Experimental Oncology 31, 127–133, 2009 (September) Exp Oncol 2009 Review 31, 3, 127–133

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RIBONUCLEASES IN TUMOR GROWTH V.A. Shlyakhovenko R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine, Kiev 03022, Ukraine This review summarizes data on ambiguous biological functions of ribonucleases (RNases) at tumor growth. In some cases the raised level of enzyme activity in biological fluids can be regarded as an additional marker of malignant growth (pancreas cancer, chronic myeloid leukemia, etc.). At  the  same time the  activity of  RNases is  often lowered in  tumor tissue. High substrate specificity of particular RNases provides metabolic balance between various kinds of RNAs with various half-time exchange turn. RNases are the important factors of epigenetic regulation of gene activity in cells. The activity of RNases is adjustable by inhibitors and other factors, and defines time of existence of different kinds of RNAs. RNases (the modified variants of RNase A, RNases of semen fluid of the cattle, RNase of amphibia oocytes) can be used as anti-tumor therapeutic agents. On the other hand, some inhibitors of RNases of natural or synthetic origin were demonstrated to be perspective drugs that inhibit tumor growth. Key Words: ribonuclease, RNA, enzymes, epigenetic regulation, cancer treatment.

Ribonucleases (RNases) are an exclusively various group of enzymes, capable to degrade various kinds of RNA. Each of principal species of RNA (ribosomal, transport and messenger) has quite certain duration of existence supervised by a certain class of RNA- splitting enzymes and corresponding inhibitors. Earlier it was considered that RNases carry out only one certain function- to degrade overaged molecules of RNA. Further, however, it was found that functions of  these enzymes are  much wider and  fall outside the  limits of  simple destruction of  RNA  molecules. RNases carry out  the  basic function in  processes of  splicing [1] and  processing [2] of  polyribonucleotides. In cases of alternative splicing RNases can considerably change the structure of synthesized protein molecules encoded by a certain gene, and therefore can be synthesized not one, but various protein products possessing the  different properties. Additional influence on  the  destiny of  various polyribonucleotides can  render ribozymes [3–5] and  numerous of  RNase inhibitors, ions of  metals, and  also local values of  pH  inside of  various cell compartments. Notorious opinion is that in rapidly proliferating cells activity of RNases are lowered, that is dictated by requirement of significant amounts of RNA for protein synthesis. Really, in the cells of many tumours activi­ty of  RNases is  lowered [6–8]. At  the same time there are exceptions. At malignant neoplasms of pancreas the  level of  RNases in  tumour tissue and  in  a  blood plasma is raised [9–11]. Moderately elevated values of  serum RNase activity was  found in  patients with 6 different neoplasms, but  strikingly abnormal elevations occur in  serum of  patients with pancreatic cancer [9]. The conclusion was made that abnormal Received: May 29, 2009. *Correspondence: Fax: +38 (044) 258-16-56 E-mail - [email protected] Abbreviations used: ALK — tyrosine kinase of anaplastic glioma; ANG — angiogenin; NSCLC — Non small cell lung carcinoma; ONC — onconase; PHPMA — poly[N-(2-hydroxypropyl) methacrylamide]; RNases — ribonucleases; RNaseL — interferon-induced latent ribonuclease.

