Synthesis, Characterization, and Cytotoxicity Evaluation of Novel Water-Soluble Cationic Platinum(II) Organometallic Complexes with Phenanthroline and Imidazolic Ligands.

Platinum-based chemotherapeutic agents are widely used in the treatment of cancer. However, their effectiveness is limited by severe adverse reactions, drug resistance, and poor water solubility. This study focuses on the synthesis and characterization of new water-soluble cationic monofunctional platinum(II) complexes starting from the [PtCl(η1-C2H4OEt)(phen)] (1, phen=1,10-phenanthroline) precursor, specifically [Pt(NH3)(η1-C2H4OEt)(phen)]Cl (2), [Pt(1-hexyl-1H-imidazole)(η1-C2H4OEt)(phen)]Cl (3), and [Pt(1-hexyl-1H-benzo[d]imidazole)(η1-C2H4OEt)(phen)]Cl (4), which deviate from traditional requirements for antitumor activity. These complexes were evaluated for their cytotoxic effects in comparison to cisplatin, using immortalized cervical adenocarcinoma cells (HeLa), human renal carcinoma cells (Caki-1), and normal human renal cells (HK-2). While complex 2 showed minimal effects on the cell lines, complexes 3 and 4 demonstrated higher cytotoxicity than cisplatin. Notably, complex 4 displayed the highest cytotoxicity in both cancer and normal cell lines. However, complex 3 exhibited the highest selectivity for renal tumor cells (Caki-1) among the tested complexes, compared to healthy cells (HK-2). This resulted in a significantly higher selectivity than that of cisplatin and complex 4. Therefore, complex 3 shows potential as a leading candidate for the development of a new generation of platinum-based anticancer drugs, utilizing biocompatible imidazole ligands while demonstrating promising anticancer properties.

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.

Immunolocalization of the AT-1R Ang II Receptor in Human Kidney Cancer.

This study aimed to evaluate AT1-R expression in normal and cancerous human kidneys, how these expressions are modified, and AT1-R functionality. AT-1R mRNA expression, determined by real-time PCR, was detected in all samples. AT-1R mRNA increased in well-differentiated cancer (G1, p < 0.01) and decreased 2.9-fold in undifferentiated cancer (G4, p < 0.001) compared with normal kidney tissues. Immunocytochemistry analysis showed that the AT-1R was expressed in the normal tubular epithelium. The glomerulus was also immunoreactive, and as expected, the smooth muscle cells of the vessel walls also expressed the receptor. A total of 35 out of 42 tumors were AT-1R positive, with the cell tumors showing varying numbers of immunoreactive cells, which were stained in a diffuse cytoplasmic and membranous pattern. Computer-assisted counting of the stained tumor cells showed that the number of AT-1R-positive cells increased in the well-differentiated cancers. The functionality of AT-1R was assessed in primary cultures of kidney epithelial cells obtained from three G3 kidney cancer tissues and corresponding histologically proven non-malignant tissue adjacent to the tumor. Indeed, Ang II stimulated, in a dose-dependent manner, the 24 h proliferation of normal kidney cells and cancer cells in the primary culture and phosphorylated extracellular regulated kinases 1 and 2. In conclusion, Ang II may be involved in the growth or function of neoplastic kidney tissue.

Trends of Phase I Clinical Trials in the Latest Ten Years across Five European Countries.

