Onconase responsive genes in human mesothelioma cells: implications for an RNA damaging therapeutic agent

<p>Abstract</p> <p>Background</p> <p>Onconase represents a new class of RNA-damaging drugs. Mechanistically, Onconase is thought to internalize, where it degrades intracellular RNAs such as tRNA and double-stranded RNA, and thereby suppresses protein synthesis. However, there may be additional or alternative mechanism(s) of action.</p> <p>Methods</p> <p>In this study, microarray analysis was used to compare gene expression profiles in untreated human malignant mesothelioma (MM) cell lines and cells exposed to 5 μg/ml Onconase for 24 h. A total of 155 genes were found to be regulated by Onconase that were common to both epithelial and biphasic MM cell lines. Some of these genes are known to significantly affect apoptosis (IL-24, TNFAIP3), transcription (ATF3, DDIT3, MAFF, HDAC9, SNAPC1) or inflammation and the immune response (IL-6, COX-2). RT-PCR analysis of selected up- or down-regulated genes treated with varying doses and times of Onconase generally confirmed the expression array findings in four MM cell lines.</p> <p>Results</p> <p>Onconase treatment consistently resulted in up-regulation of IL-24, previously shown to have tumor suppressive activity, as well as ATF3 and IL-6. Induction of ATF3 and the pro-apoptotic factor IL-24 by Onconase was highest in the two most responsive MM cell lines, as defined by DNA fragmentation analysis. In addition to apoptosis, gene ontology analysis indicated that pathways impacted by Onconase include MAPK signaling, cytokine-cytokine-receptor interactions, and Jak-STAT signaling.</p> <p>Conclusions</p> <p>These results provide a broad picture of gene activity after treatment with a drug that targets small non-coding RNAs and contribute to our overall understanding of MM cell response to Onconase as a therapeutic strategy. The findings provide insights regarding mechanisms that may contribute to the efficacy of this novel drug in clinical trials of MM patients who have failed first line chemotherapy or radiation treatment.</p

Crystal structure of Onconase at 1.1 Å resolution – insights into substrate binding and collective motion

Onconase® (ONC) is an amphibian member of the pancreatic ribonuclease superfamily that is selectively toxic to tumor cells. It is a much less efficient enzyme than the archetypal ribonuclease A and, in an attempt to gain further insight, we report the first atomic resolution crystal structure of ONC, determined in complex with sulfate ions at 100 K. The electron density map is of a quality sufficient to reveal significant nonplanarity in several peptide bonds. The majority of active site residues are very well defined, with the exceptions being Lys31 from the catalytic triad and Lys33 from the B(1) subsite, which are relatively mobile but rigidify upon nucleotide binding. Cryocooling causes a compaction of the unit cell and the protein contained within. This is principally the result of an inward movement of one of the lobes of the enzyme (lobe 2), which also narrows the active site cleft. Binding a nucleotide in place of sulfate is associated with an approximately perpendicular movement of lobe 2 and has little further effect on the cleft width. Aspects of this deformation are present in the principal axes of anisotropy extracted from C(α) atomic displacement parameters, indicating its intrinsic nature. The three lowest-frequency modes of ONC motion predicted by an anisotropic network model are compaction/expansion variations in which lobe 2 is the prime mover. Two of these have high similarity to the cryocooling response and imply that the essential ‘breathing’ motion of ribonuclease A is conserved in ONC. Instead, shifts in conformational equilibria may contribute to the reduced ribonucleolytic activity of ONC. DATABASE: Structural data have been submitted to the Protein Data Bank under accession number 3SNF

Activity of Anti-Tumor Endoribonucleases, Onconase (ranpirnase) and R-Amphinase in Chronic Lymphocytic Leukemia

Abstract Despite of evident progress in treatment of chronic lymphocytic leukemia (CLL), the disease still remains incurable. Several attempts have been made therefore to develop most effective and selective therapeutical approaches. A promising approach that involves targeting RNA either by the use of specific antisense oligonucleotides or cytostatic/cytotoxic ribonucleases is recently being promoted. Two such ribonucleases, onconase (ONC; ranpirnase) and R-amphinase (R-Amph), derived from Rana pipiens oocytes, have been developed. ONCdemonstrated preferential toxicity to tumor cells and was shown to be effective in vivo in animal tests as well as in clinical trials in treatment of malignant mesothelioma. Moreover, ONC is synergistic when used in combination with a variety of antitumor modalities including anthracyclines. R-Amph was developed only recently and thus far there is only a single report demonstrating its cytostatic and cytotoxic activity against human promyelocytic HL-60-, Jurkat T-cell- and U-937 histiomonocytic leukemic cells in vitro. In the present study we aimed to assess potential cytotoxicity of ONCand R-Amph against CLL cells. Toward this aim, leukemic cells were isolated from 36 untreated patients with CLL and were cultured for 24–72 h with either ONC or R-Amph alone and in combination with purine analogues, cladribine (2-CdA) and fludarabine (FA), two drugs routinely used in treatment of CLL, as well as with doxorubicin (DOX), the drug reported to show synergy with ONC in solid tumors. Cytotoxicity of the study drugs was assessed by the propidium iodide exclusion assay using flow cytometry. Their pro-apoptotic activity was examined by the Annexin-V (Ann-V) binding test, detection of caspase-3, -8, and -9 activation, a decrease of mitochondrial potential and the expression of apoptosis–regulating proteins from the Bcl-2 family. Compensated apoptotic index (CAI) has been calculated based on Ann-V assay as a difference in the percentage of apoptotic cells between the drug-treated sample and spontaneous apoptosis in the parallel untreated control. After preliminary experiments the optimal concentrations of both ONC and R-Amph were found to be 20 μg/ml; these were the lowest doses that induced significant cytotoxicity during 24–72 h of incubation, in comparison with parallel controls. The significant effect of ONC was evident after 48 h of treatment (median CAI=11.5%; p=0.035 versus control). After 72 h of incubation the median CAI for ONC was 17.1% (p=0.009). The significant cytotoxicity of R-Amph was seen after 72 h incubation (median CAI =19.9%; p=0.007, respectively). The mechanism of this cytotoxicity involved the induction of apoptosis along its mitochondrial pathway, with the drop of mitochondrial potential and activation of caspase-9 and caspase-3, concurrent with an increase in expression of pro-apoptotic Bax protein (p=0.035 versus control; after 72h) and a decrease of anti-apoptotic Bcl-2 expression (p=0.006; after 72 h). No significant changes in expression of Bak and Mcl-1 were observed. Synergistic effect was found for both, ONC plus 2-CDA and ONC plus FA (combination indices, CI; &amp;lt;0.8). Also the combination of R-Amph with 2-CDA or with FA exerted synergistic cytotoxiciy (both CI &amp;lt;0.8). Although, the combination of DOX with ONC or R-Amph demonstrated an increased in pro-apoptotic activity when compared to single agents, the effect was not statistically significant. In conclusion, this is the first study showing cytotoxic, pro-apoptotic affect of RNA-targeting agents, Onconase and R-Amphinase, in CLL. This promising anti-leukemic activity of both ribonucleases, especially their synergistic effects exerted in combination with purine analogues warrant further intensive preclinical and, subsequently, clinical study in this disease.</jats:p