Hsp70 confines tumor progression of rat histiocytoma and impedes the cytotoxicity induced by natural killer cells and peritoneal macrophages

ObjectiveTo study the role of inducible form of heat shock protein 70 (Hsp70) in the host tumor regression of rat tumor model.MethodsWe examined the role of Hsp70 in host tumorigenicity and in vitro cellular cytotoxicity using a rat histocytoma. The differential tumor growth and regression kinetics were studied and correlated with the expression of Hsp70, activation of macrophages and natural killer (NK) cells, and circulating or tumor infiltrating immune molecules in the host system.ResultsThe sub cuteaneous (s.c.) tumor regression was correlated with increased serum cytokines such as IL-12, TNF α, IFN γ and Hsp70. Despite of similar increase of Hsp70 in intraperitoneal (i.p.) tumor implanted animals, animals succumb to tumor growth, further, evidently, no immune molecule activation was observed. The viral promoter driven Hsp70 over expression in these tumor cells restrained solid tumor growth, however, failed to inhibit ascites growth. The NK cells from s.c. immunized animals induces cytotoxicity in the presence of anti-tumor antibody, which necessitated CD40-L expression, conversely, NK cells from i.p. immunized animals failed to induce cytotoxicity. The NK cells from s.c. or i.p. implanted animals with Hsp70 positive tumor cells failed to induce such cytotoxicity. The peritoneal macrophages isolated from s.c. tumor implanted animals when co-cultured with parental BC-8 cells lyses tumor cells, nevertheless entail macrophage specific TNF α expression. On the contrary, Hsp70 expressing BC-8 tumor cells were resistant to peritoneal macrophage induced cytolysis.ConclusionsThis study brings out that Hsp70 possibly involved in regulating the host tumor response and cellular cytotoxicity

Molekuláris chaperonok és a citoszkeleton kölcsönhatásai = Interactions of molecular chaperones and the cytoskeleton

Munkánk során azonosítottuk a citoplazma leggyakoribb stresszfehérjéje, a Hsp90 második, C-terminális nukleotid kötőhelyét, valamint e fehérje taxol-kötőhelyét, amely új Hsp90 támadáspontú gyógyszerek tervezésére ad lehetőséget. Bebizonyítottuk, hogy a Hsp90 inhibitorok hatásmódjában a megtámadott sejtek erőteljesebb lízise is részt vesz. A chaperonok rendezetlen régióin alapuló új elméletet dolgoztunk ki működésük megértésére. Elemeztük a chaperonok szerepét az öregedés során különös tekintettel az apoptózist gátló tulajdonságaikra. Az endoplazmatikus retikulum chaperonjai működési mechanizmusának számos új elemét tártuk fel egészséges, és krónikus stressznek kitett sejtekben. Számos új szempontot adtunk a chaperonok szerepének megértésére a sejt molekula-hálózataiban. Ezek legfontosabbikaként felismertük, hogy a hálózatok gyenge kölcsönhatásai részt vesznek minden komplex rendszer stabilizálásában. Kidolgoztunk és részben bizonyítottunk egy új gyógyszertervezési paradigmát, amely szerint a multi-target gyógyszerek hatékonyabbak, mint a jelenleg alkalmazott single-target gyógyszerek. Elvileg is új, rendkívül hatékony eljárás-családot dolgoztunk ki hálózatok átfedő moduljainak meghatározására és igen nagyméretű hálózatok hierarchikus ábrázolására. | We have identified a second, C-terminal nucleotide binding site, and an independent, taxol-binding site of the most abundant cytoplasmic stress protein, Hsp90. Both sites can be used as novel pharmacological targets in anticancer drug design. We demonstrated that the Hsp90 inhibitor drug candidates promote the lysis of the malignant cells by the immune system. We have discovered that chaperones contain a large amount of disordered regions and described the involvement of these regions in chaperone action. We analyzed the role of chaperones in aging with special reference to their anti-apoptotic effects. We identified several elements of chaperone action in the endoplasmic reticulum of normal cells and cells under chronic stress. We gave several new aspects to understand the role of chaperones in cellular networks. As the most important of these, we discovered that weak links stabilize all complex systems. We have developed and partially proved a novel drug design paradigm showing the efficiency of multi-target drugs. Lastly, we developed a novel, highly efficient group of methods to identify overlapping network modules and to visualize extremely large networks in a hiearachical (zoom-in) fashion

A dajkafehérjék az öregedési folyamatokban = Molecular chaperones in the ageing process

