2.3.3. Systemic Toxicological Evaluation

Systemic toxicological effects were also investigated, as previously described in ref. [37]. The mice were weighed at the beginning and end of the experiment. The animals were observed for signs of abnormalities throughout the study. Hematological analyses were performed using an Advia 60 hematology system (Bayer, Leverkusen, Germany). Their livers, kidneys, lungs, and hearts were removed, weighed, and examined for any signs of gross lesions or color changes and hemorrhage. Following macroscopic analysis, representative tissue sections of the tumors, livers, kidneys, lungs, and hearts were fixed in 4% buffered formalin and embedded in paraffin. Tissue sections with a thickness of 4 μm were stained with hematoxylin and eosin, and the analyses were performed under light microscopy.

### *2.4. Statistical Analysis*

Inhibitory concentrations of 50% (IC50) values and their 95% confidence intervals (CI 95%) were obtained via nonlinear regression. The fractional inhibitory concentration (FIC) was calculated following the formula FIC(a) = effect (a) of the compound in combination/effect (a) of the compound alone where (a) are the effects of 25%, 50%, and 75% inhibition resulting in FIC25, FIC50, and FIC75. Isobolograms were constructed using the coordinates formed by the FIC (CUR + DSS) of the 25%, 50%, and 75% effects. The line linking the number 1 in both axes was used. Points below this line indicate synergistic results of combination, and points above the line indicate antagonism. Points upon the line indicate an addictive effect [38].

Data are presented as the means ± SEMs or IC50 values. Differences between experimental groups were compared using ANOVA (analysis of variance) followed by the Student Newman-Keuls test (*p* < 0.05). Statistical analyses were performed using GraphPad software (GraphPad Software, Inc., San Diego, CA, USA).

#### **3. Results**

#### *3.1. Combination Therapy of CUR and DSS Synergistically Inhibits the Growth of B16-F10 Melanoma Cells*

CUR and DSS showed cytotoxic effects on B16-F10 and MRC-5 cells in a concentrationdependent manner after 72 h of incubation, as evaluated using the Alamar Blue method (Figure 1). The IC50 values found in B16-F10 cells were 9.69, 16.49, and 0.19 μg/mL for CUR, DSS, and DOX, respectively, while those in MRC-5 cells were 3.60, 13.63, and 1.60 μg/mL, respectively.

**Figure 1.** Effect of CUR and DSS on the viability of B16-F10 (**A**,**C**,**E**) and MRC-5 (**B**,**D**,**F**) cells measured by the Alamar Blue method after 72 h of incubation. DOX was used as a positive control. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (untreated cells) by ANOVA followed by the Student Newman-Keuls test.

Next, we tested the combination of CUR with DSS in five different ratios: 1:2, 1:3, 1:4, 2:3. and 1:10 (Figures S1–S5). Although these combinations were also toxic to noncancerous MRC-5 cells, enhanced cytotoxicity was observed in B16-F10 cells. The effect on B16-F10 cells was evaluated using FIC25, FIC50, and FIC75, revealing the magnitude of the concentration of each compound in relation to the same compound alone (Table S1). These data were also observed in isobolograms (Figure 2), where we found synergic effects for the combinations 1:2, 1:3, and 2:3 in FIC50. Therefore, these combinations were selected for further studies.

**Figure 2.** Isobolograms of the effects of the combination of CUR with DSS at ratios of 1:2 (**A**), 1:3 (**B**), 1:4 (**C**), 2:3 (**D**), and 1:10 (**E**) on the viability of B16-F10 cells. The fractional inhibitory concentration (FIC) values were calculated following the formula FIC(a) = effect (a) of the compound in combination/effect (a) of the compound alone, where (a) are the effects of 25%, 50%, and 75% inhibition resulting in FIC25, FIC50, and FIC75, respectively. Isobolograms were constructed using the coordinates formed by the FIC (CUR + DSS) of the 25%, 50%, and 75% effects. The line linking the number 1 in both axes was used. Points below this line indicate synergistic results of combination, and points above the line indicate antagonism. Points upon the line indicate an addictive effect.

