BMC Cancer
Editorial Office
October 7th, 2009
Dear Professor Alexandersson:
Thank you very much for your e-mail (September/30/2009) with the reviewer’
comments on our manuscript (MS: 8632793862883747) entitled “Role of IAPs in
prostate cancer progression: immunohistochemical study in normal and
pathological (benign hyperplastic, prostatic intraepithelial neoplasia and cancer)
human prostate”. Gonzalo Rodríguez-Berriguete, Benito Fraile, Fermín R de
Bethencourt, Ángela Prieto, Claudia Nuñez, Bruna Prati, Pilar Martínez-Onsurbe,
Gabriel Olmedilla, Ricardo Paniagua, Mar Royuela, for consideration in BMC Cancer.
After reading the reviewer’ concerns we realize that all their concerns can be
adequately performed. We have rewritten the manuscript according to these comments.
Also we included a letter to the reviewers explaining the changes made (a point-bypoint response).
I hope that the manuscript fulfils all the concerns of the referees and may be
accepted for publication.
Thank you very much for your attention.
Yours sincerely
Mar Royuela
MANUSCRIPT NUMBER: MS: 8632793862883747
TITLE: “Role of IAPs in prostate cancer progression: immunohistochemical study in
normal and pathological (benign hyperplastic, prostatic intraepithelial neoplasia and
cancer) human prostate”
AUTHORS: Gonzalo Rodríguez-Berriguete1, Benito Fraile1, Fermín R de
Bethencourt3, Ángela Prieto-Folgado1, Claudia Nuñez1, Bruna Prati1, Pilar MartínezOnsurbe2, Gabriel Olmedilla2, Ricardo Paniagua1, Mar Royuela1.
CHANGES MADE FOLLOWING THE REFEREES’ COMMENTS
Rewiewer: Atsunori Oga
Major Compulsory Revisions
In MATERIAL and METHODS
1) Antibody for ILP2 and NAIP, did you use goat anti-“mouse” antibody?. Please write correct
product name for all first-antibodies you used.
The correct product name for all the first antibodies used has been written in
Methods section (page 5, line 8-10). For more information, all the product
information of these first-antibodies as:
- Survivin: is a mouse antibody raised against amino acids 1-142 of survivin of
human origin (view Santa Cruz Biotechnology, INC.).
- NAIP: is an affinity goat polyclonal antibody raised against a peptide
mapping near the N-terminus of NAIP-1 of mouse origin (view Santa Cruz
Biotechnology, INC.).
- C-IAP ½: is an affinity goat polyclonal antibody raised against a peptide
mapping near the amino terminus of c-IAP2 of human origin (view Santa
Cruz Biotechnology, INC.).
- C-IAP-2: is a rabbit polyclonal antibody raised against amino acids 94-178
of c-IAP2 of human origin (view Santa Cruz Biotechnology, INC.).
- XIAP: is a rabbit polyclonal antibody raised against amino acids 1-202
mapping at the N-terminus of XIAP of human origin (view Santa Cruz
Biotechnology, INC.).
- ILP: is an affinity goat polyclonal antibody raised against a peptide mapping
near the C-terminus of ILP of human origin (view Santa Cruz
Biotechnology, INC.).
This information has not been written in Methods section because another review
suggests short the Methods section (“Background and Material and Methods should
be concise”). In this way, these sections have been condensed, following the
review’s suggestion.
In DISCUSSION
1) How should readers interpret the difference of the results in immunohistochemical methods
and optical density method? Please describe concisely in DISCUSSION.
We agree with your observation and explain concisely in discussion section this point
(page 8, line 1-3).
We used immunohistochemical analysis in order to know the percentage of positive
patients, the location and the expression of the protein studied and after calculate the
variation of expression between the different groups of samples studied. So, the
immunohistochemical analysis data are used to obtain the optical density data. As we
described in Methods section, we made a comparative histologic quantification of
immunolabeling among the different groups of prostates samples and for each antibody.
