Evaluation of the Antitumor Effects of Platinum-Based [Pt(η1-C2H4-OR)(DMSO)(phen)]+ (R = Me, Et) Cationic Organometallic Complexes on Chemoresistant Pancreatic Cancer Cell Lines

Pancreatic cancer is one of the most lethal malignancies with an increasing incidence and a high mortality rate, due to its rapid progression, invasiveness, and resistance to anticancer therapies. In this work, we evaluated the antiproliferative and antimigratory activities of the two organometallic compounds, [Pt(η1-C2H4-OMe)(DMSO)(phen)]Cl (1) and [Pt(η1-C2H4-OEt)(DMSO)(phen)]Cl (2), on three human pancreatic ductal adenocarcinoma cell lines with different sensitivity to cisplatin (Mia PaCa-2, PANC-1, and YAPC). The two cationic analogues showed superimposable antiproliferative effects on the tested cells, without significant differences depending on alkyl chain length (Me or Et). On the other hand, they demonstrated to be more effective than cisplatin, especially on YAPC cancer cells. For the interesting cytotoxic activity observed on YAPC, further biological assays were performed, on this cancer cell line, to evaluate the apoptotic and antimetastatic properties of the considered platinum compounds (1 and 2). The cytotoxicity of 1 and 2 compounds appeared to be related to their intracellular accumulation, which was much faster than that of cisplatin. Both 1 and 2 compounds significantly induced apoptosis and cell cycle arrest, with a high accumulation of sub-G1 phase cells, compared to cisplatin. Moreover, phenanthroline-containing complexes caused a rapid loss of mitochondria membrane potential, ΔΨM, if compared to cisplatin, probably due to their cationic and lipophilic properties. On 3D tumor spheroids, 1 and 2 significantly reduced migrated area more than cisplatin, confirming an antimetastatic ability.


Introduction
Despite its relatively low incidence, pancreatic cancer is one of the most lethal malignancies with a high mortality rate due to its invasiveness, rapid progression, and resistance to treatments [1].Pancreatic cancer is currently the seventh leading cause of cancer-related death in the world after lung, colon, liver, stomach, breast, and esophagus cancers [2], both in males and females, but it is expected to become the third in the next years [3,4].Te prognosis in patients with pancreatic tumors is the poorest of any common solid malignancy, with a 5-year overall survival of about 10% [5].Most patients are diagnosed with advanced stage, and half of them are characterized by metastases [6].Te nonspecifc symptoms associated with pancreatic cancer make the diagnosis difcult, and the important blood vessels in proximity to the tumor can be easily invaded [7,8].Since the pancreas is a multifunctional organ, it consists of diferent cell types, from which several kinds of pancreatic tumors are derived.More generally, pancreatic tumors can be either exocrine (derived by duct cells and acinar cells) or endocrine (e.g., β cells) [9].Pancreatic ductal adenocarcinoma (PDAC) and its variants represent 90% of all pancreatic carcinomas; the remaining part includes nonductal tumors, such as acinar cells and neuroendocrine tumors [7].Te pathophysiology of pancreatic adenocarcinoma is characterized by a multistep genetic alteration, which involves oncogenes that are responsible for its initiation and progression, including KRAS, CDKN2A, TP53, and SMAD4 [10].
Nowadays, gemcitabine is the frst-line therapy for pancreatic cancer, approved by the U.S. Food and Drug Administration (FDA) in 1996 [11].On the other side, cisplatin is one of the most efective and widely used chemotherapy drugs, being able to induce apoptosis in pancreatic cancer cells [12][13][14].It was observed that a combination of gemcitabine and cisplatin can enhance DNA damage and improve survival in patients with pancreatic cancer [6,15].However, severe side efects and resistance phenomena often occur after cisplatin treatment, still leaving this interesting research feld open and aiming to obtain new platinum-based compounds with an improved pharmacokinetic and pharmacodynamic profle and reduced adverse efects [16][17][18].
Recently, we obtained the [Pt(η 1 -C 2 H 4 -OMe)Cl(phen)] complex [19], through the formation of a pentacoordinate intermediate [20], starting from phenanthroline and Zeise's salt in basic methanol.From this precursor, we synthesized and characterized the new platinum complex, [Pt(η 1 -C 2 H 4 -OMe)(DMSO)(phen)] + (1), demonstrating high antiproliferative efects on various human cancer cell lines [21].Particularly, on neuroblastoma cells, we observed a fast induction of mitochondrial apoptotic process and antimigratory capacity with respect to cisplatin [22].Furthermore, 1 H-NMR-based metabolomics analyses allowed us to highlight an early alteration of glutathione (Lc-glutamyl-L-cysteinyl-glycine; GSH) metabolism pathway compared to cisplatin [23], making this complex able to bypass cancer drug resistance related to GSH antioxidant system [24].We also observed that the cytotoxic activity of this phenanthroline-containing compound can be optimized by modifying alkyl chain length since antiproliferative properties of platinum complexes can be infuenced by molecular lipophilicity.In particular, we synthesized the new cationic [Pt(η 1 -C 2 H 4 -OR)(DMSO)(phen)] + analogues {R = Me (1), Et (2), Pr (3), Bu (4)} and observed that complex 2 with the Et moiety resulted to be generally more cytotoxic in the tested series of complexes.Tis allowed us to hypothesize that an optimal activity can be obtained for specifc chain lengths [25].

