Plant-Derived Natural Products and Selective Apoptosis: A Cancer-Cell Vulnerability-State Framework from Redox Imbalance to Membrane–Ion Dysregulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review is devoted to the Natural-Product-Induced Selective Apoptosis: Cancer-Cell Vulnerability States from Redox Imbalance to Membrane–Ion Dysregulation. It is written in fine language. This article will undoubtedly be of interest to scientists and researchers in the field after thorough revisions.

 

Reviewer comments:

About the title. "Natural product" is a very broad concept. It could be the diversity of things: coal, a mollusk shell, wool, butter, and so on. Authors should reduce their claims and more specifically define the range of natural objects covered in the review. Authors should point out what they considering as a subject of the review.

It's not entirely clear what the review authors intended to convey to readers. They believe it's necessary to move away from the substance-centric approach to anticancer drug discovery. However, this concept doesn't stand up to scrutiny. First, they propose a shift to the mechanistic basis of natural substances anticancer activity, which is difficult to fulfil even in terms of roughly estimating the time which is required to confirm their precise mechanism of action. Second, many anticancer drugs, especially those from natural sources, often lack any confirmed mechanism of action. This approach can be justified only in case of extracts/decoctions from natural sources in which synergetic action of many compounds masking effect from single component.

The review could be reconfigured to focus on the mechanisms of action of natural substances on cancer cells versus normal cells.

 

Line 40 “VRAC/LRRC8” First appearance of the abbreviation/term must be followed by decipher. An unprepared reader will feel uncomfortable following the article. Authors should check the entire manuscript for all such issues.

Equation 1. No one digital descriptor was found during the review.

Line 162 “thresh-old.” Typo.

Figure 1-2. Verbose caption. It should be shortened and moved to the main text.

Line 237 “best match which” best match with?

Table 1. Caption is lost. It is difficult to follow to the table. Rows should be lined up for clarity of content.

Table 2-6. It is difficult to follow to the table. Rows should be lined up for clarity of content. Weird alignment of the text.

Abbreviations. Authors should check and revise all abbreviations around the entire manuscript. Too many ones are omitted here.

 

Conclusion:

 

Accept after major revisions.

 

 

Author Response

General comment: The review is devoted to the Natural-Product-Induced Selective Apoptosis: Cancer-Cell Vulnerability States from Redox Imbalance to Membrane–Ion Dysregulation. It is written in fine language. This article will undoubtedly be of interest to scientists and researchers in the field after thorough revisions.

Response: We sincerely thank the reviewer for the positive assessment of our manuscript, its language, and its potential relevance to researchers in natural-product oncology and cancer-cell death biology. We also appreciate the reviewer’s constructive recommendation for thorough revision. In response, we have carefully revised the manuscript to clarify its scope, strengthen the conceptual framing, moderate statements where the evidence remains emerging, improve the presentation of abbreviations, figures, and tables, and enhance the overall readability and scientific precision of the review.

 

Reviewer comments:

Comment 1: About the title. "Natural product" is a very broad concept. It could be the diversity of things: coal, a mollusk shell, wool, butter, and so on. Authors should reduce their claims and more specifically define the range of natural objects covered in the review. Authors should point out what they considering as a subject of the review.

Response 1: We thank the reviewer for this important and constructive comment. We agree that the term natural products is broad and may encompass a wide variety of naturally occurring materials beyond the intended scope of this Review. To avoid ambiguity, we have revised both the title and the Introduction to explicitly define the scope of the manuscript. We now clarify that this Review focuses on plant-derived natural products, including chemically characterized phytochemicals, standardized botanical extracts, their major bioactive constituents, and clinically relevant natural-product-derived or semisynthetic derivatives investigated in the context of cancer therapy. We further clarify that evidence derived from crude extracts is discussed separately because of the additional challenges related to chemical standardization and reproducibility. These revisions more precisely define the subject of the Review and reduce the possibility of overgeneralization.

 

Comment 2: It's not entirely clear what the review authors intended to convey to readers. They believe it's necessary to move away from the substance-centric approach to anticancer drug discovery. However, this concept doesn't stand up to scrutiny. First, they propose a shift to the mechanistic basis of natural substances anticancer activity, which is difficult to fulfil even in terms of roughly estimating the time which is required to confirm their precise mechanism of action. Second, many anticancer drugs, especially those from natural sources, often lack any confirmed mechanism of action. This approach can be justified only in case of extracts/decoctions from natural sources in which synergetic action of many compounds masking effect from single component.

Response 2: We thank the reviewer for this thoughtful comment and agree that the objective of the Review required further clarification. Our intention was not to propose abandoning compound-centered drug discovery or to suggest that complete elucidation of a compound’s molecular mechanism is a prerequisite for therapeutic development. We fully recognize that many clinically successful anticancer agents, including several natural-product-derived drugs, entered clinical practice before their mechanisms of action were completely understood.

Rather, the central message of this Review is that compound identity and conventional pathway readouts alone are often insufficient to explain why certain cancer cells undergo selective apoptosis whereas others survive or why normal cells may be spared. We therefore propose the vulnerability-state framework as a complementary interpretive model, intended to organize mechanistic observations according to pre-existing biological states of cancer cells that influence treatment response, rather than as a replacement for traditional compound-centered pharmacology.

To make this distinction clearer, we have revised the Introduction and several sections throughout the manuscript to explicitly state that the proposed framework complements existing approaches, emphasizes biological context when interpreting natural-product activity, and does not require complete mechanistic elucidation before compounds can be considered therapeutically relevant.

 

Comment 3: The review could be reconfigured to focus on the mechanisms of action of natural substances on cancer cells versus normal cells.

Response 3: We thank the reviewer for this valuable suggestion. We agree that comparing the responses of cancer and normal cells to plant-derived natural products is important for understanding therapeutic selectivity. However, the primary objective of this Review is to develop a conceptual framework explaining how pre-existing cancer-cell vulnerability states influence the response to plant-derived natural products and contribute to selective apoptosis. Accordingly, rather than restructuring the manuscript around a direct cancer-versus-normal cell comparison, we have revised the Introduction and the conceptual framework to more explicitly emphasize that many vulnerability states – including redox imbalance, mitochondrial priming, membrane remodeling, ion-channel dependence, and survival-pathway addiction – are more pronounced in malignant than in normal cells, thereby providing a biological rationale for selective cytotoxicity. We believe this clarification better reflects the intended scope of the Review while highlighting the mechanisms underlying therapeutic selectivity.

 

Comment 4: Comment Line 40 “VRAC/LRRC8” First appearance of the abbreviation/term must be followed by decipher. An unprepared reader will feel uncomfortable following the article. Authors should check the entire manuscript for all such issues.

Response 4: We thank the reviewer for this helpful comment. We have revised the manuscript to define volume-regulated anion channel (VRAC) and its leucine-rich repeat-containing protein 8 (LRRC8) subunits at their first appearance in the text. In addition, we performed a comprehensive editorial review of the manuscript to ensure that all abbreviations, acronyms, and specialized terms are defined at first mention and used consistently throughout the Review. We believe these revisions improve the readability and accessibility of the manuscript for a broader interdisciplinary audience.

 

Comment 5: Equation 1. No one digital descriptor was found during the review.

Response 5: We thank the reviewer for this valuable comment. We agree that Equation (1) should not be interpreted as a quantitative mathematical expression based on measurable numerical descriptors. Its purpose is to provide a qualitative conceptual summary of the proposed vulnerability-state framework rather than a predictive or mathematically validated model. To avoid misunderstanding, we have revised the text preceding Equation (1) to explicitly identify it as a qualitative conceptual relationship and clarified that it is intended solely to illustrate the interaction among phytochemical-induced stress, pre-existing cancer-cell vulnerability, adaptive buffering capacity, and normal-cell sparing, without implying measurable or mathematically independent variables.

 

Comment 6: Line 162 “thresh-old.” Typo.

