Highly selective toxic and proapoptotic effects of two dimeric ribonucleases on thyroid cancer cells compared to the effects of doxorubicin

Abstract

The lack of selectivity of conventional antitumour drugs against cancer cells is responsible for their high toxicity. The development of new tumour-specific drugs is therefore highly needed. We tested the cytotoxic effects and the nature of cell death induced by a naturally dimeric bovine RNase and a newly engineered dimeric human RNase upon three genetically well-defined normal and malignant thyroid cell systems. RNases effects were compared with those of doxorubicin, a conventional antineoplastic drug. Our results show significant and selective proapoptotic effects exerted on tumour cells by both RNases, the strength of their cytotoxic and apoptotic activity being directly related to the degree of cell malignancy. No toxic effects were observed upon normal cells. Doxorubicin showed, instead, cytotoxic and apoptotic effects also against normal cells. The in vitro results were corroborated by the antitumour action of both dimeric RNases towards a malignant human thyroid tumour grown in nude mice. These results indicate a selective action of dimeric RNases against cancer cells and suggest the potential application of these molecules or their derivatives to the treatment of aggressive subtypes of thyroid cancer.

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

“…17 They differ for the swapping of the N-termini (residues 1-15) within a substantially identical quaternary framework. [18][19][20] In both isoforms, the dimeric interface is formed by the hinge peptides (residues [16][17][18][19][20][21][22] and the helices (residues [23][24][25][26][27][28][29][30][31][32][33][34] that comprise the four cysteines forming the two interchain disulfide bridges (Cys31-Cys32 0 and Cys32-Cys31 0 ) and the side chains of Leu28 and 28 0 that form stabilizing hydrophobic interactions across the molecular twofold axis. 18,20 In the most abundant swapped form, MxM-BSRNase, the dimeric interface also includes the closed interface arising from the swapped elements.…”

“…30 Different approaches have been proposed to confer cytotoxicity to HP-RNase; they include (a) sitedirected mutagenesis to weaken its interaction with RI, 23 (b) conjugation of HP-RNase to protein, peptide, and antibodies, which could enhance the uptake by target cells, 31,32 (c) transformation of the protein into a stable dimer capable to evade RI. 33 Following the latter strategy, Piccoli et al 34 have mutated four residues at the helix-II region according to BSRNase sequence (Gln28!Leu, Arg31!Cys, Arg32!Cys, and Asn34!Lys) and have produced a covalent dimeric variant (HHP-RNase), enzymatically active and selectively cytotoxic for several malignant mouse and human cell lines. The elimination of a negative charge on the HHP-RNase surface (Glu111!Gly) has led to a second dimeric mutant, HHP2-RNase, which is more powerful as a cytotoxic agent than BSRNase and is selective for malignant cells.…”

Biological Activities of Secretory RNases: Focus on Their Oligomerization to Design Antitumor Drugs

“…Therefore, considering the high basicity of its native state, RNase 1 has been mutated to insert, as well as in RNase A (299), two 31 + 32 cysteine residues to make it dimerize similarly to BS-RNase: these mutants, named HHP-RNases, displayed a remarkable antitumor activity against several types of malignant cell lines, among which cells from neuroblastoma and rhabdomyosarcoma showed the highest sensibility (307). Indeed, the RNase 1 variant displaying the Cys31 + 32 couple plus the N28L/N34K/E111G mutations, and called HHP2-RNase, exerted a quite high antitumor activity against human thyroid carcinoma-derived cell lines and in vivo when malignant cells were transplanted into nude mice (308). Cytotoxicity was exerted by both swapped and unswapped isomers, contrarily to BS-RNase, and this result was ascribed to the stabilization effect of both the hinge loop and the Leu28 side chain in terms of maintaining the two Cys31/32 residues close to each other (309).…”

Binase and other microbial RNases as potential anticancer agents

“…It is generally assumed that the effect of chemotherapy on cancer cells is based on its ability to induce apoptosis. (43,60,61) To our knowledge, binase was the first bacterial RNase that demonstrated noticeable apoptogenic activity against cancer cells. (47,55) Fig.…”