Molecular Functions of Hydrogen Sulfide in Cancer
Round 1
Reviewer 1 Report
This is generally a well written review covering the incompletely understood and evolving role of H2S in cancers. Studies related to several major cancer types are reviewed, and the section addressing sulfhydration of several cancer-related proteins provides a useful mechanistic perspective. Some specific issues need to be addressed:
Line 11 typo: synthezsizing
Line 90: The authors make the important point that H2S synthesis is primarily nonenzymatic in most normal tissues, but the subsequent discussion of cancers focuses exclusively on roles of the three biosynthetic enzymes. Has nonenzymatic synthesis in malignant versus nonmalignant tissue been compared? Could this account for some of the conflicting data regarding roles for the biosynthetic enzymes in specific cancers?
Lines 137-148: is ref 44 the source for all of the statements in this paragraph? If so, it should be cited after the topic sentence.
Cited references related to specific conclusions presented in the review are consistently difficult to identify. For example, in the paragraph starting with line 150: are these studies also from ref 44, or from 45 and 46? The source is not clear. Similarly, is ref 49 the source for all the results summarized in the paragraph beginning on line 186? Which results presented in the paragraph beginning at line 218 are from ref 51 versus ref 52?
Lines 266-268: This sentence attempts to distinguish two treatments that seem to be identical. Is the wording correct? Is the first siRNA mentioned the control siRNA?
Line 394: PCNA is not defined
Section 4.1: what concentration of NaSH was used for the NFkB study? This is important to evaluate whether this reactivity is physiologically relevant.
Section 4.2: again, was the NaSH concentration appropriate?
Line 553: define PSA
Some discussion of the immunomodulatory role of H2S in relation to cancer should be included.
The sole figure is not very informative. Additional figures focusing on individual pathways that include the relevant molecular targets of H2S that mediate its effects on specific cancer pathways discussed in the review should be added.
Author Response
Reviewer 1
This is generally a well written review covering the incompletely understood and evolving role of H2S in cancers. Studies related to several major cancer types are reviewed, and the section addressing sulfhydration of several cancer-related proteins provides a useful mechanistic perspective. Some specific issues need to be addressed:
- Line 11 typo: synthezsizing
- this was corrected to “synthesizing”
Line 90: The authors make the important point that H2S synthesis is primarily nonenzymatic in most normal tissues, but the subsequent discussion of cancers focuses exclusively on roles of the three biosynthetic enzymes. Has nonenzymatic synthesis in malignant versus nonmalignant tissue been compared? Could this account for some of the conflicting data regarding roles for the biosynthetic enzymes in specific cancers?
- further recent research has demonstrated that H2S synthesis is not primarily nonenzymatic in most tissues. This part of the paper was therefore changed.
Lines 137-148: is ref 44 the source for all of the statements in this paragraph? If so, it should be cited after the topic sentence.
- this was corrected and reference 44 was cited in other parts of the paragraph
Cited references related to specific conclusions presented in the review are consistently difficult to identify. For example, in the paragraph starting with line 150: are these studies also from ref 44, or from 45 and 46? The source is not clear. Similarly, is ref 49 the source for all the results summarized in the paragraph beginning on line 186? Which results presented in the paragraph beginning at line 218 are from ref 51 versus ref 52?
- this was corrected
- Lines 266-268: This sentence attempts to distinguish two treatments that seem to be identical. Is the wording correct? Is the first siRNA mentioned the control siRNA?
– the language here was corrected
Line 394: PCNA is not defined
- this was corrected AND PCNA was spelt out fully
- Section 4.1: what concentration of NaSH was used for the NFkB study? This is important to evaluate whether this reactivity is physiologically relevant.
- this was corrected
Section 4.2: again, was the NaSH concentration appropriate?
- this was corrected
Line 553: define PSA
- this was corrected and corrected in other parts of the manuscript
- Some discussion of the immunomodulatory role of H2S in relation to cancer should be included.
- a short section on this was added to the manuscript
- The sole figure is not very informative. Additional figures focusing on individual pathways that include the relevant molecular targets of H2S that mediate its effects on specific cancer pathways discussed in the review should be added.
- three additional figures were added highlighting the pathways involving H2S in colon, ovarian, and breast cancers.
Additional changes:
- Several awkward sentences and typos in the manuscript were corrected
Author Response File: 
Author Response.pdf
Reviewer 2 Report
General comments and minor points
The present review sumarizes well the dichotomic context of H2S in diferent types of cancer. The topic and subtopic divisions of the thematic issue is clear and follows a line of reasoning that allows a general understanding of the idea throughout the review reading. Few comments listed below can be consider in order to improve the readability of the work. Perhaps the single most significant structural change suggestion point is associated with the last paragraphs within the conclusion, which made it too lengthy. Thus, it is suggested that future points of work (contradictory elements, enzymes that synthesize H2S, polysulfides and epigenetics) are shown in the discussion and the conclusion summarizes these four points in a more general and succinct way.
