A NMR-Based Metabolomic Approach to Investigate the Antitumor Effects of the Novel [Pt(η1-C2H4OMe)(DMSO)(phen)]+ (phen = 1,10-Phenanthroline) Compound on Neuroblastoma Cancer Cells

none7NMR-based metabolomics is a very effective tool to assess the tumor response to drugs by providing insights for their mode of action. Recently, a novel Pt(II) complex, [Pt(ƞ1-C2H4OMe)(DMSO)(phen)]+ (phen 1,10-phenanthroline), Pt-EtOMeSOphen, was synthesized and studied for its antitumor activity against eight human cancer cell lines. Pt-EtOMeSOphen showed higher cytotoxic effects than cisplatin in most of the cancer cell lines and in particular against the neuroblastoma cell line (SH-SY5Y). In this study, the mechanism of action of Pt-EtOMeSOphen on SH-SY5Y cells was investigated using 1H NMR-based metabolomics and compared with cisplatin. ­e observed time response of SH-SY5Y cells under treatment revealed a faster action of PtEtOMeSOphen compared with cisplatin, with a response already observed after six hours of exposure, suggesting a cytosolic target. NMR-based metabolomics demonstrated a peculiar alteration of the glutathione metabolism pathway and the diacylglycerol expression.De Castro, Federica; Stefano, Erika; De Luca, Erik; Muscella, Antonella; Marsigliante, Santo; Benedetti, Michele; Fanizzi, Francesco PaoloDe Castro, Federica; Stefano, Erika; De Luca, Erik; Muscella, Antonella; Marsigliante, Santo; Benedetti, Michele; Fanizzi, Francesco Paol

Oxidized Alginate Dopamine Conjugate: In Vitro Characterization for Nose‐to‐Brain Delivery Application

Background: The blood–brain barrier (BBB) bypass of dopamine (DA) is still a challenge for supplying it to the neurons of Substantia Nigra mainly affected by Parkinson disease. DA prodrugs have been studied to cross the BBB, overcoming the limitations of DA hydrophilicity. Therefore, the aim of this work is the synthesis and preliminary characterization of an oxidized alginate-dopamine (AlgOX-DA) conjugate conceived for DA nose-to-brain delivery. Methods: A Schiff base was designed to connect oxidized polymeric backbone to DA and both AlgOX and AlgOX-DA were characterized in terms of Raman, XPS, FT-IR, and 1H- NMR spectroscopies, as well as in vitro mucoadhesive and release tests. Results: Data demonstrated that AlgOX-DA was the most mucoadhesive material among the tested ones and it released the neurotransmitter in simulated nasal fluid and in low amounts in phosphate buffer saline. Results also demonstrated the capability of scanning near-field optical microscopy to study the structural and fluorescence properties of AlgOX, fluorescently labeled with fluorescein isothiocyanate microstructures. Interestingly, in SH-SY5Y neuroblastoma cell line up to 100 μg/mL, no toxic effect was derived from AlgOX and AlgOX-DA in 24 h. Conclusions: Overall, the in vitro performances of AlgOX and AlgOX-DA conjugates seem to encourage further ex vivo and in vivo studies in view of nose-to-brain administratio

Antitumor and antimigration effects of Salvia clandestina L. extract on osteosarcoma cells

none6noSalvia clandestina L. is a wild perennial species present in the Salento area of Italy. Here, we examined the in vitro effects of an aqueous extract of S. clandestina L. on the MG-63 osteosarcoma cell line. The extract reduced osteosarcoma cell viability mainly by way of apoptosis, as we observed (1) upregulation of gene and protein expression of p53, cyclin-dependent kinase inhibitors p21(WAF1) and p27(Kip1), and proapoptotic BAX; (2) activation of caspases; and (3) induction of a sub-G(1) peak in the cell cycle. The mitogen-activated protein kinases (MAPKs) JNK1/2 and p38 are activated and involved in the intracellular effects of the S. clandestina extract, as preincubation with the JNK1/2 inhibitor SP600125 or the p38 inhibitor SB203580 significantly decreased S. clandestina extract-induced cytotoxicity and inhibited increase in p53, p21(WAF1), p27(Kip1), and BAX. SP600125 also inhibited mRNA levels for all the aforementioned proteins, while SB203580 only affected p53 mRNA. Furthermore, S. clandestina extract treatment counteracted epithelial-to-mesenchymal transition, inhibited cell migration, and decreased the expression and activity of matrix metalloproteinase MMP2. In addition, S. clandestina extract enhanced the cytotoxic activity of cisplatin on MG-63 cells through downregulation of the Akt/PKB protein kinase. We conclude that S. clandestina extract may be a novel agent for osteosarcoma treatment.noneAntonella Muscella; Erika Stefàno; Luigi De Bellis; Eliana Nutricati; Carmine Negro; Santo MarsiglianteMuscella, Antonella; Stefano, Erika; DE BELLIS, Luigi; Nutricati, Eliana; Negro, Carmine; Marsigliante, Sant

