Anticancer drugs exert differential apoptotic and cytotoxic effects on glioblastoma primary cultures with various EGFR and bcl-2 profiles.

The aim of this study was to determine the apoptotic and cytotoxic effects induced on glioblastoma cells by various anticancer agents that possess different mechanisms of action (alkylating drugs, anti-EGFR (Epidermal Growth Factor receptor), proteasome inhibitor). Primary cell cultures were obtained from patients who underwent surgery for their glioblastoma. The cytotoxic effects of drugs were determined by MTT (dimethylthiazolyl diphenyl tetrazolium bromide) assay and apoptosis was evaluated by measuring mitochondrial potential by flow cytometry. Biological markers (EGFR, bcl-2) were studied by a immunoblotting technique to find out predictive markers of response. We found a large interindividual sensitivity, thus confirming the interest of the primary cultures. New proteasome inhibitor bortezomib had considerable cytotoxic and apoptotic potential in glioblastoma, even at very low concentrations. Moreover, the characterization of patients' cells for EGFR and bcl-2 status could constitute an interest, with the evaluation of other markers, in the study of expected chemotherapy response.

Cytotoxic and apoptotic effects of bortezomib and gefitinib compared to alkylating agents on human glioblastoma cells.

Glioblastoma is a malignant astrocytic tumor with a median survival of about 12 months for which new therapeutic strategies are required. We therefore examined the cytotoxicity of anticancer drugs with different mechanisms of action on two human glioblastoma cell lines expressing various levels of EGFR (epidermal growth factor receptor). Apoptosis induced by these anticancer agents was evaluated by flow cytometry. The cytotoxicity of alkylating drugs followed a dose-effect curve and cytotoxicity index values were lower with carboplatin than with BCNU and temozolomide. Anti-EGFR gefitinib (10 microM) cytotoxicity on DBTRG.05-MG expressing high levels of EGFR was significantly higher than on U87-MG expressing low levels of EGFR. Carboplatin and temozolomide cytotoxicity was potentiated with the addition of gefitinib on DBTRG.05-MG. Among the anticancer agents tested, the proteasome inhibitor bortezomib was the most cytotoxic with very low IC50 on the two cell lines. Moreover, all anticancer drugs tested induced apoptosis in a concentration-dependent manner. Bortezomib proved to be a more potent inductor of apoptosis than gefitinib and alkylating agents. These results show the efficacy of bortezomib and of the association between conventional chemotherapy and gefitinib on glioblastoma cells and therefore suggest the interest of these molecules in the treatment of glioblastoma.

ICP/OES application for assessing cadmium uptake (or toxicity) in glomerular cells: influence of extracellular calcium.

The risks of metals for health are highlighted by their chemical stability and their persistence in the environment. Chronic exposure to low cadmium (Cd) concentrations results in renal dysfunction mainly. Cd has been regarded primarily as a renal tubular toxicant, but glomerular structures may also be affected. Since the cellular environment may influence metal toxicity, differences concerning Cd uptake and toxicity were evaluated according to calcium (Ca) medium concentrations. An optimized inductively coupled plasma emission spectrometry method (ICP/OES) was developed under defined conditions, as a selective analytical tool to determine cadmium uptake in glomerular mesangial cells. The performance characteristics of the analytical system were evaluated for both Cd and Ca by calibration (50 to 250 microg/L and 1 to 5 mg/L), linearity (r2 .9968 and .9943), limits of detection (1 microg/L and 0.1 mg/L) and quantitation (3 microg/L and 0.3 mg/L), accuracy with spiking, and repeatability (1.2 and 2.9%) with matrix matched standards. Total intracellular Cd content was significantly threefold lower in 0.175 mM Ca medium (Ca-free Eagle's minimum essential medium [EMEM] medium with 5% fetal bovine serum [FBS]) than in EMEM medium (1.8 mM Ca) with respectively 0.16 and 0.37 microg/mg proteins after 24 h of Cd (1 microM) exposure. Similar differences were obtained in cytotoxicity studies with a fourfold reduction in the mortality index (IC50). Complementary assays using Ca-spiked medium reinforced that Cd cytotoxicity and uptake were significantly dependent on the concentration of extracellular Ca. These findings suggest direct link between Cd uptake and toxicity, underlining the relevance of the analytical method.

