Ascomylactam C Induces an Immunogenic Cell Death Signature via Mitochondria-Associated ER Stress in Lung Cancer and Melanoma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Hwang et al, addressed in vitro/in vivo anti-tumor effect of AsC and revealed its mode of action. Authors claimed that AsC exhibited anti-tumor activity by ROS-induced immunogenic cell death. The experiments appear to be well executed with suitable controls; however, authors should address following issues to support their conclusions.

Comments

1.      Figures 5A and B are not clear and support your results “the expression of ER stress-related proteins increased significantly”. Authors should provide clear results and quantify band intensity.

2.      Figure 6A and B are not clear. Authors should provide clear results and quantify band intensity.

3.      Compared to LLC, DC maturation is not significant in B16F10. Authors should discuss this discrepancy.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor,

The manuscript Ascomylactam C induces an immunogenic cell death signature via mitochondria-associated ER stress in lung cancer and melanoma has reported the in vivo and in vitro antitumorigenic effects of the cytotoxic alkaloid Ascomylactam (AsC), including the investigation of the molecular mechanisms involved against lung cancer and melanoma. However, some data descriptions are missing and the Discussion is too short. Therefore, the manuscript cannot be considered for publication as it stands.

Kindly, find below my major and minor comments:

1.      In the Abstract, the sentence related to AnnexinV/PI should be improved as “found” is not the appropriate verb.

2.      In the Introduction, examples of anticancer drugs inducing ICD should be mentioned with reference. Once the abbreviations are defined, they should be used throughout the manuscript. However, the authors forgot to define the PERK; which should be done in the Abstract and Introduction.  

3.      In the Results section, the cell viability curves depicted in the Figure 1B should be split into species categories; i.e., human cell line viability separated from mouse cell line viability. The authors should mention and describe the results obtained by the addition of oxaliplatin, regarding colony formation assays and apoptosis status determination. The effect of (10 mM) oxaliplatin on cell viability assay is strongly recommended to be added because an obvious reduction in colony formation is shown while the induction of apoptosis in oxaliplatin-treated LLC and A375 cells is negligible. Can the authors explain why oxaplatin was tested at 10 mM only? This experimental part still requires some clarifications. The molecular weight of each visualized protein should be indicated along the Western blots.

4.      The Discussion is too short and does not cover the different aspects studied, such as PERK/CHOP signaling pathway, ICD, immune cell infiltration, PD1/PDL1 expression and oxaliplatin.

Dear Editor,

The manuscript Ascomylactam C induces an immunogenic cell death signature via mitochondria-associated ER stress in lung cancer and melanoma has reported the in vivo and in vitro antitumorigenic effects of the cytotoxic alkaloid Ascomylactam (AsC), including the investigation of the molecular mechanisms involved against lung cancer and melanoma. However, some data descriptions are missing and the Discussion is too short. Therefore, the manuscript cannot be considered for publication as it stands.

Kindly, find below my major and minor comments:

1.      In the Abstract, the sentence related to AnnexinV/PI should be improved as “found” is not the appropriate verb.

2.      In the Introduction, examples of anticancer drugs inducing ICD should be mentioned with reference. Once the abbreviations are defined, they should be used throughout the manuscript. However, the authors forgot to define the PERK; which should be done in the Abstract and Introduction.  

3.      In the Results section, the cell viability curves depicted in the Figure 1B should be split into species categories; i.e., human cell line viability separated from mouse cell line viability. The authors should mention and describe the results obtained by the addition of oxaliplatin, regarding colony formation assays and apoptosis status determination. The effect of (10 mM) oxaliplatin on cell viability assay is strongly recommended to be added because an obvious reduction in colony formation is shown while the induction of apoptosis in oxaliplatin-treated LLC and A375 cells is negligible. Can the authors explain why oxaplatin was tested at 10 mM only? This experimental part still requires some clarifications. The molecular weight of each visualized protein should be indicated along the Western blots.

4.      The Discussion is too short and does not cover the different aspects studied, such as PERK/CHOP signaling pathway, ICD, immune cell infiltration, PD1/PDL1 expression and oxaliplatin.

Comments on the Quality of English Language

There are too many grammatical errors. The authors should correct their manuscript with suitable English Writer during resubmission.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, Huang et al. studied the the antitumor effects mechanisms of Ascomylactam C (AsC) on lung cancer and melanoma cells. They found that AsC suppressed growth of LLC and B16F10 tumors significantly in mice, witnessed with promoted the infiltration of CD4+T and CD8+T cells in tumor tissues. They mechanistically found that AsC increased ROS formation, which activated the PERK/eIF2α/CHOP signaling pathway to induce immunogenic cell death of tumor cells. This work is well-designed, and scientifically important by showing the potential of AsC as promising anti-tumor drug. It is recommended for publication with a revised version.

