**4. Discussion**

The treatment for patients with melanoma usually shows low response rates associated with the development of resistance and side effects with low overall survival [1–6]. In this work, we demonstrated for the first time that CUR and DSS inhibit the in vitro and in vivo development of melanoma B16-F10 cells and, when combined, presented synergistic action. Induction of apoptosis and oxidative stress was also found.

Repositioning clinically approved drugs has been considered a viable approach to building new anti-cancer drugs. The repositioning allows prior knowledge of safety factors, bioavailability, and formulations, offering advantages such as shorter development time and lower research costs, providing agility in accessing new therapeutic options for cancer patients [39,40]. The time between new drug development and clinical trials averages 9 years, with a success rate of less than 10% and an average patient cost per drug of several hundred million dollars. In contrast, drug repositioning can take 3 to 4 years for clinical trials and costs only a fraction of the amounts needed to test a new class of drug in patients [41].

CUR is a nutraceutical drug, and DSS is an FDA-approved drug, both of which are used long-term by humans. This indicates that their combination can be safe, even if it has an effect on noncancerous MRC-5 cells. Nevertheless, drug repositioning may be accompanied by side effects that have not been previously identified and described [42]. Therefore, more experiments are required to validate the safety of the CUR and DSS combination.

Apoptosis has been reported as a mechanism of programmed cell death in the presence of cytotoxic agents with chemotherapeutic potential. Treatment with high concentrations of CUR has been described as capable of inducing apoptosis, depending on the cell and tissue type, by both extrinsic and intrinsic pathways, as well as by increased endoplasmic reticulum stress [43]. The intrinsic induction of apoptosis by CUR is activated in response to cellular signals, including stress or DNA damage [34,44]. DSS is responsible for activating the extrinsic pathway of apoptosis [35]. In this work, we showed that B16-F10 cells treated with the combination of CUR with DSS for a period of 72 h showed increased externalization of phosphatidylserine, suggesting cell death by apoptosis.

Considering that the redox mechanism of melanocytes is extremely important for tumor progression, our result here was obtained by the combination of the pro-oxidant profile, favored by the use of CUR, and by the antagonistic action of the antioxidant system performed by DSS, resulting in increased oxidative stress in tumor cells, which was confirmed by the presence of superoxide radicals in B16-F10 cells. In addition, other targets, including the inhibition of the ubiquitin-proteasome system, were reported for these compounds and may contribute to their cytotoxicity [45,46].

The antitumor activities of CUR have been demonstrated in melanoma cells [15] and animal models [47]. DSS has been shown to exert protective effects on organs in experimental studies, preventing myocardial damage [48], in addition to sensitizing tumor cells to radiotherapy and increasing the cytotoxicity of antineoplastic drugs, which can be used as adjuvant therapy [49]. In our in vivo experiment, we also observed that lower doses of the combined drugs led to a significant reduction in the progression of B16F10 cells in mice.

This study provided data on the potential cytotoxic activity of the combination of CUR with DSS and may provide a useful tool for the development of novel therapeutic combinations against melanoma.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom12111600/s1, Figure S1. Effect of the combination of CUR and DSS at a ratio of 1:2 on the viability of B16-F10 (A) and MRC-5 (B) cells measured by the alamar blue method after 72 h of incubation. Figure S2. Effect of the combination of CUR and DSS at a ratio of 1:3 on the viability of B16-F10 (A) and MRC-5 (B) cells measured by the alamar blue method after 72 h of incubation. Figure S3. Effect of the combination of CUR and DSS at a ratio of 1:4 on the viability of B16-F10 (A) and MRC-5 (B) cells measured by the alamar blue method after 72 h of incubation. Figure S4. Effect of the combination of CUR and DSS at a ratio of 2:3 on the viability of B16-F10 (A) and MRC-5 (B) cells measured by the alamar blue method after 72 h of incubation. Figure S5. Effect of the combination of CUR and DSS at a ratio of 1:10 on the viability of B16-F10 (A) and MRC-5 (B) cells measured by the alamar blue method after 72 h of incubation. Figure S6. Representative dotplots of the effect of CUR and DSS on the viability of B16-F10 cells measured by annexin V-FITC/PI staining. Figure S7. Representative dotplots of the effect of the combination of CUR with DSS at a ratio of 1:2 on the viability of B16-F10 cells measured by annexin V-FITC/PI staining. Figure S8. Representative dotplots of the effect of the combination of CUR with DSS at a ratio of 1:3 on the viability of B16-F10 cells measured by annexin V-FITC/PI staining. Figure S9. Representative dotplots of the effect of the combination of CUR with DSS at a ratio of 2:3 on the viability of B16-F10 cells measured by annexin V-FITC/PI staining. Figure S10. Representative histograms of the effect of CUR and DSS on DNA fragmentation and cell cycle distribution of B16-F10 cells. Figure S11. Representative histograms of the effect of the combination of CUR with DSS at a ratio of 1:2 on DNA fragmentation and cell cycle distribution of B16-F10 cells. Figure S12. Representative histograms of the effect of the combination of CUR with DSS at a ratio of 1:3 on DNA fragmentation and cell cycle distribution of B16-F10 cells. Figure S13. Representative histograms of the effect of the combination of CUR with DSS at a ratio of 2:3 on DNA fragmentation and cell cycle distribution of B16-F10 cells. Figure S14. Representative histological analysis of hearts stained with hematoxylin and eosin and analyzed by light microscopy. Figure S15. Representative histological analysis of kidneys stained with hematoxylin and eosin and analyzed by light microscopy. Figure S16. Representative histological analysis of livers stained with hematoxylin and eosin and analyzed by light microscopy. Figure S17. Representative histological analysis of lungs stained with hematoxylin and eosin and analyzed by light microscopy. Table S1. Inhibitory concentrations of CUR and DSS alone and in combination. Table S2. Effect of CUR and DSS and their combination on body and relative organ weight from C57BL/6 mice bearing B16-F10 cells. Table S3. Effect of CUR and DSS and their combination on hematological parameters of peripheral blood from C57BL/6 mice bearing B16-F10 cells.

**Author Contributions:** S.S.F.: conceptualization, formal analysis, investigation, visualization; M.L.N.: formal analysis, investigation, visualization; R.B.D.: formal analysis, investigation, visualization; C.A.G.R.: formal analysis, writing—review and editing, funding acquisition; M.B.P.S.: writing—review and editing, supervision, funding acquisition; M.A.V.-S.: conceptualization, writing—review and editing, supervision, project administration, funding acquisition; D.P.B.: conceptualization, writing—original draft, supervision, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB). The design

of the study, collection, analysis, and interpretation of data, and the writing of the manuscript were not influenced by funding agencies.

**Institutional Review Board Statement:** The Animal Ethics Committee of the Oswaldo Cruz Foundation (Salvador, Bahia, Brazil) approved the experimental protocol (number 01/2013).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data will be made available upon reasonable request.

**Acknowledgments:** The authors are grateful to the flow cytometry and histotechnology cores of FIOCRUZ-Bahia for collecting flow cytometry data and performing histological techniques, respectively.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

