Harnessing Endophytic Fungi as a Sustainable Source of Novel Anticancer Agents: Opportunities, Challenges, and Future Directions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Major comments

The manuscript “Harnessing Endophytic Fungi as a Sustainable Source of Novel Anticancer Agents: Opportunities, Challenges, and Future Directions”, is an interesting review. I consider that the compiled information will benefit the drug researchers.

 

However, it is suggested that the authors should include more comprehensive information, such as the summarize of the information about the biological sources, structural types, and action mechanism of novel compounds which were discovered in the past five years, rather than merely focusing on the typical compounds that are commonly known to the public. The discussion and conclusion section in the manuscript needs improve to provide a more insightful analysis.

 

I consider this paper suitable for publication in Molecules upon the following revisions:

 

1. I think the author has made some omissions in compiling the literature. The authors don't mention anything about how they did the research in this review. There are guidelines, such as the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, that allow a comprehensive and systematized compilation of scientific literature on a specific topic. Authors must describe the databases used, the keywords, and the criteria for article selection and exclusion. I suggest that authors use the PRISMA guidelines and present the flow diagram of literature search based on the guidelines.
2. The manuscript mentions that the toxicity of traditional chemotherapy cannot be ignored. Therefore, when evaluating the inhibitory activity of fungal crude extracts on tumor cells, it is necessary to also consider the toxicity towards normal human cells. This will help readers determine whether the crude extracts exhibit anti-tumor activity or just cytotoxicity.
3. Line 153 states that there have been no fungal metabolites used as drugs in the field of anti-tumor treatment for nearly 20 years. It is necessary to analyze the reasons for this and propose improved research directions in the discussion section.
4. Table 1 should be placed near Section 2.2.
5. When listing the metabolites of fungi, their chemical structures should be provided to help researchers (in natural products chemistry and organic synthesis) better understand the correlation between their structures and activities.
6. For each compound mentioned in the manuscript, its source, chemical structure, structural type, the source of fungi, the IC₅₀ value towards tumor cells and normal human cells should be indicated. The positive control and its IC₅₀ value should also be provided.
7. As mentioned in section 2.4, there are three advantages of endophytic fungi. It is necessary to supplement the current research progress based on these three advantages as well as the research results obtained therefrom.
8. There is a logical flaw in Figure 1. The current image shows the discovery of anti-tumor compounds and endophytic fungi from medicinal plants. However, the main focus of this manuscript is to summarize the anti-tumor compounds produced by endophytic fungi.
9. When introducing the mechanism of action of typical compounds in section 3, whether the corresponding compounds (such as paclitaxel, jatrorrhizine, hypericin, etc.) are commercial drugs or are currently at a certain research stage (clinical, preclinical research) should be indicated. The discovery year of the relevant compounds, their initial sources (plants, microorganisms) and chemical structures also need to be explained.
10. Section 3.5 covers the compounds with strong activity discovered during the period of 2021-2022. Data for the years 2023-2025 should be added. The discovery year, initial sources, research stage (clinical, preclinical research), chemical structure, structural type, the IC₅₀ value towards tumor cells and normal human cells should be indicated.
11. The arrangement sequence of the compounds in Figure 2 may cause confusion, leading readers to believe that the compounds were obtained from different parts of the plant. Please make the necessary corrections.
12. Section 4 and Table 2 should list the specific activity values of the active compounds.
13. The bottom border is missing in Table 2.
14. Section 5 is too brief and fails to adequately analyze the cases and methods involved in each strategy. It needs to be further expanded to enhance its reference value for researchers.
15. In addition to summarizing the currently well-known anti-tumor compounds, the review should also track the natural products with anti-tumor activity which were related to plant-derived endophytic fungi that have been discovered in the past five years. In the cited literature, the number of papers published in the years 2023-2025 is insufficient. Among the 111 references, the number of published papers in 2023-2025 is only 2, 7 and 5, respectively, which is not enough to summarize the latest research progress in this field.

Author Response

Major comments: The manuscript “Harnessing Endophytic Fungi as a Sustainable Source of Novel Anticancer Agents: Opportunities, Challenges, and Future Directions”, is an interesting review. I consider that the compiled information will benefit the drug researchers.

However, it is suggested that the authors should include more comprehensive information, such as the summarize of the information about the biological sources, structural types, and action mechanism of novel compounds which were discovered in the past five years, rather than merely focusing on the typical compounds that are commonly known to the public. The discussion and conclusion section in the manuscript needs improve to provide a more insightful analysis.

 

I consider this paper suitable for publication in Molecules upon the following revisions:

 

Comment 1: I think the author has made some omissions in compiling the literature. The authors don't mention anything about how they did the research in this review. There are guidelines, such as the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, that allow a comprehensive and systematized compilation of scientific literature on a specific topic. Authors must describe the databases used, the keywords, and the criteria for article selection and exclusion. I suggest that authors use the PRISMA guidelines and present the flow diagram of literature search based on the guidelines.

