Platinum iodido drugs show potential anti-tumor activity, affecting cancer cell metabolism and inducing ROS and senescence in gastrointestinal cancer cells

Abstract

Cisplatin-based chemotherapy has associated clinical disadvantages, such as high toxicity and resistance. Thus, the development of new antitumor metallodrugs able to overcome different clinical barriers is a public healthcare priority. Here, we studied the mechanism of action of the isomers trans and cis-[PtI₂(isopropylamine)₂] (I5 and I6, respectively) against gastrointestinal cancer cells. We demonstrate that I5 and I6 modulate mitochondrial metabolism, decreasing OXPHOS activity and negatively affecting ATP-linked oxygen consumption rate. Consequently, I5 and I6 generated Reactive Oxygen Species (ROS), provoking oxidative damage and eventually the induction of senescence. Thus, herein we propose a loop with three interconnected processes modulated by these iodido agents: (i) mitochondrial dysfunction and metabolic disruptions; (ii) ROS generation and oxidative damage; and (iii) cellular senescence. Functionally, I5 reduces cancer cell clonogenicity and tumor growth in a pancreatic xenograft model without systemic toxicity, highlighting a potential anticancer complex that warrants additional pre-clinical studies.

Fig. 1. Iodido agents induce oxidative DNA damage and produce mitochondrial dysfunction in GI cancer cells.

a Oxidative DNA damage (FPG sensitive lesions) at telomeres (left up), mitochondria regions encoding for mitochondrial MT-COX1 (right up) and MT-CYB (right down) genes, as well as the nuclear 36B4 single copy gene (left down) in cells exposed, or not, to IC₅₀ concentrations (see Methods for details) of CDDP or compounds I5 or I6 during 24 h. The differences in PCR kinetics (ΔCt) between FPG-digested vs undigested DNA (Buffer) is represented for each sample. Bars represent the mean fold change ± SEM 10–12 replicates from three independent experiments normalized to the control. Statistical significance was calculated using two-tailed unpaired t-test (*p < 0.05, **p < 0.01, *** p < 0.001, ns not significant). b Mean fold change ± SD of the ratio of ΔΨm probe (CMX-ROS)/Mitochondrial mass probe (MitoGreen) as a measurement to evaluate mitochondrial functionality in AGS, MKN45 and PANC1 treated with CDDP, I5 or I6 (IC₅₀ doses for 24 h). *p < 0.05, **p < 0.01, as determined by unpaired two-sided Student’s t-test, compared to untreated (C: Control) set as 1.0.

Fig. 2. I5 and I6 affect gastric and pancreatic tumor cells oxygen consumption and mitochondrial functional properties.

a, b (upper panels) Representative plots showing mean ± SD of the oxygen consumption rate (OCR) calculated for untreated (Control) and I5- or I6-treated AGS, MKN45 and PANC1 cells (according to IC₅₀, 24 h), normalized to total protein using a BCA kit (measured as BCA absorbance). Cells were treated with distinct inhibitors of mitochondrial function: O (oligomycin), F (FCCP), A (antimycin A), and R (rotenone). Continuous OCR values (pmoles/min/µg protein) are shown. a, b (bottom panels) Mean ± SD of measured and calculated mitochondrial function parameters (n = 3 biological replicates with 3 readings). *p < 0.05, **p < 0.01, ***p < 0.001, as determined by unpaired two-sided Student’s t-test.

Fig. 3. Iodido agents induce cellular senescence in GI cancer cells.

a Mean fold change ± SD in CellEvent Senescense Green Probe detection, determined by flow cytometry, in AGS, MKN45 and PANC1 cells treated with CDDP, I5 or I6 IC₅₀ doses for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by unpaired two-sided Student’s t-test, compared to untreated (C) set as 1.0. b AGS, MKN45 and PANC1 cells were treated with CDDP, I5 or I6 (IC₅₀ concentration, see Methods) for 3 h. γ-H2AX foci (green fluorescence) were detected by immunofluorescence using DAPI to stain nuclear DNA (blue fluorescence). Representative images of each condition were taken. Graphs represent the percentage of nuclei with <15, between 16 and 30, between 31 and 45, between 46 and 60 and >60 γ-H2AX foci per nuclei for each condition. Data represent the mean values obtained in three experiments performed in duplicate. c RNA was isolated from AGS, MKN45 and PANC1 cell lines stimulated with a 24 h treatment of the complexes. CDKN1A, IL-6 and MMP1 were quantified by RT-qPCR. Target gene expression was normalized to GAPDH. All the experiments were performed three times with IC₅₀ concentrations of each compound used in all the assays. The statistical significance was evaluated with Student’s 2-tailed t-test (*p < 0.05, **p < 0.01, ***p < 0.001) compared to untreated (C) set as 1.0.