BACKGROUND: Phase 1 clinical trials represent a critical phase of drug development because new candidate therapeutic agents are tested for the first time on humans. Therefore, international guidelines and local laws have been released to mitigate and control possible risks for human health in agreement with the declaration of Helsinki and the international Good Clinical Practice principles. Despite numerous scientific works characterizing the registered clinical trials on ClinicalTrials.gov, the main features and trends of registered phase 1 clinical trials in Europe have not been investigated. This study is aimed at assessing the features and the temporal trend of distribution of phase 1 clinical studies, carried out in the five largest European countries over a ten-year period (2012-2021), and to evaluate the impact of the Italian regulatory framework on the activation of such studies. METHODS: The main data and characteristics of phase 1 clinical studies registered on the ClinicalTrials.gov database for France, Germany, Italy, Spain and the United Kingdom have been investigated and subsequently compared. The above-mentioned countries were selected based on similarities in terms of demographic and Gross Domestic Product (GDP) data available on official government websites. (3) Results: A total number of 6878 phase 1 clinical trials were registered for the five selected countries in the ClinicalTrials.gov database during the ten years analyzed; the studies were predominantly randomized (39.33%) and for-profit (76.64%). The most represented area of investigations was oncology (52.15%), followed by hematology (24.99%) and immunology (12.04%). The variability observed between the analyzed countries showed that the UK, Germany and France presented the highest reduction in the number of phase 1 clinical trials, while for Spain and Italy, a stable/increased trend was observed, although with a lower number of trials registered on the ClinicalTrials.gov database. (4) Conclusions: Italy displayed the lowest number of registered phase 1 clinical trials, even though it showed a stable trend over the years. In this regard, the Italian regulatory framework must urgently be adapted to that of other European countries (Spain has been the first country to implement the new Regulation (EU) No 536/2014) and streamline the process of clinical trial application to increase the attractiveness of the country. Moreover, nonprofit phase 1 clinical trials (which represent 19.81% of the total number of phase 1 clinical trials registered in Italy vs. 80.19% of profit phase 1 clinical studies) should be promoted and supported by the institutions, even from a financial point of view, to allow independent researchers to develop new therapeutic drugs.

Antitumor and antimigration effects of a new Pt compound on neuroblastoma cells.

Among the new Pt complexes with anticancer properties, phenanthroline derivatives have aroused great interest due to their different mode of action compared to cisplatin. We previously examined cytotoxic effects of a new Pt(II)-complex containing 1,10-phenantroline (phen), [Pt(η1-C2H4OMe)(DMSO)(phen)]Cl, in a panel of eight human cancer cell lines, and showed that it exerted the greatest cytotoxic effect in the neuroblastoma SH-SY5Y cell line. In this study, the antiproliferative and antimetastatic potential of [Pt(η1-C2H4OMe)(DMSO)(phen)]+ (in short Pt-EtOMeSOphen) was investigated in neuroblastoma SH-SY5Y, SK-N-SH and SK-N-BE(2) cells. Pt-EtOMeSOphen provoked the early signs of apoptosis induction (cleavage of PARP and activation of caspases-9 and -7); it also increased the level of proapoptotic Bax protein whereas it decreased the level of the antiapoptotic Bcl-2 protein. The effects of Pt-EtOMeSOphen on migration and invasion processes were also evaluated. A decrease of cell migration/invasion by Pt-EtOMeSOphen was observed through 2D and 3D in vitro assays. Pt-EtOMeSOphen was found to exert its actions by decreasing MMP-9 and MMP-2 expressions and activities. Pt-EtOMeSOphen provoked the phosphorylation of both ERK1/2 and p38 MAPKs. All the effects of Pt-EtOMeSOphen on SH-SY5Y cell vitality, migration and metalloproteases activities described here were due to the activation of p38 MAPK since pharmacological p38 MAPK inhibition or small interfering RNAs to p38 MAPK mRNA blocked such effects. Results suggest that Pt-EtOMeSOphen inhibits neuroblastoma cancer cells survival, motility, and invasion. This could lead to the reduction of neuroblastoma metastatic potential.

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

NMR-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. The observed time response of SH-SY5Y cells under treatment revealed a faster action of Pt-EtOMeSOphen 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.

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 administration.

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

Salvia 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 p21WAF1 and p27Kip1 , and proapoptotic BAX; (2) activation of caspases; and (3) induction of a sub-G1 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, p21WAF1 , p27Kip1 , 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.

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.

TGF-ß1 activates RSC96 Schwann cells migration and invasion through MMP-2 and MMP-9 activities.

