Institute of Immunology and Experimental Therapy
Department of Experimental Oncology
Laboratory of Experimental Anticancer Therapy
Ul. Rudolfa Weigla 12
53-114 Wroclaw,
tel.: +48-71-337 1172 ext.371,
fax: +48-71-337 1172 ext.366,
e-mail: wietrzyk@iitd.pan.wroc.pl
Wrocław, 2013-05-16
Vitamin D analogs enhance the anticancer activity of 5-fluorouracil in an in vivo
mouse colon cancer model by Magdalena Milczarek, Mateusz Psurski, Andrzej
Kutner and Joanna Wietrzyk
Dear Editor,
Please find below our corrections and comments to the reviewers critiques. The
sentences highlighted in yellow are introduced or changed in the manuscript.
First Reviewer comments:
Dear Reviewer,
Thank you very much for your remarks and valuable comments.
Major compulsory revisions:
We used transplantation of tumor tissue, because the cell line derived from this tumor
is less tumorigenic; the tumors appeared in about 70% of mice, whereas after tissue
transplantation it is 100% of takes. So in such large number of experiments, we
decided to use this type of transplantation, mainly from ethical issues.
In experiment with MC38/EGFP cells we used cultured cells to have the possibility for
monitoring the expression of EGFP before transplantation. However, the influence of
vitamin D analogs in combined treatment with 5-FU on tumor growth were monitored,
and the results were similar like after MC38 tissue transplantation. We show here the
table summarizing this experiment. Moreover, we made one experiment with the use of
wild type of MC38/0 cells from in vitro culture, and the results were also compatible
with those on cells from tumor tissue. We are showing here the figure summarizing
this experiment.
Institute of Immunology and Experimental Therapy
Department of Experimental Oncology
Laboratory of Experimental Anticancer Therapy
Ul. Rudolfa Weigla 12
53-114 Wroclaw,
tel.: +48-71-337 1172 ext.371,
fax: +48-71-337 1172 ext.366,
e-mail: wietrzyk@iitd.pan.wroc.pl
Table: Mice transplanted with MC38/EGFP cells, treated with PRI-2191 and PRI-2205 s.c. three
times a week and with 5-FU (75mg/kg)
Group
TGI (49 day)
TGI [%]
%HTGI
5,7
24,4
62,3
70,9
64,4
79,5
71,5
Control
PRI-2205
PRI-2191
5-FU
5-FU+PRI-2205
5-FU+PRI-2191
Figure: Mice transplanted with MC38/0 cells, treated with PRI-2191 and PRI-2205 s.c. three
times a week (blue arrows) and with 5-FU (75 mg/kg; black arrows).
4000
Control
PRI-2191
PRI-2205
5-FU
5-FU+PRI-2191
5-FU+PRI-2205
Tumor volume [mm3]
3500
3000
2500
2000
1500
1000
500
0
24
25
29
32
35
37
39
42
46
49
Days of experiment
In M&M section, we supplemented lacking information concerning this route of
administration in such sentence: “Both analogs were administrated p.o. by gavage,
directly into the lower esophagus using a feeding needle, three times a week on days:
12, 14, 16, 19, 21, 23, 26, 28.”
Institute of Immunology and Experimental Therapy
Department of Experimental Oncology
Laboratory of Experimental Anticancer Therapy
Ul. Rudolfa Weigla 12
53-114 Wroclaw,
tel.: +48-71-337 1172 ext.371,
fax: +48-71-337 1172 ext.366,
e-mail: wietrzyk@iitd.pan.wroc.pl
Additionally we also supplemented this information describing capecitabine dosage:
“Mice bearing MC38 tumors implanted s.c. were p.o. (by gavage, directly into the
lower esophagus using a feeding needle) administered with capecitabine…..”
This is our mistake, we showed wrong formula of calculation of TGI. We calculated
TGI from the following formula:
(TGI) [%] = 100 – [(WT/WC) × 100]
We made the same mistake in ILS formula.
In this version of manuscript both formulas are corrected.