Synthesis of Complexes.
Commercially available reagents and solvents were used as received, without further purifcation.Cisplatin was supplied by the Sigma-Aldrich Chemical Company.Te [PtCl(η 1 -C 2 H 4 OR)(phen)] (R � Me, Et) complexes were synthesized according to a previously reported procedure and gave satisfactory analytical data [25].
All NMR measurements were performed on a Bruker Avance DPX 400 NMR spectrometer or a Bruker AVANCE III 600 Ascend NMR spectrometer (Bruker, Ettlingen, Germany), equipped with a TCI cryoprobe incorporating a z-axis gradient coil and automatic tuning/matching, at 300 K. 1 H NMR monodimensional spectra and [ 1 H, 195 Pt]-HETCOR bidimensional experiments were recorded by using deuterated CDCl 3 or D 2 O as solvents. 1H NMR spectra were referenced to TMS; the residual proton signal of the solvent [CDCl 3 ; δ( 1 H) � 7.24 ppm; D 2 O; δ( 1 H) � 4.7 ppm] was used as the internal standard. 195Pt NMR chemical shifts were referenced to H 2 [PtCl 6 ] [δ ( 195 Pt) � 0 ppm] in D 2 O, as the external reference.

Cell
Cultures.YAPC (DSMZ, Braunschweig, Germany) cells were cultured in RPMI 1640 medium (EuroClone, Pero, MI) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), glutamine 2 mM, penicillin (100 U/ mL), and streptomycin (100 mg/mL).MIA PaCa-2 and PANC-1 cells (ATCC, Rockville, MD) were cultured in Dulbecco's Modifed Eagle Medium (DMEM) (4,5 mg/L glucose) (EuroClone, Pero, MI) supplemented with 10% (vol/vol) heat-inactivated FBS, glutamine 2 mM, penicillin (100 U/mL), and streptomycin (100 mg/mL).Cells were grown in a humidifed incubator containing 5% CO 2 in air at 37 °C and used for biological assays when 70-80% confuence was reached. 2 Bioinorganic Chemistry and Applications a 96-well microtiter plate (10 4 cells per well).After overnight incubation, cells were treated with diferent concentrations of cisplatin, 1 and 2 for 24 and 48 h.At that time, 100 μL of ice-cold 10% (wt/vol) trichloroacetic acid was added to each well for 30 min at 4 °C.After that, the plates were washed fve times with double distilled water and air-dried overnight.70 μL of 0.4% (wt/vol) SRB solution was added to each well and incubated for 30 min, followed by four washes with 1% (vol/vol) acetic acid.Finally, SRB was dissolved in 200 μL of 10 mM unbufered Tris base solution, and color intensity was measured fuorometrically at 560 nm.Te percentage of cell survival was calculated as the absorbance ratio of treated to vehicle-treated control cells.Te data presented are means ± standard deviation from eight replicates of three independent experiments.

