Review Reports - Mixed-Valence Pentadecavanadate with Ca<sup>2+</sup>-ATPase Inhibition Potential and Anti-Breast Cancer Activity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The research article “Mixed-valence Polyoxovanadate with Ca2+-ATPase Inhibition 2 Potential and Anti-breast Cancer Activity”, is an interesting piece of work, also for interdisciplinary research fields. I support publications upon revisions.

Main comments:

1) The authors should add to their work cytotoxicity tests on immortalized/normal cells. This is a routine control experiment and is really important to evaluate the selectivity of the POV for cancer cells.

2) The toxicity of V15, as described by Barbosa et al., reference 24 in the paper, should be properly discussed in the work.

3) The authors should introduce some references about previously described studies for polyoxidovanadate (POV) interaction with proteins, See for example: https://doi.org/10.1016/j.trechm.2024.11.005

https://doi.org/10.1038/s41586-024-07436-7

https://doi.org/10.1002/anie.202310655

Additional comments:

4) It is not clear to me how and why Ca2+-ATPase has been selected as a possible target for V15. A comment on this point should be added to the manuscript.

5) It is not clear to me how the authors discriminate the effect of V15 on Ca2+-ATPase activity with those due to the presence of other Vanadium -containing species derived from the hydrolysis of the POV in water. A comment on this point should be added to the paper.

Author Response

We sincerely thank the reviewer for the careful revision, which allowed us to improve the quality of our manuscript. During the revision process, we carefully considered all suggestions and recommendations provided. We included additional information about V₁₅, such as EPR analysis (See page 6) in aqueous solution and in enzymatic and RPMI media. As suggested, we have compared our data with related studies, particularly concerning Ca²⁺-ATPase activity, and we conducted new experiments on cytotoxicity using a non-tumorigenic, immortalized human mammary epithelial cell line HB4a (See page 11).

Reviewer #1

1) The authors should add to their work cytotoxicity tests on immortalized/normal cells. This is a routine control experiment that is crucial for evaluating the selectivity of the POV for cancer cells.

Answer: Following the reviewer's recommendation, we conducted cytotoxicity studies on HB4a (a non-tumorigenic, immortalized human mammary epithelial cell line). The results of cell viability inhibition of the cell lines treated with V₁₅ showed a dose-dependent effect for the three cell lines, with an IC₅₀ value of 1.02 μM for HB4a, 15.1 μM for MCF-7 and 17.2 μM for MDA-MB-231 (Table 2). Please see Figure 6 on page 11 of the main manuscript and Figure S3 in the supplementary material.

Table 2. IC₅₀ values determined for V₁₅ regarding Ca²⁺-ATPase inhibition, breast cancer cell lines (MCF-7 and MDA-MB-231), and normal breast cell line (HB4a) viabilities, after 24 h of incubation

Assay	IC₅₀ (µM)
Ca²⁺-ATPase	14.2
MCF-7 Cell	15.1
MDA-MB-231 Cell	17.2
HB4a	1.02

We thank the reviewer for raising this important point. These data would be useful to guide further in vivo studies. Therefore, a new paragraph was included on page 11, see the text copied below.

“The non-tumorigenic cell line showed higher sensitivity to V₁₅ compared to the two cancer cell lines, indicating that the POV does not exhibit selectivity regarding this experimental model. Despite the lack of selectivity, in our previous studies with V₁₅ using peripheral blood mononuclear cells (PBMC), an IC₅₀ value of 12.9 μM was determined in RPMI after 48 hours. These data, along with early toxicity studies in mice, may guide future in vivo research when focusing on the urgent need to develop new therapies for the triple-negative breast cancer subtype. In such studies, a V₁₅ concentration in the order of 1.0 μM (see Figure S3) would be reasonable, because it produced cytotoxic effect of approximately 30-40% on MDA-MB-231 cells.”

2) The toxicity of V₁₅, as described by Barbosa et al., reference 24 in the paper, should be properly discussed in the work.

Answer: We are sorry for not making it clear in the first version of the manuscript. Indeed, the toxicity of POMs is a significant concern in medical applications. Vanadium compounds' toxic effects are well documented in vitro, but in vivo data remain scarce, and many toxicity aspects remain unexplored. Regarding POVs, much less is known, and the few in vivo studies that we were able to find were mentioned in the manuscript. Understanding the relevance of this subject, more information about the toxicity of V₁₅ was included in both the introduction and results and discussion Sections. Please see the text below and the answer to question 1.