Following peripheral nerve injury, remnant Schwann cells adopt a migratory phenotype and remodel the extracellular matrix allowing axonal regrowth. Although much evidence has demonstrated that TGF-ß1 promotes glioma cell motility and induces the expression of extracellular matrix proteins, the effects of TGF-ß1 on Schwann cell migration has not yet been studied. We therefore investigated the cellular effects and the signal transduction pathways evoked by TGF-ß1 in rattus norvegicus neuronal Schwann RSC96 cell. TGF-ß1 significantly increased migration and invasion of Schwann cells assessed by the wound-healing assay and by cell invasion assay. TGF-ß1-enhanced migration/invasion was blocked by inhibition of MMP-2 and MMP-9. Consistently, by real-time and western blot analyses, we demonstrated that TGF-ß1 increased MMP-2 and MMP-9 mRNA and protein levels. TGF-ß1 also increased MMPs activities in cell growth medium, as shown by gelatin zymography. The selective TGF-ß Type I receptor inhibitor SB431542 completely abrogated any effects by TGF-ß1. Indeed, TGF-ß1 Type I receptor activation provoked the cytosol-to-nucleus translocation of SMAD2 and SMAD3. SMAD2 knockdown by siRNA blocked MMP-2 induction and cell migration/invasion due to TGF-ß1. TGF-ß1 also provoked phosphorylation of MAPKs extracellular regulated kinase 1/2 and JNK1/2. Both MAPKs were upstream to p65/NF-kB inasmuch as both MAPKs' inhibitors PD98059 and SP600125 or their down-regulation by siRNA significantly blocked the TGF-ß1-induced nuclear translocation of p65/NF-kB. In addition, p65/NF-κB siRNA knockdown inhibited the effects of TGF-ß1 on both MMP-9 and cell migration/invasion. We conclude that TGF-ß1 controls RSC96 Schwann cell migration and invasion through MMP-2 and MMP-9 activities. MMP-2 is controlled by SMAD2 whilst MMP-9 is controlled via an ERK1/2-JNK1/2-NF-κB dependent pathway.

[Pt(O,O'-acac)(γ-acac)(DMS)] Induces Autophagy in Caki-1 Renal Cancer Cells.

We have demonstrated the cytotoxic effects of [Pt(O,O'-acac)(γ-acac)(dimethyl sulfide (DMS))] on various immortalized cell lines, in primary cultures, and in murine xenograft models in vivo. Recently, we also showed that [Pt(O,O'-acac)(γ-acac)(DMS)] is able to kill Caki-1 renal cells both in vivo and in vitro. In the present paper, apoptotic and autophagic effects of [Pt(O,O'-acac)(γ-acac)(DMS)] and cisplatin were studied and compared using Caki-1 cancerous renal cells. The effects of cisplatin include activation of caspases, proteolysis of enzyme poly ADP ribose polymerase (PARP), control of apoptosis modulators B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and BH3-interacting domain death agonist (Bid), and cell cycle arrest in G2/M phase. Conversely, [Pt(O,O'-acac)(γ-acac)(DMS)] did not induce caspase activation, nor chromatin condensation or DNA fragmentation. The effects of [Pt(O,O'-acac)(γ-acac)(DMS)] include microtubule-associated proteins 1A/1B light chain 3B (LC3)-I to LC3-II conversion, Beclin-1 and Atg-3, -4, and -5 increase, Bcl-2 decrease, and monodansylcadaverine accumulation in autophagic vacuoles. [Pt(O,O'-acac)(γ-acac)(DMS)] also modulated various kinases involved in intracellular transduction regulating cell fate. [Pt(O,O'-acac)(γ-acac)(DMS)] inhibited the phosphorylation of mammalian target of rapmycin (mTOR), p70S6K, and AKT, and increased the phosphorylation of c-Jun N-terminal kinase (JNK1/2), a kinase activity pattern consistent with autophagy induction. In conclusion, while in past reports the high cytotoxicity of [Pt(O,O'-acac)(γ-acac)(DMS)] was always attributed to its ability to trigger an apoptotic process, in this paper we show that Caki-1 cells die as a result of the induction of a strong autophagic process.

ADP sensitizes ZL55 cells to the activity of cisplatin.