Response 1: We thank the reviewer for this important observation. To address this concern, we have substantially revised the Materials and Methods section to explicitly describe the literature search strategy and study selection process in accordance with the PRISMA 2020 guidelines. The revised manuscript now details the databases consulted (PubMed, Scopus, Web of Science, and Google Scholar), the search period, the keyword combinations used, and the inclusion and exclusion criteria applied for article selection. In addition, a PRISMA flow diagram has been included to transparently illustrate the identification, screening, eligibility assessment, and final inclusion of studies. These additions ensure methodological transparency, reproducibility, and a systematic compilation of the literature, fully aligning the review with PRISMA recommendations and MDPI Molecules standards.

Comment 2: The manuscript mentions that the toxicity of traditional chemotherapy cannot be ignored. Therefore, when evaluating the inhibitory activity of fungal crude extracts on tumor cells, it is necessary to also consider the toxicity towards normal human cells. This will help readers determine whether the crude extracts exhibit anti-tumor activity or just cytotoxicity.

Response 2: The manuscript has been revised to explicitly incorporate cytotoxicity data toward normal human cell lines wherever reported. Comparative IC₅₀ values and selectivity considerations are now discussed in the text and summarized in Tables 1 and 2, enabling clear distinction between general cytotoxicity and true anticancer selectivity.

Comment 3: Line 153 states that there have been no fungal metabolites used as drugs in the field of anti-tumor treatment for nearly 20 years. It is necessary to analyze the reasons for this and propose improved research directions in the discussion section.

Response 3: We thank the reviewer for this insightful comment. In response, we have expanded the Discussion section to critically analyze why endophyte-derived fungal metabolites have not yet achieved regulatory approval as anticancer drugs over the past two decades. The revised text now identifies key limiting factors, including reproducible yield and scale-up challenges, host-dependent biosynthesis, insufficient pharmacokinetic and toxicological validation, and late integration of formulation and regulatory considerations. We further propose future research directions to address these barriers, such as synthetic biology-based pathway engineering, multi-omics-guided dereplication, early PK/PD and selectivity assessment, and alignment with GMP-compatible production strategies. These additions clarify that the translational gap reflects technological and developmental challenges rather than a lack of biological potential, in full accordance with MDPI Molecules standards.

Comment 4: Table 1 should be placed near Section 2.2.

Response 4: Done!

Comment 5: When listing the metabolites of fungi, their chemical structures should be provided to help researchers (in natural products chemistry and organic synthesis) better understand the correlation between their structures and activities.

Response 5: We thank the reviewer for this valuable comment. To facilitate structure–activity interpretation for readers in natural products chemistry and organic synthesis, the manuscript has been revised to explicitly provide the chemical structures of representative fungal metabolites discussed throughout the review. All key compounds are now consolidated and illustrated in Figure 2, where they are grouped by structural class, and are cross-referenced in the text and tables. This presentation enables clear correlation between chemical scaffolds and reported anticancer activities, fully addressing the reviewer’s concern and aligning the manuscript with MDPI Molecules standards.

Comment 6: For each compound mentioned in the manuscript, its source, chemical structure, structural type, the source of fungi, the IC₅₀ value towards tumor cells and normal human cells should be indicated. The positive control and its IC₅₀ value should also be provided.

Response 6: We thank the editor for this important suggestion. To improve structure–activity interpretation, the manuscript has been revised to explicitly provide the chemical structures of representative fungal metabolites discussed throughout the review. All key compounds are now consolidated and illustrated in Figure 2, where they are grouped by structural class, allowing readers in natural products chemistry and organic synthesis to readily correlate chemical scaffolds with reported anticancer activities. The text and tables have been cross-referenced to this figure to avoid redundancy while ensuring structural clarity, in full alignment with MDPI Molecules standards.

Comment 7: As mentioned in section 2.4, there are three advantages of endophytic fungi. It is necessary to supplement the current research progress based on these three advantages as well as the research results obtained therefrom.

Response 7: Section 2.4 has been expanded to link the three stated advantages of endophytic fungi—cultivability, scalability, and genetic/epigenetic manipulability—to recent research outcomes, including yield optimization, chemical diversification, and preclinical validation, supported by updated literature.

Comment 8: There is a logical flaw in Figure 1. The current image shows the discovery of anti-tumor compounds and endophytic fungi from medicinal plants. However, the main focus of this manuscript is to summarize the anti-tumor compounds produced by endophytic fungi.

Response 8: We thank the reviewer for identifying this point. We agree that the original version of Figure 1 placed disproportionate emphasis on medicinal plants, which could obscure the primary focus of the manuscript. Accordingly, Figure 1 has been revised to reposition endophytic fungi as the central source of anticancer compounds, emphasizing their biosynthetic capacity, secondary metabolite diversity, and anticancer mechanisms. Medicinal plants are now depicted only as ecological hosts of endophytic fungi, rather than as discovery targets. This correction eliminates the logical inconsistency and ensures that the figure accurately reflects the fungal-centered scope and objectives of the review.

Comment 9: When introducing the mechanism of action of typical compounds in section 3, whether the corresponding compounds (such as paclitaxel, jatrorrhizine, hypericin, etc.) are commercial drugs or are currently at a certain research stage (clinical, preclinical research) should be indicated. The discovery year of the relevant compounds, their initial sources (plants, microorganisms) and chemical structures also need to be explained.

