Gene P. Siegal, M.D., Ph.D.
Editor-in-Chief,
Laboratory Investigation
November 20, 2013
Dear Dr. Siegal:
Thank you very much for your interest in reviewing our revised manuscript entitled
“Modulation of PI3K-LXR-dependent lipogenesis mediated by oxidative/nitrosative stress
contributes to inhibition of HCV replication by quercetin“, by Sandra Pisonero-Vaquero et
al, with reference 13-0320-RA.
We appreciate the comments made by the two reviewers, which we find very
reasonable. Consequently, we have made the appropriate changes in a revised version of the
manuscript, underlining the text that has been modified, and providing a point-by-point
response to each of the reviewer comments as follows:
POINT BY POINT RESPONSE TO THE COMMENTS OF REVIEWER 1
1.
As reviewer rightly stated, HCV replication inhibitory effect of quercetin at very low
concentrations are surprising. In the present research, doses of selected flavonoids were
chosen based on our previous experience with these compounds. In this regard, we have
observed that at doses higher than 10 M flavonoids, including quercetin, did not inhibit
replication in HCV replicon systems, as showed by Gonzalez O et al. in line with the
findings of our study (Hepatology, 2009, ref 21 in the revised manuscript). These results
can be explained by its antioxidant capacity itself, given that it has been described that
high rates of lipid peroxidation led to reduced HCV replication which can be blocked by
antioxidants (Huang H et al., Proc Natl Acad Sci U.S.A., 2007, ref 30 in the revised
manuscript). Moreover, it has been described that several flavonoids are able to
suppress the replication in both non-infectious replicon cells and in HCV-JFH1 cell
culture system at doses below 5 M (Sekine-Osajima Y et al., Hepatol Res, 2009; Liu
MM, Eur J Med Chem et al., 2012), supporting the effect of treatment with flavonoids
on HCV replication efficiency showed in our in vitro model.
Sekine-Osajima Y, Sakamoto N, Nakagawa M, Itsui Y, Tasaka M, Nishimura-Sakurai Y, Chen CH, Suda G,
Mishima K, Onuki Y, Yamamoto M, Maekawa S, Enomoto N, Kanai T, Tsuchiya K, Watanabe M. Two
flavonoids extracts from Glycyrrhizae radix inhibit in vitro hepatitis C virus replication. Hepatol Res. 2009.
39(1):60-9.
Liu MM, Zhou L, He PL, Zhang YN, Zhou JY, Shen Q, Chen XW, Zuo JP, Li W, Ye DY. Discovery of
flavonoid derivatives as anti-HCV agents via pharmacophore search combining molecular docking strategy.
Eur J Med Chem. 2012. 52:33-43.
2. As indicated in the revised manuscript, the vehicle used for all treatments was DMSO
(0.05%). HCV-G1 control cells were incubated with the same DMSO concentration
alone (vehicle-treated cells) (2nd paragraph, page 7). Therefore, as requested by the
reviewer, solvent controls are now indicated in the revised manuscript and
supplementary figure 1.
3. While the molecular mechanisms involved in quercetin-mediated impairment of HCV
replication seem to be very complex, in our HCV replicon system we proposed that
quercetin might exert its inhibitory effect on HCV replication at least in part through
oxidative/nitrosative stress blockage and lipid accumulation reduction, which in turn
contributes to the efficient replication of HCV (García-Mediavilla MV et al., Lab Invest,
2012, ref 15 in the revised manuscript). As indicated in the manuscript, quercetin also
may inhibit HCV replication by increasing the antiviral gene expression regulated by
the IFN-activated JAK-STAT pathway, by reducing the NS5A-driven augmentation of
IRES-mediated translation and by inhibiting both NS3 and heat shock proteins essential
for HCV replication. In addition to these molecular mechanisms, and on the basis of
experimental findings obtained in the present study, we argue that oxidative/nitrosative
stress inhibition and subsequent lipid metabolism modulation might contribute to the
indirect blockage of HCV replication and protein expression by quercetin. Along this
line, similar mechanisms have been proposed for curcumin which decrease HCV gene
expression via suppression of the AKT-SREBP-1 activation (Kim et al., FEBS Lett,
2010, ref 34 in the revised manuscript). However, we agree with the reviewer and the
conclusions have been tempered as suggested (see abstract and the discussion section of
the revised version of the manuscript).
