Validation data supporting the characterization of novel copper complexes as anticancer agents

Three copper(II) complexes, Cu(Sal-Gly)(phen), Cu(Sal-Gly)pheamine, Cu(Sal-Gly)phepoxy were synthesized and characterized for their anticancer properties and mechanism of action (Acilan et al., in press) [1]. Here, we provide supporting data on colon cancer cell lines complementing our previous findings in cervix cells. This paper also contains a data table for the fold changes and p-values of all genes analyzed in this study via a custom RT-qPCR array. All compounds induced DNA damage (based on 8-oxo-guanidine, ɣH2AX staining in cells) and apoptosis (based on elevated DNA condensation/fragmentation, Annexin V staining, caspase 3/7 activity and mitochondrial membrane depolarization) in HCT-116 colon cancer cells. The increase in oxidative stress was also further confirmed in these cells. Further interpretation of the data presented here can be found in the article entitled “Synthesis, biological characterization and evaluation of molecular mechanisms of novel copper complexes as anticancer agents” (Acilan et al., in press) [1].

& 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Chemistry, Biology More specific subject area Copper(II) complexes, Molecular biology, Cancer biology, Drug development

Value of the data
The data allows evaluating the stability of the Cu-complexes in aqueous buffers. The presented data displays evidence for the potential anticancer activity of Cu-complexes in human colon carcinoma HCT-116 cells. Therefore, the findings in our original article are not specific for only one cell line, but rather more general.
The table may be valuable in determining/comparing potential molecular targets of other Cucomplexes.
The data may give insight for researchers to design better therapeutic agents and offer a base for comparison between different compounds.

Data
There is still a great surge for potent anticancer agents with well-described activity. In today's world, most successful drugs find their place in the market among thousands of molecules, which were initially synthesized by the educated design of novel molecules with the aim of obtaining ameliorated properties. Therefore, data describing how different molecules act in different types of cells, become of great use. The desired function of anticancer drugs is inhibition of cell proliferation or survival, preferably with activity in cells with different genetic backgrounds, since every cancer is also divergent from one another. Here, we demonstrate the molecular mechanism of action of three different Cu-compounds (see Scheme 1) in HCT-116 cells supporting our previous findings with HeLa cells. We also disclose the full list of genes with fold change and exact p-values of our RT-qPCR analysis in response to Cu(Sal-Gly)(pheamine), the most specific compound against cancer cells (Table 1).

Cu(Sal-Gly)(phepoxy) Cu(Sal-Gly)(pheamine) Cu(Sal-Gly)(phen)
Scheme 1. Formulation of the complexes. Table 1 List of genes studied in the RT-qPCR array. Three housekeeping genes were used in each experiment and each gene was normalized to the average of housekeeping genes. Fold change was calculated as the fold increase compared to untreated controls. An average of two independent experiments (each done in duplicate) is shown on the Table. Standard (Std) errors represent deviations from the mean, and the p-values were calculated using paired samples t-test using SPSS 17.0 software. Only one gene, Harakiri, was found to be statistically significant above the cut-off value of 1.5 fold. Most metallodrugs are not water soluble, and a small % or organic solvent is typically used in biological studies with metal complexes. On the other hand, biomolecules and cells are very sensitive to organic solvents. Therefore it is very important to check the stability of the complexes in aqueous environments; to make sure the compounds will maintain their structure for the necessary time period, without substantial degradation or precipitation prior to the biological evaluation, and to evaluate the effect of the organic solvent in the biological molecules under study, e.g. DNA.
The complexes' stability in aqueous media (see Fig. 1) and organic solvents (Fig. 2) and was confirmed by measuring spectral changes within 1-2 h (UV-vis) and EPR (up to 24 h) after preparation of the solutions. Circular dichroism spectra of CT-DNA in the absence and presence of different % (v/v) of DMSO allowed evaluating the % range of DMSO where no changes in DNA configuration are detected (Fig. 3). The effect of adding DNA to the complexes' solutions was also evaluated by UV-vis spectroscopy (Fig. 4), which did not allow the determination of binding constants, but clearly showed occurring changes. The cytotoxicity of the Cu-complexes were verified using a different assay relying on the amount of total proteins (Sulforhodamine B (SRB) Cell Viability Assay), as an alternative to measurement of change in mitochondrial dehydrogenase enzyme (MTT assay) in A-549, HCT-116 and HeLa cells (Fig. 5).
The increase in oxidative stress was evaluated by the measurement of intracellular DCFDA (Fig. 10A) and the examination of oxidized glutathione (GSSG) by determining the ratio of GSSG/GSH (Fig. 10B) in HCT-116 cells.
Consistent with our findings in HeLa cells, the compounds also appeared to induce oxidative DNA damage as assessed by 8-oxo-Guanindine staining, the most common lesion in DNA in response to oxidative stress (Fig. 11).
In addition to 8-oxo-Guanidine, there was an evident increase in double stranded DNA breaks as judged by ɣ H2AX staining in HCT-116 cells both using flow cytometry (Fig. 12A) and microscopy (Fig. 12B).