Response 9: We thank the reviewer for this helpful suggestion. In response, Section 3 has been comprehensively revised so that each representative compound (e.g., paclitaxel, jatrorrhizine, hypericin, vinblastine, diosgenin, and toosendanin) is now introduced with clear contextual information, including its developmental status (clinically approved drug, clinical-enabling lead, or preclinical research compound), approximate discovery period, and initial biological source (plant or microorganism). The chemical structures of all compounds discussed are consolidated and illustrated in Figure 2, while Tables 1 and 2 summarize structural class, biological activity, and research stage.

Comment 10: Section 3.5 covers the compounds with strong activity discovered during the period of 2021-2022. Data for the years 2023-2025 should be added. The discovery year, initial sources, research stage (clinical, preclinical research), chemical structure, structural type, the IC₅₀ value towards tumor cells and normal human cells should be indicated.

Response 10: Section 3.5 has been revised to extend coverage through 2023–2025, incorporating newly reported endophyte-derived anticancer compounds. For representative compounds, we now indicate the discovery period, initial source, structural type (with structures shown in Figure 2), IC₅₀ values toward tumor and normal human cells where available, and research stage (preclinical), fully aligning with MDPI Molecules standards.

Comment 11: The arrangement sequence of the compounds in Figure 2 may cause confusion, leading readers to believe that the compounds were obtained from different parts of the plant. Please make the necessary corrections.

Response 11: Figure 2 has been revised to eliminate any plant-part–based visual implication. Compounds are now grouped by chemical class and biosynthetic origin, and the caption explicitly clarifies that all structures represent endophyte-derived secondary metabolites, with medicinal plants depicted only as ecological hosts.

Comment 12: Section 4 and Table 2 should list the specific activity values of the active compounds.

Response 12: Section 4 has been edited line-by-line to include quantitative activity data (e.g., IC₅₀ values) for representative compounds discussed under each enhancement strategy. In addition, Table 2 has been fully populated with specific activity values, including IC₅₀/EC₅₀ data, cancer models, and normal-cell toxicity where reported.

Comment 13: The bottom border is missing in Table 2.

Response 13: Done!

Comment 14: Section 5 is too brief and fails to adequately analyze the cases and methods involved in each strategy. It needs to be further expanded to enhance its reference value for researchers.

Comment 15: In addition to summarizing the currently well-known anti-tumor compounds, the review should also track the natural products with anti-tumor activity which were related to plant-derived endophytic fungi that have been discovered in the past five years. In the cited literature, the number of papers published in the years 2023-2025 is insufficient. Among the 111 references, the number of published papers in 2023-2025 is only 2, 7 and 5, respectively, which is not enough to summarize the latest research progress in this field.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I consider the topic novel, and the manuscript is generally well written. However, I have several comments detailed below:

General Comments

The terms "in vivo" and "in vitro" should be written in italics.

 

Keywords

There are too many keywords, and some are already included in the title. Please reduce them and ensure the selection facilitates indexing and retrieval through thesaurus-based searches.

 

Introduction

The paragraph between lines 55–64 is redundant, as it repeats previously stated information.

 

Sections 1 and 2

• Both sections contain repetitive content. The structure lacks hierarchy, and it is unclear whether the focus is on the genus level or on the morphological and physiological characteristics of endophytic fungi.
• Clarify whether endophytic fungi perform metabolite bioconversion.
• Figure 1 requires reorganization: the font sizes are inconsistent, and the box referring to sustainable production is disproportionately large.

 

Section 3

• It is necessary to clearly describe the existing anticancer mechanisms and provide a summarized, illustrative explanation relating these mechanisms to the mentioned compounds. The current text is dense, which makes the reading monotonous.
• Line 586: Remove the bold formatting.

 

References

The references, particularly those corresponding to articles, must be formatted according to the journal’s guidelines.

Author Response

General Comments; The terms "in vivo" and "in vitro" should be written in italics.

Response: Edited.

Comment: Keywords: There are too many keywords, and some are already included in the title. Please reduce them and ensure the selection facilitates indexing and retrieval through thesaurus-based searches.

Response: We thank the editor for this suggestion. The list of keywords has been revised to reduce redundancy and to remove terms already present in the title. The remaining keywords have been carefully selected to improve indexing and retrieval through thesaurus-based search systems, focusing on core concepts, mechanisms, and methodologies relevant to endophyte-derived anticancer research.

 

Editor’s Comment 1: “Sections 1 and 2 contain repetitive content. The structure lacks hierarchy, and it is unclear whether the focus is on the genus level or on the morphological and physiological characteristics of endophytic fungi.”

Response 1: We appreciate this important observation. Sections 1 and 2 have been substantially revised to remove redundancy and to improve conceptual hierarchy and focus.

• Repetitive statements regarding the importance of endophytic fungi and anticancer natural products were consolidated and streamlined.
• Section 1 (Introduction) was reorganized to clearly progress from the clinical need for anticancer agents, to the role of natural products, and finally to the specific relevance and scope of plant-derived endophytic fungi.
• Section 2 was refocused to emphasize the biological, ecological, and functional characteristics of endophytic fungi, rather than genus-level cataloguing.
• Clear subsection boundaries were introduced to distinguish definition, ecological drivers, metabolic roles, and sustainability aspects.