Fig. 4. A stress signaling pathway is activated after treatment with iodido prototypes.

a Top: Representative western blots of phosphorylated forms of p53, JNK, p38 and ERK, in AGS and PANC1 cells after the treatment with IC₅₀ concentration of CDDP, I5 or I6 at different times (1, 3, 6 and 24 h). α-Tubulin was used as an endogenous loading control. Bottom: Graphs show the mean ± SD densitometric analyses of each protein normalized with α-tubulin from three independent experiments by using ImageJ (Area under the peak method), Control cells (C, white bars), CDDP (gray), I5 (light blue) and I6 (dark blue). b RNA was isolated from AGS, MKN45 and PANC1 cell lines stimulated with a 24 h treatment of the complexes. MAP2K3 expression levels were quantified by RT-qPCR and normalized with GAPDH. Shown are the mean fold change ± SD from triplicate samples (n = 3). The statistical significance was evaluated with Student’s 2-tailed t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) compared to the untreated cells (C: Control) set as 1.0.

Fig. 5. Iodido compounds modulate redox cell response and generate ROS in GI cancer cells.

a RNA was isolated from AGS, MKN45 and PANC1 cell lines stimulated with a 24 h treatment of CDDP or the iodido complexes (IC₅₀ concentration, see Methods). SOD1, SOD2 and CAT were quantified by RT-qPCR. Target gene expression levels were normalized with GAPDH. Shown are the mean fold change ± SD from triplicate samples (n = 3). b Detection of ROS in MKN45 cells after the treatment with H₂O₂ (200 µM) and the IC₅₀ concentration of CDDP, I5 or I6 by confocal microscopy using DHE and MitoSox as O₂^•− fluorescence indicator, in cytosol and mitochondria, respectively. Cells were treated with the compounds for 1 h followed by 30 min of incubation with the probes. Left: Representative images of each condition were taken. Scale bar represents 20 μm. Right: Mean fluorescence intensity ± SD (per cell) was quantified and depicted in the graph. The experiment was performed three independent times in duplicate. a.u. = arbitrary units. c Ratio of GSH/GSSG determined with a commercial colorimetric kit (see Methods). AGS and PANC1 cells were treated with the IC₅₀ concentration of the compounds and were collected after 3 h and 24 h to perform the assay. n = 3. Shown is the ratio of GSH/GSSG ± SD. In all the experiments, statistical significance was evaluated with Student’s 2-tailed t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) compared to the untreated cells (C: Control) set as 1.0.

Fig. 6. I5 reduces in vivo PANC1 tumor growth.

a Summary of the in vivo experimental design for the pre-treated PANC1 cell xenograft in vivo studies. Schematic created with BioRender.com. b Growth curves indicating tumor take (left, defined as no. tumors formed/no. of injections at the indicated time point) or the mean tumor volume (mm³) (right) ± SD over 28 days following injection of 1 × 10⁵ Control diluent-treated or I5-treated PANC1 cells. (no. of injections = 8–9). c Quantification of the mean tumor volume and mean weights (g) ± SD for control (c) and I5-pre-treated tumors (no. of injections = 8–9). Statistical significance was evaluated with Student’s 2-tailed t-test (**p < 0.01, ***p < 0.001). d Indirect calorimetry analyses of mice treated with I5. Left: Respiratory exchange ratio (RER) was determined as V_CO2/V_O2 and right: Energy expenditure (EE) was calculated as (3.185 + 1.232 x RER) x V_O2. Shown are the mean RER and mean EE (Kcal/h/Kg) values ± SD for mice implanted treated intravenously with I5 (1.4 mg/Kg) or physiological saline (i.e., Sham) as a function of time (24 h). e Summary of the in vivo experimental design for the treatment of mice harboring PANC1 xenografted tumors. Schematic created with BioRender.com. f Average fold change in tumor volume ± SEM in mice bearing PANC1 xenografts and treated with diluent control (Control), I5 (1.4 mg/kg i.p. or r.o.; 3-times per week) and compared to d0 (n = 5-7 tumors/group). g Mean fold change in tumor volume ± SEM (left) or tumor weight ± SEM (right) determined at treatment cessation. *p < 0.05, as determined by one-way ANOVA with Dunnett post-test, compared to Control. ns not significant; g gram; d0 day 0.