A paradoxical relationship between Resveratrol and copper (II) with respect to degradation of DNA and RNA

Siddharth Subramaniam; Iqbal Vohra; Aishwarya Iyer; Naveen K Nair; Indraneel Mittra

doi:10.12688/f1000research.7202.2

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Observation Article

Revised

A paradoxical relationship between Resveratrol and copper (II) with respect to degradation of DNA and RNA

[version 2; peer review: 2 approved]

Siddharth Subramaniam¹, Iqbal Vohra¹, Aishwarya Iyer¹, Naveen K Nair¹, Indraneel Mittra¹

Siddharth Subramaniam¹, Iqbal Vohra¹, [...] Aishwarya Iyer¹, Naveen K Nair¹, Indraneel Mittra¹

PUBLISHED 02 Mar 2016

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¹ Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi-Mumbai, 410210, India

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Abstract

Resveratrol (R), a plant polyphenol, is known to reduce Cu (II) to Cu (I) generating reactive oxygen species that can cleave plasmid DNA. Here we report a surprising observation of a paradoxical relationship between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu increased progressively as the ratio of R to Cu was increased i.e., the concentration of Cu was successively reduced with respect to a fixed concentration R. Whereas cleavage of plasmid DNA occurred at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA was achieved. By further increasing the ratio, whereby the concentration of Cu was reduced to very low levels, the DNA degrading activity of R-Cu was lost. This paradoxical relationship is also seen with respect to eukaryotic genomic DNA and RNA. Since R-Cu may have anti-cancer and anti-viral activities, our findings may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.

Keywords

Resveratrol, copper, pro-oxidant activity, plasmid DNA cleavage, plasmid DNA degradation, eukaryotic DNA degradation, RNA degradation

Corresponding author: Indraneel Mittra

Competing interests: No competing interests were disclosed.

Grant information: This study was supported by the Department of Atomic Energy, Government of India, through its grant CTCTMC to Tata Memorial Centre awarded to IM.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2016 Subramaniam S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Subramaniam S, Vohra I, Iyer A et al. A paradoxical relationship between Resveratrol and copper (II) with respect to degradation of DNA and RNA [version 2; peer review: 2 approved]. F1000Research 2016, 4:1145 (https://doi.org/10.12688/f1000research.7202.2) First published: 27 Oct 2015, 4:1145 (https://doi.org/10.12688/f1000research.7202.1) Latest published: 02 Mar 2016, 4:1145 (https://doi.org/10.12688/f1000research.7202.2)

Revised Amendments from Version 1

In compliance with the Referee's suggestions, we have made substantial changes to the manuscript. In particular, we have changed the title of the manuscript whereby we have replaced the word "synergism" with "relationship". Additionally, in order for the reader to easily grasp our findings depicted in multiple gel pictures, we have provided a 'summary table' of the gel images which reflects the paradoxical relationship between R and Cu more clearly.

See the authors' detailed response to the review by Renata Cozzi

Introduction

Resveratrol (R) is a poly-phenolic stilbenoid naturally present in the skin of red grapes and other fruits and berries, peanuts and also in the roots of Japanese knotweed¹. R has been shown to have multiple health benefits that include life extension, cancer prevention, cardio-protection, neuro-protection and anti-diabetic, anti-inflammatory and anti-viral activities^2–8. These actions are thought to be mediated through its intrinsic anti-oxidant properties and the ability of R to activate SIRT1^9–11. However most of the positive effects exhibited by R could not be replicated in clinical trials possibly because of its low bio-availability^12,13.

Copper (Cu) is an essential micronutrient, and because of its role as a metal co-factor, has the ability to generate reactive oxygen species (ROS), viz., O₂^-. and ^•HO radicals¹⁴. Fukuhara and Miyata were first to show that R can act as a pro-oxidant in the presence of Cu and cause oxidative DNA cleavage in a pBR322 plasmid assay¹⁵. R forms a complex with Cu (II), leading to its reduction to Cu (I) with concomitant production of ROS which is responsible for DNA scission¹⁶. Resveratrol-copper (R-Cu) was shown to be active in biological systems as evidenced by its ability to inactivate bacteriophages⁸ and to cause fragmentation of DNA of human lymphocytes in vitro¹⁷. These findings have led to the proposal that R-Cu could be used in the prevention and treatment of cancer^17,18.

