Tesis sobre el tema "Geldanamycin"

Geldanamycin possesses various biological activities as seen in the NCI 60 cell line panel (13 nM avg., 70 nM SKBr-3 cells). The predominant mode of action providing these unique results arises from the ability of geldanamycin (GA) to bind to the chaperone heat shock protein 90 (Hsp90). Despite its complicated functionality, the first total synthesis of GA was accomplished, which included two new reactions developed specifically to address the stereochemical features. The final step in the synthesis of GA was a demethylation-oxidation sequence to generate the desired para-quinone. This step could only be accomplished with HNO3/AcOH, producing GA in 5% yield. A GA model study, which closely resembled the aromatic core, was extensively investigated to solve this critical oxidation issue. A protected hydroquinone model compound was determined to be the optimum choice. Using Pd in the presence of air with a 1,4-hydroquinone provided the desired para-quinone quickly and nearly quantitatively in 98% yield. This study formulated the recipe of success for para-quinone formation of GA and future synthetic analogs. Asymmetric glycolate alkylation has been developed using phase-transfer-catalysis (PTC). Diphenylmethoxy-2,5-dimethoxyacetophenone with trifluorobenzyl cinchonidinium catalyst and cesium hydroxide provided alkylation products at —35 °C in high yield (80-99%) and with excellent enantioselectivity (up to 90% ee). Useful α-hydroxy products were obtained using bis-TMS peroxide Baeyer—Villiger conditions and selective transesterification. The intermediate aryl esters can be obtained with >99% ee after a single recrystallization. The newly developed PTC glycolate alkylation was applied to the asymmetric syntheses of ragaglitazar and kurasoin A. Ragaglitazar is a potent antihyperglycemic and lipid modulator, currently in phase II clinical trials. Kurasoin A is a relatively potent protein farnesyltransferase (PFTase) inhibitor with an IC50 value of 59.0 micromolar. PTC glycolate alkylation was optimized to provide 4-benzyloxy glycolate intermediates in excellent overall yield and with 96% ee after recrystallization. Ragaglitazar was then synthesized after considerable experimentation to provide the potent lipid modulator with yields and enantiopurity rivaling the best-known routes produced by industry standards. Kurasoin A was produced through an α-triethylsiloxy Weinreb amide to provide the highest overall yielding route to this PFTase inhibitor currently disclosed.

Total Synthesis of 4'-ester Resveratrol Analogs and 8, 9-amido Geldanamycin Analog and toward the Total Synthesis of (-)-Englerin A Yong Wang Department of Chemistry and Biochemistry, BYU Doctor of Philosophy The phytoalexin resveratrol and its 4'-ester analogs have been prepared with a decarbonylative Heck reaction. The deprotecting step has been modified and improved to increase yield and avoid chromatography. A set of resveratrol analogs and resveratrol have been tested with melanoma and pancreatic cell assays. The 8, 9-amido Geldanamycin analog has been synthesized with a convergent route, involving 28 simplified steps in its longest linear sequence. Synthetic methodologies, such as Andrus auxiliary controlled asymmetric anti-glycolate Aldol and selective p-Quinone formation, were employed. The total synthesis of Englerin A starts from (R)-carvone, passed through the modified Farvoskii ring-contraction and ring closing metathesis to get the ring skeleton. Other routes involving isopropyl group installation before closure of the seven-member ring failed. Although there are still problems to build the isopropyl moiety and the bridged ether, several reasonable alternative routes to address the problems have been designed.

The phytoalexin resveratrol and its acetyl analogs have been made using a decarbonylative Heck reaction. The acid chloride derived from 3,5-dihydroxybenzoic acid was coupled with suitable protected 4-hydroxystyrene in the presence of palladium acetate and N,N-bis-(2,6-diisopropylphenyl)-4,5-dihydro imidazolium chloride to give the substituted stilbene in good yield as the key step. Human HL-60 cell assays showed the 4'-acetyl resveratrol variant improved activity (ED50 17 μM) relative to resveratrol (24 μM). Cinchona phase-transfer catalysts (PTC) were developed for glycolate aldol reactions to give differentially protected 1,2-diol products. Silyl enol ether of diphenylmethoxy-2,5-dimethoxyacetophenone reacted to generate benzhydryl-protected products. O-Allyl trifluorobenzyl cinchonium hydrodifluoride (20 mol %) catalyzed the addition of the silyl enol ether to benzaldehyde to give aldol product as a single syn-product in 76% yield and 80% ee. Recrystallization enriched the product to 95% ee, and a Baeyer-Villiger reaction transformed the product into useful ester intermediates. A novel unnatural product, 8,9-Methylamido-Geldanamycin, has been designed and synthesized. Using a convergent route, the total synthesis of the molecule involved only 27 longest linear steps. New synthesis methodologies, including auxiliary controlled asymmetric anti-glycolate aldol, syn-norephedrine aldol, and selective p-quinone formation, were used.