These revisions clarify that the manuscript emphasizes functional biology and biosynthetic potential, rather than taxonomic classification.

Editor’s Comment 2: “Clarify whether endophytic fungi perform metabolite bioconversion.”

Response 2: Thank you for highlighting this critical point. We have now explicitly addressed the distinction between de novo biosynthesis and metabolite bioconversion by endophytic fungi.

• A dedicated subsection has been added in Section 2 to clarify that endophytic fungi can either:
• independently biosynthesize secondary metabolites, or
• bioconvert host-derived precursors into structurally modified or bioactive derivatives.
• We further explain how modern approaches—such as paired host–endophyte metabolomics, stable-isotope labeling, and genome-based biosynthetic validation—are used to distinguish between these mechanisms.

This clarification strengthens biosynthetic attribution and improves translational rigor.

Editor’s Comment 3: “Figure 1 requires reorganization: the font sizes are inconsistent, and the box referring to sustainable production is disproportionately large.”

Response 3: We appreciate this comment and have revised Figure 1 to improve visual balance and consistency.

• Font sizes across all elements have been standardized.
• The layout has been reorganized to follow a clear hierarchical flow from plant host → endophyte → metabolite discovery → anticancer activity → sustainable production.
• The “sustainable production” box has been resized to be proportionate to the other elements and positioned as an outcome rather than a dominant focal point.
• The figure caption has been refined to avoid redundancy and to better align with MDPI Molecules graphical standards.

Editor’s Comment 4: “The references, particularly those corresponding to articles, must be formatted according to the journal’s guidelines.”

Response 4: We thank the Editor for this important comment. The reference list has been thoroughly revised and formatted in full compliance with the MDPI Molecules guidelines.

All journal articles have been standardized to the required format, including author names, article titles, journal names, year, volume, page range or article number, and DOI where applicable. In-text citations and reference list entries have been cross-checked for consistency, and formatting issues related to punctuation, italics, capitalization, and ordering have been corrected throughout the manuscript.

We believe that the revised reference list now fully meets the journal’s requirements.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is well structured, and the authors present an interesting review of endophytic fungi as a source for new antineoplasic agents.

The English throughout the manuscript seems good, however, it would be good if it could be uniformized, so who is reading it do not feel that it was just parts added individually to create a whole. To improve the readability of the manuscript, I suggest the authors to revise it, in terms of the type of English used (UK or US).

 

Line 190/191 – the authors refer “…can increase titters of the desired metabolite”, “titters” should be with one t.

 

The authors could increase the font size of figure 1 so it can be more easily read at 100% magnification, and they should mention it in the text at least one time.

 

Line 223 – the authors forgot to indicate the meaning of MAPK (mitogen-activated protein kinase), as they already have done for the others mentioned in the same line, and is also missing in the list of abbreviations.

Line 233 – where appears “Ref-34” into should appear only [34].

Lines 265/266 – the authors should define what is CHO-K1 (Chinese hamster ovary cells) the first time appearing.

Lines 279/280 – HEPG-2 and MCF-7 meaning was already mentioned, it is not necessary to repeat.

Line 292 – the references should be [51, 52] and not “[51] and [52]”.

Lines 296-301 – the authors have two phrases referring the same: “Podophyllotoxin has demonstrated therapeutic efficacy against multiple cancers, including leukaemia, testicular, prostate, lung, and ovarian malignancies, Podophyllotoxin exhibits broad-spectrum cytotoxicity against several cancer types and their corresponding cell lines, including leukaemia (HL-60), testicular carcinoma (NT2/D1), prostate cancer (PC-3), lung carcinoma (A549), and ovarian cancer (SKOV-3) by targeting early metastatic cells [33].” I suggest deleting the first part: “Podophyllotoxin has demonstrated therapeutic efficacy against multiple cancers, including leukaemia, testicular, prostate, lung, and ovarian malignancies, Podophyllotoxin exhibits broad-spectrum cytotoxicity against several cancer types and their corresponding cell lines, including leukaemia (HL-60), testicular carcinoma (NT2/D1), prostate cancer (PC-3), lung carcinoma (A549), and ovarian cancer (SKOV-3) by targeting early metastatic cells [33].”

Furthermore, I suggest the authors to delete the paragraph (Lines 239-246) since states the same as the one (Lines 295-305), but it is inserted in the wrong topic, and due to this alteration, the authors should check the references order.

Lines 313, 325, 334, 348 – ROS it was already abbreviated it is not necessary to be defined again.

Line 322 – Authors should present the meaning of EFSA (European Food Safety Authority) since it is the first time appearing.

Line 331 – There is an “and” after the reference that I think should be deleted.

Line 346 - the references should be presented as [64, 65] and not “[64] and [65]”.

Line 352 - the references should be presented as [67, 68] and not “[67] and [68]”.

Line 378 – the authors say, “has shown a lot of biological activity…”, I suggest: has shown high biological activity.

Line 386 – there is an extra space before “Further studies…”.

Line 391 – After the reference [82] it should be a comma and not a period.

 

In figure 2, I suggest increasing the font size and the compound structures size, so they can be seen clearly. The caption of the figure should be more complete describing the several compounds presented and the references from where they were adapted from, so the figure could be understood independently of the text presented in the manuscript.