The above studies have used variable molar ratios of R:Cu which have usually been of the order of 1:1 to 2:1. Here we report a surprising observation that DNA and RNA cleaving and/or degrading activity of R-Cu increases as the ratio of R to Cu is sequentially increased (i.e., the concentration of Cu is sequentially decreased with respect to a fixed concentration of R). The activity was lost when the Cu concentration was reduced to very low levels.

Methods

Isolation of DNA and RNA

Isolation of plasmid pTRIPZ DNA. Isolation of plasmid pTRIPZ DNA was performed using HiPurA plasmid DNA miniprep purification spin kit (Hi-Media) as per manufacturer’s instructions. Briefly, the transformed bacterial culture (Escherichia coli DH₅α containing plasmid pTRIPZ. Invitrogen, USA) was harvested, lysed and centrifuged. The pellet obtained was applied to a silica column and high salt (3M Potassium acetate, pH 5.5) was used to allow binding of plasmid DNA to the silica column. Washing for removal of contaminants was followed by elution of plasmid DNA in DNA binding buffer.

Eukaryotic genomic DNA. Jurkat (human lymphoblastic leukemia) cells were used for isolation of genomic DNA. Cells were procured from American Type Culture Collection and were grown in RPMI 1640 (GIBCO By Life technologies Cat No.23400-21) with 10% FBS (GIBCO By life technologies Cat No.26140-079). The Wizard® Genomic DNA purification kit (Promega) was employed for isolation of DNA. Jurkat cells (2 × 10⁶) were harvested and given three PBS washes followed by treatment with nuclei lysis solution. Genomic DNA was isolated as per manufacturer’s protocol.

Isolation of eukaryotic RNA. Jurkat cells at the exponential phase of growth (approximately 5 × 10⁶) were washed thrice in PBS and RNA was isolated using Trizol® reagent (Life Technologies, Carlsbad, CA, USA) as per manufacturer’s protocol.

Preparation of Resveratrol-Cu reaction mixture and gel electrophoresis

Stock solutions of Resveratrol (Sigma-Aldrich) (20mM) and that of CuSO₄.5H2O (MP Biomedical) (20mM) were prepared in 60% ethanol and water respectively. The reaction mixture contained a fixed amount of R and varying amounts of Cu (as specified in the text) and 500ng of plasmid or genomic DNA or 2µg RNA in a sterilized 1.5 ml micro-tube. Volume of the mixture was kept constant at 20 µl (5µl of R, 5µl of Cu and 10µl of DNA). Reaction mixtures were prepared containing varying starting concentrations of R as follows: 100µM, 500µM, 1mM and 5mM (see text). The mixture was incubated at 37°C for 1 hr. In case of plasmid and genomic DNA, electrophoreses was performed on a 1% agarose gel using a horizontal electrophoresis unit (Hoefer) at a constant voltage of 100V. In case of eukaryotic RNA, the mixture was electrophoresed on a 0.8% agarose gel at 75 volts for 90 minutes. The gel-documentation system - EC-3 Imaging system from UVP (Ultra Violet Products, USA) was used to record the images.

Observation

The table summarizes our observations under 3 separate headings: 1) observations on plasmid DNA using reducing concentrations of Cu and a constant concentration of R (Figure 1–Figure 4); 2) observations on eukaryotic DNA and RNA with reducing concentrations of Cu and a constant concentration of R (Figure 5, Figure 6), and 3) observations on plasmid DNA using different solvents with reducing concentrations of Cu and a constant concentration of R (Figure 7–Figure 9).

Figure 1. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.

Starting concentration R 100µM:Cu 100µM. Reactions were performed in 50% ethanol.

Figure 2. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.

Starting concentration R 500µM:Cu 500µM. Reactions were performed in 50% ethanol.

Figure 3. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.

Starting concentration R 1mM:Cu 1mM. Reactions were performed in 50% ethanol.

Figure 4. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.

Starting concentration R 5mM:Cu 5mM. Reactions were performed in 50% ethanol.

Figure 5. Increasing cleavage/degradation of eukaryotic genomic DNA by R-Cu in the presence of decreasing concentrations of Cu.