Les protéines de choc thermique sont essentielles au maintien de l’activité cellulaire, par leur implication dans la stabilisation de leurs protéines clientes. En raison de ce rôle clef, elles sont directement impliquées dans de nombreuses affections comme le cancer, le diabète et les processus inflammatoires. L’identification de plusieurs inhibiteurs de la protéine de choc thermique de 90kDa (HSP90), par un mode d’action unique (inhibition de la poche ATPase) permis le développement d’une nouvelle classe d’anticancéreux. La geldanamycin et le radicicol, deux composés naturels ont Eté instrumentalisés dans ces découvertes au début des années 1990. Aujourd’hui, les nombreuses recherches ont permis d’identifier de nouvelles classes de molécules. 10 molécules sont maintenant en développement clinique. Au début de ma thèse, la pochonine D avait déjà été identifié dans le laboratoire comme un analogue simplifié du radicicol, récapitulant son activité sans ses défauts. De premières études de structure-activité permirent d’identifier les pochoximes A-C, analogues oxime de la pochonine D. Ils démontrèrent une activité biologique in vivo très attractive, avec la régression de tumeurs sur des modèles animaux traités. De nouvelles études de structure-activité permirent l’identification de l’analogue le plus actif à ce jour, la 6-hydroxypochoxime C. Des modélisations numériques effectuées en collaboration avec le Pr KARPLUS et l’obtention de la structure cristalline de la pochoxime avec HSP90 ont montrées la reconformation de la poche ATPase pour accepter la molécule. La synthèse de pochoxime taguées avec une étiquette de PNA devrait permettre des screening plus large
Heat Shock Proteins (HSPs) are essential components for maintaining the correct folding, function and stability of client proteins, and to avoid aggregate formation. Given this ubiquitous role, they are directly involved in numerous diseases including cancer, diabetes and inflammation. Identification of several specific HSP90 (heat shock proteins of 90 kDa) inhibitors, through a unique mode of action (ATPase inhibition) paved the way to a potential new class of anticancer agents. Two natural products were instrumental in this endeavor: geldanamycin, a natural ansamycin, and radicicol, a 14-membered resorcylic acid lactone (RAL). Intensive research resulted in the identification of new class of inhibitor of HSP90, and 10 molecules are now in preclinical and clinical development. At the beginning of my PhD, pochonin D was identified as a simplified HSP90 inhibitor. Pochonin D proved to recapitulate radicicol properties without its drawback. First structure-activity relationships were achieved. Having in mind these preliminary results, oxime analogues of pochonin D were synthesised, yielding to the discovery of the pochoxime A-C analogues. They demonstrated very attractive biological activity in vivo, resulting in a tumor regression in treated animals. Structure-activity relationship studies resulted in the discovery of the most potent compound reported to date, 6-hydroxypochoxime C. Computer assisted modelling done in collaboration with Professor KARPLUS and a crystal structure of pochoxime with HSP90 revealed a refolding of the ATPase pocket to fit the drug. The synthesis of a PNA labelled library will allow a broad screening in order to expand the scope of pochoximes

Heat shock protein 90 (HSP90) is a molecular chaperone protein that protects proteins from degradation, repairs damaged proteins, and assists proteins in carrying out their functions. HSP90 has hundreds of clients, many of which are inflammatory signaling kinases. The mechanism by which HSP90 enables inflammatory pathways is an active area of investigation. The HSP90 inhibitors such as geldanamycin (GA) and its derivative 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) have been shown to reduce inflammation. It was hypothesized that inhibiting HSP90 would reduce inflammatory signal cascade levels. To test this, J774 mouse macrophage cells were treated with 17-DMAG and immune-stimulated with lipopolysaccharide (LPS). 17-DMAG treatment reduced nitric oxide (NO) production and the expression of pro-inflammatory cytokines interleukin (IL)-6, IL-12, and TNF-α. Inhibition of HSP90 also prevented nuclear translocation of NF-κB. To investigate the anti-inflammatory effects of HSP90 inhibition in vivo, MRL/lpr lupus mice were administered 5 mg/kg 17-DMAG for six weeks via intraperitoneal injection. Mice treated with 17-DMAG were found to have reduced proteinuria and reduced splenomegaly. Flow cytometric analysis of splenocytes showed that 17-DMAG decreased double negative T (DNT) cells. Renal expression of HSP90 was also measured and found to be increased in MRL/lpr mice that did not receive 17-DMAG. The mechanistic interactions between HSP90 and the pro-inflammatory nuclear factor-κB (NF-κB) pathway were studied and a computational model was developed. The model predicts cellular response of inhibitor of κB kinase (IKK) activation and NF-κB activation to LPS stimulation. Model parameters were fit to IKK activation data. Parameter sensitivity was assessed through simulation studies and showed a strong dependence on IKK-HSP90 binding. The model also accounts for the effect of a general HSP90 inhibitor to disrupt the IKK-HSP90 interaction for reduced activation of NF-κB. Model simulations were validated with experimental data. In conclusion, HSP90 facilitates inflammation through multiple signal pathways including Akt and IKK. Inhibition of HSP90 by 17-DMAG reduced disease in the MRL/lpr lupus mouse model. A computational model supported the hypothesis that HSP90 is required for IKK to activate the NF-κB pathway. Taken together, HSP90 is a prime target for therapeutic regulation of many inflammatory processes and warrants further study to understand its mechanism of regulating cell signaling cascades.
Ph. D.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