elevation of  serum RNase acti­vity serves as  reliable biochemical marker of  carcinoma of  the  pancreas in the presence of the normal renal function [9]. In later research by  Doran et  al. [11] serum RNase activity has been measured in 61 patients with a variety of inflammatory and malignant conditions. Serum RNase activity is elevated in patients with pancreatic carcinoma, but it is not restricted to this condition. Activity was significantly elevated in cases of disturbed liver function [11]. In blood plasma and urine of patients with a chronic myeloid leukemias RNase activity raises significanly [12]. High RNase activity of eosinophil granules allows to assume the important role of this enzyme at an eosinophilia and its participation in allergic reactions [13, 14]. Ambiguous were finding the changes of RNases activity at a prostata cancer [15–18]. Recently the interferon antiviral pathways and prostate cancer genetics have converged on a specific endoRNase. Studies from laboratories the USA, Finland and Israel suggest that mutations in RNASEL (interferon-induced latent RNase, RNaseL) gene predispose men to incidence of  prostate cancer. In  some cases such mutations reflect more aggressive disease and/or  decreased age of onset compared with non-RNASEL linked cases [15]. Being based on these data certain authors suggest a relationship between innate immunity and tumor suppression. The  RNASEL gene encodes a  singlestranded specific endoRNase involved in the antiviral actions of the interferons. Enzyme is activated after binding to unusual 2’-5’oligoadenilates (2-5A). Stable phosphorothioate analogues of  2’-5’A  synthesized chemically induced RNaseL activity and  caused apoptosis in  cultures of  metastatic human prostate cancer cell lines. The  deficiency in  RNaseL activity was correlated with a reduction its ability to dimerize into catalytically active form [16]. Because AfroCarribeans are  at  high risk of  developing prostate cancer, Shea et al. [17] re-sequenced the positional candidate gene RNASEL in 48 prostate cancer cases and genotyped the previously reported polymorphisms in 230 prostate cancer cases and 458 controls. Results

128 obtained suggest that variation in  the  putative cancer susceptibility gene RNASEL or its inhibitor does not  contribute significantly to  prostate cancer risk in this population [17]. Also Larson et al. [18] found that prostate cancer in  patients with R4  620 allelic mutation in  HPC1/RNASEL gene is  not  associated with more aggressive clinical or pathological features in specimens investigated. Angiogenin, a  protein capable of  promoting angiogenesis and possessing RNase activity was found to  be  markedly elevated among a  sub-group of  patients with progressive melanoma. High angiogenin levels were significantly associated with poor treatment response with chemoimmunotherapy. Treatment-related survival (TRS) was shorter (10 months) in patients with above-median values than in those with belowmedian levels (19 months). These data suggest that serum RNase agiogenin might be of predictive value in  the  evaluation of  treatment response for  patients with melanoma [19]. Some authors believe that RNases serve as  cytotoxic agents during host defense and  provide physiological cell death pathways. In bacteria, plants and mammals RNases can bind target cells, degrade RNA  and  kill the  cells. Such events occur in  the  interstrain competition in  bacteria, in  the  death of  incompatible pollen in  the  higher plants and  play role in the antiparasitic and anticancer activity of eosinophils in man [14, 20]. The eosinophil-derived neurotoxin (RNase) has been suggested to be the putative ani-Kaposi’s  sarcoma cells compound [14]. RNase H1 takes place in  generation of  mitochondrial DNA. Cerritelli et al. [21] found that RNaseH–/– mice resulted in significant decrease in mitochondrial DNA content leading to apoptotic cell death. Immunosuppressive cytokine interleukin-10 possess DNA and RNA hydrolyzing activity due to structural homology with endogenous differentiation factor for  the  HL-60 line of  human promyelocyte leukemia cells. The  human recombinant interkeukin-10 was  shown to  cleave all  forms of  plasmide DNA and plays significant role in apoptosis induction in monocyte cells [22]. Ability of RNases to degrade various kinds of RNAs and block protein synthesis began an occasion to attempts to use RNases as the antitumor drugs. Pancreatic RNase has appeared not suitable in this respect. Antimitotic action of enzyme was shown only at very high concentration and  was  insufficient for  a  stunt of tumour growth. At the same time RNase from bovine semen and testis (BS) exhibits anticancer activity. BS RNases exist as two dimers — swapped and unswapped. Cytotoxic and antitumor activity is a peculiar property of  the  swapped enzyme. It  was  found that in  the  reducing environment unswapped dimer dissociates into monomers whereas the  swapped one generates a metastable dimeric form with a quaternary assembly that allows the molecule to escape the protein inhibitor of RNases [23].

