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A new hydrophobic platinum(IV) complex, LA-12, a very efficient anticancer drug lacking cross-resistance with cisplatin (CDDP), is now being tested in clinical trials. Here we investigated the apoptogenic activity of LA-12 and its effect on gap-junctional intercellular communication (GJIC) in the rat liver epithelial cell line WB-F344. LA-12 induced apoptosis much more efficiently than did CDDP due to a combination of rapid penetration into the cell and attack on DNA, leading to fast activation of p53 and caspase-3. Exposure of WB-F344 cells to LA-12 led to rapid induction of the time- and dose-dependent decrease in GJIC. On the molecular level, loss of GJIC induced by LA-12 was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated by the use of inhibitors of ERK activation. Inhibition of GJIC was linked to rapid hyperphosphorylation of connexin-43 and disappearance of connexon clusters from membranes, which was not observed in the case of CDDP.

Abstract: The quartz crystal microbalance (QCM) was used to construct a piezoelectric biosensor utilizing rat liver epithelial cells (WB F344) and lung melanoma cells (B16F10) as biorecognition elements. The cells adhered on the gold surface modified by either hydrophobic polystyrene or fibronectin. The simultaneous monitoring of changes in the resonant frequency Δf and the resistance ΔR corresponded with the mass and viscoelastic properties of the adhered cells. In addition, the bound cells were stained using fluorescent probes to evaluate their viability on the non-transparent gold surface. Furthermore, this piezoelectric biosensor was used for monitoring of drug-induced apoptosis initiated by micromolar concentrations of α-tocopherol amidomalate (α-TAM), a potential anticancer drug. The characteristic features of apoptosis as shrinkage and disintegration of cell–cell contacts could be monitored by QCM. The decrease of frequency and increase of resistance characterized gradual death of cells that was confirmed by fluorescence microscopy. The results indicate that the piezoelectric biosensor can be used for the real-time study of the cell adhesion and the screening of biologically active drugs affecting cellular behaviour. [Copyright &y& Elsevier]

Abstract: The vitamin E analogue α-tocopheryl succinate (α-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of α-tocopheryl maleamide (α-TAM), an esterase-resistant analogue of α-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of α-TAM towards cancer cells (MCF-7, B16F10) compared to α-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that α-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both α-TOS and α-TAM to solve the problem with cytotoxicity of free α-TAM (neurotoxicity and anaphylaxis), as well as the low solubility of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal α-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of α-TAM and α-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of α-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of α-TOS. Thus, the liposomal formulation of α-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials. [Copyright &y& Elsevier]

Abstract: The piezoelectric sensor (quartz crystal microbalance, QCM) was used to monitor cell adhesion in real time. Two cell lines, rat epithelial cells (WB F344) and lung melanoma cells (B16F10) were used. The cells were adhered and grown on the gold surface of the sensor pre-coated with adsorbed layer of extracellular matrix proteins as vitronectin and laminin. The process of cell attachment and spreading on the gold surface was continuously monitored and displayed by changes of the resonant frequency Δf and resistance ΔR values of the piezoelectric resonators. The initial phase of cell attachment and spreading induced a decrease of frequency and increase of resistance relating viscoelastic properties of the cell monolayer on the sensing surface. The steady-state of both shifts was achieved after a few hours. The presence and state of cells on the surface was confirmed by fluorescent microscopy. The obtained results demonstrate that the piezoelectric sensor is suitable for studies of the cell adhesion processes. Thus obtained cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment. [Copyright &y& Elsevier]

α-Tocopheryl succinate (α-TOS) is a semisynthetic analogue of α-tocopherol with selective toxicity to the cancer cells and anticancer activity in vivo. Yet, no suitable formulation of α-TOS for medical application has been reported. Various formulations, for example, solutions in organic solvents, oil emulsions and vesicules prepared by spontaneous vesiculation, polyethylene glycol conjugates and liposomes of various compositions have been tested. We developed and characterised a stable lyophilised liposome-based α-TOS formulation. α-TOS (15 mol%) was incorporated into large oligolamellar vesicles (OLVs) composed of soy phosphatidylcholine (SPC) by the method of lipid film hydration followed by extrusion through polycarbonate filters. Stabilised liposomal formulation was prepared by lyophilisation in the presence of sucrose (molar ratio lipid/sucrose, 1:5). The size distribution of the liposomes (130–140 nm, polydispersity index 0.14) as well as the stable lipid and α-TOS contents were preserved during storage in the lyophilised form at 2–8°C for at least 6 months. The data indicate good physical and chemical stability of the lyophilised preparation of α-TOS liposomes that can be used in clinical medicine. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2434–2443, 2010 [ABSTRACT FROM AUTHOR]

Abstract: [(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], coded as LA-12, is an octahedral platinum(IV) complex containing a bulky hydrophobic ligand – adamantylamine. The use of bulky hydrophobic amines as non-leaving ligands, may increase uptake of the compound by the cancer cells. Therefore, the effects of LA-12 on sensitive (A2780) and cisplatin resistant (A2780cis) ovarian cancer cell lines were investigated and compared to those of cisplatin. IC50 and IC90 concentrations of LA-12 were 6- (A2780) or 18-fold (A2780cis) lower than those for cisplatin (MTT assay). Equitoxic concentrations (IC50 or IC90) of both compounds caused a significant and similar time- and dose-dependent inhibition of cell proliferation and an increase in the number of floating cells which corresponded to the decrease of total cell viability. A different type and dynamics of cell cycle perturbation after cisplatin and LA-12 treatment were detected. Exposure to LA-12 resulted in transient accumulation of A2780 and A2780cis cells in S phase, while cisplatin caused G2/M arrest in sensitive and S phase arrest in resistant cells. A relatively low rate of apoptosis after exposure to IC50 or IC90 of both complexes was observed, markedly higher in resistant A2780cis cells. Western blot analysis indicated a concentration-dependent p53 level increase in both lines (higher after cisplatin treatment). PARP cleavage was observed only in A2780cis cells. In conclusion, LA-12 was found to be significantly more efficient than cisplatin, and it was able to overcome the acquired cisplatin resistance (showing resistance factor 2.84-fold lower than those for cisplatin). In spite of the low rate of apoptosis, LA-12 caused increase of p53 level and cell cycle perturbations in the ovarian cancer cell lines studied. [Copyright &y& Elsevier]