Carotenoid-Enriched Nanoemulsions and γ-Rays Synergistically Induce Cell Death in a Novel Radioresistant Osteosarcoma Cell Line
Round 1
Reviewer 1 Report
I believe the manuscript may be interesting for publication, but needs significant revision.
The Introduction needs to be corrected. The authors pay too much attention to a detailed description of already published experiments, but do not sufficiently argue the relevance, novelty and significance of their own research. What is SAOS cell line? How is its SAOS400 derivative different and why is it used in this study? How exactly was CEN obtained and how exactly is it characterized? And why is it used in this form in this study? All these questions should be answered in the Introduction so that the expediency and meaning of this work become clear not only to the authors.
The authors state that they have studied the effect of CEN on apoptosis, autophagy, and senescence, but only the results of the study of autophagy and senescence are presented in the manuscript. I did not see the results of the study of apoptosis under the influence of the substance.
Figure 2a obviously contains an erroneous designation of statistical differences between the variants.
The densitometric analysis of the data in Figure 4 and others should be presented in the usual way, i.e. in the form of a histogram with statistical significance analysis.
Table 1 is unclear and should be changed.
A description of the cell culture (name, not just a number) and culture conditions prior to experiments should be made in Section 4.1.
All descriptions of experiments should be added with cell culture conditions (name of medium, serum, antibiotic, how often the medium was changed).
Section 4.9 should be updated with the software used for the statistical analysis.
The conclusion is very long and complex. Usually the conclusion consists of several sentences summarizing the results obtained. I suggest the authors to make the Conclusion more concise.
An analysis of the authors' publications shows that pumpkin extract has been studied by them for quite a long time. In this regard, the novelty and significance of this study is not entirely clear. also, authors need to make sure that they cite all their works, since the topic of the antitumor activity of carotenoids is quite intensively studied by various scientific groups.
Author Response
Response to Reviewer 1 Comments
Point 1.
I believe the manuscript may be interesting for publication but needs significant revision.
The Introduction needs to be corrected. The authors pay too much attention to a detailed description of already published experiments but do not sufficiently argue the relevance, novelty and significance of their own research. What is the SAOS cell line? How is its SAOS400 derivative different and why is it used in this study? How exactly was CEN obtained and how exactly is it characterized? And why is it used in this form in this study? All these questions should be answered in the Introduction so that the expediency and meaning of this work become clear not only to the authors.
Response 1. We thank Reviewer-1 for his/her observations. We largely agreed with the criticisms expressed and modified the Introduction adding the missing information and highlighting the novelty and significance of the present manuscript.
SAOS is a human osteogenic sarcoma cell line that expresses high levels of tissue-unspecific alkaline phosphatase activity, as described in reference n. 33. In this manuscript, we selected SAOS cells as a well-known preclinical model of osteosarcoma that accounts for 56% of bone neoplasms observed in children, a type of cancer that can metastasize and become resistant to chemo and radiotherapy. In a previous article (reference n.18), we showed that SAOS400 cells, derived from SAOS after two cycles of irradiation and four weeks of in vitro selection, were highly resistant to radiation-induced oxidative stress and were enriched in senescent cells with overexpression of p16INK4 and lysosomal b-GAL staining markers with respect to the parental cells.
As described in the Materials & Methods section, SAOS400 cells were obtained from the parental SAOS cells after irradiation. Following in vitro selection (lasting about 4 weeks), the subpopulations enriched in radiation-resistant cells were named SAOS400 and used to assess the role of natural flavonoids in TIS (therapy-induced senescence) after radiotherapy.
In the new version of the Introduction, we better highlighted the main novelty of the present work (lines 99-117).
Point 2. The authors state that they have studied the effect of CEN on apoptosis, autophagy, and senescence, but only the results of the study of autophagy and senescence are presented in the manuscript. I did not see the results of the study of apoptosis under the influence of the substance.