 

Table 1 is well structured and organized.

 

Lines 445-455 – The paragraph repeats several aspects; I suggest the following:

“Recent advances in endophyte-based pharmacology have reinforced the translational bridge between laboratory screening and therapeutic application, showing how in vitro assays, in vivo preclinical studies, comparative efficacy evaluations, and formulation strategies together support the clinical readiness of fungal metabolites [40,90,91]. Recent advancements in endophyte-based pharmacology have significantly enhanced the connection between laboratory discoveries and their application in medicine. This illustrates the collaboration of in vitro testing, in vivo preclinical models, comparative efficacy assessments, and enhanced formulation procedures to prepare fungal metabolites for clinical application [40,88,89]. These integrated methodologies ensure that promising compounds go beyond the initial screening phase to become validated, scalable, and safe anticancer candidates prepared for regulatory approval.”

Lines 457-467 – This paragraph is a repetition of the previous paragraph; it should be deleted.

The authors should revise the references after that.

Line 474 – the references should be [40,90-94] followed by a period, “[92]” should be deleted since it is already included in the range mentioned.

Line 484 – the reference 90 should be deleted since it is already included in the range 88-92.

Line 490 – LD₅₀ and TUNEL were never mentioned, it should be defined.

The paragraph in the Lines 489-498 should be revised; the information is being repeated within the paragraph.

Line 507 – MDR was never mentioned, it should be defined here and not in the Lines 511/512. Furthermore, the authors should also revise the whole paragraph since the information is being repeated within the paragraph.  

The same goes for the paragraph in the Lines 521-535.

Line 541 – GMP appears here for the first time; it should be defined here and not in the Lines 549/550. In the same line the references should be presented as [90, 93, 95, 97].

 

Considering table 2, I suggest reducing the width of References column and try to increase the font size in the remaining columns so they could be easier to read when in the printed version.

 

Line 568/569 – “…Fusarium, Cladosporium, Aspergillus, and Penicillium…”; it should be in italics.

Line 586 – “and”; it should not be in bold.

Line 598 – “PKS-NRPS” (Polyketide Synthase-Non-Ribosomal Peptide Synthetase) is the first and only time appearing, it should be defined here. PK here means Polyketide; however, the authors already used it to abbreviate pharmacokinetics in Line 493/494, this could be an unclear aspect.

Line 606 – “CRISPR/Cas9” is the first time and only time appearing it should be defined here.

Line 628/630 – In the phrase: “Contamination during isolation and purification remains a major issue, particularly for sterile mycelial species that are difficult to culture under laboratory conditions.”, Are the authors referring to Mycelia sterilia?, they could clarify this aspect.

The topic 6.3 that starts in the Line 677, should be revised and rewritten, since the information transmitted even though is from different sources it is being repeated.

The conclusion is well structured referring all the key points mentioned throughout the manuscript.

Considering the suggestions I gave here, and the paragraphs that should be deleted and/or rewritten, the authors need to carefully check the whole list of references and complete the list of abbreviations.

Author Response

Comment 1: Line 190/191 – the authors refer “…can increase titters of the desired metabolite”, “titters” should be with one t.

Response 1: Edited.

Comment: The authors could increase the font size of figure 1 so it can be more easily read at 100% magnification, and they should mention it in the text at least one time.

Response 2: Edited.

Comment 3: Line 223 – the authors forgot to indicate the meaning of MAPK (mitogen-activated protein kinase), as they already have done for the others mentioned in the same line, and is also missing in the list of abbreviations.

Response 3: We thank the reviewer for pointing this out. The manuscript has been revised to define MAPK (mitogen-activated protein kinase) at its first occurrence in Line 223, consistent with the definitions provided for other abbreviations in the same sentence. In addition, MAPK has been added to the list of abbreviations.

Comment 4: Line 233 – where appears “Ref-34” into should appear only [34].

Response 4: Edited.

Comment 5: Lines 265/266 – the authors should define what is CHO-K1 (Chinese hamster ovary cells) the first time appearing.

Response 5: We thank the reviewer for this comment. The manuscript has been revised to define CHO-K1 (Chinese hamster ovary) cells at their first occurrence in Lines 265/266, in accordance with MDPI Molecules abbreviation guidelines.

Comment 6: Lines 279/280 – HEPG-2 and MCF-7 meaning was already mentioned, it is not necessary to repeat.

Response 6: Edited.

Comment 6: Line 292 – the references should be [51, 52] and not “[51] and [52]”.

Response 6: Edited.

Comment 7: Lines 296-301 – the authors have two phrases referring the same: “Podophyllotoxin has demonstrated therapeutic efficacy against multiple cancers, including leukaemia, testicular, prostate, lung, and ovarian malignancies, Podophyllotoxin exhibits broad-spectrum cytotoxicity against several cancer types and their corresponding cell lines, including leukaemia (HL-60), testicular carcinoma (NT2/D1), prostate cancer (PC-3), lung carcinoma (A549), and ovarian cancer (SKOV-3) by targeting early metastatic cells [33].” I suggest deleting the first part: “Podophyllotoxin has demonstrated therapeutic efficacy against multiple cancers, including leukaemia, testicular, prostate, lung, and ovarian malignancies, Podophyllotoxin exhibits broad-spectrum cytotoxicity against several cancer types and their corresponding cell lines, including leukaemia (HL-60), testicular carcinoma (NT2/D1), prostate cancer (PC-3), lung carcinoma (A549), and ovarian cancer (SKOV-3) by targeting early metastatic cells [33].”

