Unraveling Novel Strategies in Mesothelioma Treatments Using a Newly Synthetized Platinum(IV) Compound

Malignant mesothelioma is a rare tumor associated with asbestos exposure. Mesothelioma carcinogenesis is related to enhanced reactive oxygen species (ROS) production and iron overload. Despite the recent advances in biomedical sciences, to date the only available treatments include surgery in a small fraction of patients and platinum-based chemotherapy in combination with pemetrexed. In this view, the purpose of this study was to evaluate the therapeutic potential of the newly synthetized platinum prodrug Pt(IV)Ac-POA compared to cisplatin (CDDP) on human biphasic mesothelioma cell line MSTO-211H using different complementary techniques, such as flow-cytometry, transmission electron microscopy (TEM), and immunocytochemistry. Healthy mesothelial cell lines Met-5A were also employed to assess the cytotoxicity of the above-mentioned compounds. Our in vitro results showed that Pt(IV)Ac-POA significantly interfere with iron metabolisms and more importantly is able to trigger cell death, through different pathways, including ferroptosis, necroptosis, and apoptosis, in neoplastic cells. On the other hand, CDDP triggers mainly apoptotic and necrotic cell death. In conclusion, Pt(IV)Ac-POA may represent a new promising pharmacological agent in the treatment of malignant mesothelioma.


Introduction
Malignant pleural mesothelioma (MPM) is an extremely aggressive tumor with poor prognosis (generally the survival since the diagnosis is from 6 to 14 months) arising from the serosal linings of peritoneal, pleural, or pericardial cavity [1][2][3][4] and whose main risk factor is represented by asbestos exposure.Asbestos-induced carcinogenesis is mainly associated with iron overload and oxidative stress evoked by fibers, generating reactive oxygen species (ROS) and inducing mesothelial cells transformation [5,6].Despite recent advances, treatment options for mesothelioma remain limited [1,2,7,8].Currently, the only chemotherapeutic systemic treatment approved by the U.S. Food and Drug Administration (FDA) in 2004 for mesothelioma is combination chemotherapy based on cisplatin and pemetrexed.However, many MPM patients do not respond or progress during or after firstline treatment, leading to increased use of second-line therapies.Nevertheless, there is no definitive second-line treatment regimen for MPM.For patients who have undergone firstline platinum and pemetrexed-based therapy, retreatment with pemetrexed is suggested.Alternatively, if the same combination is used, premedication and desensitization strategies should be implemented [9].
After cancer development, malignant cells are continuously exposed to this proinflammatory microenvironment and become able to survive in an iron-rich environment, which promotes cell proliferation by accumulating DNA damages and consequently resulting in ferroptosis resistance [5,6].Therefore, lowering iron levels and iron-induced ROS in cancer cells could represent a new strategy to overcome drug resistance.
On the other hand, iron exerts a fundamental role in asbestos bodies formation.Despite the fact that formation of asbestos bodies and their role in mesothelioma pathogenesis is still debated, it was suggested that their formation could have protective roles in people exposed to asbestos [10][11][12].Furthermore, cisplatin, which is used in combination with pemetrexed in the standard first-line treatment for unresectable MPM, besides its wellknown anticancer activity through DNA damage, can affect iron metabolism, leading to toxicity and cell death [13].
With the aim to combine multimodal strategy and perturbation of iron metabolism caused by platinum-based compounds, the (OC-6-44)-acetatodiamminedichlorido(2-(2propynyl)octanoato)platinum(IV) complex, nicknamed Pt(IV)Ac-POA, is proposed here.This molecule is based on cisplatin and contains, as axial ligands, the 2-(2-propynyl)octanoic acid (POA), an MCFA-HDACi (medium-chain fatty acid histone deacetylase inhibitor) along with an inert acetate ligand (Ac) (Figure 1) [14].Pt(IV) antitumor drug candidates are considered prodrugs since they can be reduced preferentially in the hypoxic (and then reducing) intracellular milieu of the tumor cells.If bioactive ligands are coordinated to the Pt(IV) core in the axial positions, the final bifunctional, hybrid, or multimodal anticancer prodrug will be able to deliver (almost selectively) two cytotoxic moieties (i.e., a Pt(II)based metabolite as cisplatin, and the axial bioactive ligands), in the same place and at the same time, after in vivo reduction [15][16][17][18][19].The possibility to co-administer cisplatin with Histone DeACetylase inhibitors (HDACi) seems to have a good effect on the increase of DNA-adducts formation [20][21][22].

