Ethanolic Extract of Ocimum sanctum Linn. Inhibits Cell Migration of Human Lung Adenocarcinoma Cells (A549) by Downregulation of Integrin αvβ3, α5β1, and VEGF

Round 1

Reviewer 1 Report

The authors of the paper " Extract Ethanolic Ocimum sanctum Linn. Inhibits cell migration of human lung adenocarcinoma by downregulation integrin αvβ3, α5β1, and VEGF", reported an anti-tumor properties for a plant-based extract.

Yet: 

• Your study must have some limitations (design, sampling, technical, etc.). Please add them to your manuscripts (preferably in the discussion section). This is an excellent practice to "ALWAYS" consider the limitations and openly discuss them in your papers. 

• There are some typographical and grammar errors in the text. Please get them checked by a fluent editor.
  

Author Response

Reviewer 1:

The authors of the paper " Extract Ethanolic Ocimum sanctum Linn. Inhibits cell migration of human lung adenocarcinoma by downregulation integrin αvβ3, α5β1, and VEGF", reported anti-tumor properties for a plant-based extract. 

Yet: 

Your study must have some limitations (design, sampling, technical, etc.). Please add them to your manuscripts (preferably in the discussion section). This is an excellent practice to "ALWAYS" consider the limitations and openly discuss them in your papers. 

Answer: thank you very much for the reviewer precious suggestion, and as the reviewer suggestion we already add the limitation according to our experiment in the discussion section lane 297 to 302  and we mark it in red in the revision manuscript

“Taken together, our finding underlines the ability of ethanolic extract of Ocimum sanctum Linn. to prevent the migration and metastasis of human lung adenocarcinoma cells (A549) however more research and discussion need to be done since our research limited only on the role of integrin and VEGF, moreover the data derived on the in vitro analysis which allow direct impact to the cells. Furthermore, the in vivo experiment need to involved as the part of basic data to complete the preclinical phase of this analysis.”

There are some typographical and grammar errors in the text. Please get them checked by a fluent editor. 

Answer: thank you very much for the reviewer suggestion and as the reviewer suggestion we already do the English revision editing on the: https://www.mdpi.com/authors/english, the certificate are attache below

Author Response File: Author Response.pdf

Reviewer 2 Report

In this paper, Kustiati et al. study the effects of extract ethanolic Ocimum sanctum Linn. (EEOS) on cell migration of human lung adenocarcinoma A549 cells in vitro. The authors claim that the effect (inhibited cell migration) is achieved through downregulation of integrin αvβ3, α5β1, and VEGF.

General comment:

The claims on the involvement of integrins αvβ3 and α5β1 and VEGF should be used with caution, since the direct experiments that show their involvement were not performed.

Specific points:

The authors say: ''Extract Ethanolic Ocimum sanctum Linn. inhibits cell migration of human lung adenocarcinoma...'' However, since this study is done using only one cell line, the ''extrapolation'' on lung adenocarcinoma in general would not be completely appropriate. One soluton to this is to include the name of the cell line (A549) in the title.

Lines 14-15: It is not clear what 'with an incidence reaching 85% in the world' means.

Lines 22-24: This would be too much of details for the Abstract.

Line 106: '100 l of Biotin-labeled antibody'

Lines 119-120, 122: It seems that the protocol was cited literally. Please, re-phrase.

Sections 2.5 and 2.7 have the same title; please, re-organize.

Sections 2.6 and 2.8 should be joined.

Figures 2 and 3 should be joined.

Figures 4, 5 and 6 should be joined. It would be good to write the name of the protein tested on the y-axis.

Author Response

Reviewer 2:

In this paper, Kustiati et al. study the effects of extract ethanolic Ocimum sanctum Linn. (EEOS) on cell migration of human lung adenocarcinoma A549 cells in vitro. The authors claim that the effect (inhibited cell migration) is achieved through downregulation of integrin αvβ3, α5β1, and VEGF.