We made toxicity studies previously, and this is published (Wietrzyk et al 2004,
Steroids and Wietrzyk et al 2007, Anticancer Drugs). However, because those studies
were not conducted in combined treatment with 5-FU we are introducing below small
table with the results of serum calcium level. These studies were performed on BALB/c
mice using 2 and 10 μg/kg/day PRI-2191 and PRI-2205, respectively. In this higher
dose of PRI-2191, which was toxic in combined treatment, serum calcium level rise:
Table 7. Serum calcium level on day 16 after first administration of PRI-2191 or PRI2205 and 5-FU
N
Calcium
level
[mEq/L]
control
5-FU
PRI-2191
PRI-2205
5
5
4
5
4.97 ±
0.08
5.02 ±
0.09
5.61 ±
0.2*
5.04 ±
0.16
PRI2191+5FU
6
PRI2205+5FU
4
5.57 ±
0.16*
5.15 ±
0.06
*Kruskal-Wallis test for multiple comparisons; P<0.05 as compared to control
N – number of mice
The method is introduced in M&M section:
Serum calcium level
Institute of Immunology and Experimental Therapy
Department of Experimental Oncology
Laboratory of Experimental Anticancer Therapy
Ul. Rudolfa Weigla 12
53-114 Wroclaw,
tel.: +48-71-337 1172 ext.371,
fax: +48-71-337 1172 ext.366,
e-mail: wietrzyk@iitd.pan.wroc.pl
Female BALB/c mice were treated i.p. with 5-FU at a dose of 50 and 75 mg/kg on days 1 and
8, respectively and/or with vitamin D analogs: PRI-2191 at a dose of 2 µg/kg/day or PRI-2205 at a dose
of 10 µg/kg/day. Both analogs were administrated s.c., three times a week on days: 1, 3, 6, 8, 10, 13.
Mice were sacrificed 16 days after the first injection of the compounds and blood sera were collected.
The calcium level was measured in each individual serum sample with the photometric Arsezano 3
method (Olympus AU400; Olympus America Inc., Melville, NY, USA).
The results are described as:
Calcemic activity of combined treatment
The results of the serum calcium level evaluation after s.c. injection of PRI-2191 or PRI-2205 (2 and 10
g/kg/day), estimated on 16 day after the first injection, are illustrated in Table 7. Calcium level in
serum of mice treated with PRI-2191 alone or combined with 5-FU was significantly higher than this in
untreated mice. However, administration of 10 g/kg/day of PRI-2205 did not affect calcium level.
In discussion we added the part of sentence (highlighted) in 7th paragraph:
“As we previously showed, both analogs used in in vivo studies were less toxic than calcitriol.
However, PRI-2191 in therapeutic doses induces a rise in serum calcium levels [19], which is not
observed in the case of PRI-2205 [12], these results are confirmed in the combined treatment with 5-FU
(Table 7). The slight increase in serum calcium levels by analog PRI-2191 without any evidence of
toxicity may favorably affect cell-cell adhesion mediated by E-cadherin which is dependent on
adequate calcium levels [38]. “
The cells of MC38/0 are almost insensitive to antiproliferative activity of vitamin D
analogs after 120 h incubation (max. 13 % of proliferation inhibition – Table 8). The
cell cycle of these cells was not influenced by vitamin D analogs. So on these cells we
showed in the manuscript only cell proliferation and caspase-3/7 activity. HT-29 cells
were the most sensitive to antiproliferative activity of vitamin D analogs from all using
cell lines, therefore other in vitro studies were performed on these cells.
Institute of Immunology and Experimental Therapy
Department of Experimental Oncology
Laboratory of Experimental Anticancer Therapy
Ul. Rudolfa Weigla 12
53-114 Wroclaw,
tel.: +48-71-337 1172 ext.371,
fax: +48-71-337 1172 ext.366,
e-mail: wietrzyk@iitd.pan.wroc.pl
CELL DISSOCIATION SOLUTIONS (Sigma) according to the producer statement: “Sigma’s
Cell Dissociation Solutions are designed for the gentle removal of cells from the growing
surface of the culture vessel. The unique formula contains no protein, and allows dislodging
of cells without the use of enzymes. Cellular proteins are preserved without enzymatic
modification or adsorption of foreign proteins. These products may be particularly useful for
immunochemical studies that are dependent upon the recognition of cell surface proteins. Cell
Dissociation Solutions are prepared in either Hanks’ balanced salts or phosphate buffered
saline. In addition each contains EDTA, glycerol, and sodium citrate; the concentration of
these components is proprietary information.”
We conducted also experiments using fluorescence microscopy without removing cells
from plates. Here are the microphotographs as an example. However we decided to
show in our manuscript quantitative data from flow cytometry.
Control+EtOH
Minor comments:
In animal experiments we used only PRI-2191 and PRI-2205, because we selected
these analogs during our previous studies, where we compared its activity with control
compounds also in vivo using mice. In presented research PRI-2201 (calcipotriol) and
calcitriol were used in in vitro studies only as control compounds.
We calculated TGI until the day in which all mice were alive. Between 28 and 30 day
of experiment the part of mice treated with PRI-2191 alone died, so we could not
calculate TGI value.
