing phase I and phase II clinical investigation. Our goal was to develop a simple limited sampling model (LSM) for AUC of 17-AAG and its active metabolite, 17-(amino)-17-demethoxygeldanomycin (17-AG) using drug concentrations from a few time points. Methods Pharmacokinetic data from 34 patients treated at 11 dose levels on a Mayo Clinic Cancer Center phase I clinical trial of 17-AAG was utilized. Blood samples were collected at 11 different time points, spanning 25 h. Graphical methods and correlations were used to assess functional forms and univariate relationships. Multivariate linear regression and bootstrap resampling were used to develop the LSM. Results Using log-transformed data, the two and three time point 17-AAG LSMs are log-AUC (17-AAG) = 0.869 + 0.653*(C_55min) +0.469*(C_5h) and log-AUC (17-AAG) = 2.449 + 0.400*(C_55min) +0.441*(C_5h) +0.142*(C_9h). The two and three time point LSMs for 17-AG are log-AUC (17-AG) = 3.590 + 0.747*(C_5h) +0.169*(C_17h), and log-AUC (17-AG) = 3.797 + 0.650*(C_5h) +0.111*(C_9h) +0.122*(C_17h). Ninety-seven percent and 94% of the predicted log-AUC values were within 5% of the observed log-AUC for the two and three time point models for 17-AAG and 17-AG respectively. Conclusions The precise calculation of AUC is cumbersome and expensive in terms of patient and clinical resources. The LSM developed using a multivariate regression approach is clinically and statistically meaningful. Prospective validation is underway.     

Efficacy of the HSP90 inhibitor 17-AAG in human glioma cell lines and tumorigenic glioma stem cells

Glioblastoma multiforme (GBM) arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Studies to date with single-agent targeted molecular therapy have revealed only modest effects in attenuating the growth of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat-shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a defined group of client proteins, many of which are deregulated in GBM. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well-characterized HSP90 inhibitor that should be able to target many of the aberrant signal transduction pathways in GBM. We assessed the ability of 17-AAG to inhibit the growth of glioma cell lines and glioma stem cells both in vitro and in vivo and assessed its ability to synergize with radiation and/or temozolomide, the standard therapies for GBM. Our results reveal that 17-AAG is able to inhibit the growth of both human glioma cell lines and glioma stem cells in vitro and is able to target the appropriate proteins within these cells. In addition, 17-AAG can inhibit the growth of intracranial tumors and can synergize with radiation both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide in any of our models of gliomas. Our results suggest that HSP90 inhibitors like 17-AAG may have therapeutic potential in GBM, either as a single agent or in combination with radiation.     

An in vitro and in vivo study of the combination of the heat shock protein inhibitor 17-allylamino-17-demethoxygeldanamycin and carboplatin in human ovarian cancer models

PURPOSE: To study the interactions of the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) and carboplatin in vitro and in vivo. EXPERIMENTAL DESIGN: The combination of 17-AAG and carboplatin on the growth inhibition of A2780, SKOV-3, IGROV-1 and HX62 human ovarian cancer cells was studied in vitro by MTT assays. The effect of the sequence of administration of both drugs was further investigated in A2780 cells by sulforhodamine B assays. The ability of 17-AAG to deplete HSP90 client proteins either alone or in combination with carboplatin was evaluated by western blotting. Tumor concentrations of 17-AAG and carboplatin alone or in combination in vivo were determined by validated liquid chromatography with ultraviolet detection and atomic absorption spectroscopy methods. The growth inhibitory effects of 17-AAG, carboplatin and the combination were studied in the A2780 xenograft model. RESULTS: The combination index (CI) at fu(0.5) for 17-AAG plus carboplatin was 0.97 (+/-0.12 SD) when A2780 cells were exposed to carboplatin followed by 17-AAG indicating additivity. The addition of carboplatin did not alter the ability of 17-AAG to cause C-RAF, CDK4 and p-AKT depletion or HSP70 induction. Tumor 17-AAG and carboplatin concentrations were not significantly different in the single agent and combination arms. Tumor weights relative to controls on day 6 (T/C) were 67% for the carboplatin, 64% for the 17-AAG and 22% for the combination. CONCLUSION: In the specified sequences of drug exposure, 17-AAG and carboplatin have additive growth inhibitory effects in vitro and beneficial effects were seen with the combination in vivo. These findings form the basis for the possible evaluation of 17-AAG and carboplatin in a clinical trial.     