Analysis by ICP-AES (Inductively Coupled Plasma Atomic Emission Spectroscopy).
For the determination of platinum concentration, cells were incubated with 30 and 50 μM, respectively, of Pt(II) compounds for 1, 6, and 18 h.After the incubation time, the cellular pellet of each sample was recovered and treated with 1 mL of 67% super-pure nitric acid at room temperature for 24 h.Ten, samples were diluted to a fnal volume of 5 mL, to obtain a suitable concentration of the acid, fltered, and analyzed, as previously reported [21].Te platinum concentration in the analyzed samples was determined by a Termo iCAP 6000 spectrometer.Te spectrophotometer was calibrated with a calibration line consisting of four points, each corresponding to a concentration of the element: 1, 10, 100, and 1000 g/L.

Cell Cycle Analysis.
Te efect of the Pt(II) complexes on the cell cycle was evaluated using the nuclear staining dye propidium iodide (PI) (Termo Fisher Scientifc Inc.) that is frequently used.After treatment, cells were washed twice in PBS and harvested by trypsinization.Ten, cells were fxed in 70% cold ethanol and kept overnight at 20 °C prior to staining.After fxation, cells were centrifuged at 2000 rpm for 5 min, and ethanol was removed.Finally, cells were resuspended in 0.5 mL 0.05% Triton X-100/PBS staining solution containing 20 μg/mL PI and 2 μg/mL RNase.Data acquisition and analyses were performed by fow cytometer (BD Biosciences, San Jose, CA, USA), and cycle distribution (sub-G1, G0/G1, S, and G2/M phase fraction) was analyzed using BD Accuri C6 Software.

3D Spheroid-Based Migration Assay.
A suspension of 2.5 × 10 5 cells/mL was used to form tumor spheroids as previously reported [31].After 4 days of incubation, tumor spheroids were transferred in 96-well plates (one tumor spheroid per well, in a fnal volume of 100 μL).After waiting for the tumor spheroids to adhere to the plate bottom (about 1 h), cells were treated with sublethal concentrations of cisplatin, 1 and 2 complexes.Images were obtained at 24 h, using an inverted microscope.Te area covered by the cells that migrated from the spheroids was measured using PhotoShop C6 software (Adobe).Data were normalized to the original size of each spheroid recorded at t0 [formula: (migrated area at t � x/migrated area at t � 0) × 100].

[Pt
complexes was evaluated by SRB assay on three human PDAC (Mia PaCa-2, PANC-1, and YAPC) to better understand, besides diferences with respect to cisplatin, the possible role of alkyl chain length on inhibition of cell proliferation.IC 50 values (concentration required for 50% growth inhibition) were calculated after 24 and 48 h of incubation with Pt(II) complexes.Exposure of PDAC cells to Pt compounds at concentrations ranging from 0 to 100 μM resulted in a concentration-dependent increase in cell death, as shown in Figure 2.Both cisplatin and phen-containing complexes signifcantly inhibited pancreatic cell viability, but in a diferent way, depending on cell line type, as shown in Figures 2(a)-2(g).In general, 1 and 2 induced a higher cytotoxic efect, especially at shorter incubation times, than cisplatin in the examined cell lines, with an IC 50 between 9.15 ± 2.02 and 31.6 ± 1.19 μM after 24 h of treatment, as shown in Figure 2(g).Cisplatin resulted in higher cytotoxicity only for MIA PaCa-2 cells (IC 50 � 3.76 ± 1.11 μM) after 48 h of incubation, compared to 1 and 2 complexes, as shown in Figures 2(b) and 2(g).Interestingly, the two [Pt(η 1 -C 2 H 4 -OR)(DMSO)(phen)] + complexes demonstrated to be highly efective in PANC-1 cells, which appeared to be the most cisplatin-resistant cells among the tested cell lines (IC 50 > 100 after 24 h; IC 50 � 87.86 ± 2.29 μM after 48 h).
Considering the cytotoxicity assay results, we decided to further investigate the cell death process induction, caused by 1 and 2 complexes with respect to cisplatin, on YAPC cells, due to the observed markedly diferent responses, as shown in Figures 2(e)-2(g).