Chemistry
The prodrug (OC-6-44)-acetatodiamminedichlorido(2-(2propynyl)octanoato)platinum(IV) (Pt(IV)Ac-POA) was prepared according to established procedures [15].Briefly, rac-2-(2-propynyl)octanoyl chloride (POA-Cl), obtained by reaction between rac-2-(2-propynyl)octanoic acid (POA) and oxalyl dichloride (1:5 molar ratio) in di-chloromethane, was reacted with (OC-6-44)acetatodiamminedichloridohydroxidoplatinum(IV), obtained by oxidation of cisplatin with aqueous H2O2.The two compounds were mixed in a 1:5 (Pt:POACl) molar ratio in anhydrous acetone containing pyridine as a base (1:1 molar ratio vs. POACl) and left to react at reflux overnight.Then, the solvent was removed by rotary evaporation, and the crude residue was washed with hexane and water.Finally, the solid was dried under Indeed, HDAC inhibition increases histone acetylation, decreasing histone-DNA interactions and allowing a higher sensitivity to DNA damage [23,24].In addition, the combination between the cisplatin-like equatorial moiety and the MCFA produce a Pt(IV) complex, which is more lipophilic than cisplatin alone and shows promising antitumor activity both in vitro and in vivo on several human cancer cell lines with fewer side effects than cisplatin [15].Recently, the interest for HDACi in mesothelioma management and other BAP1 mutated cancers, such as uveal melanoma, greatly increased, as demonstrated by several studies [25][26][27][28].Finally, it was observed that Pt(IV)-POA was able to induce ferroptosis in iron-addicted cancer cells, such as human glioblastoma U251 cells [29,30].
In this study, Pt(IV)Ac-POA will be compared to the clinically used cisplatin, on human biphasic MSTO-211H mesothelioma cells and healthy human mesothelial Met-5a cells, with a particular attention on the alteration of iron metabolism and the activated cell death pathways, as well as morphological modifications.
Forty-eight hours before the experiments, cells were seeded on glass coverslips (200,000 cells in 2 mL of media) for fluorescence microscopy or grown in 75 cm 2 plastic flasks for flow cytometric and ultrastructural analysis at transmission electron microscope (TEM).Cells were treated for 12 h and 48 h at 37 • C of continuous treatments (CT) with the compounds under investigation, then flow cytometry, ultrastructural analysis, and immunocytochemistry analysis were performed.Concentrations of 10 µM for CDDP (Teva Pharma, Milan, Italy) and Pt(IV)Ac-POA were chosen after MTS Assay and reviewing the literature (Figures S1 and S2 and Table S1) [29][30][31][32][33].

Cell Cycle Analysis
Cells were washed in PBS (phosphate-buffered saline) for 30 s, permeabilized in 70% ethanol for 10 min, treated with RNase A 100 U mL −1 (Sigma-Aldrich, Milan, Italy), and then stained at room temperature with propidium iodide (PI) 50 µg mL −1 (Sigma-Aldrich, Milan, Italy) 1 h before flow cytometric analysis.PI red fluorescence was detected with a 610 nm long-pass emission filter.At least 10,000 cells per sample were measured to obtain the distribution among the different phases of the cell cycle and the percentage of dead cells.Data were collected using Cytometer BD FACS Lyric (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with the built-in software (BD FACS Suite v1.3).

