In Vitro Evaluation of ALDH1A3-Affinic Compounds on Breast and Prostate Cancer Cell Lines as Single Treatments and in Combination with Doxorubicin

Round 1

Reviewer 1 Report

Paper is a follower of a previous publication. The previous paper characterized a group of rationally designed ALDH inhibitors and tested their cytotoxicity in tumor cells, known to have high ALDH1A1 and 1A3 activity (A549) and another lacking these activities (H1299). The present paper investigates some of these compounds in other cell types with known ALDH expression and in combination with doxorubicin to show sensitizing effect. The presented cycotoxicity assays are carefully designed and showed significant effects for only a few (or can be said to only one) of the tested substances. The conclusion is "The treatment of DOX showed an enhanced cytotoxicity when combined with the selective ALDH inhibitors, particularly compound 15 on MCF7 cell line. Other compounds did not show any promising enhancements of the DOX cytotoxicity on either cell lines. Thus, the combination treatment experiment highlighted a promising impact for the selective ALDH inhibition as a potential target to overcome the cancer resistance to chemotherapeutic agents." In the light of these results the last sentence seems to be too optimistic.

I think that this basic test system in itself is not suitable to select compounds that sensitizes tumor cells to doxorubicin based on their ALDH inhibitory action. At least a better characterization of test system is required. Also the comparison of an ALDH3A1 overexpressing and its parental cell line would be also useful, however if DOX sensitivity is dependent on ALDH1A3 activity the comparison is not easy.

 

1. ALDH expression of the used cell lines are not proved in the paper it is based only on non-self references. Western blot should be presented here to show ALDH1A3 expression

 

2. As a control a known ALDH inhibitor should be presented (like DEAB or if there is a well known specific ALDH1A3 inhibitor it would be even better) to show, that it can sensitize the given cell line to DOX in the same conditions and at what extent. Or alternatively whether sensitization can be done by siRNA of ALDH1A3 as well.

 

3. Unfortunately nothing is known about the intracellular concentration and in cell ALDH inhibitory effect of compounds. There are available assays for cellular ALDH activity - I am not on this field so can not say which is the most sensitive, but there are simple fluorescent based assays like Aldefluor or Aldered. Possibly enzimatic assays can be used also to assess intracellular actions and compair non-treated and inhibitor treated cells. It should be presented at least for compound 15 to demonstrate that the observed effect is related to ALDH inhibition.

 

4. In theory intracellular concentration of compounds can be reduced very much by cell membrane multidrug transporters. In many tumor cells ABC transporters are expressed and it is advisable to check whether the investigated inhibitor is an ABC multidrugtransporter substrate or not. Moreover DOX is also an MDR1 substrate and if the investigated compound is also a substrate due to its higher applied concentration the investigated compound can sensitize cells for cytotoxic DOX by this mechanism and not based on ALDH inhibition. It should be cleared experimentally.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Overview

Abusara et al. have presented cell proliferation data on three cancer cell lines: MCF7, MDA-MB-231, and PC-3, treated with 34 different ALDH1A3 inhibitory compounds (cpds), either singly or in combination with doxorubicin (DOX). Only one of the 34 compounds, Cpd 24, was inhibitory when given on its own to the cell lines; all the others were non-cytotoxic to the cells when given alone. In the presence of DOX, two of the cpds, 15 and 16, were able to increase the cytotoxicity above DOX alone. The authors claim that this shows a synergistic effect when given in combination, and that cpds 15 and 16 may have potential for development of drugs to be used in combination treatments. The paper lacks important information on ALDH expression in the cell lines used and the evidence that the cpds are ALDH inhibitors, either in vitro or in vivo in cells.

 

Major Comments

1.      Do the cell lines have a consistent ALDH expression? The phrase “variable expression” is used but it is not clarified which of the three cell lines have variable expression of ALDH1A3, or if they have any expression. In fact, no information is given on the ALDH expression in MCF7 and PC3. Since the 34 compounds were selected for ALDH inhibition, it is essential to state the expression levels of ALDH in the cells with and without DOX. On page 5. Line 178, the authors state that “These cell lines {MCF7 and PC-3) were chosen because they express ALDH isoforms, particularly ALDH1A3.”, but no reference is given for this comment, nor are levels of expression or variations in expression from the literature commented on. I presume ref 21 (Ibrahim  et al. 2021) would be appropriate to cite for ALDH1A3 ihibitory activity. This paper (ref. 21) presents the ALDH inhibitory activity of some of the compounds, and a short summary of the inhibitory activity of the cpds should be included in the manuscript, in particular cpds 15, 16, 24, 27, and 33 for which further data is given in Fig. 2 and Fig 3 of the present manuscript. For example, cpds 15 and 16 inhibited ALDH1A3 to 0.14% and 4.27% of the control enzyme activity. Cpd 18 also inhibited ALDH1A3 to 16.01% of the control. None of the other cpds tested in ref 21 showed any inhibitory activity for ALDH1A3, nor ALDH1A1 nor ALDH3A1.

