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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-kappa B, 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.

In this study, we used the ovarian cancer cells Skov-3,a cell line resistant to cisplatin, in order to investigate whether [Pt(O,O’-acac)(γ-acac)(DMS)] was able to induce cell death. Skov-3 cells were treated with [Pt(O,O’-acac)(γ-acac)(DMS)] or with cisplatin and cytotoxicity tests were performed, together with western blotting of several proteins involved in apoptosis and autophagy, including LC-3 I/II and Beclin-1, considered important autophagic markers. The results obtained showed that [Pt(O,O’-acac)(γ-acac)(DMS)] induced a significant decrease in Skov-3 cell viability. We assessed the activation of both apoptotic and autophagic processes since western blotting analyses showed a time-dependent increment of the expression levels of autophagic markers Beclin-1 and LC-3, together with the proteolytic activation of caspase 9 and the degradation of PARP. The formation of autophagic vacuoles was detected by visualization of monodansylcadaverine (MDC) in Skov-3 cells incubated with [Pt(O,O’-acac)(gamma-acac)(DMS)]-treated cells, but not in cisplatin-treated cells. In addition, cisplatin caused apoptosis in Skov-3 cells since it activated caspases 7, 3 and 9 with consequent PARP proteolysis whilst it did not have any effect on LC-3II and Beclin-1 activation, thus showing the absence of autophagic processess. In conclusion, both [Pt(O,O’-acac)(gamma-acac)(DMS)] and cisplatin induced apoptotic Skov-3 cell death, but only [Pt(O,O’-acac)(gamma-acac)(DMS)] was able to induce autophagy.

The aim of this study was to compare antitumor activity of [Pt(O,O’-acac)(γ-acac)(DMS)] in epithelioid and sarcomatoid type of MPM. Thus, we employed the human cell line of epithelioid derivation ZL55, and the sarcomatoid cell line ZL34 in vitro and SCID mice. In epithelioid cells, [Pt(O,O’-acac)(γ-acac)(DMS)] was approximately 12-fold more cytotoxic than cisplatin after 24 h of incubation (IC50 were 3.9±0.11 μM for [Pt(O,O’-acac)(γ-acac)(DMS)] and 46.8±0.6 μM for cisplatin, n=6). Similarly, in sarcomatoid cells cisplatin was significantly less cytotoxic than [Pt(O,O’-acac)(γ-acac)(DMS)] (IC50 48.7±1.7 μM and ND n=4, for [Pt(O,O’-acac)(γ-acac)(DMS) and cisplatin, respectively). In addition, we employed a preclinical model based on the subcutaneous injection of ZL55 and ZL34 malignant pleural mesotelioma cell lines in SCID mice. Remarkably, [Pt(O,O’-acac)(γ-acac)(DMS)] stands out for higher anticancer activity than cisplatin toward both the murine tumor models examined, inducing up to 50% inhibition of tumor growth. Mice inoculated with MPM cells showed a statistically significant reduction of tumor volume at every time point in the [Pt(O,O’-acac)(γ-acac)(DMS)] groups compared with both not treated and cisplatin-treated mice (p < 0.05). In summary, our findings show that [Pt(O,O’-acac)(γ-acac)(DMS)] seems to be more potent than cisplatin in MPM, thus providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.

We used the human mesothelioma ZL55 cells, which are a reliable model for investigating the therapeutic potential of nucleotides in MPM clinical trials. In addition we attempted to potentiate the cytotoxic effects of cisplatin by ADP. In ZL55 cells ADP is able to block cellular proliferation in a time and dose dependent manner, but evaluating the major apoptotic markers, it is seen that the mechanism underlying this effect is not apoptosis. Cell cycle analysis revealed that inhibition of proliferation of ZL55 cells by ADP was mediated by block of cell cycle progression. Flow cytometry analysis of subconfluent ZL55 cell cultures showed that treatment with ADP (10–100μM) for 24 h caused a concentration dependent increase of cells in the G0/G1 phases. ADP also induced p53 protein levels and increased the levels of p53, p21 and p27 mRNA, whilst cisplatin did not. Cisplatin plus ADP were more efficacious than cisplatin alone 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 and cleavage of PARP-1. Therefore, the antiproliferative effect of ADP is due to the cell cycle block and increases cisplatin cytotoxicity.