In the introduction Section (page 3):

“… while V₁₅ was assessed in mice through an acute 28-day repeated toxicity study. To our knowledge, no other POV has been studied so far, finding a moderated toxicity following a single oral dose of V15 from 25 to 2000 mg kg^–1, even after 14 days of exposure. However, repeated daily exposure over 28 days caused high toxicity at doses above 25 mg/kg, and V₁₅ was classified as level 5 in the oral assay. Taking all together, not only V₁₅, but also other compounds with physiologically accessible oxidation states of vanadium must be considered to better understand MV-POV chemistry, with a view to the safe future use of drugs based on vanadium. “

3) The authors should introduce some references about previously described studies for polyoxidovanadate (POV) interaction with proteins, See for example:

https://doi.org/10.1016/j.trechm.2024.11.005

https://doi.org/10.1038/s41586-024-07436-7

https://doi.org/10.1002/anie.202310655

Answer: Following the reviewer’s suggestion, the reference (https://doi.org/10.1016/j.trechm.2024.11.005) was added as reference 14. Additionally, two new references from 2025 were also added. A greater emphasis was given to the reference that reported the single crystal structure of a Cl@V₁₅-ferritin adduct. Therefore, this recent contribution was added to the timeline depicted in Figure 1 and new information was added in the introduction section, as follows:

Recently, the crystalline structure of a Cl@V₁₅-ferritin adduct was reported as obtained from a suspension containing [V^IVO(acac)₂], where acac = acetylacetonato, and H-chain ferritin in basic medium, under mild conditions[24]. This physiologically relevant protein was involved in the spontaneous formation and stabilization of the mixed-valence [Cl@V₁₅O₃₆]⁶^-, containing 8V(IV): 7V(V), which corresponds to the classical polyoxidoanion described by Muller and used in this work, differing only in the cations. These achievements should encourage further research into mixed-valence cage-like chemistry and delve into their biological properties.

Figure 1. Timeline of the cage-like MV-POV [(CH₃)₄N]₆[Cl@V₁₅O₃₆] (V₁₅), emphasizing its role in biological applications, with ball-and-stick and polyhedral representations (the cations were omitted for clarity).

References added:

New ref. 14: Giarita Ferraro, Eugenio Garribba, Antonello Merlino, Exploring polyoxidovanadate–protein interaction, Trends in Chemistry,7, ,2025, 3-6, https://doi.org/10.1016/j.trechm.2024.11.005.

New ref. 24: Rosanna Lucignano, Gabriella Tito, Giarita Ferraro, Delia Picone, Federico Pisanu, Eugenio Garribba and Antonello Merlino Spherical Mixed-Valence Pentadecavanadate Binding to Human H-chain Ferritin Inorg. Chem. Front., 2025.

4) It is not clear to me how and why Ca^2+-ATPase has been selected as a possible target for V₁₅. A comment on this point should be added to the manuscript.

Answer: As described previously, these ATPases were considered one of the several targets for POM in cancer as referred in one of the most relevant review papers about POMs and cancer in the last years (see please reference 10, in main manuscript). Still, we have now clarified in the text to overcome this concern and added a sentence highlighting the association of calcium homeostasis, ion pump ATPases, with cancer (see page 2).

New text (Page 2):

As previously described, these P-type ATPases were considered one of the potential several targets for POMs, which are expected to develop into the next generation of anticancer drugs that selectively target cancer cells [10]. Moreover, the calcium homeostasis dysfunction, a well-known hallmark of cancer, has recently been reviewed for its role in chemoresistance [11]. References added:

New ref 11: Kumari N, Pullaguri N, Rath SN, Bajaj A, Sahu V, Ealla KKR. Dysregulation of calcium homeostasis in cancer and its role in chemoresistance. Cancer Drug Resist. 2024 Mar 15;7:11. doi: 10.20517/cdr.2023.145.

5) It is not clear to me how the authors discriminate the effect of V₁₅ on Ca²⁺-ATPase activity with those due to the presence of other Vanadium -containing species derived from the hydrolysis of the POV in water. A comment on this point should be added to the paper.

Answer: As referred to on page 8, regarding the Ca²⁺-ATPase, the activity was measured within 2 minutes after the addition of the ATP. The same procedure was adopted when the POV was added. Therefore, no incubation was made with the POV in the medium to prevent any decomposition. We have now clarified this concern in the text and added a sentence to highlight that the ATPase activity was measured during the first 2 minutes of reaction. Therefore, no incubation was made with the POV in the medium to prevent any decomposition, as described elsewhere (see page 8). Besides, upon 60 minutes of incubation of the POV, no significant differences were observed in the IC₅₀ value obtained, pointing out that, for this particular experimental condition, the V₁₅ is stable.

Therefore, we now change the text (see Page 8):

“We emphasize that the IC₅₀ values were determined during the first two minutes after the POV addition. In fact, no incubation was made with the POMs in the medium to prevent any decomposition. Therefore, it is suggested that V₁₅ is the most probable species responsible for the observed inhibitory effects on the Ca²⁺-ATPase, as described elsewhere for similar experimental conditions with other POMs [12,46,47,48,49,53]. On the other hand, if putative V₁₅ decomposition into V₁ species occurs, it would lead to a lower inhibition potential activity for this ATPase, considering that the IC₅₀ values for V₁ species were reported to be lower (IC₅₀ = 80 μM) [46,49].”

References added:

New ref 49: G. Fraqueza , L. A. E. Batista de Carvalho , M. P. M. Marques , L. Maia , C. A. Ohlin , W. H. Casey and M. Aureliano , Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca²⁺-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition, Dalton Trans., 2012, 41, 12749 —12758

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive investigation into the stability and biological activity of V15. Various analytical methods were employed to assess its Ca²⁺-ATPase inhibitory potential and anti-breast cancer effects in two breast cancer cell lines: the luminal subtype (MCF-7) and the triple-negative subtype (MDA-MB-231). Given its significant biological activities, I recommend acceptance of the manuscript after minor revisions.