Starting concentration R 5mM:Cu 5mM. Reactions were performed in 50% ethanol.

Figure 6. Increasing cleavage/degradation of eukaryotic RNA by R-Cu in the presence of decreasing concentrations of Cu.

Starting concentration R 5mM:Cu 5mM. Reactions were performed in 50% ethanol.

Figure 7. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu in different solvents.

Reactions were performed in 50% Acetonitrile.

Figure 8. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu in different solvents.

Reactions were performed in 3mM NaOH.

Figure 9. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu in different solvents.

Reactions were performed in water.

We observed that when we increased the ratio of R to Cu (by reducing the concentration of Cu with respect to a fixed concentration of R) there was an enhancement of cleavage/degradation of plasmid DNA (Figure 1–Figure 4). This phenomenon was dependent on the starting concentration of R-Cu. For example, cleavage of supercoiled plasmid DNA was observed at a starting concentration of 100μM at molar ratios of 1:1 and 1:0.2 (lanes 5 and 6; Figure 1 and Table). However, with successive increases in starting concentration of R-Cu to 500μM, 1mM and 5mM, DNA cleaving activity was progressively enhanced such that complete cleavage was achieved at successively higher ratios of R to Cu (i.e., with decreasing Cu concentration) (Figure 2–Figure 4 and Table). At high starting concentrations viz., 1mM and 5mM, degradation rather than cleavage of DNA was observed. These data indicated that the DNA cleaving/degrading activity of R-Cu increases as the ratio of R to Cu is successively increased thereby suggesting the existence of a paradoxical relationship between R and Cu with respect to DNA cleavage/degradation. The data also show that the extent of cleavage/degradation is positively correlated with the starting concentrations of R and Cu. Figure 5 and Figure 6, in which genomic DNA and RNA respectively were used (starting molar ratio of R to Cu of 5mM:5mM), a similar paradoxical pattern was observed (Table).

Table. Plasmid DNA (in 50% ethanol): Variable starting concentrations (Figure 1–Figure 4).

Sr No	Starting Concentration	Cleavage/Degradation
Sr No	Starting Concentration	R	Cu	1:1	1:0.2	1:0.1	1:0.02	1:0.01	1:0.002	1:0.001	1:0.0002
1	100µM: 100µM	-	-	✔	✔	✔	-	-	-	-	-
2	500µM: 500µM	-	-	✔	✔	✔	✔	-	-	-	-
3	1mM: 1mM	-	-	✔	✔✔✔	✔✔✔	✔	✔	-	-	-
4	5mM: 5mM	-	-	✔✔✔	✔✔✔	✔✔✔	✔✔✔	✔✔✔	✔	✔	-

Genomic DNA and RNA (in 50% ethanol): Starting concentration 5mM:5mM (Figure 5, Figure 6).

Sr No	Substrate	Cleavage/Degradation
Sr No	Substrate	R	Cu	1:1	1:0.2	1:0.1	1:0.02	1:0.01	1:0.002	1:0.001	1:0.0002
1	Genomic DNA	-	-	-	✔✔✔	✔✔✔	✔✔✔	✔✔✔	✔✔✔	✔	-
2	RNA	-	-	-	-	✔✔✔	✔✔✔	✔✔✔	✔✔✔	✔✔✔	-

Plasmid DNA (in various solvents): Starting concentration 5mM:5mM (Figure 4; Figure 7–Figure 9).

Sr No	Solvent	Cleavage/Degradation
Sr No	Solvent	R	Cu	1:1	1:0.2	1:0.1	1:0.02	1:0.01	1:0.002	1:0.001	1:0.0002
1	50% Ethanol	-	-	✔✔✔	✔✔✔	✔✔✔	✔✔✔	✔✔✔	✔	✔	-
2	50% Acetonitrile	-	-	✔	✔✔✔	✔✔✔	✔✔✔	✔✔✔	✔	-	-
3	3mM NaOH	-	-	✔	✔	✔	✔✔✔	✔✔✔	✔	✔	✔
4	Water	-	-	✔	✔	✔	✔	✔	✔	✔	-

