May 8th, 2014
Dear Dr. Blagosklonny
We plan to submit the revised version of the manuscript entitled “Novel peptides
suppress VEGFR-3 activity and antagonize VEGFR-3-mediated oncogenic
effects” by Chang et al be considered for publication in Oncotarget. We have
answered every point of comments raised by reviewers. Your kind consideration of
our replies is greatly appreciated.
Based on reviewer’s suggestions, we have performed new experiments and made
substantial changes in this revised version (Figure 3E-3H, Figure 4, Figure 5A and B,
Supplemental Figure S1-S6) and these newly added description are marked by red
color in this manuscript. The suggestions and questions raised by reviewers were
addressed specifically as below.
Sincerely yours,
Jen-Liang Su, Ph. D
National Institute of Cancer Research, National Health Research Institutes
Graduate Institute of Cancer Biology, College of Medicine, China Medical University
Center for Molecular Medicine, China Medical University Hospital
1
No. 35, Keyan Road, Zhunan, Miaoli Country 35053, Taiwan.
Tel: +886-37-246-166
Fax: +886-37-586-401
E-mail: jlsu@nhri.org.tw
2
Response to Reviewer D:
Point 1:
Q: Figure 1. There is a discrepancy regarding peptide 7. It had a low binding affinity
to VEGFR-3, but a strong inhibitory effect on VEGFR-3. This needs to be explained.
Ans: The discrepancy regarding P7 peptide in Figure 1A (low binding affinity) and
Figure 1B (high inhibitory effects on VEGFR-3) may results from non-specific
inhibition and sensitivity of performed experiments. The reason we thought the effect
may due to non-specific function because we can’t detect significant inhibitory effects
of P7 peptide on VEGFR-3 activity by other VEGFR-3 activity related experiments,
for example, both KIRA-ELISA assay (Figure 1C), western blot assay (Figure 2A and
2B) shows VEGF-C-induced VEGFR-3 activation did not significantly inhibit by P7
peptide.
Point 2:
Q: Figure 3A-D. It is unclear whether the authors presented net migration and net
invasiveness rates independent of cell survival. Normalization against proliferation
rates is needed.
Ans: Thanks for reviewer’s comments. All migration and invasion assays in this
study were normalized to the proliferation rate. We have added the description in
3
the Material and Methods section on page 14, line 31 and changed Y-axis labeling to
migration/proliferation (% of control) and invasion/proliferation (% of control)
in corresponding figures.
Point 3:
Q: Fig. 4. MCF-7 cells were not tested for peptide 6.
Ans: We have tested peptide 6 (P6) in MCF-7 cells for the sphere-forming assay and
we found that P6 blocked VEGF-C-induced tumor initiating cells (TICs) formation in
A549 cells but not in MCF-7 cells (Supplemental Figure S5B). The different effects of
P6 on TICs in A549 and MCF-7 cells is an interesting issue and detailed mechanism
and cell specificity need further studies.
Point 4:
Q: English editing will improve the manuscript.
Ans: Thanks for reviewer’s suggestion, we have sent our manuscript to English
editing by Nature Publishing Group Language Editing to improve our manuscript
and enclosed with the editing certificate.
Response to Reviewer 2:
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Point 1:
Q: The paper is written with mistakes in every sentence and with using
phrases that make little sense in the context In the Abstract. “has been documented as
a gatekeeper signal molecule to trigger” “The above-mentioned results from bench
and bedside, and its widespread and specific expression in cancers strongly suggest
that VEGFR-3 is a crucial RTK and its therapeutic potential strongly merits further
investigation.” This sentence has no meaning with completely incorrect grammar: I
rewrote Abstract, all text must be edited.
Ans: Thanks for reviewer’s comments and appreciated reviewer rewrote the abstract
for us. We have sent our manuscript to English editing by Nature Publishing Group
Language Editing to improve our manuscript and enclosed with the editing
certificate.
Point 2:
Q: taxol should be paclitaxel or Taxol
Ans: We corrected “taxol” to ‘Taxol” on page 8, lines 2 and 15, page 10 line 21, and
page 12 line 6.
Point 3:
5
Q: Fig. 3 E, F – effect on drug resistance. These drugs cause cell cycle arrest and
inhibit proliferation in A549 and MDA cells. These cells do not die after 48 hrs of
treatment. MTT inhibition is just 50% (whereas cells proliferate in control). MTT test
cannot be used here. Use cell count, trypan blue, flow cytometry. VEGF cannot
possibly decrease effect of Taxol on proliferation. This is simply impossible to believe.
The data may be falsified.
