Inhibitory Effects of Metformin for Pancreatic Neuroendocrine Neoplasms: Experimental Study on Mitochondrial Function

Round 1

Reviewer 1 Report

In this article Maruzen et al described the effect of Metformin in panNEN cell line, QGP-1 and RIN-m. Authors claim that affect cell viability and cell survival (line 37), but no viability assay was shown in the article.

MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. But it was performed no differences where seen. Authors conclude that results between MTT and manual counting are different due to a possible metabolic effect (lines 185-187). For that reason, they do not rely on MTT, and they only do cell counting. But to be able to get more information and see if they really have an effect con cell viability and survival, they should repeat the counting assays using Trypan Blue. This is very easy and cheap test that allows you to determine the number of viable cells present in a cell suspension. This experiment will give us more information about the effect of metformin.

In the same line, have authors performed the MTT assay in RIN-m cells? As the effect seen when they do cell counting is higher, I am curious to know if they tried to perform an MTT assays in this cell line.

Authors can not conclude that there is an effect on activated AMPK (lines 238-240) based on results presented in Figure 2. Western blot in figure 2 must be repeated because it is very difficult to see the bands and I am not able to observe an increased in p-AMPK. Also, there is no loading control. Authors should also add cyclin D1 levels as it was done in figure 4b if they want to conclude that Metformin has an effect on it.

Author Response

April 14, 2024

     Thank you very much for your prompt and thorough review of our above-referenced manuscript entitled " Inhibitory Effects of Metformin for Pancreatic Neuroendocrine Neoplasms: Experimental Study on Mitochondrial Function ".

     According to the Editor's and the reviewers' suggestions, the manuscript has now been completely revised. We have responded to all of the reviewers' comments as follows:

Responses to comments from Reviewer 1; the comments are shown in Italic in brackets.

1. [MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. But it was performed no differences where seen. Authors conclude that results between MTT and manual counting are different due to a possible metabolic effect (lines 185-187). For that reason, they do not rely on MTT, and they only do cell counting. But to be able to get more information and see if they really have an effect con cell viability and survival, they should repeat the counting assays using Trypan Blue. This is very easy and cheap test that allows you to determine the number of viable cells present in a cell suspension. This experiment will give us more information about the effect of metformin.]

Response:

Thank you for this suggestion. According to the comment, we performed the cell counting using trypan blue as well as MTT assays, and added obtained data in Figure 3. The corresponding sentence is thus revised in results sections (lines 194-197, page 5) as follows: however, after 72 h, the cell proliferation was significantly suppressed by 0.5 and 1.0 mM metformin treatments (Figure 3B). Cellular damages were not observed by additions of metformin using the cell viability assays we employed (Figure 3A and B). We also added the following sentence in the Materials and Methods section (lines99-100, page 3): The cell viability was determined by trypan blue staining.

2. [In the same line, have authors performed the MTT assay in RIN-m cells? As the effect seen when they do cell counting is higher, I am curious to know if they tried to perform an MTT assays in this cell line.]

Response:

     Thank you for this important comment. According to the comment, we performed the MTT assay in RIN-m cells and obtained data. The corresponding sentence is thus revised in results section (lines 216-219, page 6) as follows: Figure 4C shows the cell proliferation and MTT assays. Cell proliferation was dose-dependently suppressed by the treatment of metformin at 48 and 72 h and cellular viability evaluated by MTT assay was significantly decreased when at 2.0 and 4.0 mM metformin was exposed in RIN-m cells.

3. [Authors can not conclude that there is an effect on activated AMPK (lines 238-240) based on results presented in Figure 2. Western blot in figure 2 must be repeated because it is very difficult to see the bands and I am not able to observe an increased in p-AMPK. Also, there is no loading control. Authors should also add cyclin D1 levels as it was done in figure 4b if they want to conclude that Metformin has an effect on it.]

Response:

     Thank you for the reasonable comment. According to the comment, we repeatedly performed western blotting to detect the expressions of activated AMPK, AMPK, cyclin D1 and a-Tubulin as a loading control. The band intensities were also quantified by ImageJ and the obtained relative values were shown under the bands of the new figure. We add a new Figure 2 in the revised version and insert the sentence in results section (lines183-184, page 4-5) as follows: and cyclin D1 expression was decreased by 2.0 mM metformin. The corresponding sentence “Expression of p-AMPK and T-AMPK in QGP-1 cells at 24h after addition of 1 and 2 mM metformin.” in the Figure 2 legend is replaced by “Western blotting of p-AMPK, AMPK and Cyclin D1 in QGP-1 cells after 24 h-metformin treatment. Relative expression values are shown under the bands.”

Reviewer 2 Report

In this study titled “Inhibitory effects of Metformin for pancreatic neuroendocrine neoplasms: experimental study on mitochondrial function” shows suppressed mitochondrial respiration and AMPK activation by metformin in QGP-1 and RIN-m cell lines. While the study highlights the importance of metformin in an anti-tumor effect against panNEN, some suggestions needs to be included.

1)      Fig. 2. The Western blot was visualized using Odyssey Infrared imaging systems. Is it possible to invert the image like in Fig. 4.

2)      It would be great if quantification of p-AMPK levels shown in Western blot was also included.

3)      The figures are not very clear. Quality of figures should be improved. Legend within the figure needs to be improved..

4)      Fig.4 b; Although the authors show dose dependent increase of p-AMPK levels in the Western Blot…the Cyclin D1 and Cyclin E levels appear to be normal and not downregulated. I’d suggest the authors to include quantification of the band intensity.

English language need to be improved....some sentences appear to be redundant...

Author Response

April 14, 2024

     Thank you very much for your prompt and thorough review of our above-referenced manuscript entitled " Inhibitory Effects of Metformin for Pancreatic Neuroendocrine Neoplasms: Experimental Study on Mitochondrial Function ".

     According to the Editor's and the reviewers' suggestions, the manuscript has now been completely revised. We have responded to all of the reviewers' comments as follows:

Responses to comments from Reviewer 2; the comments are shown in Italic in brackets.

1. [Fg. 2. The Western blot was visualized using Odyssey Infrared imaging systems. Is it possible to invert the image like in Fig. 4.]

Response:

     Thank you for the suggestion. According to the comment, we changed the images of Figure.

2. [It would be great if quantification of p-AMPK levels shown in Western blot was also included.]

Response:

     Thank you for the thoughtful suggestion. According to the comment, we quantified the intensity of bands appeared in Figure 2 and 4B. The relative expression values of band intensities are included in the revised version.

3. [The figures are not very clear. Quality of figures should be improved. Legend within the figure needs to be improved.]

Response:

     Thank you for the suggestion. According to the comment, we improved all figures and legends in the revised manuscript.

4. [Fig.4 b; Although the authors show dose dependent increase of p-AMPK levels in the Western Blot…the Cyclin D1 and Cyclin E levels appear to be normal and not downregulated. I’d suggest the authors to include quantification of the band intensity.]

Response:

     Thank you for the suggestion. According to the comment, we quantified the Cyclin D1 and E band intensities appeared in Figure 4 using ImageJ. The relative expression values of band intensities are shown in Figure 4 of the revised version.

Round 2

Reviewer 1 Report

Authors have adressed all comments