In order to study the expression of MMP2, MMP3 and MMP9 in breast cancer brain metastasis, we used a syngeneic rat model of distant metastasis of ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. At six weeks post inoculation we observed development of micro-metastasis in the brain. Immunohistochemistry and Western Blotting analyses showed that MMP-2, -3 and -9 proteins expressions are consistently significantly higher in neoplastic brain tissue compared to normal brain tissue. These results were confirmed by RT-PCR. In situ zymography revealed gelatinase activity within the brain metastasis. Gel zymography showed increase in MMP2 and MMP3 activity in brain metastasis. Furthermore, we were able to significantly decrease the development of breast cancer brain metastasis in animals by treatment with PD 166793, a selective synthetic MMP inhibitor. In addition, PD 166793 decreased the in vitro invasive cell behavior of ENU1546. Together our results suggest that MMP-2, -3 and -9 may be involved in the process of metastasis of breast cancer to the brain.

Matrix metalloproteinase 2 (MMP2) is important in breast cancer (BC) invasion and metastasis. We previously reported that BC brain metastases, in a rat syngeneic model developed in our laboratory, have high expression and activity of MMP2. The MMP2 mechanism of action in the brain is still under intense scrutiny. To study the role of MMP2 in the development of BC brain metastasis we transfected ENU1564 rat mammary adenocarcinoma cells with tissue inhibitor of MMP2 (TIMP2). Animals inoculated with ENU1564-TIMP2 cells had decreased orthotopic tumor growth, decreased orthotopic metastatic behavior and did not develop brain metastases. These results were associated with decreased MMP2 activity, demonstrated by gel zymography. Mitogen activated protein kinase (MAPK) pathway components, such as ERK1/2, have been correlated to MMP expression and/or astrocyte activity. We found that BC brain metastases have peripheral astrocyte reactivity and higher expression of glial fibrillary acidic protein (GFAP) and phosphorylated-ERK1/2 (p-ERK1/2). Additionally, rat astrocyte-conditioned media increased in vitro invasion of ENU1564 cancer cells and increased expression of MMP2 and p-ERK1/2. Blockage of ERK1/2 phosphorylation by treatment with MEK inhibitor (PD98059) decreased the expression of MMP2 in cancer cells grown in rat astrocyte-conditioned media. Our results are highly suggestive that MMP2 plays a role in the development of BC metastases, in particular to the brain. Furthermore, our results suggest that astrocyte factors and the ERK1/2 signaling pathway may be associated with BC brain metastasis development; and that ERK1/2 may regulate MMP2 in a way that is modifiable by astrocyte factors.

The leghemoglobin protein (LegH) from soy (Glycine max) expressed in Pichia pastoris (LegH preparation, LegH Prep) imparts a meat-like flavor profile onto plant-based food products. The safety of LegH Prep was evaluated through a series of in vitro and in vivo tests. The genotoxic potential of LegH Prep was assessed using the bacterial reverse mutation assay (Ames test) and the in vitro chromosome aberration test. LegH Prep was nonmutagenic and nonclastogenic in each test, respectively. Systemic toxicity was assessed in a 28-day dietary study in male and female Sprague Dawley rats. There were no mortalities associated with the administration of LegH Prep. There were no clinical observations, body weight, ophthalmological, clinical pathology, or histopathological changes attributable to LegH Prep administration. There were no observed effects on male reproduction in this study, but the suggestion of a potential estrous cycle distribution effect in female rats prompted a second comprehensive 28-day dietary study in female Sprague Dawley rats. This study demonstrated that female reproductive parameters were comparable between rats treated with LegH Prep and concurrent control rats. These studies establish a no observed adverse effect level of 750 mg/kg/d LegH, which is over 100 times greater than the 90th percentile estimated daily intake. Collectively, the results of the studies presented raise no issues of toxicological concern with regard to LegH Prep under the conditions tested.

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK.

Mesotheliomas are rarely reported in animal species. In this report, the occurrence of a diffuse, metastatic mesothelioma in a 6-year-old gray Arabian mare is described. The mare was presented on clinical examination with ascites, bilateral pleural effusion, and pleural roughening. Necropsy revealed abundant fluid in the abdominal and thoracic cavities. The surface of all organs was thick and fibrosed with multiple raised nodules and hemorrhages. Histology was characteristic of a generalized, biphasic mesothelioma with vascular and lymph nodes metastases. It is believed that the primary tumor developed in the pericardium and spread through lymphatics. In this report, calretinin was used as an immunohistochemical marker in the diagnosis of mesothelioma in an equine species for the first time.