Furthermore, I suggest the authors to delete the paragraph (Lines 239-246) since states the same as the one (Lines 295-305), but it is inserted in the wrong topic, and due to this alteration, the authors should check the references order.

Response 7: We thank the reviewer for this constructive suggestion. The redundant introductory phrase describing the anticancer activity of podophyllotoxin (Lines 296–301) has been removed, and the text has been streamlined to retain only the detailed description of its cytotoxic activity against specific cancer cell lines. In addition, the paragraph previously located in Lines 239–246, which duplicated the same information and was placed in an inappropriate section, has been deleted. Following these revisions, the reference order has been carefully checked and corrected to ensure consistency and proper numerical sequencing throughout the manuscript.

Comment 8: Lines 313, 325, 334, 348 – ROS it was already abbreviated it is not necessary to be defined again.

Response 8: Edited.

Comment 9: Line 322 – Authors should present the meaning of EFSA (European Food Safety Authority) since it is the first time appearing.

Response 9: Edited.

Comment 10: Line 331 – There is an “and” after the reference that I think should be deleted.

Response 10: Edited.

Comment 11: Line 346 - the references should be presented as [64, 65] and not “[64] and [65]”.

Response 11: Edited.

Comment 12: Line 352 - the references should be presented as [67, 68] and not “[67] and [68]”.

Response 12: Edited.

Comment 13: Line 378 – the authors say, “has shown a lot of biological activity…”, I suggest: has shown high biological activity.

Response 13: Edited.

Comment 14: Line 386 – there is an extra space before “Further studies…”.

Response 14: Edited.

Comment 15: Line 391 – After the reference [82] it should be a comma and not a period.

Response 15: Edited.

Comment 16: In figure 2, I suggest increasing the font size and the compound structures size, so they can be seen clearly. The caption of the figure should be more complete describing the several compounds presented and the references from where they were adapted from, so the figure could be understood independently of the text presented in the manuscript.

Response 16: Edited.

Comment 17: Table 1 is well structured and organized.

Response 17: Thanks.

Comment 18: Lines 445-455 – The paragraph repeats several aspects; I suggest the following:

“Recent advances in endophyte-based pharmacology have reinforced the translational bridge between laboratory screening and therapeutic application, showing how in vitro assays, in vivo preclinical studies, comparative efficacy evaluations, and formulation strategies together support the clinical readiness of fungal metabolites [40,90,91]. Recent advancements in endophyte-based pharmacology have significantly enhanced the connection between laboratory discoveries and their application in medicine. This illustrates the collaboration of in vitro testing, in vivo preclinical models, comparative efficacy assessments, and enhanced formulation procedures to prepare fungal metabolites for clinical application [40,88,89]. These integrated methodologies ensure that promising compounds go beyond the initial screening phase to become validated, scalable, and safe anticancer candidates prepared for regulatory approval.”

Response 18: We thank the reviewer for this helpful suggestion. The paragraph in Lines 445–455 has been revised to eliminate repetitive statements and to synthesise the overlapping content into a single, concise description. The revised text now clearly highlights the integrated role of in vitro screening, in vivo preclinical evaluation, comparative efficacy studies, and formulation strategies in advancing endophyte-derived fungal metabolites toward clinical readiness. The references have been adjusted accordingly to reflect the revised wording.

Comment 19: Lines 457-467 – This paragraph is a repetition of the previous paragraph; it should be deleted.

The authors should revise the references after that.

Response 19: Edited.

Comment 20: Line 474 – the references should be [40,90-94] followed by a period, “[92]” should be deleted since it is already included in the range mentioned.

Response 20: Edited.

Comment 21: Line 484 – the reference 90 should be deleted since it is already included in the range 88-92.

Response 21: Noted.

Comment 22: Line 490 – LD₅₀ and TUNEL were never mentioned, it should be defined.

Response 22: We thank the editor for highlighting the missing definitions. The manuscript has been revised to define LD₅₀ (median lethal dose) and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) at their first occurrence in Section 4.2. This clarification improves methodological transparency and aligns the text with MDPI Molecules standards for technical terminology without altering the scientific interpretation.

Comment 23: The paragraph in the Lines 489-498 should be revised; the information is being repeated within the paragraph.

Response 23: Edited.

Comment 24: Line 507 – MDR was never mentioned, it should be defined here and not in the Lines 511/512. Furthermore, the authors should also revise the whole paragraph since the information is being repeated within the paragraph.  

Response 24: We thank the editor for this observation. The manuscript has been revised to define multidrug resistance (MDR) at its first occurrence in the paragraph. In addition, the entire paragraph has been carefully restructured to eliminate repetitive descriptions of MDR mechanisms and apoptosis-related evaluations. The revised text now presents a concise and coherent discussion of MDR assessment strategies in preclinical models, fully aligned with MDPI Molecules editorial standards.