4. As reviewer mentioned, total AKT protein levels seem to be suppressed by quercetin in
a dose-dependent manner, though AKT/-actin ratio remains constant independently of
quercetin concentration. Moreover, we represented pAKT/AKT ratio which decreases
indicating a reduction in AKT phosphorylation levels when quercetin concentration is
increased.
5. We apologize for these confusing data. In response to the reviewer, we repeated these
experiments and included a new experimental group. Figure 7 (B-E) has been modified,
substituting results from HCV-G1 cells treated only with LY294002 for 8 h by HCV-G1
cells pretreated for 8 h with LY294002 and then incubated with vehicle for an additional
48 h, as indicated in material and methods in the revised version of the manuscript.
POINT BY POINT RESPONSE TO THE COMMENTS OF REVIEWER 2
1. In our study, quercetin inhibits HCV replication and proteins NS5A and core expression.
Nonstructural protein 5A (NS5A) has been implicated in regulation of viral genome
replication, translation from the viral IRES and viral packaging (Gonzalez O et al.,
Hepatology, 2009, ref 21 in the revised manuscript) and HCV core protein has been
shown to inhibit mitochondrial electron transport and to increase reactive oxygen species
in vitro and in vivo, which can be restored by reducing HCV replication (Ando M et al.,
Liver Int, 2008, ref 41). Thus, we agree with the reviewer that the effects of quercetin on
redox status should be mediated by reducing HCV replication and HCV NS5A and core
protein expression, as previously suggested (Harris C et al., J Biol Chem, 2011, ref 47 in
the revised manuscript). However, our results indicated that quercetin exerts an
antioxidant effect which in turn reduces lipogenesis and lipid accumulation, contributing
to HCV replication and protein expression inhibition in HCV-replicating cells, as
previously indicated (García-Mediavilla MV et al., Lab Invest, 2012, ref 15 in the
revised manuscript). Therefore, we suggested that the inhibitory effect of quercetin on
HCV replication might be exerted at least in part through these indirect mechanisms
involving redox status and lipid accumulation.
2. As reviewer requested, we studied the effect of IFNtreatment on LXR expression in
our adenoviral in vitro model. Thus, Figure 6 has been modified, including data from
Huh7 Ad-0.5 cells treated with IFN-2b (0.5 and 50 U/ml) (see material and methods,
results and discussion sections and figure 6 legend in the revised version of the
manuscript). As indicated by the reviewer, unlike quercetin, in HCV-replicating cells
IFN-2b treatment inhibited HCV-induced LXR gene expression in a PI3K-AKTindependent manner. Furthermore, in our in vitro model of LXRoverexpression in wild
type Huh7 cells IFN treatment did not repress LXR gene expression, suggesting that
the effect of IFN on lipogenesis modulation is mediated by its anti-HCV activity, as
previously indicated (Toyoda M et al., Int J Mol Med, 2011, ref 49 in the revised
manuscript).
3. and 4. As reviewer also suggested, we studied the effect of PI3K pathway inhibition
on LXR expression in our adenoviral in vitro model. Similarly, LY294002 (50M)
treatment was not able to repress the overexpression of the nuclear receptor LXR in
Huh7 cells infected with the adenoviral vector (data not shown). As reviewer rightly
mentioned, and on the basis of experimental findings obtained in the present study, we
speculated that “In our in vitro model of LXR overexpression, quercetin could exert its
inhibitory effect on LXRexpression and lipid accumulation not only through
modulation of PI3K pathway but also the expression of a large number of miRNAs
involved in cell signaling and metabolism, which could in turn cause down-regulation of
CMV-expressed LXR”. Obviously, this is merely a hypothesis which has to be
confirmed or not in future experimental studies.
Due to the above referred modifications, we are submitting a revised version of the
manuscript (reference 13-0320-RAR), in the hope it is now suitable for publication in
Laboratory Investigation.
Once again we thank you for your interest in reviewing our paper and we look forward to
hearing from you at your earliest convenience.
Yours sincerely,
Sonia Sánchez Campos Ph.D.
Department of Biomedical Sciences
Institute of Biomedicine
University of León, Spain
Phone +34 987 291266
Fax +34 987 291267
E-mail ssanc@unileon.es