Forchlorfenuron and Novel Analogs Cause Cytotoxic Effects in Untreated and Cisplatin-Resistant Malignant Mesothelioma-Derived Cells

Abstract

Malignant mesothelioma (MM) is a currently incurable, aggressive cancer derived from mesothelial cells, most often resulting from asbestos exposure. The current first-line treatment in unresectable MM is cisplatin/pemetrexed, which shows very little long-term effectiveness, necessitating research for novel therapeutic interventions. The existing chemotherapies often act on the cytoskeleton, including actin filaments and microtubules, but recent advances indicate the 'fourth' form consisting of the family of septins, representing a novel target. The septin inhibitor forchlorfenuron (FCF) and FCF analogs inhibit MM cell growth in vitro, but at concentrations which are too high for clinical applications. Based on the reported requirement of the chloride group in the 2-position of the pyridine ring of FCF for MM cell growth inhibition and cytotoxicity, we systematically investigated the importance (cell growth-inhibiting capacity) of the halogen atoms fluorine, chlorine, bromine and iodine in the 2- or 3-position of the pyridine ring. The MM cell lines ZL55, MSTO-211H, and SPC212, and-as a control-immortalized Met-5A mesothelial cells were used. The potency of the various halogen substitutions in FCF was mostly correlated with the atom size (covalent radius); the small fluoride analogs showed the least effect, while the largest one (iodide) most strongly decreased the MTT signals, in particular in MM cells derived from epithelioid MM. In the latter, the strongest effects in vitro were exerted by the 2-iodo and, unexpectedly, the 2-trifluoromethyl (2-CF₃) FCF analogs, which were further tested in vivo in mice. However, FCF-2-I and, more strongly, FCF-2-CF₃ caused rapidly occurring strong symptoms of systemic toxicity at doses lower than those previously obtained with FCF. Thus, we investigated the effectiveness of FCF (and selected analogs) in vitro in MM cells which were first exposed to cisplatin. The slowly appearing population of cisplatin-resistant cells was still susceptible to the growth-inhibiting/cytotoxic effect of FCF and its analogs, indicating that cisplatin and FCF target non-converging pathways in MM cells. Thus, a combination therapy of cisplatin and FCF (analogs) might represent a new avenue for the treatment of repopulating chemo-resistant MM cells in this currently untreatable cancer.

Keywords: FCF; cisplatin; combination therapy; forchlorfenuron; malignant mesothelioma; septin 7; septin cytoskeleton.

Figure 1

Cell proliferation/viability-decreasing effect of FCF and 10 FCF analogs in human MM cells (ZL55, MSTO-211H, SPC212) and immortalized (untransformed) Met-5A mesothelial cells. The cells were exposed to FCF and 10 analogs (for nomenclature, see Supplementary Information) at a concentration of 50 µM, and MTT assays were performed 96 h after the beginning of the treatment. The bars represent the mean (± SD) of 4–5 independent experiments, and each condition was measured in three wells. In each independent experiment, the MTT signal of the untreated cells (medium containing 0.125% DMSO) of each cell line was set to 1.0. For the ZL55 and MSTO-211H cells, the bars for the FCF analogs with halogen atoms in the 2-position of the pyridine ring are stippled; for SPC212 cells, the stippled bars represent the halogenated FCF analogs with modifications in the 3-position. In all of the MM cell lines (but not in untransformed Met-5A) the ANOVA of all of the experimental conditions including the control cells revealed significant differences (p < 0.0001) among the compounds. Pairwise comparisons (control vs. FCF compounds) showed significant decreases (Supplementary Table S1), except where they are marked as not significant (n.s.). The grouping of the four single-halide compounds in the 2-position (ZL55 and MSTO-211H) or the 3-position (SPC212) revealed significant differences (ANOVA_hal) within the group; significant differences between pairs are marked by asterisks. *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001, respectively.

Figure 2

Dose–response curves obtained with (from left to right) ZL55, MSTO-211H, SPC212 and Met-5A cells treated with FCF-2-Cl (parental FCF), FCF-2-Br, FCF-3-I and FCF-2-CF₃ for 96 h. MTT signals (normalized to non-treated cells from the control) of cells exposed to 6.25, 12.5, 25 and 50 µM FCF (and three FCF analogs). The results are the average of 3–4 independent experiments (mean ± SD); each condition was tested in three wells in each experiment. The results from the more sensitive ZL55 and MSTO-211H cells were further used to estimate the IC₅₀ values (Supplementary Figures S1 and S2).

Figure 3

Real-time growth curves of human ZL55 and MSTO-211H cells exposed to cisplatin (Cis-Pt) alone for 96 h, followed by supplemental treatment with FCF-2-Cl, FCF-3-I, FCF-2-Br and FCF-2-CF₃ for an additional 72 h at concentrations of 25 and 50 µM. The representative growth curves of non-treated MM cells are shown in black. A plateau at near 100% confluence is reached at ~100 h in MSTO-211H cells, and at >140 h in ZL55. Treatment with cisplatin (1.25 µM) strongly reduces MTT signals for the first ~60 h. From then on, an increasing number of cisplatin-resistant cells appear. At 96 h, FCF and its three analogs (FCF-3-I, FCF-2-Br and FCF-2-CF₃) were added at 25 µM (green curves) and 50 µM (magenta curves), or they were left to grow in the presence of cisplatin only (blue curves). The latter cells reached near-100% confluence at 168 h, while the addition of FCF (and its analogs) decreased the confluence to various extents. The values in the curves represent the mean ± SD from three wells obtained within one experiment. The scattered values at 96 h are the results of the brief removal of the plates from the Incucyte system to add the FCF and its analogs.

Figure 4

MTT assay reporting the decrease in proliferation/viability of either (I) naïve MM cells exposed to cisplatin alone, or (II) partially cisplatin-resistant MM cells additionally treated with FCF and its analogs for a total of 168 h. ZL55 (upper) and MSTO-211H (lower) MM cells were exposed to cisplatin for 96 h, followed by the addition of FCF and its analogs (FCF-2-I, FCF-3-I, FCF-2-Br and FCF-2-CF₃) at 25 µM (left) and 50 µM (right) for an additional 72 h. Note the concentration-dependent decrease in MTT signal intensity caused by FCF and its analogs in comparison to MM cells treated with cisplatin only at the end of the total exposure time of 168 h. The results are the averages from three to four independent experiments. Each condition was tested in three wells per experiment. The results are shown as the mean ± SD, and the values for untreated cells were set at 1.0. The ANOVA values are shown for the different treatments and cell lines. Significant differences compared to the treatment with cisplatin alone are marked by asterisks, *, **, *** and **** represent p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 respectively.