br protein bands peaks with molecular weight matching to
protein bands/peaks with molecular weight matching to that of any of the earlier reported biologically active proteins from this plant. Absence of protein bands with molecular weight as that of any of the earlier re-ported therapeutically active proteins do suggest the novelty of WSPF.
In the present study, morphological as well as biochemical charac-terization of WSFP treated cells was carried out to elucidate out its mechanism of action. As evident from cytotoxic studies, WSPF inhibited growth of all tested cancer cell lines with no significant effect on the normal cell line (Fig. 2). However, among all the cell lines WSPF showed maximum cytotoxic potential against MDA-MB-231 cells with an IC50 value of 92 μg/mL. Our results do confirm the selective toxicity of WSPF for cancer cells with no significant toxic effect on normal cells. Similar to our results, protein fractions from Licorice and Gynura procumbens have been shown to selectively kill cancer cells [23,24]. Ad-ditionally, a number of anti-cancerous proteins, like asparaginase from Withania somnifera , Polygonatum cyrtonema lectin , MAP30 from Momordica charantia  and a glycoprotein (GLP) from Codium decorticatum  have cytotoxic effects on cancer cells with little or no effect on normal cells. Growing evidences suggest that selective apopto-sis induction in cancer cells is promoted as the major focus for develop-ment of anti-cancer therapeutics [20,21].
In order to elucidate the possible molecular mechanism of WSPF in-duced apoptosis in cancer cells, effect of WSPF on various cellular pro-cesses of the cancer cells was investigated (Figs. 3–9). Cell cycle is one such process which has emerged as an important regulatory mecha-nism for cellular growth. Dysregulation of Bortezomib (PS-341) at some specific check points is the main key step in most of the malignancies [26–29].
Fig. 9. Microscopy analysis of apoptosis in WSPF treated MDA-MB-231 cells using dual acridine orange/ethidium bromide staining. Panel A in the figure is for untreated cells whereas cells treated with different concentrations of WSPF are shown in panel B (50 μg/mL), panel C (100 μg/mL) and panel D (200 μg/mL). The arrows show the apoptotic cells (blue arrows for early apoptotic and red arrows for late apoptotic).
For this, we investigated the cell cycle phases distribution using flow cy-tometry (Fig. 3). Fig. 3 shows that WSPF induced cell toxicity and reduc-tion in cell viability through G2/M phase cell cycle arrest. Our results infer that cell cycle progression was delayed due to the arrest of the cells in G2/M phase resulting in induction of apoptosis. In consistent with our results, a protein isolated from Codium decorticatum has been shown to arrest the MDA-MB-231 cancer cells in G2/M phase of cell cycle .
Mitochondrial membrane potential is a reflective of an intact and a functional mitochondria and any alteration of ΔΨm results in release of various apoptogenic factors into cytoplasm and thus induce apoptosis [30–33]. The key players that regulate mitochondrial mediated apopto-sis involve pro-apoptotic and anti-apoptotic bcl-2 family proteins (Bcl-2 and Bax) [31,34,35], which in turn maintain mitochondrial membrane integrity and permeability [36,37]. In most of the cancers, elevated ex-pression of Bcl-2 protein has been shown to render cancer cells resistant to chemotherapeutic induced apoptosis while as Bax, a cytosolic pro-apoptotic protein, upon activation by apoptotic stimuli gets oligomerised and translocated to mitochondria. Translocation of Bax in turn leads to disruption of mitochondrial membrane perme-abilization and release of apoptogenic molecules to the cytosol [30,38]. Our results clearly show a significant decrease in ΔΨm of MDA-MB-231 cancer cells with increase in concentration of WSPF (Fig. 4). In
addition to this, immunoblot analysis of WSPF treated cells revealed a dose dependent sharp up-regulation of Bax with simultaneous down-regulation of Bcl-2 protein (Fig. 5). All these observations suggest that WSPF promotes mitochondrial mediated apoptosis in MDA-MB-231 cells by dysregulation of Bax/Bcl-2 expression and simultaneous disrup-tion of ΔΨm.
A large number of studies have suggested a direct correlation be-tween ΔΨm loss and ROS generation. At highest concentration of WSPF used, a ROS generation of about 95 ± 3% was observed (Fig. 6). WSPF induced ΔΨm loss in cancer cells was found to be associated with a dose dependent generation of intracellular ROS (Figs. 4 and 6). It is an established fact that elevated ROS-induced lipid peroxidation, protein oxidation and DNA damage results in apoptosis [39–41]. Similar to our results, a glycoprotein isolated from Codium decorticatum has been shown to induce ROS generation in the same cancer cell lines as used in this study . Moreover, in most of the cell line studies, ROS de-pendent apoptosis is mediated by kinase signaling pathways [42,43]. Our results do also suggest that in WSPF induced apoptosis, ROS might play a vital role by regulating the apoptotic signaling pathways. Dose dependent sharp upregulation of Bax and simultaneously down-regulation of Bcl-2 observed under immunoblot analysis do substantiate the results. Moreover, it has been suggested that ROS induced ΔΨm loss is associated with the activation of caspase-3, which is ultimately