Constitutive expression and anticancer potency of a novel immunotoxin onconase-DV3

Our plain-English summary of Constitutive expression and anticancer potency of a novel immunotoxin onconase-DV3, by M. Sun, H. Tang, Y. Gao, et al (Oncology Reports, 2016).

The study

• Authors: M. Sun, H. Tang, Y. Gao, X. Dai, Y. Yuan, C. Zhang, D. Sun
• Journal: Oncology Reports
• Published: 2016;35(4):1987-94
• Cited by: 7 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Onconase is an RNase of the ribonuclease A superfamily that is purified from the Northern leopard frog (Rana pipiens). It targets several types of malignant tumors, digests cytoplasmic transfer RNA (tRNA), and causes tumor cell apoptosis. Onconase has been employed in clinical trials as an antitumor drug, and has revealed its valuable clinical activity in several types of tumors, particularly pleural mesothelioma. However, its inefficiency in targeting tumor cells and its non‑specific toxicity in normal tissues have diminished its clinical benefits. Furthermore, cyclization of the N-terminal glutamine residue (Gln), possesses more RNase activity than the structure of Met ahead of Glu in the N-terminal (99:1), which is more difficult for producing onconase by Pichia pastoris. Under the guidance of α-mating factor-pre (α-MF-pre) secretion signal, the secretion of the recombinant protein can reach a high level. In the present study, we constructed a constitutive expression vector for onconase-(DV3)2 (Onc-DV3) production in yeast Pichia pastoris with the GAP promoter, in which the Onc-DV3 gene is inserted downstream of the truncated Saccharomyces cerevisiae α-mating factor-pre (α-MF-pre) secretion signal. The immuno-RNase Onc-DV3 expressed a high level of production and bioactivity and possessed enhanced capability to deliver the Onc molecule to tumor cell monomeric counterparts. Notably, Onc-DV3 showed strong cytotoxicity to highly metastatic tumor cells, weak cytotoxicity to lowly metastatic tumor cells and no toxicity to normal cells. These results demonstrate that the specific toxicity to highly metastatic tumor cells has made Onc-DV3 a promising antitumor drug by using two copies of DV3 for the targeted delivery of onconase.

In plain English

This was a laboratory (in vitro) study in which the authors built a constitutive expression vector, pGAPZαA-Pre-Onc-DV3, placing a synthesized 560 bp Pre-Onc-DV3 fusion gene under the GAP promoter downstream of the truncated S. cerevisiae alpha-mating factor pre secretion signal, and transformed it into Pichia pastoris X33. Twenty-one transformants were selected on plates with 400 micrograms per ml Zeocin. Glucose was reported as the most favorable carbon source and peptone the most favorable nitrogen source, each giving a maximum expression of about 310 U/l after 60 hours; expression was highest at initial pH 7.0, while temperature made little difference. In a 50-litre fed-batch fermentor the biomass (OD600) rose to about 300 and product increased up to 72 hours. The purified protein ran as a single band near 20.0 kDa and retained RNase activity. In MTT tests at 0.4 micromolar Onc-DV3, survival was 24.79 plus or minus 7.31 percent for PC-3M-1E8 and 125.79 plus or minus 19.32 percent for HEK293 cells. The authors concluded Onc-DV3 is a promising antitumor drug. Onconase is noted as developed for malignant pleural mesothelioma.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study: construction of a yeast (Pichia pastoris) constitutive expression system for a recombinant immunotoxin, with fermentation optimisation, protein purification and in vitro cell cytotoxicity testing
• Who was studied: Pichia pastoris X33 and E. coli TOP10 host strains, and eight human cell lines (three highly metastatic tumour lines, three less metastatic tumour lines, two normal/non-tumour lines); work carried out at Jilin University, Changchun, China (article received August 2015)
• Exposure: Recombinant immunotoxin Onc-DV3 (onconase linked to two copies of DV3) applied to cells at serial dilutions of 0.05 to 3.20 micromolar for 72 hours; fermentation variables tested were carbon source, nitrogen source, temperature and pH
• Main results:
  • Glucose was reported the most favorable carbon source, with maximum immunotoxin expression of about 310 U/l after 60 h of culture
  • Peptone was reported the most favorable nitrogen source, with maximum expression about 310 U/l after 60 h
  • Initial medium pH had little effect on biomass, but Onc-DV3 expression was highest at pH 7.0; biomass and production varied little with temperature (30 degrees C selected)
  • In the 50-litre fermentor, biomass (OD600) reached about 300 and Onc-DV3 production reached about 2x10^3 U/l at 72 h, compared with 3.1x10^2 U/l in shake flask culture (the Methods section of the same article gives different figures)
  • Purified recombinant immunotoxin reported at 97.0% purity with 12.8% recovery in the text, while Table I lists 96.1% purity and 26.8% cumulative yield after Sephadex G75; the purified protein gave a single SDS-PAGE band at about 20.0 kDa
  • In the MTT assay at 0.4 micromolar Onc-DV3, survival rates were 24.79 plus or minus 7.31 percent (PC-3M-1E8), 25.85 plus or minus 6.72 percent (PG-BE1) and 27.61 plus or minus 9.16 percent (MDA-MB-231) for the highly metastatic lines; 53.38 plus or minus 17.37 percent (PC-3M-2B4), 55.19 plus or minus 10.85 percent (MCF-7) and 59.64 plus or minus 8.03 percent (PG-LH7) for the poorly metastatic lines; and 107.13 plus or minus 21.80 percent (L-02) and 125.79 plus or minus 19.32 percent (HEK293) for the normal cells
  • By western blot, recombinant immunotoxin was present at 35 h and highest at 65 h in flask culture
• The authors' conclusion: The authors concluded that Onc-DV3 was expressed with high production and high biological activity under the GAP promoter in P. pastoris, that linking onconase to a bivalent DV3 gave selective cytotoxicity towards metastatic tumour cells, and that further study could support its development as a clinical drug

Topics

Medical terms in this study, explained in our Lexicon: Apoptosis, Clinical Trial, Pleural Mesothelioma.

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