Experimental Oncology 31, 127–133, 2009 (September) Variants and  homologs of  bovine pancreatic RNase  A can  exhibit cytotoxic activity. This toxicity relies on  cellular internalization of  the  enzyme. Fuchs and  co-workers [24] found that replacing Glu49 and  Asp53 with arginine does not  affect catalytic activity or affinity for the cytosolic RNase inhibitor. This “arginine graft” does however, increase toxicity towards human cancer cells. Appending a nonargini­ne domain to this cationic variant of enzyme results in an additional increasing cytotoxicity, providing one of the most known cytotoxic wariants of RNase A [24]. These findings correlate the  potency of  a  RNase with its  deliverance of  ribonucleolytic acti­vity to  the  cytosol, and  indicate a rationale to enhance cytotoxic efficacy of RNases and other proteins. Several RNases, including onconase and  alphasarcin, are known to be toxic to tumor cells. RNase T1, although its structure is related to that of alpha-sarcin, is  noncytotoxic because of  its  inability to  internalize into tumor cells. Yuki and  others found that when RNase T1 was  internalized into human tumor cells via a novel gene transfer reagent, hemagglutinating virus of  Japan (HVJ) envelope vector this resulted in cell death. This cytotoxicity was powerfully increased by pretreatment of HVJ envelope vector with protamine sulfate and was stronger than that of onconase, which is in phase III human clinical trials as nonmutagenic cancer chemotherapeutic agent [25]. RNase T1 induced program of apoptotic cell death. Because of this cytotoxicity is  not  specific to  tumor cells, enzyme can not be developed as anticancer drug, but authors believe that other enzymes incorporated in HVJ vector will be a unique anticancer drug if HVJ envelope vector can be targeted to tumor cells. Ribotoxins are  a  family of  highly specific fungal RNases that inactivate the  ribosomes by  hydrolysis of  a  single phosphodiester bond of  the  28S rRNA. Enzyme promotes apoptosis of  human tumor cells after internalization via  endocytosis. This ability is related to its interaction with phospholipid bilayers and  shared structured core with nontoxic RNases of  the  RNase  T1 family. Garcia-Ortega et  al. [26] found that deletion of  the  NH2-terminated β-hairpin of the ribotoxin α-sarcin produces a nontoxic but active RNase. Results open a possibility of engineering RNases for the preparation of specific cytotoxins. The antiproliferative action of the guanine-specific RNase secreted by  Bacillus intermedius (binase) was studied in different chicken and mouse cell lines. The proliferation rate of chicken embryo fibroblasts, either normal or  Rous sarcoma virus transformed was significantly reduced by binase treatment. Among mouse fibroblasts V-Ras  transformed NIH  3T3 cells were sensitive to binase, whereas the growth of nontransformed, v-src-transformed or v-fms-transformed NIH  3T3 cells was  not  affected [27]. Considering the insufficient antitumor activity of natural RNases, there have been begun attempts to  modify their

