Figure 6. CFEA inhibits breast tumor growth in-vivo and mitigates tumor-induced hepato-renal toxicity.

1.0 × 10⁶ mouse breast tumor cells (4T1) were injected subcutaneously into mammary fat pad on right flank of each female Balb/c mouse and when palpable tumors were formed, mice were randomised in two groups (n = 5 in each group) and then fed with either CFEA (50 mg/kg/day) or with vehicle through oral gavage for two weeks. Tumor volume was monitored at regular intervals using an electronic digital caliper. Tumors were harvested 30 days after tumor cell injection. (a) Growth curves were shown, where Points are indicative of average value of tumor volume; bars, +/− SD. The CFEA-treated group had average tumor volumes that were significantly different from the vehicle-treated group (*p < 0.01). (b) Average body weight of vehicle and treated mice were represented as line graph. (c) Representative tumor images were shown. (d) Average tumor weight bars, +/− SD of vehicle and treated groups (^#p < 0.05) were displayed in bar diagram. (e) Immunohistochemistry were carried out to detect Hsp60 and XIAP in formalin fixed paraffin-embedded sections of both vehicle and CFEA treated mouse breast tumor tissues using anti-Hsp60 (1:100) and anti-XIAP (1:50) antibodies. Representative photomicrographs were shown in top (Hsp60) and bottom (XIAP) panels. Scale bar, 200 μm. Percentage of DAB/Nuclear staining was shown using bar graph (right panels) of respective images. (f ) Blood samples were collected before sacrificing the mice and analysed for different biochemical parameters to assess hepato-renal toxicity. ‘a’ denotes comparison between Vehicle without tumor versus Vehicle with tumor; ‘b’ signifies Vehicle without tumor versus Treated with tumor. ‘c’ indicates Vehicle with tumor versus Treated with tumor. ‘NS’ stands for no significant statistical difference between two test groups as well as with their respective controls. Abbreviations: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglycerides (TG), total cholesterol (TCHO), total protein (TP), albumin (ALB), Glucose (GLU), calcium (Ca), inorganic phosphorus (IP), total bilirubin (TBIL), creatinine (CREA) and Blood urea nitrogen (BUN). Hepatotoxicity Markers- ALT, AST, ALP, and TBIL; Nephrotoxicity Markers- CREA and BUN.