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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.

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., In this report, we examined the effects of an aqueous extract of Salvia clandestina L. on the MG‐63 osteosarcoma cell line. The extract reduced osteosarcoma cell viability mainly by inducing apoptosis; it also counteracted epithelial‐to‐mesenchymal transition, inhibited cell migration, and decreased the expression and activity of matrix metalloproteinase MMP2, as well as enhanced the cytotoxic activity of cisplatin. S. clandestina extract may therefore be a novel agent for osteosarcoma treatment.

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.

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.

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-e activation that provoked phosphorylation of p38. Both PKC-δ and PKC-e 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.

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.

The higher and selective cytotoxicity of [Pt(O,O′-acac)(γ-acac)(DMS)] towards cancer cell in both immortalized cell lines and in breast cancer cells in primary cultures, stimulated a pre-clinical study in order to evaluate its therapeutic potential in vivo. The efficacy of [Pt(O,O′-acac)(γ-acac)(DMS)] was assessed using a xenograft model of breast cancer developed by injection of MCF-7 cells in the flank of BALB/c nude mice. Treatment of solid tumor-bearing mice with [Pt(O,O′-acac)(γ-acac)(DMS)] induced up to 50% reduction of tumor mass compared to an average 10% inhibition recorded in cisplatin treated animals. Thus, chemotherapy with [Pt(O,O′-acac)(γ-acac)(DMS)] was much more effective than cisplatin. We also demonstrated enhanced in vivo pharmacokinetics, biodistribution, and tolerability of [Pt(O,O′-acac)(γ-acac)(DMS)] when compared to cisplatin administered in Wistar rats. Pharmacokinetics studies with [Pt(O,O′-acac)(γ-acac)(DMS)] revealed prolonged Pt persistence in systemic blood circulation and decreased nefrotoxicity and hepatotoxicity¬, a major target sites of cisplatin toxicity. Overall, [Pt(O,O′-acac)(γ-acac)(DMS)] turned out to be extremely promising in terms of greater in vivo anticancer activity, reduced nephrotoxicity and acute toxicity compared to cisplatin.

Thyroid hormone 3,5,3′-triiodo- l -thyronine (T3) is known to affect cell metabolism through both the genomic and non-genomic actions. Recently, we demonstrated in HepG2 cells that T3 controls the expression of SREBP-1, a transcription factor involved in the regulation of lipogenic genes. This occurs by activation of a cap-independent translation mechanism of its mRNA. Such a process is dependent on non-genomic activation of both MAPK/ERK and PI3K/Akt pathways. The physiological role of 3,5-diiodo- l -thyronine (T2), previously considered only as a T3 catabolite, is of growing interest. Evidences have been reported that T2 rapidly affects some metabolic pathways through non-genomic mechanisms. Here, we show that T2, unlike T3, determines the block of proteolytic cleavage of SREBP-1 in HepG2 cells, without affecting its expression at the transcriptional or translational level. Consequently, Fatty Acid Synthase expression is reduced. T2 effects depend on the concurrent activation of MAPKs ERK and p38, of Akt and PKC-δ pathways. Upon the activation of these signals, apoptosis of HepG2 cells seems to occur, starting at 12 h of T2 treatment. PKC-δ appears to act as a switch between p38 activation and Akt suppression, suggesting that this PKC may function as a controller in the balance of pro-apoptotic (p38) and anti-apoptotic (Akt) signals in HepG2 cells.

