Fungal vincristine from Eutypella spp-CrP14 isolated from Catharanthus roseus induces apoptosis in human squamous carcinoma cell line-A431

Background: Catharanthus roseus, a medicinal plant, is known to produce secondary metabolites, vincristine and vinblastine, which are terpenoid indole alkaloids. Previously we have reported that Eutypella spp - CrP14 isolated from stem cutting of this plant had shown significant antiproliferative activity when tested in vitro against HeLa cell line. The present study was conducted to identify the anticancer compound responsible for the anti-proliferative activity of the fungal extract and to evaluate its in vitro anticancer and apoptotic effects. Methods: The anti-proliferative activity of the fungal anticancer compound, vincristine was analyzed by MTT assay against different cancer cell lines. We examined its efficacy of apoptotic induction on A431 cells. The parameters examined included cell cycle distribution, loss of mitochondrial membrane potential (MMP), DNA fragmentation and reactive oxygen species (ROS) generation. Results: The presence of vincristine in fungal culture filtrate was confirmed through chromatographic and spectroscopic analyses, and the amount was estimated to be 53 +/- 5.0 mu g/l. The partially purified fungal vincristine had strong cytotoxic activity towards human squamous carcinoma cells - A431 in the MTT assay. Furthermore, we showed that the fungal vincristine was capable of inducing apoptosis in A431 cells through generation of reactive oxygen species and activation of the intrinsic pathway leading to loss of MMP. Conclusions: We have demonstrated for the first time that the vincristine from Eutypella spp - CrP14 is an efficient inducer of apoptosis in A431 cells, meriting its further evaluation in vivo