Figures & data
Figure 1. Experimental protocol and animal growth during the entire term of the study. A: Schematic representation of the experimental design utilized to investigate the effect of pomegranate emulsion (PE) on 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinogenesis. B: Effect of dietary PE on body weight gain during DMBA-initiated mammary tumorigenesis in rats. Each data point indicates mean ± SEM (n = 12 for Group A, 11 for Group B, 8 each for Groups C and D, 7 for Group E, and 5 for Group F). No significant difference in body weights was observed among various rat groups at any time-point of the study.
Table 1. Effect of oral PE administration on DMBA-induced mammary tumorigenesis in Sprague-Dawley rats.
Figure 2. Chemoprevention of 7,12-dimethylbenz(a)anthracene (DMBA)-initiated rat mammary tumorigenesis by pomegranate. Effects of pomegranate emulsion (PE) on the size of mammary tumors (A–D), intratumor histopathological profiles (E–H), cell proliferation (I–L), apoptosis (M–P), Bax (Q–T), and Bcl2 (U–X) protein expression. The rats were treated with oral PE 2 wk prior to and 16 wk following DMBA administration. All animals were sacrificed 16 wk following DMBA exposure. The mammary tumors were subjected to morphological observation as well histopathological (H&E) and immunohistochemical analysis using anti-PCNA, anti-Bax, and anti-Bcl2 antibodies. Apoptosis was detected by DNA fragmentation assay. Magnification: 100× for tumor and H&E and 200× for PCNA, apoptosis, Bax, and Bcl2.
Figure 3. Quantitative analysis of mammary tumor cell proliferation and apoptosis during 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in rats in the presence or absence of pomegranate emulsion (PE). Effects of PE on intra-tumor PCNA LI as determined by immunohistochemistry (A) and apoptotic index (AI) as measured by DNA fragmentation assay (B). The labeling index (LI) or AI was expressed as the number of immunopositive cells 100×/total number of tumor cells analyzed. Results are expressed as mean ± SEM (n = 4). *P < 0.001 compared with DMBA control.
Figure 4. Effects of pomegranate emulsion (PE) on intratumor Bax and Bcl2 expression as determined by immunohistochemistry. Quantitative analysis of Bax-immunopositive cells (A), Bcl2-immunopositive cells (B), and Bax/Bcl2 ratio (C) in mammary tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) in rats. Each bar represents the mean ± SEM (n = 4). A,B: *P < 0.001; C: +P < 0.05 and *P < 0.001 as compared to DMBA control.
Figure 5. Effects of pomegranate emulsion (PE) treatment on transcriptional expressions of genes related to apoptosis in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated rat mammary tumorigenesis. Rats were sacrificed 18 wk following the commencement of the study and mammary tumor samples were harvested from various experimental groups. Total RNA was extracted. The resultant complementary DNA following reverse transcription was subjected to PCR using specific sequences for various genes: BAX, BCL2, BAD, CASPASE 3 (CASP3), CASPASE 7 (CASP7), CASPASE 9 (CASP 9), poly (ADP-ribose) polymerase (PARP), and cytochrome c (CYT. C). Representative reverse-transcriptase-PCR pictures are shown with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene.