Transmission Electron Microscopy (TEM)
Control and treated MSTO-211H cells were harvested by mild trypsinization (0.25% trypsin in PBS containing 0.05% EDTA) and centrifugated at 800 rpm for 5 min in fresh tubes.The samples were immediately fixed with 2.5% glutaraldehyde in culture medium (2 h at room temperature) and washed several times with PBS.Samples were stained in 1% OsO4 for 2 h at room temperature (rt) and washed in distilled water.The cell pellets were pre-embedded in 2% agar, dehydrated with increasing concentrations of acetone (30%, 50%, 70%, 90%, and 100%), and finally were embedded in epoxy resin (Electron Microscopy Sciences, Hatfield, PA, USA, EM-bed812) and polymerized at 60 • C for 48 h.Ultrathin sections (60-80 nm) were cut on a Reichert OM-U3 ultramicrotome, collected on nickel grids, and stained with uranyl acetate and lead citrate.Lastly, sections were observed under a JEM 1200 EX II (JEOL, Peabody, MA, USA) electron microscope, equipped with a MegaView G2 CCD camera (Olympus OSIS, Tokyo, Japan) and operating at 100 kV.

Immunocytochemical Reactions: Fluorescence Microscopy Evaluation
Control and treated MSTO-211H and Met-5a cells were grown on glass coverslips, fixed with 4% formalin for 20 min, and post-fixed with 70% ethanol at −20 • C for at least 24 h.Samples were rehydrated for 10 min in PBS, and then unspecific sites were blocked by using a blocking solution of PBS supplemented with 4% BSA (bovine serum albumin) and 0.2% tween for 15 min at rt. Next, cells were immunolabeled with primary antibodies diluted in PBS-Tween 0.2% for 1 h, at rt in a dark moist chamber.After 3 washes in PBS of 5 min each, coverslips were incubated with secondary antibodies in PBS-Tween 0.2% (1:200, Alexa Fluor, Molecular Probes, Invitrogen, Waltham, MA, USA) for 45 min.At the end of the incubation and after other washing in PBS, sections were counterstained for DNA with 0.1 µg/mL Hoechst 33258 (Sigma-Aldrich, Milano, Italy), washed with PBS, and mounted in a drop of Mowiol (Calbiochem, Inalco, Italy) for fluorescence microscopy analysis.Primary and secondary antibodies used for immunocytochemical reactions at the fluorescence microscope are reported in Table 1.An Olympus BX51 microscope equipped with a 100 W mercury lamp was used under the following conditions: 330-385 nm excitation filter (excf), 400 nm dichroic mirror (dm) and 420 nm barrier filter (bf) for Hoechst 33258; 450-480 nm excf, 500 nm dm, and 515 nm bf for the fluorescence of Alexa 488; 540 nm excf, 580 nm dm, and 620 nm bf for Alexa 594.Images were recorded with an Olympus MagnaFire camera system and processed with the Olympus Cell F software version 3.1 (Olympus Italia Srl, Segrate, MI, Italy).To make the fluorescence intensity comparable, during image acquisition, the exposure time to detect every single fluorescence was selected based on the control sample and then maintained constant for the respective experimental conditions, thus avoiding the insertion of any variables in the analysis.The fluorescence intensity of the proteins of interest was analyzed with the CellProfiler software version 4.2.6 [34].

Statistical Analysis
For immunofluorescence quantification, at least 100 cells from 11 quadrants were evaluated for a random analysis, and the values obtained were expressed as mean ± SEM (standard error of the mean) in arbitrary fluorescence unit (a.f.u.) and normalized to control conditions.One-way ANOVA and post hoc Dunnet's test were used to analyze data differences for Ki-67 and acetyl-H3 fluorescent staining in mesothelioma cells.Data differences between MSTO-211H and Met-5a cell lines were analyzed for statistical significance using two-way ANOVA and post hoc Turkey's test (software package GraphPad Prism Inc., version 10.2.3, La Jolla, CA, USA).p-values < 0.05 were considered statistically significant.

