The new platinum(IV) derivative LA-12 shows stronger inhibitory effect on Hsp90 function compared to cisplatin.

BACKGROUND: Cisplatin and its derivatives are commonly used anti-cancer drugs. However, cisplatin has clinical limitations including serious side effects and frequent emergence of intrinsic or acquired resistance. Thus, the novel platinum(IV) complex LA-12 represents a promising treatment modality, which shows increased intracellular penetration resulting in improved cytotoxicity in various cancer cell lines, including cisplatin resistant cells. RESULTS: LA-12 disrupts cellular proliferation regardless of the p53 status in the cells, however the potency of the drug is greatly enhanced by the presence of a functional p53, indicating several mechanisms of action. Similarly to cisplatin, an interaction of LA-12 with molecular chaperone Hsp90 was proposed. Binding of LA-12 to Hsp90 was demonstrated by Hsp90 immunoprecipitation followed by platinum measurement using atomic absorption spectrometry (AAS). An inhibitory effect of LA-12 on Hsp90 chaperoning function was shown by decrease of Hsp90-assisted wild-type p53 binding to p21WAF1 promoter sequence in vitro and by accelerated ubiqutination and degradation of primarily unfolded mutant p53 proteins in cells exposed to LA-12. CONCLUSIONS: To generalize our findings, LA-12 induced degradation of other Hsp90 client proteins such as Cyclin D1 and estrogen receptor was shown and proved as more efficient in comparison with cisplatin. This newly characterised molecular mechanism of action opens opportunities to design new cancer treatment strategy profitable from unique LA-12 properties, which combine DNA damaging and Hsp90 inhibitory effects.

The effect of cellular environment and p53 status on the mode of action of the platinum derivative LA-12.

In this study, we characterized the effects of LA-12 on tumor cell lines possessing wild type p53 and on p53-deficient/mutant cell lines and the results were compared to those obtained using cisplatin. We have determined changes of p53 levels, of its transcriptional activity, of its posttranscriptional modifications and the effect of the treatment on the cell cycle, on the induction of apoptosis and on gene expression. LA-12 induces weak accumulation of both transcriptionally active p53 tumor suppressor and of p21(WAF1/CIP1) protein. LA-12 and cisplatin also significantly differ in their effects on apoptosis and cell cycle and on gene expression spectra in studied cell lines. LA-12 induces higher apoptosis levels in comparison with those induced by cisplatin, especially in p53-deficient H1299 cells and in MCF-7DD cells with transcriptionally inactive p53. We suggest that LA-12-mediated apoptosis is not fully dependent on p53. This confirms the therapeutic potential of LA-12 as a more potent cytostatic agent for both tumor cells expressing wild type p53 and for p53-deficient or mutant cells.

Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles.

BACKGROUND: Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects. METHODS: A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 - 10(9) copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively. RESULTS: Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242-292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes. CONCLUSION: Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.

The novel platinum(IV) complex LA-12 induces p53 and p53/47 responses that differ from the related drug, cisplatin.

The platinum(II)-based complex cisplatin is one of the most frequently used antitumour agents; however, a high incidence of harmful side effects and the frequent emergence of acquired resistance are the major clinical problems. The novel platinum(IV)-based complex LA-12 exhibits a high efficacy against cancer cell lines, including cisplatin-insensitive cells, but the mechanisms by which LA-12 perturbs cell growth are unclear. We tested the effects of LA-12 on the p53 response and demonstrate that LA-12 induces unique changes in the profile of gene expression compared with cisplatin and doxorubicin. Furthermore, the ability of LA-12 to disrupt cellular proliferation is greatly enhanced by the expression of p53 and p53/47 indicating both p53-dependent and p53-independent effects of LA-12. Exposure of the human cancer cell lines H1299, A2780, BT549 and BT474 to LA-12 alters the expression of p53 and p53/47 in both a time-dependent and dose-dependent manner. Treatment of cells with a low concentration of the drug results in accumulation of p53 and p53/47 concomitant with their posttranslational modification, whereas a high dose results in the disappearance of both the forms of p53. The distinct p53 activation profile of LA-12 compared with cisplatin and doxorubicin provides a molecular explanation for the ability of this drug to treat cisplatin-resistant cells and indicates its potential usefulness as an alternative antitumour agent in first-line therapy or as a second-line therapy in patients with acquired cisplatin resistance.

New platinum(IV) complex with adamantylamine ligand as a promising anti-cancer drug: comparison of in vitro cytotoxic potential towards A2780/cisR cisplatin-resistant cell line within homologous series of platinum(IV) complexes.

The aim of this study was to compare anti-tumor potency of platinum(IV) complexes with increasing hydrophobicity of their ligands. Cytotoxic potential of the new platinum(IV) complex, coded as LA-12 [(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], was compared within the series of complexes of the general formula (OC-6-43)-bis(acetato)(alkylamine)amminedichloroplatinum(IV). Alkylamine ligands with increasing hydrophobicity were: isopropylamine, cyclohexylamine, 1-adamantylamine and 3,5-dimethyl-1-adamantylamine. Particular platinum(IV) complexes were coded as LA-4, LA-2 (known as JM-216), LA-12 and LA-15, respectively. Cytotoxicity was tested with the microplate tetrazolium (MTT) assay on the panel of cancer cell lines and the results were verified by microscopy. HPLC was used to measure hydrophobicity, stability of complexes in various buffers and velocity constants for their reactivity with glutathione. Platinum(IV) complexes with bulky hydrophobic ligands (LA-12 and LA-15) demonstrated about one order higher velocity constant for pseudo-first-order reaction with glutathione in comparison to cisplatin, LA-4 and LA-2, whose velocity constants were close to those measured for cisplatin and related platinum(II) complexes. Cytotoxicities of LA-12 and LA-15 towards cisplatin-resistant epithelial carcinoma A2780/cisR were superior to cisplatin, LA-4 and LA-2 in both 24- and 72-h continuous exposure MTT tests. Rapid induction of apoptosis in the treated cancer cell lines and no cisplatin cross-resistance were found for LA-12, which is a candidate for clinical testing.