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  • br Fig Schematic illustration for


    Fig. 10. Schematic illustration for possible mechanism of WSPF mediated apoptosis in MDA-MB-231 cells. WSPF on coming in contact with cancer cell membrane may either lead to activation of receptor mediated signaling cascade or enter the cell through receptor mediated endocytosis. Both these activation mechanisms may in turn lead to ROS generation, ΔΨm loss, SB203580 arrest and destabilization of nuclear membrane and chromatin disintegration which ultimately will result in apoptosis.
    responsible for cellular suicide [44]. Caspase-3, being important for some typical hallmarks of apoptosis, is indispensable for apoptotic DNA fragmentation and chromatin condensation in all cell types [45]. Capase-3 involves degradation of wide range of cellular substrates in-cluding DNA repair enzymes poly-(ADP-ribose) polymerase (PARP) and structural proteins (lamins) [46,47]. Thus, caspase-3 is indispens-able for certain apoptotic processes connected with the dismantling of the cell and the apoptotic bodies formation [45]. It is quite evident from our results that WSPF treated MDA-MB-231 cells induce cleavage of capase-3 in a dose dependent manner (Fig. 5A). Altogether, these re-sults suggest that WSPF induced apoptosis in the MDA-MB-231 cancer cell lines is mitochondrial as well as intrinsic pathways mediated apo-ptosis. Similar kind of results have been observed with other biologically active protein fractions [23,24]. r> Furthermore, it has been suggested that in late-stage apoptosis, sev-eral morphological and biochemical changes like chromatin condensa-tion, cytoplasmic blebbing, overall cellular and nuclear membrane damage, DNA fragmentation, and apoptotic body formation do take place [48–51]. In this context, fluorescence microscopy using DAPI staining of MDA-MB-231 was carried out in presence WSPF (Fig. 8A). It was observed that majority of the apoptotic cellular end-points were found to be significantly elevated with increase in WSPF concen-tration (Figs. 7 and 8A). As compared to untreated cells, WSPF treated MDA-MB-231 cells exhibited nuclear changes like chromatin condensa-tion, nuclear shrinkage and apoptotic body formation (Fig. 8A). In addi-tion to this, fluorescent microscopic images of WSPF treated cancer cell nuclei deciphered an altered nuclear DNA staining pattern with a de-formed nuclear architecture (Fig. 8A). These finding were further sup-ported by evaluating the effect of WSPF on lamin A/C nuclear membrane protein using immunoblot analysis (Fig. 8B). As can be seen in Fig. 8B, immunoblot analysis revealed WSPF induced cleavage of lamin A/C in a dose dependent manner. Nuclear lamins (lamin A/C) are important structural components of nucleus, which form a filamen-tous meshwork alongside the nuclear membrane and provide mechan-ical stability to the nucleus [52]. Lamin filamentous network has been suggested to be important for many nuclear processes like DNA replica-tion, transcription etc. [52,53]. Based on our results, it seems that WSPF induced degradation of lamin A/C leads to alteration of biochemical pa-rameters of nucleus like nuclear fragmentation, DNA fragmentation and chromatin condensation. In addition to this, overall morphology of the MDA-MB-231 cancer cells was found to be altered in presence of WSPF (Fig. 7). It can be seen that as compared to the spindle like angular shaped morphology of untreated cells, WSPF exposed MDA-MB-231 cells exhibited senescence-like phenotype characterized by increased cell size, flattened shape, fragmentation and formation of apoptotic bod-ies and cytoplasmic condensation. WSPF induced apoptosis, indicated by morphological assessment of cells, was confirmed by AO/EtBr stain-ing where chromatin condensation and DNA fragmentation was ob-served with least or no necrosis (Fig. 9). It is quite interesting that WSPF largely induces apoptosis as drugs inducing cell death through necrosis are usually toxic and have limited clinical applications. Alto-gether these results suggest that WSPF did affect the cell membrane ar-chitecture as well as its nuclear integrity which could ultimately lead to apoptosis. Results obtained from the morphological and biochemical analysis suggest that WSPF induces cell death through ROS-induced mi-tochondria mediated apoptosis with altered expression of apoptosis as-sociated proteins.
    6. Conclusion
    We report for the first time a novel anti-cancerous protein fraction from Withania somnifera roots. The protein fraction induced apoptosis was observed to be mitochondria mediated where in mitochondrial de-stabilization led to ROS generation, ΔΨm loss and altered expression of pro-apoptotic and anti-apoptotic proteins with activation of caspase-3. In addition to this, WSPF induced cell shrinkage, G2/M phase cell cycle