## 4 Discussion  
The treatment of NSCLC requires improvement and additional drugs are necessary. Those could be used alone or in combination with chemotherapy to boost its efficacy and simultaneously overcome resistance and reduce its adverse side effects. A promising therapeutic strategy relies on using SODm, particularly MnPs, to increase ROS levels in cancer cells towards marked cytotoxic levels and induction of cell death. This strategy is based on the knowledge that malignant cells typically have higher levels of ROS and lower levels of some key antioxidant enzymes when compared to healthy cells [14; 15; 16; 17]. Currently, one Mn porphyrin, the analog of MnTnHex, is being evaluated in Phase I/II cancer clinical trials (MnTnBuOE-2-PyP\({}^{5+}\), BMX-001) to assess its efficacy when combined with chemotherapy or radiation. The advantages afforded by this redox-active drug in terms of healthy tissues are also one of the outcome measures evaluated (www.clinicaltrials.gov, accessed on 22 July 2022). The trials on BMX-001 are being conducted on glioma, head & neck cancer, anal cancer, and multiple brain metastases [25]. The in vitro and in vivo studies indicate that both Mn porphyrins are powerful anticancer drugs and protectors of normal tissues. The other analog, MnTE-2-PyP\({}^{5+}\) (AEOL10113, BMX-010), is in Phase II clinical trial (NCT03381625) on atopic dermatitis and itch [25]. Our data on MnTnHex would not only contribute to the knowledge of the therapeutic potential of Mn porphyrins in other cancers but also to our understanding of their mechanism of action. MnPs have already been tested in several types of cancer in cellular and animal models. Yet, there is still a lack of information regarding the cytotoxic effects of these compounds in general and most so in lung cancer. Therefore, the goal of the present work was to assess the effect of MnTnHex on several aspects of lung cancer biology, including cell viability and motility. The innovative aspects of this study also include the in vitro evaluation of the combinatory effects of MnTnHex with cisplatin. Finally, we used exometabolome to  
Figure 7: GC-MS-based metabolomics analysis of the extracellular media of H1975 cells exposed to MnTnHex and cisplatin, alone and combined. (**A**) Volcano plot of the GC-MS data of H1975 cells exposed to MnTnHex (1 uM) 72 h compared with controls. (**B**) Volcano plot of GC-MS data of H1975 cells co-exposed to Cisplatin (1 uM) and MnTnHex (1 uM) compared with controls, indicating the statistical significance (\(p\)-value) vs. the magnitude of change (fold change). Statistical significance was assessed using the Mann-Whitney test and \(p\)-values \(<\) 0.05 were considered significant. The grey dots indicate non-significant m/z-RT pairs, while the red dots indicate statistically significant m/z-RT pairs corresponding to isobutanal, 3-methylpentanal and benzaldehyde as marked. (**C**) Boxplots representing the normalized peak areas of the three significant VCCs. Statistical significance was assessed using the Kruskal-Wallis ANOVA test (* \(p<\) 0.05, ** \(p<\) 0.01). address the oxidative stress-based lipid peroxidation induced predominantly by MnTnHex, via the analysis of the VCCs in a culture medium of NSCLC cells. The work presented here was carried out using two NSCLC cell lines. The A549 cell line is one of the most used cell lines, and as such, it allows us to compare our results to other reports. A549 cells display a K-RAS mutation [51] and are known for their sensitivity to cisplatin [52]. The H1975 cell line originated from a non-smoker female with lung adenocarcinoma and is considered highly invasive [53], with a mutation that confers resistance to EGFR inhibitors [54]. To better characterize both cell lines in terms of basal antioxidant gene expression, the relative expression of the genes (RT-qPCR) that code for the main H2O2 processing enzymes was carried out. Our results showed that both NSCLC cell lines exhibited very low levels of CAT and relatively low levels of GPX1 and PRDX2.