Response 2. Accordingly with Reviewer-1’s request, we added in the supplementary figures (Fig. S1) the results of the caspase-3 assay. As reported in Fig. S1, we did not measure any increase in the enzymatic activity of caspase-3 in SAOS400 cells treated with radiation (10 Gy), CEN and their combination. Therefore, we excluded a key role of apoptosis in the processes under investigation, as reported in the R1 version of the manuscript (line 268).
Point 3. Figure 2a obviously contains an erroneous designation of statistical differences between the variants.
Response 3. We corrected this figure aligning correctly the symbols referring to statistical differences.
Point 4. The densitometric analysis of the data in Figure 4 and others should be presented in the usual way, i.e. in the form of a histogram with statistical significance analysis.
Response 4. To implement the quality and scientific correctness of the densitometric analysis, we added the values referring to ± standard deviation and statistical significance (where present). We kindly request Reviewer-1 to overlook the query of presenting the densitometric analysis in the form of histograms. Our preference is justified by the need to not complicate excessively the figures with extra panels that necessarily will reduce the size of the immunoblottings. Presenting densitometric analysis as numeric data below the bands is applied in many published articles and probably facilitates readability.
Point 5. Table 1 is unclear and should be changed.
Response 5. We respectfully do not understand why or where Table 1 is unclear; therefore, we are not sure how we can modify it to satisfy Reviewer-1 request. We simply elaborated the style and content of data reported in Table 1 based on similar examples published in other manuscripts.
Table 1 resumes the results of complex data obtained by Combosyn software after the immission of experimental values relative to the dose of drugs (in our paper CEN 100 or 200 mg/ml w/v) or radiations (in our paper -g rays doses expressed as 250 rad corresponding to 2.5 Gy or 500 rad corresponding to 5 Gy) and the effect expressed as a percentage of dead cells (fa) after single treatments or combined treatments in a constant ratio of CEN and radiation doses (2.5/1=250 rad/100 mg/ml CEN; 500 rad/ 200 mg/ml CEN). Combosyn software is based on the median effect equation (MEE). Combosyn calculates several data. As an example, the parameter m is the dynamic order signifying the shape of the dose-effect curve; Dm is the median-effect dose signifying potency, the universal reference point, and the common link for different dynamic orders. The dynamics of interactions of multiple entities resulted in the general combination index equation (CIE), algorithm and computer simulation which quantitatively determine ‘synergism (CI <1), additive effect (CI = 1) and antagonism (CI>1) automatically. To avoid too much information that may confuse the readers, we resumed the most important values in Table 1: effects (relative to fa) and CI, e.g. if the value for “effect” was 0.44, dead cells in combined treatments corresponded to 44%, and so on. Data in Table 1, clearly indicate that CEN plus radiation induce a synergistic effect being CI <1.
To increase clarity, we added part of the considerations reported above in the revised text (lines 259-268) in the Materials & Methods section; lines 628-636).
Point 6. A description of the cell culture (name, not just a number) and culture conditions prior to experiments should be made in Section 4.1.
All descriptions of experiments should be added with cell culture conditions (name of medium, serum, antibiotic, how often the medium was changed).
Response 6. We improved this part with a more detailed description in Material & Methods of cell culture conditions (lines 501-514) of the revised manuscript).
Point 7 Section 4.9 should be updated with the software used for the statistical analysis.
Response 7. Differences between the two groups were analyzed using the Students’ t-test (Excel MS software) and significance was set at p < 0.05 with specific values indicated in figure legends. This was clearly evidenced in the Materials & Methods section and in the new legends of the figures (where necessary).
Point 8. The conclusion is very long and complex. Usually, the conclusion consists of several sentences summarizing the results obtained. I suggest the authors to make the Conclusion more concise.
An analysis of the authors' publications shows that pumpkin extract has been studied by them for quite a long time. In this regard, the novelty and significance of this study is not entirely clear. also, authors need to make sure that they cite all their works, since the topic of the antitumor activity of carotenoids is quite intensively studied by various scientific groups.
