Jessica Castro

A human ribonuclease induces apoptosis associated with p21 WAF1/CIP1 induction and JNK inactivation

BackgroundRibonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.MethodsCytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.ResultsWe show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAPConclusionsWe conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.

A cytotoxic ribonuclease reduces the expression level of P-glycoprotein in multidrug-resistant cell lines

SummaryWe have previously described a cytotoxic human pancreatic-ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells. We have investigated whether PE5 could specifically inhibit the accumulation of P-glycoprotein in multidrug-resistant cells, since P-glycoprotein overexpression is one of the most important mechanisms contributing to the multiple drug resistance phenotype. We show that PE5 is able to reduce the amount of P-glycoprotein in two different multidrug-resistant cell lines, NCI/H460-R and NCI/ADR-RES, while glutathione S-transferase-л is not affected. We also show that onconase, an amphibian ribonuclease that is undergoing phase II/III clinical trials as an antitumor drug, does not affect the expression of these proteins. The reduction of P-glycoprotein accumulation, which has been functionally confirmed by flow cytometry analysis, may be caused by the previously reported underphosphorylation of JNK induced by PE5. We also show that PE5 has synergistic cytotoxicity with doxorubicin on the NCI/ADR-RES multidrug-resistant cell line. In conclusion, PE5 is a cytotoxic ribonuclease that cleaves nuclear RNA and decreases the expression of P-glycoprotein, showing anticancer activity in multidrug-resistant cell lines.

Mini-review: nucleus-targeted ribonucleases as antitumor drugs.

Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. This global burden of cancer continues to increase largely because of the aging and growth of the world population. Although very much progress has been attained in the development of new therapies, there is a clear need of more efficient and selective antitumor drugs for the effective treatment of many types of cancer. Among the different strategies developed to create new antitumor drugs, pleiotropic non-genotoxic effectors have gained interest since this approach is less susceptible to known resistance mechanisms. The cell nucleus is the subcellular compartment where the genetic information and the transcription machinery reside and accordingly where numerous therapeutic agents efficiently work. Hence, nuclear-targeted drugs are expected to kill cancer cells more directly and efficiently. In this review, we discuss the potential of nuclear-targeted drugs as antineoplastic therapeutics and reason the benefits of the strategy to endow ribonucleases with cytotoxic properties based on its targeting into the nucleus.

Contribution of the C30/C75 disulfide bond to the biological properties of onconase.

Abstract Onconase, a member of the pancreatic type ribonuclease family, is currently used as a chemotherapeutic agent for the treatment of different types of cancer. It is widely accepted that one of the properties that renders this enzyme cytotoxic is its ability to evade the cytosolic ribonuclease inhibitor (RI). In the present work, we produced and characterized an onconase variant that lacks the disulfide bond C30/C75. This variant mimics the stable unfolding intermediate des(30–75) produced in the reductive unfolding pathway of onconase. We found that the reduction of the C30/C75 disulfide bond does not significantly alter the cytotoxic properties of onconase, although the variant possesses a notably reduced conformational stability. Interestingly, both its catalytic activity and its ability to evade RI are comparable to wild-type onconase under mild reductive conditions in which the three disulfide containing intermediate des(30–75) is present. These results suggest that the C30/C75 disulfide bond could easily be reduced under physiological redox conditions.

Activating transcription factor 3 is crucial for antitumor activity and to strengthen the antiviral properties of Onconase.

Onconase is a ribonuclease that presents both antitumor and antiviral properties linked to its ribonucleolytic activity and represents a new class of RNA-damaging drugs. It has reached clinical trials for the treatment of several cancers and human papilloma virus warts. Onconase targets different RNAs in the cell cytosol but Onconase-treated cells present features that are different from a simple arrest of protein synthesis. We have used microarray-derived transcriptional profiling to identify Onconase-regulated genes in two ovarian cancer cell lines (NCI/ADR-RES and OVCAR-8). RT-qPCR analyses have confirmed the microarray findings. We have identified a network of up-regulated genes implicated in different signaling pathways that may explain the cytotoxic effects exerted by Onconase. Among these genes, activating transcription factor 3 (ATF3) plays a central role in the key events triggered by Onconase in treated cancer cells that finally lead to apoptosis. This mechanism, mediated by ATF3, is cell-type independent. Up-regulation of ATF3 may also explain the antiviral properties of this ribonuclease because this factor is involved in halting viral genome replication, keeping virus latency or preventing viral oncogenesis. Finally, Onconase-regulated genes are different from those affected by nuclear-directed ribonucleases.

