[Pt(O,O'-acac)(γ-acac)(DMS)]: Alternative Strategies to Overcome Cisplatin-Induced Side Effects and Resistance in T98G Glioma Cells

Abstract

Cisplatin (CDDP) is one of the most effective chemotherapeutic agents, used for the treatment of diverse tumors, including neuroblastoma and glioblastoma. CDDP induces cell death through different apoptotic pathways. Despite its clinical benefits, CDDP causes several side effects and drug resistance.[Pt(O,O'-acac)(γ-acac)(DMS)], namely PtAcacDMS, a new platinum(II) complex containing two acetylacetonate (acac) and a dimethylsulphide (DMS) in the coordination sphere of metal, has been recently synthesized and showed 100 times higher cytotoxicity than CDDP. Additionally, PtAcacDMS was associated to a decreased neurotoxicity in developing rat central nervous system, also displaying great antitumor and antiangiogenic activity both in vivo and in vitro. Thus, based on the knowledge that several chemotherapeutics induce cancer cell death through an aberrant increase in [Ca²⁺]_i, in the present in vitro study we compared CDDP and PtAcacDMS effects on apoptosis and intracellular Ca²⁺ dynamics in human glioblastoma T98G cells, applying a battery of complementary techniques, i.e., flow cytometry, immunocytochemistry, electron microscopy, Western blotting, qRT-PCR, and epifluorescent Ca²⁺ imaging. The results confirmed that (i) platinum compounds may induce cell death through an aberrant increase in [Ca²⁺]_i and (ii) PtAcacDMS exerted stronger cytotoxic effect than CDDP, associated to a larger increase in resting [Ca²⁺]_i. These findings corroborate the use of PtAcacDMS as a promising approach to improve Pt-based chemotherapy against gliomas, either by inducing a chemosensitization or reducing chemoresistance in cell lineages resilient to CDDP treatment.

Keywords: Apoptosis; Ca2+ signaling; Cisplatin; Pt(O,O'-acac)(γ-acac)(DMS); T98G glioblastoma cells.

Fig. 1

Flow cytometry after Annexin V/PI staining. a Representative cytograms of control and treated cells. Panel B: Histograms showing the percentage distribution of different categories and the percentage of apoptotic cells (right panel). CTRL vs CDDP (mean diff. − 7.803) **ρ < 0.001, CTRL vs PtAcacDMS (mean diff. − 19.47) ***ρ < 0.0001, and CDDP vs PtAcacDMS (mean diff. − 11.66) ***ρ < 0.0001; (F 99.62, R² 0.9708)

Fig. 2

TEM ultrastructural analysis. a Ultrastructural morphology of a control cell. Arrowheads indicated mitochondria with a physiological, normal feature; asterisk identified a well-visible nucleolus. b 10 µM PtAcacDMS-treated cell showing necroptotic features: chromatin condensation (asterisk) and a partially preserved cytoplasmic membrane (thin arrows). c Autophagic cell after 10 µM PtAcacDMS treatment. Several autophagic vacuoles were detectable (arrowheads). d 40 µM CDDP-treated cell displayed autophagic vacuoles (arrowheads). e 40 µM CDDP-treated necrotic cell showing marked cytoplasmic components degradation as well as plasma membrane disassembling (thin arrows). f 40 µM CDDP-treated apoptotic cell displaying typical blebs formation (asterisks); elongated mitochondria were also observed in other neighboring cells (arrowhead). Bar: 4 µm

Fig. 3

Fluorescence microscopy: intracellular organelles analysis. Immunocytochemical detection of mitochondria and Golgi apparatus (red fluorescence) with α-tubulin and actin cytoskeleton (green fluorescence) respectively, in control cells and after 48 h exposure with CDDP or PtAcacDMS. DNA counterstaining with Hoechst 33,258 (blue fluorescence). Bar: 20 µm

