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Local mediator prostaglandins and bradykinin are involved in inflammation and pain. We explored bradykinin effects on prostaglandin E2 (PGE2) release from fibroblasts derived from human skeletal muscular biopsies. Bradykinin induced PGE2 release through bradykinin B2 receptors, since PGE2 release was blocked by the bradykinin B2 receptor selective antagonist FR173657 and B2 receptor agonist (Hyp3)-bradykinin showed effects comparable to bradykinin. Consistently, bradykinin induced both mRNA cyclooxygenase 2 (COX-2) and protein. Bradykinin also induced ERK1/2 and p38 phosphorylation and provoked the translocation from the cytosol to the nucleus of p65/NF-kB. The release of PGE2 by bradykinin could be blocked inhibiting COX-2 and p65/NF-kB, ERK1/2 or p38 activation. Both ERK1/2 and p38 were upstream to NF-kB inasmuch siRNAs significantly blocked the p65/NF-kB activation induced by bradykinin. Thus, bradykinin, acting via B2 receptors, induced PGE2 release through ERK1/2 and p38-dependent pathways and consequent p65/NF-kB translocation to nucleus. p65/NF-kB induced COX-2 transcription. The release of PGE2 provide a possible explanation for the role of bradykinin in inflammatory diseases.

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.

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.

Local mediator prostaglandins and bradykinin are involved in inflammation and pain. We explored bradykinin effects on prostaglandin E2 (PGE2) release from fibroblasts derived from human skeletal muscular biopsies. Bradykinin induced PGE

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-β1 promotes glioma cell motility and induces the expression of extracellular matrix proteins, the effects of TGF-β1 on Schwann cell migration has not yet been studied. We therefore investigated the cellular effects and the signal transduction pathways evoked by TGF-β1 in rattus norvegicus neuronal Schwann RSC96 cell. TGF-β1 significantly increased migration and invasion of Schwann cells assessed by the wound-healing assay and by cell invasion assay. TGF-β1-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-β1 increased MMP-2 and MMP-9 mRNA and protein levels. TGF-β1 also increased MMPs activities in cell growth medium, as shown by gelatin zymography. The selective TGF-β Type I receptor inhibitor SB431542 completely abrogated any effects by TGF-β1. Indeed, TGF-β1 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-β1. TGF-β1 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-β1-induced nuclear translocation of p65/NF-kB. In addition, p65/NF-κB siRNA knockdown inhibited the effects of TGF-β1 on both MMP-9 and cell migration/invasion. We conclude that TGF-β1 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. (Figure presented.).

We have demonstrated the cytotoxic effects of [Pt(O,O&prime, acac)(&gamma, 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&rsquo, 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&prime, 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&rsquo, acac)(DMS)] did not induce caspase activation, nor chromatin condensation or DNA fragmentation. The effects of [Pt(O,O&rsquo, 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&rsquo, acac)(DMS)] also modulated various kinases involved in intracellular transduction regulating cell fate. [Pt(O,O&rsquo, 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&rsquo, 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.

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 [Ca(2+) ]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 p21(WAF1) and p27(Kip) . 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, p21(WAF1/CIP1) , and p27(kip1) . 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.

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.

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.

We previously showed that [Pt(O,O’-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-e/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibites cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-e and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-e activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-e activation.

Background and Purpose The aim of this study was to determine whether [platinum (Pt)(O,O′-acetylacetonate (acac))(γ-acac)(dimethylsulphide (DMS))] is differentially cytotoxic in normal and cancer cells, and to measure comparative levels of cytotoxicity compared with cisplatin in the same cells. Experimental Approach We performed experiments on cancerous and normal epithelial breast cells in primary culture obtained from the same patients. The apoptotic effects [Pt(O,O′-acac)(γ-acac)(DMS)] and cisplatin in cancerous and normal breast cells were compared. Key Results Cancer cells were more sensitive to [Pt(O,O′-acac)(γ-acac)(DMS)] (IC50 = 5.22 ± 1.2 μmol·L−1) than normal cells (IC50 = 116.9 ± 8.8 μmol·L−1). However, the difference was less strong when cisplatin was used (IC50 = 96.0 ± 6.9 and 61.9 ± 6.1 μmol·L−1 for cancer and normal cells respectively). Both compounds caused reactive oxygen species (ROS) production with different mechanisms: [Pt(O,O′-acac)(γ-acac)(DMS)] quickly activated NAD(P)H oxidase while cisplatin caused a slower formation of mitochondrial ROS. Cisplatin and [Pt(O,O′-acac)(γ-acac)(DMS)] caused activation of caspases, proteolysis of PARP and modulation of Bcl-2, Bax and Bid. [Pt(O,O′-acac)(γ-acac)(DMS)] also caused leakage of cytochrome c from the mitochondria. Overall, these processes proceeded more quickly in cells treated with [Pt(O,O′-acac)(γ-acac)(DMS)] compared with cisplatin. [Pt(O,O′-acac)(γ-acac)(DMS)] effects were faster and quantitatively greater in cancer than in normal cells. [Pt(O,O′-acac)(γ-acac)(DMS)] caused a fast decrease of mitochondrial membrane potential, especially in cancer cells. Conclusions and Implications [Pt(O,O′-acac)(γ-acac)(DMS)] was specific to breast cancer cells in primary culture, and this observation makes this compound potentially more interesting than cisplatin.