Response 6: We thank the reviewer for pointing this out. The apparent typographical error results from the automatic word hyphenation applied by the journal’s manuscript template during text justification, rather than from the manuscript itself. We have nevertheless carefully checked the revised version to ensure that no unintended typographical errors remain.

 

Comment 7: Figure 1-2. Verbose caption. It should be shortened and moved to the main text.

Response 7: We thank the reviewer for this helpful suggestion. We have substantially shortened the captions of both Figures 1 and 2 to include only the essential information required to interpret each figure. The explanatory text describing the conceptual framework, its biological rationale, and the interpretation of the vulnerability states has been incorporated into the main text at the points where the figures are introduced. This revision improves readability and aligns the figure legends with standard scientific publishing practice.

 

Comment 8: Line 237 “best match which” best match with?

Response 8: We thank the reviewer for identifying this ambiguity. We have revised the sentence to explicitly state that the proposed framework seeks to determine which phytochemical stresses best match specific cancer-cell vulnerability states, thereby clarifying the intended relationship between phytochemical-induced stress and pre-existing cellular vulnerabilities.

 

Comment 9: Table 1. Caption is lost. It is difficult to follow to the table. Rows should be lined up for clarity of content.

Table 2-6. It is difficult to follow to the table. Rows should be lined up for clarity of content. Weird alignment of the text.

Response 9: We thank the reviewer for these helpful suggestions. To improve readability, we have revised the title of Table 1 to “Conceptual interpretation of major phytochemical classes within the proposed vulnerability-state framework of selective apoptosis,” making its purpose clearer and more immediately identifiable. In addition, horizontal table borders have been added to separate individual rows and improve visual organization and readability. We also carefully reviewed the formatting of Tables 2–6 and improved their layout where possible within the manuscript template. We note that some alignment and spacing issues arise from the journal’s formatting template, and we will work with the Technical Editor during the production stage to ensure that all tables are properly aligned and presented according to the journal’s publication standards.

 

Comment 10: Abbreviations. Authors should check and revise all abbreviations around the entire manuscript. Too many ones are omitted here.

Response 10: We thank the reviewer for this helpful comment. We have carefully reviewed the manuscript and revised the use of abbreviations throughout the text. Abbreviations for technical and biological terms, including IC₅₀, ROS, MOMP, and other commonly used terminology, have been defined at their first appearance where appropriate. Standard gene/protein symbols (e.g., BAX, BCL2, TP53, NRF2) were retained according to accepted gene nomenclature conventions and were therefore not expanded, as these symbols are internationally standardized and routinely used in the biomedical literature. This revision improves the readability and consistency of the manuscript.

 

Conclusion: Accept after major revisions.

Response: We sincerely thank the reviewer for the thorough and constructive evaluation of our manuscript. We appreciate the insightful comments and suggestions, which have substantially improved the clarity, organization, and scientific rigor of the Review. We have carefully addressed all comments and revised the manuscript accordingly. We hope that the revised version satisfactorily addresses the reviewer’s concerns and is now suitable for publication.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is comprehensive, well illustrated, and appropriately emphasizes matched normal-cell controls, orthogonal cell-death assays, chemical standardization, pharmacokinetic plausibility, and the need to distinguish mechanistic observations from true vulnerability-state evidence. The figures are conceptually strong, particularly the comparison between compound-centered and vulnerability-centered pharmacology and the summary of five proposed vulnerability states.

However, the manuscript is currently overly long, repetitive, and sometimes more assertive than the available evidence permits. Significant restructuring and clarification are needed before publication.

1. The review requires a clearly described literature-search methodology. The manuscript presents itself as a broad and authoritative synthesis, but no transparent search strategy is provided. The authors should add a short methodology section describing databases searched, date range, key search terms, inclusion and exclusion criteria, how clinical, in vivo, organoid, and cell-line studies were prioritized, how crude extracts were distinguished from purified compounds, whether the review was narrative, scoping, or systematic. Without this information, it is difficult to assess whether the evidence supporting each vulnerability state was selected comprehensively or selectively.
2. The novelty of the “vulnerability-state model” should be distinguished more clearly from established concepts. The proposed framework is interesting, but many elements overlap with existing concepts such as mitochondrial priming, oncogene addiction, redox adaptation, synthetic lethality, therapeutic windows, stress-buffer dependence, and cancer-selective vulnerability.

The authors should explicitly explain which elements are established, which elements are newly integrated, what is genuinely novel about applying the model specifically to natural products, how the framework differs from existing cancer vulnerability and stress-adaptation models.

A comparison table contrasting the proposed model with established frameworks would strengthen the manuscript.

3. The conceptual equation should not be presented as if it were mathematically defined. The manuscript expresses selective apoptosis as phytochemical stress × pre-existing vulnerability × insufficient adaptive buffering × normal-cell sparing.

This is useful as a conceptual mnemonic, but multiplication implies measurable, independent, quantitative variables. The terms are not operationally defined or mathematically validated.

The authors should either clearly label the equation as a qualitative conceptual relationship or replace it with a schematic logic model.

“Normal-cell sparing” is also not a causal driver of apoptosis but an observed requirement for selectivity. It may therefore be better placed as an outcome criterion rather than a multiplicative component.

4. Evidence supporting each vulnerability state should be graded. The manuscript often moves between established mechanisms, plausible hypotheses, and largely untested proposals. This is especially important for membrane-ion dysregulation, which is repeatedly described as a “missing” or distinctive layer between phytochemical stress and apoptosis. The authors should classify evidence as, for example clinically validated, supported by in-vivo studies, supported by organoid or advanced models, supported only by conventional cell lines, mechanistically inferred, hypothetical or underexplored.

This could be incorporated into the major tables. Without such grading, readers may interpret speculative mechanisms as equally established.

5. The membrane-ion section is potentially novel but currently overemphasized relative to the evidence. The manuscript repeatedly suggests that ion flux, apoptotic volume decrease, VRAC/LRRC8 activity, aquaporins, and membrane potential may provide the missing biophysical connection between natural-product exposure and apoptotic commitment. This is a valuable hypothesis, but the cited evidence appears substantially less developed than that supporting redox imbalance or mitochondrial priming.

The authors should moderate claims such as “missing layer” unless directly supported, identify specific natural products for which causal membrane–ion evidence exists, distinguish early causal events from secondary consequences of apoptosis, discuss pharmacological and genetic rescue experiments required to establish causality, explain how nonspecific membrane damage would be excluded.

The present manuscript itself acknowledges that most phytochemical studies do not directly test these mechanisms.

6. The manuscript is too long and contains extensive repetition. At approximately 54 pages, the review repeatedly restates the same concepts as pathway modulation does not equal vulnerability, IC₅₀ values alone are insufficient, matched normal cells are required, ROS can be protective or toxic depending on context, pharmacokinetic plausibility is essential, membrane effects must be separated from nonspecific toxicity.

These points are important but appear in the abstract, introduction, framework section, individual vulnerability sections, translational sections, and conclusion. The conclusion also repeats material presented immediately before it.

The manuscript should be shortened substantially, possibly by 25–35%. Each section should focus on evidence specific to that vulnerability state rather than restating the general framework.

7. The tables are highly detailed but difficult to use. Table 1 and subsequent tables contain very long text entries and combine mechanistic, experimental, and translational information. This creates dense blocks of information that may overwhelm readers.

The authors should consider, shortening each cell, moving detailed assay recommendations to a supplementary table, adding an “evidence level” column, separating natural-product classes from experimental validation requirements, highlighting only representative compounds with the strongest evidence.

Table 1 should not imply that all members of a phytochemical class share the same properties. The manuscript recognizes this problem, but the table structure may still promote class-level generalization.