General comment:

1. The claims on the involvement of integrins αvβ3 and α5β1 and VEGF should be used with caution, since the direct experiments that show their involvement were not performed.

Answer: Thank you very much for the reviewer valuable suggestion, and as the reviewer suggestion we already put new reference in the discussion section to strengthen the role of avb3, a5b1 and VEGF on the cancer regulation mainly in the migration and adhesion activity of the human lung adenocarcinoma cells. Furthermore we also already describe on our results the decreasing expression of avb3, a5b1, and VEGF thus its prevent the adhesion and migration of the A549 cells thus may impaired the migration and metastasis of the A549 cells. We mark it in red the additional sentences on the discussion section of the revision manuscript.

Reference:

Lino, R; Dos Santos, L.B; Pisani, P.K.; Altei, G. F. D.; Cominetti, W. F.; Selistre-de-Araújo, H. S. Alphavbeta3 integrin blocking inhibits apoptosis and induces autophagy in murine breast tumor cells. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2019, 12, 1-13 118536. doi:10.1016/j.bbamcr.2019.118536.

Goel, H.L.; Mercurio, A.M. VEGF targets the tumour cell. Nature Review Cancer 2013, 12, 871-82. doi: 10.1038/nrc3627.

Hou, J.; Yan, D.; Liu, Y.; Huang, P.; Cui, H. The Roles of Integrin α5β1 in Human Cancer. Onco Targets Theraphy 2020, 13,13329-13344. doi: 10.2147/OTT.S273803.

Specific points: 

2. The authors say: ''Extract Ethanolic Ocimum sanctum Linn. inhibits cell migration of human lung adenocarcinoma...'' However, since this study is done using only one cell line, the ''extrapolation'' on lung adenocarcinoma in general would not be completely appropriate. One soluton to this is to include the name of the cell line (A549) in the title.

Answer: thank you very much for the reviewer suggestion, and as the reviewer suggestion we already revise the title of the manuscript into:

“Ethanolic Extract of Ocimum sanctum Linn. Inhibits cell migration of human lung adenocarcinoma cells (A549) by downregulation of integrin αvβ3, α5β1, and VEGF ”

In the revision manuscript we already mark it in red

3. Lines 14-15: It is not clear what 'with an incidence reaching 85% in the world' means.

Answer: Thank you very mich for the reviewer correction and as the reviewer correction we already revise the sentence in the revision manuscript as:

“Adenocarcinoma lung cancer is a type of non-small cell lung carcinoma (NSCLC), which accounts for 85% of lung cancer incidence globally”

and we mark it in red on revision version

4. Lines 22-24: This would be too much of details for the Abstract.

Answer: Thank you very much for the reviewer valuable suggestion as the reviewer suggestion we already revise the abstract and we mark it in red on the revision manuscript

„Adenocarcinoma lung cancer is a type of non-small cell lung carcinoma (NSCLC), which accounts for 85% of lung cancer incidence globally. The therapies that are being applied, both conventional therapies and antibody-based treatments, are still found to have side effects. Several previous studies have demonstrated the ability of the ethanolic extract of Ocimum sanctum Linn. (EEOS) as an ethnomedicine with anti-tumor properties. The aim of this study was to determine the effect of Ocimum sanctum Linn. ethanolic extract in inhibiting the proliferation, angiogenesis, and migration of A549 cells (NSCLC). The adhesion as well as the migration assay was performed, furthermore Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure the expression of αvβ3 integrins, α5β1 integrins, and VEGF. The cells were divided into treatment groups: control (non-treated/NT), positive control (AP3/inhibitor β3 80 µg/ml), cisplatin (9 µg/ml), and EEOS at concentrations of 50, 70, 100, and 200 µg/ml. The results showed that EEOS inhibits adhesion ability and migration in A549 cells, with an optimal concentration of 200 µg/ml. ELISA testing showed that the group of A549 cells given EEOS 200 µg/ml presented a decrease in the optimal expression of integrin α5β1, integrin αvβ3, and VEGF.”

