*2.3. Intracellular- and Mitochondrial ROS Levels of HCT116 Cells Are Differentially Induced by the Three Naphthalimide-NHC Complexes*

The involvement of excessive ROS generation has been repeatedly mentioned in the anti-cancer mechanisms of organometallic drugs, including that of metal NHC complexes [18]. We therefore sought to evaluate the influence of the three compounds on intracellular ROS formation in HCT116 cells using dihydroethidium (DHE) staining. After 24 h of treatment with various concentrations of each compound, we found that all of the complexes produced a modest but consistent increase in ROS levels, which occurred concentration-dependently (Figure 3A,B). When comparing the three molecules, the highest fold change (1.7) was observed after treatment with 50 μM of the metal-free ligand (MC5), followed by Ru(II) (MC6) and Rh(I) (MC7) compounds which caused a 1.2-fold increase in ROS levels at the highest used concentrations, 12 and 50 μM, respectively (Figure 3A). Pre-treatment with the ROS scavengers, *N*-acetyl-L-cysteine (NAC) and reduced glutathione (GSH) clearly prevented the

MC6-triggered ROS production (Figure 3C). To gain further insight into the source of ROS, we sought to analyze the role of mitochondrial respiration and included co-treatment with either the complex I inhibitor, rotenone, or the uncoupling agent, CCCP. As shown in Figure 3C, blocking complex I was found to significantly decrease MC6-induced ROS, whereas co-treatment with CCCP (2.5 μM, 2 h) caused a slight increase in the total cellular ROS levels.

**Figure 3.** Total cellular reactive oxygen species (ROS) levels are moderately increased by naphthalimide-NHC analogues. (**A**) HCT116 cells were treated with various concentrations of the respective compound for 24 h, after which flow cytometric analysis of ROS generation was performed using the superoxide indicator, dihydroethidium (DHE). A 24 h treatment with the gold(I) NHC complex, MC3 [13] as well as the rapid apoptosis inducer, raptinal [16] was included as positive control. Cellular ROS levels were found to be concentration-dependently induced in response to all the three complexes, with the metal-free ligand (MC5) showing the highest induction. Data were normalized to mock (0.1% DMSO) treatment. Error bars ± SD; *n* = 4. Statistical significance between the respective treatment and mock was determined by two-tailed student's *t*-test. (**B**) Representative density plots of one out of four biological replicates shown in (**A**); (**C**) ROS levels induced by MC6 (6 μM, 24 h) were found to be significantly decreased in HCT116 cells pre-treated for 1 h with the anti-oxidants, *N*-acetyl-L-cysteine (NAC) and glutathione (GSH), as well as the mitochondrial complex I inhibitor, rotenone at the concentrations indicated. A 2 h co-treatment with the mitochondrial uncoupling reagent (CCCP), and Ru(II) complex (MC6) caused a mild increase in the latter's effects on ROS generation stained by DHE. Data were normalized to the values of mock (0.1% DMSO) as well as the corresponding single treatments. Statistical significance between MC6 in the absence/presence of each of the inhibitors was determined by two-tailed student's *t*-test. \*, \*\*, \*\*\*, and \*\*\*\* represent *p*-values less than or equal to 0.05, 0.01, 0.001, and 0.0001, respectively.

One of the main sources of intracellular ROS is mitochondria, known as mitochondrial ROS (mtROS), which are produced in the form of superoxide anions (O2 −) as a by-product of oxidative metabolism [19]. To obtain further insight into the ROS inducing ability of naphthalimide-NHC conjugates, we performed a live cell analysis of MitoSox Red staining. In contrast to intracellular ROS levels, mtROS production was found to have the highest induction with the Ru(II) complex (MC6) at all of the tested time points, followed by MC5, and finally the Rh(I) analogue (Figures 4A,B and S3). As early as 3 h, mtROS were elevated up to 3.1-fold upon treatment with 12 μM of MC6 and reached the maximum after 12 h (5.4-fold), followed by a slight decrease at 24 h (Figure 4B). A similar decrease could be also observed with a concentration of 50 μM of MC5 (Figure 4B). Such a reduction after long-term treatments or higher concentrations might be due to the activation of anti-oxidant defense mechanisms by cancer cells, as previously reported for gold(I) NHC complexes [13].

**Figure 4.** Mitochondrial ROS (mtROS) generation is strongly influenced by naphthalimide-NHC analogues. (**A**) Live cell imaging of mitochondrial superoxide generation stained with MitoSox Red and associated quantification (**B**) as described in the methods' section. MitoTracker Green was used to indicate mitochondria; (**C**) The mitochondria-targeted anti-oxidant, Mito TEMPO, attenuated the ROS induced by 12 μM of MC6, determined by flow cytometric analysis of MitoSox Red staining. Mito TEMPO (10 μM) was pre-incubated with HCT116 cells 2 h before the exposure to MC6 for 24 h. Data are shown as mean ± SD of three biological replicates. Comparison of ROS fold change between the two groups was performed by two-tailed student's *t*-test where a *p*-value less than or equal to 0.05 is denoted by \*; (**D**) Fluorescence micrographs showing mitochondrial localization of the three complexes in HCT116 cells upon treatment with MC5 (50 μM), MC6 (12 μM), and MC7 (50 μM) for 4 h. Mitochondria were stained by MitoTracker Green. Scale bar: 40 μm. 0.1% DMSO was used as mock.