Fig. 4

Fluorescence microscopy: intrinsic and extrinsic apoptotic pathway evaluation. a immunocytochemical detection of caspase 9, caspase 8, and caspase 3 (red fluorescence) and actinic cytoskeleton (green fluorescence) in control cells and after 48 h exposure to CDDP or PtAcacDMS. DNA counterstaining with Hoechst 33,258 (blue fluorescence). Bar: 40 µm. b histograms showing the percentage of fluorescence intensity for each caspase in control and treated cells. Caspase 9: CTRL vs CDDP (mean diff. − 29.89) **ρ < 0.001, CTRL vs PtAcacDMS (mean diff. − 32.28) ***ρ < 0.0001, CDDP vs PtAcacDMS (mean diff. − 11.66) ns, (F 32.01, R² 0.9143). Caspase-8: CTRL vs CDDP (mean diff. − 58.19) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. − 66.80) ***ρ < 0.0001, CDDP vs PtAcacDMS (mean diff. − 8.607) *ρ < 0.05; (F 347.6, R² 0.9914). Caspase 3: CTRL vs CDDP (mean diff. − 25.79) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. − 19.79) ***ρ < 0.0001, CDDP vs PtAcacDMS (mean diff. 5.999) *ρ < 0.05; (F 147.9, R² 0.9801). ns not significant

Fig. 5

Immunofluorescence detection of PARP-1. a Immunocytochemical detection of PARP-1 (red fluorescence) and actin cytoskeleton (green fluorescence) in control cells (a) and after 48 h exposure to CDDP (b) or PtAcacDMS (c). DNA counterstaining with Hoechst 33,258 (blue fluorescence). Arrowheads indicated apoptotic cells in which p89 fragment translocated to the cytoplasm. Bar: 40 µm. b Western blotting analysis of PARP-1 reveals the presence of the cleaved 89 kDa fragment

Fig. 6

Flow cytometry: analysis of mitochondrial membrane potential changes using JC-1. Graphs show red and green fluorescence of mitochondria stained in control and CDDP- or PtAcacDMS-treated cells. Compared to control, the green fluorescence increase reveals the presence of apoptotic cells after exposure to both platinum compounds. Histogram showing the percentage of green fluorescence positive cells. CTRL vs CDDP (mean diff. − 70.53) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. − 81.70) ***ρ < 0.0001, and CDDP vs PtAcacDMS (mean diff. − 11.17) ***ρ < 0.0001; (F 5382, R² 0.9994)

Fig. 7

PtAcacDMS and CDDP induce extracellular Ca²⁺ entry. In a, b representative tracings of the Ca²⁺ responses to CDDP and PtAcacDMS, respectively, show that the acute addition of PtAcacDMS caused a larger increase in [Ca²⁺]_i as compared to CDDP. In c mean ± SE of the percentage of T98G cells responding to PtAcacDMS and CDDP. In d mean ± SE of the amplitude of the Ca²⁺ signal induced by PtAcacDMS and CDDP in T98G cells. PtAcacDMS vs CDDP (mean diff. 0.03693) ***ρ < 0.0001; (F 3.553, R² 0.1324). e Tracings of the Ca²⁺ responses to PtAcacDMS and CDDP, respectively, in the absence (0 Ca²⁺) and in the presence of extracellular Ca²⁺. f Mean ± SE of the amplitude of Ca²⁺ release and Ca²⁺ entry induced by PtAcacDMS and CDDP in T98G cells PtAcacDMS vs CDDP (mean diff. 0.06093) ***ρ < 0.0001; (F 4.164,, R² 0.3646).

Fig. 8

Orai1 mediates CDDP- and PtAcacDMS-induced extracellular Ca²⁺ entry. a The pharmacological blockade of Orai1 channels with either Pyr6 (10 μM) or La³⁺ (10 μM) inhibited CDDP-induced Ca²⁺ entry in T98G cells. CDDP was administered at 40 μM. b mean ± SE of the percentage of inhibition of CDDP-induced Ca²⁺ entry by, respectively, Pyr6, and La³⁺. c. The pharmacological blockade of Orai1 channels with either Pyr6 (10 μM) or La³⁺ (10 μM) inhibited PtAcacDMS-induced Ca²⁺ entry in T98G cells. PtAcacDMS was administered at 10 μM. d mean ± SE of the percentage of inhibition of CDDP-induced Ca²⁺ entry by, respectively, Pyr6 and La³⁺

Fig. 9

PtAcacDMS causes a larger increase in resting [Ca²⁺]_i as compared to CDDP after 48 h continuous exposure. Calibration of Fura-2 fluorescence revealed that resting [Ca²⁺]_i was significantly higher upon 48 h exposure to 10 μM PtAcacDMS compared to 40 μM CDDP. CTRL vs CDDP (mean diff. − 370.0) *ρ < 0.05, CTRL vs PtAcacDMS (mean diff. − 780.8) ***ρ < 0.0001, PtAcacDMS vs CDDP (mean diff. 410.8) *ρ < 0.05. (F 12.79, R² 0.05518)