A stresszválasz fontos szerepet játszik az öregedésben, és terápiás célpont a korfüggő betegségek kezelésében. Pályázatunkban kimutattuk, hogy (1) az öregedés kóroki tényezőjeként ismert oxidatív stressz osztódó sejtekben nem vezet fehérje denaturációhoz és kompenzatorikus stresszfehérje indukcióhoz, így a globális fehérje homeosztázisra gyakorolt akut hatása elhanyagolható; (2) a kitekeredő és szuszceptibilis oldalláncokat exponáló fehérjék oxidáltsága megnövekszik, mely felveti, hogy az öregedés során felszaporodó aggregátumok másodlagosan (is) oxidálódnak; (3) oxidatív stressz a Hsp90 módosulását okozza in vitro, a Hsp90 izoformaspecifikus funkciókat mutat; (4) a perifériás limfociták nyugalmi és hőindukált Hsp70 szintje idős emberekben számottevően csökken, valamint (5) Hsp70 indukálhatósága pozitív korrelációt mutat a nyugalmi Hsp70 szinttel, ami az enyhe stresszek pozitív hatását sejteti; (6) a polifenol resveratrol emlős sejtekben és C. elegans-ban a stresszválasz indukciójához, hősokkal mutatott szinergizmushoz és termotoleranciához vezet, melyet nem antioxidáns és proteotoxikus hatása, hanem a hősokk faktor-1 aktivációja által fejt ki. Összefoglalva, az oxidatív stressz és az antioxidánsok nem módosítják a fehérje homeosztázist és a stresszválaszt, ellenben hatnak a Hsp90-re. A resveratrol chaperon induktor tulajdonsága révén biztosíthatja és fenntarthatja a stresszválasz robusztusságát, mely hozzájárulhat terápiás és élettartam növelő hatásaihoz. | The heat-shock response is an important player in aging and is a therapeutic target in the treatment of age-related diseases. We demonstrated that (1) oxidative stress, the major causative factor in aging failed to induce protein misfolding and heat-shock response in replicating cells, therefore it did exert a significant short-term effect on protein homeostasis; (2) misfolded proteins displayed higher oxidation due to the exposure of susceptible residues, suggesting that increased protein aggregation may also secondarily lead to increased protein oxidation during aging; (3) oxidative stress modified Hsp90 in vitro, Hsp90 displayed isoform-specific functions; (4) basal and heat-induced Hsp70 levels declined in human lymphocytes from aged subjects and (5) Hsp70 inducibility showed a strong positive correlation with basal Hsp70 level, suggesting a priming role for mild stresses; (6) the polyphenol resveratrol induced the heat-shock response, synergized with mild heat stress and conferred thermotolerance both in mammalian cells and in C. elegans. It was neither mediated by proteotoxicity nor its antioxidant property, but a selective activation of heat shock factor-1. Thus, oxidative stress and antioxidants do not seem to modify protein homeostasis and the heat-shock response, while affect Hsp90. Resveratrol as a chaperone inducer may ensure and maintain a robust heat-shock response, that may contribute to its therapeutic and longevity-promoting potential

Repercussion of Mitochondria Deformity Induced by Anti-Hsp90 Drug 17AAG in Human Tumor Cells

Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects on tumor cell mitochondria were largely unknown. Here, we show that Hsp90 inhibition with 17AAG first affects mitochondrial integrity in different human tumor cells, neuroblastoma, cervical cancer and glial cells. Using human neuroblastoma tumor cells, we found the early effects associated with a change in mitochondrial membrane potential, elongation and engorgement of mitochondria because of an increased matrix vacuolization. These effects are specific to Hsp90 inhibition as other chemotherapeutic drugs did not induce similar mitochondrial deformity. Further, the effects are independent of oxidative damage and cytoarchitecture destabilization since cytoskeletal disruptors and mitochondrial metabolic inhibitors also do not induce similar deformity induced by 17AAG. The 1D PAGE LC MS/MS mitochondrial proteome analysis of 17AAG treated human neuroblastoma cells showed a loss of 61% proteins from membrane, metabolic, chaperone and ribonucleoprotein families. About 31 unmapped protein IDs were identified from proteolytic processing map using Swiss-Prot accession number, and converted to the matching gene name searching the ExPASy proteomics server. Our studies display that Hsp90 inhibition effects at first embark on mitochondria of tumor cells and compromise mitochondrial integrity

Proteomic Analyses Reveal High Expression of Decorin and Endoplasmin (HSP90B1) Are Associated with Breast Cancer Metastasis and Decreased Survival

BACKGROUND: Breast cancer is the most common malignancy among women worldwide in terms of incidence and mortality. About 10% of North American women will be diagnosed with breast cancer during their lifetime and 20% of those will die of the disease. Breast cancer is a heterogeneous disease and biomarkers able to correctly classify patients into prognostic groups are needed to better tailor treatment options and improve outcomes. One powerful method used for biomarker discovery is sample screening with mass spectrometry, as it allows direct comparison of protein expression between normal and pathological states. The purpose of this study was to use a systematic and objective method to identify biomarkers with possible prognostic value in breast cancer patients, particularly in identifying cases most likely to have lymph node metastasis and to validate their prognostic ability using breast cancer tissue microarrays. METHODS AND FINDINGS: Differential proteomic analyses were employed to identify candidate biomarkers in primary breast cancer patients. These analyses identified decorin (DCN) and endoplasmin (HSP90B1) which play important roles regulating the tumour microenvironment and in pathways related to tumorigenesis. This study indicates that high expression of Decorin is associated with lymph node metastasis (p<0.001), higher number of positive lymph nodes (p<0.0001) and worse overall survival (p = 0.01). High expression of HSP90B1 is associated with distant metastasis (p<0.0001) and decreased overall survival (p<0.0001) these patients also appear to benefit significantly from hormonal treatment. CONCLUSIONS: Using quantitative proteomic profiling of primary breast cancers, two new promising prognostic and predictive markers were found to identify patients with worse survival. In addition HSP90B1 appears to identify a group of patients with distant metastasis with otherwise good prognostic features

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