To further elucidate the source of ROS, Mito TEMPO, which is a mitochondria-specific ROS scavenger, was pre-incubated with the cells 2 h before MC6 treatment for 24 h. As shown in Figure 4C, anti-oxidant treatment was capable of reducing mitochondrial superoxide levels induced by 12 μM of MC6.

The rich photophysical properties of naphthalimides make them useful tools for monitoring their uptake and localization in living cells [2]. Using fluorescence microscopy, we detected a clear mitochondrial accumulation of all the three compounds in HCT116 cells after 4 h of treatment (Figure 4D). This may explain the stronger effect of the compounds on mtROS generation rather than that of cytosolic ROS.

#### *2.4. Naphthalimide-NHC Derivatives Impact Mitochondrial Membrane Potential (MMP) in Different Ways*

MtROS production is determined by a number of factors, one of which is MMP (Δψm) [20]. Flowcytometric analysis of Δψm in HCT116 cells revealed that, among the three complexes, MC5 and MC6 have the lowest and the highest potentials at all the tested time points, respectively (Figures S4 and 5A). It has been proposed by a "redox-optimized ROS balance hypothesis" that physiological ROS signaling occurs at optimized MMP levels, whereas oxidative stress can happen at either extreme (low or high) of Δψm [21]. This is in line with our results, showing the highest mtROS production in case of MC6 and MC5, which exhibit the most oxidized and reduced redox potentials, respectively. Accordingly, MC7, whose MMP does not move far from the basal levels (Figures S4 and 5A), shows only a moderate increase in mtROS generation, as compared to those of the other two compounds (Figures S4 and 5A).

Bcl-xL is known to govern the integrity of the mitochondrial outer membrane through protecting it from Bax-induced permeabilization, which leads to the release of cytochrome *c* and activation of caspases [22]. In this regard, we observed a concentration-dependent decrease in the protein expression of the pro-survival Bcl-2 member, Bcl-xL after 24 h of treatment, with MC6 and MC7 showing the highest and lowest reduction, respectively (Figure 5C,D). This was in parallel to the transcriptional activation of the apoptogenic factors, *Bax* and *Bad* (Figure 5B). The pro-apoptotic function of Bad is known to be mediated via its interaction with Bcl-2/Bcl-xL, which neutralizes the pro-survival activity of the latter proteins, thereby sensitizing cells to apoptosis [23]. In support of this, we observed elevated Bad protein levels upon 24 h of treatment with all three compounds at the indicated concentrations (Figure 5C,D). Of note, it has been repeatedly shown that only the unphosphorylated Bad is able to heterodimerize with Bcl2/Bcl-xL and that phosphorylation of the protein at either of the three serine residues, S112, S136, and S155 sequesters Bad away from mitochondrial membrane [23]. We therefore evaluated Bad phosphorylation status in response to the compounds and observed a reduction in two of the aforementioned phosphorylation sites (S112 and S136), suggesting the potential role of Bad in promoting cell death in response to the three analogues (Figure 5C,D). Taken together, all the above findings demonstrate the involvement of ROS and mitochondrial death pathway in the anti-tumor effects of the naphthalimide-NHC conjugates.

**Figure 5.** *Cont.*

**Figure 5.** Naphthalimide-NHC analogues reduce, induce and have no clear effect on the Mitochondrial Membrane Potential (MMP) (Δψm) in HCT116 cells. (**A**) MMP was time-dependently decreased by MC5 (25 μM); it was increased in the earlier time points by MC6 (12 μM) followed by a decrease at 24 h; and was found to be mostly unaltered in response to MC7 (25 μM). Error bars are the SD of three biological replicates; (**B**) mRNA expression analysis of the pro-apoptotic Bcl-2 family members, *Bad* and *Bax*, after 24 h of treatment with the compounds at indicated concentrations. Relative expression was calculated by the ΔΔ Ct method where the Ct values of the target genes were normalized to those of vinculin. Lower and upper ends of the bars denote the minimum and maximum values, respectively and "+" in the middle represents the mean. Error bars ± SD; *n* = 4; (**C**) 24 h of treatment with the respective compound led to a concentration-dependent decrease and increase in Bcl-xL and Bad protein levels, respectively, while it had no clear effect on Bax protein expression. Additionally, different phosphorylated forms of Bad were found to decrease in response to treatment; (**D**) Densitometric quantification of Bcl-2 family members normalized to the respective loading control (vinculin). The values of phosphorylated Bad were normalized to those of vinculin as well as total protein levels. Data are expressed as mean ± SEM of two independent experiments, one of which is presented in (**C**). Statistical comparisons were made between mock (0.1% DMSO), and the respective treatment using two-tailed student's *t*-test. \*, \*\*, \*\*\*, and \*\*\*\* represent *p*-values less than or equal to 0.05, 0.01, 0.001, and 0.0001, respectively.