Tumor growth, tumor cell proliferation and microvessel density, in a xenograft model of RCC, developed by injection of Caki-1 cells in BALB/c nude mice, were investigated. Exposure of the Caki-1 cells to cisplatin and to [Pt(O,O’-acac)(γ-acac)(DMS)] resulted in a dose-dependent inhibition of cell survival. [Pt(O,O′-acac)(γ-acac)(DMS)] was much more effective than cisplatin in the inhibition of tumor growth, proliferation and angiogenesis in vivo as well as migration, tube formation and MMP-1, MMP-2 and MMP-9 secretion of endothelial cells, in vitro. Wherease, cisplatin exerted a higher cytotoxic activity on Huvecs, but did not affect tube formation and migration of endothelial cells. In addition, treatment of xenograft mice with [Pt(O,O′-acac)(γ-acac)(DMS)] decreased, VEGF, MMP-1 and MMP-2 expression in tumors.The anti-angiogenic and anti-tumor activities of [Pt(O,O’-acac)(γ-acac)(DMS)] providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.

This study aimed to evaluate the effect of TGF-β1 on RSC96 Schwann cell migration and to study its intracellular transduction pathways. We found that TGF-β1 induced the activation of key signaling molecules involved in non-Smad (Akt and MAPK) and focal adhesion signaling pathways (FAK and Src) that cooperatively regulated the process of RSC96 cells migration together with MMP-2 and MMP-9 upregulation. These findings indicate a regulatory pathway of TGF-β1 in MMP-9/-2 expression which may be significant in regulating Schwann cell migration and physiology in peripheral nervous sytem injury.

The selectivity of [Pt(O,O’-acac)(γ-acac)(DMS)] stimulates a more detailed study aimed at pre-clinical investigation of its therapeutic potential in vivo. In this context, we employed a preclinical model based on the subcutaneous injection of MCF-7 breast cancer and ZL55 malignant pleural mesotelioma cell lines in SCID mice. Remarkably, [Pt(O,O’-acac)(γ- acac)(DMS)] stands out for higher anticancer activity than cisplatin toward both the murine tumor models examined, inducing up to 50% inhibition of tumor growth. We also demonstrated enhanced in vivo pharmacokinetics (PK), biodistribution and tolerability of [Pt(O,O’-acac)(γ-acac)(DMS)] when compared to cisplatin administered in Wistar rats. Altogether, these findings suggest that [Pt(O,O’-acac)(γ-acac)(DMS)] is a promising therapeutic agent for preventing growth of cancer, thus providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.

In the present study, we aimed to investigate the anti-angiogenesis effects of [Pt(O,O’-acac)(γ-acac)(DMS)]. [Pt(O,O′-acac)(γ-acac)(DMS)] significantly inhibited human umbilical vein endothelial cell (HUVEC) proliferation. Then, the ex vivo rat aortic ring capillary-network sprouting has been carried out. It was observed that after one week of treatment under regular growth conditions, [Pt(O,O′-acac)(γ-acac)(DMS)] potently inhibited the sprouting as well as capillary-network formation from rat aortic ring in a dose-dependent manner. Compared to control 0,5 and 1 μM doses of [Pt(O,O′-acac)(γ- acac)(DMS)] suppressed capillary-network formation by 68 to 100%, respectively. In addition, in mouse xenograft model of breast cancer, treatment of mice with [Pt(O,O′-acac)(γ-acac)(DMS)], decrease MMP-1 content in tumours. These observations demonstrated that [Pt(O,O′- acac)(γ-acac)(DMS)] could inhibit angiogenesis, which warrants further studies in other in vivo models.

We here demonstrated the expression of 4 (P2Y1,4,6,11) purinoceptors mRNAs coupled to Gq protein. The extracellular ATP, UTP, ADP and UDP caused a transient [Ca2+]i peak followed by a sustained lower phase. Removal of extracellular Ca2+ decreased the initial transient and abolished the plateau phase. Ca2+ signal was blocked by the inhibitor of PLCβ, U73122. Through experiments of fluorescence quenching, it was seen that nucleotides increased plasmamembrane Ca2+ permeability. These results indicate that [Ca2+]i increase was due to activation of P2Y receptors exclusively and that Ca2+ is released from intracellular stores and also enters from the extracellular liquid. We also investigated the effects of purinoceptors on cell proliferation. UTP, ATP and UDP had no significant effects, while ADP causes a drastic cell proliferation decrease in a dose- and time-dependent manner. In conclusion, these results indicate that the expressed purinergic receptors act via PLC activation and that ADP may have a therapeutic potential in MM.