Synergistic inhibition of the growth of MDA-MB-231 cells in triple-negative breast cancer by salinomycin combined with 17-AAG and its mechanism

Salinomycin (SAL), a typical ion carrier antibiotic, inhibits tumor growth and metastasis by inducing apoptosis or autophagy in cancer or cancer stem cells and thus overcomes drug resistance. 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein Hsp90 competitive inhibitor, also has a role in inhibiting tumor development. However, their combination on the growth of breast cancer cells and its specific mechanism remains to be elucidated. The present study tested the influence of SAL and 17-AAG on cell growth, apoptosis and autophagy by MTT assays, Annexin V-FITC and propidium iodide double staining assay and immunoelectron microscopy. The influence of SAL and 17-AAG on proteomics was investigated by isobaric tag for relative and absolute quantitation. It was found that SAL combined with 17-AAG synergistically inhibited the cell growth and induced the apoptosis in a concentration-dependent manner, with the expression of caspase 3 and Bcl-2 were decreased while the expression of Bax was increased. In addition, SAL combined with 17-AAG inhibited autophagy, with the expression of microtubule-associated protein 1 light chain 3, Beclin1, p62 being decreased. Mechanistically, SAL combined with 17-AAG synergistically inhibited the reactive oxygen species/JNK signaling pathway. In conclusion, SAL combined with 17-AAG had a synergistic inhibitory effect on cell growth of breast cancer via inducing apoptosis and inhibiting autophagy. The present study might provide a new strategy for potential clinical application of SAL as a new anti-tumor drug especially as a drug combination with other molecular targeting therapeutics.     

The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin modulates radiosensitivity by downregulating serine/threonine kinase 38 via Sp1 inhibition

The ansamycin-based HSP90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin) combats tumors and has been shown to modulate cellular sensitivity to radiation, prompting researchers to use 17-AAG as a radiosensitizer. 17-AAG causes the degradation of several oncogenic and signaling proteins. We previously demonstrated that oxidative stress activates serine/threonine kinase 38 (STK38), a member of the protein kinase A (PKA)/PKG/PKC-like family. In the present study, we investigated how 17-AAG affects STK38 expression, and evaluated STK38’s role in the regulation of radiosensitivity. We found that 17-AAG depleted cellular STK38 and reduced STK38’s kinase activity. Importantly, 17-AAG downregulated the stk38 gene expression. Deletion analysis and site-directed mutagenesis experiments demonstrated that Sp1 was required for the stk38 promoter activity. Treatment with 17-AAG inhibited Sp1’s binding to the stk38 promoter by decreasing the amount of Sp1 and knocking down Sp1 reduced STK38 expression. Moreover, 17-AAG treatment or STK38 knockdown enhanced the radiosensitivity of HeLa cells. Our data provide a novel mechanism, mediated by stk38 downregulation, by which 17-AAG radiosensitizes cells.     