The above experiments were done in 50% ethanol (Figure 1–Figure 4). We undertook similar experiments under different solvent conditions, namely, 50% acetonitrile (Figure 7), 3mM NaOH (Figure 8) and water (Figure 9). We observed a similar paradoxical relationship under all three conditions (Table). Cleavage of plasmid DNA was most efficient in 50% acetonitrile wherein cleavage was seen to commence at a R:Cu ratio of 1:1 while complete degradation occurred in all ratios between 1:0.2 and 1:0.01. Cleavage/degradation was less efficient in 3mM NaOH wherein cleavage of plasmid DNA was seen between 1:1 and 1:0.1; complete degradation being observed at ratios of 1:0.02 and 1:0.01. Water proved to be the least efficient medium where degradation was not seen under any R-Cu ratios although cleavage was observed at all ratios of R-Cu between 1:1 and 1:0.0002. The above findings suggested that reduction of Cu(II) to Cu(I) to generate free radicals can occur under diverse conditions leading to cleavage/degradation of DNA.

Discussion

Spectroscopic studies using an analogue of Resveratrol, namely Piceatannol (3,3',4,5'-tetrahydroxy-trans-stilbene; Pice), have shown that Pice-Cu(II) induced DNA cleavage involves the Haber Weiss and Fenton reactions¹⁹. DNA cleavage is a result of hydroxyl radical formation and the Cu (II) to Cu (I) redox cycle generated ROS production¹⁹. Our experiments using R-Cu reported here suggest that the Cu (II) – mediated oxidation of R is in a catalytic mode via Cu (II) – Cu (I) redox cycling; Cu (II) acts as a catalyst with an optimum dosage depending on the starting concentration of R-Cu. However, our unexpected finding of increasing DNA and RNA cleavage/degradation with decreasing concentration of Cu remains currently unexplained and requires further investigation.

Since R-Cu may have anti-cancer and anti-viral activities^8,18, our finding may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.

Author contributions

SS, IV and AI performed the experiments and interpreted the results. NKN wrote the manuscript. IM designed the experiments, interpreted the results and wrote the manuscript. All authors have seen and agreed to the final content of the manuscript.

Competing interests

No competing interests were disclosed.

Grant information

This study was supported by the Department of Atomic Energy, Government of India, through its grant CTCTMC to Tata Memorial Centre awarded to IM.

I confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgement

We thank Dr. K. G. Akamanchi for helpful discussions and comments on the manuscript.