Calf thymus DNA binding experiments
UV-Visible absorption (UV-vis) spectra were recorded on a Perkin-Elmer Lambda 35 spectrophotometer at room temperature. Circular dichroism (CD) spectra were recorded at 25°C on a Jasco J-720 spectropolarimeter with an UV-vis (180-800 nm) photomultiplier (EXEL-308). EPR spectra were measured on a Bruker ESP 300E spectrometer at 77 K.
Millipore water was used for the preparation of TRIS and Phosphate Saline Buffer (PBS) buffers (0.10 M, pH ¼7.4). Calf thymus DNA (CT-DNA) was from Sigma (#D3664) and used as received. DNA stock solutions were prepared by dissolution in TRIS or PBS buffer. The stock solutions of the

Sulforhodamine B (SRB) cell viability assay
For SRB assay, 5-8 Â 10 3 cells ( $ 70-80% confluency depending on the cell line) were seeded in 96well plates in regular culture medium in triplicate, overnight. The following day, serial dilutions of complexes, Cu(Sal-Gly)(pheamine), Cu(Sal-Gly)(phepoxy) and Cu(Sal-Gly)(phen) (0.19-12.5 mM), were freshly prepared and added to the cells. After 24 or 72 h, viable cells were fixed with the 50% trichloroacetic acid (TCA) at a final concentration of 10%. Plates were kept at 4°C for 1 h, the supernatant was discarded and the plate was washed with deionized water five times. TCA-fixed cells were stained with SRB solution (0.4% in 1% acetic acid) for 30 min at room temperature (RT). Unbound SRB was removed by washing with 1% acetic acid and air-dried. Bound SRB stain was solubilized with Tris base solution (10 mM, pH:10.0), and plates were left on a shaker (10 min, 150 rpm). Absorbance was read by a spectrophotometer at 570 nm.

Detection of apoptosis
Apoptosis was detected though DNA condensation/fragmentation analysis using immunofluorescence staining (Leica DMI 6000 microscope) as described in [3]. Increase in Annexin V staining, caspase 3/7 activity and mitochondrial membrane depolarization were determined using the Muse Cell Analyzer (Millipore, Hayward, CA, USA) following manufacturer's protocols (Annexin V/Dead Cell kit MCH100105, Caspase 3/7 kit MCH100108, Muse MitoPotential kit MCH100110 respectively) and the details of the protocols are defined further in [1].
2.7. ɣ H2AX assay for the assessment of DNA damage using flow cytometry HCT-116 cells were exposed to the Cu-complexes at the IC 90 concentration for 12 h and stained using Muse ɣ H2AX Activation Dual Detection (kit MCH200101, Millipore, Darmstadt, Germany) as described previously [1]. The data were acquired on the Muse Cell Analyzer (Millipore, Hayward, CA, USA).