Erica spiculifolia Extract Potentiates Cisplatin Cytotoxicity by Reactivating p53 and Caspase-3-Dependent Apoptosis in Colorectal Carcinoma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Remarks

The authors should clarify how many independent biological replicates were performed for the MTT, combination, and proteomic assays.

The authors should be clearer about the solvent/vehicle used for the extract and the inclusion of a vehicle-only control.

Although cleaved caspase-3 is activated in all treated groups, only the combination leads to strong and sustained cytotoxicity. The discussion should be expanded regarding a more explicit link to concepts such as incomplete apoptosis, apoptotic escape, or anastasis.

The interpretation of Smac/DIABLO and XIAP modulation is plausible, but somewhat speculative, and should be expressed with more cautious language (e.g., "suggests," "may indicate").

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Comment 1: The authors should clarify how many independent biological replicates were performed for the MTT, combination, and proteomic assays.
Response: We thank the reviewer for raising this point. The MTT cell viability assays, including all combination experiments, were performed in triplicate independent measurements. Proteome profiling was conducted in duplicate for each protein, in accordance with the manufacturer’s assay design, whereby each target is spotted and quantified twice on the membrane. This clarification has now been added to the Materials and Methods section.

Comment 2: The authors should be clearer about the solvent/vehicle used for the extract and the inclusion of a vehicle-only control.
Response: We thank the reviewer for this comment. Erica spiculifolia extract (ESE) was dissolved in dimethyl sulfoxide (DMSO), a standard solvent routinely used in cell culture studies for poorly water-soluble compounds. The final DMSO concentration in the wells did not exceed 2 µL per 100 µL (2% v/v), a range widely reported to be non-toxic to mammalian cells. This information has now been clarified in the Materials and Methods section to improve transparency regarding solvent use.

Comment 3: Although cleaved caspase-3 is activated in all treated groups, only the combination leads to strong and sustained cytotoxicity. The discussion should be expanded regarding a more explicit link to concepts such as incomplete apoptosis, apoptotic escape, or anastasis.
Response: Thank you for your note. In the revised manuscript, we have expanded the Results and Discussion sections to explicitly relate our findings to the concepts of incomplete apoptosis, apoptotic escape, and anastasis. In particular, we now emphasize that, despite similarly high levels of cleaved caspase‑3 in all treatment arms, ESE alone and cisplatin alone are compatible with transient or reversible apoptosis when p53 signaling remains only partially engaged, whereas the combination restores a fully competent p53 response that prevents apoptotic reversal and leads to durable cytotoxicity.

Comment 4: The interpretation of Smac/DIABLO and XIAP modulation is plausible, but somewhat speculative, and should be expressed with more cautious language (e.g., "suggests," "may indicate").
Response: We thank the reviewer for the constructive suggestion. We have revised the relevant text to adopt more cautious language, replacing definitive statements with phrasing such as “suggests”, “may indicate”, “may reflect”, etc. to better reflect the interpretative nature of the Smac/DIABLO and XIAP modulation in our findings.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the anti-cancer activity of Erica spiculifolia extract (ESE) on colorectal cancer cell line HT-29 and the mechanism by which it enhances the cytotoxicity of cisplatin. Through MTT assay and Chou-Talalay combined analysis method, it was found that the combination of ESE and cisplatin has a significant synergistic effect and can reduce the dose of cisplatin. Mechanistically, the combined treatment activates p53 phosphorylation (Ser15, Ser46, Ser392) and caspase-3-dependent apoptotic pathways, restoring the apoptotic signal. UHPLC-HRMS analysis showed that ESE mainly contains flavonols (kaempferol 3-O-glucoside, myricetin) and phenolic acids (chlorogenic acid, quinic acid, etc.), providing a material basis for its activity.