Response 8. We modified the conclusions to summarize the results and evidence some important concepts. For the novelties of this paper and the role of carotenoids see the revised Introduction and Discussion sections.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Summary
In the manuscript titled, ‘Carotenoid enriched nanoemulsions and γ rays synergistically induce cell death in a novel radio-resistant osteosarcoma cell line’, the authors investigate the role of carotenoid enriched nanoemulsions on the survival of an osteosarcoma cell line. The authors employ a cell line throughout the study and show the effect of the nanoemulsions different processes like autophagy by determining cell viability and the expression of different markers. They also determine the combined effect of the nanoemulsions and irradiation on cell viability.
Strengths
The authors have explained the basis of the study and the methods in good detail. The figures are appropriate and help the reader understand the results for the most part. The study is interesting.
Weaknesses
1. There are many grammatical errors that sometimes make it difficult to understand different aspects of the study. It is highly recommended to have the manuscript proof read before submission.
2. The blots should be shown in a better way, especially in figure 1. They are too small and even though the entire blot picture is provided in a supplemental file, some of them do not match. For example, figure 1e SOAS4000 blots. It is difficult to agree with the authors when the blots are shown in this manner.
3. In relation to figure 4b, the authors claim that the expression of Beclin-1 was significantly overexpressed. It is not clear whether statistical tests were done or not to support this statement.
4. In the last sentence in section 2.5, the authors should elaborate more on why they think a senolytic effect is observed.
5. Figure 6c needs more explanation. The figure legend has no mention of SF. The reader needs to go to the Materials and Methods to find it out. There also needs to be more explanation for the isobologram and it’s significance. It is not clear to the reader what is meant by constant and non-constant concentrations.
6. The figure legend for figure 8 mentions that Autophagy induced by CEN in SAOS400 cell was related to intracellular ATP levels and activation of AMPK. We cannot conclude this from the data provided as there could be mechanisms other than autophagy that could lower ATP levels and increase pAMPK levels. The authors need to block autophagy and show that these changes do not occur to confidently state that there is a link between autophagy and changes in ATP and pAMPK. It is also unclear why the authors did not determine the ATP levels for the IR+CEN condition.
Author Response
Response to Reviewer 2 Comments
Point 1: There are many grammatical errors that sometimes make it difficult to understand different aspects of the study. It is highly recommended to have the manuscript proof read before submission.
Response 1: Thanks for this observation. We carefully reviewed the manuscript to correct grammar errors and improved the form of our manuscript using specific software (Writefull) and with the support of a colleague who is a native English speaker. All the typos and other grammatical corrections are shown in red in the revised text.
Point 2: The blots should be shown in a better way, especially in figure 1. They are too small and even though the entire blot picture is provided in a supplemental file, some of them do not match. For example, figure 1e SOAS4000 blots. It is difficult to agree with the authors when the blots are shown in this manner.
Response 2: We understand Reviewer-2 complaint but the size of the figures only partially depends on our will. The MDPI journals provide a format for the manuscripts that need to be respected. To help the comprehension of Fig. 1, we tried to differently align the panels (vertically) hoping to magnify them and facilitate the analysis of the results shown in the figure. In addition, according to the request received from Reviewer- 1, we added the value of the densitometric analysis ± s.d. and statistical significance in the immunoblots. In such a way, we hope that the differences described in the text can match those reported in panels d-e.
Point 3. In relation to figure 4b, the authors claim that the expression of Beclin-1 was significantly overexpressed. It is not clear whether statistical tests were done or not to support this statement.
Response 3. As described in point 2 above, we implementation of the densitometric analysis with an indication of ± s.d. and significance should help to positively solve the proper issue raised here by Reviewer-2.
Point 4. In the last sentence in section 2.5, the authors should elaborate more on why they think a senolytic effect is observed.
