Role of epidermal growth factor receptor signaling in a Pt(II)-resistant human breast cancer cell line.

Platinum complexes are currently used for breast cancer therapy, but, as with other drug classes, a series of intrinsic and acquired resistance mechanisms hinder their efficacy. To better understand the mechanisms underlying platinum complexes resistance in breast cancer, we generated a [Pt(O,O'-acac)(γ-acac)(DMS)]-resistant MCF-7, denoted as [Pt(acac)2]R. [Pt(O,O'-acac)(γ-acac)(DMS)] was chosen as previous works showed that it has distinct mechanisms of action from cisplatin, especially with regard to cellular targets. [Pt(acac)2]R cells are characterized by increased proliferation rates and aggressiveness with higher PKC-δ, BCL-2, MMP-9 and EGFR protein expressions and also by increased expression of various genes covering cell cycle regulation, invasion, survival, and hormone receptors. These [Pt(acac)2]R cells also displayed high levels of activated signaling kinases Src, AKT and ERK/2. [Pt(acac)2]R cells incubated with [Pt(O,O'-acac)(γ-acac)(DMS)], showed a relevant EGFR activation due to PKC-δ and Src phosphorylation that provoked proliferation and survival through MERK1/2/ERK1/2 and PI3K/Akt pathways. In addition, EGFR shuttled from the plasma membrane to the nucleus maybe acting as co-transcriptional factor. The data suggest that growth and survival of resistant cells rely upon a remarkable increase in EGFR level which, in collaboration with an enhanced role of PKC-δ and Src kinases support [Pt(acac)2]R cell. It could therefore be assumed that combination treatments targeting both EGFR and PKC-δ/Src can improve therapy for breast cancer patients.

Antitumour and antiangiogenic activities of [Pt(O,O'-acac)(γ-acac)(DMS)] in a xenograft model of human renal cell carcinoma.

BACKGROUND AND PURPOSE: It is thought that the mechanism of action of anticancer chemotherapeutic agents is mainly due to a direct inhibition of tumour cell proliferation. In tumour specimens, the endothelial cell proliferation rate increases, suggesting that the therapeutic effects of anticancer agents could also be attributed to inhibition of tumour angiogenesis. Hence, we investigated the potential effects of [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(DMS)]), a new platinum drug for non-genomic targets, on human renal carcinoma and compared them with those of the well-established anticancer drug, cisplatin. EXPERIMENTAL APPROACH: Tumour growth, tumour cell proliferation and microvessel density were investigated in a xenograft model of renal cell carcinoma, developed by injecting Caki-1 cells into BALB/c nude mice. The antiangiogenic potential of compounds was also investigated using HUVECs. KEY RESULTS: Treatment of the Caki-1 cells with cisplatin or [Pt(DMS)] resulted in a dose-dependent inhibition of cell survival, but the cytotoxicity of [Pt(DMS)] was approximately fivefold greater than that of cisplatin. [Pt(DMS)] was much more effective than cisplatin at inhibiting tumour growth, proliferation and angiogenesis in vivo, as well as migration, tube formation and MMP1, MMP2 and MMP9 secretion of endothelial cells in vitro. Whereas, cisplatin exerted a greater cytotoxic effect on HUVECs, but did not affect tube formation or the migration of endothelial cells. In addition, treatment of the xenograft mice with [Pt(DMS)] decreased VEGF, MMP1 and MMP2 expressions in tumours. CONCLUSIONS AND IMPLICATIONS: The antiangiogenic and antitumour activities of [Pt(DMS)] provide a solid starting point for its validation as a suitable candidate for further pharmacological testing.

Effect of cisplatin on proteasome activity.

Cisplatin is a widely used chemotherapy drug which exerts cytotoxic activity by affecting both nuclear and cytosolic pathways. Herewith, we report, for the first time, that cisplatin inhibits proteasome activity in vitro. Cisplatin induces a dose dependent inhibition of the three enzymatic activities of proteasome (i.e., the chymotrypsin-like activity, the trypsin-like activity and the caspase-like activity). Moreover, cisplatin administration to neuroblastoma cells brings about a fast loss of proteasome particle activity, which is followed by a de novo synthesis of proteasome. Lastly, we report that the simultaneous administration of lactacystin and cisplatin enhances the cytotoxicity of cisplatin alone. The overall bulk of data opens to an intriguing scenario, concerning the biological effects of cisplatin in the control of cellular life, which goes beyond the well established genotoxic effect.