Comment 25: Line 541 – GMP appears here for the first time; it should be defined here and not in the Lines 549/550. In the same line the references should be presented as [90, 93, 95, 97].

Response 25: We thank the editor for this comment. The manuscript has been revised to define Good Manufacturing Practice (GMP) at its first occurrence (Line 541), and the redundant later definition has been removed. In addition, the references cited in this sentence have been reordered and reformatted as [90, 93, 95, 97] in accordance with MDPI Molecules citation guidelines.

Comment 26: Considering table 2, I suggest reducing the width of References column and try to increase the font size in the remaining columns so they could be easier to read when in the printed version.

Response 26: Edited.

Comment 27: Line 568/569 – “…Fusarium, Cladosporium, Aspergillus, and Penicillium…”; it should be in italics.

Response 27: Edited.

Comment 28: Line 586 – “and”; it should not be in bold.

Response 28: Edited.

Comment 29: Line 598 – “PKS-NRPS” (Polyketide Synthase-Non-Ribosomal Peptide Synthetase) is the first and only time appearing, it should be defined here. PK here means Polyketide; however, the authors already used it to abbreviate pharmacokinetics in Line 493/494, this could be an unclear aspect.

Response 29: We thank the editor for pointing out this potential ambiguity. The manuscript has been revised to define polyketide synthase–non-ribosomal peptide synthetase (PKS–NRPS) at its first and only occurrence (Line 598). To avoid confusion with the previously defined abbreviation PK (pharmacokinetics), the term “polyketide” is now written in full, and the standalone abbreviation “PK” is no longer used in this context. This revision improves clarity and aligns the text with MDPI Molecules abbreviation guidelines.

Comment 30: Line 606 – “CRISPR/Cas9” is the first time and only time appearing it should be defined here.

Response 30: We thank the editor for this comment. The manuscript has been revised to define clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) at its first and only occurrence (Line 606), in accordance with MDPI Molecules abbreviation guidelines.

Comment 31: Line 628/630 – In the phrase: “Contamination during isolation and purification remains a major issue, particularly for sterile mycelial species that are difficult to culture under laboratory conditions.”, Are the authors referring to Mycelia sterilia?, they could clarify this aspect.

Response 31: We thank the editor for this valuable clarification request. The manuscript has been revised to explicitly state that the term “sterile mycelial species” refers to sterile mycelial endophytic fungi (commonly described as mycelia sterilia), which lack diagnostic reproductive structures and present challenges in taxonomic identification and laboratory cultivation. This clarification has been incorporated directly into the text to avoid ambiguity.

Comment 32: The topic 6.3 that starts in the Line 677, should be revised and rewritten, since the information transmitted even though is from different sources it is being repeated.

Response 32: We thank the editor for this comment. Section 6.3 has been fully revised and rewritten to eliminate repetitive information and to synthesise overlapping concepts derived from different sources. The revised text now presents a concise and structured discussion of key translational challenges—namely metabolite production instability, isolation and dereplication limitations, and insufficient in vivo validation—followed by a forward-looking perspective on integrated discovery and development strategies. All references are cited consistently in author–year format, in accordance with MDPI Molecules guidelines.

Comment 33: The conclusion is well structured referring all the key points mentioned throughout the manuscript.

Response 33: Thanks.

Comment 34: Considering the suggestions I gave here, and the paragraphs that should be deleted and/or rewritten, the authors need to carefully check the whole list of references and complete the list of abbreviations.

Response 34: Noted.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded excellently to the suggestions and incorporated into the manuscript the improvements that had been recommended. At this stage, the only remaining recommendation is to increase the font size in Figures 2 and 3.

Author Response

Reviewer 2:

Comments and Suggestions for Authors

The authors responded excellently to the suggestions and incorporated into the manuscript the improvements that had been recommended. At this stage, the only remaining recommendation is to increase the font size in Figures 2 and 3.

Response: We thank the reviewer for the positive assessment of the revised manuscript. In response to the suggestion regarding Figures 2 and 3, we increased the overall size of both figures so that each now occupies approximately 80% of a page. We elected to enlarge the entire figures rather than adjusting font size alone, as this approach improves overall readability while preserving visual balance and structural clarity. We trust that this modification adequately addresses the reviewer’s recommendation.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The English compared to the previous manuscript version has improved substantially. The manuscript overall is well structured and readable, and gives important information on the endophytic fungi as novel anticancer drugs.

The title of the section 2 does not seem the best fit for the information the authors are giving, suggest an alteration of the section title.

Line 88/89 - there is an unnecessary space between “with” and “MDPI Molecules…”. The same happens for the lines 101/102 between “Google Scholar” and “Citation management…”.

Lines 162,322,446,553/554,615 - ROS was already mentioned.

The authors should be careful with Figure 1, it seems there is information missing from the boxes, at least in the box on the up-right side of the flow diagram, is missing something after “(n=”, and in the last box of the flow diagram there is no “n=” information.

In the Figure 2, the font size in the printed version is not readable and the quality/definition of the image in the pdf digital version could be improved.

Line 290/291 – the fungi mentioned by the authors should be presented in italics.