A truncated apoptin protein variant selectively kills cancer cells

SummaryApoptin is a nonstructural protein encoded by one of the three open reading frames of the chicken anemia virus genome. It has attracted a great deal of interest due to its ability to induce apoptosis in multiple transformed and malignant mammalian cell lines without affecting primary and non-transformed cells. However, the use of Apoptin as an anticancer drug is restricted by its strong tendency to aggregate. A number of methods to overcome this problem have been proposed, including transduction techniques to deliver the Apoptin gene into tumor cells, but all such methods have certain drawbacks. Here we describe that a truncated variant of Apoptin, lacking residues 1 to 43, is a soluble, non-aggregating protein that maintains most of the biological properties of wild-type Apoptin when transfected into cells. We show that the cytotoxic effect of this variant is also present when it is added exogenously to cancer cells, but not to normal cells. In addition to the interest this protein has attracted as a promising therapeutic strategy, it is also an excellent model to study the structural properties of Apoptin and how they relate to its mechanism of action.

Insights into the mechanism of Apoptin's exquisitely selective anti-tumor action from atomic level characterization of its conformation and dynamics

Apoptin is a 121 residue protein which forms large, soluble aggregates and possesses an exceptionally selectively cytotoxic action on cancer cells. In the accompanying paper, we described the design, production and initial characterization of an Apoptin truncated variant called H6-ApopΔProΔLeu. Whereas both the variant and wild type protein possess similar selective cytotoxicity against cancer cells following transfection, only the variant is cytotoxic when added externally. Remarkably, as observed by gel filtration chromatography and dynamic light scattering, H6-ApopΔProΔLeu lacks the tendency of wild type Apoptin to form large aggregates, which greatly facilitated the study of its biological properties. Here, we characterize the conformation and dynamics of H6-ApopΔProΔLeu. Using a battery of 2D, 3D and (4,2)D NMR spectra, the essentially complete 1H, 13C and 15N resonance assignments of H6-ApopΔProΔLeu were obtained. The analysis of these data shows that the variant is an intrinsically disordered protein, which lacks a preferred conformation. This conclusion is corroborated by a lack of protection against proteolytic cleavage and hydrogen/deuterium exchange. Moreover, the CD spectra are dominated by random coil contributions. Finally, 1H-15N NOE ratios are low, which indicates flexibility on the ps-ns time scale. Interestingly, H6-ApopΔProΔLeus intrinsically disordered ensemble is not significantly altered by the redox state of its Cys residues or by Thr phosphorylation, which has been proposed to play a key role in Apoptins selective cytotoxicity. These results serve to better comprehend Apoptins remarkably selective anticancer action and provide a framework for the future design of improved Apoptin variants.

A nuclear-directed human pancreatic ribonuclease (PE5) targets the metabolic phenotype of cancer cells

Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells.

Approaches to Endow Ribonucleases with Antitumor Activity: Lessons Learned from the Native Cytotoxic Ribonucleases

Typical antitumor drugs disrupt the flow of biochemical information from DNA to proteins with the aim of precluding uncontrolled cell proliferation and inducing cancer cell apoptosis. However, most of the currently used small antitumor drugs are genotoxic because they act over DNA. Pharmaceutical industry is now searching for a new line of cancer chemotherapeutics without genotoxic effects. Ribonucleases (RNases) are small basic proteins, present in all life forms, which belong to this kind of chemotherapeutics. Some of them present with remarkable selective antitumor activity linked to their ability to destroy RNA, a powerful way to control gene expression, leaving DNA unharmed. In the last two decades, the knowledge gained on the cytotoxic mechanism of these RNases has been used to engineer more powerful and selective variants to kill cancer cells. In this chapter, we describe the advances reached in endowing an RNase with antitumor abilities.

Transcriptional profiling of NCI/ADR-RES cells unveils a complex network of signaling pathways and molecular mechanisms of drug resistance

Background Ovarian cancer has the highest mortality rate among all the gynecological cancers. This is mostly due to the resistance of ovarian cancer to current chemotherapy regimens. Therefore, it is of crucial importance to identify the molecular mechanisms associated with chemoresistance. Methods NCI/ADR-RES is a multidrug-resistant cell line that is a model for the study of drug resistance in ovarian cancer. We carried out a microarray-derived transcriptional profiling analysis of NCI/ADR-RES to identify differentially expressed genes relative to its parental OVCAR-8. Results Gene-expression profiling has allowed the identification of genes and pathways that may be important for the development of drug resistance in ovarian cancer. The NCI/ADR-RES cell line has differential expression of genes involved in drug extrusion, inactivation, and efficacy, as well as genes involved in the architectural and functional reorganization of the extracellular matrix. These genes are controlled through different signaling pathways, including MAPK–Akt, Wnt, and Notch. Conclusion Our findings highlight the importance of using orthogonal therapies that target completely independent pathways to overcome mechanisms of resistance to both classical chemotherapeutic agents and molecularly targeted drugs.