## 3 Results
### MnTnHex Enhances the Cytotoxicity of Cisplatin  
To assess the effect of MnTnHex combined with cisplatin, NSCLC cells were exposed to both compounds simultaneously, and the effects were evaluated using the CV assay. For all combinations tested, a significant decrease in cell viability when compared to the cisplatin-treated cells was observed (Figure 4). In A549 cells, the cytotoxicity of cisplatin alone was slight to moderate at 1 and 2 uM, respectively, which was increased when combined with MnTnHex. In absolute percentage values, the decrease in cell viability observed for 1 uM of cisplatin, when combined with 0.5 and 1 uM of MnTnHex, was 18.8% (_p_ < 0.01) and 39.2% (_p_ < 0.001), respectively, and for 2 uM of cisplatin, when combined with the same MnTnHex concentrations, 17.0% (_p_ < 0.05) and 31.2% (_p_ < 0.001) (Figure 4A).  
Figure 2: Gene expression analyses of the main H\({}_{2}\)O\({}_{2}\) detoxifying enzymes. Values represent mean \(\pm\) SD (_n_ = 3) and are normalized to the reference gene \(\beta\)-ACTIN. ***_p_ < 0.001 (Student’s _t_-test).  
Figure 3: Cytotoxic effects of MnTnHex (0.5–25 \(\upmu\)M) in A549 and H1975 cells. The decrease in cell viability upon exposure to MnTnHex, for 72 h, was assessed by CV and MTS assays in (**A**) A549 and (**B**) H1975 cells. Values represent mean \(\pm\) SD (\(n\) = 3–4) and are expressed as percentages of the vehicle-treated control cells. (**C**) A549 cell morphology upon treatment with MnTnHex. (**D**) H1975 cell morphology upon treatment with MnTnHex. Scale bar = 100 \(\upmu\)m.  
In H1975 cells, the lowest cisplatin concentration used (1 \(\upmu\)M) did not show any cytotoxicity, while the higher concentration (5 \(\upmu\)M) displayed clear cytotoxic effects. The concomitant treatment of cisplatin with MnTnHex significantly decreased cell viability when compared with cisplatin alone. In absolute percentages, the combination of 1 \(\upmu\)M of cisplatin with 0.5 and 1 \(\upmu\)M of MnTnHex reduced cell viability by 46.4% (\(p<0.001\)) and 62.1% (\(p<0.001\)), respectively. Combining 5 \(\upmu\)M cisplatin with the same MnP concentrations led to a reduction in cell viability of 29.7% (\(p<0.05\)) and 37.8% (\(p<0.01\)), respectively (Figure 4B). The morphology of A549 and H1975 cells upon treatment with MnTnHex and cisplatin are depicted in Figures 4C and 4D, respectively. Overall, by combining MnTnHex with cisplatin, the cytotoxicity of this chemotherapeutic drug was significantly enhanced in NSCLC in vitro, this combination being more efficient in H1975 cells.  
Figure 4: Cytotoxic effect of MnTnHex combined with cisplatin in A549 and H1975 cells. Cells were simultaneously treated with (**A**) MnTnHex (0.5 and 1 \(\upmu\)M) and cisplatin (1 and 2 \(\upmu\)M) in A549 cells and (**B**) MnTnHex (0.5 and 1 \(\upmu\)M) and cisplatin (1 and 5 \(\upmu\)M) in H1975 cells, for 72 h. This effect was evaluated using a CV assay. (**C**) A549 cell morphology upon treatment with MnTnHex and/or cisplatin. (**D**) H1975 cell morphology upon treatment with MnTnHex and/or cisplatin. Values represent mean \(\pm\) SD (n = 4) and are expressed as percentages relative to control cells. * \(p<0.05\), **\(p<0.01\), *** \(p<0.001\) and ****\(p<0.0001\) (one-way ANOVA with [PERSON]’s multiple comparisons test) when compared with control and cisplatin-treated cells. Scale bar = 100 \(\upmu\)m.