1. The title should specify the particular type of mixed-valence polyoxovanadate under discussion. For instance, "Mixed-Valence Polyoxovanadate V15 with Ca²⁺-ATPase Inhibition Potential and Anti-Breast Cancer Activity."

2. The synthesis method for V15 should be described in detail in the Materials and Methods section.

3. While the manuscript discusses the biological activity of V15, a more in-depth exploration of the underlying mechanisms would strengthen the conclusions drawn. This could involve a discussion of how different vanadium species interact with biological systems.

Author Response

Reviewer #2

1) The title should specify the particular type of mixed-valence polyoxovanadate under discussion. For instance, "Mixed-Valence Polyoxovanadate V₁₅ with Ca²⁺-ATPase Inhibition Potential and Anti-Breast Cancer Activity."

Answer: We agree with the suggestion. We changed the title to:

“Mixed-Valence Pentadecavanadate with Ca²⁺-ATPase Inhibition Potential and Anti-Breast Cancer Activity”

2) The synthesis method for V₁₅ should be described in detail in the Materials and Methods section.

Answer: We respectfully disagree with the reviewer's suggestion. Since the synthesis is already available in the literature, we believe it is unnecessary to include it in the main text. However, in the attempt to meet the suggestion, we provided a synthetic description along with data of two common characterization techniques (IR and PDRX) in the Supplementary Material. New data on V₁₅ stability in solid state over a period of 2 years were obtained by the infrared spectroscopic analysis, indicating that this MV-POV remained stable for a long time of storage.

Therefore, we now change the text (see Page 16):

“The (Me₄N)₆[Cl@V₁₅O₃₆], V_15, was synthetized following the procedure described in the literature and in the Supplementary Material. A detailed scaled-up synthesis was described for some of us in reference [27]. IR spectra were registered for a freshly synthesized sample, after two months and two years of aging. Main bands in IR spectra (Figure S6) of dark green crystals: ν_as and ν_s (CH₃) weak bands at 3024 and 2952 cm^-1 respectively, strong δ_as and δ_s(CH₃) absorptions at 1485 cm^-1 and 1446 cm^-1 respectively, and ν(V=O) at 983 cm^-¹; ν(V−O−V) at 580, 660 and 725 cm⁻¹. The diffraction peaks at angles 2q of 7.4, 8.6, 15.4, 17.6, 19.9, 22.3, 23.1, 25.7, 26.7, 27.2, 30.7, 32.8, 33.2, 41.2 and 41.7° (Figure S7)”

3) While the manuscript discusses the biological activity of V₁₅, a more in-depth exploration of the underlying mechanisms would strengthen the conclusions drawn. This could involve a discussion of how different vanadium species interact with biological systems.

Answer: We explored the understanding of the mechanism of action of V₁₅, being a MV-POV and belonging to the POM class of compounds, starting to describe one of the first studies performed by Yamase about forty years ago (page 15). In order to emphasize the V₁₅ effects in biological systems, we further made a comparison with other POMs, and we now include additional information on page 15, after reference 10. A new schematic figure illustrating the biological activities of V₁₅, which were assessed in this manuscript, was also added. See Figure 10 (page 20)

New text (page 15):

POMs might affect cancer cells through several mechanisms, including the ones referred above, such as inducing apoptosis and affecting bioenergetics processes, but other mechanisms were described, such as interfering with DNA replication and gene expression, cell cycle arrest, generating reactive oxygen species (ROS), disrupting membrane transports, and/or inactivating essential enzymes within cancer cells [10,75]. Of course, those specific mechanisms for anticancer activity might depend on the POM's structure and composition in order to selectively target cancer cells and reduce toxicity to healthy cells. When comparing and sorting the cell viability IC₅₀ values in ascending order for 20 human cancer cell lines tested for different types of POMs, it was obtained first polyoxovanadates (POVs), polyoxomolybdates (POMos), polyoxopaladates (POPds) and polyoxotungstates (POTs) [75]. When comparing clinically approved drugs and POMs, in many cases, better results were observed for POMs in relation to the drugs [75]. Therefore, POVs such as V₁₅ could become in the future an alternative to existing drugs in cancer therapy [75].

References added:

New ref. 75: Carvalho F, Aureliano M. Polyoxometalates Impact as Anticancer Agents. Int J Mol Sci. 2023 Mar 6;24(5):5043. doi: 10.3390/ijms24055043. PMID: 36902473; PMCID: PMC10003337.

Figure 10. Compilation of the results obtained for V₁₅ in this work: a) Inhibition of Ca²⁺-ATPase activity; b) Cytotoxicity to breast cancer cell lines MCF-7 and MDA-MB-231; c) Inhibition of MDA-MB-231 cell migration; d) Potentially reverse epithelial-mesenchymal transition and e) Showed necroptosis mechanism of cell death.