Intracellular Accumulation of Pt(II)
Complexes.Total intracellular Pt(II) content was evaluated in YAPC, by ICP-AES, after incubation with 50 μM (IC 50 value calculated after treatment with cisplatin) of cisplatin, 1, and 2 for 1, 6, 18, and 24 h.As already noted for [Pt(η 1 -C 2 H 4 -OMe)(DMSO)(phen)] + (1) [21], [Pt(η 1 -C 2 H 4 -OEt)(DM-SO)(phen)] + (2) seemed to enter cells via a passive or facilitated passive transport very quickly.Te intracellular accumulation of 1 and 2 complexes was much higher than cisplatin starting from 1 h of incubation (p < 0.05), as shown in Figure 3.During the frst 1-6 h of treatment, complex 2 accumulation in treated cells was also slightly higher, if compared to complex 1, but both complexes showed comparable accumulation of platinum values after 18 h, as shown in Figure 3(b).Terefore, these two Pt(II) complexes exhibited very similar uptake profles and cytotoxicity levels in YAPC cells, suggesting that the cytotoxicity of complexes 1 and 2 on YAPC cells after 24 h of incubation may be related to a high intracellular uptake with respect to cisplatin (about 9-fold), as shown in Figures 2 and 3.

[Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) Induces Apoptosis and Causes Cell Cycle
Arrest in YAPC Cells.Cell death after exposure to cisplatin, 1, and 2 complexes was also investigated by fow cytometry, measuring the percentage of cells that exhibited Annexin V-FITC and/or PI fuorescence.Since complexes 1 and 2 gave comparable results, only fow cytometry results obtained after treatment with 2 are shown in Figures 4 and 5.
YAPC cells were exposed to phen-containing complexes to assess the apoptosis induction compared to the control and cisplatin-treated, as shown in Figure 4. Incubation with cisplatin and [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) resulted in signifcant apoptosis enhancement compared to the untreated cells (p < 0.0001).In addition, complex 2 induced cell death more than cisplatin after 18 h of treatment (p < 0.05).In the control group, only 1.4% apoptosis was observed while 35.4 and 46.7% apoptosis and necrosis were found in the groups treated with 50 μM cisplatin and 2, respectively.Te infuence of Pt complexes on YAPC cell proliferation was evaluated by fow cytometry in PI (propidium iodide)-stained cells after treatment for 18 h.Here, the classical antineoplastic drug cisplatin was used as positive controls.
G1 represents the longest phase of the cell cycle; thus, the largest fraction of cells is usually in the G1 phase.Te sub-G1 method for death cell detection is based on the principle that after DNA endonucleolytic cleavage, low-molecular-weight DNA fragments are released from cells during prolonged fxation.Tat will yield a population of cells that bind a quantitative DNA stain (PI) to a lesser extent than what is characteristic of G1 cells [32].In YAPC cells, [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) caused a signifcant and timedependent (data not shown) increase in the percentage of YAPC cells in the sub-G1 and decrease in the percentage of cells in G2/M, compared to cisplatin, as shown in Figures 4(a) and 4(b).Bioinorganic Chemistry and Applications disruption of mitochondrial transmembrane potential (ΔΨ M ) in YAPC cells using the cation dye JC-1.Mitochondrial membrane potential is an important parameter of mitochondrial function, acting as an indicator of cell health [33].In healthy cells (high ΔΨ M ), JC-1 forms J-aggregate complexes with intense red fuorescence.Instead, in cells with low ΔΨ M , JC-1 remains in the monomeric form, which exhibits green fuorescence.Depending on whether JC-1 exists as a monomer or J-aggregate, it emits at 520 nm (green) and 590 nm (red), respectively, after excitation at 488 nm [33,34].Te red/green fuorescence intensity ratio indicates the change in ΔΨ M and then the occurrence of apoptosis.

