In vitro cytotoxic activity of two potential anticancer drugs isolated from Strychnos: strychnopentamine and usambarensine.

The cytotoxicity and the selective antiprotozoal activity of some Strychnos alkaloids, namely strychnopentamine (SP) and usambarensine (US) (7) led us to analyze and compare their effects with emetine (EM) by using mouse B16 melanoma cells cultivated in vitro. We observed by cytological analysis and proliferation rate studies that these substances induce analogous cytotoxic effects in B16 cells, but at different concentrations i.e. formation of lamellar bodies in the cytoplasm, the which contain pre-melanosomes in the case of SP and US, vacuoles and blebs. At concentrations near their respective IC50, SP and US, but not EM, decreased colony formation. We showed by incorporation of labelled precursors that SP and US first inhibit RNA synthesis while EM initially acts on protein synthesis. These alkaloids increased melanin synthesis. Furthermore, only EM and SP caused hemolysis of sheep red blood corpuscles. This could explain why the rate of antiplasmodial activity is higher for SP and EM.

Characterization and tumorigenicity of spheroids composed of pigmented or non pigmented B16 melanoma cells.

A parental line of mouse B16 melanoma cells (B16) and two derived cloned lines, either pigmented (B16P) or non pigmented (B16NP), were cultured in vitro as spheroids. After 48 hrs, the pigmented cells (B16, B16P) formed smaller and looser aggregates, with higher rates of cell proliferation and lower amounts of extracellular matrix as compared to B16NP spheroids. The three lines were more tumorigenic when inoculated subcutaneously as spheroids than as isolated cells. Furthermore, B16P or B16 spheroids developed richly vascularized subcutaneous tumors and metastases more rapidly than B16NP aggregates. After intravenous injection of spheroids, the measurement with an image analyzer of the area of sections in lung colonies indicated that B16P colonies were larger and more numerous than those induced by B16NP cells.

Laminin and 67 kD laminin binding protein in mouse B16 melanoma cells and 3T3 fibroblast spheroids.

Multicellular spheroids which promote cell-cell and cell-matrix interactions were prepared in culture with mouse B16 melanoma cells (pigmented or non pigmented) alone or mixed with mouse 3T3 fibroblasts. Their volume and proliferation or necrosis rate were evaluated. As measured by dot blot immunoassay, laminin was mainly produced by fibroblasts rather than by melanoma cells. High levels of laminin B1 chain mRNA were detected only in spheroids composed of 3T3 fibroblasts. The levels of 67 kD laminin binding protein mRNA were high in all cell populations studied here.