Biotechnological potential of the seaweed Cladophora rupestris (Chlorophyta, Cladophorales) lipidic extract.

Recently, with the advent of modern technologies, various marine organisms including algae are being studied as sources of natural substances effective on classical microorganisms and able to also combat the new trend of acquired resistance in microbes. In the present study the antimicrobial activity of the lipidic extract of the green seaweed Cladophora rupestris collected in a Mediterranean area, in two sampling periods (January and April), was assayed. The chemical characterization of the lipidic fractions was performed by gas-chromatography and multinuclear and multidimensional NMR spectroscopy. In the lipidic extract of C. rupestris collected in January an antibacterial activity against Enterococcus sp., Streptococcus agalactiae and Vibrio cholerae non-O1 was recorded; by contrast, bacterial inhibition was measured on several Vibrio species only in April. The fatty acid profile of C. rupestris lipidic extract, analyzed by gas chromatography, resulted mainly composed of palmitic, myristic, oleic, α linolenic, palmitoleic and linoleic acids. Moreover, since α-linolenic acid was the predominant ω3 fatty acid in April, we suggest its involvement in the antibacterial activity observed in this month, taking also into account that pure α-linolenic acid resulted effective towards some vibrios strains. C. rupestris fatty acid profile revealed also an interesting composition in polyunsaturated fatty acids in both the considered periods with the ω6/ω3 ratio lower than 1, leading to conclude that this macroalga may be employed as a natural source of ω3. Finally, the (1)H NMR spectrum in CDCl3 of algal lipid fractions showed the characteristic signals of saturated (SAFAs) and unsaturated fatty acids (UFAs) as well as other metabolites and a marked difference in free fatty acids (FFAs) content for the two examined algal lipid fractions. It is noteworthy that C. rupestris lipidic extracts show, by NMR spectroscopy, the signal pattern of polyhydroxybutyrate, a natural biocompatible and biodegradable polymer. In conclusion, on account of its antimicrobial activity, nutritional value and bioplastic content, C. rupestris lipidic extract can be considered a promising source for future biotechnological applications.

Antitumor activity of [Pt(O,O'-acac)(γ-acac)(DMS)] in mouse xenograft model of breast cancer.

The higher and selective cytotoxicity of [Pt(O,O'-acac)(γ-acac)(DMS)] toward cancer cell in both immortalized cell lines and in breast cancer cells in primary cultures, stimulated a pre-clinical study so as 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 with 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 with 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, 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 with cisplatin.

A new platinum(II) compound anticancer drug candidate with selective cytotoxicity for breast cancer cells.

[Pt(O,O'-acac)(γ-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-ζ and PKC-α translocations; (3) activated antiapoptotic pathways based on the PKC-α, ERK1/2 and Akt kinases; (4) activated PKC-ζ and, only in cancer cell PKC-δ, 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.

The lipidic extract of the seaweed Gracilariopsis longissima (Rhodophyta, Gracilariales): a potential resource for biotechnological purposes?

