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; <0.8). Also the combination of R-Amph with 2-CDA or with FA exerted synergistic cytotoxiciy (both CI <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.

Disclosures: No relevant conflicts of interest to declare.

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