1,720,972 research outputs found
Purinergic receptors in a pleural mesotelioma cell line.
No full textWe 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
Bradykinin stimulates prostaglandin E2 release in human skeletal muscular fibroblasts
No full textLocal 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
Immunolocalization of the AT-1R Ang II Receptor in Human Kidney Cancer
Full text linkThis 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
PKC-δ/PKC-α activity balance regulates the lethal effects of cisplatin
No full textCisplatin 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
Protein kinase C-delta mediates cisplatin-induced apoptosis of ZL55 mesothelioma cells
No full textWe 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
Cytotoxic effects of [ Pt( O,O' – acac)(γ-acac)(DMS)] and cisplatin in Caki-1 renal cancer cells
No full textADP sensitizes ZL55 cells to the activity of anticancer cisplatin
No full textWe 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
[Pt(O,O'-acac)(γ-acac)(DMS)] induces autophagy and apoptosis in Skov-3 ovarian carcinoma cells
No full textIn 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
Adenosine diphosphate regulates MMP2 and MMP9 activity in malignant mesothelioma cells
Full text linkAlthough 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
[Pt(O,O′-acac)(γ-acac)(DMS)] inhibits malignant pleural mesothelioma cells in vitro and in vivo
No full textThe 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
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