Determination of Mitochondrial Changes Using
To observe the shift in fuorescence emission of JC-1, cancer cells were treated or not with 50 μM of Pt complexes for 24 h and stained with the fuorescence probe.Figure 6(a) shows images obtained using fuorescence microscopy.Te quantifcation of cells with green and red fuorescence is displayed as a bar graph, as shown in Figure 6(c).Red fuorescence, the sign of preserved ΔΨ M , was observed in almost all untreated controls (about 85%), whereas YAPC cells incubated with Pt compounds displayed green fuorescent signals, index of mitochondrial membrane depolarization, as shown in Figures 6(a   to control (p < 0.0001), and about 3-fold with respect to cisplatin (p < 0.01), as shown in Figure 6(b).Superimposable efects were observed when cells were exposed to complex 1 (data not shown).
Mitochondrial membrane depolarization (ΔΨ M ) was also detected fuorometrically by a shift in fuorescence emission of the cationic probe JC-1.YAPC cells were stained with JC-1 and then incubated with cisplatin, 1 and 2 complexes, and the shift in fuorescence emission of JC-1 was followed for 12 to 72 min.Also, in this case, results obtained after treatment with the two phenanthrolinecontaining complexes were similar (p > 0.05).Te aggregate/monomeric JC-1 ratio is comparable in 1 and 2 complexes (only results regarding 2 are shown in Figure 6).Fluorometric analysis showed that treatment of YAPC cells with [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) caused an early transition of JC-1 fuorescence, with a shift toward green fuorescence that was detected as early as the frst minutes after exposure to Pt complex, as shown in Figure 6(c).ΔΨ M signifcantly decreased 12 min after the addition of complex 2 and reached a minimum after 24 min, as shown in Figure 6(c).Diferently from phenanthroline-containing complexes, cisplatin treatment decreased ΔΨ M similar to 2 only 1 h after incubation, as shown in Figure 6(c).

Discussion and Conclusions
Pancreatic cancer is one of the most lethal malignancies, with a rising incidence and a high mortality rate.According to GLOBOCAN estimates of cancer incidence and mortality worldwide (produced by the International Agency for Research on Cancer) in 2020, pancreatic cancer accounts for 496,000 cases with 466,000 deaths [2].Te high mortality associated with pancreatic cancer is due to its rapid progression, invasiveness, and resistance to current treatments [1].Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic cancer type, with a 5-year survival rate of less than 5% [5].Te high mortality rate is often related to late diagnosis, and the aggressive nature of malignant cells disseminate to nearby tissues at an early stage of the disease, making treatment difcult [35].Cisplatin is one of the best chemotherapeutic drugs and the frst metal-based drug, which has demonstrated a high efcacy in the treatment of various cancers (testicular, ovarian, head and neck, bladder, lung, cervical cancer, melanoma, lymphomas, and others) [36].Te use of cisplatin for PDAC treatment in combination with chemotherapy was evaluated in several clinical trials, but the response was limited by intrinsic and acquired drug resistance [37].Tis latter can be developed by cancer cells in diferent ways: (i) reduced accumulation due to increased extrusion of cisplatin by transporters; (ii) sequestration and inactivation by GSH and other cytoplasmic scavengers with nucleophilic properties; (iii) reduced DNA damage recognition and apoptotic response; (iv) other unspecifc adaptive responses resulting in strong antiapoptosis ability and resistance of cancer cells to cisplatin (e.g., dysregulation of TP54, MAPK, PI3K/AKT, NF-κB, and Stat3 pathways) [38].Organometallic complexes containing 1,10-phenanthroline (phen) showed high antitumoral activity  against cisplatin-resistant cells, thanks to the capability of phenanthroline to interact with DNA and intercalate between nucleic acid base pairs, inhibiting DNA synthesis and DNA repair systems [39][40][41][42].Moreover, intercalating agents can induce cytotoxicity by inhibiting RNA synthesis and topoisomerase II (TOPO-II) [43,44].We previously synthesized the [Pt(η 1 -C 2 H 4 -OMe) Cl(phen)] precursor leading by direct reaction with DMSO, to the formation of [Pt(η 1 -C 2 H 4 -OMe)(DM-SO)(phen)] + (1) [21].Tis complex (1) demonstrated a broad spectrum of antiproliferative activity toward several cancer cell lines and antimetastatic properties often superior to those exhibited by cisplatin [21][22][23].Furthermore, (1) was also able to reduce glutathione (GSH) levels possibly as a specifc efect of the drug-induced stress [23].Tis result was interesting, since GSH is the most abundant antioxidant in eukaryotic cells, showing