Response 4. Thanks for this keen observation. We try to better explain in the revised version of the manuscript the concept of ‘senolytic effects’ in Section 2.5 (lines 242-249). In Figure 5b it is shown that the expression level of p16INK4 decreased (about 20%) after treatment with
Point 5. Figure 6c needs more explanation. The figure legend has no mention of SF. The reader needs to go to Materials and Methods to find it out. There also needs to be more explanation for the isobologram and its significance. It is not clear to the reader what is meant by constant and non-constant concentrations.
Response 5. We added the missing information requested by Figure-3 in the legend of Fig. 6c. Briefly, S.F. indicates the surviving fractions, the colonies that survived after each treatment (CEN, radiation or combinations) with respect to untreated cells considering plating efficiency (P.E. in other words, how many cells can form colonies after plating), and calculated with a standard formula: S.F. = colonies counted/(cells seeded x P.E./100) (lines 294-297of the revised manuscript).
Point 6. The figure legend for figure 8 mentions that Autophagy induced by CEN in SAOS400 cells was related to intracellular ATP levels and activation of AMPK. We cannot conclude this from the data provided as there could be mechanisms other than autophagy that could lower ATP levels and increase pAMPK levels. The authors need to block autophagy and show that these changes do not occur to confidently state that there is a link between autophagy and changes in ATP and pAMPK. It is also unclear why the authors did not determine the ATP levels for the IR+CEN condition.
Response 6. We thank Reviewer-2 for this important observation that certainly needs a better explanation from our side regarding the results reported in Fig. 8.
One of the most potent inducers of autophagy is AMPK kinase, which plays a key role in cell energy homeostasis, primarily by activating glucose and fatty acid uptake and oxidation when cellular energy is low. Therefore, when a drop in intracellular ATP and a change in the ATP/AMP ratio are observed, the AMPK kinase is activated. For these reasons, we used the well-known AMPK activator, AICAR, to simulate the effects of CEN in our experimental system to mimic the effect of the “physiological modulator”, AMP.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The Introduction was revised and now i think it write very good.
Line 150 "FITC / DAP ratio" - Please, correct this typos.
Figure 4a. The first line on the immunoblot has the density data "0.36 +/- 23". Is it typos?
The additional file contains only one picture concerning the activity of caspase 3. I believe that it can be transferred to the main file without any damage and not complicate the reading of the article by opening additional files.
The manuscript contains comments, probably in the native language of the authors. I hope those to whom these comments were addressed take them into account.
In general, the revised manuscript began to look more suitable for publication.
Author Response
Response to Reviewer 1 Comments
Point 1.
Line 150 "FITC / DAP ratio" - Please correct this typos.
Figure 4a. The first line on the immunoblot has the density data "0.36 +/- 23". Is it typos?
Response 1. We corrected these typos, as evidenced in the new version of the manuscript.
Point 2.
The additional file contains only one picture concerning the activity of caspase 3. I believe that it can be transferred to the main file without any damage and not complicate the reading of the article by opening additional files.
Response 2. We are happy to add the data showing the results of the apoptotic effects of CEN. To avoid renumbering all figures in the text, we inserted the graph of caspase-3 activity as a new panel in Fig. 5 (panel a). The comments on these results are reported in the R2 version of the manuscript (lines 224-229 and lines 242-248). We also added the method used to measure caspase-3 activity in the Material and Method section (lines 603-617).
Point 3.
The manuscript contains comments, probably in the native language of the authors. I hope those to whom these comments were addressed take them into account.
Response 3. We feel sorry for the mishap due to the left-over notes in the comments boxes. Those comments were referring to one of the initial drafts of the R1 version and, despite the embarrassing situation, they demonstrate that the authors seriously took into consideration the criticism expressed by Reviewer-1 about the Conclusions paragraph. By the way, we further modified the Conclusions as reported in the R2 version of the manuscript (lines 678-690).
