br It is a well established
It is a well-established fact that mitochondrial dysfunction is strongly associated with enhanced intracellular ROS generation. WSPF induced loss in ΔΨm prompted us to monitor ROS generation in the cancerous cells. For that, MDA-MB-231 3X FLAG Peptide were treated with different concentrations of WSPF and then stained with DCFH-DA, a ROS scaven-ger. On treatment of MDA-MB-231 cells with WSPF, a right shift in DCF-DA fluorescence was observed indicating an increase in intracellular ROS production (Fig. 6A). This increase in ROS generation in presence of different concentrations of WSPF was found to be dose dependent (Fig. 6B). At highest concentration of WSPF used, ROS levels of about 92 ± 3.0% were generated in the cells which are quite significant for cell death (Fig. 6B).
4.7. Induction of morphological changes in MDA-MB-231 cells
In phase contrast microscopy, remarkable morphological changes were observed in MDA-MB-231 cells on treatment with different con-centrations of WSPF (Fig. 7). Untreated MDA-MB-231 cells displayed typical spindle shape with no morphological alteration (Fig. 7A). How-ever, on exposure of cells to different concentrations of WSPF for 72 h, drastic morphological alterations in MDA-MB-231 cells were observed
(Fig. 7B–D). It was observed that as compared to untreated MDA-MB-231 cells, the morphology of WSPF treated cells changed from spindle to circular flattened shape, a characteristic feature of dying cells. Senescence-like phenotype characteristic features were exhibited by the WSPF treated MDA-MB-231 cells with increased cell size and flat-tened shape as compared to untreated cells (Fig. 7B–D). Moreover, WSPF treated MDA-MB-231 cells also exhibited cytoplasmic vacuolation.
Alterations in nuclear morphology and apoptotic body formation are the hallmarks of apoptosis. Fluorescence microscopic analysis, using DAPI as a fluorescence probe, was carried out for monitoring nuclear al-teration. From Fig. 8A, it can be seen that as compared to untreated cells, WSPF treated MDA-MB-231 cells exhibited nuclear morphological changes due to induced chromatin condensation, nuclear shrinkage and apoptotic body formation. In the untreated MDA-MB-231 cells, the nuclei were homogeneously stained with DAPI and appeared round in shape indicating no alteration in nuclear morphology while as WSPF treated cancer cells exhibited an altered nuclear DNA staining pattern with deformed nuclear architecture (Fig. 8A). For elucidation of possible mechanism, effect of WSPF on cleavage of nuclear mem-brane lamins was investigated (Fig. 8B). As can be seen in Fig. 8B, immu-noblotting expression analysis revealed cleavage of lamin A/C by WSPF in a dose-dependent fashion. To further confirm the WSPF induced ap-optosis and distinguish between various apoptotic cell populations, AO/ EtBr staining of the WSPF-treated cells was carried out. As can be seen in Fig. 9A, no apoptosis was detected in the untreated cells as all live cells were uniformly stained green with organized chromatin (Fig. 9A). How-ever, in treated MDA-MB-231 cells, percentage of apoptotic cells in-creased with increase in WSPF concentration (50 to 200 μg/mL). In fact, with increase in concentration of WSPF apoptotic body formation
Fig. 8. Nuclear morphology and lamin A/C expression analysis of MDA-MB-231 cells by using fluorescence microscopy with 20× magnification and immunoblot assay, respectively.
(A) Arrows indicate the condensed nuclei and apoptotic bodies. Untreated cells show intact nuclei while as WSPF treated cells (50 μg/mL–200 μg/mL) show increased nuclei deformation with increase in concentration of WSPF. (B) Observed degradation of lamin A/C in WSPF treated MDA-MB-231 cells.
in treated cells increases, as observed by orange colour, while as density of overall cell population decreases. In addition to this, both early and late apoptotic cells were observed at 50 μg/mL dose (Fig. 9B). Early-stage apoptotic cells manifest granular yellow-green AO nuclear stain-ing as detected in the experimental group. With increasing concentra-tions of WSPF, the number of late-stage apoptotic cells increased as detected by concentrated and asymmetrically localized orange nuclear EtBr staining (Fig. 9B–D).
Despite large number of anti-cancerous studies conducted on Withania somnifera, we could not find any such study with respect to its proteinaceous constituents. This is the first kind of such study with respect to the Withania somnifera. Since apoptotic induction in cancer cells is considered as a universal target mechanism for treatment of can-cer [20,21], WSPF was evaluated for its cytotoxic and apoptotic effects on cancer cell lines. As shown in Fig. 1A, WSPF exhibited two major pro-tein bands of molecular weight of approximately 41 and 21 kDa with some less abundant small molecular weight proteins. Further intact pro-tein mass spectral analysis of WSPF by mass spectrometry also con-firmed the presence of only these two major proteins with molecular weight peaks of about 41 and 21 kDa (Fig. 1B). So far, therapeutically ac-tive proteins isolated from Withania somnifera include Withania somnifera glycoprotein (WSG), Withania lectin like protein and asparaginase [17–19,22]. However, as observed in electrophoretic and intact protein mass spectrometric analysis (Fig. 1), we did not find any