M 12th day of tumor development on every alternate day until 45thday, 2 g/kg), a significant reduction in the tumor volume was observed compared to untreated control animals bearing tumor (Fig. 2A, 3A). By 45th day of treatment, most of the MESB treated animals showed no tumor, unlike untreated tumor animals (Fig. 2A and Fig. 3B). More importantly, we observed a significant 301-00-8 increase in the lifespan of MESB treated animals (Fig. 2B). When chemopreventive effect of MESB was studied on tumors induced by breast adenocarcinoma cells, following oral feeding of MESB for 20 days prior to injection of tumor inducing cells, results showed a significant reduction in solid tumor formation asCancer Therapeutic Effects of StrawberryFigure 7. Expression of apoptotic proteins in T47D cells following MESB treatment. Whole cell extracts (A-C) and cyotosolic extracts (D) were prepared from T47D cells following treatment with MESB (0, 0.1, 0.4, 0.7 mg/ml for 48 h). Western blotting studies were performed using primary antibodies against (A) MCL-1, BCL-xL, BAX and BID, (B) p53, MDM2, p73 and PARP1, (C) SMAC/DIABLO, CYTOCHROME C, APAF1, CASPASE 3 and CASPASE 9, (D) SMAC/DIABLO and CYTOCHROME C. In panels A-C, TUBULIN was used as an internal loading control, while in D, ACTIN was used. doi:10.1371/journal.pone.0047021.gcompared to controls (Fig. 2C). Further, we observed a significant increase in the life span of MESB pretreated animals as compared to control group of animals (Fig. 2D). These 25837696 results indicate that strawberry extracts can provide significant chemoprevention in mice. Gross anatomical appearance of thigh tissue containing tumor, liver and spleen of control and experimental animals on 30th and 45th day after tumor development further confirmed the effect of MESB in regression of tumor (Fig. 3A, B). The appearance of the treated animals after 45 days as well as morphology of their dissected organs were comparable with those of normal animals indicating that MESB treatment did not lead to visible alterations (Fig. 3). Histopathological studies were performed on sections from thigh or thigh bearing tumor and liver tissues of normal, tumor bearing and MESB treated animals after 30th and 45th days of treatment using haematoxylin-eosin staining (Fig. 4). Thigh tissue from tumor bearing mouse showed damages in muscle architecture and tumor cell proliferation with very high nuclear staining [Fig. 4A(a ), B(a )]. After treatment with MESB, damages in muscle architecture and tumor cell proliferation were limited indicating the reduction in tumor Vitamin D2 chemical information growth [Fig. 4A(e, f), B(e, f)]. The adverse effect of MESB treatment on other tissues wasanalysed by taking liver as a model organ. Studies using hematoxylin and eosin stained liver sections showed infiltration of inflammatory cells in animals bearing tumors compared to no tumor controls [Fig. 4C(a-d), D(a )]. However, upon treatment with MESB, the liver exhibited mostly normal morphology, with no or limited infiltration in hepatocytes [Fig. 4C(e,f), D(e,f)]. Therefore, the above results suggest that treatment with strawberry fruit crude extracts did not adversely affect the morphology, anatomy or physiology of the other organs. In order to evaluate 
side effects of MESB, normal mice were fed with MESB for 10 days and results showed similar levels of serum profile (alkaline phosphatase, creatinine and urea) compared to untreated controls (Fig. 5B). Further there was no significant difference in RBC and WBC counts i.M 12th day of tumor development on every alternate day until 45thday, 2 g/kg), a significant reduction in the tumor volume was observed compared to untreated control animals bearing tumor (Fig. 2A, 3A). By 45th day of treatment, most of the MESB treated animals showed no tumor, unlike untreated tumor animals (Fig. 2A and Fig. 3B). More importantly, we observed a significant increase in the lifespan of MESB treated animals (Fig. 2B). When chemopreventive effect of MESB was studied on tumors induced by breast adenocarcinoma cells, following oral feeding of MESB for 20 days prior to injection of tumor inducing cells, results showed a significant reduction in solid tumor formation asCancer Therapeutic Effects of StrawberryFigure 7. Expression of apoptotic proteins in T47D cells following MESB treatment. Whole cell extracts (A-C) and cyotosolic extracts (D) were prepared from T47D cells following treatment with MESB (0, 0.1, 0.4, 0.7 mg/ml for 48 h). Western blotting studies were performed using primary antibodies against (A) MCL-1, BCL-xL, BAX and BID, (B) p53, MDM2, p73 and PARP1, (C) SMAC/DIABLO, CYTOCHROME C, APAF1, CASPASE 3 and CASPASE 9, (D) SMAC/DIABLO and CYTOCHROME C. In panels A-C, TUBULIN was used as an internal loading control, while in D, ACTIN was used. doi:10.1371/journal.pone.0047021.gcompared to controls (Fig. 2C). Further, we observed a significant increase in the life span of MESB pretreated animals as compared to control group of animals (Fig. 2D). These 25837696 results indicate that strawberry extracts can provide significant chemoprevention in mice. Gross anatomical appearance of thigh tissue containing tumor, liver and spleen of control 
and experimental animals on 30th and 45th day after tumor development further confirmed the effect of MESB in regression of tumor (Fig. 3A, B). The appearance of the treated animals after 45 days as well as morphology of their dissected organs were comparable with those of normal animals indicating that MESB treatment did not lead to visible alterations (Fig. 3). Histopathological studies were performed on sections from thigh or thigh bearing tumor and liver tissues of normal, tumor bearing and MESB treated animals after 30th and 45th days of treatment using haematoxylin-eosin staining (Fig. 4). Thigh tissue from tumor bearing mouse showed damages in muscle architecture and tumor cell proliferation with very high nuclear staining [Fig. 4A(a ), B(a )]. After treatment with MESB, damages in muscle architecture and tumor cell proliferation were limited indicating the reduction in tumor growth [Fig. 4A(e, f), B(e, f)]. The adverse effect of MESB treatment on other tissues wasanalysed by taking liver as a model organ. Studies using hematoxylin and eosin stained liver sections showed infiltration of inflammatory cells in animals bearing tumors compared to no tumor controls [Fig. 4C(a-d), D(a )]. However, upon treatment with MESB, the liver exhibited mostly normal morphology, with no or limited infiltration in hepatocytes [Fig. 4C(e,f), D(e,f)]. Therefore, the above results suggest that treatment with strawberry fruit crude extracts did not adversely affect the morphology, anatomy or physiology of the other organs. In order to evaluate side effects of MESB, normal mice were fed with MESB for 10 days and results showed similar levels of serum profile (alkaline phosphatase, creatinine and urea) compared to untreated controls (Fig. 5B). Further there was no significant difference in RBC and WBC counts i.
