A human ribonuclease induces apoptosis associated with p21WAF1/CIP1induction and JNK inactivation

Abstract

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.

RNase MC2: a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKs and induction of caspase pathways

“…Here, our data showed that RNase MC2 selectively induced cell death in MCF-7 cells by virtue of its cytostatic and cytotoxic activities. This finding is reminiscent of the observation of Castro and colleagues that both onconase and PE5 possess cytostatic and cytotoxic activities [9]. Our further investigation disclosed that RNase MC2 could induce tumor cell apoptosis as exemplified by the appearance of stereotypic apoptotic features, including karyorrhexis, chromatin condensation, and DNA fragmentation (Fig.…”

“…After incubation for 24 h, the cells were treated with increasing concentrations (0, 0.195 to 25 lM) of RNase MC2 for 24-48 h, and cell viability was determined by MTT assay [13]. In addition, total cell numbers as well as cell death in different group were counted using a haemocytometer combined with the trypan blue exclusion assay [9]. SPSS 11.0 software was used for IC 50 analysis.…”

“…The IC 50 values at 24 and 48 h were 13 and 6.25 lM, respectively. Since the MTT method does not allow a discrimination of cytostatic effect and cytotoxic effect [9], we examined the effect of RNase MC2 on the proliferation and cell death of MCF-7 cells. Our results demonstrated that RNase MC2 exerted both cytostatic (Fig.…”

“…Onconase has demonstrated some advantages for potential clinical applications, including (a) evading human RNase inhibitors in cytosol, (b) inhibitory activity against a broad types of human tumors, (c) without any untoward immune response and exerting only weak and reversible renal toxicity [6]. The phase III clinical trial of onconase prompts the genetic engineering of known RNases as well as searching for new medicinal RNases [6,8,9].…”

Rana catesbeiana ribonuclease induces cell apoptosis via the caspase-9/-3 signaling pathway in human glioblastoma DBTRG, GBM8901 and GBM8401 cell lines

“…In particular, Onconase has been used as an antitumor agent in clinical trials (7,19,25). Additionally, a previous study reported that Onconase exerts a cytotoxic effect on the human glioma SHG-44 cell line (26).…”