Cytotoxicity of new organometallic Pt(II)-complexes containing 1,10-phenantroline

Among the emerging anti-cancer compounds, phenanthroline derivatives are of high interest. In contrast to cisplatin, phenanthrolines and their metal complexes are potentially intercalant molecules that can interact with DNA by aromatic π-stacking between base pairs. In this study, two new organometallic Pt(II)-complexes containing 1,10-phenantroline (phen), [Pt(phen)(DMSO)(η1CH2CH2OMe)]+, 1, [Pt(phen)(NH3)(η1-CH2CH2OMe)]+, 2, have been taken into consideration in order to evaluate their cytotoxicity in different human cancer cell lines. In addition, maximal intracellular uptake (MIU) was assayed by ICP-AES after incubation of cells with 100 μM 1 and 2 for 0.5-12 hours. Ten different human cancer cell lines (Caco-2, Caki-1, HeLa, Hep-G2, MCF-7, MG-63, SH-SY5Y, Skov-3, ZL-34 and ZL-55) were treated with 1, 2 and cisplatin at increasing concentrations (0.1-200 μM) from 12 to 72 hours to assess their effect on cell viability. While 2 did not show significantly greater cytotoxic effects than cisplatin in any cell line, 1 proved to be highly effective in almost all cell lines and mainly in the first 12-24 hours of treatment (Figure 1). The greater effects were observed in neuroblastoma cells SH-SY5Y (IC50 (12-24 h) between 8.23 ± 1.11 μM and 19.8 ± 3.26 μM) and ovarian adenocarcinoma cells SKOV-3 (IC50 (12-24 h) between 39.8 ± 3.56 μM and 92.13 ± 7.81 μM). ICP-AES in SH-SY5Y and SKOV-3 demonstrated a high intracellular uptake of compound (1) (MIUSH-SY5Y 430.5 ± 40.1 ng Pt/mg protein; MIUSKOV-3 497.6 ± 59.5 ng Pt/mg protein) compared to cisplatin (MIUSH-SY5Y 155.9 ± 31.4 ng Pt/mg protein; MIUSKOV-3 30 ± 10.2 ng Pt/mg protein). Total Pt concentration of compound 2 (MIUSH-SY5Y 300 ± 39.2 ng Pt/mg protein; MIUSKOV-3 140.4 ± 46.3 ng Pt/mg protein) was also higher than cisplatin despite not having significantly greater cytotoxic effects. Further studies are needed in order to evaluate the mechanism of action of both 1 and 2 compounds and therefore understand why compound 1 is more toxic than compound 2. Finally, it is desirable to use healthy cell lines corresponding to the tumor lines used here in order to verify any cellular specificity towards cancer cells of the two compounds

Oxidized Alginate Dopamine Conjugate: A Study to Gain Insight into Cell/Particle Interactions

Background: We had previously synthetized a macromolecular prodrug consisting of oxidized Alginate and dopamine (AlgOx-Da) for a potential application in Parkinson disease (PD). Methods: In the present work, we aimed at gaining an insight into the interactions occurring between AlgOx-Da and SH-SY5Y neuronal cell lines in view of further studies oriented towards PD treatment. With the scope of ascertaining changes in the external and internal structure of the cells, multiple methodologies were adopted. Firstly, fluorescently labeled AlgOx-Da conjugate was synthetized in the presence of fluorescein 5(6)-isothiocyanate (FITC), providing FITC-AlgOx-Da, which did not alter SH-SY5Y cell viability according to the sulforhodamine B test. Furthermore, the uptake of FITC-AlgOx-Da by the SH-SY5Y cells was studied using scanning near-field optical microscopy and assessments of cell morphology over time were carried out using atomic force microscopy. Results: Notably, the AFM methodology confirmed that no relevant damage occurred to the neuronal cells. Regarding the effects of DA on the intracellular reactive oxygen species (ROS) production, AlgOx-Da reduced them in comparison to free DA, while AlgOx did almost not influence ROS production. Conclusions: these findings seem promising for designing in vivo studies aiming at administering Oxidized Alginate Dopamine Conjugate for PD treatment