It was previously demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)] exerted toxic effects at high doses, whilst sub-cytotoxic concentrations induced anoikis and decreased cell migration. Aim of this study was to investigate the hypothesis that [Pt(O,O'-acac)(γ-acac)(DMS)] alters the [Ca(2+)](i) and that this is linked to its ability to trigger rapid apoptosis in MCF-7 cells. Thus, cells were treated with [Pt(O,O'-acac)(γ-acac)(DMS)] and its effects on some of the systems regulating Ca(2+) homeostasis were studied, also in cells dealing with the complex changes occurring during the Ca(2+) signalling evoked by extracellular stimuli. [Pt(O,O'-acac)(γ-acac)(DMS)] caused the decrease of PMCA activity (but not SERCA or SPCA) and Ca(2+) membrane permeability. These two opposite effects on [Ca(2+)](i) resulted in its overall increase from 102±12nM to 250±24nM after 15min incubation. The effects of [Pt(O,O'-acac)(γ-acac)(DMS)] were also evident when cells were stimulated with ATP: the changes in Ca(2+) levels caused by purinergic stimulation resulted altered due to decreased PMCA activity and to the closure of Ca(2+) channels opened by purinergic receptor. Conversely, [Pt(O,O'-acac)(γ-acac)(DMS)] did not affect the store-operated Ca(2+) channels opened by thapsigargin or by ATP. [Pt(O,O'-acac)(γ-acac)(DMS)] provoked the activation of PKC-α and the production of ROS that were responsible for the Ca(2+) permeability and PMCA activity decrease, respectively. The overall effect of [Pt(O,O'-acac)(γ-acac)(DMS)] is to increase the [Ca(2+)](i), an effect that is likely to be linked to its ability to trigger rapid apoptosis in MCF-7 cells. These data reinforce the notion that [Pt(O,O'-acac)(γ-acac)(DMS)] would be a promising drug in cancer treatment.

It was previously shown that [Pt(O,O′-acac)(γ-acac)(DMS)] induces apoptosis in various cancer cells and exerts antimetastatic responses in vitro. In rats, [Pt(O,O′-acac)(γ-acac)(DMS)] reaches the central nervous system in quantities higher than cisplatin causing less excitotoxicity. The aim of the present paper was to investigate whether [Pt(O,O′-acac)(γ-acac)(DMS)] is able to exert cytotoxic effects on SH-SY5Y human neuroblastoma cell line, and to study the intracellular transduction mechanisms underlying these effects. Here we have demonstrated that [Pt(O,O′-acac)(γ-acac)(DMS)] was more effective than cisplatin in provoking apoptosis characterized by: (a) mitochondria depolarization, (b) decrease of Bcl-2 expression and increase of BAX expressions with cytosol-to-mitochondria translocation, (c) activation of caspase-7 and -9 and (d) generation of reactive oxygen species (ROS). [Pt(O,O′-acac)(γ-acac)(DMS)] provoked the activation of the following signalling kinases that were interacting with each other: PKC-δ and -ɛ, ERK1/2, p38MAPK, JNK1/2, NF-κB, c-src and FAK. We found that ROS generated by NADPH oxidase was responsible for the [Pt(O,O′-acac)(γ-acac)(DMS)]-mediated PKC-δ and -ɛ activation and consequential phosphorylation of all MAPKs. [Pt(O,O′-acac)(γ-acac)(DMS)]-induced mitochondrial apoptosis was blocked when p38MAPK and JNK1/2 were inhibited, whilst the effects on Bax/Bcl-2 mRNA and protein levels were blocked inhibiting NF-κB. NF-κB nuclear translocation was blocked inhibiting MEK1/2 activity. In addition to the induction of apoptosis [Pt(O,O′-acac)(γ-acac)(DMS)] downregulated pro-survival pathway. Survival inhibition started from mitochondrial ROS generation which induced c-src, FAK and Akt activation. In conclusion, our results suggest that [Pt(O,O′-acac)(γ-acac)(DMS)] may be considered a promising compound for the treatment of neuroblastoma. Further studies are warranted to explore in detail the therapeutic potential of this compound.