From each sample, six histologic sections were selected at random. In each section, the
staining intensity (optic density) per unit surface area was measured with an automatic
image analyzer (Motic Images Advanced version 3.2, Motic China Group Co., China)
in 5 light microscopic fields per section, using the X20 objective. From the average
values obtained (by the automatic image analyzer) for each prostate, the means ± SD for
each prostate type (NP, BPH, PIN and PC) were calculated. The results were
corroborated by two different observers. The statistical significance between means of
the different prostate group samples was assessed by the one way ANOVA test at
p0.05, by multiple pairwise comparisons (GraphPad PRISMA 3.0 computer program).
2) It is necessary to describe the limit of the description in your experiment a little more
carefully.
To determine whether the limit of our results description several sentences has been
modified in discussion section (page 8, lines 15-19; page 8, lines 29-30; page 8, lines
31-34; as example).
3) In the prostatic cancer, if amount of the caspase family, especially caspase 7, 9 and 3, is
increasing, could the IAPs effects cancel?
Molecularly, classical apoptosis is caused by the activation of caspases, a family of
intracellular cysteine proteases that cleave substrates at aspartic acid residues (Cryns et
al., 1999; Thornberry et al., 1998). Caspases lie in a latent (zymogen) state in cells but
become activated in response to a wide variety of cell death stimuli. Through a
proteolytic cascade, caspases are functionally connected to each other, with upstream
(initiator) caspases cleaving and activating downstream (effectors) caspases (Salvesen et
al., 1997). At present, IAPs inhibit at least two of the major pathways for initiation of
caspase activation: (a) the intrinsic or mitochondrial pathway related with cytochrome c,
and (b) the extrinsic or death receptor pathway related with the tumour necrosis factor
(TNF) family of death receptors. The two pathways converge at the activation of
downstream effector caspases such as caspase 3.
The IAPs are a family of caspase inhibitors that specifically inhibit caspases 3, 7 and 9
and thereby prevent apoptosis. Subsequent studies identified IAPs in a diverse range of
species and discerned that IAPs inhibit apoptosis by blocking caspases (Salvesen et al.,
2002; or view manuscript references). In this way, for exemple, its know that XIAP,
cIAP1 and c-IAP-2 prevented the proteolytic processing of Pro-caspases-3, -6, and -7
by blocking the cytochrome c induced activation of pro-caspase-9 by binding directly to
(pro)-caspase-9. But they did not prevent caspase-8 induced activation of pro-caspase-3;
however they subsequently inhibit processing of caspase-3 directly, thus blocking
downstream apoptotic events such as further activation of caspases (Deveraux et al.,
1998). Therefore it was confirmed that survivin and XIAP act on the level of the
executioner caspases-3 and -7, but do not act on the level of initiator caspases (since
they did not interacted with caspase-8).
In this way, the increase in the expression of IAPs could be cancelled caspases effects
but at the same time, since PC is a heterogeneous disease in which multiple transduction
pathways, cytokines, oncogenes, or other mitogenic signals may interact in the
uncontrolled apoptosis/cell proliferation, additional fundamental research and their
correlation with clinical experimentation will be required in this field.
4) p.11: How is it written in other laboratories manuscripts though the self-quotation looks as
excess in the discussion,?
According to the manuscripts of McEleny et al and Krajewska et al quoted by you, c-IAP-1/2 or
c-IAP are important contribution to apoptotic resistance in PC. However, in your observations
expression level of c-IAP-1/2 or c-IAP in normal tissue is higher than in PC. You observed the
expression of IAPs in prostatic cancer specimens and connected the results to the increasing
tendency of prostatic cancer cells.
We are rewritten the sentence (page 9, lines 9-13). We observed that the percentage of
positive patients in PC is similar than BPH to c-IAP1/2; and minor to c-IAP-2. But, the
optical density is similar (c-IAP-1/2) or more elevated in PC than in BPH (c-IAP-2) but
c-IAP-2 did not increase with Gleason grade. After we compared with the results of
these authors because McEleny et al. observed similar localization to c-IAP-1, c-IAP-2
and XIAP in PC3 cells (in vitro study). In this way, Krajewska et al. also by
immunohistochemical analysis of prostate tumour tissues (in vivo study, similar to those
results present here) reveals elevations in the expression of cIAP2 but immunostaining
data did not correlate with Gleason scores (the same results that we presents here).
5) Which IAPs are important for keeping of excessive cell number in BPH or PC?. Is it c-IAP1/2, c-IAP or XIAP?
