Synthesis and evaluation of apoptosis induction of thienopyrimidine compounds on KRAS and BRAF mutated colorectal cancer cell lines.

Monoclonal antibodies (MoAb) and tyrosine kinase inhibitors (TKI) targeting the EGFR (Epidermal Growth Factor Receptor) pathways are currently used in colorectal cancer treatment. Despite the improvement of median overall survival, resistance is observed notably due to KRAS and BRAF gene mutations. We synthesized four series of thienopyrimidines whose scaffold is structurally close to TKI used in clinical practice. We evaluated apoptosis induced by these compounds using flow cytometry on KRAS and BRAF mutated cell lines. Our results confirm that the mutated cell lines (HCT116 and HT29) are more resistant to apoptosis than the non-mutated cell line (Hela). Interestingly, among the 13 compounds tested, three of them (5b, 6b and 6d) and gefitinib exhibited a noteworthy pro-apoptotic effect, especially on mutated cell lines with an IC(50) value between 70 and 110µM. These three compounds seem particularly attractive for the development of novel treatments for colorectal cancer patients harboring EGFR pathway mutations.

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.

Interindividual differences in anticancer drug cytotoxicity in primary human glioblastoma cells.

Glioblastoma multiforme is a malignant astrocytic tumor characterized by rapid growth, extensive invasiveness and high vascularity. Despite advances in surgical techniques and in the development of new protocols in radio- and chemotherapy, the prognosis for patients suffering from this malignancy remains poor. Since the clinical response to chemotherapy varies greatly owing to different interindividual gene expression profiles, it would be of considerable interest to develop an in vitro model able to evaluate anticancer drug toxicity and the effectiveness of therapeutic strategies on cells obtained from individual patients. In the protocol for obtaining primary cultures of glioblastoma cells described in this report, a confluent monolayer of cells can be obtained within 1 or 2 weeks. A complementary immunocytochemical assay using glial fibrillary acidic protein (GFAP) to reliably mark glial cells confirms the glial origin of the cultured cells. A cytotoxicity test based on mitochondrial activity is then used to evaluate in vitro drug efficacy. Cell dedifferentiation as evidenced by loss of GFAP expression after a few passages requires determination of drug toxicity before the fourth passage. Data show a wide range of response to temozolomide (1000 microM) after 72 h with 24-81% cell death depending on patients. Results presented confirm the heterogeneity of response to anticancer drugs between the patients and methods described allow to carry out cytotoxicity studies in order to determine the individualized most effective treatment.

Synthesis and study of antiproliferative activity of novel thienopyrimidines on glioblastoma cells.

The receptor tyrosine kinases (for example EGFR, PDGFR, VEGFR) are a transmembrane protein family which plays a crucial role in tumor growth, survival, metastasis dissemination and angiogenesis. During the past 10 years, many tyrosine kinase inhibitors (TKIs) have been approved for cancer treatment (imatinib, gefitinib, erlotinib, sunitinib, sorafenib). These compounds generally possess a pyrrolo- or pyrimido- pyrimidine scaffold or approaching molecular structure. We synthesized 10 thienopyrimidine compounds (including 5 newly synthesized) whose scaffold is very similar to the agents cited above. The cytotoxicity of these agents was evaluated using a MTT assay and a flow cytometry technique on glioblastoma cell lines. Two compounds showed a similar cytotoxicity to the standard anti-EGFR gefitinib (IC50: gefitinib=51.9 microM, 6b=61.8 microM, 6c=41.2 microM), suggesting a blockade of the EGFR pathway by binding to the TK receptor.