5. Line 106: '100 l of Biotin-labeled antibody'

Answer: Thank you very mich for the reviewer correction and as the reviewer correction we already revise in to 100 ul in the revision manuscript, and we mark it in red

6. Lines 119-120, 122: It seems that the protocol was cited literally. Please, re-phrase.

Answer: Thank you very much for the reviewer correction and as the reviewer correction we already rephrase in the revision manuscript and mark it in red

7. Sections 2.5 and 2.7 have the same title; please, re-organize.

Answer: Thank you very much for the reviewer valuable suggestion and as teh reviewer suggestion we already reorganize section 2.5 and 2.7 and we mark it in red on the revision manuscript

“2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

2.5.1. Integrin human ITG αvβ3 dan VEGF

This test was carried out using sandwich ELISA for both human integrin αvβ3 and VEGF (Fine Test, Wuhan, China). The procedure was performed according to the manual in the kit (Fine Test, Wuhan, China). The plate was washed twice before adding the sample in the form of lysate, along with the negative control, to a 96-well plate. A total of 100 ml of sample and standard was added to each well and incubated for 90 minutes at 37°C. The plate then washed for 2 times. A volume of 100 ul of Biotin-labeled antibody was added to each well and incubated for 60 min at 37°C. The plate then wash for 3 times. Working solution of 100 ml HRP–streptavidin conjugate (SABC) was added to each well and incubated for 30 minutes at 37°C, plate then washed for 5 times. TMB substrate (90 ml) was added and incubated for 15–30 minutes at 37°C. A 50 ml stop solution was added, and the well plates were immediately read at a wavelength of 450 nm (Bio-Rad, California, USA).

2.5.2. Integrin human ITG α5β1

The test was performed using competitive ELISA human integrin α5β1 (MyBioSource, Southern California, USA). The procedure was carried out according to the manual in the kit (MyBioSource, Southern California, USA). A total of 100 ml of standard and lysate sample was added to each, then 10 ml of balanced solution was added and homogenized; no bubbles were formed. A total of 50 ml of the conjugate was added in the well, then homogenized, and incubated at 37°C for 60 minutes. After 60 minutes, we drained the liquid on the plate and washed it with wash buffer five times, for one minute each time. Volumes of 50 ml of substrates A and B were added to the wells. Plate then closed tightly and incubated at 37°C for 15 minutes. Stop solution (50 ml) was added, and the well plate was immediately read at a wavelength of 450 nm (Bio-Rad, California, USA).

2.5.3. Enzyme-Linked Immunosorbent Assay (ELISA) Data Analysis

ELISA data analysis of human ITG αvβ3, human ITG α5β1, and vascular endothelial growth factor (VEGF) was carried out quantitatively using an ELISA reader to determine the optical density value of each test, then the concentration value were calculated based on the standard value. Data were analyzed via one-way ANOVA using the GraphPad Prism 7 software (La Jolla, California, USA).”

8. Sections 2.6 and 2.8 should be joined.

Answer: Thank you very much for the reviewer valuable suggestion and as the reviewer suggestion we already reorganize section 2.6 and 2.8 and we mark it in red on the revision manuscript

9. Figures 2 and 3 should be joined.

Answer: Thank you very much for the reviewer valuable suggestion and as the reviewer suggestion we already reorganize Figure 2 and 3  and for the description or figure legend we mark it in red on the revision manuscript,

10. Figures 4, 5 and 6 should be joined. It would be good to write the name of the protein tested on the y-axis.

Answer: Thank you very much for the reviewer valuable suggestion and as the reviewer suggestion we already reorganize Figure 4, 5 and 6 and we put also the name of the protein tested on the y-axis, and for the description or figure legend we mark it in red on the revision manuscript,

Author Response File: Author Response.pdf