8. The distinction among apoptosis, ferroptosis, necrosis, pyroptosis, necroptosis, and cytostasis needs more consistent treatment. The manuscript appropriately warns against interpreting metabolic viability assays as proof of apoptosis. However, some sections still group ROS production, lipid peroxidation, mitochondrial dysfunction, and caspase changes under a broad selective-apoptosis narrative.

The authors should provide a concise experimental decision framework indicating the minimum evidence needed to claim apoptosis, ferroptosis, mixed death phenotypes, necrosis or membrane lysis, cytostasis.

For apoptosis, Annexin V/PI and caspase cleavage alone may not always be sufficient. Time-course analysis, morphological evidence, caspase dependence, and appropriate rescue controls should be considered.

9. “Natural products” is treated too broadly. The manuscript includes, purified phytochemicals, crude extracts, dietary constituents, metabolites, optimized synthetic derivatives, approved natural-product-derived drugs.

These categories have very different standards of chemical identity, exposure, reproducibility, and translational relevance.

The authors should establish a hierarchy and consistently distinguish, crude or partially characterized extracts, purified natural compounds, active metabolites, semisynthetic or optimized derivatives, clinically approved natural-product-derived drugs.

Claims based on paclitaxel or vinca alkaloids should not automatically validate unrelated investigational phytochemicals.

10. More quantitative translational criteria are needed. The manuscript repeatedly calls for pharmacokinetic and pharmacodynamic plausibility, which is appropriate, but it does not define how readers should evaluate this.

The authors should recommend comparison of in vitro active concentration, unbound plasma exposure, tumor tissue exposure, metabolite exposure, protein binding, achievable duration of exposure, formulation-dependent delivery, maximum tolerated dose.

This would make the framework more practical and prevent unrealistic interpretation of high micromolar in vitro effects.

11. The review should address chemical liabilities more systematically. The manuscript mentions aggregation, impurities, assay interference, instability, and promiscuity, particularly for curcumin. These issues deserve a more organized section discussing PAINS-related concerns, colloidal aggregation, redox cycling, covalent nonspecific reactivity, fluorescence or absorbance interference, instability in culture media, contamination and batch variability, active metabolite formation.

A concise checklist for validating natural-product activity would be useful.

12. Claims of cancer selectivity require stronger definition. The manuscript correctly emphasizes matched normal-cell models, but “normal-cell sparing” is repeatedly used without defining an acceptable therapeutic window.

The authors should discuss appropriate normal comparators, tissue matching, proliferating versus quiescent normal cells, immune-cell compatibility, selectivity-index calculations, clinically meaningful versus statistically significant differences, whether transformed and non-transformed cells have comparable growth rates.

A cancer cell being more sensitive than an unrelated normal cell line does not necessarily establish therapeutically relevant selectivity.

13. The framework should be demonstrated through a small number of case studies. The review would be more convincing if the authors selected three or four well-supported examples and applied the full model step by step. For each case, they could show, chemical identity and quality, pre-existing vulnerability, induced stress, buffering failure, rescue experiment, cancer versus normal-cell comparison, in vivo or pharmacokinetic support, remaining uncertainty.

This would demonstrate the practical usefulness of the framework more effectively than repeated general statements.

14. The title and scope should be reconsidered. The title emphasizes “selective apoptosis,” but the manuscript extensively discusses ferroptosis, autophagy, membrane injury, metabolic stress, and other death pathways. The authors should either narrow the review more strictly to apoptosis or broaden the title to reflect cancer-cell vulnerability and regulated cell death more generally.
15. The first-page header states “Pharmaceuticals 2029,” whereas subsequent pages state 2026. This should be corrected.
16. Author initials in the contribution statement should be checked for consistency with the author list. 17. Terminology should be standardized: “membrane–ion dysregulation,” “ion-volume dysregulation,” and “ion-channel and cell-volume dysregulation” appear to be used interchangeably.
17. “Cancer-cell vulnerability architecture” should be defined once and then used consistently.
18. The manuscript should avoid describing natural products as inherently safer than synthetic agents.
19. “Phytochemical” and “natural product” should not always be treated as synonyms.
20. The authors should define whether “selective apoptosis” requires direct comparison with normal cells or may also refer to differential sensitivity among cancer subtypes.
21. The number of figures and tables may be reduced by consolidating overlapping material.
22. Several sentences are overly long and should be divided for clarity.
23. The English is generally understandable but requires editorial polishing to reduce repetition and improve readability.

Overall, the manuscript has a strong conceptual foundation and addresses important weaknesses in natural-product anticancer research. The emphasis on pre-existing vulnerability, adaptive buffering, normal-cell sparing, chemical standardization, and mechanistic validation is valuable. The proposed membrane-ion dimension is also thought-provoking.

Nevertheless, the review currently reads as an expansive conceptual essay rather than a sufficiently disciplined evidence-based synthesis. The authors should shorten the manuscript, provide a transparent literature methodology, distinguish established evidence from hypothesis, moderate claims regarding membrane-ion biology, and demonstrate the framework through well-supported case studies.

 

Author Response

General comment: The manuscript is comprehensive, well illustrated, and appropriately emphasizes matched normal-cell controls, orthogonal cell-death assays, chemical standardization, pharmacokinetic plausibility, and the need to distinguish mechanistic observations from true vulnerability-state evidence. The figures are conceptually strong, particularly the comparison between compound-centered and vulnerability-centered pharmacology and the summary of five proposed vulnerability states.

         However, the manuscript is currently overly long, repetitive, and sometimes more assertive than the available evidence permits. Significant restructuring and clarification are needed before publication.

Response: We sincerely thank the reviewer for the thoughtful and constructive evaluation of our manuscript and for recognizing its conceptual framework, figures, and emphasis on rigorous interpretation of mechanistic evidence. We appreciate the reviewer’s concerns regarding the manuscript’s length, repetition, and the strength of some of the conclusions. In response, we have thoroughly revised the manuscript to improve clarity, reduce redundancy, and present the proposed vulnerability-state framework in a more balanced and evidence-based manner. Specifically, we have shortened several sections, streamlined repetitive discussions, clarified the distinction between established evidence and conceptual hypotheses, moderated statements that could be interpreted as overly assertive, and revised the figure captions by moving explanatory text into the main body of the manuscript. We believe these revisions have substantially improved the readability, organization, and scientific rigor of the Review while preserving its central conceptual message.

 

Comment 1: The review requires a clearly described literature-search methodology. The manuscript presents itself as a broad and authoritative synthesis, but no transparent search strategy is provided. The authors should add a short methodology section describing databases searched, date range, key search terms, inclusion and exclusion criteria, how clinical, in vivo, organoid, and cell-line studies were prioritized, how crude extracts were distinguished from purified compounds, whether the review was narrative, scoping, or systematic. Without this information, it is difficult to assess whether the evidence supporting each vulnerability state was selected comprehensively or selectively.

Response 1: We thank the reviewer for this valuable suggestion. We agree that greater transparency regarding the literature selection process strengthens the manuscript. Accordingly, we have added a dedicated “Literature Search Strategy” subsection to clarify the methodology used in preparing this Review. We explicitly state that this manuscript is a narrative, concept-driven review rather than a systematic or scoping review. The new subsection describes the databases searched, publication period, representative search terms, general inclusion and exclusion criteria, and the principles used to prioritize evidence, including clinical studies, in vivo models, organoid studies, and mechanistic cell-based investigations. We also clarify how evidence derived from chemically defined phytochemicals was distinguished from studies using crude botanical extracts and explain that the vulnerability-state framework was developed through thematic synthesis of the available literature rather than formal systematic evidence grading. We believe these additions improve the transparency and reproducibility of the review while remaining consistent with its conceptual objective.

 

Comment 2: The novelty of the “vulnerability-state model” should be distinguished more clearly from established concepts. The proposed framework is interesting, but many elements overlap with existing concepts such as mitochondrial priming, oncogene addiction, redox adaptation, synthetic lethality, therapeutic windows, stress-buffer dependence, and cancer-selective vulnerability.