Faculty Opinions recommended

References

1. Burns J, Yokota T, Ashihara H, et al.: Plant foods and herbal sources of resveratrol. J Agric Food Chem. 2002; 50(11): 3337–3340. PubMed Abstract | Publisher Full Text
2. Hector KL, Lagisz M, Nakagawa S: The effect of resveratrol on longevity across species: a meta-analysis. Biol Lett. 2012; 8(5): 790–793. PubMed Abstract | Publisher Full Text | Free Full Text
3. Suh DH, Kim Mi-K, Kim HS, et al.: Cancer-specific Therapeutic Potential of Resveratrol: Metabolic Approach against Hallmarks of Cancer. J FFHD. 2013; 3(8): 332–343. Reference Source
4. Pollack RM, Crandall JP: Resveratrol: therapeutic potential for improving cardiometabolic health. Am J Hypertens. 2013; 26(11): 1260–1268. PubMed Abstract | Publisher Full Text
5. Menard C, Bastianetto S, Quirion R: Neuroprotective effects of resveratrol and epigallocatechin gallate polyphenols are mediated by the activation of protein kinase C gamma. Front Cell Neurosci. 2013; 7: 281. PubMed Abstract | Publisher Full Text | Free Full Text
6. Bhatt JK, Thomas S, Nanjan MJ: Resveratrol supplementation improves glycemic control in type 2 diabetes mellitus. Nutr Res. 2012; 32(7): 537–541. PubMed Abstract | Publisher Full Text
7. Das S, Das DK: Anti-inflammatory responses of resveratrol. Inflamm Allergy Drug Targets. 2007; 6(3): 168–173. PubMed Abstract | Publisher Full Text
8. Ahmad A, Farhan Asad S, Singh S, et al.: DNA breakage by resveratrol and Cu(II): reaction mechanism and bacteriophage inactivation. Cancer Lett. 2000; 154(1): 29–37. PubMed Abstract | Publisher Full Text
9. Leonard SS, Xia C, Jiang BH, et al.: Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses. Biochem Biophys Res Commun. 2003; 309(4): 1017–1026. PubMed Abstract | Publisher Full Text
10. Gusman J, Malonne H, Atassi G: A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis. 2001; 22(8): 1111–1117. PubMed Abstract | Publisher Full Text
11. Lagouge M, Argmann C, Gerhart-Hines Z, et al.: Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell. 2006; 127(6): 1109–1122. PubMed Abstract | Publisher Full Text
12. Ponzo V, Soldati L, Bo S: Resveratrol: a supplementation for men or for mice? J Transl Med. 2014; 12: 158. PubMed Abstract | Publisher Full Text | Free Full Text
13. Walle T, Hsieh F, DeLegge MH, et al.: High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos. 2004; 32(12): 1377–1382. PubMed Abstract | Publisher Full Text
14. Li Y, Kuppusamy P, Zweier JL, et al.: ESR evidence for the generation of reactive oxygen species from the copper-mediated oxidation of the benzene metabolite, hydroquinone: role in DNA damage. Chem Biol Interact. 1995; 94(2): 101–120. PubMed Abstract | Publisher Full Text
15. Fukuhara K, Miyata N: Resveratrol as a new type of DNA-cleaving agent. Bioorg Med Chem Lett. 1998; 8(22): 3187–3192. PubMed Abstract | Publisher Full Text
16. Fukuhara K, Nagakawa M, Nakanishi I, et al.: Structural basis for DNA-cleaving activity of resveratrol in the presence of Cu(II). Bioorg Med Chem. 2006; 14(5): 1437–1443. PubMed Abstract | Publisher Full Text
17. Azmi AS, Bhat SH, Hadi SM: Resveratrol-Cu(II) induced DNA breakage in human peripheral lymphocytes: implications for anticancer properties. FEBS Lett. 2005; 579(14): 3131–3135. PubMed Abstract | Publisher Full Text
18. Hadi SM, Ullah MF, Azmi AS, et al.: Resveratrol mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for chemoprevention of cancer. Pharm Res. 2010; 27(6): 979–988. PubMed Abstract | Publisher Full Text
19. Li Z, Yang X, Dong S, et al.: DNA breakage induced by piceatannol and copper (II): Mechanism and anticancer properties. Oncol Lett. 2012; 3(5): 1087–1094. PubMed Abstract | Publisher Full Text | Free Full Text

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¹ Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi-Mumbai, 410210, India

Competing interests

No competing interests were disclosed.

Grant information

This study was supported by the Department of Atomic Energy, Government of India, through its grant CTCTMC to Tata Memorial Centre awarded to IM.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Subramaniam S, Vohra I, Iyer A et al. A paradoxical relationship between Resveratrol and copper (II) with respect to degradation of DNA and RNA [version 2; peer review: 2 approved]. F1000Research 2016, 4:1145 (https://doi.org/10.12688/f1000research.7202.2)

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Reviewer Report 14 Apr 2016

Asfar S. Azmi, Department of Oncology, School of Medicine, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA

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Renata Cozzi, Department of Science, Roma Tre University, Rome, Italy

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Reviewer Report 12 Nov 2015

Renata Cozzi, Department of Science, Roma Tre University, Rome, Italy

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https://doi.org/10.5256/f1000research.7758.r11205

The topic of this paper is very important and has interesting implications for the purpose of using resveratrol as chemopreventive agent. In fact the authors rightly refer to the paper by Azmi et al. where the problem of the potential breaking activity of resveratrol in human lymphocytes is addressed.

However in my opinion the interpretation of the results of this manuscript is not always supported by the data presented.

Referring to figures from 1 to 4 the authors state: “with successive increases in starting concentration of R-Cu to 500μM, 1mM and 5mM, DNA cleaving activity was progressively enhanced such that complete cleavage was achieved at successively higher ratios of R to Cu”. This is partially true in the sense that starting from the ratio R-Cu 1:1 the cleavage of supercoiled increases until 1:0.01 with the highest dose of resveratrol (5mM). At lower ratios no cleavage/degradation is observed. In other word it seems that the cleaving activity is due to resveratrol concentration increase rather than the ratio R-Cu.