Cooperative inhibitory effect of ZD1839 (Iressa) in combination with 17-AAG on glioma cell growth

ZD1839 ("Iressa") is an orally active, selective epidermal growth factor (EGF) receptor-tyrosine kinase inhibitor. We evaluated the antitumor activity of ZD1839 in combination with HSP90 antagonist, 17-AAG in malignant human glioma cell lines. ZD1839 independently produced a dose-dependent inhibition of cellular proliferation in glioma cells grown in culture with time- and dose-dependent accumulation of cells in G(1) phase of the cell cycle on flow cytometric analysis, although the concentrations required for optimal efficacy were at or above the limits of clinically achievable levels. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the HSP90 inhibitor 17-AAG would potentiate ZD 1839-mediated glioma cytotoxicity by decreasing the activation status of EGF receptor, as well as down regulating the levels of other relevant signaling effectors. We, therefore, examined the effects of ZD1839 and 17-AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death and quantitative analysis revealed that interaction between ZD1839 and 17-AAG-induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase-3 and PARP cleavage. No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of EGFR receptor, Raf-1 and mitogen activated protein kinase (MAPK). Cells exposed to 17-AAG and ZD1839 displayed a significant reduction in cell cycle regulatory proteins, such as CDK4 and CDK6. Taken together, these findings suggest that ZD1839, an EGF receptor tyrosine kinase inhibitor, plays a critical role in regulating the apoptotic response to 17-AAG and that multi-site targeting of growth signaling and cell survival pathways could provide a potent strategy to treat patients with malignant gliomas.     

Preclinical toxicity of a geldanamycin analog, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), in rats and dogs: potential clinical relevance

Purpose 17-DMAG is a hydrophilic derivative of the molecular chaperone inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG; NSC-330507), which is currently being evaluated for the treatment of cancer in clinical trials. 17-DMAG offers a potential advantage over 17-AAG because its aqueous solubility eliminates the need for complicated formulations that are currently used for administration of 17-AAG. In addition, 17-DMAG undergoes only limited metabolism compared to 17-AAG. The present results are from preclinical toxicity studies evaluating 17-DMAG in rats and dogs.Methods Doses of 0, 2.4, 12 and 24 mg/m² per day were administered to rats, while dogs received doses of 0, 8 or 16 mg/m² per day. In both species, 17-DMAG was administered i.v. (slow bolus for rats; 1-h infusion for dogs) daily for 5 days. An additional cohort of dogs received 16 mg/m² per day orally for 5 days. Clinical observations were noted, and standard hematology and clinical chemistry parameters were monitored. Selected tissues were evaluated microscopically for drug-related lesions. Tissue and plasma 17-DMAG concentrations were measured by HPLC/MS at selected time-points on days 1 and 5.Results Daily i.v. administration of 17-DMAG at doses of 24 mg/m² per day in rats or 16 mg/m² per day in dogs produced lethality on day 6, approximately 24 h following the last dose. Body weight loss was common in rats and dogs. Drug-related gastrointestinal, bone marrow and hepatic toxicities were also common in rats and dogs. Dogs also exhibited signs of renal and gallbladder toxicity. Plasma concentrations of 17-DMAG increased proportionately with dose in rats and disproportionately with dose in dogs. In rat tissues, however, only fourfold to sixfold increases in 17-DMAG concentrations were observed with a tenfold increase in dose. The highest concentrations of 17-DMAG were found in the liver of rats, with progressively lower concentrations in the spleen, lung, kidney and plasma. Regardless of the route of administration, higher drug concentrations were present in plasma (rat and dog) and tissue (rat) samples obtained on day 5 compared to those obtained on day 1. Although plasma concentrations decreased with time, 17-DMAG was still detected in dog plasma for at least 24 h after drug administration.Conclusions With the recent approval of 17-DMAG for clinical use, the data generated from these preclinical studies will provide guidance to clinicians as they administer this drug to their patients. The MTD of 17-DMAG was 12 mg/m² per day in rats and 8 mg/m² per day in dogs; therefore, the recommended starting dose for phase I trial is 1.3 mg/m² per day for 5 days. Gastrointestinal and bone marrow toxicity were dose-limiting in rats, and gastrointestinal, renal, gallbladder and bone marrow toxicity were dose-limiting in dogs. All adverse effects were fully reversible in surviving animals after treatment was complete.E.R. Glaze and A.L. Lambert contributed equally to this work.