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: 1Date:1 July 2010
Reviewer: Beat Schwaller
Reviewer's report:
Comments to authors
Khan et al. have investigated the putative link between keratin 8 (K8) expression
levels, butyrate concentration and colon cancer progression. In an initial part they
investigated whether K8 expression depends on the site, mid-sigmoid (MS) or
contralateral wall (CO) and found no differences between K8 expression in MS
and CO. For this they used immunohistochemistry (IHC) for K8 by applying a IHC
score sheet and additionally Western blot analysis. The authors report that K8
levels in colorectal tumor samples were significantly reduced in samples from
patients with high butyrate concentration in the stool. A similar trend appeared to
be present also in the non-tumor tissues. From their study the authors conclude
that “butyrate may down-regulate the expression of K8 in the cancerized colon.”
Major Compulsory Revisions
1) First of all, the number of samples (e.g. 14 cancer patients) appears to be
rather small to make any significant conclusions. Also based on the fact that
authors state that “There was a wide variation in staining patterns probably
because of regional heterogeneity of the tissue”.
2) Is there any reason (physiological or other) to separate into two groups (high
and low butyrate), with 4 mM being the separation line? The data should be
presented as a scatter plot with butyrate concentration on the x-axis and the
score on the Y-axis. From this plot a regression line should be calculated.
In addition, when the 3 scores were determined for the High and Low butyrate
group as shown in Table 3, none of the 6 values (3 from MS, 3 from CO) were
significantly different between the groups, also shedding some doubt on the
sturdiness of the results presented in Fig. 3B.
3) As reported here, the butyrate level was determined from one stool sample per
patient. How representative is this value for the average butyrate level to be
found in the stool of this person? It is expected that depending on the diet this
value may change considerably. In another study with the involvement of some
of the co-authors of this study, the tested subjects will use a high fiber diet for 8
weeks before experiments will be carried out to assess the role of butyrate (Corfe
et al. (2009) BMC Cancer 9: 332). Thus, selecting just a singular point (butyrate
concentration) is considered to be insufficient to make a statement about the
putative role of butyrate on K8 levels, even less that “butyrate may down-regulate
the expression of K8 in the cancerized colon.”
4) In the Western blot shown in Fig. 4, besides a major band at around 40 kDa,
many lower bands are detected by the antibody. No information is provided about
the identity of these bands. Moreover, the pattern in the sample with 2.4 mM
butyrate looks completely different with respect to size distribution of bands
recognized by the antibody. Which signals were analyzed in this sample? No
information is given how the bands were quantified: Only the upper band
assuming it to be the full-length K8? All bands? What was used as the loading
control for the Western blots? Without this information, Fig. 4B can’t be
interpreted.
5) To demonstrate that butyrate directly affects K8 expression, the authors need
to perform these experiments in a controlled in vitro system, e.g. using colon
cancer cells. There they should show that butyrate down-regulates K8 in a
butyrate-concentration dependent way. Furthermore, they could also use a
reporter assay to show that the promoter region of K8 has elements that act as
butyrate-dependent repressors.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
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