Targeting eukaryotic translation in mesothelioma cells with an eIF4E-specific antisense oligonucleotide.

BACKGROUND: Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO) is assessed as a therapy for mesothelioma. METHODS: Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. RESULTS: eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. CONCLUSION: 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity

Triptolide and its prodrug minnelide suppress Hsp70 and inhibit in vivo growth in a xenograft model of mesothelioma

Malignant mesothelioma is a devastating disease with a poor prognosis for which there is a clear need for more successful therapeutic approaches. Triptolide, a diterpenoid triepoxide, is a highly effective agent against several cancer types in animal models. Owing to triptolide's poor solubility in water, a water-soluble analog, minnelide, was synthesized. Minnelide is a prodrug of triptolide and is activated by exposure to phosphatases that are found in all body tissues, including blood. Mesothelioma cells were treated in vitro with minnelide or its parent compound, triptolide. Minnelide and triptolide were both found to significantly reduce mesothelioma cell viability and induce apoptosis. The level of Hsp70, a protein that promotes cancer cell survival, was measured in mesothelioma cells before and after treatment with triptolide. Hsp70 levels were decreased in a dose-dependent manner. In addition, triptolide sensitized cells to gemcitabine and pemetrexed as measured by cell viability. Mice bearing mesothelioma flank tumors were treated with daily injections (28 d) of minnelide or saline solution and xenograft tumor growth recorded. Mice displayed significantly reduced tumor burden. These findings support the clinical evaluation of minnelide therapy for mesothelioma

4EASO transfection induces apoptosis in mesothelioma.

<p>Mesothelioma cell lines H2373 and H2461 along with human mesothelial cells (LP9) were treated with the indicated concentration of 4EASO for 48 hours. Lysates were immunoblotted with anti-PARP antibody. In mesothelioma cell lines PARP cleavage was substantially increased in 4EASO treated compared to untreated cells.</p

eIF4E expression is reduced by 4EASO treatment in mesothelioma.

<p>Cultured normal mesothelial and mesothelioma cells were transfected with mmASO or 4EASO and eIF4E and β-actin protein expression was evaluated by immunoblot analysis from lysates harvested after 72 hours. The mmASO does not alter eIF4E levels in mesothelial cells or mesothelioma cells. The band intensity levels for eIF4E following 4EASO treatment was normalized to untreated cells for each cell line and was determined using ImageJ. β-actin level does not change upon treatment and serves as a loading control.</p

Enhanced susceptibility of mesothelioma cells treated with 4EASO to cytotoxic drugs.

<p>Mesothelioma cell lines transfected with mmASO or 4EASO were treated with the indicated concentration of gemcitabine (GEM) or pemetrexed (PEM) and viable cells were counted. Elevated cell death was found for cells treated in combination with 4EASO and pemetrexed or gemcitabine compared to each treatment alone. Concurrent treatment of mesothelioma cells with 4EASO combined with pemetrexed or gemcitabine suppresses proliferation compared to treatment with mmASO combined with pemetrexed or gemcitabine. <i>Columns</i>, the mean of three independent determinations of cell number normalized to untreated cells, <i>bars</i>, s.d. Averages of combination treatment was compared to either agent alone by Student’s <i>t</i>-test. * denotes a p value <0.05. NS = not statistically significant.</p

4EASO transfection suppresses mesothelioma proliferation.

<p>Normal mesothelial cells (LP9) and mesothelioma cell lines H2373, H2461 and H2596 were treated with the indicated concentration of mmASO (open columns) and 4EASO (filled columns), and viable cells were counted. Normal LP9 cells were most resistant to 4EASO treatment, while growth of mesothelioma cell lines was reduced extensively. <i>Columns</i>, the mean of three independent determinations of cell number normalized to untreated cells, <i>bars</i>, s.d.</p

PDTC inhibits picornavirus polyprotein processing and RNA replication by transporting zinc ions into cells.

Contains fulltext : 52333.pdf (publisher's version ) (Closed access)Previously, it was shown that pyrrolidine dithiocarbamate (PDTC) inhibits proteolytic polyprotein processing and replication of human rhinovirus by transporting metal ions into cells. Here, it is shown that PDTC also inhibits replication of two other picornaviruses: coxsackievirus B3 (CVB3), a closely related virus that belongs to the genus Enterovirus, and mengovirus, an encephalomyocarditis virus strain that belongs to the genus Cardiovirus, and that this inhibition is due to the dithiocarbamate moiety of the compound. Making use of subgenomic replicons, evidence is provided that PDTC inhibits replication of these two viruses by disturbing viral RNA synthesis. Furthermore, it is shown that PDTC transports zinc ions into cells and that these zinc ions play an important role in the antiviral activity mediated by PDTC. Finally, it is shown that PDTC interferes with proteolytic processing of the polyproteins of both CVB3 and mengovirus, but that the underlying mechanism between these two viruses differs. In CVB3-infected cells, PDTC interferes strongly with the proteolytic activity of 3CD(pro), as shown by the impaired production of the mature capsid proteins as well as the autocleavage of 3CD(pro) into 3C(pro) and 3D(pol). In mengovirus-infected cells, however, PDTC had no effect on the proteolytic production of capsid proteins or the autocleavage of 3CD(pro). Instead, PDTC caused the accumulation of a high-molecular-mass precursor protein, due to an impairment in the primary 'break' that normally occurs at the 2A-2B junction. Thus, PDTC disturbs polyprotein processing and replication of two groups of picornaviruses, enteroviruses and cardioviruses, but the underlying mechanism is different