Induction of apoptosis in Ehrlich ascites tumour cells via p53 activation by a novel small-molecule MDM2 inhibitor - LQFM030.

OBJECTIVE: The activation of the p53 pathway through the inhibition of MDM2 has been proposed as a novel therapeutic strategy against tumours. A series of cis-imidazoline analogues, termed nutlins, were reported to displace the recombinant p53 protein from its complex with MDM2 by binding to MDM2 in the p53 pocket, and exhibited an antitumour activity both in vitro and in vivo. Thus, the purpose of this study was to evaluate the antitumour properties of LQFM030 (2), a nutlin analogue created by employing the strategy of molecular simplification. METHODS: LQFM030 (2) cytotoxicity was evaluated in Ehrlich ascites tumour (EAT) cells, p53 wild type, by the trypan blue exclusion test, and the mechanisms involved in EAT cell death were investigated by light and fluorescence microscopy, flow cytometry, real-time PCR and Western blotting. KEY FINDINGS: Our results demonstrate that LQFM030 has dose-dependent antiproliferative activity and cytotoxic activity on EAT cells, induces the accumulation of p53 protein and promotes cell cycle arrest and apoptosis. p53 gene transcription was unaffected by LQFM030 (2); however, MDM2 mRNA increased and MDM2 protein decreased. CONCLUSIONS: These results suggest that the small-molecule p53 activator LQFM030 (2) has the potential for further development as a novel cancer therapeutic agent.

Liposomal Entrapment of 4-Nerolidylcatechol: Impact on Phospholipid Dynamics, Drug Stability and Bioactivity.

Liposomes containing 4-nerolidylcatechol (4-NC), the major metabolite isolated from Pothomorphe umbellata, were obtained and characterized. Influence of liposomal encapsulation on chemical stability of 4-NC and on cytotoxicity profile of this drug was evaluated. Soybean phosphatidylcholine liposomes were prepared by lipid film hydration followed by extrusion. Entrapment efficiency for 4-NC was approximately 92%. Mean diameter of liposomes was 100 nm with a polydispersity index below 0.13. Liposomal 4-NC (L4-NC) and free drug (F4-NC) were submitted to forced degradation assays, monitored by HPLC. Photodegradation assay followed ICH Guidelines, using a photostability chamber equipped with both UV and white light sources. Liposomal encapsulation was able to markedly reduce 4-NC degradation rates under all the conditions tested. L4-NC showed a half-live approximately 15% higher than F4-NC under light exposure. After 72 hours, acid and base hydrolysis of F4-NC lead to 13 and 16% of degradation, respectively. However, no degradation was observed in L4-NC. EPR spectra of liposomal membrane showed that greatest changes in membrane properties were obtained when 5-doxyl stearic acid was used as the spin label, indicating a marked decrease in the fluidity of the bilayer. Following incubation with K562 cells, 4-NC showed a concentration-dependent cytotoxicity profile, while L4-NC exhibited a time and concentration-dependent profile, consistent with a controlled drug release system. F4-NC induced extensive hemolysis under isotonic conditions; conversely liposomal encapsulation protected erythrocytes from 4-NC induced lysis. Liposomal 4-NC resulted in a hemocompatibility and stable formulation, representing a viable drug delivery system to further investigate in vivo performances of 4-NC in pre clinical studies.

Antitumoral and antiangiogenic activity of Synadenium umbellatum Pax.

AIM OF THE STUDY: Synadenium umbellatum Pax (SU), a plant used in the Midwestern region of Brazil, was tested for its antitumor and antiangiogenic activities in vitro, using K-562 and Ehrlich ascites tumor (EAT) cells, and in vivo, using the EAT-bearing model. MATERIALS AND METHODS: The viability of tumor cells was evaluated by MTT and trypan blue exclusion assays, after incubation with the ethanolic extract of SU (EESU) (0.15-20mg/mL) or equivalent concentrations of its partitioned fractions (chloroformic, hexanic, and methanolic). In vivo studies were performed in EAT-bearing mice treated intraperitoneally with 5, 10, and 25mg/kg of the EESU or equivalent doses of the fractions for 10 days. The methotrexate (1.5mg/kg), for 10 days, was used as control. RESULTS: SU and fractions, except the methanolic, decreased the viability of the cells in a concentration-dependent manner. In vivo results showed a significant dose-dependent antitumoral efficacy of SU against EAT growth. The best results in prolonging life span were produced by 25mg/kg of EESU. In these animals, the levels of vascular endothelial growth factor were markedly decreased after the treatment. CONCLUSIONS: The data presented herein could open interesting perspectives for further research of SU as a candidate anticancer agent.