Download Response to reviewer 1 - 2694

Response to reviewer 1 - 2694-16107
Marko Živanović, PhD
Novel Seleno-Hydantoin Palladium(II) Complex – Antimigratory, Cytotoxic and Prooxidative
Potential on Human Colon HCT-116 and Breast MDA-MB-231 Cancer Cells
Dear professor, at first we want to thank for many reasonable suggestions on this Manuscript. The
most of suggestions actually helped us to better understand this field of our investigations.
The Comments from reviewers:
---- REVIEW #1 ---The manuscript deals with preparation and testing of anticancer activities of a novel
selenohydantoin Pd(II) complex. The topic of manuscript is relevant and the most important result
should be emphasized – the complexation of a selenohydantoin with Pd(II) enhanced an
antimigratory potency of the Pd(II) on metastatic MDA-MB-231 cells. Accordingly, this
manuscript is suitable to be published in GPB. However, several revisions are needed as follows:
It is not clear how stock solutions of the Pd(II) complex and the ligand were prepared. Has been
used DMSO or water-DMSO solution of the tested substances?
Answer 1
The stock solutions were dissolved in DMSO at concentration of 100 mM.
Explanation is added to the part Chemicals:
“The stock solution was prepared in 100% DMSO at final concentration of 100 mM. Subsequent
dilution was performed with cell growing medium, where in the highest treatment concentration
(500 µM) DMSO concentration was at 0.05%, which is non-toxic to investigated cells”.
We treated all cell lines many times with DMSO to prove its effect as control. In our Laboratory,
this procedure is mandatory because of SRPS ISO/IEC 17025:2006 standard. This standard
requires such analysis through internal and external control of Laboratory in every 6 months. The
concentrations we used are extremely non-toxic to cells, i.e. as we start with stock solution of 100
mM in 100% DMSO, the DMSO content in the highest concentration of substance (500 µM) is
only 0.05%, while in the lowest concentration (0.1 µM) DMSO is at 0.00001%. These DMSO
concentrations have been many times proved to be non-toxic, i.e. DMSO possessed no influence
on MTT result.
The data (Fig. 1 and Table 1) are not expressed as mean ± SE, therefore it is very difficult to
compare cytotoxicity of the ligand with the Pd(II) complex. A correct determination of the IC50
values is not possible from the presented data.
Answer 2
Data in Figure 1 and Table 1 corrected.
Physicochemical properties of the metal complex should be given to understand the mechanism of
its cytotoxicity. The basic properties, e.g. solubility, lipophilicity, and stability of complexes in the
water-solution could help to consider a cellular uptake of the tested Pd(II) complex.
Answer 3
The presented results have been controlled, i.e. from June 2015 to February 2016 we repeated our
assays to prove the obtained results, as we usually do. Our data was the same in ± 5% and all
experiments we performed with the same stock solution prepared in June 2015. As our Laboratory
is a key part of Centre for Preclinical Testing of Active Substances (http://cpctaslcmb.pmf.kg.ac.rs/lcmb/index.php?jezik=english), all assays are performed in accordance with our
SRPS ISO/IEC 17025:2006 standard. According to standardized procedures all tested substances
must be absolutely soluble in water or 0.1% DMSO because of comparison of all data. These basic
properties are the first step in investigations. After cell treatment we monitor the plate wells
microscopically to check whether applied substances have been precipitated of crystalized. With
the substances investigated in this article, we monitored no precipitation, crystallization or forming
of colloids. Thus, we concluded that substances are well dissolved and our concentrations are
correct.
The cytotoxicity of the ligand and Pd(II) complex has been observed, could the authors give a
comparison of these effects with the toxicity of cisplatin or some other metal-complex drug?
Answer 4
Corrected in Discussion part as follows:
At the beginning it was considered that palladium(II) complexes possess no anti-tumor properties.
Compared to cisplatin, cispalladium, cis-[Pd(NH3)2Cl2] complex does not show anti-tumor
activity. Coordination chemistry of Pd(II) and Pt(II) is very similar whereas their structural and
equilibrium behavior of the solutions are very similar (Bugarčić et al. 2015), but introduction of
specific ligands in coordination with Pd(II) can result with great anti-tumor activities (Ulukaya et
al. 2011, Divsalar et al. 2011). Compared to action of the cisplatin presented in our previous article
(Petrovic et al. 2015), our results showed that Hid-Se exerts lower cytotoxic effects, while (HidSe)2Pd showed greater effect 24 h after treatment on HCT-116 cells and lower but comparable
effect on MDA-MB-231 cells.
DTNB has been used for determination of thiols in the cells. These results (Table 4) should be
verified because they do not correspond with the intracellular level of GSH determined by the
other scientists (5- 10 nmol GSH/mg prot. in HCT-116 and 60 - 70 nmol GSH/mg prot. in MDAMB-231).
Answer 5
Our Laboratory use DTNB for determination of GSH in cancer cells according to Baker`s (1990y)
methodology. In many of our published data GSH was expressed as nmol/ml or as µM, and the
most commonly our results were in the range from 15 to 40 µM of GSH. Sometimes, we express
results in relation of survived cells, and if the active substance is toxic, the GSH level is greater.
Unfortunately, at this moment, if this methodology is not acceptable for GPB, we can exclude
GSH results and modify conclusions.
---- END REVIEW #1 ----
.
Response to reviewer 2 - 2694-16107
Marko Živanović, PhD
Novel Seleno-Hydantoin Palladium(II) Complex – Antimigratory, Cytotoxic and Prooxidative
Potential on Human Colon HCT-116 and Breast MDA-MB-231 Cancer Cells
Dear professor, at first we want to thank for many reasonable suggestions on this Manuscript. The
most of suggestions actually helped us to better understand this field of our investigations. We
tried to correct all grammatical issues.
Comments to manuscript
Title: Novel Seleno-Hydantoin Palladium(II) Complex – Antimigratory, Cytotoxic and
Prooxidative Potential on Human Colon HCT-116 and Breast MDA-MB-231 Cancer Cells
Running title: Seleno-Hydantoin and its Pd(II) Complex Biological Activity
Create date: 2016-06-14
Authors: Živanović et al.
Comments and recommendations:
Please check English wording and conjugation (especially use of “are” and “were”).
Examples:
“Cancer (malignant neoplasia) represents a large group of diseases, which are characterized by
unregulated cell differentiation and formation of malignant tumors, which may invade other parts
of the body affecting the basic physiological functions (Craig A. Almeida 2010).”
“Cell Preparation and Culturing” – recommendation: “Cultivation”
“Human colorectal HCT-116 and breast MDA-MB-231 cancer cell lines are were purchased from
the American Tissue Culture Collection (Manassas, VA, USA).“
On MDA-MB-231 cells, investigated substances showed cytotoxic character exhibited also in dose
and time dependent manner, i.e. with increasing increase of the substance concentration, cell
viability decreased.
While tested substances, in general, decreased O2•– in HCT-116 cells, on MDA-MB-231 cells
they acted as prooxidants, i.e. induced significant increasing increase in O2•– content in both
treatment periods.