IAPs is a gene family that comprises different proteins such as cIAP1, cIAP2, Survivin
or XIAP. When we use the term IAPs we name the family in general (IAPs) but when
we name a specific proteins of this family we use the protein name (c-IAP-1/2, for
example). BPH consists of overgrowth of the epithelium and fibromuscular tissue of the
transition zone and periurethral area. In this manuscript we related IAPs with the
increase of proliferation cells typical of BPH since are proteins that inhibit the cellular
death, promoting the proliferation effects typical of BPH. BPH is frequently seen in
association with PC and there are a number of compelling similar, including increasing
with age or hormonal requirements for growth and development. The antiapoptotic
properties of IAPs have also been linked to NF-kB and mitogen-activated protein kinase
signal transduction (Hofer-Warbinek et al., 2000). NF-kB is a new predictive marker of
prostate cancer, which promotes survival factors such as bcl-2 and bcl-XL (Tamatani et
al., 1999). Different report (Zou et al., 2004; Jim et al., 2009) described IAPs family as
downstream targets of activated NF-kB. At the same time, several authors have
proposed the use of NF-kB inhibitors as therapeutic agents, either alone or combined
with other agents (Ross et al., 2004; Domingo-Domenech, 2005).
6) “Isn’t there possibility that overexpression of caspase family member proteins make the level
of IAPs increase. “
No, at the present and in our knowledge, IAPs is a potent suppressor of apoptosis owing
to its ability to bind and inactivate caspases. But the overexpression of caspase family
does not make an increase in the IAPs level. For example, activation of the caspase
cascade is involved in the execution of apoptosis in a variety of cellular systems.
Several studies demonstrated that caspase-1 and 3 activations were required for human
prostate cancer cells to undergo apoptosis in response to transforming growth factor-b
(Guo and Kyprianou, 1999; Winter et al., 2001). Inmunohistochemical analyses
demonstrate a diminished detection of caspase-1 and -3 proteins in human prostate
cancer compared with the normal gland (Winter et al., 2001). Therefore it was
confirmed that survivin and XIAP act on the level of the executioner caspases-3 and as
we show in our manuscript the expressions of IAPs (survivin and XIAP) increase in PC
(survivin is not expresses in PN and XIAP is scantly expressed).
7) In the previous paper, you described that both the apoptotic index of the normal and BPH
tissue were similar. However, in the present manuscript, you thought the apoptotic index (in
BPH?) was lower than in normal. Does not this give a false impression to the audience?
We agree with your observation and we have changed this paragraph in the revised
manuscript according to your suggestion (page 8, line 28-30). If you read De Miguel
et al (2000) is true that the apoptotic index measured by TUNNEL in BPH (1.5±0.5)
is lower than in normal prostate (2.0±0.4) but it is not a good interpretation because
these values are not statistical significatives. For this, the good interpretation is that
“in BPH the apoptotic index (measured by TUNEL) was similar than normal
prostate”.
8) There is a sentence that this is the first manuscript that describes ILP-2 and NAIP in human
prostate.
Have not McEleny et al observed NAIP level using prostatic cancer cell line?
We agree with your comment and in order to clarify the sentences (background and
discussion sections) we added a short comment (page 4, line 19 in background; page
9, lines 11 and 21 in discussion section). Our manuscript is the first that describes
ILP-2 and NAIP in human prostate in vivo tissue. McEleny et al described ILP-2
and NAIP in in vitro experiments, because work in different prostate cancer cell
lines (DU145, PC3 and LNCaP) with different characteristic as example some
cellular line is androgen dependent cells (LNCaP) and other is androgen
independent cells (PC3). The manuscript of McEleny et al, compared the
expressions of ILP-2 and NAIP between the three cell lines but not with in vivo
prostate tissue.
Minor Essential Revisions
1) Your English especially in the summary seems not to be enough, I think.
The manuscript has been revised by an English-speaking colleague, and we will
thank of receiving editorial assistance. In this way, several typographical errors have
been corrected.
SUMMARY
1) L-3: IAPs is….., L-3: In this study was investigate IAPs …..L-8: analyses were performed
…….. in 27 men, L-10: “epithelial” epithelial cells?,
These typographical errors have been corrected.