In the paragraph: “The prolongation of 5-FU antitumor activity by PRI-2191 or PRI2205” on page 15, table 6 (Tumor growth delay values for each group and survival
Institute of Immunology and Experimental Therapy
Department of Experimental Oncology
Laboratory of Experimental Anticancer Therapy
Ul. Rudolfa Weigla 12
53-114 Wroclaw,
tel.: +48-71-337 1172 ext.371,
fax: +48-71-337 1172 ext.366,
e-mail: wietrzyk@iitd.pan.wroc.pl
analysis of mice) is a proper table. In the table 7 (in corrected manuscript: Table 8)
are showed the results of in vitro experiments.
In this version of manuscript this is corrected.
In this version of manuscript this is corrected:
“CPC was administered p.o. at a dose of 450 mg/kg/day and/or vitamin D analogs: PRI-2191 at a dose
of 1 µg/kg/day and PRI-2205 at a dose of 10 µg/kg/day, both were administrated s.c., three times a
week. Black arrows indicate the days of CPC administration and gray – days of vitamin D analog
administration. The number of mice: control - 8; PRI-2191 – 4, PRI-2205: 3, 5-FU: 6, 5-FU+PRI-2191
– 6; 5-FU+PRI-2205: 6. Statistical analysis: Kruskal-Wallis ANOVA Multiple Comparisons P values
(2-tailed) test. P<0.05 as compared to the control: CPC from days 14 to 35; PRI-2191 in combined
treatment with CPC from days 10 to 35; PRI-2205 in combined treatment with CPC from days 10 to
33.”
Institute of Immunology and Experimental Therapy
Department of Experimental Oncology
Laboratory of Experimental Anticancer Therapy
Ul. Rudolfa Weigla 12
53-114 Wroclaw,
tel.: +48-71-337 1172 ext.371,
fax: +48-71-337 1172 ext.366,
e-mail: wietrzyk@iitd.pan.wroc.pl
Second Reviewer comments:
Dear Reviewer,
Thank you very much for your remarks and valuable comments.
Major Revisions:
This is very important issue in the studies on everyone potential therapeutic agent. To
address it, we extended the discussion of our paper (highlighted):
“Our data suggest that these vitamin D analogs might be potentially applied to clinical use in
order to enhance the anticancer effect of 5-FU and also prolong its activity in patients suffering from
colorectal cancer. However, the attention should be paid on such conclusions, because, the
relationship between vitamin D and cancer is not easy to interchangeable interpretation. A lot
of studies have shown a relationship between vitamin D and cancer, however the direct
movement from preclinical to clinical conditions is impossible [57]. The dosage of vitamin D
analogs appears to be one of the important reasons of indirect translation of preclinical to
clinical results. In our studies, lower doses of PRI-2191 act antagonistically with 5-FU
(summarized in Figure 9). In clinical studies, in general, single agent vitamin D has not been
directly associated with objective tumor response in phase I–II clinical trials. It is probably the
result of the fact, that very few clinical studies have been conducted using doses attaining the
maximum tolerated dose. There are also questions, if there is known the maximal tolerated
dose for patients [58]. Second important clue, is the heterogeneity of patient tumors, which
frequently lead to misinterpretation of the results. The animal studies are conducted on
particular histological type of tumor, and some clinical studies showed, that consideration of
the histological type of tumors during interpretation of the results may change the conclusions
[59]. As it is concluded in many papers analyzing the preclinical and clinical studies on
vitamin D compounds, the further studies should be pointed to preclinical analysis of the
possible large panel of vitamin D targets and the clinical studies should be better planned to
eliminate the known defects of those conducted [57,58].”
Institute of Immunology and Experimental Therapy
Department of Experimental Oncology
Laboratory of Experimental Anticancer Therapy
Ul. Rudolfa Weigla 12
53-114 Wroclaw,
tel.: +48-71-337 1172 ext.371,
fax: +48-71-337 1172 ext.366,
e-mail: wietrzyk@iitd.pan.wroc.pl
We are presenting the diagram illustrating the work-flow of in vivo studies as Figure
9. We are grateful for this comment, because it clarified our results. In “Discussion”
we added the sentence in the second paragraph: Work-flow of in vivo studies on combined
treatment with vitamin D analogs and 5-FU is summarized in Fig. 9.
Minor revisions:
We can remove this figure from the manuscript, however we conducted experiments
concerning E-cadherin expression by two different methods (immunochemistry and
flow cytometry) obtaining similar results, so we decided to show the results of one
from these techniques. These results are important in the interpretation of
antimetastatic effect of vitamin D analogs. On the other hand, the cell cycle analyses
showed the differences between both analogs tested.