The authors should explicitly explain which elements are established, which elements are newly integrated, what is genuinely novel about applying the model specifically to natural products, how the framework differs from existing cancer vulnerability and stress-adaptation models.

Response 2: We thank the reviewer for this important observation. We agree that the novelty of the proposed framework should be distinguished more explicitly from the well-established concepts on which it builds. We have therefore revised the Introduction and Discussion to clearly state that the individual biological concepts underlying the framework—including mitochondrial priming, oncogenic survival-pathway dependence, redox adaptation, therapeutic windows, and cancer-cell vulnerability—are established in the cancer biology literature and are not presented as novel discoveries. Rather, the novelty of this Review lies in their integration into a unified vulnerability-state framework specifically designed to interpret plant-derived natural-product-induced selective apoptosis. We further emphasize that the proposed framework shifts the focus from describing molecular responses after phytochemical exposure to identifying the pre-existing biological states that determine whether phytochemical-induced stress results in selective apoptosis, adaptive survival, or non-selective toxicity. Finally, we explicitly compare the proposed framework with existing cancer vulnerability and stress-adaptation concepts, highlighting that it is intended as a complementary conceptual model for phytopharmacology rather than a replacement for established theories.

 

Comment 3: A comparison table contrasting the proposed model with established frameworks would strengthen the manuscript.

Response 3: We thank the reviewer for this helpful suggestion. To improve the clarity of the conceptual contribution of the proposed framework, we have added Table 1, “Relationship between the proposed vulnerability-state framework and established concepts in cancer biology.” The new table compares the proposed framework with well-established concepts, including mitochondrial priming, redox adaptation, oncogene addiction/survival-pathway dependence, stress-buffer dependence, therapeutic windows/cancer-selective vulnerability, and synthetic lethality. For each concept, we summarize its established role in cancer biology and explain how it relates to the proposed vulnerability-state framework. We also clarify that the framework does not introduce new biological mechanisms, but rather integrates these complementary concepts into a unified phytopharmacology-oriented model for interpreting plant-derived natural-product-induced selective apoptosis. We believe that this addition more clearly distinguishes the conceptual novelty of the Review while highlighting its relationship to existing cancer biology frameworks.

 

Comment 4: The conceptual equation should not be presented as if it were mathematically defined. The manuscript expresses selective apoptosis as phytochemical stress × pre-existing vulnerability × insufficient adaptive buffering × normal-cell sparing.

This is useful as a conceptual mnemonic, but multiplication implies measurable, independent, quantitative variables. The terms are not operationally defined or mathematically validated.

The authors should either clearly label the equation as a qualitative conceptual relationship or replace it with a schematic logic model.

Response 4: We thank the reviewer for this valuable suggestion and agree that the equation should not be interpreted as a mathematically defined or predictive model. Following the reviewer’s recommendation, we retained the equation but revised the manuscript to explicitly describe it as a qualitative conceptual relationship intended solely to illustrate the interaction among the biological components of the proposed framework. We now state that Equation (1) should not be interpreted as a quantitative mathematical or predictive model. In addition, we added explanatory text immediately following the equation clarifying that it represents a conceptual heuristic rather than a mathematically validated model, and that the individual components represent interconnected biological processes rather than independent, operationally defined quantitative variables. We believe these revisions clarify the illustrative purpose of the equation while preserving its value as a concise conceptual summary of the proposed vulnerability-state framework.

 

Comment 5: “Normal-cell sparing” is also not a causal driver of apoptosis but an observed requirement for selectivity. It may therefore be better placed as an outcome criterion rather than a multiplicative component.

Response 5: We thank the reviewer for this insightful observation and agree that normal-cell sparing is conceptually distinct from the biological determinants of apoptotic commitment. In response, we have revised the text accompanying Equation (1) to clarify that normal-cell sparing is included as the defining criterion for selective apoptosis rather than as a causal biological driver of apoptosis. We further explain that phytochemical-induced stress, pre-existing vulnerability, and adaptive buffering represent the principal biological determinants within the framework, whereas normal-cell sparing serves to distinguish selective anticancer activity from non-selective cytotoxicity. Because Equation (1) is now explicitly presented as a qualitative conceptual relationship rather than a mathematical model, we believe its inclusion remains appropriate while the revised explanation more accurately reflects the conceptual role of normal-cell sparing.

 

Comment 6: Evidence supporting each vulnerability state should be graded. The manuscript often moves between established mechanisms, plausible hypotheses, and largely untested proposals. This is especially important for membrane-ion dysregulation, which is repeatedly described as a “missing” or distinctive layer between phytochemical stress and apoptosis. The authors should classify evidence as, for example clinically validated, supported by in-vivo studies, supported by organoid or advanced models, supported only by conventional cell lines, mechanistically inferred, hypothetical or underexplored.

This could be incorporated into the major tables. Without such grading, readers may interpret speculative mechanisms as equally established.

Response 6: We thank the reviewer for this important recommendation. In response, we comprehensively revised the major synthesis tables to distinguish established evidence from mechanistic inference and emerging hypotheses. An “Evidence level” column has now been incorporated into Table 2 and into each vulnerability-state table covering redox imbalance, mitochondrial priming, membrane remodeling, ion-channel and cell-volume dysregulation, and survival-pathway dependence. The revised tables classify the evidence using descriptors such as clinically validated, preclinical, mechanistically supported, emerging/mechanistically inferred, hypothesis-generating, and hypothetical/underexplored, according to the maturity and causal strength of the available evidence.

         We also added an explanation of the qualitative evidence-grading approach to the Literature Search Strategy. This clarification distinguishes evidence derived from human or clinically established contexts, advanced preclinical and in vivo models, conventional cell-line studies, pathway-specific mechanistic experiments, and largely untested proposals. We emphasize that this grading is intended as a transparent narrative evidence classification rather than a formal systematic-review or GRADE assessment, and that the assigned level does not necessarily apply uniformly to every compound within a phytochemical class.

         Particular care was taken in revising the membrane–ion and ion–volume sections. Table 6 now explicitly identifies apoptotic volume decrease and K⁺-dependent mechanisms as mechanistically supported but incompletely validated, regulatory volume decrease and store-operated Ca²⁺ entry as emerging or hypothesis-generating, and VRAC/LRRC8 and aquaporin-related mechanisms as hypothetical or underexplored in the context of natural-product-induced selective apoptosis. We also moderated the narrative wording by replacing the expression “missing biophysical layer” with “an underexplored candidate biophysical link” and explicitly stating that causal natural-product-specific evidence remains limited. These revisions are intended to prevent speculative mechanisms from being interpreted as equivalent in evidentiary strength to redox imbalance, mitochondrial apoptosis, or other more extensively studied vulnerability states.

 

Comment 7:  The membrane-ion section is potentially novel but currently overemphasized relative to the evidence. The manuscript repeatedly suggests that ion flux, apoptotic volume decrease, VRAC/LRRC8 activity, aquaporins, and membrane potential may provide the missing biophysical connection between natural-product exposure and apoptotic commitment. This is a valuable hypothesis, but the cited evidence appears substantially less developed than that supporting redox imbalance or mitochondrial priming.

The authors should moderate claims such as “missing layer” unless directly supported, identify specific natural products for which causal membrane–ion evidence exists, distinguish early causal events from secondary consequences of apoptosis, discuss pharmacological and genetic rescue experiments required to establish causality, explain how nonspecific membrane damage would be excluded.

Response 7: We thank the reviewer for this thoughtful suggestion and agree that the membrane–ion section should be presented more cautiously than the more extensively validated redox and mitochondrial vulnerability states. Accordingly, we substantially revised this section to emphasize that membrane–ion dysregulation represents an emerging and underexplored component of the proposed framework rather than an established mechanism of phytochemical-induced selective apoptosis. We replaced wording suggesting a “missing biophysical layer” with more conservative terminology (“underexplored candidate biophysical context”) and explicitly acknowledge throughout the manuscript that direct causal evidence remains limited.

