Synergistic efficacy of RLIP inhibition and 2'-hydroxyflavanone against DMBA-induced mammary carcinogenesis in SENCAR mice

Abstract

Substantial evidence suggests that 7,12-dimethylbenzanthracene (DMBA)-induced mammary carcinogenesis in mice mimics human breast cancer (BC) in many respects. Therefore, it has been used extensively to evaluate preventive and therapeutic agents for human BC. Mammary carcinogenesis induced by DMBA administration in female SENsitive to CARcinogen (SENCAR) mice was characterized by histopathological analysis of the mammary glands and alterations to the phosphatidylinositol 3-kinase/protein kinase B/cyclin-dependent kinase 1 (PI3K/Akt/CDK1) pathway. We recently reported that 2'-hydroxyflavanone (2HF) is a promising diet-derived chemotherapeutic agent that suppresses BC growth in vitro and in vivo by targeting a 76 kDa ral-interacting protein (RLIP). The objective of the current study was to investigate the synergistic anticarcinogenic effects of RLIP inhibition/depletion and 2HF in an in vivo model of DMBA-induced mammary carcinogenesis in SENCAR mice. Mice were given 2HF (50 mg/kg, bw, orally on alternate days), RLIP antibody (Rab; 5 mg/kg, bw, ip weekly), RLIP antisense (RAS; 5 mg/kg, b.w., ip weekly), or a combination of 2HF + Rab + RAS. Animals were monitored daily, and 7 days after the first appearance of moribund behavior, tissues were harvested for morphological and immunohistological analysis. Western blot analyses were performed to determine the expression of anti- and proapoptotic proteins in the mammary glands. Our results reveal that 2HF, RAS, and Rab significantly prevented the carcinogenic effects of DMBA administration in the mammary glands and other organs. Further, mice treated with a combination of 2HF + RAS + Rab exhibited no carcinogenic effect of DMBA as compared to either or the single agent-treated mice. This study demonstrates for the first time the anticarcinogenic effects of 2HF and RLIP inhibition/depletion in vivo in a novel DMBA-induced model of BC in SENCAR mice and provides the rationale for further clinical investigation.

Keywords: DMBA; RLIP; chemotherapeutics; mammary gland; mercapturic acid pathway transporter; phytochemicals.

Figure 1. Whole mount preparations of mammary gland fat pads from SENCAR mice

Mammary gland fat pads, excised from DMBA-administered SENCAR mice that received a control treatment (A, corn oil; B, CAS; C, PIS) or an experimental treatment (D, 2HF; E, RAS; F, Rab; G, a combination of 2HF+RAS+Rab), and stained with Mayer’s hematoxylin. Mammary gland fat pads from the control treatment groups were slightly small and edematous, making it difficult to achieve adequate spread on glass slide. The lymph nodes associated with the fat pads were present in all groups (yellow arrow). The development of the duct system (green arrows) was markedly attenuated in all control treatment groups when compared with any of the experimental treatment groups. Fat pads in all of the experimental treatment groups are similar to each other. The stained glands were placed in 70% ethanol until photographed using a Leica MZ 10F Stereomicroscope (Chicago, IL). Abbreviations: 2HF, 2'-hydroxyflavanone-treated; CAS, scrambled antisense-treated; RAS, RLIP antisense-treated; PIS, preimmune serum-treated; Rab, RLIP antibody-treated.

Figure 2. Effect of RLIP-targeting treatments on tissue morphology in DMBA-administered SENCAR mice

Tissue sections from DMBA-administered female SENCAR mice that received a control treatment (corn oil, CAS, or PIS) or an experimental treatment (2HF, RAS, Rab, or a combination of 2HF+RAS+Rab) were used for H&E staining. Hemorrhagic areas involving the liver, duodenum, spleen, kidney, bone marrow, mammary gland, brain, lung, and heart tissues were observed in the corn oil-, CAS-, and PIS-treated mice whereas tissues from 2HF-, RAS-, Rab-, and combination-treated mice were histologically normal. Representative images at x400 magnification are shown. 2HF, 2'-hydroxyflavanone-treated; CAS, scrambled antisense-treated; RAS, RLIP antisense-treated; PIS, preimmune serum-treated; Rab, RLIP antibody-treated.

Figure 3. Effects of 2HF and RLIP-targeting agents on protein expression in the mammary glands of DMBA-administered SENCAR mice

Mammary gland fat pads excised from DMBA-administered SENCAR mice that received a control treatment (corn oil, CAS, or PIS) or an experimental treatment (2HF, RAS, Rab, or combination of 2HF+RAS+Rab) were analyzed for changes in the expression of RLIP, pAkt, maspin, PCNA, survivin, CDK1, Bax, and Bcl2. β-actin was used as a loading control. Numbers below the blots represent the fold change in the levels of protein expression, compared to expression in control tissues, as determined by densitometry. 2HF, 2'-hydroxyflavanone-treated; CAS, scrambled antisense-treated; RAS, RLIP antisense-treated; PIS, preimmune serum-treated; Rab, RLIP antibody-treated.

Figure 4. Immunohistochemistry of mammary gland tissue from DMBA-administered SENCAR mice

Immunohistochemistry was performed to detect RLIP, Ki67, CD31, E-cadherin, and vimentin expression in mammary gland tissue isolated from DMBA-administered mice that received a control treatment (corn oil, CAS, or PIS) or an experimental treatment (2HF, RAS, Rab, or 2HF+RAS+Rab). The intensity of antigen staining was quantified by digital image analysis using Pro Plus software. Bars represent mean ± S.E. (n = 5). One representative image for each treatment group is shown. *Statistically significant differences were determined by two-tailed Student’s t tests; p < 0.04, tissues from 2HF-, RAS-, and Rab-treated mice compared with their respective controls. 2HF, 2'-hydroxyflavanone-treated; CAS, scrambled antisense-treated; RAS, RLIP-antisense-treated; PIS, pre-immune serum-treated; Rab, RLIP antibody-treated.