Suppression of inflammatory cascade is implicated in methyl amooranin-mediated inhibition of experimental mammary carcinogenesis

Abstract

Breast cancer represents the second leading cause of cancer-related deaths among women worldwide and preventive therapy could reverse or delay the devastating impact of this disease. Methyl-amooranin (methyl-25-hydroxy-3-oxoolean-12-en-28-oate, AMR-Me), a novel synthetic oleanane triterpenoid, reduced the incidence and burden of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats through antiproliferative and proapoptotic effects. Since chronic inflammation plays an important role in the pathogenesis of breast cancer and several synthetic oleanane compounds are known potent anti-inflammatory agents, we aim to investigate anti-inflammatory mechanisms of AMR-Me by monitoring various proinflammatory and stress markers, such as cyclooxygenase-2 (COX-2) and heat shock protein 90 (HSP90), and nuclear factor-κB (NF-κB) signaling during DMBA mammary tumorigenesis in rats. Mammary tumors were harvested from a chemopreventive study in which AMR-Me (0.8-1.6 mg/kg) was found to inhibit mammary carcinogenesis in a dose-response manner. The expressions of COX-2, HSP90, NF-κB, and inhibitory κB-α (IκB-α) were determined by immunohistochemistry and reverse transcription-polymerase chain reaction. AMR-Me downregulated the expression of intratumor COX-2 and HSP90, suppressed the degradation of IκB-α, and reduced the translocation of NF-κB from cytosol to nucleus. Our present study provides the first in vivo evidence that NF-κB-evoked inflammatory cascade is a major target of AMR-Me in breast cancer. Our current results together with our previous findings suggest that disruption of NF-κB signaling contributes to anti-inflammatory, antiproliferative, and apoptosis-inducing mechanisms involved in AMR-Me-mediated chemoprevention of rat mammary carcinogenesis. These encouraging mechanistic results coupled with a safety profile should facilitate the clinical development of AMR-Me as breast cancer chemopreventive drug.

Keywords: COX-2; DMBA; HSP90; NF-κB; breast tumor; inflammation; oleanane triterpenoid.

Figure 1

Effects of AMR-Me on COX-2 expression in breast tumors induced by DMBA in female Sprague-Dawley rats. The rats were treated with various oral doses of AMR-Me (three times a week) 2 weeks prior to and 16 weeks following DMBA administration. All animals were sacrificed 18 weeks following the commencement of the study, tumor tissue was harvested and subsequently used for various assays. (A) Immunohistochemical localization of COX-2-positive cells in tumor samples. Arrows indicate immunohistochemical staining of COX-2 (magnification: × 200). (a) Intense COX-2 immunoreactivity in DMBA control; (b) marginal decrease in COX-2 expression in AMR-Me (0.8 mg/kg) plus DMBA group; and (c and d) very limited expression of COX-2 in AMR-Me (1.2 mg/kg) plus DMBA group. (B) Quantitative analysis of COX-2-immunopositive cells during DMBA mammary carcinogenesis in rats in the presence or absence of AMR-Me treatment. Results are based on 1,000 cells per animal and 4 animals per group. Each bar represents the mean±SEM (n = 4). *P < 0.001 compared to DMBA control. (C) COX-2 mRNA expression in mammary tumors isolated from rats exposed to DMBA in the presence or absence of AMR-Me treatment. Total RNA was extracted from tumor samples, subjected to reverse transcription, and resulting cDNA was used for RT-PCR analysis using specific primer sequence. The 18s RNA was used as the loading control.

