Name: an in vitro enzymatic assay to measure transcription inhibition by gallium(iii) and h3 5,10,15-tris(pentafluorophenyl)corroles
Uploaded: 2015-03-18T13:00:00
Description: Watch this Scientific Journal Video about An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles at JoVE.com

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 &#38; Johnson and USC Y86786.

NOTE: When working with RNA maintain a clean working environment to avoid contamination by DNase and RNase enzymes that degrade DNA and RNA. Ensure that pipette tips and tubes are DNase and RNase free. It is also helpful to wipe down lab surfaces and equipment such as pipettes, tube holders, etc. with a decontamination solution.
1. RNA Transcription with Corrole Treatment
<ol>
	<li>Prepare the corrole and inhibitor compounds in a 0.01:1 molar ratio of [complex]:[DNA]. 
	NOTE: In our cas...

This assay demonstrates that the addition of Ga(tpfc) inhibits RNA transcription comparably to the known DNA-binding anticancer complexes Actinomycin D and triptolide. The cytotoxic behavior of Ga(tpfc) (GI50 = 58.1-154.7 &#956;M) may due to its inhibitive properties. Since no transcription inhibition was observed in tpfc, the cytotoxicity of tpfc is not due to RNA transcription inhibition but is caused by other means not yet studied.
In the four transcription reactions performed, D...

Chemotherapy often involves broad-spectrum cytotoxic agents with undesired side effects and limited targeting, yet with greater understanding of cancer biology, there is an ever increasing demand for anticancer agents with higher cancer-targeting efficacy and fewer side effects.1 Human cancer cells frequently become dependent on a single activated or overexpressed oncogene for survival.2 Thus, many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Treatments that block the expression of these transforming genes are effective in eliminating cancer cells and lead to cell death.3 Transformed cells are more sensitive to disruptions in RNA transcription than are corresponding normal cells.4 Anticancer drugs that inhibit transcription are expected to selectively inhibit the expression of the oncogenes which are necessary for the cancer cell to survive.5 Consequently, RNA transcription inhibition is a useful way to identify potential anticancer drug candidates and learn more about their mechanism of action. This protocol demonstrates that Ga(tpfc) inhibits RNA transcription on the same order as the chemotherapy drugs Actinomycin D and triptolide; similar comparisons can be made using this protocol with other anticancer drug candidates. Actinomycin D is a RNA transcription inhibitor commonly used to treat gestational trophoblastic cancer, testicular cancer, Wilm&#8217;s tumor, rhabdomyosacoma, and Ewing&#8217;s sarcoma6. Actinomycin D has been used in cancer therapy for nearly fifty years since it was first approved by the FDA in 1964.Triptolide is a selective transcription inhibitor that has been investigated in vitro and in various tumor-bearing animal models for 30 years.7
The amphiphilic macrocyclic nature of corroles imparts significant advantages over other drug classes such as small molecules or biologics.8-14 The macrocyclic character allows for cellular permeability that is greater than expected for such large molecules, and they are large enough to interact with macromolecular surfaces, such as those of proteins.8 Corroles are known to form tight noncovalent complexes with biomolecules and drugs.10 In addition to the inherent cytotoxicity of the corrole framework, we have demonstrated that a sulfonated corrole acts as a carrier molecule for chemotherapeutic agents, specifically the DNA-intercalating anthracycline drug doxorubicin. When the sulfonated corrole was coadministered with doxorubicin, a 3-fold enhancement in the IC50 of doxorubicin was observed for DU-145 cells.9 The corrole framework is stable and has inherent absorbance and fluorescence properties that, when functionalized, undergo unique absorbance shifts that can be used for characterization.10 Functionalization of the scaffold does not inherently affect the photophysical properties of the corrole,9-15 but, as seen with a sulfonated corrole, selectively modifying the framework of the corrole can substantially change its biological properties.16 We previously evaluated six metallocorroles against seven human cancer cell lines. The results indicate that toxicity toward human cancer cells is dependent on the specific metal ion, as well as functional group substitution. For instance, sulfonated gallium corroles experienced high cellular uptake and penetrated selectively into the nucleus of brain metastatic prostate cancer cells (DU-145); the same corrole, though it does not penetrate into the nucleus of other cell lines, exhibits greater cytotoxicity for breast (MDA-MB-231), melanoma (SK-MEL-28), and ovarian (OVCAR-3) cancer cells than for prostate cancer.9
Initial cell-based assays indicate that these compounds show promise as anti-cancer therapeutic agents, which merits further investigation into the mechanism of action. Transcription inhibition is observed with certain organometallic complexes17-27 and we sought to examine this process as a possible mechanism for the cytotoxic behavior of the corrole family. This transcription assay provides a straightforward, inexpensive, and facile method for assessing transcription inhibition, which will lead to more detailed information about the effects of these molecules in live cells.
Here, the transcription inhibition of gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and its freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc) (Figure 1) are tested. Unlike some transition metal complexes, gallium(III) is redox inactive and therefore is not directly involved in the redox process of redox-based metabolic pathways.28 Regardless, gallium(III) does exhibit cytotoxic properties and has been investigated for therapeutic purposes. Gallium is the second most promising metal for anticancer therapeutics after platinum and has undergone many studies and investigations; nitrate and chloride gallium salts have been evaluated in clinical trials against hepatoma, lymphoma, bladder cancers, and other diseases.29-34 Gallium(III) is therefore ideal for anticancer corrole studies. Initial data show Ga(tpfc) and tpfc have low GI50, the drug concentration necessary to inhibit 50% of maximal cell proliferation, with various cancer cell lines (see Figure 2); this affirms the validity of further experiments on these two compounds to determine their inhibitive properties. We compare these compounds with the common anticancer drugs Actinomycin D and triptolide. Actinomycin D intercalates DNA, inhibits RNA elongation, and induces apoptosis in certain cell line at picomolar concentrations.6,35-37 Triptolide has shown to inhibit tumor growth; it binds to human XPB/ERCC3, a subunit of transcription factor TFIIH, leading to inhibition of RNA polymerase II activity.6-7,38-40
While it is commonly known that corroles exhibit cytotoxic properties, there exists little information about the different mechanisms arising from functionalization. Corrole inhibition of RNA transcription would offer greater insight on their interactions with biomacromolecules. Other complexes known to bind to DNA, such as dirhodium(II,II) complexes, chromium(III) complexes, ruthenium(II) polypyridyl complexes, rhodium(III) complexes, and various others, were subjected to RNA transcription assays,18-27 resulting in greater understanding of their interactions with biomacromolecules. This facile and widely available experiment is also a good initial test to assess the cytotoxicity properties of a given molecule and determine whether it merits further biological testing. The RNA transcription assay also allows for many modifications, such as varying the quantity of compound or enzymes used; varying the incubation period, reaction time and sample time points; and varying the DNA template length and sequence, among other variables of interest, thus potentially providing a large amount of data. This transcription assay is also readily available as affordable kits with all necessary reaction components provided, although components can be bought and prepared individually. In these experiments, we use a commercially available kit known to have high yield. 41
To assess transcription inhibition, we use two methods: agarose gel electrophoresis and UV-Vis spectroscopy. Agarose gel electrophoresis is a simple and effective method for separating, identifying, and purifying 0.5- to 25-kb DNA and RNA fragments.42 UV-Vis spectroscopy can be used to determine the concentration and purity of RNA.43

