Claudie Madoulet

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 µM) inhibits the growth of a doxorubicin‐resistant B16 melanoma cell subline (B16/DOX) (IC50 = 25 µM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G1–S phase transition, as demonstrated by cell‐cycle analysis and down‐regulation of cyclin D1/cdk4 and increased of p53 expression level. The G1‐phase arrest of cell cycle in resveratrol‐treated (10–100 µM) 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 µM 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. J. Cell. Biochem. 110: 893–902, 2010.

Immunosuppressors as multidrug resistance reversal agents.

Multidrug-resistance (MDR) is the major reason for failure of cancer therapy. ATP-binding cassette (ABC) transporters contribute to drug resistance via ATP-dependent drug efflux. P-glycoprotein (Pgp), which is encoded by MDR1 gene, confers resistance to certain anticancer agents. The development of agents able to modulate MDR mediated by Pgp and other ABC transporters remained a major goal for the past 20 years. The calcium blocker verapamil was the first drug shown to be a modulator of Pgp, and since many different chemical compounds have been shown to exert the same effect in vitro by blocking Pgp activity. These included particularly immunosuppressors. Cyclosporin A (CSA) was the first immunosuppressor that have been shown to modulate Pgp activity in laboratory models and entered very early into clinical trials for reversal of MDR. The proof of reversing activity of CSA was found in phase II studies with myeloma and acute leukemia. In phase III studies, the results were less convincing regarding the response rate, progression-free survival, and overall survival, which were detected in advanced refractory myeloma. The non-immunosuppressive derivative PSC833 (valspodar) was subsequently developed. This compound showed tenfold higher potency in reversal of MDR mediated by Pgp. However, pharmacokinetic interactions required reductions in the dose of the concurrently administered anticancer agents. The pharmacokinetic interactions were likely because of decreased clearance of the anticancer agents, possibly as a result of Pgp inhibition in organs such as the gastrointestinal tract and kidney, as well as inhibition of cytochrome P450. Finally, CSA and PSC833 have been shown also to modulate the ceramide metabolism which stands as second messenger of anticancer agent-induced apoptosis. In fact, CSA and PSC833 are also able to respectively inhibit ceramide glycosylation and stimulate de novo ceramide synthesis. This could enhance the cellular level of ceramide and potentiate apoptosis induced by some anticancer agents.

Triterpenoid glycosides from the leaves of Meliosma henryi.

Seven triterpenoid glycosides, named meliosmosides A-G, were isolated from the leaves of Meliosma henryi Diels (Sabiaceae). Their structures were elucidated by different spectroscopic methods including 1D and 2D NMR experiments as well as HRESIMS analysis. Isolated compounds were evaluated for their cytotoxic activity against KB cell line.

Cytotoxicity and Apoptosis Induced by Alfalfa (Medicago sativa) Leaf Extracts in Sensitive and Multidrug-Resistant Tumor Cells

Alfalfa (Medicago sativa) has been used to cure a wide variety of ailments. However, only a few studies have reported its anticancer effects. In this study, extracts were obtained from alfalfa leaves and their cytotoxic effects were assessed on several sensitive and multidrug-resistant tumor cells lines. Using the mouse leukaemia P388 cell line and its doxorubicin-resistant counterpart (P388/DOX), we showed that the inhibition of cell growth induced by alfalfa leaf extracts was mediated through the induction of apoptosis, as evidenced by DNA fragmentation analysis. The execution of programmed cell death was achieved via the activation of caspase-3, leading to PARP cleavage. Fractionation of toluene extract (To-1), the most active extract obtained from crude extract, led to the identification of 3 terpene derivatives and 5 flavonoids. Among them, (-)-medicarpin, (-)-melilotocarpan E, millepurpan, tricin, and chrysoeriol showed cytotoxic effects in P388 as well as P388/DOX cells. These results demonstrate that alfalfa leaf extract may have interesting potential in cancer chemoprevention and therapy.

Accumulation of lactosylceramide and overexpression of a PSC833-resistant P-glycoprotein in multidrug-resistant human sarcoma cells

The selection pressure for resistance to chemotherapy is accompanied by the enhanced expression of ABC proteins and increased cellular glycosphingolipid content. Thus, a possible connection between glycosphingolipid metabolism and ABC proteins in drug resistance has been suggested. In the present study, we established two human multidrug-resistant (MDR) cell lines derived from MESSA sarcoma cells by culturing with increasing concentrations of doxorubicin (DX5 cells) or doxorubicin together with cyclosporin A (GARF cells). Both resistant cell lines overexpressed the MDR1 gene and the wild-type P-glycoprotein at the same level. The cyclosporin derivative PSC833, a potent inhibitor of P-glycoprotein, sensitized DX5 but not GARF cells to the cytotoxic effects of daunorubicin. Moreover, PSC833 increased the nuclear accumulation of daunorubicin and the cellular accumulation of [3H]vinblastine in the DX5 but not in the GARF cells. The cellular incorporation of [3H]-cyclosporin A was lower in DX5 cells compared to MESSA and GARF cells, which incorporated the same level of [3H]-cyclosporin A. Sphingolipid analysis showed that the lactosylceramide level was 2.5- and 5-fold higher in DX5 and GARF cells, respectively, than in MESSA cells. Whereas the pharmacological inhibition of lactosylceramide synthesis was able to reverse only partially the resistance of GARF cells to daunorubicin without significant increase in nuclear accumulation of the drug, the same treatment before the co-treatment with PSC833 and daunorubicin increased the cytotoxic effect of daunorubicin and its nuclear accumulation. These data suggest a possible relationship between lactosylceramide levels and the resistance of P-glycoprotein to modulation by MDR modulators.

In vivo anti-melanoma activities of the Melan-A/MART-1101–115 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.