         We further strengthened the section by identifying representative phytochemicals for which membrane- or ion-associated effects have been reported (e.g., saponins as the strongest membrane-directed example, together with selected evidence for curcumin, berberine, resveratrol, quercetin, EGCG, and withaferin A), while clearly stating that direct evidence linking these compounds to VRAC/LRRC8- or aquaporin-dependent mechanisms remains sparse. We also distinguished early membrane–ion events from secondary consequences of apoptosis, emphasized the need for temporal analyses to establish causality, expanded the discussion of pharmacological and genetic rescue experiments required to validate membrane–ion mechanisms, and clarified how nonspecific membrane injury should be excluded through appropriate controls, including membrane-integrity assays, hemolysis assessment for membrane-active compounds, matched non-malignant cells, and pathway-specific rescue experiments. Finally, the evidence grading in Table 6 and the accompanying table note were revised to reflect the current maturity of evidence for each membrane–ion mechanism. These revisions better align the emphasis of this section with the available evidence while preserving its value as a hypothesis-generating component of the proposed framework.

 

Comment 8: The present manuscript itself acknowledges that most phytochemical studies do not directly test these mechanisms.

Response 8: We appreciate the reviewer’s observation and agree that the available evidence supporting membrane–ion biology is substantially less mature than that supporting redox imbalance or mitochondrial priming. Our intention was not to present membrane–ion dysregulation as an equally established vulnerability state, but rather to identify it as an emerging area that may merit greater investigation in future phytochemical studies. To avoid overinterpretation, we have revised the manuscript throughout to emphasize its hypothesis-generating nature. Specifically, we now explicitly state that membrane–ion biology is included because of its potential biological relevance suggested by apoptosis and cancer-cell physiology, despite the fact that most natural-product studies have not directly examined these mechanisms. We further distinguish established evidence from speculative mechanisms through qualitative evidence grading, identify representative phytochemicals for which preliminary membrane-related evidence exists, and emphasize that causal validation will require time-resolved studies together with pharmacological and genetic rescue experiments. These revisions better align the emphasis of this section with the current strength of the available evidence.

 

Comment 9: The manuscript is too long and contains extensive repetition. At approximately 54 pages, the review repeatedly restates the same concepts as pathway modulation does not equal vulnerability, IC₅₀ values alone are insufficient, matched normal cells are required, ROS can be protective or toxic depending on context, pharmacokinetic plausibility is essential, membrane effects must be separated from nonspecific toxicity.

These points are important but appear in the abstract, introduction, framework section, individual vulnerability sections, translational sections, and conclusion. The conclusion also repeats material presented immediately before it.

The manuscript should be shortened substantially, possibly by 25–35%. Each section should focus on evidence specific to that vulnerability state rather than restating the general framework.

Response 9: We appreciate this important suggestion and agree that several overarching concepts were reiterated throughout the manuscript to maintain continuity across the vulnerability-state sections. In the revised version, we substantially reduced repetition by consolidating the general conceptual framework within the Introduction and Section 2, while revising the individual vulnerability-state sections to focus primarily on evidence specific to each biological domain. Repeated discussions of pathway modulation versus vulnerability, the limitations of IC₅₀ values, the importance of matched non-malignant controls, the context-dependent nature of ROS signaling, pharmacokinetic considerations, and the distinction between selective membrane signaling and nonspecific toxicity were consolidated into dedicated sections rather than repeated throughout the manuscript.

 

Comment 10: The tables are highly detailed but difficult to use. Table 1 and subsequent tables contain very long text entries and combine mechanistic, experimental, and translational information. This creates dense blocks of information that may overwhelm readers.

The authors should consider, shortening each cell, moving detailed assay recommendations to a supplementary table, adding an “evidence level” column, separating natural-product classes from experimental validation requirements, highlighting only representative compounds with the strongest evidence.

Table 1 should not imply that all members of a phytochemical class share the same properties. The manuscript recognizes this problem, but the table structure may still promote class-level generalization.

Response 10: We thank the reviewer for this valuable suggestion and have substantially revised all major tables to improve readability and reduce the risk of overgeneralization.

         First, an Evidence level column has been added to each mechanistic table to distinguish the maturity of supporting evidence, ranging from clinically validated or advanced preclinical evidence to mechanistic, emerging, or hypothesis-generating evidence, as appropriate. Second, the tables have been reorganized to separate the biological rationale, representative natural-product relevance, evidence level, and key validation criteria, thereby improving conceptual organization and reducing the mixing of mechanistic, experimental, and translational information.

         To avoid class-level generalization, Table 2 (previously Table 1) has been revised to emphasize that phytochemical classes are presented as conceptual examples rather than uniform biological entities. The title, column headings, and table notes now consistently refer to representative phytochemicals, and we explicitly state that representative compounds and mechanisms should not be generalized to all members of a phytochemical class. Likewise, the evidence-level notes clarify that the assigned evidence reflects the highest level of support available for representative compounds and should not be interpreted as applying uniformly across an entire phytochemical class.

         In addition, we shortened numerous table entries by removing repetitive explanatory text and retaining only the information essential for the conceptual framework.

 

Comment 11: The distinction among apoptosis, ferroptosis, necrosis, pyroptosis, necroptosis, and cytostasis needs more consistent treatment. The manuscript appropriately warns against interpreting metabolic viability assays as proof of apoptosis. However, some sections still group ROS production, lipid peroxidation, mitochondrial dysfunction, and caspase changes under a broad selective-apoptosis narrative.

The authors should provide a concise experimental decision framework indicating the minimum evidence needed to claim apoptosis, ferroptosis, mixed death phenotypes, necrosis or membrane lysis, cytostasis.

Response 11: We thank the reviewer for this valuable suggestion. We agree that reactive oxygen species accumulation, lipid peroxidation, mitochondrial dysfunction, caspase activation, and reduced metabolic viability are not individually sufficient to define a specific cell-death modality. Accordingly, we have revised the manuscript to provide a clearer and more consistent distinction among apoptosis, ferroptosis, necroptosis, pyroptosis, primary necrosis or direct membrane lysis, cytostasis, and mixed death phenotypes.

         Specifically, we substantially revised Section 9 (“When selective apoptosis fails: necrosis, non-selective toxicity and hormesis”) and added Table 8, which provides a concise decision framework summarizing the minimum evidence required to support each cellular outcome. Rather than relying on individual biomarkers, the framework emphasizes the integration of morphological, molecular, functional, and pathway-specific rescue evidence. It also identifies findings that are insufficient on their own to establish a particular death modality, thereby helping readers distinguish regulated cell-death programs from nonspecific cytotoxicity or growth inhibition.

         We additionally revised the surrounding text to avoid implying that ROS production, lipid peroxidation, mitochondrial depolarization, or caspase modulation automatically indicate selective apoptosis. Instead, these events are consistently presented as pathway-associated or upstream stress responses that require context-specific validation before assigning a specific cell-death mechanism. The revised section and Table 8 are aligned with the current recommendations of the Nomenclature Committee on Cell Death and recent consensus reviews on regulated cell death, thereby improving the conceptual clarity and methodological rigor of the proposed vulnerability-state framework.

 

Comment 12: For apoptosis, Annexin V/PI and caspase cleavage alone may not always be sufficient. Time-course analysis, morphological evidence, caspase dependence, and appropriate rescue controls should be considered.

Response 12: We thank the reviewer for this important comment. We agree that Annexin V/propidium iodide staining and caspase activation alone are insufficient to establish apoptosis. In the revised manuscript, we strengthened the discussion of apoptosis classification by emphasizing that apoptosis should be supported by integrated morphological, molecular, temporal, and functional evidence rather than individual biomarkers.