Experimental Oncology 31, 127–133, 2009 (September) structure on purpose to increase their transmembrane permeability, or cytotoxicity concerning tumor cells. Chemically coupling RNases to  new  binding moieties or  fusing RNase genes to  antibody genes results in chimeric molecules with specified cell-type cytotoxicity. This allows the  use  of  human enzymes instead of plant and bacterial toxins, in the construction of  immunotoxins . RNases can  be  engineered to  kill cells by  cytosolic expression or  to  kill viruses by packaging into viruses. Egineering RNases into cell-type-specific cytotoxins may  result in  a  new  class of  therapeutic agents. De  Lorenzo et  al. [28] reports the  preparation and  charac­terization of  fully human antitumor immuno-RNase (IR). Human RNase and  fusion protein made up of a human single chain of variable immunoglobulin fragment is directed to the ErbB2 receptor that overexp­ressed in many carcinomas [28]. Anti-ErbB-2 immuno-RNase retains the  enzymatic activity of the wild-type enzyme and specifically binds to ErbB-2 — positive cells with high affinity. Enzyme behaves as an immunoprotoxin. Administrated in five doses of 1.5 mg/kg to mice bearing an ErbB-2-positive tumor hErb-hRNase induced a dramatic reduction in tumor volume [28]. Two  anti-Erb2 immunoconjugates, called ErbhRNase (erbicin) and Erb-hc Ab have been prepared by De Lorenzo et al. [29]. It was found that conjugates were selectively cytotoxic on  ErbB2-positive cancer cells in  vitro and  in  vivo. In  Erb-hRNase, erbicin was linked to the key structural and functional regions of  a  human IgG  [29]. Authors found that antitumor action of Herceptin and that of the novel agents were significantly increased in  an  additive fashion. An  inspection of the mechanism of action of Erb-hRNase or  Erb-hcAb combined with Herceptin provided evidence that the antibody combinations engendered an increased downregulation of the ErbB2 receptor, and led to an enhanced apoptotic cell death. Pouckova et al. [30] show that polymer-conjuga­ted bovine pancreatic and bovine seminal RNases inhibit growth of human tumors in nude mice. Suzuki et al. [31] mutated two human RNases — pancreatic RNase and  eosinophil-derived neurotoxin to  incorporate cysteine residues at  putative sites of  close contact to  RNase inhibitor but  distant to  catalytic sites. Coupling of  Cys89 of  RNase and Cys87 of eosinophil-derived neurotoxin to proteins at these sites via thioether bond produced enzymatically active conjugates that were resistant to inhibitor. The transferrin-recombinant human RNase thioether conjugate was 5000 fold more toxic to U251 cells than recombinant wild-type hRNase. Transferrin-targeted eosinophil-derived neurotoxin exhibited tumor cell toxi­cities similar to those of hRNase. So, authors endowed two human RNases sensitive to inhibitor with greater cytotoxicity by  increasing their resistance to inhibitor. This strategy shows the possibility to gene­ rate a novel set of RNases useful for targeted strategy of cancer therapy [31].

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Rutkosky et  al. [32] designed variants of  RNase  A  that evade of  RNase inhibitor.Three related variants of  RNase A  were more resistant to cytosolic inhibitor and more toxic to human cancer cells than was  amphibian RNase (onconase). Results obtained by researchers demonstrate the utility of the new techno­logy in the examination of proteinprotein interfaces and represent a landmark towards the  goal of  developing chemotherapeutics based on mammalian RNases. Recently, hydrophilic poly[N-(2-hydroxypropyl) methacryl-amide] (PHPMA) was used for BS-RNase modifications to  prevent its  degradation in  bloodstream or fast elimination [33]. Polymer-conjugated bovine seminal RNase preparation proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was  ineffective. RNase unimer was  conjugated with two  HPMA polymers and  their antitumor effects both in  vitro and  in  vivo were compared with those of  BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates were also pronounced. The RNase conjugates injected intravenously to  mice bearing melanoma tumor caused significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A, while native BS-RNase (50 μg/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125-I labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an 1 h, whereas the  polymer conjugate (S-BS) was  not  internalized. On  the  contrary, the  in  vivo experiments showed that whereas 40% of S-BS conjugate persisted in  blood stream for  24 h  after administration, 98% of the native BA-RNase was already eliminated [33]. Interestingly, RNases selectively kill tumor cells via  induction of  caspases activity and  promoting apoptosis [34–37]. Ranpirnase is an amphibian oocyte or early embryo RNase of  105 amino acids in  length that is  capable of controlling tumor growth by degrading RNA withing cancer cells, resulting in inhibition of protein synthesis and arresting mitosis in G1 phase. It represents the first successful isolation, purification and characterization of the embryonic factor capable controlling cell growth of  the  early embryonic tissues [38]. This enzyme is a novel class of cancer chemotherapeutic agents, based on homologs and variants of bovine pancreatic RNase. Ranpirnase in combination with doxorubicin is in clinical trials for the treatment of nonresectable malignant mesothelioma and other cancers. The putative mechanism for rainpirnase cytotoxicity involves binding to  anionic components of  the  extracellular membrane, cytosolic internalization and degradation of transfer RNA leading to apoptosis [39]. The  maintenance of  ribonucleolytic activi­t y in  the  presence of  the  cytosolic RNase inhibitor is a key aspect of the cytotoxic activity of ranpirnase.

