Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript Ramos-Silva and co-workers systematically evaluate the anticancer and chemosensitizing effects of two commercially available plant/algae-derived formulations combined wth doxorubicin (DOX) against colorectal cancer (CRC) using 2D monolayer cell models and 3D tumor spheroids. The authors determined IC₅₀ values of all test agents used alone or in combination across multiple cancer cell lines and normal colon epithelial cells, demonstrated that caspase-3/7 activation as a core apoptotic mechanism, and confirmed the synergistic anti-tumor efficacy of combined treatments in physiologically relevant 3D spheroid models. This research topic fits well with the scope of Molecules, focusing on natural bioactive compounds and their adjuvant application in cancer chemotherapy. The experimental design is logical, data are basically complete, and the main conclusions are supported by multiple in vitro assays. I would like to recommend publication of the manuscript subjected to a major revision.

Specific comments:

1. In Table 1 the authors listed IC₅₀ values marked with superscripts a, b, and c, but they did not explain what the marks represent. Please add footnotes in the table or explain these marks in corresponding Results section.
2. For AO/EB staining results in 3D spheroids shown in Figure 7, only qualitative images are provided. If possible, please add semi-quantitative statistical data of apoptotic/necrotic cell ratios to support the conclusions.
3. The authors claim synergistic anti-tumor effects between natural formulations and DOX, but no synergistic analysis (e.g., combination Index, CI) was performed. It would strengthen the manuscript if the authors could calculate CI values via relevant methods to quantitatively judge synergism, additivity or antagonism.
4. This work only detected caspase-3/7 activation (executive apoptosis pathway), but did not explore upstream apoptotic signaling pathways. It is suggested to add discussion or preliminary detection on classic pathways related to the tested compounds, such as PI3K/Akt, Bcl-2 family, ROS and mitochondrial membrane potential.
5. In the Discussion section the authors mention redox regulation of natural compounds, but no oxidative stress-related indicators (e.g. ROS level) were detected. Please state this limitation and propose targeted follow-up research directions.
6. Formulation 10.0 shows stronger cytotoxicity to normal CCD-481 colon cells than other formulations. The authors did not discuss this phenomenon. Please explain the underlying reasons and evaluate the biosafety risk of this formulation as a potential adjuvant.
7. The two cell lines, Caco2 and HT-29, exhibited distinct sensitivity to solo and combined treatments. The current explanation is too superficial. It would strength the manuscript if the authors could combine the intrinsic differences in cell differentiation, drug resistance phenotype and signaling pathways of the two CRC cell lines to further elaborate the mechanism of differential responses.
8. The authors state that the combination of formulations 2.1 + 10.0 has an inhibitory effect equivalent to DOX alone in HT-29 spheroids. It would strengthen the work if you further discuss the advantages of natural compound combinations, and analyze the application potential of mixed formulations in reducing DOX dosage and toxic side effects.
9. The manuscript contains numerous grammatical errors, typos, please check throughout and correct.

Author Response

Please find attached our detailed point-by-point responses to reviewers comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The work is interesting, the experiments are well and clearly described, but I have, in my opinion, significant comments on the article.

• The material presented in the article partially repeats the material already published by the same authors in the Journal Nutrients for 2024 (Anticancer Activity of Plant Tocotrienols, Fucoxanthin, Fucoidan, and Polyphenols in Dietary Supplements. Nutrients 2024, 16, 4274. https://doi.org/10.3390/nu16244274)
• The in vitro researches display that the plant metabolites in extracts causes inhibition of cancer cell

through DNA mutilation as well as stimulation of apoptosis-tempting enzymes in different models.

• Did the authors study the effect on cell DNA? If not, why?
• Did decreasing the concentration of doxorubicin change the effect of the extracts on tumor cells? Have such experiments been conducted? After all, it is precisely this effect that would be desirable to obtain.
• It is desirable to improve the quality of the photos submitted.
• In the article's references, 21 out of 56 references are older than 2020, which is 37.5%, which, in my opinion, is too many.

Author Response

Please find attached our detailed point-by-point responses to reviewers comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors;

The manuscript addresses an interesting and relevant topic, namely the potential use of plant- and algae-derived formulations to enhance the anticancer effects of chemotherapy in colorectal cancer models. The use of both 2D cultures and 3D spheroids is a strength, and the results suggest that formulations 2.1 and 10.0 may increase doxorubicin-associated cytotoxicity and caspase-3/7 activation in Caco2 and HT-29 cells. However, several major issues should be addressed before the manuscript can be considered for publication.