#### *3.2. Combination Therapy with CUR and DSS Causes Apoptotic Cell Death in B16-F10 Melanoma Cells*

In a new set of experiments, apoptosis quantification was evaluated in B16-F10 cells by annexin-V/PI double staining using flow cytometry after 48 and 72 h incubation. The numbers of viable (annexin-V/PI double-negative cells), apoptotic (all annexin-V-positive cells), and necrotic cells (annexin-V-negative/PI-positive cells) were quantified. The IC50 values of each compound were used (CUR 10 μg/mL and DSS 18 μg/mL). The ratios of 1:2 (CUR 2 μg/mL and DSS 4 μg/mL), 1:3 (CUR 2 μg/mL and DSS 6 μg/mL), and 2:3 (CUR 2 μg/mL and DSS 3 μg/mL) were also tested.

In B16-F10, CUR treatment led to 56.6% and 71.5% apoptosis after 48 and 72 h incubation, respectively, whereas DSS caused 39.7% and 36.3% apoptosis (Figures 3 and S6). The combinations tested significantly increased apoptosis after 72 h of incubation. Combination 1:2 led to 32.0% apoptosis in B16-F10 cells (Figures 4 and S7), while combination 1:3 caused 22.7% apoptosis (Figures 5 and S8), and combination 2:3 caused 31.6% apoptosis (Figures 6 and S9) after 72 h of incubation. A statistically significant proportional reduction in the viable cells was also observed.

**Figure 3.** Effect of CUR and DSS on the viability of B16-F10 cells measured by annexin V-FITC/PI staining after 48 (**A**) and 72 (**B**) h of incubation. DOX was used as a positive control. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test.

**Figure 4.** 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 after 48 (**A**) and 72 (**B**) h of incubation. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test.

**Figure 5.** 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 after 48 (**A**) and 72 (**B**) h of incubation. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test.

**Figure 6.** 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 after 48 (**A**) and 72 (**B**) h of incubation. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test.

Next, the DNA content was measured by flow cytometry to quantify the internucleosomal DNA fragmentation and cell cycle distribution in B16-F10 cells treated with CUR and DSS alone or in combination after 48 and 72 h incubation. All DNA that was subdiploid (sub-G0/G1) was considered fragmented. CUR induced 33.5% and 34.4% DNA fragmentation in B16-F10 cells, while DSS caused 34.0% and 32.7% DNA fragmentation after 48 and 72 h of incubation, respectively (Figures 7 and S10). After 72 h of incubation, the 1:2 combination induced 24.9% DNA fragmentation in B16-F10 cells (Figures 8 and S11), while the 1:3 (Figures 9 and S12) and 2:3 (Figures 10 and S13) combinations caused 23.4% DNA fragmentation. No significant changes were observed after 48 h of incubation. A proportional reduction in the cell cycle phase was also observed. An increase in the cell cycle phase G2/M was found after 48 h of incubation with CUR, as well as in the combination 1:2 after 72 h of incubation. DOX caused cell cycle arrest at the G2/M phase, which was followed by DNA fragmentation in B16-F10 cells.

**Figure 7.** Effect of CUR and DSS on DNA fragmentation and cell cycle distribution of B16-F10 cells after 48 (**A**) and 72 (**B**) h of incubation. DOX was used as a positive control. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test.

#### *3.3. Combination Therapy with CUR and DSS Induces Oxidative Stress in B16-F10 Melanoma Cells*

MitoSOX™ Red was used to quantify mitochondrial superoxide levels in B16-F10 cells treated with CUR and DSS alone or in combination after 24 h of incubation (Figure 11). CUR or DSS significantly increased MitoSOX™ Red staining in B16-F10 cells, showing an MFI of 3313 ± 204.3 for CUR and 3532 ± 661.9 for DSS, against 1641 ± 292.6 found for the negative control, indicating increased oxidative stress. A significant increase in oxidative stress was also observed in B16-F10 cells treated with the combinations 1:2 (MFI of 3168 ± 156.7) and 1:3 (MFI of 3571 ± 360.1).

