N MESB treated mice compared to the controls (Fig. 5B). Besides, there was no HIF-2��-IN-1 custom synthesis significant change in body weight measured after 10 days of MESB treatment (Fig. 5A).Effect of MESB Treatment on the Expression of Ki67, p53BP1, BID and t-BID in Tumor TissuesKi67 is a cell proliferation marker for tumor progression [31]. Immunohistochemical staining of Ki67 protein tumor section showed increased cell proliferation in untreated animals bearingCancer Therapeutic Effects of StrawberryFigure 8. Proposed model for mechanism of MESB induced cytotoxicity. MESB treatment resulted in activation of intrinsic pathway of apoptosis. This is mediated through activation of p73. This activation leads to changes in the level of mitochondrial apoptotic protein, BAX. This may result in the imbalance of proapoptotic/antiapoptotic proteins. The activation of BAX, get 60940-34-3 further leads to cleavage of MCL-1 and release of CYTOCHROME C, which along with APAF1 helps in cleavage of CASPASE 9. Cleaved CASPASE 9 activates CASPASE 3 which further initiates PARP1 cleavage and cell death. doi:10.1371/journal.pone.0047021.gtumor, while it decreased upon treatment with MESB (Fig. 6A). An enhanced expression of p53 binding protein 1(p53BP1), a DNA damage sensor, was observed upon treatment with MESB (Fig. 6B). We have also observed activation of proapoptotic proteins, BID and t-BID following treatment with MESB compared to untreated tumor tissues (Fig. 6C and D) suggesting the induction of apoptosis in tumor cells in mice. Therefore, our results suggest that MESB treatment inhibits the proliferation of tumor cells by activating apoptosis in mice bearing breast adenocarcinoma allograft.MESB Activates Intrinsic Pathway of Apoptosis in Breast Cancer CellsIn order to understand the mechanism by which MESB induces cell death, we chose the breast cancer cell line, T47D, for further investigation. T47D cells were treated with increasing concentrations of MESB, cell extracts were prepared and used for immunoblotting analysis. Results showed activation of apoptotic marker, MCL-1, which acts as a proapoptotic protein upon cleavage. We find that MESB treatment resulted in prominent cleavage of MCL-1 as compared to the control (Fig. 7A). MESB treatment also resulted in downregulation of BCL-xL, an antiapoptotic protein, at the highest concentration studied (Fig. 7A). Results also showed a significant upregulation of expression of proapoptotic proteins such as BAX and BID (Fig. 7A). Previously, it has been shown that the tumor suppressor gene, p53, is mutated in T47D cells [32,33]. Consistent to this, we could not find any significant change in p53 expression in this cell line, even upon addition of MESB (Fig. 7B). MDM2 is a modulator of p53 and we observed no considerable difference in its expression when treated with MESB (Fig. 7B). Interestingly in case of p73, a paralogue of p53, we observed a dose-dependent increase in expression (Fig. 7B and 8).p73 can induce apoptosis through both intrinsic as well as extrinsic pathways [34]. Results 
showed a low level of PARP cleavage and activation of CASPASE 3 and CASPASE 9 indicating the activation of intrinsic pathway of apoptosis (Fig. 7B, C). A significant increase in the expression of SMAC/ DIABLO, CYTOCHROME C and APAF1 upon treatment with MESB as compared to control, also confirmed activation of the intrinsic pathway of apoptosis (Fig. 7C). More importantly, western blotting using cytosolic fractions of MESB treated T47D cells, showed release of.N MESB treated mice compared to the controls (Fig. 5B). Besides, there was no significant change in body weight measured after 10 days of MESB treatment (Fig. 5A).Effect of MESB Treatment on the Expression of Ki67, p53BP1, BID and t-BID in Tumor TissuesKi67 is a cell proliferation marker for tumor progression [31]. Immunohistochemical staining of Ki67 protein tumor section showed increased cell proliferation in untreated animals bearingCancer Therapeutic Effects of StrawberryFigure 8. Proposed model for mechanism of MESB induced cytotoxicity. MESB treatment resulted in activation of intrinsic pathway of apoptosis. This is mediated through activation of p73. This activation leads to changes in the level of mitochondrial apoptotic protein, BAX. This may result in the imbalance of proapoptotic/antiapoptotic proteins. The activation of BAX, further leads to cleavage of MCL-1 and release of CYTOCHROME C, which along with APAF1 helps in cleavage of CASPASE 9. Cleaved CASPASE 9 activates CASPASE 3 which further initiates PARP1 cleavage and cell death. doi:10.1371/journal.pone.0047021.gtumor, while it decreased upon treatment with MESB (Fig. 6A). An enhanced expression of p53 binding protein 1(p53BP1), a DNA damage sensor, was observed upon treatment with MESB (Fig. 6B). We have also observed activation of proapoptotic proteins, BID and t-BID following treatment with MESB compared to untreated tumor tissues (Fig. 6C and D) suggesting the induction of apoptosis in tumor cells in mice. Therefore, our results suggest that MESB treatment inhibits the proliferation of tumor cells by activating apoptosis in mice bearing breast adenocarcinoma allograft.MESB Activates Intrinsic Pathway of Apoptosis in Breast Cancer CellsIn order to understand the mechanism by which MESB induces cell death, we chose the breast cancer cell line, T47D, for further investigation. T47D cells were treated with increasing concentrations of MESB, cell extracts were prepared and used for immunoblotting analysis. Results showed activation of apoptotic marker, MCL-1, which acts as a proapoptotic protein upon cleavage. We find that MESB treatment resulted in prominent cleavage of MCL-1 as compared to the control (Fig. 7A). MESB treatment also resulted in downregulation of BCL-xL, an antiapoptotic protein, at the highest concentration studied (Fig. 7A). Results also showed a 
significant upregulation of expression of proapoptotic proteins such as BAX and BID (Fig. 7A). Previously, it has been shown that the tumor suppressor gene, p53, is mutated in T47D cells [32,33]. Consistent to this, we could not find any significant change in p53 expression in this cell line, even upon addition of MESB (Fig. 7B). MDM2 is a modulator of p53 and we observed no considerable difference in its expression when treated with MESB (Fig. 7B). Interestingly in case of p73, a paralogue of p53, we observed a dose-dependent increase in expression (Fig. 7B and 8).p73 can induce apoptosis through both intrinsic as well as extrinsic pathways [34]. Results showed a low level of PARP cleavage and activation of CASPASE 3 and CASPASE 9 indicating the activation of intrinsic pathway of apoptosis (Fig. 7B, C). A significant increase in the expression of SMAC/ DIABLO, CYTOCHROME C and APAF1 upon treatment with MESB as compared to control, also confirmed activation of the intrinsic pathway of apoptosis (Fig. 7C). More importantly, western blotting using cytosolic fractions of MESB treated T47D cells, showed release of.