Figure 2

Expression of HSP90 during DMBA-initiated mammary gland tumorigenesis in rats in the presence or absence of AMR-Me. The rats were treated with various oral doses of AMR-Me (three times a week) 2 weeks prior to and 16 weeks following DMBA administration. All animals were sacrificed 18 weeks following DMBA treatment. The mammary tumors were subjected to immunohistochemical analysis using anti-HSP90 antibody. (A) Immunohistochemical localization of HSP90-positive cells in tumor samples. Arrows indicate immunohistochemical staining of HSP90 (magnification: × 200). Various treatment groups are: (a) DMBA control; (b and c) AMR-Me (0.8 mg/kg body weight) plus DMBA; and (d) AMR-Me (1.2 mg/kg body weight) plus DMBA. (B) Quantitative analysis of HSP90-positive cells during DMBA mammary carcinogenesis in rats in the presence or absence of AMR-Me treatment. Results are based on 1,000 cells per animal and 4 animals per group. Each bar represents the mean±SEM (n = 4). *P < 0.001 compared to DMBA control.

Figure 3

Effect of AMR-Me on NF-κB p65 activation during DMBA-evoked mammary neoplasia in female Sprague-Dawley rats. The rats were treated with various oral doses of AMR-Me (three times a week) 2 weeks prior to and 16 weeks following DMBA administration. All animals were sacrificed 18 weeks following DMBA treatment. The mammary tumors were subjected to immunohistochemical analysis using anti-NF-κB p65 antibody. (A) Representative immunohistochemical localization of NF-κB p65 in nucleus (red arrows) and cytoplasm (black arrows) are depicted (magnification: × 200). Various treatment groups are: (A) DMBA control; (b) AMR-Me (0.8 mg/kg body weight) plus DMBA; and (c and d) AMR-Me (1.2 mg/kg body weight) plus DMBA. Quantitative analysis of (B) nuclear and (C) cytoplasmic NF-κB-immunopositive cells in rat mammary tumors induced by DMBA in the presence or absence of AMR-Me treatment. Results are based on 1,000 cells per animal and 4 animals per group. Each bar represents the mean±SEM (n = 4). *P<0.001 compared to DMBA control. (D) Alteration of NF-κB mRNA expression in mammary tumors isolated from rats exposed to DMBA in the presence or absence of AMR-Me treatment. Total RNA was extracted from tumor samples, subjected to reverse transcription, and resulting cDNA was subjected to RT-PCR analysis using specific primer sequence. The 18s RNA was used as the loading control.

Figure 4

Representative immunohistochemical expression of cytosolic IκB-α during DMBA-induced mammary gland carcinogenesis in rats in the presence or absence of AMR-Me. The rats were treated with various oral doses of AMR-Me (three times a week) 2 weeks prior to and 16 weeks following DMBA administration. All animals were sacrificed 18 weeks following DMBA treatment. The mammary tumors were subjected to immunohistochemical analysis using anti-IκB-α antibody. (A) Immunohistochemical localization of IκB-α-positive cells in tumor samples. Arrows indicate immunohistochemical staining of IκB-α in cytoplasm (magnification: × 200). Various treatment groups are: (a) DMBA control; (b) AMR-Me (0.8 mg/kg body weight) plus DMBA; and (c and d) AMR-Me (1.2 mg/kg body weight) plus DMBA. (B) Quantification of cytoplasmic IκB-α-immunopositive cells in rat mammary tumors induced by DMBA in the presence or absence of AMR-Me treatment. Results are based on 1,000 cells per animal and 4 animals per group. Each bar represents the mean±SEM (n = 4). *P<0.001 compared to DMBA control.

Figure 5

Schematic representation of the possible molecular mechanisms of AMR-Me-mediated chemoprevention of experimental mammary carcinogenesis. DMBA activates NF-κB signaling pathway, which in turn, results in elevated expression of target genes, including COX-2, Bcl-2 and cyclin D1. Activation of all these molecular pathways contributes to sustained inflammation, accelerated proliferation, and evasion of apoptosis which consequently lead to the development of mammary tumor. AMR-Me blocks activated NF-κB signaling cascade and thereby facilitates suppression of subsequent cellular events, including anti-inflammatory, antiproliferative, and apoptosis-inducing effects, contributing to chemoprevention of mammary tumorigenesis. ↓, activation and ⊥, downregulation.