JC-1 ratio (% of t0)
Figure 6: Analysis of mitochondrial membrane potential using the cationic dye JC-1 in YAPC cells.(a) Double staining of nuclei (DAPI) and mitochondria (JC-1) was evaluated in YAPC cells by fuorescence microscopy (scale bar: 60 μm).(a, b) Cells were incubated or not with 50 μM cisplatin and complex 2 for 24 hours and stained with DAPI and JC-1 dyes.(b) Quantifcation of cells observed with green and red fuorescence (expressed as a percentage) was carried out using Image J. Data are represented as the mean ± SD from three independent experiments.Asterisks ( * * p < 0.01; * * * * p < 0.0001) indicate values of green fuorescence that are signifcantly higher than untreated cells.(c) Te sums of J-aggregate and J-monomer fuorescence from the measurements were obtained from spectrophotometry data and were used for calculating the respective JC-1 ratios.Te results represent the mean ± SD from three independent measurements.Values with shared letters are not signifcantly diferent according to Tukey's multiple comparisons test.
particularly high levels in mitochondria, where it contributes to redox balance through ROS detoxifcation and phospholipidic membrane protection [45].Moreover, GSH mediates many other physiological reactions, including cellular signaling, cell cycle regulation, proliferation, and apoptosis [23].
On this basis, we further optimized the general structure of ( 1 Digital images of the spheroids were then captured at 0, 6, 18, and 24 h (scale bar � 300 μm).(A-J) Tumor spheroids treated or not treated with 1 μM cisplatin and 2 at 6, 18, and 24 h.(J) Migrating areas were measured and reported on the graph as a percentage of t0.(K) Tumor spheroids treated or not treated with 0.1 to 1 μM cisplatin and 2 at 24 h.Migrating areas were measured and reported on the graph as a percentage of the control.(J,K) All data were expressed as the mean ± standard deviation (SD) values of three independent experiments.Values with shared letters are not signifcantly diferent according to post-hoc Tukey's test.Bioinorganic Chemistry and Applications OR)(DMSO)(phen)] + {R � Me (1), Et (2), Pr (3), Bu (4)} complexes with diferent alkyl chain length [25].Indeed, we observed that the variation of the alkyl chain length could improve the antiproliferative properties of these complexes with an optimal length depending on the cancer cell type.In fact, the cytotoxicity of complexes 2, 3, and 4 resulted higher in hepatocarcinoma cells where complex 1 was less efective [21,25].On the other hand, antiproliferative properties of platinum complexes seem related to molecular lipophilicity [46,47].Tus, the gradual modifcation of the Pt complex structure may lead to the optimization of antitumor activity and drug selectivity.Among the four above-described phenanthroline-containing complexes, 2 was generally the most cytotoxic [25].For these reasons, we decided to study in this work the antiproliferative activity of [Pt(η 1 -C 2 H 4 -OMe)(DMSO)(phen)] + (1) and [Pt(η 1 -C 2 H 4 -OEt)(DM-SO)(phen)] + (2) on three PDAC cell lines (Mia PaCa-2, PANC-1, and YAPC) and compare their proapoptotic efects with those of cisplatin.Cytotoxicity assays allowed us to verify the correlation between the compounds' hydrophobic properties and their antiproliferative activity on pancreatic cell lines with diferent phenotypic and genotypic characteristics [29,48].Te cytotoxicity of phen-containing complexes was determined by SRB assay in the three PDAC lines, using cisplatin as a control.Generally, the two cationic complexes had superimposable efects on cell viability, except for MIA PaCa-2 cells where complex 2 was more efective.Meanwhile, a rapid and higher antiproliferative efect was observed for both complexes 1 and 2, with respect to cisplatin, as shown in Figure 2. Complexes 1 and 2, after 24 h of incubation with YAPC cells, showed IC 50 values about 6 times lower than cisplatin, indicating their high cytotoxic efect also on cisplatin-resistant tumor lines.Moreover, the cationic complexes 1 and 2 exhibited very similar uptake profles and cytotoxicity levels in the examined PDAC lines, suggesting that the cytotoxicity of complexes 1 and 2 on YAPC cells may be related to a high intracellular uptake with respect to cisplatin, as shown in Figures 2 and 3. Since the induction of apoptosis on YAPC cell line after exposure to cisplatin, [Pt(η 1 -C 2 H 4 -OMe)(DMSO)(phen)] + (1) and [Pt(η 1 -C 2 H 4 -OEt)(DM-SO)(phen)] + (2), gave similar results for the two phenanthroline-containing complexes (p > 0.05), we only focused on complex 2 (showing slightly higher activity) for some of our further investigations.Flow cytometry analyses demonstrated that complex 2 treatment signifcantly increased levels of apoptosis in YAPC cells, even more than cisplatin, as shown in Figure 4. Furthermore, [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) exposure caused a high accumulation of sub-G1 phase cells compared to cisplatin, which is indicative of cell death, as shown in Figure 5. Te accumulation in the sub-G1 phase after treatment with cisplatin could be related to the occurrence of DNA repair system defects, resulting in altered cell cycle regulation and increased cell death [49].We recently hypothesized a cytosolic target for [Pt(η 1 -C 2 H 4 -OMe)(DMSO)(phen)] + (1), which caused a faster and greater inhibition of cell proliferation and metabolic alteration [22,23].In this study, diferently from cisplatin, complex [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) demonstrated an early induction of apoptosis and cell cycle block at sub-G1 phase, thus confrming that these type of Pt(II) compounds have a cellular target diferent from that of cisplatin.Te lipophilic cation JC-1 was also used to assess whether complex 2 induced alterations of mitochondrial membrane potential (ΔΨ M ) in PDAC cells.Te aggregation of JC-1 in the mitochondria is driven by the transmembrane potential.Yellow-orange fuorescence of JC-1 dimers is present in cell areas with high mitochondrial membrane potential, while green fuorescence of JC-1 monomers is prevalent in cell areas with low mitochondrial membrane potential.As shown in Figure 6 in control cells with intact nuclei, JC-1 aggregates inside healthy mitochondria and fuoresces red while, in apoptotic cells with fragmented nuclei, the monomeric form of JC-1 fuoresces green.In the YAPC line, progressive loss of ΔΨ M as measured by the green/red emission pattern of the mitochondria-selective dye JC-1 was induced by cisplatin.On the other hand, in [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) treated cells, ΔΨ M signifcantly decreased during the frst minutes of incubation with Pt compound as observed by fuorescence data, as shown in Figure 6(c).Te electron transport chain generates an electrochemical gradient across the inner mitochondrial membrane, which is essential for ATP production by mitochondrial ATP synthase and also allows cargo delivery into mitochondria using cations [50,51].It was demonstrated that various molecules with anticancer properties, including metal-based compounds, are able to act directly on mitochondria, inducing loss of membrane potential and release of apoptotic proteins [52][53][54][55].Cisplatin may induce changes in ΔΨ M and activate damage pathways by activating several proteins and binding to mitochondrial DNA [51,53,[56][57][58].In the present case, given the rapid reduction of ΔΨ M , the cationic phenanthroline-containing complexes could cause alterations in mitochondrial morphology and function by interacting with mitochondrial membrane proteins.Moreover, it has been reported that the membrane potential of the mitochondria in cancer cells is higher than in normal cells, resulting in the accumulation of cationic lipophilic compounds down an electrochemical gradient [55].Terefore, the efect of alkyl chain length and related hydrophobic properties could allow these Pt(II) compounds to be readily taken up by mitochondria, a possible example of passive drug delivery.

