Mikhail Blagosklonny
Editor Oncotarget
blagosklonny@oncotarget.com
Dear Prof Mikhail Blagosklonny
Thank you for a professional review process, and for the opportunity to
address the criticism raised by the Reviewers. In preparing the revised
version of the manuscript, we addressed the comments raised by the referees
by performing new experiments and modifying the text. A list of the
corrections incorporated in the revised MS is attached to this letter.
In essence, we performed the experiments and the text modifications as
requested by the Reviewers.
It is my hope you will find our corrections appropriate, and consequently
approve publication of this work in the Oncotarget Journal.
Sincerely,
Pinkas-Kramarski Ronit
LIST OF CORRECTIONS INCORPORATED IN THE REVISED
MANUSCRIPT
(Note: Reviewers' comments are in bold type)
Reviewer # 1:
The study is too preliminary. Numerous control experiments are necessary:
1.
Long term clonogenic assay should be additionally used in
experiments shown in figures 5 and 6.
We performed clonogenic assay and the results are presented in Figure 7 of
the revised manuscript. We also described the experiment in the text of the
revised manuscript (see specific corrections at the end of this letter).
2.
Why such high doses of FTS were used?
We used several doses 30-100M FTS in the experiments, which are within
the range used in other studies. (Weisz B, et al. Oncogene. 1999; 18(16):25792588; Elad G, et al. Biochim Biophys Acta. 1999; 1452(3):228-242; Erlich S et al.
Biochem Pharmacol. 2006; 72(4):427-436)
3.
The additive effect of methyladenine in fig. 6 is not impressive,
given that this compound is toxic by itself.
We agree with the referee comment. In the revised manuscript we included
results obtained using chloroquine as suggested by the referee. The new
results were inserted in Figure 6. Also the text was modified accordingly.
4.
Clinical relevant inhibitors of autophagy such as quinacrine and
chloroquine should be also used in experiments shown in fig. 6
We agree with the referee that is why we added results using additional
inhibitor, chloroquine (Figure 6). The text of the revised manuscript was also
modified (see specific corrections below).
5. Will inhibition of autophagy increase toxicity of FTS against MEFs?
Then side effects to normal cells will preclude the combination. Some
additional normal human cellc should be used for this experiment.
We agree with the referee comment. Previously it was shown that FTS is
more effective in cell growth inhibition of transformed cells compared to
normal cells. We performed experiments using Rat-1 fibroblasts and Ras
transformed Rat-1 fibroblasts (EJ). As shown in Figure 6, the effect of the
treatment is more pronounced in the Ras transformed cells compared to the
normal cells.
6.
Effects of FTS on PI3K/mTOR pathway should be demonstrated by
immunoblot such as pS6
We performed the requested experiments (see revised Figure 5). Under the
conditions tested, p-S6K was reduced in MEF and HCT-116 cells but not in
Hela and DLD-1 cells. Indicating that the mechanism of FTS induced
autophagy may differ in different cell lines and may be mTOR dependent or
independent. We inserted the new results in the revised manuscript.
7.
Would rapamycin also induce autophagy in these cells? Effects of
rapamycin on FTS toxicity
Rapamycin is a known inducer of autophagy since it inhibits mTOR.
However, rapamycin has immunosuppressive and antiproliferative
properties (related to its effect on downstream signaling components that
affect gene transcription and translation). Thus in order not to complicate the
interpretation of the results, we did not examine the effect of combined
treatment of FTS and rapamycin yet.
Reviewer #2:
There are too many unneeded words and general statments that do not
convey information. For example, the Abstract can be reduced ……..
The text of the manuscript was changed as requested and as listed below.
Specific corrections:
1. Tow new authors that helped performing the experiments, were added:
Sari Schokoroy and Eya Wolfson,
2. The text was modified as requested: in the abstract, “Autophagy, a process
of regulated turnover of cellular constituents, is essential for normal growth
control but may be defective under pathological conditions. The
Ras/PI3K/mTOR signaling pathway negatively regulates autophagy. Ras
signaling has been documented in a large number of human cancers. In this
in-vitro study we examined the effect of the Ras inhibitor Salirasib (S-trans,
trans-farnesylthiosalicylic acid; FTS) on autophagy induction and cell
viability. We show that Ras inhibition by FTS induced autophagy in several
cell lines, including mouse embryonic fibroblasts and the human cancer cell
lines HeLa, HCT-116 and DLD-1. The autophagy induced by FTS seems to
inhibit the cell death induced by FTS, since in the absence of autophagy the
death of FTS-treated cells was enhanced. Therefore, inhibition of autophagy
may promote the inhibition of tumor cell growth and the cell death
mediated by FTS.”
3. Page 3 line 6 text was deleted: “It permits disposal of damaged organelles
in the cell and allows free amino acids and nutrients to be recycled under
conditions of nutrient deprivation or other insults.”