Synthesis and Evaluation of the Cytotoxic Activity of Water-Soluble Cationic Organometallic Complexes of the Type [Pt(η1-C2H4OMe)(L)(Phen)]+ (L = NH3, DMSO; Phen = 1,10-Phenanthroline)

Starting from the [PtCl(η1-C2H4OMe)(phen)] (phen = 1,10-phenanthroline, 1) platinum(II) precursor, we synthesized and characterized by multinuclear NMR new [Pt(η1-C2H4OMe)(L)(phen)]+ (L = NH3, 2; DMSO, 3) complexes. These organometallic species, potentially able to interact with cell membrane organic cation transporters (OCT), violating some of the classical rules for antitumor activity of cisplatin analogues, were evaluated for their cytotoxicity. Interestingly, despite both complexes 2 and 3 resulting in greater cell uptake than cisplatin in selected tumor cell lines, only 3 showed comparable or higher antitumor activity. General low cytotoxicity of complex 2 in the tested cell lines (SH-SY5Y, SK-OV-3, Hep-G2, Caco-2, HeLa, MCF-7, MG-63, ZL-65) appeared to depend on its stability towards solvolysis in neutral water, as assessed by NMR monitoring. Differently, the [Pt(η1-C2H4OMe)(DMSO)(phen)]+ (3) complex was easily hydrolyzed in neutral water, resulting in a comparable or higher cytotoxicity in cancer cells with respect to cisplatin. Further, both IC50 values and the uptake profiles of the active complex appeared quite different in the used cell lines, suggesting the occurrence of diversified biological effects. Nevertheless, further studies on the metabolism of complex 3 should be performed before planning its possible use in tissue- and tumor-specific drug design

Dopamine and Antioxidant Grape Seed Extract loaded chitosan nanoparticles: A preliminary in vitro characterization

Neuronal cell model line SHSY-5Y is extensively adopted when in vitro investigations are related to Parkinson disease (PD) application. Herein, chitosan nanoparticles (CS NPs) were formulated for the co-administration of dopamine (DA) and Grape Seed Extract (GSE) with the aim to gain insight into the interactions occurring between SHSY-5Y and NPs. Following the ionic gelation technique, the mean particle size of the NPs resulted in the range 310–330 nm and the zeta measurements were in the range +16.4 – +35.5 mV. The presence of CS chains on the surface suggested by positive zeta values was also confirmed by FT-IR analysis, whereas storage stability studies upon different temperatures evidenced that, although aggregation occurred, DA autoxidation was prevented because no black suspensions were detected over the time, irrespectively of the temperature assayed. From a biological viewpoint, release studies of CS NPs loaded with DA and GSE showed that in SHSY-5Y cell lines DA accumulation was time-dependent, irrespectively of the presence of GSE. Furthermore, ROS levels and carbonylated proteins both decreased in SHSY-5Y cell line once NPs administering both DA and GSE were incubated, suggesting a significative reduction of oxidative stress which plays a significative role for PD development

Compatibility of Nucleobases Containing Pt(II) Complexes with Red Blood Cells for Possible Drug Delivery Applications

The therapeutic advantages of some platinum complexes as major anticancer chemotherapeutic agents and of nucleoside analogue-based compounds as essential antiviral/antitumor drugs are widely recognized. Red blood cells (RBCs) offer a potential new strategy for the targeted release of therapeutic agents due to their biocompatibility, which can protect loaded drugs from inactivation in the blood, thus improving biodistribution. In this study, we evaluated the feasibility of loading model nucleobase-containing Pt(II) complexes into human RBCs that were highly stabilized by four N-donors and susceptible to further modification for possible antitumor/antiviral applications. Specifically, platinum-based nucleoside derivatives [PtII(dien)(N7-Guo)]2+, [PtII(dien)(N7-dGuo)]2+, and [PtII(dien)(N7-dGTP)] (dien = diethylenetriamine; Guo = guanosine; dGuo = 20-deoxy-guanosine; dGTP = 50-(20-deoxy)-guanosine-triphosphate) were investigated. These Pt(II) complexes were demonstrated to be stable species suitable for incorporation into RBCs. This result opens avenues for the possible incorporation of other metalated nucleobases analogues, with potential antitumor and/or antiviral activity, into RBCs

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