Fig. 10

Platinum compounds increase ER-dependent Ca²⁺ release and SOCE upon 48 h treatment. a Representative tracing showing the Ca²⁺ response to CPA (30 μM) after 48 h treatment with CDDP (40 μM) and PtAcacDMS (10 μM) under 0Ca²⁺ conditions and upon Ca²⁺ restitution to the bath, which is indicative of SOCE activation. The baselines of the Ca²⁺ tracings have been realigned in order to overlap to facilitate the comparison of the different amplitudes of ER Ca²⁺ release and SOCE under the three different conditions. b Mean ± SE of CPA-induced ER Ca²⁺ release and SOCE under the designated treatments. Ca²⁺ release: CTRL vs CDDP (mean diff. − 0.02739) *ρ < 0.05, CTRL vs PtAcacDMS (mean diff. − 0.07424) ***ρ < 0.0001, PtAcacDMS vs CDDP (mean diff. 0.04685) **ρ < 0.001 (F 20.08, R² 0.1266). SOCE: CTRL vs CDDP (mean diff. − 0.05557) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. − 0.06347) ***ρ < 0.0001, PtAcacDMS vs CDDP (mean diff. 0.007897) ns, (F 29.08, R² 0.1887). c Representative tracing showing the Ca²⁺ response to ADP (100 μM) after 48 h treatment with CDDP (40 μM) and PtAcacDMS (10 μM) under 0Ca²⁺ conditions. d Mean ± SE of ADP-induced ER Ca²⁺ release under the designated treatments. CTRL vs CDDP (mean diff. − 0.05434) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. − 0.05685) ***ρ < 0.0001, PtAcacDMS vs CDDP (mean diff. 0.002510) ns, (F 35.97, R² 0.1982). ns not significant

Fig. 11

Platinum compounds alter the expression of SERCA2B, InsP₃Rs, STIM and Orai transcripts after 48 h exposure. Transcript levels of SERCA2B (a), InsP₃R1-3 (b–e), Orai1-3 (f–h), Stim1-2 (I, j) in T98G cells under control conditions and upon 48 hexposure to 40 μM CDDP and 10 μM PtAcacDMS. Data are expressed as mean ± SE of qRT-PCR runs performed at least in triplicate. a SERCA2B transcript was significantly enhanced by 40 µM PtAcacDMS as compared to control and 10 µM CDDP. CTRL vs CDDP (mean diff. − 0.005946) ns, CTRL vs PtAcacDMS (mean diff. − 0.02163) *ρ < 0.0099, CDDP vs PtAcacDMS (mean diff. − 0.01568) *ρ < 0.0099 (F 6.722, R² 0.5084). b InsP₃R2 was the most abundant InsP₃R subtype expressed in T98G cells, followed by InsP₃R3 and InsP₃R1. InsP₃R1 vs InsP₃R2 (mean diff. − 0.003327) ***ρ < 0.0001, InsP₃R1 vs InsP₃R3 (mean diff. − 0.001047) *ρ < 0.05, InsP₃R2 vs InsP₃R3 (mean diff. 0.002280) ***ρ < 0.0001 (F 35.97, R² 0.1982). c InsP₃R1 transcript was significantly downregulated only in the presence of 10 µM CDDP. CTRL vs CDDP (mean diff. 0.0002341) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. − 0.00005955) ns, CDDP vs PtAcacDMS (mean diff. − 0.0002936) ***ρ < 0.0001 (F 63.87, R² 0.9076). d InsP₃R2 transcript was significantly downregulated by both 40 µM PtAcacDMS and 10 µM CDDP. CTRL vs CDDP (mean diff. 0.002764) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. 0.001623) ***ρ < 0.0001, CDDP vs PtAcacDMS (mean diff. − 0.001141) ***ρ < 0.0001 (F 55.60, R² 0.8953). e InsP₃R3 transcript was not affected by either 40 µM PtAcacDMS or 10 µM CDDP. CTRL vs CDDP (mean diff. 0.0002891) ns, CTRL vs PtAcacDMS (mean diff. − 0.0001312) ns, CDDP vs PtAcacDMS (mean diff. − 0.0004203) ns, (F 3.189, R² 0.3291); ρ = 0.0747. f Orai1 transcript was significantly upregulated by 40 µM PtAcacDMS. CTRL vs CDDP (mean diff. − 0.0005407) ns, CTRL vs PtAcacDMS (mean diff. − 0.001134) *ρ < 0.02585, CDDP vs PtAcacDMS (mean diff. − 0.0005936) ns, (F 5.003, R² 0.4764). g Orai2 transcript was not affected by either 40 µM PtAcacDMS or 10 µM CDDP. CTRL vs CDDP (mean diff. − 0.00003147) ns, CTRL vs PtAcacDMS (mean diff. − 0.000006621) ns, CDDP vs PtAcacDMS (mean diff. 0.00002485) ns, (F 0.6013, R² 0.1939); ρ = 0.5834. h Orai3 transcript was significantly downregulated in T98G cells pretreated with 40 µM PtAcacDMS as compared to control cells and cells pretreated with 10 µM CDDP. CTRL vs CDDP (mean diff. − 0.000007510) ns, CTRL vs PtAcacDMS (mean diff. 0.00007403) *ρ < 0.0113, CDDP vs PtAcacDMS (mean diff. 0.00008154) *ρ < 0.0113 (F 12.55, R² 0.83390). i STIM1 transcript was significantly downregulated in T98G cells pretreated with 10 µM CDDP as compared to control cells and cells pretreated with 40 µM PtAcacDMS. CTRL vs CDDP (mean diff. 0.0008951) **ρ < 0.001, CTRL vs PtAcacDMS (mean diff. − 0.0003403) ns, CDDP vs PtAcacDMS (mean diff. − 0.001235) ***ρ < 0.0001 (F 24.60, R² 0.7910). j STIM2 transcript was not affected by either 40 µM PtAcacDMS or 10 µM CDDP. CTRL vs CDDP (mean diff. − 0.00002792) ns, CTRL vs PtAcacDMS (mean diff. 0.00005410) ns, CDDP vs PtAcacDMS (mean diff. 0.00008202) ns, (F 1.743, R² 0.4108); ρ = 0.2665. ns not significant

