Water-soluble 4-(dimethylaminomethyl)heliomycin exerts greater antitumor effects than parental heliomycin by targeting the tNOX-SIRT1 axis and apoptosis in oral cancer cells

Joint Public Review:

Previous findings by authors show that heliomycin induces autophagy to inhibit cancer progression, while its water-soluble analogs induce apoptosis. Here, they show that one of the analogs, 4-dmH, binds to tNOX, a NADH oxidase which supports SirT1 activity, in addition to SirT1, while heliomycin only binds to SirtT1 but not tNOX, using CETSA and in silico molecular docking studies, in human oral cancer cells. The additional binding activity of 4-dmH to tNOX might explain the different biological outcome from heliomycin. 4-dmH induces ubiquitination and degradation of tNOX protein, in dependent of p53 status. The tumor suppressive effect of 4-dmH (by intra-tumoral injections) is better than heliomycin. TCGA data base analysis suggests that high tNOX mRNA expression is correlated with poor prognosis of oral cancer patients.

This group has been a leading lab of chemical and biological characterization of heliomycin and its analogs. Although their findings are interesting and advance their previous findings, they arbitrarily focus on tNOX as a potential new target of 4-dmH without clear rationale. Moreover, it remains unclear if the different biological outcomes caused by heliomycin and 4-dmH are indeed due to 4-dmH's ability to bind to tNOX in addition to SirT1. Moreover, molecular biological analyses to establish the proposed tNOX-SirT1 axis on inducing autophagy vs apoptosis are insufficiently performed.

Comments on the current version:

1. The rationale of selecting tNOX/ENOX2 as a potential target of 4-dmH, but not heliomycin, is unclear by taking a biased approach. Thus, there is high possibility that 4-dmH binds to other proteins involved in apoptosis inhibition. An unbiased screen to identify 4-dmH-binding proteins would be a better approach unless there is a clear and logical rationale.

2. The authors should show whether heliomycin indeed does not induce apoptosis, while 4-dmH cannot induce autophagy.

3. They should demonstrate whether genetic knockdown of tNOX, SirT1, or both tNOX and SirT1 induces apoptosis or autophagy and also reduces malignant properties of oral cancer cells.

4. The authors should examine whether overexpression of SirT1 or tNOX in cells treated with heliomycin or 4-dmH could nullify heliomycin-induced autophagy and 4-dmH-induced apoptosis. Also, instead of overexpressing tNOX, they can supplement NAD into cells treated with 4-dmH.

5. Related to Fig. 5C and 6a, the authors should examine the effects of heliomycin and 4-dmH on the cell cycle profiles, Annexin V positivity, and colony formation.

6. They should also examine whether either or both heliomycin and 4-dmH induce reactive oxygen species (ROS).

7. Related to Fig. 9d, they should mutate amino acid residue(s) in tNOX that are crucial for the 4-dmH-tNOX binding, including Ile 90, Lys98, Pro111, Pro113, Leu115, Pro117, and Pro118, to examine whether these mutants lose the binding to 4-dmH and fail to rescue 4-dmH-induced apoptosis, unlike wild-type tNOX.

8. Related to Fig. 10a, heliomycin appears to also reduce tNOX levels (although the extent is not as robust as 4-dmH), which is not expected since heliomycin does not bind to tNOX. They should compare the effects of heliomycin and 4-dmH on reducing the protein levels of tNOX. If heliomycin does not change the tNOX protein levels, then they need to discuss why heliomycin reduces tNOX levels in vivo.

9. Related to Fig. 10F, if tNOX is an upstream regulator of SirT1 and both heliomycin and 4-dmH ultimately target SirT1, it is unclear why heliomycin and 4-dmH cause different biological outcomes. One explanation is that tNOX has apoptosis-inhibiting function other than supporting (or independent of) SirT1 and hence 4-dmH-mediated tNOX inhibition causes apoptosis rather than autophagy. They should explain and discuss more about whether tNOX-inhibiting/binding function of 4-dmH is sufficient to explain the different biological outcomes from heliomycin.

10. They should examine the effects of heliomycin and 4-dmH on cell viability of non-tumor cells to examine their toxicities.

11. They should consistently use either tNOX or ENOX2 to avoid confusion.