Cell Cycle Analysis in MSTO-211H Cell Line
To investigate the effect of treatments with CDDP and Pt(IV)Ac-POA on the cell cycle, DNA was stained with IP and analyzed using flow cytometry in mesothelioma cells.As reported in Figure 2A,B, CDDP-and Pt(IV)Ac-POA-treated samples showed strong alterations in terms of DNA content distribution.After 12 h of CT with CDDP, cells in G2-S phases tend to accumulate, while after 12 h and 48 h of Pt(IV)Ac-POA exposure, cells in S and G2 strongly decreased.Moreover, a sub-G1 peak was clearly present only after treatments with Pt(IV)Ac-POA.Conversely, after 48 h of CT with both CDDP and Pt(IV)Ac-POA, the G1, S, and G2 peaks were totally absent with the exception of a small G2 peak detected in CDDP treated samples.
Ki-67 and acetyl-H3 expression was then evaluated by double immunofluorescence reaction after treatments with CDDP and Pt(IV)Ac-POA (Figure 2C).The amount of Ki-67-positive cells was significantly lower compared to the control condition after 48 h of exposure to CDDP (p < 0.0001) and after both 12 and 48 h of treatment with Pt(IV)Ac-POA (p < 0.0001 and p < 0.0001, respectively) (Figure 2D).
As reported in Figure 2E, the acetyl-H3 fluorescence intensity was significantly higher after 48 h of CT (p < 0.0001).Notably, the acetylation of H3 was significantly stronger after treatment with Pt(IV)Ac-POA compared to CDDP (p < 0.0001).

Ultrastructural Analysis
In order to better understand the effects of the studied compounds, morphological changes in MSTO-211H cells were analyzed by transmission electron microscopy.Untreated cells (Figure 3A) were characterized by the nucleus in a central position (indicated by the letter "n"), a decondensed chromatin, and a well-organized Golgi apparatus (indicated by the letter "g") in the perinuclear zone.While in CDDP-treated samples necrosis (Figure 3B) and apoptosis (Figure 3C) were observed, in Pt(IV)Ac-POA samples we detected both apoptosis (Figure 3E) and necroptosis (Figure 3D,F).The latter is characterized by both apoptotic features (i.e., karyorrhexis, indicated by the letter "n"), and necrotic ones (i.e., cytoplasmic vacuolations, indicated by "v" in Figure 3D and plasma membrane rupture).Enlargement of perinuclear spaces (indicated by letter "p" in Figure 3D-F) and nuclear envelope detachment were also observed (insert Figure 3E).After 48 h of continuous treatment with Pt(IV)Ac-POA, cells with an extreme chromatin decondensation (letter "n"), a destroyed mitochondria cristae, and cytoplasm charged of vesicles (letter "v") were observed, indicating the presence of ferroptotic cells (Figure 3G).

BAP1 and NF2 Modulation
Since BAP1 (BRCA1-Associated Protein 1) and NF2 (Neurofibromin 2) are frequently mutated in mesothelioma and exploit fundamental functions in regulating cell death, proliferation, and chromatin organization [35][36][37][38][39], their expression was evaluated by immunofluorescence in MSTO-211H and Met-5a cells.As expected, under the control condition and in both cell lines, the expression of NF2 and BAP1 was maintained Figure 4) while the cytoskeleton was well structured in actin and tubulin filaments within all cytoplasm.In mesothelioma cells, NF2 expression was found to be significantly downregulated in all tested conditions apart from 12 h of CT with Pt(IV)Ac-POA, and the signal was localized mainly in the cytoplasm (Figure 4).Conversely, in normal cell lines, NF2 expression was found to be significantly upregulated in respect to the control condition after long-lasting treatment with both compounds (the results of the statistical analysis are shown in Figure 4).
BAP1 signal localized mainly in the nucleus (Figure 4), and its expression was significantly higher in CDDP-and Pt(IV)Ac-POA-treated mesothelioma cells at 12 h compared to untreated mesothelioma cells (p < 0.0001).On the other hand, Met-5a cells displayed again an opposite trend, even though it was statistically significant only after the longer

BAP1 and NF2 Modulation
Since BAP1 (BRCA1-Associated Protein 1) and NF2 (Neurofibromin 2) are frequently mutated in mesothelioma and exploit fundamental functions in regulating cell death, proliferation, and chromatin organization [35][36][37][38][39], their expression was evaluated by immunofluorescence in MSTO-211H and Met-5a cells.As expected, under the control condition and in both cell lines, the expression of NF2 and BAP1 was maintained Figure 4) while the cytoskeleton was well structured in actin and tubulin filaments within all cytoplasm.In mesothelioma cells, NF2 expression was found to be significantly downregulated in all tested conditions apart from 12 h of CT with Pt(IV)Ac-POA, and the signal was localized mainly in the cytoplasm (Figure 4).Conversely, in normal cell lines, NF2 expression was found to be significantly upregulated in respect to the control condition after long-lasting treatment with both compounds (the results of the statistical analysis are shown in Figure 4).BAP1 signal localized mainly in the nucleus (Figure 4), and its expression was significantly higher in CDDP-and Pt(IV)Ac-POA-treated mesothelioma cells at 12 h compared to untreated mesothelioma cells (p < 0.0001).On the other hand, Met-5a cells displayed again an opposite trend, even though it was statistically significant only after the longer lasting treatments with cisplatin with respect to the negative control.

