Review for 10.1186/1471-230X-11-2 (2011)

Reviewer's report
Title:Effect of butyrate and cancer progression on keratin 8 expression in the
colon
Version: 2Date:22 October 2010
Reviewer: Beat Schwaller
Reviewer's report:
While the authors have replied to all of my points raised in the first review, in my
view, not all have been adequately addressed. Since I am not a clinician, I have
made no strict recommendations with respect to the number of cases. I see the
point that it may be difficult to obtain tissue samples and stool specimen to carry
out the analysis. However, there were a total of 17 patients from which the
following analyses were performed: IHC for scoring in normal tissue (15), IHC
scoring in tumor tissue (14); the correlation of butyrate levels vs. K8 expression
(10) and Western blot analysis (8) was only done in a rather small subset of
cases.
1) Starting with the Western blots: the “trend” towards higher K8 levels in the low
butyrate group is essentially the result of 2 samples: the low value of the 5.1 mM
butyrate sample and the extremely high value of the 2.4 mM butyrate sample.
Thus, when eliminating those 2 values, the trend would likely disappear.
Moreover, the 2.4 sample is the only one, where there is almost no full-length K8.
The author’s explanations that these lower bands result from activation of the
degradome in the cancer tissue is plausible, nonetheless it is disturbing that this
single sample is essentially responsible for the apparent effect. Thus, the only
statistically significant effect is observed in Fig. 3B on the K8 expression scores
when dividing into 2 haptile groups with different butyrate levels. The authors
“have undertaken a systematic analysis by scatter plot and correlations for
butyrate versus each K8 score at each location relative to the lesion”. The fact
that the data was not significantly different could be “most likely due to the very
small sample number in this study” (author’s comments) or might be, because
there is no correlation. Only increasing the sample number will allow to testing
these two hypotheses.
2) I appreciate the effort of the authors to carry out experiments with colon
cancer cells in vitro to investigate the effect of butyrate on K8 expression as also
recommended by one of the other reviewers. In contrary to the author’s
expectations, they have observed that butyrate increases K8 expression in
HCT116 cells and have stated that this appears “counter-intuitive”. The
difference between rapidly growing cells in vitro and the rather slow proliferation
of cancer cells in vivo is used as an argument to explain these differences. This
could be tested experimentally by treating the cells when they have reached near
confluency. Under these conditions cell/cell contacts are established and
proliferation is strongly diminished. Since butyrate was shown previously to
strongly reduce cell proliferation of colon cancer cells in vitro, that is a experiment
that could address this question. I completely agree with the argumentation of
increased butyrate concentration perceived by the colonocytes along the
crypt-villus axis, but that might then lead to a different interpretation of the data
provided in the manuscript. That is, under normal conditions, butyrate increases
K8 expression. That would be in line with their Figs. 1A and 2A (stronger staining
in the villi as compared to the crypts). For whatever reason, the colon cancer
cells might become more resistant towards butyrate’s anti-proliferative and
pro-differentiating action resulting in lower K8 expression. This would be in
agreement with the finding that “low K8 expression is associated with poor
prognosis”. Where do now the observed low butyrate feces level and the high K8
expression fit in? As stated by the authors in their reply, “there is no
dose-response relationship between fiber intake and SCFA level”. One could
hypothesize that there is a crosstalk between the SCFA-producing bacteria
(responsible for the butyrate levels in the lumen) and the colonocytes
metabolizing butyrate. In this model, colon cancer cells would fail to differentiate
(e.g. reflected by the lower K8 expression) despite high levels of butyrate in the
lumen. Whether low K8 in colon cancer cells is the cause or the consequence of
impaired differentiation would need to be addressed experimentally. In my view
this an equally well supported statement as the one by the authors that “butyrate
may down-regulate the expression of K8 in the cancerized colon. If further
validated these findings may suggest the chemopreventive value of butyrate is
limited to early stage carcinogenesis as low K8 expression is associated with a
poor prognosis.”
In summary, the data presented in the revised manuscript doesn’t allow to
deducing neither “Effect of butyrate … on K8 expression” nor “Effect of … cancer
progression on K8” in the colon. The only rather weak statement based on the
rather arbitrary haptile groups would be that low K8 expression in colon cancer is
correlated with high butyrate levels in the feces of cancer patients. If the reporting
of this finding is considered sufficient to warrant publication, then the title and the
abstract should be modified accordingly.
Level of interest: An article of limited interest
Quality of written English: Acceptable
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I declare that I have no competing interests