Section 4.1.2, in the title, if the authors only mention Vinblastine in this section, it is not necessary to have Vincristine in the title as well.

Line 350, 439 – the plant mentioned should be in italics.

Section 4.2.1, if the authors do not abbreviate once in the section there is no need to have the abbreviation in the section title.

Line 504 – “…reinforcing endophytic fungi…”, endophytic is not necessary to be in italics.

The Figure 3 is not visible, in terms of font size and chemical structures, at least in the printed version. In the pdf digital version, the chemical structures are not visible except for the chemical structure of Asparaginase, the authors could improve greatly the quality/definition of the images.

It is missing a paragraph between the sections 5.4 and 5.5.

OSMAC should be mentioned once besides in the abstract the first time appearing in line 78.

Lines 776/777 - OSMAC was already mentioned.

Lines 818/819 – PKS-NRPS only appearing here. It should appear first the complete name without brackets followed by PKS-NRPS in brackets.

Concerning the references, I do not understand why the authors decided to remove references 2 and 31 from the previous manuscript version. Reference 2 was from the World Health Organization, WHO, which seems to me extremely important to include and reference 31 which was also included in the previous manuscript and seemed to me to be an important contribution from the authors to this review and to the knowledge about endophytic fungi.

Author Response

Reviewer 3:

Comments and Suggestions for Authors

The English compared to the previous manuscript version has improved substantially. The manuscript overall is well structured and readable, and gives important information on the endophytic fungi as novel anticancer drugs.

Response: We are grateful that the reviewer finds the technical content of the review relevant and of interest to MDPI Molecules.

The title of the section 2 does not seem the best fit for the information the authors are giving, suggest an alteration of the section title.

Response: As suggested, the “Materials and Methods” section has been retitled “Search Strategy and Selection Criteria.”

 

Line 88/89 - there is an unnecessary space between “with” and “MDPI Molecules…”. The same happens for the lines 101/102 between “Google Scholar” and “Citation management…”.

Response: The typographical issue involving unnecessary spacing has been identified and corrected in the revised manuscript.

Lines 162,322,446,553/554,615 - ROS was already mentioned.

Response: Reactive oxygen species (ROS) has been defined at its first occurrence, and the abbreviation ROS has been used consistently throughout the manuscript thereafter.

The authors should be careful with Figure 1, it seems there is information missing from the boxes, at least in the box on the up-right side of the flow diagram, is missing something after “(n=”, and in the last box of the flow diagram there is no “n=” information.

Response: Typo issue: Solved.

In the Figure 2, the font size in the printed version is not readable and the quality/definition of the image in the pdf digital version could be improved.

Response: In response to the comment on Figures 2 and 3, we increased the overall figure size to approximately 80% of a page each. The figures were scaled in their entirety to enhance readability while preserving clarity and proportionality.

Line 290/291 – the fungi mentioned by the authors should be presented in italics.

Response: Done!

Section 4.1.2, in the title, if the authors only mention Vinblastine in this section, it is not necessary to have Vincristine in the title as well.

Response: Vincristine has been deletd.

Line 350, 439 – the plant mentioned should be in italics.

Response: Done!

Section 4.2.1, if the authors do not abbreviate once in the section there is no need to have the abbreviation in the section title.

Response: Abbreviation has been deleted.

Line 504 – “…reinforcing endophytic fungi…”, endophytic is not necessary to be in italics.

Response: Done!

The Figure 3 is not visible, in terms of font size and chemical structures, at least in the printed version. In the pdf digital version, the chemical structures are not visible except for the chemical structure of Asparaginase, the authors could improve greatly the quality/definition of the images.

Response: In response to the comment on Figure 3, we increased the overall figure size to approximately 80% of a page each. The figures were scaled in their entirety to enhance readability while preserving clarity and proportionality.

 

It is missing a paragraph between the sections 5.4 and 5.5.

Response: To address the reviewer’s comment, an additional paragraph has been added between Sections 5.4 and 5.5, incorporating Zaferanloo et al. as a reference.

OSMAC should be mentioned once besides in the abstract the first time appearing in line 78.

Response: Done!

Lines 776/777 - OSMAC was already mentioned.

Response: The abbreviation has been retained, and repeated explanations have been removed throughout the manuscript.

Lines 818/819 – PKS-NRPS only appearing here. It should appear first the complete name without brackets followed by PKS-NRPS in brackets.

Response: Done!

Concerning the references, I do not understand why the authors decided to remove references 2 and 31 from the previous manuscript version. Reference 2 was from the World Health Organization, WHO, which seems to me extremely important to include and reference 31 which was also included in the previous manuscript and seemed to me to be an important contribution from the authors to this review and to the knowledge about endophytic fungi.

Response: Response: We thank the reviewer for highlighting this important point. References 2 (World Health Organization) and 31, which represents a significant contribution by the authors to the field of endophytic fungi, have now been reinstated in the revised manuscript. In addition, two further relevant references have been incorporated to strengthen the contextual and scientific foundation of the review. As a result, the total number of references has increased from 160 to 164. We appreciate the reviewer’s observation, which has helped improve the completeness and rigor of the reference framework.

 

Author Response File: Author Response.pdf