BACKGROUND
1) L-7: “Inhibitor” inhibitor?, L-17: “cervical” (uterine) cervix?, L-25: “ILP2-“
These typographical errors have been corrected.
DISCUSSION
1) p.10 ki-27?
2) p.11-L12, Using the same PC samples than in this study…than?
These typographical errors have been corrected:

“Ki-27” has been written as “Ki-27 nuclear antigen” (page 8, line 27; page
10, line 4).

This sentence has been modified (page 10, lines 5-8).
MANUSCRIPT NUMBER: MS: 8632793862883747
TITLE: “Role of IAPs in prostate cancer progression: immunohistochemical study in
normal and pathological (benign hyperplastic, prostatic intraepithelial neoplasia and
cancer) human prostate”
AUTHORS: Gonzalo Rodríguez-Berriguete1, Benito Fraile1, Fermín R de
Bethencourt3, Ángela Prieto-Folgado1, Claudia Nuñez1, Bruna Prati1, Pilar MartínezOnsurbe2, Gabriel Olmedilla2, Ricardo Paniagua1, Mar Royuela1.
CHANGES MADE FOLLOWING THE REFEREES’ COMMENTS
Rewiewer: Shigefumi Yoshino
Major Compulsory Revisions
Authors concluded that IAPs and NF-kB could be involved in prostate disorder
developement. But I can not see the data concerning on NF-kB in this article.
Authors shoud show the results concerning on NF-kB in prostatic carcinoma and
shoud investigate the relationship between IAPs and NF-kB in cancer
developement.
We also added a new table (Table 3) with all data concerning to NF-kB, Elk-1 and
ATF-2 (other review suggest also included Elk-1 and ATF-2 data). We added or
modified in discussion section sentences in order to discuss the relation of these data
with the manuscript (page 8, lines 5-6, 21, 33-34; page 9, lines 1-6, 30-33; page 10,
lines 1-12; for example).
MANUSCRIPT NUMBER: MS: 8632793862883747
TITLE: “Role of IAPs in prostate cancer progression: immunohistochemical study in
normal and pathological (benign hyperplastic, prostatic intraepithelial neoplasia and
cancer) human prostate”
AUTHORS: Gonzalo Rodríguez-Berriguete1, Benito Fraile1, Fermín R de
Bethencourt3, Ángela Prieto-Folgado1, Claudia Nuñez1, Bruna Prati1, Pilar MartínezOnsurbe2, Gabriel Olmedilla2, Ricardo Paniagua1, Mar Royuela1.
CHANGES MADE FOLLOWING THE REFEREES’ COMMENTS
Rewiewer: Michiya Kobayashi
1) Author should check the English including the typological or grammatical point of view.
For one of the examples:
Page 4, line4: “is know” should be “is known”
Page 6, line last 5: “swine swine” should be “swine”
Page 8, line 9: 59.25%
Page 10, line 17: “was described” should be “described.
etc.
The manuscript has been revised by an English-speaking colleague, and we will
thank of receiving editorial assistance. In this way, several typographical errors have
been corrected.
2) Background and Material and Methods should be concise.
The Background and Methods sections have been condensed, following the review’s
suggestion.
3) Author should add the statistical analysis about the immunohistochemical reactions (Table 1)
Statistical analysis about the immunohistochemical reactions (optical densities) has
been added in all the Tables. Average optical densities were evaluated only in
patients showing positive immunoreactions. Statistical analysis refers to each
antibody separately. Values denoted by different superscripts are significantly
different from each other. Significance was determined by the one way ANOVA test
at p0.05, by multiple pairwise comparisons (GraphPad PRISMA 3.0 computer
program).
4) For the comprehension of the readers, author should describe the brief results of the
previous data, such as NF-kB, ATF-2, and Elk-1, in the discussion parts. And discuss the
relation of the immunohistochemical data in this manuscript with them.
We also added a new table (Table 3) with all data concerning to NF-kB, Elk-1 and
ATF-2 (other review suggest also included Elk-1 and ATF-2 data). This data also
has been included in discussion section. We added in discussion section new
sentences in order to discuss the relation of these data with the manuscript (page 8,
lines 5-6, 21, 33-34; page 9, lines 1-6, 30-33; page 10, lines 1-12; for example).