Impairment of Iron Metabolisms and Ferroptotic Pathway
Next, oxidative stress, iron metabolism, and ferroptotic pathways were evaluated by immunofluorescence.
Firstly, COX2 (Cyclooxygenase 2) expression was investigated since both CDDP and Pt(IV)Ac-POA can result in the production of ROS (Figure 5).Considering both cell lines, a significant downregulation of COXI2 was observed across all the tested conditions, apart from 48 h of CT with CDDP and 48 h of Pt(IV)Ac-POA exposure in Met-5a cells (Figure 5).Consequently, other ROS responsive elements, namely NRF2 (NF-E2-related factor 2) and Aco2 (Aconitase 2), also related to the ferroptotic pathways were investigated.While NRF2 signal was found to be significantly higher in Met-5a cells, mitochondrial aconitase-2 was upregulated in MSTO-211H cells (Figure 5).

Impairment of Iron Metabolisms and Ferroptotic Pathway
Next, oxidative stress, iron metabolism, and ferroptotic pathways were evaluated by immunofluorescence.
Firstly, COX2 (Cyclooxygenase 2) expression was investigated since both CDDP and Pt(IV)Ac-POA can result in the production of ROS (Figure 5).Considering both cell lines, a significant downregulation of COXI2 was observed across all the tested conditions, apart from 48 h of CT with CDDP and 48 h of Pt(IV)Ac-POA exposure in Met-5a cells (Figure 5).Consequently, other ROS responsive elements, namely NRF2 (NF-E2-related factor 2) and Aco2 (Aconitase 2), also related to the ferroptotic pathways were investigated.While NRF2 signal was found to be significantly higher in Met-5a cells, mitochondrial aconitase-2 was upregulated in MSTO-211H cells (Figure 5).Finally, the relative expression of ferroptosis indicators was assessed by immunofluorescence analysis.As illustrated in Figure 6, in the control condition, cells exhibit a mild immunopositivity for GPX4 (Glutathione peroxidase 4) that colocalized with mitochondria, which showed their characteristic spotted-like shape.Moving to samples treated with platinum-based compounds, as shown in Figure 6, we observed a significant decrease in GPX4 expression after 12 and 48 h of CT of Pt(IV)Ac-POA in both cell lines (pvalues are illustrated in Figure 6).In particular, cells lost their morphology, and mitochondria distribution was severely impaired, with formation of clusters around the nucleus, especially in cells that showed apoptotic appearance (Figure 7).Finally, the relative expression of ferroptosis indicators was assessed by immunofluorescence analysis.As illustrated in Figure 6, in the control condition, cells exhibit a mild immunopositivity for GPX4 (Glutathione peroxidase 4) that colocalized with mitochondria, which showed their characteristic spotted-like shape.Moving to samples treated with platinum-based compounds, as shown in Figure 6, we observed a significant decrease in GPX4 expression after 12 and 48 h of CT of Pt(IV)Ac-POA in both cell lines (p-values are illustrated in Figure 6).In particular, cells lost their morphology, and mitochondria distribution was severely impaired, with formation of clusters around the nucleus, especially in cells that showed apoptotic appearance (Figure 7).Moreover, FTH1 (Ferritin Heavy chain 1) expression was severely altered in all CDDP-and Pt(IV)Ac-POA-treated cells (p < 0.0001), thereby suggesting a severe iron metabolism impairment (Figures 6 and 7).However, MSTO-211H cells treated for 48 h with Pt(IV)Ac-POA also exhibited significantly higher levels of a ferroptosis inhibitor Aconitase-2 (Figures 6 and 7) in respect to untreated samples (p < 0.001).SLC7A11 (cystine transporter Solute Carrier family 7 member 11) was found to be significantly upregulated only in Met-5a cells in all the tested conditions, with the exception of the longer timepoints of Pt(IV)Ac-POA.The ANOVA and post hoc comparisons are shown in Figure 6.