Ans: According to reviewer’s suggestion, we used trypan blue exclusion assay and
DNA flow cytometric assay to detect the effects of VEGF-C on drug resistance in
A549 cells (Figure 3E-3H). As shown in Figure 3E and 3F, VEGF-C significantly
decreased Taxol and cisplatin-mediated cell death by flow cytometric analysis. By
trypan blue exclusion assay, we also found that VEGF-C treatment increases the cell
viability in the present of Taxol and cisplatin (Figure 3G and 3H). In addition, P5 and
P6 peptides significantly abolished VEGF-C-induced drug resistance was also
verified by both flow cytometry and trypan blue exclusion assays. We have carefully
searched references and found that previous studies also detect the effects of Taxol
and cisplatin on cell viability by MTT assay [1-6]. In fact, it has been reported that
VEGF-C helps cancer cells to survive Taxol treatment via downregulation of
mTORC1 function in prostate and pancreatic cancers [7]; additionally, VEGF-C
was shown to enhance cisplatin-resistance through NF-κB activation in gastric
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cancer [8]. Moreover, VEGF-C-induced Akt activation has been suggested to
promote prostate cancer cell survival [9]. Depletion of VEGF-C has further been
shown to inhibit cell proliferation and cell invasion in non-small cell lung cancer
(NSCLC)
[10].
VEGFR-3
overexpression
or
activation
also
confers
chemo-resistance to treatment in leukemia cancer by enhancing Bcl-2 expression
[11]. According to above studies and our experimental data, VEGF-C increased
cancer cell survival, proliferation and resistant to Taxol and cisplatin treatment is
reasonable and believable. We have added above data into Results section (page 8
lines 1-9), the description of these two assays (trypan blue exclusion assay and DNA
flow cytometric assay) were included in Material and Methods section on page 14
lines 32-34 to page 15 lines 1-7 and we moved MTT results into Supplemental Figure
S3.
Figure 3
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Point 4:
Q: Labeling and especially labeling on Y-axis is too small to be visible in print.
Ans: We have refined the Y-axis labeling of our figures to make it more clearly.
Point 5:
Q: Figure 4. the effect is small. I suggest to move it to supplement and not to
emphasize
Ans: According to reviewer’s suggestion, we have moved these data to Supplemental
Figure S5.
Major points:
Major Point 1:
Q: Structures of the peptides must be provided.
Ans: We have provided the structures of peptides in Supplemental Figure S1 and
added description on page 6 line 12.
Major Point 2:
Q: Endothelial cells, including lymphatic endothelial cells, should be tested
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Ans: According to reviewer’s suggestion, we invegestigated the effects of
VEGFR-3-inhibitroy peptides on VEGF-C-induced VEGFR-3 phosphorylation,
migration and proliferation in lymphatic endothelial cells (LECs) in Figure 4A. Our
data indicated that VEGF-C-induced VEGFR-3 phosphorylation, cell migration
and proliferation of LECs were abolished by the P5 and P6 peptides. We have
added the description of above data into Results section on page 8 lines 28-35.
Figure 4
Major Point 3:
Q: A wide panel of cancer and normal cells must be tested
Ans: As suggested by the reviewer, we have examined the effects of P5 and P6
peptides on VEGFR-3 phosphorylation and cell migration and invasion in various
cancer cells lines (breast cancer cell lines: MDA-MB-231, BT474, HS578T, HBL100,
MDA-MB-361, and MCF-7; lung cancer cell lines: CL1-5, H460 and H322) ( Figure
9
4B-4E and Supplemental Figure S4). Our results indicated that P5 and P6 peptides
significantly inhibit VEGF-C-induced VEGFR-3 phosphorylation, cell migration
and invasion ability in all tested cancer cell lines. We also examined the effect of
VEGFR-3-inhibitory peptides on cell proliferation and migration of LECs. As shown
in Figure 4A, our data indicated that P5 and P6 peptides inhibit VEGF-C-induced
VEGFR-3 phosphorylation, cell migration and proliferation in LECs. We have
added above description in Results section on page 8 lines 28-36 to page 9 lines 1-5
and cell line information in Material and Methods section on page 12 lines 14-26.
Figure 4
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Major Point 4:
Q: The effect on tumor model must be shown in animal model, in mice
Ans: According to reviewer’s suggestion, we performed the expermental metastasis
animal model to confirm our in vitro findings. Briefly, we stably transfected
VEGF-C-expressing
vector
(MDA-MB-231/VEGF-C)
or
control
vector
(MDA-MB-231/vector) into MDA-MB-231 cells for experimental metastasis assay by
tail-vein injection. Control (P4), P5 and P6 peptides were i.v. injected into mice one
day before the cancer cell injection. After injected with indicated cancer cells into
mice, peptides were administered twice weekly for 8 weeks and detect the lung
metastatic nodules by bioluminescent imaging system. Our data demonstrated that
VEGF-C-induced metastasis was significantly inhibit by P5 and P6 peptides in
animal model (Figure 5A and 5B). These data was described on page 9 lines 24-33
and experimental procedures was incorporated into Material and Methods section on
page 15 lines 33-34 to page 16 lines 1-11.
Figure 5
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