**Figure 8.** 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 after 48 (**A**) and 72 (**B**) h of incubation. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test.

**Figure 9.** 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 after 48 (**A**) and 72 (**B**) h of incubation. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test.

**Figure 10.** 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 after 48 (**A**) and 72 (**B**) h of incubation. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test.

**Figure 11.** Effect of CUR and DSS (**A**) and their combinations at ratios of 1:2 (**B**), 1:3 (**C**), and 2:3 (**D**) on the mitochondrial superoxide level of B16-F10 cells measured by MitoSOX™ Red staining after 24 h of incubation. Data are shown as the mean ± S.E.M. of three independent experiments carried out in duplicate. \* *p* < 0.05 compared with control (0.5% DMSO) by ANOVA followed by Student Newman-Keuls test. MFI = Mean Fluorescence Intensity.

#### *3.4. Combination Therapy with CUR and DSS Inhibits B16-F10 Melanoma Cells Grown In Vivo*

The antitumor activities of CUR and DSS alone and in combination were evaluated in C57BL/6 mice bearing B16-F10 cells (Figure 12). The treatment was performed by intraperitoneal injection of 20 mg/kg CUR, 60 mg/kg DSS and their combination at a ratio of 1:3 (20 mg/kg CUR + 60 mg/kg DSS) for 15 days. DOX was used as a positive control at 1 mg/kg. At the end of treatment, the mean tumor mass weight of the control group was 6.9 ± 0.3 g. CUR and DSS showed mean tumor mass weights of 5.7 ± 0.5 and 5.6 ± 0.3 g, respectively, while an average tumor mass of 4.9 ± 0.6 g was found for the combination of CUR with DSS. Tumor mass inhibition rates were 17.0%, 19.8%, and 28.8% for CUR, DSS, and the combination, respectively. DOX (1 mg/kg) reduced tumor weight by 43.9%.

**Figure 12.** Effect of CUR and DSS and their combination at a ratio of 1:3 on the in vivo development of B16-F10 cells measured by tumor weight (**A**) and tumor inhibition (**B**). DOX was used as a positive control. Data are shown as the mean ± S.E.M. of 7–10 animals. \* *p* < 0.05 compared with control (5% DMSO) by ANOVA followed by Student Newman-Keuls test.

In the histological analysis of tumors (Figure 13), a highly proliferative tumor exhibiting rounded and disrupted cells was observed. Atypical mitosis, apoptosis, and necrosis were frequent features. In CUR-treated animals, we observed more delimitated and less vascularized tumors.

**Figure 13.** Representative histological analysis of B16-F10 tumor tissues stained with hematoxylin and eosin and analyzed by light microscopy. The animals were treated with 5% DMSO (**A**), 1 mg/kg DOX (**B**), 20 mg/kg CUR (**C**), 60 mg/kg DSS (**D**), or 20 mg/kg CUR + 60 mg/kg DSS (**E**). Bars = 20 μm (**A**,**D**), 50 μm (**B**), 200 μm (**C**), or 100 μm (**E**).

The systemic toxic effect of the CUR and DSS treatments or their combination was also evaluated. Three animals died in the combination group, and one animal died in each group, with the exception of the control group. No significant changes were found in the relative mass of the organs or body weights (Table S2). In the hematological parameter analyses, an increase in erythrocytes, hemoglobin, MCV, and platelets was found in the groups treated with DOX and DSS compared to the control (Table S3).

The histopathological examinations of hearts and kidneys showed well-preserved structures in all experimental groups (Figures S14 and S15). In the liver, areas of hydropic degeneration, vascular hyperemia and inflammation were frequent in the control groups, although swelling of hepatocytes was seen in the CUR and DSS groups (Figure S16). Single treatment with CUR or DSS resulted in mild inflammation and areas of fibrosis in the kidney and liver. In the combination treatment group, mild edema was identified. Focal areas of microgoticular steatosis were observed in some animals of the control and groups treated with DOX, CUR, and the combination treatment. In the lungs, areas of inflammation, vascular hyperemia, and alveolar septal thickening were observed in all groups (Figure S17).