130 But the real basis of the specific toxicity against cancer cells is not known. In cell culture studies ONC significantly inhibited tumor growth of  A549 human non-small cell lung carcinoma (NSCLC) cells without damaging non-cancerous cells (HLF-1 human lung fibroblasts). Multiple small doses of ONC significantly prolonged tumor growth of  A549 tumors, with increased apoptosis in vivo from 0.5 ± 0.3 to 70 ± 1.1%. Interestingly, multiple small doses of ONC were more effective than a single large dose for the tumor growth inhibition with minimal side effects [38]. Alfacell Corporation is conducting phase III of registration trials of  ranpirnase plus doxorubicin versus doxorubicin alone in more than 360 patients with nonresectable malignant mesothelioma, and  will asses survival as  the  primary endpoint. The  targeted treatment group in this trial represents 90% of malignant mesothelioma patients at the time of diagnosis. The trial is being conducted in the US, Canada, Poland, Italy, Germany, Australia, New Zeland, Russia, Romania, Mexico, and Brasil [38–41]. Cytotoxic­ RNases such as ranpirnase, represent a novel mechanism-based approach to  anticancer therapy. Based on  phase I  study data the  maximum tolerated dose (MTD) was found 960 microgr/m2 with the dose limiting toxicity (DLT) characterized by proteinuria with or without azotemia, peripheral edema and fatigue. Ranpirnase did not induce myelosuppression, mucositis, alopecia, cardiotoxicity,coagulopathy, hepatotoxicity or adverse metabolic effects. Phase II tumor-specific trials investigated the  activity of  ranpirnase in  malignant mesothelioma, breast cancer, non-small cell lung cancer, and renal cell cancer. A phase III randomized study in malignant mesothelioma patients compares the  combination of  ranpirnase plus doxorubicin to doxorubicin monotherapy [40, 42]. Onconase (ONC), isolated from amphibian oocytes was used to study its effect on the radiation response in A549 human NSCLC in vitro and in vivo [41]. In cell culture studies authors found that ONC  increased the  radiation response by  ONC-induced inhibition of  O2 consumption. The  occurrence of  apoptosis was increased by ONC and was dependent on dosa­ ges and  time exposure (measured by  a  TUNNEL assay). Moreover, ONC  inhibited sublethal damage repair in  a  split-dose experiment. In  animal studies ONC  significantly increased the  radiation-induced tumor growth delay of  A549 tumors in  vivo. Authors concluded that the  ONC-induced enhancement in tumor oxygenation was mainly due to the reduction in  QO2 rather than an  increase in  tumor blood flow [40]. Administration of  1.67 µM  ONC  into cultures of HL60 cells led to appearance of cells that had features characteristic of  apoptosis. Studies indicated that ONC induces apoptosis of the target cells most likely along the mitochondrial pathway involving caspase-9 as  the  initiator caspase [39]. Interestingly, that during apoptosis caspases and  Ser-proteases may transactivate each other [37].

