Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

Abstract

BackgroundRNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.Methodology/Principal FindingsIn search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC50) ranging from 0.2 to 13 µM and to leukemia cell lines with IC50 values ranging from 2.4 to 82 µM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC50 = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.Conclusions/SignificanceThese results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

“…A recombinant single-chain fragment of this humanized monoclonal antibody, 4D5scFv, has been confirmed to be an effective targeting moiety for use in various applications, such as the verification of therapeutic principles: immunotoxins, immuno-RNases, etc. (16,17). Generally, scFvs are considered promising for medical applications because of superior tissue penetration, the absence of side reactions involving the constant domains, and facile engineering of fusion proteins, such as scFv-coupled toxins, enzymes for prodrug activation (ADEPT), or the creation of multivalent or bispecific proteins (18).…”

Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells

“…[1][2][3][4][5] The antitumor activity was shown for BS-RNase from bovine seminal, [6][7][8][9] onconase from oocytes of Rana pipiens, [10][11][12] bovine pancreatic RNase A, 13 cSBL and jSBL RNases from Rana catesbeiana and Rana japonica, 14 microbial RNases [15][16][17] and conjugates of RNases with various molecules. [18][19][20] One of the properties that are important for potential therapeutic application of RNases is their ability to induce cancer cell death by apoptosis.…”

Protein-assisted self-assembly of multifunctional nanoparticles

“…For QD, molar ratios were QD∶EDC∶Protein ¼ 1∶1000∶40. The conjugation efficiency was studied by RNA-hydrolyzing activity of barnase and inhibitary properties of barstar (29).…”