Cytotoxic properties of unfractionated and fractionated bromelain alone or in combination with chemotherapeutic agents in colorectal cancer cells

Kuei-Yen Tsai; Po-Li Wei; Mohamed Azarkan; Nasiha M’Rabet; Precious Takondwa Makondi; Hsin-An Chen; Chien-Yu Huang; Yu-Jia Chang

doi:10.1371/journal.pone.0285970

Peer Review History

Original SubmissionFebruary 6, 2023
29 Mar 2023 Decision Letter - Sreeparna Banerjee, Editor PONE-D-23-03463Cytotoxic Properties of Unfractionated and Fractionated Bromelain Alone or in Combination with Chemotherapeutic Agents in Colorectal Cancer CellsPLOS ONE Dear Dr. Chang, 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. Please submit your revised manuscript by May 13 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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. 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Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Additional Editor Comments: Both reviewers have identified several points for further clarification, including how bromelain itself contributes towards cytotoxicity, details on the methodology and the role of the lectin moeity. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: 1. When F3 bromelain has shown the cytotoxic properties with that of unfractinated bromelain, what is the point in fractionating bromelain? 2. Since Bromelain is a complex of proteolytic enzymes, it is necessary to identify single molecule or individual proteolytic enzyme involved in cytotoxicity in CRC cells. How do the authors justify this? 3. The study has evaluated F3 bromelain in combination with routine chemotherapeutic agents and found out that synergistic cytotoxicity. In this evaluation, how quantitatively F3 bromelain contributes to cytotoxicity? 4. Methods: Is it correct that authors have not isolated bromelain naturally from pineapple, instead they have used commercially available bromelain from Sigma-Aldrich?. Reviewer #2: The MS deals with the purification of an active fraction from bromelain and the comparison of anti tumor effects with unfractionated sample, using colorectal cancer cells. The topic is interesting and follows some other papers (i.e. Badar et al 2021, PMID: 34150016). However, in my opinion the MS is very difficult to be read. I suggest to join Results and Discussion sections. The splitting make the results more difficult to be interpreted. Moreover the Discussion is too long, repeating a lot of information from Introduction. So, in my opinion, during the merging of these two sections the authors should shorten these parts, and conversely better highlight the relationships between their results and the conclusions that they draw. In the present form it is very difficult to appreciate the MS and focus on the Results presented. The authors state that F3 contains a lectin: did they check the hemagglutination activity of the fraction? MINOR POINTS: Page 4: please, insert more data about chromatographic runs (for example flow rate and CV for each step). Page 9 line 28: “chromatographically”. Figure 1: uL (capitol L) Figure 2 is very difficult to be read. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [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.] 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0285970.r001
Revision 1
20 Apr 2023 Author Response Response to the critiques: Reviewer #1: Q1. When F3 bromelain has shown the cytotoxic properties with that of unfractionated bromelain, what is the point in fractionating bromelain? Response: Bromelain is a long-established drug with high absorption rate, safety, and few side effects. However, it is a complex of a group of enzymes. In such a drug we cannot know which molecule its special curative effect comes from, and it cannot be further purified to maximize its efficacy. Because previous studies have found that bromelain has anti-tumor properties, the purpose of chromatographic fractionation is to try to further separate individual parts, and to find out the part that really has a curative effect. It is better to perform a study with a homogeneous and well-characterized fraction than with a mixture containing proteases, other proteins and non-protein compounds. The interpretation of the results and sample standardization will be easier when using a well-characterized sample. A previous study has found that the combined use of F3 and chemotherapeutic drugs has a more significant inhibitory effect on pancreatic cancer and HCC than the combination of bromelain and chemotherapeutic drugs (Ref 7). The results in the current study indicate that the cytotoxicity of F3 bromelain on CRC is comparable to that of unfractionated bromelain; thus, the anticancer effects may be exclusively confined to F3 bromelain. In addition, we can check the contribution of the proteolytic activity to the cytotoxic effect by using specific inhibitors of basic bromelain (F3 fraction). This will be difficult to assess by using the complex mixture of stem bromelain. Q2. Since Bromelain is a complex of proteolytic enzymes, it is necessary to identify single molecule or individual proteolytic enzyme involved in cytotoxicity in CRC