In fact the authors confirm this reading of the results (“The data also show that the extent of cleavage/degradation is positively correlated with the starting concentrations of R and Cu”) but suggest also “the existence of a paradoxical synergistic relationship” (even in the title) that is not at all supported by the data. The word “synergism” does not seem appropriate.

As far as eukaryotic genomic DNA a similar behavior is showed: the cleavage/degradation of DNA is evident from ratio 1:0.2 to 1:0.002,not at lower ratios.

I suggest:

To use (certainly in the title and throughout the text) a less demanding word than “synergism”. Furthermore a synergistic effect would require also a statistical evaluation.
To provide an explanation and/or an interpretative hypothesis about the showed effect present only in the middle ratios.
To provide a more accurate description of figure 7, 8 and specially 9.

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Indraneel Mittra, Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, 410210, India

02 Mar 2016

Author Response
1. As suggested by the Reviewer, we have replaced the word "synergism" with "relationship" in the title and throughout the paper.
2. As mentioned in the 'discussion' section, we are unable to provide
... Continue reading
As suggested by the Reviewer, we have replaced the word "synergism" with "relationship" in the title and throughout the paper.

As mentioned in the 'discussion' section, we are unable to provide any explanation for this paradoxical relationship between Resveratrol and copper. This is the reason why we have submitted the manuscript under "Observation" section of the Journal which stipulates "We welcome Observation Articles describing a novel observation that may be unexpected, and possibly currently without explanation". We would be delighted if the Referee could provide us some clues.

We have now provided an accurate descriptions of Figures 7, 8 and 9.

In order for the reader to easily grasp our findings depicted in multiple gel pictures, we have provided a 'summary table' of all the gel figures in a tabular format (Table) which reflects the paradoxical relationship between R and Cu more clearly.
We trust that we have satisfactorily responded to all the suggestions of the referee and hope that she would now find the paper worthy of approval.
As suggested by the Reviewer, we have replaced the word "synergism" with "relationship" in the title and throughout the paper.

As mentioned in the 'discussion' section, we are unable to provide any explanation for this paradoxical relationship between Resveratrol and copper. This is the reason why we have submitted the manuscript under "Observation" section of the Journal which stipulates "We welcome Observation Articles describing a novel observation that may be unexpected, and possibly currently without explanation". We would be delighted if the Referee could provide us some clues.

We have now provided an accurate descriptions of Figures 7, 8 and 9.

In order for the reader to easily grasp our findings depicted in multiple gel pictures, we have provided a 'summary table' of all the gel figures in a tabular format (Table) which reflects the paradoxical relationship between R and Cu more clearly.
We trust that we have satisfactorily responded to all the suggestions of the referee and hope that she would now find the paper worthy of approval.
Competing Interests: No competing interests were disclosed. Close
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Author Response 02 Mar 2016

Indraneel Mittra, Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, 410210, India

02 Mar 2016

Author Response
1. As suggested by the Reviewer, we have replaced the word "synergism" with "relationship" in the title and throughout the paper.
2. As mentioned in the 'discussion' section, we are unable to provide
... Continue reading
As suggested by the Reviewer, we have replaced the word "synergism" with "relationship" in the title and throughout the paper.

As mentioned in the 'discussion' section, we are unable to provide any explanation for this paradoxical relationship between Resveratrol and copper. This is the reason why we have submitted the manuscript under "Observation" section of the Journal which stipulates "We welcome Observation Articles describing a novel observation that may be unexpected, and possibly currently without explanation". We would be delighted if the Referee could provide us some clues.

We have now provided an accurate descriptions of Figures 7, 8 and 9.

In order for the reader to easily grasp our findings depicted in multiple gel pictures, we have provided a 'summary table' of all the gel figures in a tabular format (Table) which reflects the paradoxical relationship between R and Cu more clearly.
We trust that we have satisfactorily responded to all the suggestions of the referee and hope that she would now find the paper worthy of approval.
As suggested by the Reviewer, we have replaced the word "synergism" with "relationship" in the title and throughout the paper.

As mentioned in the 'discussion' section, we are unable to provide any explanation for this paradoxical relationship between Resveratrol and copper. This is the reason why we have submitted the manuscript under "Observation" section of the Journal which stipulates "We welcome Observation Articles describing a novel observation that may be unexpected, and possibly currently without explanation". We would be delighted if the Referee could provide us some clues.