         Specifically, we added Table 8, which provides a practical framework for distinguishing major cellular outcomes following natural-product exposure. For apoptosis, the table now emphasizes coordinated apoptotic morphology, temporally ordered mitochondrial and caspase events, demonstration of caspase dependence, and pathway-specific rescue or genetic validation where appropriate. We also revised the accompanying text in Section 9 to clarify that Annexin V positivity, mitochondrial depolarization, caspase activation, and reduced metabolic viability should be interpreted as supportive rather than definitive evidence of apoptosis in the absence of time-course analysis and functional validation. These revisions are consistent with current recommendations of the Nomenclature Committee on Cell Death.

 

Comment 13: “Natural products” is treated too broadly. The manuscript includes, purified phytochemicals, crude extracts, dietary constituents, metabolites, optimized synthetic derivatives, approved natural-product-derived drugs.

These categories have very different standards of chemical identity, exposure, reproducibility, and translational relevance.

Response 13: We thank the reviewer for this important observation. We agree that the term “natural products” encompasses chemically and translationally heterogeneous entities that differ substantially in chemical definition, reproducibility, pharmacokinetics, regulatory status, and clinical relevance.

         To address this concern, we further clarified the scope of the Review throughout the manuscript. In the Introduction, we explicitly define plant-derived natural products as referring primarily to chemically characterized phytochemicals, standardized botanical extracts, their major bioactive constituents, and clinically relevant natural-product-derived or semisynthetic derivatives, while emphasizing that mechanistic interpretation is preferentially based on chemically defined agents. We further distinguish evidence derived from crude botanical extracts because of the additional challenges associated with chemical standardization, batch-to-batch reproducibility, and attribution of biological activity.

         We also revised the text and tables to consistently distinguish chemically defined phytochemicals from crude extracts, dietary constituents, approved natural-product-derived drugs, and optimized derivatives. Table 2 now explicitly presents these categories as conceptually distinct examples with different levels of translational maturity, and the accompanying notes emphasize that representative compounds should not be generalized across entire phytochemical classes. Throughout the manuscript, mechanistic conclusions are preferentially drawn from chemically characterized compounds, whereas evidence from crude extracts and dietary exposures is interpreted more cautiously.

 

Comment 14: The authors should establish a hierarchy and consistently distinguish, crude or partially characterized extracts, purified natural compounds, active metabolites, semisynthetic or optimized derivatives, clinically approved natural-product-derived drugs.

Response 14: Thank you for this valuable suggestion. We agree that distinguishing different categories of plant-derived materials is essential because they differ substantially in chemical identity, reproducibility, pharmacokinetics, regulatory maturity, and translational relevance. We have therefore revised the Introduction to explicitly establish this conceptual hierarchy. The revised text distinguishes chemically characterized phytochemicals, standardized botanical extracts, major bioactive constituents, semisynthetic or natural-product-derived derivatives, and clinically approved natural-product-derived anticancer drugs. Throughout the manuscript, mechanistic interpretation is presented in the context of the specific material under investigation rather than treating all plant-derived agents as a homogeneous class. We also clarify that evidence derived from crude botanical extracts, dietary exposures, and metabolites is interpreted more cautiously because of the additional challenges associated with chemical standardization, exposure assessment, and attribution of biological activity. This distinction has been applied consistently throughout the manuscript.

 

Comment 15: Claims based on paclitaxel or vinca alkaloids should not automatically validate unrelated investigational phytochemicals.

Response 15: Thank you for this important comment. We agree that clinically approved natural-product-derived drugs should not be used to validate unrelated investigational phytochemicals. We have revised the manuscript to explicitly distinguish these categories and clarify that agents such as paclitaxel and vinca alkaloids represent optimized and clinically validated natural-product-derived drugs, whereas most phytochemicals remain investigational. The revised text emphasizes that the clinical success of these drugs supports the therapeutic potential of natural-product scaffolds but should not be interpreted as evidence for the efficacy or translational readiness of unrelated plant-derived compounds. This distinction is now applied consistently throughout the manuscript.

 

Comment 16: More quantitative translational criteria are needed. The manuscript repeatedly calls for pharmacokinetic and pharmacodynamic plausibility, which is appropriate, but it does not define how readers should evaluate this.

Response 16: Thank you for this constructive suggestion. We agree that translational plausibility should be evaluated using explicit criteria rather than discussed only conceptually. We have therefore revised the methodological roadmap to define practical benchmarks for translational assessment. Specifically, we state that studies should demonstrate (i) achievable biologically active exposures under clinically relevant conditions, (ii) target engagement or pathway modulation at those exposures, (iii) concordance between pharmacokinetic and pharmacodynamic responses, and (iv) validation of the proposed mechanism in physiologically relevant experimental models. These additions provide readers with a practical framework for assessing the translational potential of investigational phytochemicals while remaining applicable across diverse classes of plant-derived compounds.

 

Comment 17: The authors should recommend comparison of in vitro active concentration, unbound plasma exposure, tumor tissue exposure, metabolite exposure, protein binding, achievable duration of exposure, formulation-dependent delivery, maximum tolerated dose.

This would make the framework more practical and prevent unrealistic interpretation of high micromolar in vitro effects.

Response 17: Thank you for this excellent suggestion. We agree that translational interpretation should extend beyond reporting in vitro activity alone. We have therefore expanded the methodological framework to recommend explicit comparison of biologically active in vitro concentrations with key pharmacokinetic and pharmacodynamic parameters, including unbound plasma exposure, target-tissue (or intratumoral) exposure where available, active metabolite exposure, plasma protein binding, achievable duration of pharmacologically active exposure, formulation-dependent delivery, and maximum tolerated dose or clinically achievable exposure. We further emphasize that target engagement should be demonstrated at clinically relevant exposures and that pharmacokinetic, pharmacodynamic, and efficacy data should be interpreted together. These additions provide a practical framework for evaluating whether experimentally active concentrations are realistically attainable in vivo and help prevent overinterpretation of high-micromolar in vitro findings.

 

Comment 18: The review should address chemical liabilities more systematically. The manuscript mentions aggregation, impurities, assay interference, instability, and promiscuity, particularly for curcumin. These issues deserve a more organized section discussing PAINS-related concerns, colloidal aggregation, redox cycling, covalent nonspecific reactivity, fluorescence or absorbance interference, instability in culture media, contamination and batch variability, active metabolite formation.

A concise checklist for validating natural-product activity would be useful.

Response 18: Thank you for this valuable suggestion. We agree that potential chemical liabilities should be presented in a more systematic manner. We have therefore expanded the methodological section by introducing a dedicated discussion on analytical validation of natural-product activity. The revised text summarizes major sources of experimental artifacts, including chemical purity and batch variability, compound stability, colloidal aggregation, redox cycling, nonspecific covalent reactivity, fluorescence and absorbance interference, contamination, and active metabolite formation. In addition, we have added a concise checklist (Table 9) summarizing these potential liabilities together with recommended validation approaches. This addition complements the biological validation framework and provides readers with a practical guide for distinguishing genuine pharmacological activity from experimental artifacts.

 

Comment 19: Claim of cancer selectivity require stronger definition. The manuscript correctly emphasizes matched normal-cell models, but “normal-cell sparing” is repeatedly used without defining an acceptable therapeutic window.

The authors should discuss appropriate normal comparators, tissue matching, proliferating versus quiescent normal cells, immune-cell compatibility, selectivity-index calculations, clinically meaningful versus statistically significant differences, whether transformed and non-transformed cells have comparable growth rates.

A cancer cell being more sensitive than an unrelated normal cell line does not necessarily establish therapeutically relevant selectivity.