O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

Glutathione S-transferase M1 and multidrug resistance protein 1 act in synergy to protect melanoma cells from vincristine effects.

Previous studies have shown that glutathione S-transferases (GSTs) can operate in synergy with efflux transporters, multi-drug resistance proteins (MRPs), to confer resistance to several carcinogens, mutagens and anticancer drugs. To address the poorly documented role of the GSTM1 in cancer chemoresistance, we used CAL1 human melanoma cells expressing no endogenous GSTM1 and a high level of MRP1. Cells were transfected with an expression vector containing the GSTM1 cDNA, and different clones were selected expressing different levels of GSTM1 (RT-PCR, Western blot, and enzyme activity). Cells overexpressing GSTM1 displayed a 3- to 4-fold increase in resistance to anticancer drugs vincristine (VCR) and chlorambucil (CHB) in proliferation, cytotoxic, and clonogenic survival assays. Inhibitors of MRP1 (sulfinpyrazone, verapamil) and GST (dicumarol, curcumin) completely reversed the GSTM1-associated resistance to VCR, indicating that a MRP efflux function is necessary to potentiate GSTM1-mediated resistance to VCR. Conversely, MRP1 inhibitors had no effect on the sensitivity to CHB. Using immunofluorescence assay, GSTM1 was also shown to protect microtubule network integrity from VCR-induced inhibition of microtubule polymerization. In conclusion, these results show that GSTM1 alone is involved in melanoma resistance to CHB, whereas it can act in synergy with MRP1 to protect cells from toxic effects of VCR.

Cytotoxicity, DNA damage, and apoptosis induced by new fotemustine analogs on human melanoma cells in relation to O6-methylguanine DNA-methyltransferase expression.

Fotemustine is a third generation chloroethylnitrosourea that has demonstrated significant antitumoral effects in malignant melanoma. However, its use is somewhat limited by its toxic side effects and chemoresistance caused by direct repair of O6-alkyl groups by the enzyme O6-methylguanine DNA-methyltransferase (MGMT). The aim of this work was to determine to what extent the expression of MGMT influences cytotoxicity, DNA damage, and apoptosis induced by new nitrososulfamide analogs of fotemustine (compounds 4 and 8), which have previously demonstrated interesting antiproliferative properties. We carried out complementary strategies that consisted of MGMT cDNA transfection in CAL77 Mer- melanoma cells and of MGMT inhibition with O6-benzylguanine (BG) in A375 Mer+ melanoma cells. MGMT-transfected cells were 7 to 9 times less sensitive to fotemustine than parent cells, whereas no difference between the transfected and parent cells was observed for nitrososulfamide analogs. The cytotoxicity of these analogs vis à vis a MGMT-proficient A375 melanoma cell line was approximately 3 times greater than that of fotemustine. Coincubation of these cells with O6-benzylguanine significantly increased the cytotoxicity of fotemustine and compound 8, whereas BG had little effect on the cytotoxicity of compound 4. Furthermore, DNA fragmentation determined by a comet assay was greater with nitrososulfamide analogs than with fotemustine. O6-benzylguanine increased DNA fragmentation for fotemustine and compound 8, but not for compound 4, which induced comets with a typical apoptotic appearance. The ability of this compound to induce apoptosis in the absence of BG was confirmed by a specific enzyme-linked immunosorbent assay apoptotic assay using a single-stranded DNA monoclonal antibody.

Synthesis and biological evaluation of Fotemustine analogues on human melanoma cell lines.

Two new analogues of Fotemustine have been synthesized and tested on two melanoma cell lines. Compounds 4 and 8 proved to be more potent than the reference compound on A375 cell line which express the MGMT enzyme involved in the chemoresistance of tumoral cells.