br Fig Effect of the protein fraction
Fig. 5. Effect of the protein fraction on expression of Bcl-2, Bax and caspase-3 of MDA-MB-231 cells. (A) Immunoblots show dose dependent WSPF induced down regulation of Bcl-2 and up-regulation of Bax and cleaved caspase-3 protein. (B) Densitometry analysis of expression of apoptosis related proteins in untreated and WSPF treated MDA-MB-231 cells.
3. Statistical analysis
One-way ANOVA followed by the Bonferroni t-test was employed to evaluate the inter group differences and the data obtained was pre-sented as mean ± SD. All experiments were repeated three times. p ≤ 0.05 was taken as statistically significant.
4.1. Preparation and identification of the protein fraction
SDS-PAGE result confirmed two protein bands, with a molecular weight of approximately 41 and 21 kDa, to be most prominent though additional low intensity bands were visible (Fig. 1A). Fraction contain-ing the two prominent proteins named, as WSPF, was collected and
studied further. Intact mass spectrum analysis of WSPF also revealed two abundant peaks with a molecular weight of 41 and 21 kDa. Results obtained in mass spectrometric analysis of the extracted protein frac-tion were in conformity to that of the SDS-PAGE (Fig. 1B). Together these results do suggest WSPF to be a novel protein fraction as none of the earlier biologically active reported proteins from this plant have such molecular weights [17–19].
4.2. WSPF induced inhibition of cell proliferation
MTT assay was performed for evaluation of in vitro cytotoxicity of WSPF against human cancer cell lines of different tissues such as breast (MDA-MB-231, MCF-7, T47D and MBA-MB-435), lung (A549), colon (HCT-116) and non-cancerous human breast cell line (MCF-10A). Among all the cell lines, WSPF was found to be most cytotoxic to
human breast cancer cell line MDA-MB-231 with an IC50 value of 92 μg/mL (Fig. 2). In addition to this, no significant toxic effect was ob-served against non-cancerous MCF-10A cell line. Further studies were therefore carried out with MDA-MB-231 breast cancer cell line only.
4.3. WSPF induced Ruxolitinib (INCB018424) arrest
Subsequent to the observed WSPF induced inhibition of cell prolifer-ation, we examined the cell cycle progression to determine if WSPF in-duced a cell cycle stall in progression. For this, flow cytometric analysis was carried out by measuring DNA content of MDA-MB-231 cells using Propidium Iodide dye. Fig. 3 demonstrates that the G2 arrest is in a dose dependent manner, indicating that the WSPF fraction is stalling mitotic progression, likely to the point that it is catastrophic to the cell.
4.4. WSPF treatment induces mitochondrial membrane potential loss
Loss in ΔΨm, used as a marker for inner mitochondria membrane disruption, is considered as a point of no return for cells and is thus an important assay for investigation of apoptosis. For this, effect of WSPF on ΔΨm of MDA-MB-231 cells was studied using rhodamin123
fluorescent dye. Fig. 4A shows concentration dependent loss of ΔΨm in presence of WSPF. Results obtained do confirm a dose dependent loss in ΔΨm which in turn leads to activation of mitochondrial medi-ated apoptotic pathway in the cells (Fig. 4B). At highest concentration of WSPF (200 μg/mL), a significant ΔΨm loss of N30% was observed which indicates disruption of inner mitochondrial membrane (Fig. 4B).
4.5. Expression analysis of apoptosis associated proteins
A hierarchical interaction between pro-apoptotic and anti-apoptotic proteins has been shown to govern the mitochondrial mediated apopto-tic pathway. In order to elucidate whether WSPF has any effect on ex-pression of Bcl-2 gene family, expression profile of anti-apoptotic (Bcl-
2) and pro-apoptotic (Bax) markers in WSPF treated MDA-MB-231 cells was carried out by using immunoblotting (Fig. 5). As shown in Fig. 5A, exposure of MDA-MB-231 cells to different concentrations of WSPF led to dose dependent reduction in Bcl-2 expression with an in-crease in Bax expression. Additionally, expression analysis of caspase-3, an important apoptosis associated protein protein marker was carried out. As can be seen in Fig. 5A, exposure of MDA-MB-231 cells to different concentrations of WSPF led to dose dependent elevation in cleaved
Fig. 7. Morphological analysis of MDA-MB-231 cells in absence and presence of WSPF by phase contrast microscopy with 20× magnification. 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).
caspase-3 expression. At 200 μg/mL dose, a significant elevation in ex-pression level of caspase-3 was observed. Altered gene expression pro-file of WSPF treated cells was further confirmed by quantitative analysis of the apoptosis associated proteins using densitometry calculation (Fig. 5B). Quantification results revealed significant dose dependent re-duction in Bcl-2 level along with increased Bax level and activated caspase-3 level.