1. Are "EAE" and "ESE" referring to the same extract in the article? Please unify the terms (such as "EAE" in the title of Table 2 and "ESE" in the main text).
2. The protein expression level is expressed as "percentage of maximal signal". Please explain how "maximal signal" is defined (is it the highest value among all samples?) 。
3. In the protein expression results, the expression of Smac/DIABLO was decreased in the combined group. The authors explained it as "degradation after release", and it is suggested that experimental evidence or references to relevant literature should be provided for support.
4. There is a lack of further validation experiments on cell apoptosis (such as flow cytometry for detecting apoptosis rate, caspase activity detection, etc.). Relying solely on protein chip results is somewhat insufficient.
5. In Figure 5, the heat map or array diagram of protein expression should provide more detailed annotations, such as protein names, up-regulation and down-regulation multiples, etc.
6. In the discussion section, it should further elaborate on how the main components in ESE (such as kaempferol-3-O-glucoside, myricetin triol) enhance cisplatin sensitivity by regulating p53 phosphorylation or related pathways, in combination with existing literature.
7. When introducing the p53 signal, it can briefly mention the functional significance of the phosphorylation sites (Ser15, Ser46, Ser392) that were focused on in this study.

Author Response

Comment 1: Are "EAE" and "ESE" referring to the same extract in the article? Please unify the terms (such as "EAE" in the title of Table 2 and "ESE" in the main text).
Response: Thank you for pointing out this typographical inconsistency. “EAE” and “ESE” refer to the same extract; we have corrected this typo and now use a single, consistent abbreviation (“ESE”) throughout the text, tables and figure legends.

Comment 2: The protein expression level is expressed as "percentage of maximal signal". Please explain how "maximal signal" is defined (is it the highest value among all samples?)
Response: We thank the reviewer for requesting this clarification. The “maximal signal” refers to the positive control signal provided on the protein array membranes by the manufacturer, which serves as the internal normalization reference, rather than the highest value among experimental samples. This has now been explicitly clarified in the Methods section of the revised manuscript.

Comment 3: In the protein expression results, the expression of Smac/DIABLO was decreased in the combined group. The authors explained it as "degradation after release", and it is suggested that experimental evidence or references to relevant literature should be provided for support.
Response: We thank the reviewer for this important note. We agree that the interpretation requires literature support and have revised the text to use more cautious wording. Specifically, we now state that the reduced Smac/DIABLO signal in the combination group may reflect enhanced turnover/degradation of cytosolic Smac after mitochondrial release, consistent with reports that mitochondrial-released Smac can undergo ubiquitin-proteasome-mediated degradation, including via IAP E3 ligase activity (e.g., XIAP- and Livin-dependent ubiquitination of Smac).

Comment 4: There is a lack of further validation experiments on cell apoptosis (such as flow cytometry for detecting apoptosis rate, caspase activity detection, etc.). Relying solely on protein chip results is somewhat insufficient.
Response: We thank the reviewer for this constructive comment and agree that additional validation strengthens the interpretation of the apoptotic findings. In response, we have conducted supplementary apoptosis validation experiments using AO/PI fluorescent staining to directly assess apoptotic and necrotic cell populations following the various treatments. As these experiments required additional time for completion and analysis, we have formally requested an extension from the journal to incorporate the new data into the revised manuscript. The results will be included and discussed in the updated version (Section 3.5) to provide further experimental support for the protein array findings in mono- as well as combined treatment regimens.

Comment 5: In Figure 5, the heat map or array diagram of protein expression should provide more detailed annotations, such as protein names, up-regulation and down-regulation multiples, etc.
Response: We thank the reviewer for this helpful suggestion. The names of all detected proteins (1-13) have been annotated in the caption of Figure 5. Changes in absolute protein expression levels normalized to the manufacturer’s positive control reference spots and expressed as a percentage of maximal signal in Figure 6, as now explicitly clarified in the Materials and Methods section, as well as in the caption of Figure 5.

Comment 6: In the discussion section, it should further elaborate on how the main components in ESE (such as kaempferol-3-O-glucoside, myricetin triol) enhance cisplatin sensitivity by regulating p53 phosphorylation or related pathways, in combination with existing literature.
Response: We thank the reviewer for this valuable suggestion. In the revised Results and Discussion section, we have expanded the mechanistic rationale linking the major ESE constituents kaempferol-3-O-glucoside and myricetin to enhanced cisplatin sensitivity, with emphasis on p53-centered stress signaling.

Comment 7: When introducing the p53 signal, it can briefly mention the functional significance of the phosphorylation sites (Ser15, Ser46, Ser392) that were focused on in this study.
Response: We thank the reviewer for this helpful suggestion. In the revised manuscript, we have expanded the corresponding Section 3.4. to briefly outline the functional relevance of the specific p53 phosphorylation sites analyzed (Ser15, Ser46, and Ser392).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The paper have been well revised.