Activation of Different Cell Death Mechanisms
Finally, to better understand the results obtained by transmission electron microscope, other cell death mechanisms were evaluated.First, to analyze the activation of the intrinsic apoptotic pathway, immunocytochemical reaction to cleaved caspase-3 was conducted.As shown in Figure 8, in the control conditions of both cell lines, no expression of active caspase-3 was detected, consistently with the presence of only viable cells.The actin cytoskeleton was well structured in filaments within all cytoplasm.After treatments with both platinum-based compounds, MSTO-211H cells underwent apoptosis; the percentage of immuno-positive cleaved caspase-3 cells was significantly increased, and cell morphology was altered: cells developed a round shape and their nuclei appeared fragmented.The actin cytoskeleton collapsed, with inhomogeneous distribution localized around the nucleus (Figure 8).Conversely, only the longer lasting Pt(IV)Ac-POA treatment triggered the intrinsic apoptotic pathway in Met-5a cells (p < 0.0001).Of note, the percentage of cleaved caspase-3-positive mesothelioma cells was significantly higher after 48 h of CDDP and Pt(IV)Ac-POA exposures compared to the healthy cells (Figure 8).Moreover, FTH1 (Ferritin Heavy chain 1) expression was severely altered in all CDDP-and Pt(IV)Ac-POA-treated cells (p < 0.0001), thereby suggesting a severe iron metabolism impairment (Figures 6 and 7).However, MSTO-211H cells treated for 48 h with Pt(IV)Ac-POA also exhibited significantly higher levels of a ferroptosis inhibitor Aconitase-2 (Figures 6 and 7) in respect to untreated samples (p < 0.001).SLC7A11 (cystine transporter Solute Carrier family 7 member 11) was found to be significantly upregulated only in Met-5a cells in all the tested conditions, with the exception of the longer timepoints of Pt(IV)Ac-POA.The ANOVA and post hoc comparisons are shown in Figure 6.

