For example, Hh signaling is often activated in melanomas and might have a critical role in determining the stem-like characteristics of melanoma initiating cells, contributing to the acquisition of a more undifferentiated and aggressive state through a process similar to reprogramming [48, 49]. cancer cells to some chemotherapeutic agents [25]. Indeed, human Patched expressed in yeast conferred resistance to several ACX-362E chemotherapeutic agents used to treat metastatic cancers (doxorubicin, methotrexate, temozolomide, 5-FU) and increases doxorubicin efflux. This yeast model has been extended to fibroblasts (often used to study Hh signaling) and human cancer cell lines which endogenously express Patched such as melanoma cell lines. The presence of Shh, the ligand of Patched which induces Patched internalization and degradation, was shown ACX-362E to increase the accumulation of doxorubicin into these cells and its cytotoxicity. Altogether, these results suggest that the Hh receptor Patched participates to chemotherapy resistance, and they prompted us to propose Patched as a new target for anti-cancer therapy. Discovering compounds able to inhibit the drug efflux activity of Patched would then lead to an increase in the efficiency of chemotherapy and thus to a reduction of the risk of metastasis and recurrence for patients with cancers expressing Patched. We then generated innovative yeast- and cell-based screenings to identify molecules able to inhibit the drug efflux activity ACX-362E of Patched. For this purpose, we decided to screen natural compounds produced by marine sponges commonly found in the Mediterranean Sea. Indeed, sponge natural products have already been identified as promising and original leads for therapeutic applications [26C28], and the high biodiversity ACX-362E of marine sponges growing in the diverse Mediterranean ecosystems is a guarantee of a large chemodiversity, enabling us to explore a significant volume of a bioactive chemical space. In the present study, we show that four known paniceins isolated from the species [29] significantly inhibit the resistance to the chemotherapeutic agent doxorubicin of yeast expressing human Patched. One of these compounds, namely panicein A hydroquinone (1), enhances the accumulation and the cytotoxicity of doxorubicin for two melanoma cell lines, and we show that these effects are due to the inhibition of Patched doxorubicin efflux activity. RESULTS Paniceins isolated from the sponge are inhibitors of the resistance to doxorubicin of yeast expressing Patched In a previous study, we showed that the expression of human Patched allowed yeast to grow in the presence of a concentration of doxorubicin (dxr) that inhibits the growth of control yeast, indicating that Patched confers resistance to dxr [25]. From these results, we developed a screening test in ACX-362E 96-well plates to identify compounds capable of inhibiting the resistance of yeast expressing human Patched to dxr. First, the methanolic fractions of fifteen representative Northwestern Mediterranean Mouse monoclonal to CD45RA.TB100 reacts with the 220 kDa isoform A of CD45. This is clustered as CD45RA, and is expressed on naive/resting T cells and on medullart thymocytes. In comparison, CD45RO is expressed on memory/activated T cells and cortical thymocytes. CD45RA and CD45RO are useful for discriminating between naive and memory T cells in the study of the immune system sponges ([30], completely repressed the growth of yeast expressing Patched even in the absence of dxr and appeared to be cytotoxic for yeast. The methanolic fractions obtained from and were the only that clearly inhibit the growth of yeast expressing Patched in the presence of dxr without significant effect in the absence of dxr. We decided to focus our study on the methanolic fraction obtained from (Figure ?(Figure2a)2a) which significantly inhibited the resistance of yeast expressing Patched to dxr with only a small effect on basal yeast growth (in the absence of dxr) (Figure ?(Figure2b).2b). In order to identify the compounds responsible for this bioactivity, the methanolic fraction obtained from was purified by C18 preparative HPLC to yield 9 peaks (P1-P9) (Supplementary Figure 1). Compounds present in these collected peaks were added singly to the yeast growth medium in the presence or in the absence of dxr. Five of these peaks (namely P1, P3, P4, P6 and P7) were shown to strongly inhibit the resistance to dxr of yeast expressing Patched (Figure ?(Figure2c).2c). The effects of P2 and P9 on yeast growth were lower, and P8 had no effect. Surprisingly,.
