Resveratrol induces cell-cycle disruption and apoptosis in chemoresistant B16 melanoma.

Resveratrol, a naturally occurring polyphenol, has been shown to possess chemopreventive activities. In this study, we show that resveratrol (0-500 microM) inhibits the growth of a doxorubicin-resistant B16 melanoma cell subline (B16/DOX) (IC(50) = 25 microM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G(1)-S phase transition, as demonstrated by cell-cycle analysis and down-regulation of cyclin D1/cdk4 and increased of p53 expression level. The G(1)-phase arrest of cell cycle in resveratrol-treated (10-100 microM) B16/DOX cells was followed by the induction of apoptosis, which was revealed by pyknotic nuclei and fragmented DNA. Resveratrol also potentiated at subtoxic dose (25 microM for 24 h) doxorubicin cytotoxicity in the chemoresistant B16 melanoma (P < 0.01). When administered to mice, resveratrol (12.5 mg/kg) reduced the growth of an established B16/DOX melanoma and prolonged survival (32% compared to untreated mice). All these data support a potential use of resveratrol alone or in combination with other chemotherapeutic agents in the management of chemoresistant tumors.

Enhanced immune response induced by BSA loaded in hydroxyethylstarch microparticles.

Microparticles and nanoparticles represent promising carriers for the in vivo delivery of peptides, proteins or deoxyribonucleic acid (DNA). In this study, new hydroxyethylstarch (HES) microparticles were obtained by interfacial cross-linking with terephtaloyl chloride. These microparticles exhibit the characteristics required to improve antigen release and presentation to antigen presentating cells compared to free antigens. The adjuvant activity of HES microparticles as vaccine carrier was investigated in mice using bovine serum albumin (BSA) as model antigen. We showed HES microparticles were phagocyted by peritoneal mononuclear cells. The immunization with BSA-microparticles induced antibody synthesis that was predominantly immunoglobulin G1 (IgG1). Aluminium hydroxide remained more efficient to induce IgG synthesis. The analysis of the cytokine profile from spleen cells revealed that BSA-microparticles induced the secretion of both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). However, the immune responses induced by BSA-microparticles were qualitatively and quantitatively affected by the route of injection. Taken together, these results demonstrate that HES microparticles induce a mixed T helper 1/T helper 2 response against BSA and may be a suitable delivery and presentation system in the field of vaccine development.

Immunization with liposome-anchored pegylated peptides modulates doxorubicin sensitivity in P-glycoprotein-expressing P388 cells.

The clinical use of chemotherapy in cancer treatment is limited by the occurrence of multidrug resistance (MDR) associated with the overexpression of membrane transporters, one of the best known is P-glycoprotein (Pgp), that actively expels drugs out of tumor cells. To overcome Pgp-mediated MDR, synthetic peptides corresponding to fragments from extracellular loops 1, 2 and 4 of the murine Pgp were coupled to polyethylene glycol-distearoylphosphatidylethanolamine and inserted into empty or monophosphoryl lipid A-containing liposomes. This formulation elicited specific antibodies which blocked Pgp-mediated efflux of doxorubicin, resulting in increased intracellular drug accumulation and subsequent potentiation of the cytotoxic effect of doxorubicin on multidrug-resistant P388 (P388R) cells. Previous immunizations with MDR1 peptides improved the efficiency of chemotherapy against P388R cells in vivo, with an increase of 83% of mice survival time. Overall, these results suggest that this approach can modulate Pgp activity by blocking drug efflux and may have clinical relevance as an alternative strategy to toxic chemosensitizers in drug-resistant cancer therapy.

In vivo anti-melanoma activities of the Melan-A/MART-1(101-115) T CD4+ cell peptide.

Malignant melanoma causes significant health problems. The identification of tumour-associated antigens has led to novel approaches to increase T cell mediated anti-tumour immune response. Melan-A/MART-1 has been use as target antigen for several T cell based immunotherapeutic treatments. More recently, the critical role of CD4+ T cells in inducing and maintaining anti-tumour immunity has been increasingly recognized. In order to optimize tumour immunotherapy, greater efforts have been concentrated on the identification of tumour antigens presented by MHC class II molecules to CD4+ T cells. In a publication, Tiwari et al. (2004) [1] have identified by a computational approach the 15-mer amino-acid sequence 101-115 (PPAYEKLSAEQSPPP) of the Melan-A/MART-1 as a good target for a vigorous and safe immunotherapy. Therefore, we have investigated the in vivo anti-tumour activity of this peptide in a murine melanoma model. For the prophylactic treatment, 20microg or 50microg peptide was subcutaneously injected in mice once a week during 3 weeks before tumour induction. Treatment with 50microg peptide significantly affected tumour development. Thus, our preliminary data demonstrate potential in vivo prophylactic activity of the 101-115 peptide-based vaccine to control melanoma growth.

Induction of autoantibodies to murine P-glycoprotein: consequences on drug sensitivity in MDR cancer cells and on the expression of mdr genes in organs.

Overexpression of the 170 kDa plasma membrane P-glycoprotein (P-gp) represents the most common MDR mechanism in chemotherapy. In this work, specific autoantibodies to fragments from extracellular loops 1, 2, and 4 of the murine MDR1 P-gp were elicited in mice using synthetic palmitoylated peptides reconstituted in liposomes and alum. The highest IgG level was observed after the third immunization and the immune response against lipopeptides was still detected more than 200 days after immunizations. Immunocytochemichal studies revealed that these antibodies were specific for P-gp. When incubated with P-gp-expressing MDR cell lines, serum from immunized mice restored sensitivity to either doxorubicin or vinblastine, or had no effect in a cell type specific manner, suggesting that several mechanisms may occur in the establishment of the MDR phenotype. The expression of mdr1 and mdr3 genes was unchanged in organs from mice immunized with palmitoylpeptides grafted on liposomes. These results suggest that the induction of autoantibodies to P-gp is a safe strategy to overcome MDR in cancer chemotherapy.