碩士
國立清華大學
生命科學系
90
Capacitation and acrosome reaction are of fundamental importance in the fertilization of oocyte by spermatozoon. The process of capacitation consists of a series of functional and biophysical modifications that render the ejaculated spermatozoa competent for fertilization of the oocyte. Nitric oxide (NO) has been shown to have multifunction in male reproduction system. The objectives of the present study were to investigate whether boar spermatozoa can generate NO and the effects of NO and heat-shock protein 90 (HSP90) on the capacitation of boar spermatozoa. The major results were as follows: Boar spermatozoa possess endothelial nitric oxide synthase (eNOS) in the acrosome and middle piece, and spermatozoa can generate NO. Under noncapacitating conditions, spermatozoa produced low levels of NO. However, there was an up to 2-fold increase of NO generation under capacitating conditions (P<0.001). When boar spermatozoa were incubated with capacitation medium in the presence of NOS inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) or geldanamycin (GA) for 4 h, L-NAME treatment reduced 30% to 40% of NO production in capacitating spermatozoa (P<0.05). However, GA promoted 23% to 75% of NO production in a dose-dependent manner (P<0.05). When spermatozoa were capacitated in the presence or absence of NO-releasing compound (SNP), L-NAME or GA, and then the percentage of acrosome reaction induced by calcium ionophore (A23187) was determined. The results indicated that L-NAME decreased the percentage of acrosome reaction, whereas SNP and GA increased the percentage of acrosome reaction (P<0.05). Since GA treatment did not decrease but increase NO production as expected, HSP90 may not directly involve in regulating NOS activity during boar spermatozoa capacitation in vitro. On the other hand, the phenomenon of increasing NO production is similar to the effect of A23187, we suggest that acrosome reaction induced by GA may be triggered the increase of intracellular calcium concentration and NO production.

碩士
國立清華大學
生命科學系
89
Geldanamycin (GA), a benzoquinone ansamycin, is an antitumor drug which originally described as a tyrosine kinase inhibitor. Herein, we show that exposure of 9L rat brain tumor cells to 5 mM GA for different time durations induces reorganization of cytoskeleton¸ G2/M arrest and apoptosis. In our previous studies, we showed that the intracellular level of reactive oxygen species (ROS) is significantly increased in GA-treated cells. By using confocal microscopy we demonstrated for the first time that after treatment of GA the structure of cytoskeleton in 9L RBT cells was dramatically reorganized and disrupted, and this phenomenon was not reversible by removal of GA and allowed for recovery. Using flow cytometry we showed that after GA treatment significant percentage of cells were arrested in G2/M phase and subdiploid apoptotic status. To investigate whether ROS involved in the aforementioned phenomenon, N-acetylcysteine (NAC), a free radical scavenger, was added prior to GA treatment. We found that pretreatment of NAC could partially prevent GA-induced irreversible reorganization of cytoskeleton and cell arrest into G2/M phase suggesting that the oxidative stress was exerted by GA treatment and that part of the GA-induced ROS occurred upstream of reorganization of the cytoskeleton, cell cycle G2/M arrest and/or apoptosis. We conclude that GA-induced apoptosis, cytoskeletal disruption, and G2/M arrest are, in part, a consequence of a critical GA-induced ROS in 9L RBT cells. ABSTRACT (Chinese)………………………………………………………1 ABSTRACT ……………………………………………………………….2 INTRODUCTION…………………………………………………………..3 MATERIALS AND METHODS……………………………………………….5 RESULTS………………………………………………………………….7 DISCUSSION……………………………………………………………..10 REFERENCES……………………………………………………………..13 FIGURE LEGENDS………………………………………………………..21 FIGURES………………………………………………………………………23

Fibronectin is an extracellular matrix glycoprotein with key roles in cell adhesion and migration. Hsp90 binds directly to fibronectin and Hsp90 depletion regulates fibronectin matrix stability. Where inhibition of Hsp90 with a C-terminal inhibitor, novobiocin, reduced the fibronectin matrix, treatment with an N-terminal inhibitor, geldanamycin, increased fibronectin levels. Geldanamycin treatment induced a stress response and a strong dose and time dependent increase in fibronectin mRNA via activation of the fibronectin promoter. Three putative heat shock elements (HSEs) were identified in the fibronectin promoter. Loss of two of these HSEs reduced both basal and geldanamycin-induced promoter activity, as did inhibition of the stress-responsive transcription factor HSF1. Binding of HSF1 to one of the putative HSE was confirmed by ChIP under basal conditions, and occupancy shown to increase with geldanamycin treatment. These data support the hypothesis that fibronectin is stress-responsive and a functional HSF1 target gene. COLA42 and LAMB3 mRNA levels were also increased with geldanamycin indicating that regulation of extracellular matrix (ECM) genes by HSF1 may be a wider phenomenon. Taken together, these data have implications for our understanding of ECM dynamics in stress-related diseases in which HSF1 is activated, and where the clinical application of N-terminal Hsp90 inhibitors is intended.