We have now provided an accurate descriptions of Figures 7, 8 and 9.

In order for the reader to easily grasp our findings depicted in multiple gel pictures, we have provided a 'summary table' of all the gel figures in a tabular format (Table) which reflects the paradoxical relationship between R and Cu more clearly.
We trust that we have satisfactorily responded to all the suggestions of the referee and hope that she would now find the paper worthy of approval.
Competing Interests: No competing interests were disclosed. Close
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The studies in this article demonstrate that the requirement of Cu becomes minimal with increasing concentrations of resveratrol in mediating DNA damage to plasmid DNA. These results are in line with previous reports where copper-resveratrol complex was shown to induce oxidative DNA breakage in plasmid DNA as well as in human peripheral lymphocytes. The article is well written, however, it can be enhanced by the addition of a paragraph on the bioavailability of resveratrol. This is especially important given that the doses being used to show DNA cleavage are quite high to be pharmacologically achievable. Will such high concentrations be achieved in vivo? Also is there a saturating concentration where the role of Cu becomes insignificant and resveratrol alone induces oxidative DNA damage. Addition of these points will certainly enhance the readability of the manuscript.

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Reviewer Report

14 Views

10 Mar 2016 | for Version 2

Renata Cozzi, Department of Science, Roma Tre University, Rome, Italy

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Approved

I have read the new version of the manuscript and I find that the authors have properly answered to my comments and requests.

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Reviewer Report

39 Views

12 Nov 2015 | for Version 1

Renata Cozzi, Department of Science, Roma Tre University, Rome, Italy

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Approved With Reservations

To use (certainly in the title and throughout the text) a less demanding word than “synergism”. Furthermore a synergistic effect would require also a statistical evaluation.
To provide an explanation and/or an interpretative hypothesis about the showed effect present only in the middle ratios.
To provide a more accurate description of figure 7, 8 and specially 9.

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Author Response

02 Mar 2016

Indraneel Mittra, Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, 410210, India

As suggested by the Reviewer, we have replaced the word "synergism" with "relationship" in the title and throughout the paper.
As mentioned in the 'discussion' section, we are unable to provide any explanation for this paradoxical relationship between Resveratrol and copper. This is the reason why we have submitted the manuscript under "Observation" section of the Journal which stipulates "We welcome Observation Articles describing a novel observation that may be unexpected, and possibly currently without explanation". We would be delighted if the Referee could provide us some clues.
We have now provided an accurate descriptions of Figures 7, 8 and 9.

In order for the reader to easily grasp our findings depicted in multiple gel pictures, we have provided a 'summary table' of all the gel figures in a tabular format (Table) which reflects the paradoxical relationship between R and Cu more clearly.

We trust that we have satisfactorily responded to all the suggestions of the referee and hope that she would now find the paper worthy of approval.

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No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. Burns J, Yokota T, Ashihara H, et al.: Plant foods and herbal sources of resveratrol. J Agric Food Chem. 2002; 50(11): 3337–3340. PubMed Abstract | Publisher Full Text

[2] 2. Hector KL, Lagisz M, Nakagawa S: The effect of resveratrol on longevity across species: a meta-analysis. Biol Lett. 2012; 8(5): 790–793. PubMed Abstract | Publisher Full Text | Free Full Text

[3] 3. Suh DH, Kim Mi-K, Kim HS, et al.: Cancer-specific Therapeutic Potential of Resveratrol: Metabolic Approach against Hallmarks of Cancer. J FFHD. 2013; 3(8): 332–343. Reference Source

[4] 4. Pollack RM, Crandall JP: Resveratrol: therapeutic potential for improving cardiometabolic health. Am J Hypertens. 2013; 26(11): 1260–1268. PubMed Abstract | Publisher Full Text

[5] 5. Menard C, Bastianetto S, Quirion R: Neuroprotective effects of resveratrol and epigallocatechin gallate polyphenols are mediated by the activation of protein kinase C gamma. Front Cell Neurosci. 2013; 7: 281. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Bhatt JK, Thomas S, Nanjan MJ: Resveratrol supplementation improves glycemic control in type 2 diabetes mellitus. Nutr Res. 2012; 32(7): 537–541. PubMed Abstract | Publisher Full Text

