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Published March 18, 2015 | public
Journal Article

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H_3 5,10,15-tris(pentafluorophenyl)corroles

Abstract

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy. Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UVVis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action. Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control. The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide. This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

Additional Information

© 2015 JoVE. Date Published: 3/18/2015. We sincerely thank Dr. Cindy N. Chiu for help with gel electrophoresis, and Andy Zhou and Michael Grodick for their generous donation of DNA and restriction enzyme. We gratefully acknowledge Professor J. Heath and Professor D. Prober for generous access to equipment and materials. We thank Dr. Karn Sorasaenee for helpful suggestions. We thank Mary H. Tang for creating the illustration used in the schematic overview in the video. Funding was provided by Johnson & Johnson and USC Y86786.

Additional details

Created:
August 20, 2023
Modified:
October 23, 2023