Ex Vivo Cytotoxic Activity of Endoribonucleases, Onconase (ranpirnase) and R-Amphinase, against Acute Myeloblastic Leukemia Cells
Authors: Smolewski Piotr , Cebula Barbara , Zwolinska Malgorzata , Linke-Szewczyk Anna , Wozniak Dominika , Shogen Kuslima , Ardelt Wojciech , Darzynkiewicz Zbigniew , Robak Tadeusz 
Published: April 2020

Abstract
Most patients with acute myeloblastic leukemia (AML) to whom allogeneic stem cell transplantation is unavailable cannot be cured by the standard chemotherapy regimen. Because cytostatic agents used for AML treatment induce damage to DNA of both tumor and proliferating normal cells significant toxicity to the patients is observed. Therefore, there is a need to develop new strategies that would be more effective in terms of selectively targeting tumor cells. A promising therapeutic approach may involve the use of agents targeting cellular RNA namely cytostatic/cytotoxic ribonucleases derived from Rana pipiens oocytes. One of them, onconase (ranpirnase, ONC) has been found to be effective in clinical trials for treatment of malignant mesothelioma and other solid tumors, as well as in preclinical in vitro studies on several leukemia lines. More recently, antitumor activity of R-amphinase (R-Amph) has also been reported. As of yet, none of those agents have been tested ex vivo in hematological malignancies. The goal of this study was to assess the anti-tumor properties of both these endoribonucleases in leukemic cells derived from patients with AML. Toward this aim, leukemic cells isolated from 22 patients with newly diagnosed AML were cultured for 24 – 72h with either ONC or R-Amph alone and also in combination with the cytostatic drugs used in routine treatment of AML, namely doxorubicin (DOX) and cytarabine arabinoside (ARA-C). In pilot experiments, the cytostatic/cytotoxic effects of ONC and R-Amph were tested on AML and HL-60 cell lines. Cytotoxicity of the study drugs was assessed by the propidium iodide exclusion assay. Their pro-apoptotic activity was examined by the Annexin-V (Ann-V) binding test, detection of caspase-3, -8, and -9 activation, and a reduction of mitochondrial potential as an expression of apoptosis–regulating proteins from Bcl-2 family. Compensated apoptotic index (CAI) has been calculated based on Ann-V assay a difference in the percentage of apoptotic cells between drug-treated sample and parallel untreated control. The results indicate that ONC and R-Amph were effective anti-leukemic drugs against AML cells in both in vitro and ex vivo settings at comparable concentrations. ONC at 10mg/ml and R-Amph also at 10mg/ml concentration were found to induce significant cytotoxicity for the studied cells. The effect was evident after 48 h of the treatment (both drugs versus control – p &lt;0.03). After 72 h of incubation with the drugs CAI for ONC was 15.9%; p = 0.011 and for R-Amph 34.3%; p = 0.0005, respectively. In the ex vivo testing the combination of ONC or R-Amph with DOX have shown an increased pro-apoptotic effect compared to each of these agents when used alone. The synergistic effect was observed for both ONC and DOX (combination index, CI, 0.67) and R-mph and DOX (CI 0.58). Induction of apoptosis was the mechanism of cytotoxicity. The mitochondrial pathway of apoptosis was manifested by a reduction of mitochondrial potential and activation of caspase-9 and caspase-3. Concurrently, an increase in expression of the pro-apoptotic Bax (p=0.001; versus control) and decrease anti-apoptotic Bcl-2 expression (p=0.027; versus control) was observed after 72 h of treatment. In conclusion, this is the first study showing in ex vivo experiments that both endoribonucleases, ONC and R-Amph, are significantly effective against AML cells. The main mechanism of this action is triggering the caspase-dependent apoptosis by activation of the mitochondrial pathway. The combination of ONC or R-Amph with DOX exerts synergistic cytotoxicity. The data clearly indicate that study on ONC and R-Amph activity in AML should be continued in in vivo settings and then, eventually, in clinical trials.