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor in which cisplatin therapy is commonly used, although its effectiveness is limited. It follows that research efforts dedicated to identify promising combinations that can synergistically kill cancer cells are needed. Because we recently demonstrated that ADP inhibits the proliferation of ZL55 cells, an MPM-derived cell line obtained from bioptic samples of asbestos-exposed patients. Our objective in this study was to investigate the hypothesis that ADP also potentiates the cytotoxic activity of cisplatin. Results show that in ZL55 cells ADP enhanced (a) the cytotoxicity of cisplatin by 12-fold, (b) the restraint of cell clonogenic potential cisplatin-mediated, and (c) the number of apoptotic cells. Cisplatin, but not ADP, caused caspases activation; nevertheless, poly(ADP-ribose) polymerase-1 was not only cleaved in cisplatin-treated cells but also in cells treated with ADP alone. Furthermore, ADP, but not cisplatin, decreased mTOR and 6SK phosphorylations. Both ADP and cisplatin increased p53 protein, but ADP was also able to enhance p53 messenger RNA. P53 silencing resulted in a very large decrement of cell death induced by ADP or by cisplatin and reverted ADP effects on mTOR/S6K phosphorylation, suggesting that activated p53 may act as a negative regulator of mTOR. Consistently, the inhibition of mTOR by rapamycin also sensitized cells to cisplatin, and the effects of cisplatin plus rapamycin were identical to those obtained with cisplatin plus ADP. These findings suggest that the combination of ADP and cisplatin may be a promising strategy for the clinical treatment of cisplatin-resistant MPM.

Response of Cisplatin Resistant Skov-3 Cells to [Pt(O,O'-Acac)(γ-Acac)(DMS)] Treatment Revealed by a Metabolomic ¹H-NMR Study.

The novel [Pt(O,O'-acac)(γ-acac)(DMS)], Ptac2S, Pt(II) complex has recently gained increasing attention as a potential anticancer agent for its pharmacological activity shown in different tumor cell lines, studied both in vitro and in vivo. The mechanism of action of Ptac2S, operating on non-genomic targets, is known to be very different from that of cis-[PtCl2(NH3)2], cisplatin, targeting nucleic acids. In this work, we evaluated the cytotoxicity of Ptac2S on the cisplatin resistant Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells, by the MTT assay. A ¹H-NMR metabolomic approach coupled with multivariate statistical analysis was used for the first time for Ptac2S to figure out the biological mechanisms of action of the complex. The metabolic variations of intracellular metabolites and the composition of the corresponding extracellular culture media were compared to those of cisplatin (cells were treated at the IC50 doses of both drugs). The reported comparative metabolomic analysis revealed a very different metabolic profile between Ptac2S and cisplatin treated samples, thus confirming the different mechanism of action of Ptac2S also in the Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells line. In particular, higher levels of pyruvate were observed in Ptac2S treated, with respect to cisplatin treated, cells (in both aqueous and culture media). In addition, a very different lipid expression resulted after the exposure to the two drugs (Ptac2S and cisplatin). These results suggest a possible explanation for the Ptac2S ability to circumvent cisplatin resistance in SKOV-3 cells.

Adenosine diphosphate regulates MMP2 and MMP9 activity in malignant mesothelioma cells.

Although an association between cancer progression and matrix metalloproteinase (MMP) 2 and MPP9 expression has been known, the expression, nuclear localization, and physiologically controlled activation of these two MMPs have not been investigated in malignant mesothelioma cells. We examined the expression and intracellular localization of MMP2/9 in ZL55 malignant mesothelioma cells, as well as their regulation by ADP. Using real-time PCR, we showed that activation of the P2Y1 receptor by ADP increased the expression of MMP2/9 mRNAs; MMP2/9 collected from conditioned media also showed an increase in activity; and ADP induced the nuclear localization of MMP2/9. The effects of ADP on transcription of the MMPs were due to activation of c-Src, Akt, and NF-κB, while ERK1/2 phosphorylation was needed for the increase in enzymatic activity and the regulation of nuclear import. We also showed that the nuclear localization of MMP2/9 induced by ADP causes the cleavage and inactivation of poly-ADP-ribose polymerase-1. These findings may help to elucidate the mechanisms regulating MMP2/9 activation in ZL55 human epithelioid mesothelioma cells, and perhaps other cells. Therapeutic approaches that promote ADP accumulation in a tumor environment may constitute an effective means to induce anticancer activity.