博士
國立清華大學
生命科學系
94
Experiments in this thesis were designed to investigate the upstream signaling of GA-induced stress response and elucidate the inductive mechanisms of Hsp90 upon GA treatment. Geldanamycin (GA), an ansamycin-derivative benzoquinone compound, was originally isolated as a natural product with anti-fungal activity. GA specifically inhibits the essential ATPase activity of Hsp90 results in inactivation, destabilization, and degradation of Hsp90 client proteins, including a wide variety of signal-transducing proteins that regulate cell growth and differentiation, such as protein kinases and steroid hormone receptors. Recently, several reports have shown that treatment with GA induces a heat-shock response and intracellular calcium may involve in the process. In this study, we found changes in upstream signaling mediators, including HSF1 and calcium, as well as possible involvement of protein kinase in human non-small cell lung cancer H460 cells treated with Hsp90 binding agents (i.e., GA and radicicol). We further found that GA was able to provoke a rapid calcium influx and thereby resulted in an instant induction of Hsp70. Our results demonstrated that calcium mobilization, a calcium dependent and H7-sensitive protein kinase, along with HSF1 activation by phosphorylation, were all involved in the Hsp70 induction process triggered by the drug. On the other hand, treatment of cells with GA also induced the expression level of Hsp90 other than Hsp70. In mammals, two major cytoplasmic isoforms of Hsp90, known as Hsp90alpha and Hsp90beta, have been identified and found to be highly conserved among different species. In order to examine the expression of Hsp90 isoforms, the levels of mRNA and nascent protein of Hsp90alpha and Hsp90beta in GA-treated rat brain tumor 9L cells were quantitatively examined using real-time qPCR and metabolic labeling analyses. It is found that Hsp90alpha was more inducible compared to Hsp90beta after GA treatment. Interestingly, the hsp90alpha mRNA was, on the contrary, lower than that of hsp90beta. The relative mRNA level of hsp90beta was respectively about 8.8-fold and 3.7-fold that of hsp90alpha under normal condition and upon GA treatment. Furthermore, we examine whether distinct expression of Hsp90alpha and�nHsp90beta is a result of post-transcriptional regulation. The translational efficiency of Hsp90 isoforms employing in vitro translation was further measured. We found that hsp90alpha mRNA revealed highly effective translation. The translational efficiency of hsp90alpha mRNA was respectively about 8.9-fold and 5.8-fold that of hsp90beta under normal condition and upon GA treatment. Our results indicate that differential expression of Hsp90alpha and Hsp90beta is a consequence involving both the distinct mRNA profiles and the differential translation processes. In conclusion, treatment with GA was able to activate HSF1 and bring about the induction of Hsp70. Intracellular calcium mobilization and related protein kinase activities are involved in signaling for GA. The primary effect of GA on expression of Hsp90 isoforms is according to promote the transcription of both hsp90 genes. The differential expression of�nHsp90alpha and�nHsp90beta is a consequence of both distinct mRNA profiles and differential translation processes.

碩士
國立清華大學
生命科學系
89
In the previous study, it has been found that the syntheses of the 78kDa glucose-regulated protein (GRP78) were vigorously induced by stresses, such as A23187, thapsigargin (TG) and geldanamycin (GA) in 9L rat brain tumor (RBT) cell line. In this study, we have found that an 8h treatment with either 2 μM A23187 or 300 nM TG caused the redistribution of GRP78 in 9L RBT cells. GRP78 exhibited the pattern outside the ER when respectively treated with A23187 and TG, but this phenomenon was less obvious after 5 μM GA treatment for 8h. Although GRP78 are primarily localized in the endoplasmic reticulum (ER), subcellular fractionation study showed that the stress-induced GRP78 was in the mitochondria-enriched fraction in from 9L RBT cells. Furthermore, confocal microscopic and immunogold electron microscopic studies confirmed that the stress-induced GRP78 was indeed localized in mitochondria. These results indicate that GRP78 is not present exclusively in the ER after stressed. This is the first report demonstrating that GRP78 is localized in mitochondria in 9L RBT cells after being exposed to GRP78-inducing stresses.

碩士
國立清華大學
生命科學系
90
Geldanamycin (GA), a benzoquinone ansamycin, is an inhibitor of HSP90 and has been implicated as a potent anti-cancer drug. Previous studies reported that endoplasmic reticulum stress (ER stress) was evoked with GA, and in our laboratory reactive oxygen species (ROS) was found to play important signal transduction roles. Glucose regulated protein 78 (GRP78) is mainly located in lumen of ER and has chaperone function to assist new synthesized proteins folding to correct conformations, and it was induced in response to ER stress. In this study, we investigated the roles of calcium, ROS and their interrelationship in GA-induced GRP78 expression in 9L rat brain tumor (RBT) cells. The induction of GRP78 by GA requires intact process of protein synthesis and is regulated mainly in transcription level. By monitoring cytoplasmic calcium contents with fluorescence microscopy, the calcium signaling evoked by GA was influenced by depletion of organelle-stored calcium or by preventing the influx of calcium across the plasma membrane. Chelation of intracellular calcium with 1,2-bis(2-amino- phenoxy)ethane-N,N,N’,N’,-tetraacetic acid (BAPTA/AM) or extracellular calcium with ethylene glyco-bis(β-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA) reduces GRP78 induction in GA-treated 9L RBT cells, implying that calcium signaling is required for GRP78 induction. The compound 1-96-[17beta- 3-methoxyestra-1,3,5(10)-trien-17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122) decreases the GA-induced GRP78 expression, suggesting that this calcium mobilization might be dependent on phospholipase C. To assess the possible kinases involved, inhibitors were used and coupled with calcium monitoring. We found that the increase of cytoplasmic calcium concentration in GA signal transduction pathway may act through protein kinase C (PKC) and the specific subtype involved may be PKCβII. Significant reduction of GRP78 induction was also evident when genistein was applied, indicating the involvement of protein tyrosine kinase. On the other hand, antioxidants prevent GRP78 induction by GA, implying ROS generation in vivo under GA treatment. To distinguish the contribution of diverse chemical species of ROS, our results indicate that GA-induced GRP78 expression was prominently blocked by antioxidants specific for hydroxyl radical. To delineate the causal effects of these intermediates, we show that increase in cytoplasmic calcium concentration activating PKCbII precedes the generation of ROS. In conclusion, we investigated the regulation of inductive expression of GRP78 in 9L RBT cells by GA treatment and identified that this signal transduction pathway is acting through calcium, PLC, PKCβII, and protein tyrosine kinase and then driving the ROS generation to concert this induction.

