Turcotte RF, Raines RT.

Medical Scientist Training Program and Biophysics Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA.

Abstract

One of the tightest known protein–protein interactions in biology is that between members of the ribonuclease A superfamily and the ribonuclease inhibitor protein (RI). Some members of this superfamily are able to kill cancer cells, and the ability to evade RI is a major determinant of whether a ribonuclease will be cytotoxic. The archetypal cytotoxic ribonuclease, onconase (ONC), is in late-stage clinical trials for the treatment of malignant mesothelioma. We present here the first measurement of the inhibition of the ribonucleolytic activity of ONC by RI. This inhibition occurs with K_i = 0.15 μM in a solution of low salt concentration.

Keywords: Cancer; Cytotoxin; Enzyme inhibition; Onconase; Ribonuclease; Salt concentration

Abbreviations: RNase A, bovine pancreatic ribonuclease; RI, ribonuclease inhibitor protein; PBS, phosphate-buffered saline; RNase 1, human pancreatic ribonuclease; ONC, onconase

Zhao H, Ardelt B, Ardelt W, Shogen K, Darzynkiewicz Z.

Department of Pathology, Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10595, USA.

Abstract

Onconase (Onc), a ribonuclease from oocytes of Northern Leopard frogs (Rana pipiens) is cytostatic and cytotoxic to a variety of tumor lines in vitro, inhibits growth of tumors in animal in vivo models and enhances sensitivity of tumor cells to a number of other cytotoxic agents with diverse mechanism of action. In Phase III clinical trials Onc demonstrated significant efficacy in patients with malignant mesothelioma that failed prior chemotherapy. We previously postulated that the antitumor activity of Onc and the observed synergisms with other antitumor modalities at least in part may be mediated by targeting RNA interference (RNAi). In the present study we observed that the silencing of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene in human lung adenocarcinoma A549 cells by siRNA was effectively prevented by Onc. While transfection of cells with GAPDH siRNA reduced expression of this protein by nearly 70%, the expression was restored in the cells exposed to 0.8 muM Onc for 48 or 72 h. The data thus provide evidence that one of the targets of Onc is siRNA, likely within the RNA-induced silencing complex (RISC). In light of the findings that microRNAs are involved in tumor pathogenesis as well as in enhancing cell resistance to anticancer therapy the present data may provide explanation for both, the antitumor Onc activity and its propensity to enhance effectiveness of cytotoxic drugs.

Keywords: microRNAs, ranpirnase, gene regulation, RISC, glyceraldehyde 3-phosphate dehydrogenase, laser scanning cytometry, mesothelioma

Rutkoski TJ, Raines RT.

Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706 1544, USA.

Abstract

Onconase® (ONC) is an amphibian member of the bovine pancreatic ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma, and is poised to become the first chemotherapeutic agent based on a ribonuclease. Investigations into the mechanism of ribonuclease-based cytotoxicity have elucidated several important determinants for cytotoxicity, including efficient deliverance of ribonucleolytic activity to the cytosol and preservation of conformation stability. Nevertheless, the most striking similarity between ONC and bovine seminal ribonuclease, another naturally cytotoxic ribonuclease, is their insensitivity to inhibition by the potent cytosolic ribonuclease inhibitor protein (RI). RI typically binds to its ribonuclease ligands with femtomolar affinity — an extraordinary feat considering the lack of sequence identity among the bound ribonucleases. Mammalian ribonucleases such as RNase A or its human homologue, RNase 1, have the potential to be more desirable chemotherapeutic agents than ONC owing to their higher catalytic activity, low potential for immunogenicity, favorable tissue distribution, and high therapeutic index, but are limited by their sensitivity to RI. These non-toxic mammalian ribonucleases can be transformed into potent cytotoxins by engendering them with RI-evasion using protein engineering strategies such as site-directed mutagenesis, multimerization, fusion to a targeting moiety, and chemical modification. In several instances, these engineered ribonucleases exhibit greater cytotoxicity in vitro than does ONC. Herein, we review the biochemical characteristics of RI ribonuclease complexes and progress towards the development of mammalian ribonuclease-based chemotherapeutics through the elicitation of RI-evasion.

Keywords: Cancer, cytotoxin, pancreatic ribonuclease, ribonuclease A, ribonuclease inhibitor

Ardelt W, Shogen K, Darzynkiewicz Z.

