PONE-D-19-23209

Application of non-invasive low-intensity pulsed electric field with thermal cycling-hyperthermia for synergistically enhanced anticancer effect of chlorogenic acid on PANC-1 cells

PLOS ONE

Dear Dr. Chao,

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Academic Editor

PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: The paper proposes a novel treatment modality includes non-invasive treatment CGC, coupled with thermal-cycling hyperthermia (TC-HT) and low-intensity pulsedelectric field (LIPEF). The triple combination leads to excessive apoptotic cell death with known signatures for apoptosis in Panc-1 pancreatic cancer cells. Although MS includes non-malign cell data, due to genetic heterogenety of pancreatic tumors, other cell lines carry different mutations for p53 or KRas is missing. MS skips the mutational loss of functional p53 status of Panc-1 cells in discussion part. Therefore discussion part should be revised according to p53 burden in tumors and effect on therapy. There are number of typo errors in the text.

Reviewer #2: Title: "non-invasive low-intensity pulsed electric field" - The description of PEF is lacking in the M&M and why use it in their studies. It is noteworthy that there are electric fields being larger fields (1.8 and 1.26 kV/cm) can produced a high drug intake rate during the first 10 ms that can decrease significantly thereafter. On the other hand, the intake rate during the first 10 ms for the two smaller fields (0.63 and 0.45 kV/cm) maybe undetectable and may gradually increased afterward. The rates for these two fields can be similar to one another until the end of the analysis. In addition, the optimal PEF levels for in vitro tests need to be described in detail; e.g., how many pulses at 1 kHz and 0.45 kV/cm to be nonlethal to normal cells but effectively kill cancer cells.

Abstract: "In this study, we propose a novel non-invasive treatment calling for exposure of cells to CGA, coupled with thermal-cycling hyperthermia (TC-HT) and low-intensity pulsed electric field (LIPEF)." Why use non-invasive here? No definition for CGA here. The authors published a previous article (PLoS One. 2019; 14(5): e0217676.Published online 2019 May 31. doi: 10.1371/journal.pone.0217676) using CGA and TC-HT treatment of PANC1 cells. There is no rationale for using LIPEF in this study.

Introduction: As written, it does not provide a rationale for the study. The authors have published a paper on CGA and TC-HT which needs to be articulated at the beginning with the findings. Then, why use LIPEF as a combination treatment with CGA and TC-HT in this study. This section is poorly written. For example, "We have recently shown that the thermal cycling-hyperthermia (TC-HT) has an anticancer synergy via coupling with the polyphenols, chlorogenic acid (CGA) and epigallocatechin gallate (EGCG) in combating human pancreatic cancer PANC-1 cells [7], thanks to the effect of the cell cycle arrest and the induction of mitochondria-mediated apoptosis, which were not found in the separate treatments."

Discussion: Poorly written. "Unlike alternating current signal stimulus, non-invasive LIPEF can be viewed as the composition of many sinusoidal subcomponents with multiple frequencies [30]. When the electric pluses enter and interact with a biological sample, we propose that the LIPEF is dispersed electrically to multiple frequencies due to the encounter with biological dielectrics. Thus, the LIPEF’s multiple frequencies, caused by electrical dispersion in biological dielectrics, can simultaneously interact with molecules, proteins, organelles, and cells to induce bioeffects during the LIPEF exposure. The study points out that the application of LIPEF could intensify the disturbance of mitochondria triggered by double treatment with CGA and TC-HT, which in turn stimulated the depolarization of MMP." Unfortunately, the description of LIPEF is lacking in the study which needs to be addressed in detail.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-23209R1

Application of non-invasive low-intensity pulsed electric field with thermal cycling-hyperthermia for synergistically enhanced anticancer effect of chlorogenic acid on PANC-1 cells

PLOS ONE

Dear Dr. Chao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

PLOS ONE now requires that submissions reporting blots or gels include original, uncropped blot/gel image data as a supplement or in a public repository. Although not uncropped, the images for PARP and corresponding GAPDH would have satisfied PLOS ONE's request. However, the rest of the blot images often as closely cropped as in the figures only presented on a larger clipboard. Please provide images for original blots of  Bcl-2, Bax, their corresponding GAPDH and all +NAC blots that meet PLOS ONE's requirement.

We would appreciate receiving your revised manuscript by Jan 04 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Ferenc Gallyas, Jr., Ph.D., D.Sc.

Academic Editor

PLOS ONE

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Application of non-invasive low-intensity pulsed electric field with thermal cycling-hyperthermia for synergistically enhanced anticancer effect of chlorogenic acid on PANC-1 cells

PONE-D-19-23209R2

Dear Dr. Chao,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Ferenc Gallyas, Jr., Ph.D., D.Sc.

Section Editor

PLOS ONE

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