4. Page 7 4 lines from the end: “in all three cell lines” and “In all three cancer cell
lines, “ text was deleted
5. Page 7 2 lines from the bottom: “We also examined the effect of FTS treatment
on S-6-kinase (S6K) phosphorylation (Figure 5C). Phosphorylated S6K was
reduced in MEF and HCT-116 cells but not in HeLa and DLD-1 cells.”
6. Page 8 line 4: text was added: “As shown in Figure 6, cell viability in response to
FTS was significantly lower in the presence than in the absence of 3-methyladenine
or chloroquine. Of note, the effect of FTS and 3-methyladenine treatment on Rat-1
fibroblasts viability was significantly lower compared to the effect of the treatment
on Ras transformed Rat-1 fibroblasts (EJ).”
7. Page 8 second paragraph a repetitive text was deleted: “There is evidence for a role
of autophagy in tumor suppression, but also for its participation in oncogenesis.”
8. Page 8 line 9, text was added: “To further examine the effect of Ras and autophagy
inhibition on cell viability, we have used the long-term clonogenic assay (Figure 7).
As shown, the combined treatment significantly reduced the number of colonies.
Taken together, these results…”
9. Page 9 line 3 text was added: “polyclonal rabbit anti-phospho-Thr389-S6 kinase
(S6311; Sigma-Aldrich), polyclonal rabbit anti-S6 kinase (S4047; Sigma-Aldrich)
and polyclonal rabbit anti p62 (PM045; MBL Intenational, Woburn, MA). Salirasib
(FTS, S-trans, trans-farnesylthiosalicylic acid), 3-methyladenine (3-MA; M9281),
chloroquine (CQ, C6628) and bafilomycin A1 (B1793) were from Sigma.”
10. Page 9 line 13 text was added: “Rat-1 fibroblast cells and H-Ras-transformed
Rat-1 cells (EJ cells) were grown in DMEM. ”
11. Page 11 clonogenic assay description was added to M&M: “Clonogenic
assay
HCT-116, DLD-1 and HELA cells were plated at a density of 60*103, 50*103,
75*103 cells, respectively, onto 6-well plate, grown for 24 hr and then treated as
indicated. After treatment, the cells were detached and replated on 10-cm plates
(1:10, 1:400, 1:40 dilution, respectively). 5,7,10 days later, respectively, the cells
were fixed with 0.1% acetic acid in PBS and then stained with 0.4% crystal violet in
acetic acid. Total colonies area was calculated using the ImageJ program”
12. Figure 5 legend: “Figure 5: FTS induces autophagy in cancer cell lines (A) HCT116, HeLa and DLD-1 cell lines were treated for the indicated times with FTS (75
µM or 90 µM), with or without 10 nM bafilomycin A1. The treated cells were
subjected to immunoblot analysis using anti-LC3 Abs. (B, C) HCT-116, HeLa,
DLD-1 and MEF (WT and Atg5-/-) cells were treated for 18 h with FTS at the
indicated concentrations and were then subjected to immunoblot analysis using antiLC3, anti-p62, anti-phospho-S6K and anti-S6K Abs. Upper panels: Representative
blots. Lower panels: Densitometric analysis of the results, presented as fold
induction over induction assessed in control untreated cells. *, p < 0.05 and **,
p<0.01; n=3. Values are means ± S.D.
13. Figure 6 legend was modified: “ 3-methyladenine and chloroquine, enhance FTSinduced inhibition of cell growth. (A) HCT-116, HeLa, DLD-1 cells were treated
for 4 days with FTS at the indicated concentrations, with or without the addition
of 10 mM 3-MA for the last 24 h. (B) EJ and Rat-1 cells were treated for 4 days
with 90µM FTS, with or without the addition of 10 mM 3-MA for the last 24 h.
The cells were then tested for cell viability using the methylene blue staining
assay. Results are presented as percent of control; values are means ± S.D. of 4‒6
determinations. **, p < 0.01. (C) HCT-116, HeLa and DLD-1 cells were treated
with 75 µM FTS with or without the addition of chloroquine (CQ) at the indicated
concentrations for 4 days (DLD-1) or 5 days (HCT-116 and HeLa). The cells were
then tested for cell viability using the methylene blue staining assay. Results are
presented as percent of control; values are means ± S.D. of 4‒6 determinations. **,
p < 0.01.”
14. Figure 7 legend was added: “3-methyladenine enhances FTS-induced
inhibition of cancer cell colonies formation. (A) HCT-116, DLD-1 and HeLa
cells were treated for 4 days with FTS (50, 63 and 75 µM, respectively) with or
without the addition of 10 mM 3-MA for the last 24 h. The cells were then
detached and recultured for another 5-10 days. Colonies were stained and the total
colonies area was calculated as described in materials and buffers. Results are
presented as percent of control; values are means ± S.D. of 10 determinations. **,
p < 0.001 (scale bars, 5 mm). “