Activation of Different Cell Death Mechanisms
Finally, to better understand the results obtained by transmission electron microscope, other cell death mechanisms were evaluated.First, to analyze the activation of the intrinsic apoptotic pathway, immunocytochemical reaction to cleaved caspase-3 was conducted.As shown in Figure 8, in the control conditions of both cell lines, no expression of active caspase-3 was detected, consistently with the presence of only viable cells.The actin cytoskeleton was well structured in filaments within all cytoplasm.After treatments with both platinum-based compounds, MSTO-211H cells underwent apoptosis; the percentage of immuno-positive cleaved caspase-3 cells was significantly increased, and cell morphology was altered: cells developed a round shape and their nuclei appeared fragmented.The actin cytoskeleton collapsed, with inhomogeneous distribution localized around the nucleus (Figure 8).Conversely, only the longer lasting Pt(IV)Ac-POA treatment triggered the intrinsic apoptotic pathway in Met-5a cells (p < 0.0001).Of note, the percentage of cleaved caspase-3-positive mesothelioma cells was significantly higher after 48 h of CDDP and Pt(IV)Ac-POA exposures compared to the healthy cells (Figure 8).Autophagy and necroptosis were also evaluated by IF.
In the control conditions, LC3B (Microtubule-associated proteins light chain 3B) and p62/SQSTM1 (Sequestosome 1) are localized in the nucleus and cytoplasm within lysosomes (Figure 9).However, the LC3B fluorescent signal was found to be dysregulated only in Met-5A cells, after CDDP exposure and the longest Pt(IV)Ac-POA treatment.In contrast, the p62/SQSTM1 signal was found significantly higher in MSTO-211H cell line, especially after 48 h of CT with both platinum-based compounds.Met-5A displayed an opposite trend, reaching statistical significance after 48 h of both CDDP and Pt(IV)Ac-POA exposures (Figure 9).
Pharmaceutics 2024, 16, x FOR PEER REVIEW 1 Autophagy and necroptosis were also evaluated by IF.
In the control conditions, LC3B (Microtubule-associated proteins light chain 3B p62/SQSTM1 (Sequestosome 1) are localized in the nucleus and cytoplasm within somes (Figure 9).However, the LC3B fluorescent signal was found to be dysregu only in Met-5A cells, after CDDP exposure and the longest Pt(IV)Ac-POA treatme contrast, the p62/SQSTM1 signal was found significantly higher in MSTO-211H ce especially after 48 h of CT with both platinum-based compounds.Met-5A display opposite trend, reaching statistical significance after 48 h of both CDDP and Pt(I POA exposures (Figure 9).RIP1 (Receptor-Interacting Protein 1) immunolabelling showed that in control conditions, contrary to what was observed in the samples subjected to CT, the fluorescent signal was diffused homogeneously along the entire cytoplasm and was not revealed in or around the nucleus (Figure 10).
RIP1 (Receptor-Interacting Protein 1) immunolabelling showed that in control co tions, contrary to what was observed in the samples subjected to CT, the fluorescent si was diffused homogeneously along the entire cytoplasm and was not revealed i around the nucleus (Figure 10).
Even in CDDP and Pt(IV)Ac-POA treated MSTO-211H samples, RIP1 was found nificantly upregulated (p < 0.0001).MLKL (Mixed Lineage Kinase domain-Like prot seemed to localize mainly in perinuclear spots in all the samples; its expression was nificantly upregulated only after 48 h of Pt(IV)Ac-POA treatment (p < 0.0001), while in others conditions the signal levels remained similar to the controls.Conversely, a gen downregulation of MLKL has been detected in Met-5a cells, regardless of the exp mental condition (Figure 10).Even in CDDP and Pt(IV)Ac-POA treated MSTO-211H samples, RIP1 was found significantly upregulated (p < 0.0001).MLKL (Mixed Lineage Kinase domain-Like protein) seemed to localize mainly in perinuclear spots in all the samples; its expression was significantly upregulated only after 48 h of Pt(IV)Ac-POA treatment (p < 0.0001), while in the others conditions the signal levels remained similar to the controls.Conversely, a general downregulation of MLKL has been detected in Met-5a cells, regardless of the experimental condition (Figure 10).