Epidermal growth factor receptor (EGFR) signal transduction pathway has been reported to play a vital role in the biologic progression of several tumours and as targets for therapeutic intervention. We have investigated the role of EGFR in the thyroid PC Cl3 cells response to the chemo-therapeutic agent cisplatin. It was found that cisplatin provoked (1) the activation (phosphorylation) and internalization of EGFR, (2) the phosphorylation of mitogen-activated protein kinase (MAPK)/p38, (3) the activation of PKC-epsilon, (4) the enhancement of matrix metalloproteinase-2 (MMP-2) expression and activity, (5) the generation of reactive oxygen species (ROS) and (6) the activation of the apoptotic intrinsic pathway. Inhibition or down regulation of EGFR reduced (1) the phosphorylation of MAPK/p38, (2) the cisplatin-provoked activation of PKC-varepsilon, and (3) the activation of caspase-7 and PARP cleavage and the overall cells sensitivity to cisplatin. PKC-varepsilon inhibition achieved by siRNA blocked MAPK/p38 activation and significantly increased the cell resistance to cisplatin. Finally, when the cisplatin-induced ROS generation was blocked by using NAD(P)H oxidase inhibitors, a decrease in cisplatin-induced MMP-2 enhancement, MAPK/p38 and EGFR activation, and caspase-7 proteolysis occurred. In conclusion, these findings supported a model in which cisplatin provokes an oxidant-induced MMP-2-dependent EGFR transactivation responsible for the induction of cell apoptosis, a process ascribable to the intracellular signalling of PKC-epsilon and MAPK/p38.

We report the cytotoxic effects obtained in HeLa cells of three newly synthesized platinum complexes containing both an O,O'-chelated acetylacetonate ligand and a sulfur ligand in the platinum coordination sphere, which show, by (1)H NMR, negligible reactivity with purine bases. These compounds induce cell death with [Pt(O,O'-acac)(gamma-acac)(DMS)] being the most effective (IC(50)=0.98+/-0.056 and 1.82+/-0.023 microM for [Pt(O,O'-acac)(gamma-acac)(DMS)] and cisplatin, respectively). About 50% of cells died after 5h treatment with 100 microM [Pt(O,O'-acac)(gamma-acac)(DMS)] whilst a 16 h incubation was required to get the same results using 100 microM cisplatin. Cellular accumulation measurements, after treatment with equimolar drug concentrations, indicated the major lipophilicity and cellular uptake of the new compounds. While the cytotoxicity of cisplatin was due to both intracellular accumulation and DNA binding, that of [Pt(O,O'-acac)(gamma-acac)(DMS)] was associated with intracellular Pt accumulation only, since it has low reactivity to DNA in intact cells and in vitro. The reaction of the new complexes with guanosine and 5'-GMP was negligible, whereas the L-methionine instantly reacted with the initial Pt complexes. Both cisplatin and [Pt(O,O'-acac)(gamma-acac)(DMS)] induced apoptosis in HeLa cells. [Pt(O,O'-acac)(gamma-acac)(DMS)] provoked the early signs of apoptosis induction (cleavage of PARP and activation of caspases-9, -3 and -7) only 1h after addition of the drug. However, in cisplatin-treated cells, cleavage of PARP was seen after 9h with activation of caspases also proceeding more slowly. In conclusion, these results indicate that the newly synthesized platinum(II) complexes have high and rapid cytotoxic activity in vitro, and suggest that DNA may not be their primary target.

[Pt(O,O'-acac)(gamma-acac)(DMS)] (PtAcD) is able to induce apoptosis in various human cancer cells, including the cisplatin-resistant human breast carcinoma MCF-7 cells. Here, to confirm that PtAcD has the potentiality for therapeutic intervention, we studied its effects in primary cultured epithelial breast cells obtained from cancers and also from the corresponding histologically proven non-malignant tissue adjacent to the tumor. We demonstrated that PtAcD (1) is more cytotoxic in cancer than in normal breast cells; (2) activated NAD(P)H oxidase, leading to PKC-zeta and PKC-alpha tanslocations; (3) activated antiapoptotic pathways based on the PKC-alpha, ERK1/2 and Akt kinases; (4) activated PKC-zeta and, only in cancer cell PKC-delta, responsible for the sustained phosphorylation of p38 and JNK1/2, kinases both of which are involved in the mitochondrial apoptotic process. Moreover, crosstalk between ERK/Akt and JNK/p38 pathways affected cell death and survival in PtAcD-treated breast cell. In conclusion, this study adds and extends data that highlight the pharmacological potential of PtAcD as an anti breast cancer drug.