Response 19: Thank you for this insightful comment. We agree that cancer selectivity should be defined using explicit biological and experimental criteria rather than qualitative terminology alone. We have substantially expanded the sections on normal-cell sparing and cytotoxicity testing to clarify that selectivity should be interpreted as a comparative property requiring biologically relevant normal-cell comparators under matched experimental conditions. The revised manuscript discusses tissue-matched comparator cells, proliferating versus quiescent non-transformed cells, immune-cell compatibility, interpretation of selectivity indices together with concentration-response relationships and biological effect size, the distinction between statistical significance and biologically meaningful therapeutic windows, and the potential influence of differences in proliferation kinetics between transformed and non-transformed cells. We intentionally avoid proposing a universal numerical selectivity threshold because no consensus exists across tumor types or experimental systems. Instead, we provide a practical framework for evaluating cancer selectivity using multiple complementary criteria, consistent with the overall methodological roadmap developed in this Review.

 

Comment 20: The framework should be demonstrated through a small number of case studies. The review would be more convincing if the authors selected three or four well-supported examples and applied the full model step by step. For each case, they could show, chemical identity and quality, pre-existing vulnerability, induced stress, buffering failure, rescue experiment, cancer versus normal-cell comparison, in vivo or pharmacokinetic support, remaining uncertainty.

This would demonstrate the practical usefulness of the framework more effectively than repeated general statements.

Response 20: Thank you for this insightful suggestion. We agree that demonstrating the practical application of the proposed framework strengthens its translational value. Accordingly, we have added a new subsection (Section 12.6, “Illustrative application of the vulnerability-state framework”) presenting three representative case studies (β-lapachone, piperlongumine, and parthenolide/DMAPT). Rather than providing descriptive summaries, these examples systematically apply the framework by evaluating: (i) chemical identity and quality, (ii) the pre-existing cancer-cell vulnerability, (iii) the induced stress, (iv) failure of adaptive buffering, (v) evidence from rescue or mechanistic perturbation experiments, (vi) comparisons with biologically relevant normal cells, (vii) available in vivo and pharmacokinetic support, and (viii) remaining mechanistic or translational uncertainties. The selected examples represent different levels of mechanistic and translational maturity, illustrating how the framework can be used to distinguish biomarker-supported, pharmacologically plausible candidates from investigational phytochemicals that require additional chemical, biological, or pharmacokinetic validation. We believe this addition provides a practical demonstration of the framework while avoiding repetition elsewhere in the manuscript.

 

Comment 21: The title and scope should be reconsidered. The title emphasizes “selective apoptosis,” but the manuscript extensively discusses ferroptosis, autophagy, membrane injury, metabolic stress, and other death pathways. The authors should either narrow the review more strictly to apoptosis or broaden the title to reflect cancer-cell vulnerability and regulated cell death more generally.

Response 21: Thank you for this thoughtful comment. We agree that the scope of the Review should be clearly aligned with its title. Our intention is not to provide a comprehensive review of regulated cell death, but rather to explain selective apoptosis through the broader biological context of cancer-cell vulnerability states. Accordingly, we have revised the manuscript to sharpen this focus throughout. Discussions of ferroptosis, autophagy, membrane injury, metabolic stress, and other regulated cell death pathways have been substantially condensed and are presented only where they directly contribute to understanding the mechanisms, boundaries, or differential diagnosis of selective apoptosis. In particular, the integrative model (Section 12) now emphasizes that these processes primarily represent adaptive responses, upstream vulnerability states, or alternative outcomes that influence whether cells ultimately undergo selective apoptosis. Thus, apoptosis remains the central biological endpoint of the Review, whereas other regulated cell death mechanisms are discussed only insofar as they help explain why selective apoptosis occurs in some cellular contexts but not others. We therefore believe that the current title accurately reflects the revised scope of the manuscript.

 

Comment 22: The first-page header states “Pharmaceuticals 2029,” whereas subsequent pages state 2026. This should be corrected.

Response 22: Thanks. Corrected.

 

Comment 23: Author initials in the contribution statement should be checked for consistency with the author list.

Response 23: Thanks. Checked.

 

Comment 24: Terminology should be standardized: “membrane–ion dysregulation,” “ion-volume dysregulation,” and “ion-channel and cell-volume dysregulation” appear to be used interchangeably.

Comment 25: Thank you for identifying this inconsistency. We have standardized the terminology throughout the manuscript by adopting “membrane–ion dysregulation” as the primary name of this vulnerability state, consistent with the manuscript title and the proposed conceptual framework. At its first mention, we define this term as encompassing altered ion-channel activity, membrane potential, ion transport, and cell-volume regulation. We have revised the text accordingly to eliminate interchangeable terminology and improve conceptual consistency throughout the Review.

 

Comment 26: “Cancer-cell vulnerability architecture” should be defined once and then used consistently.

Response 26: Thank you for this helpful suggestion. We agree that the term should be explicitly defined before being used throughout the manuscript. Accordingly, we have added a formal definition in Section 2 when introducing the vulnerability-centered framework. We define cancer-cell vulnerability architecture as the integrated configuration of pre-existing biological vulnerabilities – including redox imbalance, mitochondrial priming, membrane remodeling, membrane–ion dysregulation, survival-pathway addiction, and adaptive-buffering capacity – that collectively determines susceptibility to phytochemical-induced stress. We have subsequently used this terminology consistently throughout the manuscript to improve conceptual clarity and avoid repeated implicit definitions.

 

Comment 27: The manuscript should avoid describing natural products as inherently safer than synthetic agents.

Response 27: Thank you for this important comment. We agree that botanical origin should not be interpreted as implying superior safety. The manuscript has been revised to clarify that plant-derived compounds are not inherently safer than synthetic anticancer agents. We now explicitly state that their therapeutic value depends on the same criteria applied to all anticancer therapeutics, including chemical characterization, pharmacokinetic properties, toxicological evaluation, therapeutic window, and preferential activity against malignant relative to matched normal cells. Throughout the manuscript, we have also ensured that statements regarding selectivity refer to experimentally demonstrated normal-cell sparing rather than presumed safety based on natural origin. This clarification is consistent with the overall emphasis of the Review on evidence-based vulnerability exploitation rather than assumptions regarding the source of a compound.

 

Comment 28: “Phytochemical” and “natural product” should not always be treated as synonyms.

Response 28: Thank you for this important observation. We agree that these terms describe related but distinct concepts. To improve precision, we have clarified the terminology in the Introduction by explicitly defining plant-derived natural products as the overarching category encompassing chemically characterized phytochemicals, standardized botanical extracts, major bioactive constituents, semisynthetic or natural-product-derived derivatives, and approved natural-product-derived drugs. We further specify that phytochemicals refers only to naturally occurring plant-derived chemical constituents. The terminology has been standardized throughout the manuscript so that the broader term is used when referring to the overall field or conceptual framework, whereas phytochemicals is reserved for discussions of defined plant-derived compounds and their mechanisms of action. This revision improves conceptual clarity and avoids treating the two terms as interchangeable.

 

Comment 29: The authors should define whether “selective apoptosis” requires direct comparison with normal cells or may also refer to differential sensitivity among cancer subtypes.

Response 29: Thank you for this valuable comment. We agree that this distinction should be stated explicitly. The manuscript has been revised to clarify that, within the proposed framework, selective apoptosis refers to the preferential induction of apoptosis in malignant cells relative to biologically relevant matched non-malignant cells under comparable exposure conditions. By contrast, differential sensitivity among cancer cell lines or molecular subtypes is interpreted as evidence of differences in cancer-cell vulnerability architecture rather than as evidence of selective apoptosis by itself. Such comparisons remain important for identifying vulnerability states but should ideally be complemented by normal-cell controls when therapeutic selectivity is inferred. This clarification has been incorporated into the conceptual framework and is reinforced in the section discussing cancer selectivity and normal-cell sparing.

 

Comment 30: The number of figures and tables may be reduced by consolidating overlapping material.

Response 30: Thank you for this suggestion. We carefully reviewed all figures and tables to identify potential overlap. Because each visual element serves a distinct purpose within the conceptual progression of the Review, we retained the current number while reducing redundancy. Specifically, we streamlined figure captions, shortened table notes, removed repetitive explanations from the main text, and ensured that each figure and table contributes unique conceptual or translational information without duplicating material presented elsewhere

 

Comment 31: Several sentences are overly long and should be divided for clarity.

