*2.5. p38 MAPK Signaling Is Activated in Response to Naphthalimide-NHC Complexes, with the Ru(II) Analogue Exhibiting the Strongest Effect*

Several reports have demonstrated the activation of p38 signaling by a number of organometallic drugs, including gold(I) [13]- and Rh(I) [24] NHC complexes. MtROS generation, on the other hand, has been repeatedly associated with MAPK activation, leading to inflammatory responses, apoptosis, and autophagy [20]. We thus evaluated the involvement of this signaling molecule in the mode of action of naphthalimide-NHC analogues in HCT116 cells. After 24 h of treatments, the levels of phospho-p38 MAPK (pp38 MAPK; T180/Y182) were clearly increased across the three compounds, with the Ru(II) analogue (MC6) showing the highest fold change (~7) at a concentration of 12 μM (Figure 6A,B). The activation of p38 signaling by the Rh(I) and Ru(II) complexes was further confirmed as its downstream genes, *ATF2* and *Stat1* were found to be transcriptionally activated after 24 h of treatment with the respective concentrations of the compounds (Figure 6C). A time-dependent analysis of pp38 MAPK (T180/Y182) protein levels upon treatment with 12 μM of MC6 showed that p38 activation occurs as early as 1 h and it persists over the test period of 24 h (Figure 6D,E). Additionally, we observed a rapid and consistent activation of ATF2 in response to MC6, as determined by increased levels of its phosphorylated form (pATF2; T71) (Figure 6D,E).

**Figure 6.** Naphthalimide-NHC derivatives activate the p38 pathway in human breast- and colon cancer cell lines. (**A**) Immunoblots as well as densitometric quantification (**B**) showing the accumulation of pp38 mitogen activated protein kinase (MAPK) (T180/Y182) protein levels by the three complexes in HCT116 CRC cells upon 24 h treatment with increasing concentrations of the respective compound as indicated. The induction appeared to be more profound in case of the Ru(II) analogue, MC6. Data in (**B**) are presented as mean ± SEM of three independent experiments, one of those is shown in (**A**); (**C**) qRT-PCR analysis of p38-associated signaling molecules, *ATF2* and *Stat1* in HCT116 cells treated with the compounds at indicated concentrations for 24 h. Lower and upper ends of the bars denote the minimum and maximum values, respectively, with the "+" sign representing the mean of four biological replicates. Error bars ± SD; (**D**) Time-course analyses of pp38 MAPK (T180/Y182) as well as its down-stream effector, pATF2 (T71) in HCT116 cells treated with 12 μM of MC6, determined by immunoblotting; (**E**) Densitometric analyses of pATF2 (T71) and pp38 (T180/Y182) bands obtained from three independent experiments, one of which is depicted in (**D**). Error bars ± SEM; (**F**) Regulation of p38 MAPK signaling was compared among HCT116, MCF-7 and MDA-MB-231 cancer cell lines treated for 24 h with the metal-containing analogues (MC6 and MC7). In case of MDA-MB-231 where the basal levels of pp38 (T180/Y182) are high, treatments did not profoundly impact the molecule's phosphorylation; (**G**) Densitometric quantifications illustrate no significant change in pp38 (T180/Y182) levels in MDA-MB-231 cells. Error bars ± SEM; n = 3. Statistical comparisons were made between mock (0.1% DMSO) and the respective treatment using two-tailed student's *t*-test. *p*-values less than or equal to 0.05, 0.01, 0.001, and 0.0001 are indicated as \*, \*\*, \*\*\*, and \*\*\*\*, respectively.

Next, we aimed to assess whether p38 activation is reproducible in cancer cell lines other than HCT116 CRC cells. As shown in Figure 6F,G, MC6 and MC7 were able to induce the levels of phospho-p38 MAPK (pp38 MAPK(T180/Y182)) in the breast cancer cell lines, MCF-7 and MDA-MB-231, however with a much lower efficiency in the case of the latter. With regards to this, it has been reported that breast cancer cell lines with a triple-negative (ER negative, PR negative and HER-2 negative) molecular profile exhibit enhanced expression and activity of p38 MAPK, which has been correlated with poor prognosis and survival in patients [25]. Thus, the minor effect on p38 in MDA-MB-231 is most likely due to the endogenous higher activity. In this context, p38 may act as a tumorigenic factor rather than a tumor suppressor. Therefore, its inhibition rather than its activation is shown to exert anti-proliferative effects in invasive breast cancer models [26].

p38 and other members of the MAPK family of proteins, ERK and JNK, have been shown to crosstalk at several levels, determining whether the cell survives or undergoes apoptosis in response to chemotherapeutic drugs [27,28]. We therefore evaluated the phosphorylation status of ERK and JNK using immunoblotting, as well as protein ELISA-microarray analysis [29]. We observed a mild inhibitory effect of the compounds, in particular MC6 and MC7, on the phospho-activation of ERK (Figure S5A–C), however the difference was not found to be statistically significant. With regards to JNK, 24 h treatment with increasing concentrations of the compounds did not show a clear alteration in the phosphorylated protein levels (Figure S5C), which most probably rules out the involvement of JNK signaling in the pro-apoptotic response of HCT116 cells to naphthalimide-NHC analogues.