博士
國立清華大學
生命科學系
90
In a previous study (Lee et al., 1996), we found that the expression of HSP90 in cardiac tissue was substantially reduced in sudden-death pigs with hypertrophic cardiomyopathy (HCM). This was the first evidence correlating a reduced HSP90 level in ventricular tissue with cardiac arrest in HCM pigs. To realize the biochemical characterization, we cloned porcine hsp90 cDNA and determined its heat-inducibility. In addition, we further purified HSP90 to test its phosphorylation ability and using geldanamycin (GA), an inhibitor of HSP90, to study if GA indeed blocks HSP90 function in vitro and in vivo. We have isolated and sequenced cDNA clones encoding a 90-kDa heat shock protein (HSP90) from a porcine brain cDNA library. The sequence of the 2,202- nucleotide coding region showed 88.6% homology with that of the human homologue. Moreover, the deduced amino acid sequence of the porcine hsp90 cDNA was 99.7% identical to that of the human counterpart, with differences of only three amino acids in a total of 733 residues. Expression of the gene was greatly elevated in cultured cells during recovery from heat shock treatment at 45℃ for 60 min. Three major transcripts with 2.2, 3.0, and 4.1 kb in size were detected by Northern blot hybridization. These transcripts were further identified in a whole pig hyperthermia experiment. These three hsp90 transcripts were constitutively expressed in porcine tissues including kidney, liver, brain, and heart, and their levels were markedly enhanced during recovery from 30 min hyperthermia treatment at 43℃. Furthermore, we found that HSP90 was preferentially expressed in pituitary gland, brain, adrenal gland, and testis, in comparison to the other tissues. Therefore, we purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90a. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [γ-32P]ATP.Mg2+ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [γ-32P]ATP.Mg2+ alone but could be phosphorylated by various protein kinases. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3α. Geldanamycin (GA) is HSP90-specific inhibitor, which binds the ATP-binding site of HSP90. Therefore, we test if GA could inhibit the phosphorylation of HA-HSP90. The result showed that GA did not inhibit the phosphorylation of HA-HSP90. However, heparin severely inhibited the phosphorylation. This result implied that the phosphorylation of HA-HSP90 was due to the associated CKII. Moreover, high temperatures almost completely inhibited the phosphorylation of HA-HSP90. Accordingly, we suggest that the HA-HSP90 cannot autophosphorylate in our system. The phosphorylated function of HSP90 did not affect by GA in our in vitro system. Therefore, we use COS-7 cells as our in vivo system to test whether GA affect the cellular functions in trichostatin A (TSA)-treated cells. TSA is an inhibitor of histone deacetylase, results in histone H4 hyperacetylation and cell cycle arrest. When using TSA treatment, although caused COS-7 cell death, pretreatment of 0.5 mg/ml GA for 30 min and an addition of 50 ng/ml TSA (GA+TSA) apparently averted cell death. Our results indicated that the cell survival rate was only approximately 20% when prolonged treatment was undertaken with 50 ng/ml TSA (TSA) alone for 24 h. In contrast, the cell survival rate was enhanced by two folds when treating with GA+TSA. Furthermore, DNA fragmentation assay revealed that fragmented DNA was produced 8 h after prolonged treatment with TSA alone. Within 16 h, the apoptotic percentages of TSA-treated cells were between 15-25%. In contrast, the other treatments did not exceed 6%. Furthermore, GA inhibited TSA-induced histone H4 hyperacetylation. Western blotting analysis further demonstrated that the HSP70 levels did not significantly increase in TSA-treated cells. However, the accumulated 70-kDa heat shock protein (HSP70) markedly increased up to 2 to 3 folds at 8 h in GA- and GA+TSA-treated cells, and the maximum amount up to 5 to 7 folds at 20 h. Conversely, HSP90 did not markedly increase in all treatments. Based on the results in this study, we suggest that apoptosis induced by TSA can be prevented by GA-induced increment of heat shock proteins, particularly HSP70.

碩士
國立陽明大學
藥理學研究所
95
Arsenic trioxide (ATO) was effective in treatment of acute promyelocytic leukemia. Induction of mitotic apoptosis is a potential mechanism underlying the therapeutic effect of ATO. Numerous reports have demonstrated that the therapeutic efficacy of ATO can be enhanced by combined treatment of ATO with other anticancer drugs. Heat shock proteins, highly expressed in many tumors, can protect cells from ATO-induced injuries. 17-Dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG), a newly developed heat shock protein 90 (Hsp90) inhibitor, was shown to enhance the cytotoxicity of a variety of chemotherapeutic agents. In this report, the combined effect of 17DMAG and ATO was studied in CGL-2 cells. As compared to treatment with ATO alone, cotreatment of CGL-2 cells with 17DMAG and ATO significantly enhanced mitotic arrest, phosphorylation of BubR1 and accumulation of Pds1, mitotic abnormalities, and mitotic apoptosis. Akt, one of Hsp90 client proteins, plays important roles on regulation of G2/M cell cycle transition and involves in anti-apoptotic survival pathway. Western blot analysis showed that cellular Akt is dose-dependently reduced by cotreatment with 17DMAG and ATO. In addition, inhibition of phosphoinositide 3-kinase (PI3K)-Akt pathway by Akt inhibitor V or LY294002 also significantly increased ATO-induced mitotic arrest and apoptosis. Overexpression of constitutively active Akt reduced ATO-induced mitotic arrest and apoptosis, but decrease the expression of Hsp90 or Akt induced complicate effects and is different from the inhibition of 17DMAG. These results indicated that enhanced induction of mitotic arrest and apoptosis in cells cotreated with 17DMAG and ATO is mediated through 17DMAG-induced degradation of Akt protein.