2.      Why do all the compounds, with the exception of 24, have no effect on the proliferation of the three cell lines. Is there no expression of ALDH1A3 by the cell lines? Since it is claimed that ALDH is over-expressed in cancerous cells (or DOX -resistant cells), why is growth of MCF7, MDA-MB-231, and PC-3 cells not inhibited by the cpds? In Ref 21, Ibrahim et al. tested the cytotoxicity of the cpds in two cell lines, a ALDH1A3-positive cell line (A549) and an ALDH1A3-negative cell line (H1299). Only 6, 24, and 32 showed significantly low IC50 values for growth, and all of these were in the ALDH1A3-negative cell line. None of the cpds showed lower IC50 values in the ALDH1A3-positive cell line. Ibrahim et al. concluded that either the compounds were degraded in the cells or that ALDH was not involved in the mechanism of action of the cpds. Based on these conclusions, Abusara et al. should be careful not to imply that ALDH-inhibitory compounds are affecting the DOX sensitivity of their cells by a mechanism that includes inhibition of ALDH1A3.

3.      The manuscript discusses a synergistic effect between the compounds and DOX without any formal testing for synergism (Chou, Talalay paper, not cited). Synergism is shown by calculating a combination index (CI value) which is equal to [(D1/Dx1) + (D2/Dx2)], in which D1 and D2 are the concentrations of the cpds, i.e. the ALDH inhibitor and DOX, when given in combination that give the same effect as Dx1 and Dx2, i.e. the concentrations of the compounds when given alone. Since there is no concentration for the ALDH inhibitors that inhibits growth of the cells when given alone (except for cpd 24) no synergy can be calculated because DX1 for the cpds cannot be determined. The authors state this in their response to reviewers letter. Unfortunately, cpd 24, which does have activity when tested alone, does not seem to increase DOX cytotoxicity at the higher concentrations when given in combination with DOX in either of the two tested cell lines.

4.      The positive control, DEAB, also does not inhibit the growth of the cells? If given in combination with DOX, would it be expected to increase the inhibitory effect of DOX? This was not tested in the project.

5.      In the Ibrahim et al. paper (ref 21), 35 cpds were synthesised and tested for ALDH activity. However, some of the cpds in ref 21 are structurally different from some of the same-numbered cpds in the Abusara manuscript. Specifically, cpds 3-6, 15-19, 24 are the same in both papers, whereas 1-2, 23, 25-28, and 32-34 are different between the two. Presumably the compounds should all be the same between the two papers.

6.      In the combination treatment figures, Fig. 2 and Fig. 3, the increased inhibition of viability induced by the added cpds on DOX inhibition only occurs at the higher concentrations, and at the highest concentrations for cpd 24, cpd 24 added alone is more effective than when added in combination with DOX. This is true for both the MCF7 and the PC-3 cell line. Perhaps DOX is increasing the sensitivity to cps 15 and 16, rather than the cpds increasing the sensitivity to DOX.

7.      Abusara et al. seem to be claiming in the manuscript that the inhibitory activity of ALDH1A3 is involved in the mechanism of action of the cpds; however, the results do not support this conclusion. In fact, in their earlier paper (ref 21, Ibrahim et al.), it is concluded, based on effects only seen in the ALDH1A3-negative cells, that the mechanism of action of the compounds is unknown. The Abusara et al. manuscript is important because some of the cpds do increase the activity of DOX, which would be helpful in DOX treatment regimes in the clinic, especially in cancers that have acquired resistance to DOX, but the mechanism is unknown and does not seem to involve the ALDH1A3 expression in the cells. A favourable further development of cpds 15 and 16 would be helpful since the compounds are non-toxic at rather high concentrations but increase the sensitivity of the cancer cells to DOX.

Author Response

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Round 2

Reviewer 1 Report

Comments for author File: Comments.docx

Author Response

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Reviewer 2 Report

The authors have made some of the suggested changes to the text and figures, but other changes were not made.The English has not been improved at all.

They have added some of the information requested on ALDH1A3 expression in their cell lines and have added specific data on the ALDH1A3 inhibition by their compounds.

They introduce the new concept that the ALDH1A3-affinic cpds may be substrates for the drug efflux pumps such as P-gp, and by competing for these pumps, extra DOX may enter the cell when the cpds are present.

In Fig. 1, cpds 1 and 2 have been changed to match those in reference 21 (Ibrahim et al.), but cpds 23, 26-28, and 32-34 are still different from the same numbered cpds in ref. 21.

In their Discussion, they don’t mention the major finding in the Ibrahim et al. paper that cytotoxicity was only seen in a cell line that doesn’t express ALDH1A3 (H1299 cells) whereas in a cell line that expresses ALDH1A3 (A549 cells), no effects were seen.

There was also no discussion of the actual levels of ALDH1A3 in the cell lines, only a relative hierarchy.

Author Response

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Round 3

Reviewer 2 Report

Version 3 of the manuscript by Abasura et al. has been substantially improved over version 1 and 2. The results support the conclusions and the English is very high quality.

 

Specifically, in Fig. 1, of version 2, cpds 1 and 2 were changed to match those in reference 21 (Ibrahim et al.). Now, in version 3, cpds 23, 26-28, and 32-34 have also been changed to match reference 21 (Ibrahim et al.). Therefore, the structures are now in line with the reference 21.

English has been very much improved in revision V3 and reads well.

They have added new data on WBs of ALDH1A3 in MDA-MB-231, MCF7, and PC3 cell lines (New Fig. 2).

Minor comments:

Line 209, Change “IC50S” to “IC50 values”.

Line 424, remove the extra spacing of the lines between the “and” and the “which”

Line 503, It might be nice to acknowledge the person or firm who did such excellent work on improving the English in the manuscript. It was a first-class effort.