Correction: Apoptosis by [Pt(O,O'-acac)(γ-acac)(DMS)] requires PKC-δ mediated p53 activation in malignant pleural mesothelioma.

[This corrects the article DOI: 10.1371/journal.pone.0181114.].

Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling pathway.

Extracellular nucleotides can regulate cell proliferation in both normal and tumorigenic tissues. Here, we studied how extracellular nucleotides regulate the proliferation of ZL55 cells, a mesothelioma-derived cell line obtained from bioptic samples of asbestos-exposed patients. ADP and 2-MeS-ADP inhibited ZL55 cell proliferation, whereas ATP, UTP, and UDP were inactive. The nucleotide potency profile and the blockade of the ADP-mediated inhibitory effect by the phospholipase C inhibitor U-73122 suggest that P2Y1 receptor controls ZL55 cell proliferation. The activation of P2Y1 receptor by ADP leads to activation of intracellular transduction pathways involving [Ca2+ ]i , PKC-δ/PKC-α, and MAPKs, ERK1/2 and JNK1/2. Cell treatment with ADP or 2-MeS-ADP also provokes the activation of p53, causing an accumulation of the G1 cyclin-dependent kinase inhibitors p21WAF1 and p27Kip . Inhibition of ZL55 cell proliferation by ADP was completely reversed by inhibiting MEK1/2, or JNK1/2, or PKC-δ, and PKC-α. Through the inhibition of ADP-activated transductional kinases it was found that PKC-δ was responsible for JNK1/2 activation. JNK1/2 has a role in transcriptional up-regulation of p53, p21WAF1/CIP1 , and p27kip1 . Conversely, the ADP-activated PKC-α provoked ERK1/2 phosphorylation. ERK1/2 increased p53 stabilization, required to G1 arrest of ZL55 cells. Concluding, the importance of the study is twofold: first, results shed light on the mechanism of cell cycle inhibition by ADP; second, results suggest that extracellular ADP may inhibit mesothelioma progression.

Apoptosis by [Pt(O,O'-acac)(γ-acac)(DMS)] requires PKC-δ mediated p53 activation in malignant pleural mesothelioma.

Mesothelioma cancer cells have epithelioid or sarcomatoid morphology. The worst prognosis is associated with sarcomatoid phenotype and resistance to therapy is affected by cells heterogeneity. We recently showed that in ZL55 mesothelioma cell line of epithelioid origin [Pt(O,O'-acac)(γ-acac)(DMS)] (Ptac2S) has an antiproliferative effect in vitro and in vivo. Aim of this work was to extend the study on the effects of Ptac2S on ZL34 cell line, representative of sarcomatoid mesothelioma. ZL34 cells were used to assay the antitumor activity of Ptac2S in a mouse xenograft model in vivo. Then, both ZL34 and ZL55 cells were used in order to assess the involvement of p53 protein in (a) the processes underlying the sensitivity to chemotherapy and (b) the activation of various transduction proteins involved in apoptosis/survival processes. Ptac2S increases ZL34 cell death in vivo compared with cisplatin and, in vitro, Ptac2S was more efficacious than cisplatin in inducing apoptosis. In Ptac2S-treated ZL34 and ZL55 cells, p53 regulated gene products of apoptotic BAX and anti-apoptotic Bcl-2 proteins via transcriptional activation. Ptac2S activated PKC-δ and PKC-ε; their inhibition by PKC-siRNA decreased the apoptotic death of cells. PKC-δ was responsible for JNK1/2 activation that has a role in p53 activation. In addition, PKC-ε activation provoked phosphorylation of p38MAPK, concurring to apoptosis. In ZL34 cells, Ptac2S also activated PKC-α thus provoking ERK1/2 activation; inhibition of PKC-α, or ERK1/2, increased Ptac2S cytotoxicity. Results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, giving a substantial starting point for its further validation.

CCL20 promotes migration and invasiveness of human cancerous breast epithelial cells in primary culture.