Alfacell Corporation, 300 Atrium Drive, Somerset, NJ 08873, USA. wardelt@alfacell.com

Abstract

Rana pipiens oocytes contain two homologues of pancreatic ribonuclease A that are cytostatic and cytotoxic to human cancer cells. Extensively studied Onconase is in advanced Phase IIIb clinical trials against malignant mesothelioma, while Amphinase is a novel enzyme in pre-clinical development. Onconase is the smallest (104 amino acid residues) member of the ribonuclease A superfamily while Amphinase (114 residues) is the largest among amphibian ribonucleases. Both enzymes share the characteristic frog ribonucleases C-terminal disulfide bond but another signature of this group, the N-terminal pyroglutamate, an integral part of Onconase active site is not conserved in Amphinase.

Although Onconase and Amphinase are weak catalysts their enzymatic activities are required for cytostatic and cytotoxic activity. While it was postulated that tRNA is the primary substrate of Onconase in vivo there is also extensive indirect evidence that suggests other RNA species, in particular micro RNAs, may actually be the critical target of these ribonucleases. The cytostatic effects of Onconase and Amphinase are manifested as cell arrest in the G1 cell cycle phase. Apoptosis then follows involving activation of endonucleases(s), caspases, serine proteases and transglutaminase. Onconase was shown to be strongly synergistic when combined with numerous other antitumor modalities. Onconase and Amphinase are highly cationic molecules and their preferential toxicity towards cancer cells (having distinctly higher negative charge compared to normal cells) may depend on increased binding efficiency to the cell surface by electrostatic interactions.

Here we will discuss the structures of Onconase and Amphinase and the molecular basis for their enzymatic and anticancer functions.

Keywords: Onconase, amphinase, cytotoxic ribonucleases, Rana pipiens, structure and function, apoptosis, cell cycle, microRNA

Rybak SM.

Bionanomics, LLC, 411 Walnut Street, Green Cove Springs, FL 32043, USA. rybak@mindspring.com

Abstract

Onconase, a member of the pancreatic ribonuclease A superfamily, is currently in Phase III clinical trials for treatment of unresectable malignant mesothelioma. The anticancer effect of onconase may relate to its intracellular target, a non-coding RNA. Some non-coding RNAs are aberrantly expressed in cancer cells. This discovery is creating new interest in drugs that target RNA. Conjugating onconase to agents that recognize tumor associated molecules further increases its potency and specificity. Analysis of onconase activity when directed to two different internalizing and one non-internalizing receptor reveals that the ideal targeting agents would rapidly enter lysosomal compartments before onconase escaped to the cytosol. Antibody-onconase conjugates are effective in preclinical models, cause little non-specific toxicities in mice and have favorable formulation properties. Understanding the reason for their potency coupled with understanding novel RNA-based mechanisms of tumor cell death will lead to improved variations of targeted onconase.

Krauss J, Arndt MA, Dübel S, Rybak SM.

Natinal center for tumor diseases (NCT), Universit6y of Heidelberg, Im Neuenheimer Fild 350, D-69120 Heidleberg, Germany. juergen.krauss@med.uni-heidelberg.de

Abstract

Ribonucleases (RNases) of the superfamily A exhibit potent antineoplastic activity yet do not mediate appreciable immunogenicity or non-specific toxicity in both animal models and cancer patients. Ranpirnase (Onconase), the first ribonuclease being evaluated as a therapeutic in humans, has progressed to phase III clinical trials in patients with unresectable mesothelioma. Conjugation of RNases to internalizing tumor-targeting monoclonal antibodies was shown to enhance specific cell killing by several orders of magnitude both in vitro and in animal models. In this review we describe the development and current status of genetically engineered 2(nd) generation immunoRNases as promising novel anti-cancer therapeutics.

Keywords: Rnase, Onconase®, immunoRNase, antibody-RNase fusion protein, immunotherapeutics

Ramos-Nino ME, Littenberg B.

Departments of Pathology and Medicine and Nursing, University of Vermont, Burlington, Vermont.

Abstract

Accumulating evidence supports the idea that two known phosphatidylinositol 3′-kinase (PI3K) downstream proteins, Fra-1 and Survivin, are potential targets for cancer therapy. Increased expression of Fra-1, a Fos family member of the transcription factor activator protein-1, has been implicated in both the maintenance and the progression of the transformed state of several cancer cells. In addition, high Survivin expression in tumors correlates with more aggressive behavior, lower response to chemotherapeutic drugs, and shortened survival time. Previously, we reported that, in malignant mesothelioma cells with increased PI3K activity, small-molecule inhibitors of the PI3K/AKT pathway acted cooperatively with the amphibian RNase chemotherapeutic drug ranpirnase to inhibit cell growth. Because the thiazolidinedione antidiabetic drug rosiglitazone targets the PI3K/AKT pathway, we investigated the effect of the combination of these two drugs in cell survival in several cancer cell lines. We show here that the combination of ranpirnase and rosiglitazone synergistically decreases cell viability and increases cell apoptosis in several cancer cell lines. Cell killing is associated with decreased Fra-1 and Survivin expression and knockdown of Fra-1 increases cell killing by ranpirnase in a dose-dependent manner but not by rosiglitazone. The drug combination does not have a synergistic effect on killing in Fra-1 knockdown cells, showing that Fra-1 modulation accounts in part for the synergism. The novel drug combination of ranpirnase and rosiglitazone is a promising combination to treat cancers with increased PI3K-dependent Fra-1 expression or Survivin.