Fig. 12

Platinum compounds cause PMCA-1 downregulation upon 48 h treatment. a Immunocytochemical detection of PMCA-1 (red fluorescence) and α-tubulin (green fluorescence). DNA counterstaining with Hoechst 33,258 (blue fluorescence) in control cells (a), after 48 h exposure to CDDP (b) or PtAcacDMS (c). Bar: 40 µm. b Histogram showing the percentage of fluorescence intensity in control and CDDP- or PtAcacDMS-treated cells. CTRL vs CDDP (mean diff. 30.58) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. 19.88) ***ρ < 0.0001, CDDP vs PtAcacDMS (mean diff. − 10.70) **ρ < 0.001; (F 125.4, R² 0.9766)

Fig. 13

Platinum compounds alter the expression of Ca²⁺-binding proteins upon 48 hexposure. a Immunocytochemical detection of calcium-binding proteins, namely calmodulin, calretinin, calbindin and parvalbumin (red fluorescence) and actin cytoskeleton (green fluorescence) in control cells (a), after 48 hexposure to CDDP (b) or PtAcacDMS (c). Bar: 40 µm. b Histogram showing the percentage of fluorescence intensity for each calcium-binding protein in control and treated cells. Calmodulin: CTRL vs CDDP (mean diff. − 13.88) **ρ < 0.001, CTRL vs PtAcacDMS (mean diff. − 8.539) *ρ < 0.05, CDDP vs PtAcacDMS (mean diff. 5.337) ns, (F 18.65, R² 0.8614). Calbindin: CTRL vs CDDP (mean diff. − 24.37) **ρ < 0.001, CTRL vs PtAcacDMS (mean diff. − 24.05) **ρ < 0.001, CDDP vs PtAcacDMS (mean diff. 0.3203) ns, (F 18.25, R² 0.8588). Calretinin: CTRL vs CDDP (mean diff. 47.55) ***ρ < 0.0001, CTRL vs PtAcacDMS (mean diff. 52.14) ***ρ < 0.0001, CDDP vs PtAcacDMS (mean diff. 4.591) ns, (F 62.16, R² 0.9540). Parvalbumin: CTRL vs CDDP (mean diff. 0.2374) ns, CTRL vs PtAcacDMS (mean diff. 6.592) **ρ < 0.001, CDDP vs PtAcacDMS (mean diff. 6.431) **ρ < 0.001; (F 14.14, R² 0.8250). ns not significant