[7] 7. Das S, Das DK: Anti-inflammatory responses of resveratrol. Inflamm Allergy Drug Targets. 2007; 6(3): 168–173. PubMed Abstract | Publisher Full Text

[8] 8. Ahmad A, Farhan Asad S, Singh S, et al.: DNA breakage by resveratrol and Cu(II): reaction mechanism and bacteriophage inactivation. Cancer Lett. 2000; 154(1): 29–37. PubMed Abstract | Publisher Full Text

[9] 9. Leonard SS, Xia C, Jiang BH, et al.: Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses. Biochem Biophys Res Commun. 2003; 309(4): 1017–1026. PubMed Abstract | Publisher Full Text

[10] 10. Gusman J, Malonne H, Atassi G: A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis. 2001; 22(8): 1111–1117. PubMed Abstract | Publisher Full Text

[11] 11. Lagouge M, Argmann C, Gerhart-Hines Z, et al.: Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell. 2006; 127(6): 1109–1122. PubMed Abstract | Publisher Full Text

[12] 12. Ponzo V, Soldati L, Bo S: Resveratrol: a supplementation for men or for mice? J Transl Med. 2014; 12: 158. PubMed Abstract | Publisher Full Text | Free Full Text

[13] 13. Walle T, Hsieh F, DeLegge MH, et al.: High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos. 2004; 32(12): 1377–1382. PubMed Abstract | Publisher Full Text

[14] 14. Li Y, Kuppusamy P, Zweier JL, et al.: ESR evidence for the generation of reactive oxygen species from the copper-mediated oxidation of the benzene metabolite, hydroquinone: role in DNA damage. Chem Biol Interact. 1995; 94(2): 101–120. PubMed Abstract | Publisher Full Text

[15] 15. Fukuhara K, Miyata N: Resveratrol as a new type of DNA-cleaving agent. Bioorg Med Chem Lett. 1998; 8(22): 3187–3192. PubMed Abstract | Publisher Full Text

[16] 16. Fukuhara K, Nagakawa M, Nakanishi I, et al.: Structural basis for DNA-cleaving activity of resveratrol in the presence of Cu(II). Bioorg Med Chem. 2006; 14(5): 1437–1443. PubMed Abstract | Publisher Full Text

[17] 17. Azmi AS, Bhat SH, Hadi SM: Resveratrol-Cu(II) induced DNA breakage in human peripheral lymphocytes: implications for anticancer properties. FEBS Lett. 2005; 579(14): 3131–3135. PubMed Abstract | Publisher Full Text

[18] 18. Hadi SM, Ullah MF, Azmi AS, et al.: Resveratrol mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for chemoprevention of cancer. Pharm Res. 2010; 27(6): 979–988. PubMed Abstract | Publisher Full Text

[19] 19. Li Z, Yang X, Dong S, et al.: DNA breakage induced by piceatannol and copper (II): Mechanism and anticancer properties. Oncol Lett. 2012; 3(5): 1087–1094. PubMed Abstract | Publisher Full Text | Free Full Text

A paradoxical relationship between Resveratrol and copper (II) with respect to degradation of DNA and RNA

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Methods

Isolation of DNA and RNA

Preparation of Resveratrol-Cu reaction mixture and gel electrophoresis

Observation

Figure 1. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.

Figure 2. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.

Figure 3. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.

Figure 4. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.

Figure 5. Increasing cleavage/degradation of eukaryotic genomic DNA by R-Cu in the presence of decreasing concentrations of Cu.

Figure 6. Increasing cleavage/degradation of eukaryotic RNA by R-Cu in the presence of decreasing concentrations of Cu.

Figure 7. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu in different solvents.

Figure 8. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu in different solvents.

Figure 9. Increasing cleavage/degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu in different solvents.

Table. Plasmid DNA (in 50% ethanol): Variable starting concentrations (Figure 1–Figure 4).

Genomic DNA and RNA (in 50% ethanol): Starting concentration 5mM:5mM (Figure 5, Figure 6).

Plasmid DNA (in various solvents): Starting concentration 5mM:5mM (Figure 4; Figure 7–Figure 9).

Discussion

Author contributions

Competing interests

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Acknowledgement

References

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