Beck AK, Pass HI, Carbone M, Yang H.

New York University School of Medicine, 530 First Ave., Suite 9V, New York, NY 10016, USA,

Abstract

Ranpirnase, originally isolated from oocytes of the northern leopard frog (Rana pipiens), is a member of the pancreatic RNase A superfamily of ribonucleases. Ranpirnase exerts antiproliferative and cytotoxic effects in vitro and in vivo and has been shown to act synergistically with different cancer therapeutic agents. The cytotoxic and cytostatic effects of ranpirnase are the consequence of tRNA degradation that results in the disruption of protein translation and the induction of programmed cell death (apoptosis). Ranpirnase has been shown to target malignant cells both in human cancer cell lines and in animal models, and has demonstrated efficacy in the treatment of several human cancers in clinical studies. Most clinical studies have been conducted in patients with malignant mesothelioma, and a confirmatory Phase IIIb trial is currently underway for the treatment of this disease. Owing to its selective destruction of malignant cells and favorable toxicology profile, ranpirnase is a promising antitumor agent with ideal attributes that are generally lacking in conventional cytotoxic drugs.

Assaly M, Bongiovanni M, Kumar N, Egger JF, Pelte MF, Genevay M, Finci V, Tschanz E, Pache JC.

Department of Pathology, Geneva University Hospital, Geneva, Switzerland.

Abstract

Background: Ranpirnase, a cytotoxic amphibian ribonuclease, is effective against cancer cells, inducing apoptosis independently of p53 protein. Onconase® (the smallest member of the RNase A superfamily) has moved into clinical testing in the US and Europe.

Objective: The main focuses of this review are to examine the manipulation of tumour physiological parameters by ranpirnase and discuss its molecular, pharmacological and physiological roles in preclinical and clinical trials in terms of benefits and toxicity.

Methods: Relevant literature, including the author’s unpublished presentations at recent conferences, was examined.

Results/ Conclusion: In animal studies, improvements in tumour physiology (i.e., increased blood flow, inhibited oxygen consumption, increased oxygenation and decreased tumour hypertension) and selectively enhanced radiation responses (i.e., increased radiation sensitivity and inhibited repair of sublethal and potentially lethal damage) were observed after ranpirnase treatment in preclinical tumour models. Ranpirnase is a promising candidate as an enhancer for radiation- and chemotherapy. Ongoing clinical trials promise to further improve the treatment of mesothelioma and lung cancer.

Ita M, Halicka HD, Tanaka T, Kurose A, Ardelt B, Shogen K, Darzynkiewicz Z.

Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, NY, 10595; Ita Dental Clinic, Kama-city, Fukuoka 820-0501, Japan.

Abstract

Onconase (Onc), a ribonuclease from oocytes or early embryos of Northern Leopard frog (Rana pipiens), is cytostatic and cytotoxic to a variety of tumor lines in vitro, inhibits growth of tumors in animal in vivo models and is currently in Phase IIIb clinical trials for malignant mesothelioma where it displays antitumor activity with minor overall toxicity to the patient. One of the characteristic features of Onc is a synergism with a variety of other antitumor modalities. Cepharanthine (Cep), a biscoclaurine alkaloid from Stephania cepharantha Hayata, is widely used in Japan to treat variety of ailments. It also shows low toxicity to patients. The aim of the present study was to assess the interaction of these two drugs on different tumor cell lines. When human promyelocytic leukemia HL-60, histiomonocytic lymphoma U937, multiple myeloma RPMI-8228, prostate carcinoma DU 145 and prostate adenocarcinoma LNCaP cells were exposed to relatively low concentrations of Onc or Cep their growth rates were somewhat suppressed but the cells were still able to proliferate. Cell growth, however, was totally abolished in each of these cell lines when treated with Onc and Cep combined. The frequency of apoptosis was also many-fold higher in cultures treated with a combination of Onc and Cep than in respective cultures treated with Onc or Cep alone. The mechanism of the observed synergism is unclear but it may be associated with the Onc activity in targeting microRNAs and/or NFkappaB and Cep activity also targeting NFkappaB. The data suggest that the combination of these two drugs, that individually express a low toxic profile, may have strong antitumor potential.