The relation between the tumor and its microenvironment is one of the most interesting and less understood issues. Recently, we showed a role of CCL20 chemokine in proning the healthy tissue neighboring the tumor to carcinogenesis. Besides, tumor-secreted CCL20 induced proliferation, migration, and EMT of healthy cells. In this context, we have studied here if CCL20 had effects on the migration of cancer cells and the intracellular pathways used in breast epithelial cells in primary culture. Using molecular (siRNA) and pharmacological (inhibitors) techniques, we found multiple signaling kinases to be activated and involved in CCL20-induced tumor breast cell migration. CCL20 provoked a 2.5-fold increase of cell migration and invasion; CCL20 also enhanced MMP- 2 and MMP-9 mRNAs/protein expression and activities. Cell migration and invasiveness due to CCL20 significantly decreased when MMP-2 and MMP-9 were inhibited in CCL20-stimulated cells. CCL20 controlled MMP-2 expression through the JAK2/STAT3 pathway, while the expression of MMP-9 occurred by PKC-α that activated, consequently, c-Src, Akt, and finally NF-kB. These results reveal a role for CCL20 also in tumor breast cell and point to CCL20 as a novel therapeutic target in cancer.

In Vitro and In Vivo Antitumor Activity of [Pt(O,O'-acac)(γ-acac)(DMS)] in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy highly resistant to chemotherapy. There is an urgent need for effective therapy inasmuch as resistance, intrinsic and acquired, to conventional therapies is common. Among Pt(II) antitumor drugs, [Pt(O,O'-acac)(γ-acac)(DMS)] (Ptac2S) has recently attracted considerable attention due to its strong in vitro and in vivo antiproliferative activity and reduced toxicity. The purpose of this study was to examine the efficacy of Ptac2S treatment in MPM. We employed the ZL55 human mesothelioma cell line in vitro and in a murine xenograft model in vivo, to test the antitumor activity of Ptac2S. Cytotoxicity assays and Western blottings of different apoptosis and survival proteins were thus performed. Ptac2S increases MPM cell death in vitro and in vivo compared with cisplatin. Ptac2S was more efficacious than cisplatin also in inducing apoptosis characterized by: (a) mitochondria depolarization, (b) increase of bax expression and its cytosol-to-mitochondria translocation and decrease of Bcl-2 expression, (c) activation of caspase-7 and -9. Ptac2S activated full-length PKC-δ and generated a PKC-δ fragment. Full-length PKC-δ translocated to the nucleus and membrane, whilst PKC-δ fragment concentrated to mitochondria. Ptac2S was also responsible for the PKC-ε activation that provoked phosphorylation of p38. Both PKC-δ and PKC-ε inhibition (by PKC-siRNA) reduced the apoptotic death of ZL55 cells. Altogether, our results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.

PKC-δ/PKC-α activity balance regulates the lethal effects of cisplatin.

Cisplatin is commonly employed in therapy of mesothelioma but its efficacy is limited and the mechanisms by which induces its effects are not clearly understood. PKCs can regulate cisplatin sensitivity. PKCs effects on cellular sensitivity/resistance depend on the pattern of active PKC isozymes as well as on cellular context. The present study was undertaken to determine if specific PKC isoforms regulate cisplatin-induced apoptosis in the human mesothelioma ZL55 cells. Cells were treated with cisplatin at various concentrations and for different incubation periods. Cytotoxicity assays and Western blottings of various proteins involved in apoptosis and survival were then performed. Exposure of ZL55 cells to cisplatin at concentrations ranging from 1 to 200 µM resulted in a dose-dependent inhibition of cell survival and the activation of the mitochondrial apoptotic pathway. Cisplatin activated full-length PKC-δ and generated a PKC-δ fragment. PKC-δ inhibition (by PKC-δ-siRNA) decreased ZL55 cell apoptosis. Full-length PKC-δ translocated to the nucleus and activated caspase-3 expression, whereas PKC-δ fragment preferentially localized to mitochondria. Cisplatin also provoked the generation of reactive oxygen species (ROS) by NADPH oxidase. ROS increment was responsible for the PKC-α activation that provoked EGFR transactivation and consequential phosphorylation of ERK1/2. The inhibition of this pathway at various level (PKC-α, EGFR or ERK1/2) increased cisplatin-induced cytotoxicity. The results suggest that PKC-δ is an essential part of the apoptotic program in mesothelioma cells, whereas PKC-α mediates a pro-survival response to cisplatin.