碩士
國立清華大學
生命科學系
89
The benzoquinoid ansamycin geldanamycin (GA) interferes with many cell-signaling pathways and is currently being evaluated as an anticancer agent. Recent studies have shown that GA induces the expression of heat shock proteins (HSPs), including the HSP70s. The family of 70-kDa heat-shock proteins (HSP70s) is evolutionarily highly conserved and is assumed to serve an important protective function under a variety of adverse envirnmental conditions. In human cells, two stress inducible hsp70 genes, hsp70a and hsp70b, have often been studied. By using metabolic labeling followed by gel electrophoresis, Western and Northern blotting techniques, we found that hsp70a is much more inducible than hsp70b in GA-treated human non-small cell lung cancer H460 cells. The induced expression of HSP70A by GA is dependent on both concentration and the duration. And both actinomycin D and cycloheximide block HSP70A induction. This shows that the effect of GA on HSP70A induction is in transcription level. On the other hand, GA causes intracellular calcium ([Ca2+]i) mobilization, we further analyzed the effect of [Ca2+]i on HSP70A induction in the above processes by exploiting a set of drugs that affect the [Ca2+]i mobilization, including EGTA, BAPTA-AM, A23187, and thapsigargin (TG). It was found that the induced synthesis of HSP70A in GA-treated H460 cells could be attenuated by these drugs, but failed to completely block the HSP70A production and hsp70a mRNA accumulation. Taken together, our results lead us to conclude that GA transactivated hsp70a in H460 cells through calcium-dependent and -independent pathways. ABSTRACT ………………………………………………………………………….3 INTRODUCTION ……………………………………………………………………4 MATERIALS AND METHODS……………………………………………………...7 RESULTS ……………………………………………………………………………12 DISCUSSION ……………………………………………………………………….16 FOOTNOTES ………………………………………………………………………..20 REFFERENCES ……………………………………………………………………..21 FIGURE LEGENDS ………………………………………………………………...28

碩士
國立清華大學
生命科學系
88
ABSTRACT Geldanamycin (GA) specifically binds to HSP90 and disrupts the interaction of HSP90 and its target proteins. The binding will lead to substrate protein dissociation from HSP90 and may affect their structures and functions. Herein, we showed that exposure of non-small lung cancer H460 cells to 0.5 mM GA leads to enhancement of the synthesis of the 70 kDa heat shock proteins (HSP70s). The induction of HSP70s by GA is concentration- and time-dependent and this process coincides to the accumulation of its mRNA. By using the specific probes for hsp70-1 and hsp70b, we found that HSP70-1 is abundantly involved in GA-induced HSP70s. Furthermore importantly, we demonstrated for the first time that the PKC pathway is significant in this process since it is inhibited by H-7 and H-8 but not affected by HA1043. Similar results can be found in the protein and mRNA level. These data lead us to conclude that PKC pathway plays a major role in the GA-induced HSP70-1 expression in H460 cells. By using the electrophoretic mobility shift assay (EMSA), we showed that proximal Heat shock element (HSE1) was responsible for the inducible expression of HSP70. We found that the nuclear extracts prepared from GA-treated cells exhibited a significant increase in binding activity toward Heat shock element. Moreover, this increase in binding activity toward the Heat shock element is reduced by H-7 and H-8 but not affected by HA1004. We concluded that the PKC pathway is the major pathway involved in GA-induced expression of HSP70-1 in H460 cells.

Heat shock protein 90 (HSP90), which is implicated in post-translational folding, stability, and maturation of proteins, controls several key cell cycle regulators. Thus, the hypothesis was raised that geldanamycin, a specific and potent inhibitor of HSP90 function, may have pronounced effects on cell cycle progression. The objective of this study was to test this hypothesis in normal and cancer cells of human origin. The experiments performed on human lymphocytes mitogenically stimulated by phytohemagglutinin (PHA) indicated that 100 nM or 150 nM geldanamycin induces transition of cells to the G0 state of cell cycle. This was documented utilizing acridine orange, a metachromatic dye which differentially stains DNA versus RNA. The same experimental protocol allowed demonstration that geldanamycin is a potent inducer of apoptosis in PHA-activated cells. Importantly, both the block in G0 and induction of apoptosis were reversible and returned to control values upon removal of geldanamycin. Similar conclusions were reached when cell number in cultures was analyzed, excluding the possibility that a relevant fraction of cells was disintegrated during the incubation period. Experiments on Jurkat line of acute T-cell leukemia were performed next. Jurkat cells were used here as a model system in which the cytostatic and cytotoxic properties of geldanamycin on cancer cells can be tested. Initial experiments determined the time course and concentration-dependence of geldanamycin-induced alterations in cell cycle distribution and apoptosis. In contrast to human lymphocytes, geldanamycin did not induce G0 arrest in Jurkat cells, but inhibited them initially in the G2 phase, and at later time points in the G1 phase. The G2 was distinguished from mitosis by the absence of phosphorylation of histone H3, a specific marker of mitotic cells. The inhibition of Jurkat cells in G1 was linked to a decrease in phosphorylation of retinoblastoma protein. Finally, the exposure of Jurkat cells to geldanamycin resulted in induction of apoptosis, predominantly in cells being arrested in G1 and G2/M phases of the cell cycle. Finally, to address the possibility that stimulation of nuclear factor kappa-B (NF-kB), downstream of HSP90, modulates the effects of geldanamycin on cancer cells, Jurkat IkBaM line was employed. These cells cannot activate their NF-kB-mediated responses because of the mutation in its inhibitory protein, IkB. In the absence of functional NF-kB, geldanamycin-mediated induction of apoptosis and loss of cycling cells were markedly higher than when NF-kB was functional. These results were further corroborated by experiments in which parthenolide, a plant-derived inhibitor of NF-kB was employed. Geldanamycin-treated Jurkat cells responded to the parthenolide challenge by partial arrest in S phase and increased cell death by apoptosis. In conclusion, inhibition of HSP90 by geldanamycin blocks cell cycle progression and induces apoptosis of Jurkat cells, and NF-kB mediates these effects. The newly identified network of interactions may facilitate understanding of the mechanism of cytostatic and cytotoxic action of geldanamycin derivatives used currently in clinical trials.