cells. How do the authors justify this? Response: Stem bromelain contains, in addition to non-protein compounds and non-proteolytic enzymes, at least eight cysteine proteases of the papain family (Ref 6, Matagne et al. 2017, Phytochemistry 138: 29-51). We already demonstrated that these proteases display different substrate specificity and inhibitory properties. We assessed the different fractions on cytotoxicity in CRC cells and the results clearly showed that the fraction containing basic bromelain (F3) gives the expected cytotoxic properties. The reason why we opted for the use of basic bromelain (F3) in our study. Q3. The study has evaluated F3 bromelain in combination with routine chemotherapeutic agents and found out that synergistic cytotoxicity. In this evaluation, how quantitatively F3 bromelain contributes to cytotoxicity? Response: Bromelain is a compound with multiple proteolytic enzymes and has diverse functions, but its cytotoxic effect on tumor cells is still unclear. Our current research points out that Bromelain/F3 can induce apoptosis in CRC cells, increase oxidative stress, and regulate autophagy. SRB assay in the current study detected that Bromelain/F3 has 50% inhibitory ability to the growth of CRC cells at 20~40 μg/mL. Q4. Methods: Is it correct that authors have not isolated bromelain naturally from pineapple, instead they have used commercially available bromelain from Sigma-Aldrich?. Response: Yes, we used a commercially available bromelain from Sigma-Aldrich. Reviewer #2: Q1. The MS deals with the purification of an active fraction from bromelain and the comparison of antitumor effects with unfractionated sample, using colorectal cancer cells. The topic is interesting and follows some other papers (i.e. Badar et al 2021, PMID: 34150016). However, in my opinion the MS is very difficult to be read. I suggest to join Results and Discussion sections. The splitting makes the results more difficult to be interpreted. Moreover the Discussion is too long, repeating a lot of information from Introduction. So, in my opinion, during the merging of these two sections the authors should shorten these parts, and conversely better highlight the relationships between their results and the conclusions that they draw. In the present form it is very difficult to appreciate the MS and focus on the Results presented. Response: Fractionation of bromelain has been reported in a previous study (Ref 7). Therefore we cannot present repetitive results in the current study. The focus of this study is on the anti-tumor effects in CRC comparing F3 and unfractionated bromelain. We have reorganized and abridged the introduction and discussion sections in the study and tried to make it easier to read and catch. Q2. The authors state that F3 contains a lectin: did they check the hemagglutination activity of the fraction? Response: Yes, we published data dealing with Ananas comosus mannose-binding lectin crystal structure and hemagglutination activity (Azarkan et al., Scientific Reports (2018) 8:11508 DOI:10.1038/s41598-018-29439-x). Hemagglutinating assays revealed that this lectin showed no hemagglutination activity of human, rabbit, and sheep erythrocytes. In contrast, the lectin readily hemagglutinates rat erythrocytes. Q3. MINOR POINTS: Page 4: please, insert more data about chromatographic runs (for example flow rate and CV for each step). Page 9 line 28: “chromatographically”. Figure 1: uL (capitol L) Figure 2 is very difficult to be read. Response: We have added the asked information at page 4 and revised the errors at pages 9 (line 28) and in Figure 1. The quality of Figure 2 was improved for more clarity. https://doi.org/10.1371/journal.pone.0285970.r002
7 May 2023 Decision Letter - Sreeparna Banerjee, Editor Cytotoxic Properties of Unfractionated and Fractionated Bromelain Alone or in Combination with Chemotherapeutic Agents in Colorectal Cancer Cells PONE-D-23-03463R1 Dear Dr. Chang, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Sreeparna Banerjee, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): The authors have satisfactorily addressed the comments of both reviewers. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Authors have cleared all queries and manuscript is improved. I would recommend to accept this research paper... Reviewer #2: The main suggestion during the first Revision was to merge Results and Discussion, in order to make the MS more clear and simple to be read. However, this modification has not be done. Moreover, the Authors did not highlighted the various corrections during R1. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Nagaraj K Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0285970.r003
Formally Accepted
23 May 2023 Acceptance Letter - Sreeparna Banerjee, Editor PONE-D-23-03463R1 Cytotoxic Properties of Unfractionated and Fractionated Bromelain Alone or in Combination with Chemotherapeutic Agents in Colorectal Cancer Cells Dear Dr. Chang: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sreeparna Banerjee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0285970.r004

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