In recent years seaweeds increasingly attracted interest in the search for new drugs and have been shown to be a primary source of bioactive natural products including antibiotics. In the present investigation the antimicrobial activity of Gracilariopsis longissima lipidic extract was assayed and its chemical characterization was carried out by means of advanced analytical techniques such as gas-chromatography and multinuclear and multidimensional NMR spectroscopy. G. longissima lipidic extract showed an antibacterial activity against several Vibrio species. These results are interesting considering both the resistance against antibiotics developed by vibrios and the need to control fish and shellfish diseases due to vibriosis. Analysis of fatty acid methyl esters performed by gas-chromatography showed that palmitic acid methyl ester (16:0) was the predominant saturated fatty acid (42%), while, among monounsaturated fatty acids, oleic acid methyl ester (18:1) prevailed (8.5%). Because the palmitic acid represents the main component of fatty acids we hypothesized its involvement in the antibacterial activity observed. However, a pure sample of palmitic acid did not show an antibacterial activity. The fatty acid profile of G. longissima revealed also an interesting composition in polyunsaturated fatty acids and in particular the ratio of ω-3 to ω-6 fatty acids was >1 thus suggesting that this macroalga may be used as a natural source of ω3. Moreover, the (1)H NMR spectrum in CDCl(3) of algal lipid fraction shows the characteristic signals of saturated and unsaturated fatty acids as well as other metabolites. Interestingly, in the lipid extract the presence of polyhydroxybutyrate, a linear biodegradable and biocompatible polyester, was clearly identified by NMR spectroscopy. In conclusion, the lipidic extract of G. longissima on account of its antimicrobial activity, nutritional value and content in biodegradable and biocompatible polyester represents an interesting potential biotechnological resource.

Developing central nervous system and vulnerability to platinum compounds.

Comparative studies on the effects of the platinum complexes in use or in clinical trials are carried out in order to discover differences in the neurotoxic potential and the reversibility of neurotoxicity. In this paper, we summarized the current literature on neurotoxicity and chemoresistance of cisplatin (cisPt) and discussed our recent efforts on the interference of cisPt and a new platinum compound [Pt(O,O'-acac)(γ-acac)(DMS)] (PtAcacDMS), with high specific reactivity with sulphur ligands instead of nucleobases as cisPt, on some crucial events of rat postnatal cerebellum development. The acute effects of drug treatments on cell proliferation and death in the external granular layer and granule cell migration and the late effects on the dendrite growth of Purkinje cells were evaluated. Together with the demonstrated antineoplastic effectiveness in vitro, compared with cisPt, data suggest a lower neurotoxicity of PtAcacDMS, in spite of its presence in the brain that involves considerations on the blood brain barrier permeability.

[Pt(O,O'-acac)(gamma-acac)(DMS)], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway.

BACKGROUND AND PURPOSE: We showed previously that a new Pt complex containing an O,O'-chelated acetylacetonate ligand (acac) and a dimethylsulphide in the Pt coordination sphere, [Pt(O,O'-acac)(gamma-acac)(DMS)], induces apoptosis in HeLa cells. The objective of this study was to investigate the hypothesis that [Pt(O,O'-acac)(gamma-acac)(DMS)] is also cytotoxic in a MCF-7 breast cancer cell line relatively insensitive to cisplatin, and to gain a more detailed analysis of the cell death pathways. EXPERIMENTAL APPROACH: Cells were treated with Pt compounds and cytotoxicity tests were performed, together with Western blotting of various proteins involved in apoptosis. The mitochondrial membrane potential was assessed by fluorescence microscopy and spectrofluorometry and the Pt bound to cell fractions was measured by atomic absorption spectrometry. KEY RESULTS: In contrast to cisplatin, the cytotoxicity of [Pt(O,O'-acac)(gamma-acac)(DMS)] correlated with cellular accumulation but not with DNA binding. Also, the Pt content in DNA bases was considerably higher for cisplatin than for [Pt(O,O'-acac)(gamma-acac)(DMS)], thus excluding DNA as a target of [Pt(O,O'-acac)(gamma-acac)(DMS)]. [Pt(O,O'-acac)(gamma-acac)(DMS)] exerted high and fast apoptotic processes in MCF-7 cells since it provoked: (a) mitochondria depolarization; (b) cytochrome c accumulation in the cytosol; (c) translocation of Bax and truncated-Bid from cytosol to mitochondria and decreased expression of Bcl-2; (d) cleavage of caspases -7 and -9, and PARP degradation; (e) chromatin condensation and DNA fragmentation. CONCLUSIONS AND IMPLICATIONS: [Pt(O,O'-acac)(gamma-acac)(DMS)] is highly cytotoxic for MCF-7 cells, cells relatively resistant to many chemotherapeutic agents, as it activates the mitochondrial apoptotic pathway. Hence, [Pt(O,O'-acac)(gamma-acac)(DMS)] has the potential to provide us with new opportunities for therapeutic intervention.