碩士
國立清華大學
生命科學系
90
The cellular activity of several regulatory and signal transduction proteins, which depend on the heat shock protein 90 (HSP90) molecular chaperone for folding, is markedly decreased by geldanamycin (GA) and by radicicol (RA), and the mechanisms of these down-regulations are well-studied. However, the upstream mechanism for GA and RA to induce heat shock protein-70 (HSP70) expression through disrupting a chaperone complex of the inactive transcription factor, heat shock factor-1 (HSF1), subsequently activating and binding to the heat shock elements in the promoter region of hsp70 is largely unknown. This study is to investigate the differential effects of GA and RA on HSP70 synthesis for short-term treated with human non-small lung cancer H460 cells (H460 cells). Although both GA and RA induced the expression of HSP70 in H460 cells, only GA caused the induction of HSP70 synthesis within 5 min while RA takes for 2 h. RT-PCR and immunoblotting analysis showed that the GA-induced prompt HSP70 expression associated with the mRNA accumulation and protein hyperphosphorylation of HSF1. The potent protein kinase C (PKC) inhibitor H-7 dramatically suppresses HSP70 expression and HSF1 hyperphosphorylation in GA and RA-treated cells, suggesting that PKC is at least one of the common mediator for both drugs. Furthermore, GA produces a prompt intracellular calcium ([Ca2+]i) spike while RA did not show the obvious raise in [Ca2+]i. To test the importance of calcium in these pathways, cells were cultured with calcium free medium, and these results in suppression of HSP70 induced synthesis by GA and RA. This indicates that calcium is required in both pathways. I hypothesize that the prompt induction of calcium might be responsible for the fast induction of HSP70 in GA, if so, the ecotopic addition of calcium might force the otherwise slow-inductive RA to elicit a prompt HSP70 induction just as that GA does. As cells were treated with CaCl2, RA acquired the prompt induction ability like GA as I expected. Taken together, GA possesses the prompt induction ability to increase HSP70 expression through prompt induction of calcium and activation of PKC and HSF1. The fact that RA can mimic the prompt induction ability of GA after exogenous high calcium is provided, suggesting that common signaling pathways are shared by these HSP90 binding drugs, GA and RA, and that the differential calcium induction in the beginning might contribute to the differential HSP70 induction patterns between GA and RA.