Response 31: Thank you for this helpful suggestion. We carefully re-read the entire manuscript with particular attention to sentence structure and readability. Several long or multi-clause sentences have been divided into shorter, more focused statements throughout the manuscript, particularly in the Introduction and conceptual framework sections. These revisions improve clarity and readability while preserving the scientific precision and logical progression of the Review.

 

Comment 32: The English is generally understandable but requires editorial polishing to reduce repetition and improve readability.

Response 32: Thank you for this helpful suggestion. We carefully edited the manuscript throughout to improve clarity, readability, and linguistic consistency. Repetitive wording has been reduced where appropriate, several long or multi-clause sentences have been divided into shorter statements, and transitions between sections have been refined to improve the overall flow of the Review. We also standardized terminology and phrasing throughout the manuscript to ensure greater consistency while preserving scientific precision.

 

General comment: Overall, the manuscript has a strong conceptual foundation and addresses important weaknesses in natural-product anticancer research. The emphasis on pre-existing vulnerability, adaptive buffering, normal-cell sparing, chemical standardization, and mechanistic validation is valuable. The proposed membrane-ion dimension is also thought-provoking.

Nevertheless, the review currently reads as an expansive conceptual essay rather than a sufficiently disciplined evidence-based synthesis. The authors should shorten the manuscript, provide a transparent literature methodology, distinguish established evidence from hypothesis, moderate claims regarding membrane-ion biology, and demonstrate the framework through well-supported case studies.

Response: We sincerely thank the Reviewer for the thoughtful and constructive overall assessment of our work and for recognizing the conceptual value of the proposed vulnerability-state framework. We appreciate the positive evaluation of the manuscript’s emphasis on pre-existing cancer-cell vulnerabilities, adaptive buffering, normal-cell sparing, chemical standardization, mechanistic validation, and the inclusion of membrane–ion biology as an emerging dimension of selective apoptosis.

         In response to the Reviewer’s recommendations, we have substantially revised the manuscript to improve its scientific rigor, balance, and readability. Specifically, we have:

- Shortened and streamlined the manuscript by reducing redundant discussions, simplifying figure captions and table notes, and improving the overall organization.

- Added a dedicated Literature Search Strategy section, clearly describing the databases searched, search period, thematic approach, inclusion priorities, and qualitative evidence-grading strategy.

- Systematically distinguished established evidence from hypothesis or emerging concepts throughout the text and summary tables by explicitly indicating the maturity of evidence and carefully moderating speculative statements.

- Refined the discussion of membrane–ion biology, presenting it as an emerging and underexplored vulnerability state supported by increasing mechanistic evidence while acknowledging that many proposed mechanisms require further experimental validation.

- Expanded the translational perspective by strengthening discussion of chemical characterization, analytical validation, pharmacokinetic and pharmacodynamic considerations, assay limitations, and the central importance of matched normal-cell controls and normal-cell sparing when interpreting selective apoptosis.

- Added illustrative case studies demonstrating how the proposed vulnerability-state framework can be applied to representative plant-derived compounds with different levels of mechanistic and translational evidence.

- Improved conceptual clarity by explicitly defining key terms, including cancer-cell vulnerability architecture and selective apoptosis, and by standardizing terminology throughout the manuscript.

- Performed comprehensive editorial revision to improve readability, reduce repetition, and enhance the logical progression of the Review.

         We believe these extensive revisions have transformed the manuscript from a predominantly conceptual perspective into a more disciplined, evidence-based synthesis while preserving the central conceptual contribution of the vulnerability-state framework. We are grateful for the Reviewer’s insightful comments, which have substantially strengthened the scientific quality, clarity, and translational relevance of the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors Well-written and scientifically sound review. But author should also discuss following points-  

1. How does the proposed vulnerability-state model differ from existing concepts such as oncogene addiction, synthetic lethality, non-oncogene addiction, or stress-response vulnerabilities?
2. Cancer-cell vulnerabilities differ across tumor types, molecular subtypes, and treatment-resistant populations. A discussion of these differences would improve the translational applicability of the framework.
3. A dedicated discussion acknowledging potential limitations, such as dynamic vulnerability states, tumor evolution, and challenges in clinical implementation, would strengthen the review.

Author Response

General comment: Well-written and scientifically sound review.

Response: We sincerely thank the Reviewer for the positive evaluation of our manuscript and for recognizing its scientific quality and clarity. We greatly appreciate your encouraging assessment that the Review is well written and scientifically sound. Your thoughtful evaluation has been valuable throughout the peer-review process, and we are grateful for your time and constructive consideration of our work.

 

But author should also discuss following points- 

Comment 1: How does the proposed vulnerability-state model differ from existing concepts such as oncogene addiction, synthetic lethality, non-oncogene addiction, or stress-response vulnerabilities?

Response 1: Thank you for this important conceptual question. We agree that the distinction between the proposed framework and established concepts should be stated more explicitly. Accordingly, we have revised the Introduction and Section 2 to clarify that the vulnerability-state framework is complementary to, rather than a replacement for, existing concepts in cancer biology. Specifically, we explain that oncogene addiction, non-oncogene addiction, synthetic lethality, mitochondrial priming, redox adaptation, and stress-response dependence each describe specific biological mechanisms or forms of cellular dependence. In contrast, the proposed vulnerability-state framework is an integrative interpretive model that organizes these complementary concepts according to the pre-existing biological state of the cancer cell and explains how diverse phytochemical-induced stresses converge on common vulnerability states to determine selective apoptotic responses. We have also expanded Table 1 to explicitly compare the proposed framework with these established concepts and emphasize that the novelty of the Review lies in their integration rather than in proposing new biological mechanisms.

 

Comment 2: Cancer-cell vulnerabilities differ across tumor types, molecular subtypes, and treatment-resistant populations. A discussion of these differences would improve the translational applicability of the framework.

Response 2: We thank the Reviewer for this valuable suggestion. We agree that cancer-cell vulnerabilities are context-dependent and may vary across tumor types, molecular subtypes, and treatment-resistant populations. Accordingly, we have expanded the discussion to clarify that the proposed vulnerability-state framework is intended as a dynamic, context-dependent interpretive model rather than a fixed hierarchy of vulnerabilities. The revised text highlights that distinct cancers may preferentially depend on different vulnerability states according to their genetic, epigenetic, metabolic, and microenvironmental contexts, and that therapeutic pressure can reshape these dependencies during disease progression, thereby creating new opportunities for vulnerability-directed intervention. This addition strengthens the translational applicability of the proposed framework.

 

Comment 3: A dedicated discussion acknowledging potential limitations, such as dynamic vulnerability states, tumor evolution, and challenges in clinical implementation, would strengthen the review.

Response 3: We thank the Reviewer for this valuable suggestion. We agree that the conceptual scope and translational boundaries of the proposed framework should be explicitly acknowledged. Accordingly, we have expanded the translational discussion by adding a dedicated paragraph outlining the principal limitations of the framework. Specifically, we emphasize that vulnerability states are dynamic and context-dependent, varying across tumor types, molecular subtypes, and treatment-resistant populations during disease evolution. We also clarify that the framework is intended as an integrative, hypothesis-generating conceptual model rather than a predictive clinical algorithm and discuss the need for biomarker development, standardized vulnerability-state assessment, longitudinal validation, and prospective clinical studies before routine implementation. Finally, we acknowledge that several proposed interactions, particularly those involving membrane–ion biology, remain supported predominantly by preclinical evidence and require further mechanistic and translational validation. This addition provides a more balanced discussion of both the strengths and current limitations of the proposed framework.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made sighnificant improvements of the manuscript. Now this one can be accepted by scientific community and published in humble opinion of the reviewer.

Conclution

Accept in current form.