1) “Hydrogen sulfide (H2S) is a recently identified…” (L10). Of the three best known gas transmitters, H2S was certainly the most recently described. However, given that other gas transmitters have been described in the last 10 years (HCN, for example), we can suggest that the degree of importance and relevance of H2S has increased a lot since 1996. Thus, it is suggested that this expression be changed to something that describes the high physiological relevance of H2S.
2) “H2S damages many organs…” (L25-26). The ideal was to make sure that high concentrations of H2S tend to be harmful to the functioning of organs.
3) “The -2 H2S” (L45). Are the authors referring to the smallest form of H2S, S2-? If so, my suggestion is that this be expressed as such.
4) "Nonenzymatic H2S synthesis accounts for most of the H2S in human tissues, save for the liver and kidneys. It occurs by iron and vitamin B6-mediated catalysis of thiol and thiol-containing compounds, such as cysteine .[38]” (L90-92). The elegant work carried out by Yang et al., 2020 describes the formation of H2S by non-enzymatic pathways as dependent on Vitamin B6 (on Iron), with the most relevant results also being noticed in the plasma of animals. This statement, however, may not be entirely in line with the rest of the literature. This is because in many organs, but especially in the cardiovascular system, the endogenous production of H2S is essentially based on enzymatic activity. Perhaps because of the method used by Yang and his collaborators that offers some limitations (lead acetate), but mainly because of the various findings in KO animals for H2S-producing enzymes, in which, a significant reduction in the production of H2S can be seen in different organs. Perhaps it would be interesting to comment on this work (reference 38), but not as the main source of H2S in most organs. In this sense, one should not forget the phylogenetic past of H2S, without which life would very likely not have developed as it did.
5) “CBS knockdown or pharmacologic inhibition by aminooxyacetic acid (AOAA)…” (L117-118). As it is common to observe in previous works, aminoxoacetic acid is commonly pointed out as a specific inhibitor for CBS. However, more recent findings show that it is not that specific, as it can inhibits both CSE and 3-MST as well. This does not disqualify the citation, as the information obtained by the CBS KO matches what was observed in the pharmacological blockade. This observation is valid for the rest of the text, when AOAA is referred to as a specific CBS inhibitor. Check http://doi.org/10.1111/bph.12171
6) “thymidylate synthase. Further analysis revealed that CBS-synthesized H2S suppressed the colon cancer tumor suppressor miR-215-5p, increasing thymidylate synthase and…” (L177-179). The sentence appears to be unformatted like the rest of the text.
7) “decreased with increased AOAA dosages…” (L221). In terms of experiments with cell culture, from a pharmacological point of view, it makes no sense to use dosages, but rather concentrations.
8) “vascular endothelia growth factor” (L320-321). The author was probably referring to Vascular Endothelial Growth Factor.
9) “3.7 Thyroid” (L399). The text does not appear to be finished.Something is missing...
10) “25-50 µM NaS increased CBS, sulfide:quinone oxidoreductase...” (L413-414). Check the used donor. Probably the author refers to sodium sulfide (Na2S). Also present in (L417) and table 1.
11) “Treatment of these cells with DATS…” (L465). The author refers to the donor diallyl tri-sulfide, which has never been mentioned before throughout the text. Perhaps a short introduction to garlic-derived compounds would complement the differences between exogenous sources of H2S.
12) The description on lines 494-495 is confusing, and authors must rewrite. “where a sulfur molecule is added...”. The authors are probably referring to the addition of an atom and not a sulfur molecule. However, it is important to note that polysulfides can be formed by n additions of sulfur atoms and, depending on the amount, the biological effect can be different. This was not explicitly commented.
13) "Taken together, these studies indicate that H2S and related sulfur compounds can be elevated in gas and fluids and may have utility in cancer diagnostics." (L561-563). Perhaps listing what the possible uses would be interesting, from the authors' point of view, as H2S could be incorporated as a marker in cancer.
14) “mitochondrial bioenergetics” (L583). As shown in Figure 1, at some points in the table it is not clear that mitochondrial bioenergetics is increased by changes in enzyme expression associated with H2S in cancer. Considering the metabolic importance of this pathway, especially in this case, it would be interesting to make this information explicit also in table 1.
Author Response
Reviewer 2
- The present review summarizes well the dichotomic context of H2S in different types of cancer. The topic and subtopic divisions of the thematic issue is clear and follows a line of reasoning that allows a general understanding of the idea throughout the review reading. Few comments listed below can be consider in order to improve the readability of the work. Perhaps the single most significant structural change suggestion point is associated with the last paragraphs within the conclusion, which made it too lengthy. Thus, it is suggested that future points of work (contradictory elements, enzymes that synthesize H2S, polysulfides and epigenetics) are shown in the discussion and the conclusion summarizes these four points in a more general and succinct way.