Experimental Oncology 31, 127–133, 2009 (September) The  generation of  micro RNAs is  dependent on the RNase III enzyme DICER, the levels of which vary in different normal cells and in desease states. Type I interferons repress DICER protein in contrast to  IFN-gamma, which induces DICER [43]. In  this connection ribonomic profiling and strategic merging genomic technologies was proposed for the induction or interruption of cellular growth in cancer [44]. The discovery that RNA can act as hydrolytic enzyme apart from carrying genetic information has gi­ ven a new direction to the gene therapy. Ribozymes can be used to down-regulate by RNA cleavage or repair by RNA transsplicing unwanted gene expression involved in cancer [45, 46]. Powers et al. [4] reduced tyrosine kinase of  anaplastic glioma (ALK) by  ribozyme targeting and demonstrated that this prevents pleiotrophin-stimulated phosphorylation of  the  antiapoptotic protein Akt. This depletion of ALK reduced tumor growth of the xenografts in athymic nude mice and prolonged survival of the animals because of increasing of apoptosis in the tumors. There is, however, a number of certificates in favor of  that some RNases can  stimulate tumor growth. Some of  them are  produced by  tumor cells. There is a number of certificates that suppression of activity of certain intracellular RNases can have inhibiting effect on a cellular proliferation and tumor growth. Angiogenin (ANG), a  14.4 kDa  monomeric basic protein was originally identified as tumor-derived angiogenesis factor [47]. However, further it was shown that angiogenin possesses RNase activity. Monoclonal antibodies and an antisense oligonucleotides directed against angiogenin inhibited growth of human colon, prostate, breast, lung and fibroblast tumors in athymic mice [48–50]. ANG mRNA and protein was elevated in  colorectal [51, 52], gastric [51], pancreatic [53], breast [54, 55], prostate cancers [56], and melanoma [57], comparing with corresponding normal tissues. In some cases high level of ANG was shown to correlate with cancer progression or poor prognosis [52, 53, 58]. ANG was significantly increased in serum of patients with gastric [59], pancreatic [53], ovarian [60], renal cancer [61], and melanoma [62]. An attractive strategy for develo­ping anti-angiogenic drugs is a ribonucleolytic activity of ANG. Enzymic activity is essential for angiogenesis [63-65]. ANG is a member of bovine pancreatic RNase A superfamily and possess 33% sequence identity to pancreatic enzyme [66]. Kao et al. [67] have chosen from 18310 compounds [8-amino5-(41-hydroxybiphenyl-4-ylazo) naphthalene-2sulfonate] (NCI  65828) that possessed the  RNase inhibiting activity. Local treatment with modest doses of  NCI  65828 significantly delayed the  formation of tumors from two distinct human cancer cells in athymic mice [67, 68]. At  the  same time the  compound 65828 at concentrations up to 100 µM did not inhibit culture growth of PC-3, HT-29, or any of 57 other tumor cell lines tested. Thus, antitumor activity of this compound does not  depend on  the  direct effect

Experimental Oncology 31, 127–133, 2009 (September) on tumor cells but are consistent with inhibition of angiogenesis. Polakowski et  al. [69] found that recombinant RNase inhibitor could inhibit angiogenesis and  reduce tumor growth in  adult mice. Cell penetration into a polyvinyl alcohol sponge was reduced to 29% of control (PBS or heat inactivated RNase inhibitor). Inhibitor caused significant reduction in  mammary tumor growth that autors devoted to  angiogenesis inhibition. Human RNase inhibitor is  an  acid protein with molecular weight of  50 kDa. Fu  et  al. [66] cloned cDNA gene RNase inhibitor (RI) and inserted in  retroviral vector pLNCX. The  combined vector pLNCX-RI  was  transfected into retroviral packa­ging cells, and  a  clone producing a  high titer of  virus was obtained. Next, isolated hematopoietic cells from mice bone marrow were infected with virus carrying the pLNCX-RI. After administration of hematopoietic cells, carrying the RI gene mice were implanted with B16 melanoma cells. The results showed that tumors of control groups became large and well vascularized. In contrast, tumors in mice treated with hematopoietic cells carrying RI gene were small and possessed a re­ latively low density of blood vessels. The rate of tumor growth inhibition was 47%. Thus, we  can  summarize that RNases represent an extensive group of the essential enzymes for a cell which function provide necessary balance between processes of  synthesis, operation and  destruction of various classes of RNA in the cells on various phases of a cell cycle. Function of these enzymes as the critical epigenetic regulators, in some cases provides signaling tools between cells [1, 70]. Therefore, working out the new ways of influence on RNase activity as well as discovery the new hydrolytic enzymes highly speci­fic on the certain sites of ribonucleic acids, can become the important approach to creation the new directions in cancer treatment.

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