First, the rationale for using doxorubicin in colorectal cancer models needs to be more clearly justified. Doxorubicin is not a standard chemotherapeutic backbone in colorectal cancer, and this substantially limits the translational relevance of the study. The authors should either provide a strong experimental rationale for its use as a reference cytotoxic agent or, preferably, include combinations with clinically relevant colorectal cancer drugs such as 5-fluorouracil, oxaliplatin, or irinotecan.

Second, the manuscript repeatedly implies synergistic or chemosensitizing effects, but no formal drug-interaction analysis is presented. Combining compounds at their individual IC50 concentrations is not sufficient to demonstrate synergy. The authors should either remove the term “synergy” and use more cautious wording, such as “combined cytotoxic effect,” or perform appropriate combination analyses using accepted models such as Bliss independence, Loewe additivity or Chou–Talalay combination index.

Third, the selectivity and safety profile require further attention. Although formulation 2.1 appears less cytotoxic toward the non-tumorigenic colon epithelial cell line, formulation 10.0 shows relevant activity in normal colon cells. The authors should calculate selectivity indices and test the most relevant combinations in non-tumorigenic colon cells to determine whether the observed effects are tumor-selective.

Fourth, the mechanistic conclusions should be toned down or experimentally strengthened. Caspase-3/7 activation supports an apoptosis-associated response, but it is not sufficient to establish apoptosis as the main mechanism of cell death. The authors should confirm apoptosis using an independent method, such as Annexin V/PI staining, cleaved PARP/caspase-3 analysis, or a caspase-inhibition assay. In addition, claims related to oxidative stress, mitochondrial dysfunction, or specific signaling pathways should be presented as hypotheses unless these mechanisms are directly measured.

Overall, the manuscript has potential, but substantial revisions are required to improve its mechanistic rigor, translational relevance, chemical characterization, and statistical support.

Author Response

Please find attached our detailed point-by-point responses to reviewers comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate that the authors have made effort to address all my concerns on their original manuscript, and would like to recommend publication of the revised manuscript subjected to a monir revision.

For my comment 1, the authors response that different superscript letters with each column in Table 1 indicate statistically significant differences, but do not still explain what each superscript letter represents. Does the letter a represent p<0.05, letter b represent p<0.01.... ?   

Author Response

Comment 1:

For my comment 1, the authors response that different superscript letters with each column in Table 1 indicate statistically significant differences, but do not still explain what each superscript letter represents. Does the letter a represent p<0.05, letter b represent p<0.01.... ?  

Response:
We thank the Reviewer for pointing out this lack of clarity. The superscript letters do not represent different p-value thresholds. Instead, they indicate statistical groupings resulting from the post hoc multiple-comparison analysis. Values with different superscript letters are significantly different from each other at p < 0.05, whereas values sharing the same letter are not significantly different. To avoid confusion, the footnote of Table 1 has been revised to explicitly explain the meaning of the superscript letters and the statistical test employed.

Location in revised manuscript: Page 5, Lines 153–157.

Change in the manuscript: IC₅₀ values of dietary supplements (2.0, 2.1, 3.0, 4.0, and 10.0) and doxorubicin (DOX) after 24 h treatment in cancer and normal colon (CCD-481) cell lines. Values represent mean ± SEM (n = 3) determined by MTT assay. Different superscript letters within the same column indicate statistically significant differences among treatments, as determined by one-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.05). The superscript letters (a, b, c, d) are used solely as statistical grouping labels and do not represent different p-value thresholds; all comparisons were evaluated using the same significance level (p < 0.05). Values sharing at least one common superscript letter are not significantly different, whereas values with different superscript letters differ significantly (p < 0.05).

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed all of my comments satisfactorily and transparently. In my opinion, the manuscript can be accepted for publication in its current form. However, for future studies, I would encourage the authors to use drugs that are employed in the clinical management of the tumor under investigation, as this would further enhance the translational relevance of their findings.

Author Response

Comment 1:

The authors have addressed all of my comments satisfactorily and transparently. In my opinion, the manuscript can be accepted for publication in its current form. However, for future studies, I would encourage the authors to use drugs that are employed in the clinical management of the tumor under investigation, as this would further enhance the translational relevance of their findings.

Response:
We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the constructive comments provided throughout the review process. We greatly appreciate the Reviewer’s positive assessment and recommendation for publication.

We also appreciate the valuable suggestion regarding the use of clinically relevant chemotherapeutic agents in future studies. We fully agree that evaluating these formulations in combination with drugs commonly employed in the clinical management of colorectal cancer, such as 5-fluorouracil, oxaliplatin, and irinotecan, would further strengthen the translational relevance of the findings. This recommendation will be carefully considered in the design of our future investigations.

 

We are grateful for the Reviewer’s insightful comments, which have significantly contributed to improving the quality and clarity of the manuscript.