– this was corrected and the conclusion was significantly shortened and the discussion of the papers reporting contradictions was removed
1) “Hydrogen sulfide (H2S) is a recently identified…” (L10). Of the three best known gas transmitters, H2S was certainly the most recently described. However, given that other gas transmitters have been described in the last 10 years (HCN, for example), we can suggest that the degree of importance and relevance of H2S has increased a lot since 1996. Thus, it is suggested that this expression be changed to something that describes the high physiological relevance of H2S.
– this was corrected
2) “H2S damages many organs…” (L25-26). The ideal was to make sure that high concentrations of H2S tend to be harmful to the functioning of organs.
– this was corrected and an emphasis was placed on high H2S concentrations
3) “The -2 H2S” (L45). Are the authors referring to the smallest form of H2S, S2-? If so, my suggestion is that this be expressed as such.
– this ambiguous sentence was corrected
4) "Nonenzymatic H2S synthesis accounts for most of the H2S in human tissues, save for the liver and kidneys. It occurs by iron and vitamin B6-mediated catalysis of thiol and thiol-containing compounds, such as cysteine.[38]” (L90-92). The elegant work carried out by Yang et al., 2020 describes the formation of H2S by non-enzymatic pathways as dependent on Vitamin B6 (on Iron), with the most relevant results also being noticed in the plasma of animals. This statement, however, may not be entirely in line with the rest of the literature. This is because in many organs, but especially in the cardiovascular system, the endogenous production of H2S is essentially based on enzymatic activity. Perhaps because of the method used by Yang and his collaborators that offers some limitations (lead acetate), but mainly because of the various findings in KO animals for H2S-producing enzymes, in which, a significant reduction in the production of H2S can be seen in different organs. Perhaps it would be interesting to comment on this work (reference 38), but not as the main source of H2S in most organs. In this sense, one should not forget the phylogenetic past of H2S, without which life would very likely not have developed as it did.
– this was corrected
5) “CBS knockdown or pharmacologic inhibition by aminooxyacetic acid (AOAA)…” (L117-118). As it is common to observe in previous works, aminoxoacetic acid is commonly pointed out as a specific inhibitor for CBS. However, more recent findings show that it is not that specific, as it can inhibits both CSE and 3-MST as well. This does not disqualify the citation, as the information obtained by the CBS KO matches what was observed in the pharmacological blockade. This observation is valid for the rest of the text, when AOAA is referred to as a specific CBS inhibitor. Check http://doi.org/10.1111/bph.12171
- this point was added to the manuscript
6) “thymidylate synthase. Further analysis revealed that CBS-synthesized H2S suppressed the colon cancer tumor suppressor miR-215-5p, increasing thymidylate synthase and…” (L177-179). The sentence appears to be unformatted like the rest of the text.
- this was corrected
7) “decreased with increased AOAA dosages…” (L221). In terms of experiments with cell culture, from a pharmacological point of view, it makes no sense to use dosages, but rather concentrations.
– this was corrected and the word “concentrations” was used
8) “vascular endothelia growth factor” (L320-321). The author was probably referring to Vascular Endothelial Growth Factor.
– this was corrected
9) “3.7 Thyroid” (L399). The text does not appear to be finished. Something is missing...
– this was also corrected
10) “25-50 µM NaS increased CBS, sulfide:quinone oxidoreductase...” (L413-414). Check the used donor. Probably the author refers to sodium sulfide (Na2S). Also present in (L417) and table 1.
– this was corrected to NaHS
11) “Treatment of these cells with DATS…” (L465). The author refers to the donor diallyl tri-sulfide, which has never been mentioned before throughout the text. Perhaps a short introduction to garlic-derived compounds would complement the differences between exogenous sources of H2S.
– corrected and three references and a short discussion were added to the manuscript
12) The description on lines 494-495 is confusing, and authors must rewrite. “where a sulfur molecule is added...”. The authors are probably referring to the addition of an atom and not a sulfur molecule. However, it is important to note that polysulfides can be formed by n additions of sulfur atoms and, depending on the amount, the biological effect can be different. This was not explicitly commented.
– this was corrected to “sulfur atom”
13) "Taken together, these studies indicate that H2S and related sulfur compounds can be elevated in gas and fluids and may have utility in cancer diagnostics." (L561-563). Perhaps listing what the possible uses would be interesting, from the authors' point of view, as H2S could be incorporated as a marker in cancer.
– a short description of the possible therapeutic uses of these cancer markers was added to the manuscript
14) “mitochondrial bioenergetics” (L583). As shown in Figure 1, at some points in the table it is not clear that mitochondrial bioenergetics is increased by changes in enzyme expression associated with H2S in cancer. Considering the metabolic importance of this pathway, especially in this case, it would be interesting to make this information explicit also in table 1.
– this change was made to Table 1
Additional changes:
- Several awkward sentences and typos in the manuscript were corrected
- Three additional figures were added to the manuscript to make the descriptions of colon, ovarian, and breast cancers more clear
- A section on H2S, immune-modulation, and cancer was added to the manuscript
Author Response File: Author Response.pdf