1. Why AsC can induce ROS inside tumor cells? 

2. What's the type of ROS induced by AsC? Singlet oxygen? Hydroxyl radical? or superoxide radical?

3. Gate in Figure 7d is not specified for DCs. Many immune cell lines, such as DCs, macrophages, granulocytes, B cells, can express CD11c+ marker. Even though this is GMCSF induced DCs, it is suggested to add some markers to gate DCs, such as MHC-II.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript “Ascomylactam C Induces an Immunogenic Cell Death Signature Via Mitochondria-associated ER stress in Lung cancer and Melanoma” described in vitro and in vivo anticancer activity of a new 13-membered-ring macrocyclic alkaloid from mangrove endophytic fungus Didymella sp. (Ascomylactam C (AsC)) against lung cancer and melanoma cells. The molecular mechanism of its effect was found to be associated with increasing of ROS formation, inducing ofendoplasmic reticulum stress, up-regulation of the phosphorylation of PERK, eukaryotic initiation factor 2α (eIF2α) and the expression of the transcription factor CHOP,and finaly inducing of immunogenic cell death of cancer cells. The manuscript seems well written, and contents look important. I recommend this paper to be published in Marine Drugs after some minor modification as follows:

Figure 1. It is not clear why the concentration of 20 µM which is higher than IC50 was used for colony formation and apoptosis assays? Why the time of cells’ treatment with AcS was different in cell viability and apoptosis assay? In the Section of Results the explanation of concentration and time treatment conditions should be given.

Line 32. The term “Annenin” should be exchanged by “Annexin” one.

Line 61. The full name of PERK protein should be given.

Line 123. The word “Inhibited” should be lower case.

Line 191. The word “not” is missing.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor,

The manuscript Ascomylactam C induces an immunogenic cell death signature via mitochondria-associated ER stress in lung cancer and melanoma has been considerably improved. Overall, I am satisfied with the answers and with most of the changes. However, there are still minor corrections to be made:

-          Addition of the hyphen to “danger associated molecular patterns”. Once the acronym/abbreviation has been defined in the Abstract and Introduction, the acronym/abbreviation (i.e.,  ATF4, eIF2a) should be used only in the remainder of the text. However, DC (l 84), NK (l 168) cells, and PD-L1 (l 179) remain to be defined.

-          For consistency, the authors should choose the spelling of anticancer with or without hyphen.

-          The authors must complete this statement “the inhibitory of Asc was dose-dependent” (l 104). Remove s from “cells proliferation” (l 116), same remark from “cells viability” (l 130, 2.2) and “DCs maturation”, replace “every” (i.e., “every tumor model) with each (l 132), and correct “could significantly inhibited”.

Comments on the Quality of English Language

There are still too many grammatical errors.

Author Response

The author thanks the Reviewer for the meticulous revision of the manuscript. To address the comments raised by the Reviewer, the amendments have been made to the manuscript text and the responses for each point have been represented as below:

1. Addition of the hyphen to “danger associated molecular patterns”. Once the acronym/abbreviation has been defined in the Abstract and Introduction, the acronym/abbreviation (i.e., ATF4, eIF2a) should be used only in the remainder of the text. However, DC (l 84), NK (l 168) cells, and PD-L1 (l 179) remain to be defined.

Response: 

(1) “danger associated molecular patterns” has been modified “danger-associated molecular patterns”;

(2) DC (l 84), NK (l 168) cells, and PD-L1 (l 179) have been defined and the acronym/abbreviation are used in accordance with the specification throughout the text.

2. For consistency, the authors should choose the spelling of anticancer with or without hyphen.

Response:  The authors chose the spelling of anticancer without hyphen and remained consistent throughout the text

3. The authors must complete this statement “the inhibitory of Asc was dose-dependent” (l 104). Remove s from “cells proliferation” (l 116), same remark from “cells viability” (l 130, 2.2) and “DCs maturation”, replace “every” (i.e., “every tumor model) with each (l 132), and correct “could significantly inhibited”.

Response:

(1) The statement “the inhibitory of Asc was dose-dependent” has been completed as “the inhibitory of AsC was in a dose-dependent manner”;

(2) ‘s’ has been removed from “cells proliferation” (l 116)

(3) ‘s’ has been removed from “cells viability” (l 130, 2.2) and “DCs maturation”

(4) ‘s’ has been removed from “DCs maturation”

(5) “every” has been replaced (i.e., “every tumor model) with each (l 132)

(6) “could significantly inhibited” has been corrected by “could significantly inhibit”.