Antitumor activities of substances from natural sources apart from vascular plants and micro-organisms have been poorly investigated. Here we report on a pharmacological screening of a bryophyte extract library using a phenotypic cell-based assay revealing microtubules, centrosomes and DNA. Among the 219 moss extracts tested, we identified 41 extracts acting on cell division with various combinations of significant effects on interphasic and mitotic cells. Seven extracts were further studied using a cell viability assay, cell cycle analysis and the phenotypic assay. Three distinct pharmacological patterns were identified including two unusual phenotypes.
The search of new pharmacological targets with original mechanism of action within the ubiquitin-proteasome pathway is still a goal to be reached in oncopharmacology. Modification by phosphorylation/dephosphorylation has been found to be involved in cancer and to regulate functional activity of proteasome. Until now, phosphorylated forms of alpha subunits of the 20S human proteasome have been mostly reported. Here, we have rationally designed a polyclonal antibody specifically directed against a phosphorylated peptide sequence bearing the beta7 subunit Ser249 residue of the human 20S proteasome. This anti-beta7 phosphoSer249 antibody appeared to be a probe of choice to detect the presence of a phosphorylated isoform of the beta7 subunit of the human 20S proteasome using mono or two-dimensional gel electrophoresis. PhosphoSer249 was sensitive to acid phosphatase treatment of native 20S proteasome. Dephosphorylation affected the peptidylglutamyl-peptide hydrolyzing activity whereas the chymotrypsin-like and trypsin-like activities remained unchanged. A comparative analysis between human normal and tumor cells showed a differential expression of the phosphoSer249 beta7 isoform with a significantly lower detection in the proteasome isolated from tumor cells, suggesting its possible use as a biomarker.
Cell surface expression of MHC class I molecules by tumor cells is determinant in the interplay between tumor cells and the immune system. Nevertheless, the mechanisms which regulate MHCI expression on tumor cells are not clear. We previously showed that immune innate cells from the spleen can regulate MHCI expression on MHCI(low) tumor cells. Here, using the murine model of B16 melanoma, we demonstrate that the MHCI status of tumor cells in vivo is regulated by the microenvironment. In subcutaneous grafts, induction of MHCI molecules on tumor cells is concomitant to the recruitment of lymphocytes and relies on an IFNgamma-mediated mechanism. gammadelta T and NK cells are essential to this regulation. A small proportion of tumor-infiltrating NK cells and gammadelta T cells were found to produce IFNgamma, suggesting a possible direct participation to the MHCI increase on the tumor cells upon tumor cell recognition. Depletion of gammadelta T cells increases the tumor growth rate, confirming their anti-tumoral role in our model. Taken together, our results demonstrate that in vivo, NK and gammadelta T cells play a dual role during the early growth of MHCI(low) tumor cells. In addition to controlling the growth of tumor cells, they contribute to modifying the immunogenic profile of residual tumor cells.
Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5(+) cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.
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