The new b-diketonate compounds ([Pt(O,O’-acac)(gamma-acac)(NH3)] and [Pt(O,O’-acac)(g-acac)(py)]) were chemical characterized by NMR spectroscopy and preliminary cytotoxic assays of [Pt(O,O’-acac)(g-acac)(NH3)] on cisplatin resistant MCF-7 breast cancer cell line were performed. Interestingly, [Pt(O,O’-acac)(gamma-acac)(NH3)] resulted in dose dependent inhibition of cell survival comparable to [Pt(O,O’-acac)(γ-acac)(DMS) and approximately 90-fold greater than that observed for cisplatin.

We assessed the therapeutic potential of [Pt(O,O’-acac)(γ-acac)(DMS)] in vivo using a xenograft model of breast cancer developed by injection of MCF-7 cells in the flank of BALB/c nude mice. Solid tumor-bearing mice showed up to 50% reduction of tumor mass after [Pt(O,O’-acac)(γ-acac)(DMS)] treatment, whereas an average 10% inhibition was recorded in cisplatin treated animals. Moreover, enhanced in vivo pharmacokinetics (PK), biodistribution and tolerability for [Pt(O,O’-acac)(γ-acac)(DMS)] administered Wistar rats have been observed with respect to cisplatin treated.

We used ZL55 mesothelioma cell line in order to study the cytotoxicity of cisplatin and the cellular pathways eventually ending to the apoptotic process. To this end, ZL55 cells were treated with increasing doses (1-200 μM) and for different incubation times (1-72 hours) with cisplatin and then the number of viable cells was evaluated by the MTT test. The cleavage patterns of caspase-3, -7 and -9, and Poly-ADP ribose polymerase (PARP) by cisplatin were assessed by Western methods. The results showed here suggest that activation of PKC-δ is an essential part of the apoptotic program provoked by cisplatin in mesothelioma cells and that PKC-δ transduces a pro-apoptotic signal in these cells.

The effects of a Pt-based drug on human mesothelioma cells NCI-H28 and H2373

[Pt(O,O′-acac)(γ-acac)(DMS)] can inhibit the metastatic diffusion of breast carcinoma

[Pt(O,O′-acac)(γ-acac)(DMS)] change the expression of 84 proteins as assessed by a proteomic approach in SH-SY5Y cells

BACKGROUND AND PURPOSE: We showed previously that a new Pt(II) complex ([Pt(O,O'-acac)(gamma-acac)(DMS)]) exerted high and fast apoptotic processes in MCF-7 cells. The objective of this study was to investigate the hypothesis that [Pt(O,O'-acac)(gamma-acac)(DMS)] is also able to exert anoikis and alter the migration ability of MCF-7 cells, and to show some of the signalling events leading to these alterations. EXPERIMENTAL APPROACH: Cells were treated with sublethal doses of [Pt(O,O'-acac)(gamma-acac)(DMS)], and the efficiency of colony initiation and anchorage-independent growth was assayed; cell migration was examined by in vitro culture wounding assay. Gelatin zymography for MMP-2 and -9 activities, Western blottings of MMPs, MAPKs, Src, PKC-epsilon and FAK, after [Pt(O,O'-acac)(gamma-acac)(DMS)] treatment, were also performed. KEY RESULTS: Sub-cytotoxic drug concentrations decreased the: (i) anchorage-dependent and -independent growth; (ii) migration ability; and (iii) expression and activity of MMP-2 and MMP-9. [Pt(O,O'-acac)(gamma-acac)(DMS)] provoked the generation of reactive oxygen species (ROS), and the activation of p38MAPK, Src and PKC-epsilon. p38MAPK phosphorylation, cell anoikis and migration due to [Pt(O,O'-acac)(gamma-acac)(DMS)] were blocked by PKC-epsilon inhibition. Furthermore, Src inhibition blocked the [Pt(O,O'-acac)(gamma-acac)(DMS)]-provoked activation of PKC-epsilon, while ROS generation blockage inhibited the activation of Src, and also the decrement of phosphorylated FAK observed in detached [Pt(O,O'-acac)(gamma-acac)(DMS)]-treated cells. CONCLUSIONS AND IMPLICATIONS: Sublethal concentrations of [Pt(O,O'-acac)(gamma-acac)(DMS)] induced anoikis and prevented events leading to metastasis via alterations in cell migration, anchorage independency, stromal interactions and MMP activity. Hence, [Pt(O,O'-acac)(gamma-acac)(DMS)] may be a promising therapeutic agent for preventing growth and metastasis of breast cancer.