Anti-Cancer Effects of Protein Extracts fromCalvatia lilacina,Pleurotus ostreatusandVolvariella volvacea

Calvatia lilacina(CL),Pleurotus ostreatus(PO) andVolvariella volvacea(VV) are widely distributed worldwide and commonly eaten as mushrooms. In this study, cell viabilities were evaluated for a human colorectal adenocarcinoma cell line (SW480 cells) and a human monocytic leukemia cell line (THP-1 cells). Apoptotic mechanisms induced by the protein extracts of PO and VV were evaluated for SW480 cells. The viabilities of THP-1 and SW480 cells decreased in a concentration-dependent manner after 24 h of treatment with the protein extracts of CL, PO or VV. Apoptosis analysis revealed that the percentage of SW480 cells in the SubG1phase (a marker of apoptosis) was increased upon PO and VV protein-extract treatments, indicating that oligonucleosomal DNA fragmentation existed concomitantly with cellular death. The PO and VV protein extracts induced reactive oxygen species (ROS) production, glutathione (GSH) depletion and mitochondrial transmembrane potential (ΔΨm) loss in SW480 cells. Pretreatment withN-acetylcysteine, GSH or cyclosporine A partially prevented the apoptosis induced by PO protein extracts, but not that induced by VV extracts, in SW480 cells. The protein extracts of CL, PO and VV exhibited therapeutic efficacy against human colorectal adenocarcinoma cells and human monocytic leukemia cells. The PO protein extracts induced apoptosis in SW480 cells partially through ROS production, GSH depletion and mitochondrial dysfunction. Therefore, the protein extracts of these mushrooms could be considered an important source of new anti-cancer drugs.

Proliferation and differentiation properties of permanent Schwann cell lines immortalized with a temperature-sensitive oncogene.

Permanent Schwann cells lines have been established in culture after stable transfection of newborn rat Schwann cells with the pJC-SVLTtsA vector, expressing a thermosensitive oncogene driven by the early promoter-enhancer region of the gliotropic GS/B variant of the papovavirus JC. The proliferation and differentiation of two clonal cell lines have been studied. The cells of these lines display the morphology of primary Schwann cells and express Schwann cell differentiation markers such as the S-100 protein, laminin, the low-affinity receptor to nerve growth factor and the glial fibrillary acidic protein. One of the lines is able to differentiate further. Indeed, in the presence of dorsal root ganglion neurones, the cells synthesize the myelin Po protein and are capable of some myelination, although to a lesser extent than secondary Schwann cells.

Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain.

The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when coexpressed with truncated Po, appears to cluster in the membrane. We suggest that truncated Po exerts its dominant-negative effect by preventing clustering of full-length Po. We also show that colchicine, which disrupts microtubules, prevents adhesion of cells expressing only the full-length Po. This strengthens our suggestion that an interaction of Po with the cytoskeleton, either directly or indirectly, is required for adhesion to take place.