<table border="0" cellpadding="0" cellspacing="0" width="809">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Name of Material/ Equipment</td>
			<td style="width:219px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Actinomycin D</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1410</td>
			<td>Store at 2-8 &#176;C , protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triptolide</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3652</td>
			<td>Store at 2-8 &#176;C , protect from light</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">nuclease-free H2O&#160;</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">AM9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript T7 Transcription Kit</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">AM1334</td>
			<td>Store at &#8211;20 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Ethidium Bromide</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7637</td>
			<td>CAUTION: For proper handling procedures of ethidium bromide, please see: http://www.sciencelab.com/msds.php?msdsId=9927667</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Tris Acetate</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T6025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">EDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">UltraPure Agarose</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">16500-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">mini Quick Spin RNA Columns</td>
			<td style="width:219px;">Roche Life Science</td>
			<td align="right" style="width:119px;">11814427001</td>
			<td>Store at 2-8 &#176;C , do not freeze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">1 kb DNA Ladder</td>
			<td style="width:219px;">New England Biolabs</td>
			<td style="width:119px;">N3232S</td>
			<td>Store at &#8211;20 &#176;C&#160;</td>
		</tr>
	</tbody>
</table>

an in vitro enzymatic assay to measure transcription inhibition by gallium(iii) and h3 5,10,15-tris(pentafluorophenyl)corroles

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 UV-Vis 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.

RNA Transcription Qualitatively Assessed by Agarose Gel Electrophoresis
Agarose gel electrophoresis is used to image the transcribed RNA. Ethidium bromide fluoresces upon binding (&#955;em = 605 nm, &#955;ex = 210 nm, 285 nm)46 allowing imaging of RNA. Darker bands in the gel correspond to higher concentrations of RNA. If Actinomycin D, triptolide, or either corrole complex inhibits RNA transcription, the production of RNA is reduced and the ba...

Watch this Scientific Journal Video about An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles at JoVE.com

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Research

JoVE Journal

Bioengineering

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...