Molecular chaperones have emerged in recent years as major players in many aspects of cell biology. Molecular chaperones are also known as heat shock proteins (HSPs) since many were originally discovered due to their increased synthesis in response to heat shock. They were initially identified when Drosophila salivary gland cells were exposed to a heat shock at 37°C for 30 min and then returned to their normal temperature of 25°C for recovery. A “puffing” of genes was found to have occurred in the chromosome of recovering cells, which was later shown to be accompanied by an increase in the synthesis of proteins with molecular masses of 70 and 26 kDa. These proteins were hence named “heat shock proteins”. The first identification of a function for HSPs was the discovery in Escherichia coli that five proteins synthesized in response to heat shock were involved in λ phage growth. The products of the groEL and groES genes were found to be essential for phage head assembly while the dnaK, dnaJ and grpE gene products were essential for λ phage replication. It was later shown that GroEL and GroES are part of a chaperonin system for protein folding in the prokaryotic cytosol while DnaK is a member of the Hsp70 family that works in conjunction with the DnaJ (Hsp40) co-chaperone and the nucleotide exchange factor GrpE to promote phage replication by dissociating the DnaB helicase from the phage-encoded P protein. Since then, a large number of other proteins collectively referred to as HSPs have been discovered. However, heat shock is not the only signal that induces synthesis of heat shock proteins. Stress of any kind, such as nutrient deprivation, chemical treatment and oxidative stress among others causes increased production of HSPs and therefore, they are also known as stress proteins. The term “molecular chaperone” was originally used to describe the function of nucleoplasmin, a Xenopus oocyte protein that promotes nucleosome assembly by binding tightly to histones and donating the bound histone to chromatin. However, since then, chaperones have been defined as “a family of unrelated classes of proteins that mediate the correct assembly of other proteins, but are not themselves components of the final functional structure”. This view of molecular chaperones, though undoubtedly correct, doesn’t capture the multifaceted roles they have since been discovered to play in cellular processes. In recent years, molecular chaperones have been shown to perform other functions in addition to the maintenance of protein homeostasis: translocation of proteins across organelle membranes, quality control in the endoplasmic reticulum, turnover of misfolded proteins as well as signal transduction. As a result, many chaperones are also essential under non-stress conditions and play crucial roles in cell growth and development, cell-cell communication and regulation of gene expression. Heat shock protein 90 (Hsp90) is one of the most abundant and highly conserved molecular chaperones in organisms ranging from bacteria to all branches of eukarya. It has been shown to be essential for cell viability in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster. Although the bacterial homolog HtpG is dispensable under normal conditions, it is important for cell survival during heat shock. In addition to its role as general chaperone in protein folding following stress, Hsp90 has a more specialized role as a chaperone for several protein kinases and transcription factors. Many Hsp90 client proteins are signaling proteins involved in regulation of cell growth and survival. These proteins are critically dependent on Hsp90 for their maturation and conformational maintenance resulting in a key role for Hsp90 in these processes. Recent reports have also highlighted a role for Hsp90 in linking the expression of genetic and epigenetic variation in response to environmental stress with morphological development in Drosophila melanogaster and Arabidopsis thaliana. In Candida albicans, Hsp90 augments the development of drug resistance, implicating a role for Hsp90 in the evolution of infectious diseases. The malarial parasite, Plasmodium falciparum, is the causative agent of the most lethal form of human malaria. The parasite life cycle involves two hosts: an invertebrate mosquito vector and a vertebrate human host. As the parasite moves from the mosquito to the human body, it experiences an increase in temperature resulting in a severe heat shock. The mechanisms by which the parasite adapts to changes in temperature have not been deciphered. Our laboratory has been interested in investigating the role of heat shock proteins during acclimatization of the parasite to such temperature fluctuations. Heat shock proteins of the Hsp40, Hsp60, Hsp70 and Hsp90 families have been characterized in the parasite and are being examined in our laboratory. This thesis pertains to understanding the functional role of Plasmodium falciparum Hsp90 (PfHsp90) during adaptation of the parasite to fluctuations in environmental temperature. The parasite expresses a single gene for cytosolic Hsp90 on chromosome 7 (PlasmoDB accession no.: PF07_0029) coding for a protein of 745 amino acids with a pI of 4.94 and Mw of 86 kDa. Eukaryotic Hsp90 regulates several protein kinases and transcription factors involved in cell growth and differentiation pathways resulting in a crucial role for Hsp90 in developmental processes. A role for PfHsp90 in parasite development, therefore, seems likely. Indeed, PfHsp90 has previously been implicated in parasite development from the ring stage to the trophozoite stage during the intra-erythrocytic cycle. Pharmacological inhibition of PfHsp90 function using geldanamycin (GA), a specific inhibitor of Hsp90 activity, abrogates stage progression. These experiments suggest that PfHsp90 may play a critical role in parasite development. This is further substantiated by the fact that several pathogenic protozoan parasites such as Leishmania donovani, Trypanosoma cruzi, Toxoplasma gondii and Eimeria tenella depend on Hsp90 function during different stages of their life cycles. It appears, therefore, that a principal role of Hsp90 in protozoan parasites may be the regulation of their developmental cycles. However, the precise functions of PfHsp90 during the intra-erythrocytic cycle of the malarial parasite are not clear. In this study we have carried out a functional analysis of PfHsp90 in the malarial parasite. We have examined the role of PfHsp90 in parasite development during repeated exposure to febrile temperatures. We have investigated its involvement in parasite development during a commonly used synchronization protocol involving cyclical changes in temperature. We have examined the interaction of GA with the Hsp90 multi-chaperone complex from P. falciparum as well as the human host. Finally, we have carried out a systems level analysis of chaperone networks in the malarial parasite as well as its human host using an in silico approach. We have analyzed the protein-protein interactions of PfHsp90 in the chaperone network and predicted putative cellular processes likely to be regulated by parasite chaperones, particularly PfHsp90.

碩士
國立清華大學
生命科學系
87
Geldanamycin (GA), a benzoquinone ansamycin, is a potent inducer of ER stress pathway by affecting the folding of proteins in the ER. Herein, we show that exposure of 9L rat brain tumor cell to 5 mM GA leads to enhance the synthesis of the 78 kDa glucose-regulated protein (GRP78). The induction of GRP78 by GA is concentration- and time-dependent and this process coincides to the accumulation of its mRNA. We demonstrate for the first time that the intracellular level of reactive oxygen species (ROS) is significant increase in GA-treated cells and this process is inhibited by N-acetylcysteine (NAC) and partially by pyrrolidine dithiocarbamate (PDTC). Similar results can be found in the GRP78 protein and mRNA level. These data lead us to conclude that generation of ROS plays a major role in the GA-induced grp78 expression in 9L rat brain tumor cells. By using the electrophoretic mobility shift assay (EMSA), we showed that all of the three previous identified regulatory elements including CORE, CRE, and C1 are involved in the basal expression of GRP78. However, we found that the nuclear extracts prepared from GA-treated cells exhibit an significant increase in binding activity toward the cis-regulatory elements including CORE and C1. Moreover, this increase in binding activity toward the CORE element is reduced by NAC. We conclude that the oxidative stress is activated by GA treatment and this process is crucial in the GA-induced expression of GRP78 and the CORE element plays the important role in 9L RBT cells.