Discussion
Despite the recent advances in biomedical research, the only approved mesothelioma treatments include cisplatin-pemetrexed doublet and immunotherapy combination with Nivolumab and Ipilimumab [40].However, platinum-based drugs are associated with heavy side effects and the onset of drug resistance mechanisms.Furthermore, it is already noted that the induction of oxidative stress and hypoxia due to cisplatin treatment, as well as tobacco smoke exposure, could result in cancer development, progression, and the onset of drug resistance [41,42].Novel strategies to overcome pharmacological resistance of cancer cells is represented by the usage of HDACi, as explained before [20][21][22].Accordingly with our previous studies, the most marked effects have been observed after Pt(IV)Ac-POA treatments [14,29,30,33].Namely, after treatment with this compound, we observed high cell mortality and lower proliferation.Further experiments revealed that several cell death pathways were altered: apoptosis, ferroptosis, and autophagy.As expected, CDDP exerted much lower cytotoxic effects in terms of cell mortality.The higher cytotoxicity of Pt(IV)Ac-POA compared to cisplatin was confirmed, in both flow-cytometry and ultrastructural microscopy analysis.More precisely, the association of the HDAC inhibitory molecule on the cisplatin sketch resulted in a more decondensed chromatin that enhanced the antitumor effects of the alkylating core.With the ultrastructural analysis, we confirmed as well that the newly synthetized prodrug activated multiple mechanisms of cell death, thus confirming our previous studies on glioblastoma cells [29,30,33].Importantly, while cisplatin triggered necrotic cell death, after Pt(IV)Ac-POA exposure, more apoptotic, necroptotic, and ferroptotic cells were detected.This means that Pt(IV)Ac-POA preferentially induces a regulated cell death and, therefore, it is characterized by a lower systemic toxicity for the human organism.This result is of particular importance since the currently available therapies, based on platinum compounds, provoke devastating side effects on the patients due to their high toxicity; thus, Pt(IV)Ac-POA might represent a step toward a more tolerable mesothelioma therapy.
To better characterize the properties of the tested pharmacological treatments, here we investigated their effect on ferroptosis, given its importance in mesothelioma.In ferroptosis, cell death is executed by ROS-mediated oxidation of polyunsaturated fatty acids (PUFAs).Lipid peroxidation is prevented by glutathione peroxidase 4 (GPX4), which uses reduce glutathione (GSH), synthetized from cystine, as reducing agent [43].BAP1 suppresses SLC7A11, a cystine transporter, while NF2 prevents LATS2 accumulation into the nucleus thereby inhibiting the transcription ASLC4, a ferroptosis promoter [43,44] In the present work, we demonstrated that, after treatments with the new prodrug, significantly lower levels of GPX4 and FTH1 were detected in mesothelioma cells, indicating the activation of ferroptosis, supporting the ultrastructural analysis.
Next, we investigated whether the tested compounds were able to induce other forms of cell death, such as apoptosis and necroptosis by immunofluorescence on both cell lines.It is known that enhanced ROS production could trigger apoptosis by disrupting the mitochondrial membrane, phenomena observed during our ultrastructural analysis; immunocytochemical staining indeed confirmed the activation of caspase-3 in CDDP-and Pt(IV)Ac-POA-treated samples as well as a significant downregulation of COX2, especially in mesothelioma cells.Importantly, the downregulation of COX2 pointed to a general ROS accumulation, especially after both 12 h and 48 h of CT with Pt(IV)Ac-POA, therefore explaining the higher cytotoxicity of the new prodrug compared to CDDP.Of note, the showing the concentration of Pt(IV)Ac-POA able to induce cell death, causing a significant decrease (about 50%-IC50) in different cancer cell lines.* literature data.

Figure 2 .
Figure 2. CDDP and Pt(IV)Ac-POA affect cell cycle and histone acetylation.(A) Cytofluorimetric analysis.Graphs showing DNA content after PI staining human MSTO-211H cells in control condition and after 12 h and 48 h of CT with CDDP and Pt(IV)Ac-POA treatments (10µM).(B) Stacked histograms displaying the percentage of events recorded across the different cell cycle phases after treatments.(C) Control samples vs. treated cells after 12 h and 48 h of CT to CDDP and Pt(IV)Ac-POA (10 µM).Double immunofluorescence reaction, with the relative quantification, for Ki-67 (red) and acetyl-H3 (green), nuclei were counterstained with Hoechst 33258 (blue) Bar = 40 µm; magnification: 60×.(D,E) Histograms illustrating the results of imaging and statistical analysis for (D) Ki-67 positive cells and (E) Acetyl-H3 fluorescence quantification.One-way ANOVA test: p < 0.0001.* statistical significance respect to control condition is marked by an asterisk; p-values: (****) p < 0.0001.

Figure 2 .
Figure 2. CDDP and Pt(IV)Ac-POA affect cell cycle and histone acetylation.(A) Cytofluorimetric analysis.Graphs showing DNA content after PI staining human MSTO-211H cells in control condition and after 12 h and 48 h of CT with CDDP and Pt(IV)Ac-POA treatments (10µM).(B) Stacked histograms displaying the percentage of events recorded across the different cell cycle phases after treatments.(C) Control samples vs. treated cells after 12 h and 48 h of CT to CDDP and Pt(IV)Ac-POA (10 µM).Double immunofluorescence reaction, with the relative quantification, for Ki-67 (red) and acetyl-H3 (green), nuclei were counterstained with Hoechst 33258 (blue) Bar = 40 µm; magnification: 60×.(D,E) Histograms illustrating the results of imaging and statistical analysis for (D) Ki-67 positive cells and (E) Acetyl-H3 fluorescence quantification.One-way ANOVA test: p < 0.0001.* statistical significance respect to control condition is marked by an asterisk; p-values: (****) p < 0.0001.

Table 1 .
Primary and secondary antibodies used for immunofluorescence reactions.