Managing Editor from BMC Compplementary and Alternative Medicine

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1 Author s response to reviews Title: Euphorbia tirucalli modulates gene expression in larynx squamous cell carcinoma Authors: Gabriela Franco-Salla (gabi.bfranco@gmail.com) Janesly Prates (janeslyprates@hotmail.com) Laila Cardin (lailinha_7@hotmail.com) Anemari dos Santos (anedinarte@gmail.com) Wilson da Silva Júnior (wilsonjr@usp.br) Bianca da Cunha (biancarc@usp.br) Eloiza Tajara (etajara@hotmail.com) Sonia Oliani (smoliani@ibilce.unesp.br) Flavia Cristina Rodrigues-Lisoni (lisoni@bio.feis.unesp.br) Version: 1 Date: 03 Feb 2016 Author s response to reviews: Ilha Solteira, February 1st, 2016 Chanpen Chanchao, PhD Managing Editor from BMC Compplementary and Alternative Medicine Dear Professor, Re: Ref. No.: BCAM-D

2 Title: Euphorbia tirucalli modulates gene expression in larynx squamous cell carcinoma Gabriela Bueno Franco-Salla, MsC; Janesly Prates, MsC; Laila Toniol Cardin, MsC; Anemari Ramos Dinarte dos Santos; Wilson Araujo da Silva Júnior, PhD; Bianca Rodrigues da Cunha, MsC; Eloiza Helena Tajara, PhD; Sonia Maria Oliani, PhD; Flavia Cristina Rodrigues-Lisoni, PhD Thank you for your editorial comments on this manuscript and the Referees reports that we found to be helpful and allowed us, in our opinion, to improve our revised manuscript. As you will see from the resubmitted copy we have addressed the issues raised by the Referee in a constructive manner, clarifying some issues. Below we list our responses to each individual point raised by the Referee. We have uploaded our revised manuscript hoping that it now reaches the standard for publication in BMC Compplementary and Alternative Medicine. Yours sincerely, Professor Flávia Cristina Rodrigues-Lisoni, Correspondingt Author at: Department of Biology and Animal Science, Faculty of Engineering of Ilha Solteira - FEIS/UNESP Av. Brasil, 56 CEP: , Ilha Solteira, São Paulo, Brazil. flavialisoni@hotmail.com ; lisoni@bio.feis.unesp.br Tel: 55 (18) Fax:55 (18)

3 Author's response to reviewer comments Manuscript Ref. No.: BCAM-D Euphorbia tirucalli modulates gene expression in larynx squamous cell carcinoma Version: 2 Date: 1 February 2016 Reviewer: #1 Reviewer's report: 1) Use "sense, antisense" instead of :right, left". We agree with the editor that the terms sense and antisense will be better in the manuscript, we change in the Table1. 2) Describe in more detail the method used in plant extraction. We have insert this information in the methods (Page 6 in Pharmacological treatment). 3) There are some English errors. We thank the editor for the valuable comments. We have rewritten parts of the paper on the basis of these suggestions.

4 4) Where is the statistics? The statistics analysis of the Proliferation Assay and the Quantitative PCR was performed using GraphPad Prism software and it is possible see the standard deviation in the Figures 1 and 3. 5) Provide the HPLC chart and LC-MS chart if applied? We had the chromatogram in the patent of the active fraction of a polar solvent extract from the latex of Euphorbiaceae plants (Doc 1, reference n. 22), but I can not insert HPLC chart in the manuscript, because It was not realized by my group. 6) In the method "eight fractions were separated from the butanol fraction of the latex extract", however, Why the authors examined only Aveloz? Further, the authors used E. tirucalli instead of Aveloz throughout the manuscript. We agree with the editor that there should be an explanation about the use of only one fraction of the latex extract, then we add in in the methods (Page 6 in Pharmacological treatment). We used E. tirucalli instead of aveloz throughout the manuscript because it is a scientifically name of the plant, aveloz is only a popularly name and in pubmed, for example, you do not find many articles with a popularly name. 7) The manuscript should have prepared according to author guidelines. e.g. Aquino et al. (2008) We change in the discussion some phrases to remake the manuscript according to author guidelines (Pages 13, 14 and 15 in Discussion) 8) Please use full names of abbreviations before using abbreviations. There are also duplication in the abbreviations.

5 We add some full names of abbreviations and remove some duplication of the abbreviations. 9) Discussion is to telegraphic and uses mostly textbook information, without discussion of results. We change something in the discussion to became better, highlighting in the discussion. 10) 70 references are too much. Please use total 40 citations. We excluded some references, now the manuscript has 40 citations. (Reference pages 19-24) Reviewer #4: Major comments: 1) Morphology figure should be added. Thank you for your comments. The morphology was add in Figure 1A and 1B. 2) Fig.2 seems to be included in Fig.1. Please explain the different between them. Fig.1 has the same 25 ug/ml like that of Fig. 2. Yes, Figure 2 has the same 25 ug/ml that Figure 1, but the account of the others concentrations are not so significant statistically than the 25 ug/ml, so we only show the results of this concentration. But I think that it would be better insert together the Figure 2 and the Figure 1. Then, we created a new Figure 1, that show the morphology (1A e 1B), the growth curve with E. tirucalli latex (1C) and with the ethanol (1D) together!! But if you think better take out the Figure 1D, because it was not contributed to the manuscript, please let me know!

6 3) Is the authors make all samples with the same ethanol concentration for experiment? Yes, the authors made all samples with the same ethanol concentration (Page 6 in Pharmacological treatment). 4) The detail information for this extracts is not enough. Please add more information in the introduction. We have insert this plant extraction in the methods (Page 6 in Pharmacological treatment). 5) The fingerprint like NMR or HLPS may be provided to illustrate the possible components in the active fraction of an extract from E. tirucalli latex. We had the chromatogram in the patent of the active fraction of a polar solvent extract from the latex of Euphorbiaceae plants (Doc 1, reference n. 22), but I can not insert HPLC chart in the manuscript, because It was not realized by my group, but we add an explanation about that in the methods (Page 6 in Pharmacological treatment). 6) More larynx cancer cell lines or one normal cells are suggested to be tested in the survival data. We agree that more larynx cancer cell lines or one normal cells could be tested in the survival data, but in Brazil we have some problems with the reagents and cell lines, it has delayed import, almost three month, these facts delay the publish of the results. We will do all approaches again with more cell lines, but in other project, in a second moment. Therefore, our results open innovative perspectives for the use of this phytotherapy in the treatment of larynx cancer and, in our opinion, make this study interesting. In this way we have reviewed other cell lines studies and new studies will be developed in our laboratory concerning this issue.

7 Minor comments: 1)Fig.1 & 2 (label of Y-axis): > 10^4 The label of Y-axis in the Figures 1 and 2 were substitute 104 for ) Statistics for Fig. 4 is needed. Thank you for your comments, in the Fig. 4 (now Fig. 3) we had the standard deviation, but the pure statistics analysis would be born to read, because we prepared according to Pfaffl (2001) and calculated the log2 (attachment 1)