Bioinorganic Chemistry and Applications
In conclusion, both cationic complexes [Pt(η 1 -C 2 H 4 -OR)(DMSO)(phen)] + {R � Me (1) and Et (2)} were found to contrast PDAC progression by inducing apoptosis and inhibiting metastatic processes.In the case of YAPC cells' response to complexes 1 and 2, signifcant diferences were observed compared to cisplatin, with both complexes exhibiting signifcantly higher intracellular accumulation within the frst 24 hours of treatment, with complex 2 displaying a slightly higher intracellular accumulation than 1.Despite the diferences in cell accumulation, similar anticancer efects were observed, not related to the alkyl chain length as previously observed in other tumor cell lines [25].Tese phenanthroline organometallic compounds entered YAPC cells and mitochondria very rapidly, causing a fast induction of cell death, likely due to the activation of mitochondrial apoptotic pathways [22].It is well-established that cationic platinum complexes can be actively transported by organic cation transporters (OCTs) of the SLC22 family, enhancing intracellular accumulation and antitumor activity, with phen-related ligands generally favoring this process, as observed for phenanthriplatin [61,62].Terefore, the results of this study, which demonstrate a rapid intracellular accumulation of both complexes 1 and 2, appear to further suggest an involvement of cell membrane OCTs in the cell membrane crossing of these organometallic species, thus improving their antiproliferative and antimetastatic efects.

