Trianthema portulacastrum Linn. displays anti-inflammatory responses during chemically induced rat mammary tumorigenesis through simultaneous and differential regulation of NF-κB and Nrf2 signaling pathways

Abstract

Trianthema portulacastrum, a medicinal and dietary plant, has gained substantial importance due to its various pharmacological properties, including anti-inflammatory and anticarcinogenic activities. We have recently reported that a characterized T. portulacastrum extract (TPE) affords a considerable chemoprevention of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumorigenesis though the underlying mechanisms are not completely understood. The objective of this study was to investigate anti-inflammatory mechanisms of TPE during DMBA mammary carcinogenesis in rats by monitoring cyclooxygenase-2 (COX-2), heat shock protein 90 (HSP90), nuclear factor-kappaB (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2). Mammary tumors were harvested from our previous study in which TPE (50-200 mg/kg) was found to inhibit mammary tumorigenesis in a dose-response manner. The expressions of intratumor COX-2, HSP90, NF-κB, inhibitory kappaB-alpha (IκBα) and Nrf2 were determined by immunohistochemistry. TPE downregulated the expression of COX-2 and HSP90, blocked the degradation of IκBα, hampered the translocation of NF-κB from cytosol to nucleus and upregulated the expression and nuclear translocation of Nrf2 during DMBA mammary carcinogenesis. These results in conjunction with our previous findings suggest that TPE prevents DMBA-induced breast neoplasia by anti-inflammatory mechanisms mediated through simultaneous and differential modulation of two interconnected molecular circuits, namely NF-κB and Nrf2 signaling pathways.

Figure 1

Effect of TPE on COX-2 expression in DMBA-induced breast tumors in female Sprague-Dawley rats. The rats had free access to food with or without TPE two weeks prior to, and 16 weeks following, DMBA administration. All animals were sacrificed 18 weeks following the commencement of the study, tumor tissues were harvested and used for the assay using anti-COX-2 antibody. (A) Immunohistochemical localization of COX-2-positive cells in tumor sections. Arrows indicate immunohistochemical staining of COX-2 (magnification: ×200). (a) Intense COX‑2 immunoreactivity in DMBA control; (b) minimal increase in COX‑2 expression in TPE (50 mg/kg) plus DMBA group; (c) extensive decrease in COX‑2 expression in TPE (100 mg/kg) plus DMBA group; and (d) very limited expression of COX-2 in TPE (200 mg/kg) plus DMBA group; (B) Quantitative analysis of COX-2-immunopositive cells during DMBA mammary tumorigenesis in rats in the presence or absence of TPE treatment. Results are based on 1000 cells per animal and 4 animals per group. Each bar represents the mean ± SEM (n = 4). * p < 0.01 and ** p < 0.001 compared to DMBA control.

Figure 2

Expression of HSP90 during DMBA-initiated mammary gland tumorigenesis in rats in the presence or absence of TPE treatment. The animals were treated as indicated in legend to Figure 1. The mammary tumor sections 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) TPE (50 mg/kg) plus DMBA; (c) TPE (100 mg/kg) plus DMBA; and (d) TPE (200 mg/kg) plus DMBA; (B) Quantitative analysis of HSP90-positive cells during DMBA mammary tumorigenesis in rats in the presence or absence of TPE treatment. Results are based on 1000 cells per animal and four animals per group. Each bar represents the mean ± SEM (n = 4). * p < 0.01 and ** p < 0.001 compared to DMBA control.

Figure 3

Effect of TPE on NF-κB p65 activation during DMBA-induced mammary gland carcinogenesis in female Sprague-Dawley rats. The animals were treated as indicated in the legend to Figure 1. The mammary tumor sections were subjected to immunohistochemical analysis using anti-NF-κB p65 antibody. (A) Representative immunohistochemical localization of NF-κB p65 in nucleus (yellow arrows) and cytoplasm (black arrows) are depicted (magnification: × 200). Various treatment groups are: (a) DMBA control; (b) TPE (50 mg/kg) plus DMBA; (c) TPE (100 mg/kg) plus DMBA; and (d) TPE (200 mg/kg) plus DMBA. The nuclear expression of NF-κB p65 in the designated area marked by the white box is shown as an inset (magnification: ×1000) for each treatment group. 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 TPE treatment. Results are based on 1000 cells per animal and 4 animals per group. Each bar represents the mean ± SEM (n = 4). (B) * p < 0.05 and ** p < 0.001 compared to DMBA control. (C) * p < 0.01 and ** p < 0.001 compared to DMBA control.

Figure 4

The immunohistochemical expression of IκBα during DMBA-evoked mammary neoplasia in rats in the presence or absence of TPE. The animals were treated as indicated in the legend to Figure 1. The mammary tumor sections were subjected to immunohistochemical analysis using anti-IκBα antibody. (A) Immunohistochemical localization of IκBα-positive cells in tumor samples. Arrows indicate immunohistochemical staining of IκBα in cytoplasm (magnification: ×200). Various treatment groups are: (a) DMBA control; (b) TPE (50 mg/kg) plus DMBA; (c) TPE (100 mg/kg) plus DMBA; and (d) TPE (200 mg/kg) plus DMBA; (B) Quantification of cytoplasmic IκBα-immunopositive cells in rat mammary tumors induced by DMBA in the presence or absence of TPE treatment. Results are based on 1000 cells per animal and four animals per group. Each bar represents the mean ± SEM (n = 4). * p < 0.05 and ** p < 0.001 compared to DMBA control.

Figure 5

Effect of TPE on intratumor Nrf2 expression during DMBA-initiated breast carcinogenesis in rats. The animals were treated as indicated in legend to Figure 1. The mammary tumor sections were subjected to immunohistochemical analysis using anti-Nrf2 antibody. (A) Representative immunohistochemical localization of Nrf2 in nucleus. Arrows indicate immunohistochemical staining of Nrf2 (magnification: ×200). Various treatment groups are: (a) DMBA control; (b) TPE (50 mg/kg) plus DMBA; (c) TPE (100 mg/kg) plus DMBA; and (d) TPE (200 mg/kg) plus DMBA; (B) Quantification of nuclear Nrf2-immunopositive cells in rat mammary tumors induced by DMBA in the presence or absence of TPE treatment. Results are based on 1000 cells per animal and four animals per group. Each bar represents the mean ± SEM (n = 4). * p < 0.01 and ** p < 0.001 compared to DMBA control.