trichostatin-a and geldanamycin

Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein-dependent retrograde trafficking to a juxtanuclear location. We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation. These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.

Topics: Acetylation; Benzoquinones; Cell Extracts; Cell Line; Cell Nucleus; Epithelial Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Lactams, Macrocyclic; Leupeptins; Pigment Epithelium of Eye; Protein Structure, Quaternary; Tubulin; Ubiquitination

Estrogen has a variety of neuroprotective effects but the molecular basis of its function is still mainly unclear. Estrogen receptor (ER) signaling is highly dependent on posttranslational modifications and the assembly of coactivator and corepressor complexes. Several proteins involved in ER alpha signaling have recently been found to be acetylated, including ER alpha itself and Hsp90, a key chaperone in the functional regulation of ER alpha. ER alpha complexes also contain histone deacetylases (HDAC) which repress transactivation. Our purpose was to clarify the role of protein acetylation and Hsp90 function in the ERE-mediated ER alpha signaling in neuronal HN10 cells. We observed that increasing protein/histone acetylation status with trichostatin A, a potent HDAC inhibitor, increased the 17beta-estradiol (E2)-induced transactivation of ERE-driven luciferase in non-transfected cells, and even more extensively in pER alpha-transfected cells. E2-induced ERE-driven transactivation was blocked by ICI 182.780. Several ER antagonists, such as raloxifene and tamoxifen, were unresponsive. Valproate, an antiepileptic drug which is recently characterized as a HDAC inhibitor, was also able to potentiate the E2-induced ERE-transactivation. Inhibition of the function of Hsp90 chaperone with geldanamycin strongly inhibited the E2-induced ERE-transactivation. Overexpression of SIRT2 protein deacetylase did not inhibit the acetylation-potentiated ERE-driven transactivation indicating that SIRT2 deacetylase is not involved in ER alpha signaling. Our results reveal that ER alpha signaling is dependent on protein acetylation and epigenetic regulation.

Topics: Acetylation; Animals; Benzoquinones; Cell Line; Dactinomycin; Dehydroepiandrosterone; Diethylstilbestrol; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Estrogens, Non-Steroidal; Fulvestrant; Genes, Reporter; Histone Deacetylase Inhibitors; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Lactams, Macrocyclic; Neurons; Protein Synthesis Inhibitors; Selective Estrogen Receptor Modulators; Signal Transduction; Sirtuin 2; Sirtuins; Transcriptional Activation; Valproic Acid

Pancreatic cancer is lethal because of its invasiveness, rapid progression, and profound resistance to chemotherapy and radiation therapy. To identify the molecular mechanisms underlying this, we have examined the expression and potency of three major death receptors: tumor necrosis factor receptor (TNF-R), TNF-related apoptosis-inducing ligand receptor (TRAIL-R), and Fas in mediating cytotoxicity in four invasive pancreatic cancer cell lines. We have analyzed the expression of major antiapoptotic factors, cell cycle regulators and death receptor decoys (DcR) in comparison with normal pancreas tissues and five other human malignant tumor cell lines. We have found that different pancreatic cancer cell lines coexpress high-level TRAIL-R, Fas, and TNF-R1 but are strongly resistant to apoptosis triggered by the death receptors. DcR2 and DcR3 overexpression may partly contribute to the resistance of pancreatic cancer cells to TRAIL-R- and Fas-mediated cytotoxicity. Bcl-XL and Bcl-2 are predominantly overexpressed in pancreatic cancer cell lines, respectively. Bcl-XL is also predominantly overexpressed in prostate, colorectal, and intestinal cancer cells. The knockdown of the predominant Bcl-XL overexpression significantly reduces the viability of pancreatic cancer cells to TNFalpha- and TRAIL-mediated apoptosis by sublethal-dose single and combined antitumor drugs, including geldanamycin, PS-341, Trichostatin A, and doxorubicine. Geldanamyin and PS-341 synergistically block NFkappaB activation, suppress Akt/PKB pathway, and down-regulate Bcl-XL, Bcl-2, cIAP-1, and cyclin D1 expression. This combined regimen dramatically enhances TRAIL cytotoxic effects and breaks through chemoresistance. Bcl-XL plays a vital role in pancreatic cancer chemoresistance. Geldanamycin, PS-341, and TRAIL triple combination may be a novel therapeutic strategy for pancreatic cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Benzoquinones; Boronic Acids; Bortezomib; Cell Line, Tumor; Doxorubicin; Drug Synergism; Gene Expression Regulation, Neoplastic; Gene Silencing; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Lactams, Macrocyclic; Membrane Glycoproteins; Pancreatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Quinones; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

As widely believed treating cells with trichostatin A (TSA), an inhibitor of histone deacetylase, results in histone H4 hyperacetylation and cell cycle arrest. This compound is often compared with other potential anticancer drugs in cell cycle, proliferation and differentiation research. Furthermore, geldanamycin (GA), a 90-kDa heat shock protein (HSP90) specific inhibitor, is a well-known potential anticancer agent. This study examines whether GA can affect the cellular functions induced by TSA. When using TSA treatment, although caused COS-7 cell death, pretreatment of 0.5 microg/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.

Topics: Acetylation; Animals; Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Cell Survival; COS Cells; DNA Fragmentation; Drug Antagonism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; HSP70 Heat-Shock Proteins; Hydroxamic Acids; Lactams, Macrocyclic; Quinones; Transfection

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.

Topics: Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Animals; Benzoquinones; Binding Sites; Cattle; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Lactams, Macrocyclic; Lactones; Macrolides; Macromolecular Substances; Precipitin Tests; Protein Binding; Quinones; Substrate Specificity; Tandem Repeat Sequences; Two-Hybrid System Techniques; Yeasts