Figure 5 :
Figure 5: Efects of cisplatin and [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) on the cell cycle.(a) Flow cytometry (BD Accuri C6 fow cytometer) was used to investigate the cell cycle distribution of propidium iodide-stained cells after treatment with YAPC with or without cisplatin and complex 2 for 18 hours.(b) Te pie charts indicate the percentages of cells in the G1, S, or G2/M phases of the cell cycle.Tese images are representative of three independent experiments.

Figure 7 :
Figure 7: Efects of cisplatin and [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) on cell migration.(A-K) Tumor spheroids were transferred into a 96-well fat-bottomed migration plate and treated with 0.1, 0.5, 1.0, and 1.5 μM [Pt(η 1 -C 2 H 4 -OEt)(DMSO)(phen)] + (2) or cisplatin for 24 h.Digital images of the spheroids were then captured at 0, 6, 18, and 24 h (scale bar � 300 μm).(A-J) Tumor spheroids treated or not treated with 1 μM cisplatin and 2 at 6, 18, and 24 h.(J) Migrating areas were measured and reported on the graph as a percentage of t0.(K) Tumor spheroids treated or not treated with 0.1 to 1 μM cisplatin and 2 at 24 h.Migrating areas were measured and reported on the graph as a percentage of the control.(J,K) All data were expressed as the mean ± standard deviation (SD) values of three independent experiments.Values with shared letters are not signifcantly diferent according to post-hoc Tukey's test.
S.D. for each experimental group.Te normality of data before the analyses was confrmed by the Kolmogorov-Smirnov tests.Statistical analysis was carried out using ANOVA associated with Tukey's multiple comparisons test.A p value <0.05 was considered to achieve statistical signifcance.
Analyses.Statistical analyses were performed with GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA).Experimental points represent Bioinorganic Chemistry and Applications means ±