**5. Conclusions**

Several studies have supported the therapeutic use of high ROS levels present in tumor cell lines for the induction of pro-apoptotic pathways. In this study, we show that altered cellular redox balance induced by a novel Ru(II) naphthalimide-NHC complex—MC6—plays an important role in its in vitro anti-tumor efficacy, most likely via mediating the activation of p38 MAPK signaling. Nonetheless, several issues remain to be addressed with regards to the activation of ROS-p38 axis by naphthalimide-NHC analogues, such as: (i) if the pro-apoptotic functions of MC6-induced p38 MAPK could be extrapolated to other cellular contexts, in particular, the ones with enhanced p38 expression and (ii) whether the MC6-triggered elevation in mtROS levels is through an increase in ROS production at the mitochondrial respiratory chain complexes and/or an alteration in cellular/mitochondrial anti-oxidant systems, for instance, superoxide dismutases (SODs), glutathione peroxidases (GPXs), and thioredoxins (TRXs).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/19/12/3964/s1. Figure S1: Human fibroblasts are less susceptible to the cytotoxic effects of naphthalimide-NHC analogues. Figure S2: Cytotoxic efficacy of naphthalimide-NHC analogues in CRC cells harboring deficient- or mutant p53. Figure S3: MtROS is strongly influenced by naphthalimide-NHC analogues. Figure S4: Naphthalimide-NHC analogues reduce, induce and have no clear effect on the MMP (Δψm) in HCT116 cells. Figure S5: Naphthalimide-NHC analogues do not appear to profoundly impact the activation of ERK and JNK MAPKs.

**Author Contributions:** Y.D. and X.C. designed the study. I.O. and W.S. designed and synthesized the complexes. Y.D., A.S., J.T., and B.B. performed experiments. Y.D., A.S., X.C. and S.W. analyzed and interpreted the data. Y.D. and X.C. wrote the manuscript. All authors revised and approved the final version of the manuscript prior to submission.

**Funding:** Y.D. is supported by the Landesgraduiertenförderung (LGF) fellowship program for individual doctoral training from Heidelberg University. B.B. is a recipient of a DAAD doctoral fellowship. S.W. is supported by BMBF (SysToxChip, FKZ 031A303E). X.C. is a recipient of the DFG grant program (CH 1690/2-1). We acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding program Open Access Publishing, by the Baden-Württemberg Ministry of Science, Research and the Arts and by Ruprecht-Karls-Universität Heidelberg.

**Conflicts of Interest:** Authors declare no conflict of interest.
