alpha 1-Antitrypsin- and anchorage-independent growth of MCF-7 breast cancer cells.

Abstract

alpha 1-Antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) are closely related protease inhibitors, synthesized primarily by the liver, which play major roles in modulation of the inflammatory response. Previously, we had shown that MCF-7 human breast cancer cells were able to synthesize active alpha 1-AT and alpha 1-ACHY and that the synthesis of both inhibitors varied among different MCF-7 sublines. We now show that when MCF-7(ML) cells (a subline synthesizing low levels of alpha 1-AT) are grown in soft agar in medium depleted of its trypsin inhibitory capacity (i.e. alpha 1-AT-free), addition of alpha 1-AT (50 micrograms/ml) significantly reduces colony formation in both the presence and absence of estradiol (34% and 44%, respectively). Under these conditions, incubation with 10(-7) M estradiol alone increased colony formation 2- to 3-fold. Colony formation was also significantly reduced by serum leukocyte protease inhibitor, which, like alpha 1-AT, is a potent inhibitor of elastase-like enzymes. We also found that a variety of inflammatory mediators, cytokines, and steroid hormones are able to stimulate synthesis of alpha 1-AT and alpha 1-ACHY by MCF-7 cells. Stimulation by interleukin-6 (IL-6; 200 U/ml), epidermal growth factor (4 nM), and estradiol (10(-7) M) was 2- to 3-fold, whereas stimulations by tetradecanoylphorbol-13-acetate (TPA; 80 nM) and IL-1 (10 U/ml) were 2- to 5-fold and 5- to 10-fold, respectively. In each instance, protein synthesis, monitored by immunoprecipitation of 35S-labeled proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and steady state mRNA levels, monitored by Northern blot analysis with specific cDNA probes, increased to the same extent. Consistent with their ability to stimulate alpha 1-AT synthesis, TPA and IL-1 reduced colony formation in the absence of estradiol by 65% and 63%, respectively. In addition, the effects of both TPA and IL-1 could be reversed by antibody to alpha 1-AT. These results suggest that local synthesis of alpha 1-AT and possibly other protease inhibitors may be important in regulating the tumorigenic potential of MCF-7 breast cancer cells.

Association of polymorphism in the alpha-1-antitrypsin gene with milk production traits in Holstein dairy cows

“…Possible roles of A1AT in the immune response include inhibition of lymphocyte toxicity and of chemotaxis (Blank & Brantly, 1994). A1AT is synthesized primarily in the liver, but is also expressed by blood monocytes and macrophages (Perlmutter et al, 1985), intestinal epithelium cells (Molmenti et al, 1993), breast carcinoma cells (Finlay et al, 1993) and the cornea (Twining et al, 1994). In addition, it has been reported that A1AT protein is produced by the human mammary gland and may increase the survival of milk proteins such as lactoferrin and lysozyme (Chowanadisai & Lonnerdal, 2002).…”

Alpha1-antitrypsin, an endogenous immunoregulatory molecule: distinction between local and systemic effects on tumor immunology

“…Such an outcome is highly unexpected when considering that AAT caused an increase in VEGF expression, as corroborated in angiogenesis-related studies. In line with these findings, a series of studies centered on the MCF-7 human breast cancer cell line and revealed a negative correlation between AAT production by tumor cells and tumor cell proliferation; this was accompanied by a reduction in active TGFβ release and in in vivo invasiveness [40][41][42].…”

“…While AAT may directly inhibit proteases responsible for cleavage and activation of mature TGFβ, AAT may also indirectly increase local TGFβ levels by promoting the polarization of macrophages towards M2 profile. Indeed, while in vitro treatment of isolated monocultures of human breast cancer cells MCF-7 with AAT resulted in lower levels of mature TGFβ [40,41], in vivo studies demonstrate that AAT treatment results in an elevation of TGFβ levels [48], correlating with a shift in macrophage polarization towards M2 profile [20]. An example for the important of context in interpreting the activities of AAT involves an in vivo model of liver fibrosis, where hepatic synthesis of TGFβ is found to be significantly higher in transgenic mice carrying the Z allele of AAT [49]; in order to fully interpret this outcome one should take into consideration that the model incorporates acute liver inflammation, as instigated by intracellular aggregates of AAT, rather than a possible effect of reduced circulating levels of AAT.…”

“…TGFβ was found to be associated with EMT in a variety of tumor models [90]. Nonetheless, while AAT treatment was shown to elevate TGFβ expression in models of allogeneic transplantation [20], production of TGFβ by MCF-7 cells was blocked during treatment with AAT [40][41][42], resulting in reduced in vivo invasiveness. As AAT may also strongly block the cleavage of pro-TGFβ, a more thorough investigation of TGFβ-associated EMT responses and their relation to AAT therapy may prove highly informative.…”

Protease Inhibitors in Chemoprevention of Cancer: An overview

“…BBI was shown to be internalized by colonic epithelial cells grown in culture which would allow the protease inhibitor to inhibit critical intracellular proteases and thus suppress malignant transformation (68). Chang et al (69) in their in vitro studies found that antipain could affect c-myc expression in C, H/ 10T1/2 cells and Garte et al (70) (74) noted that colony formation could be reduced by protease inhibitors in vitro, suggesting that local synthesis of alpha-1-antitrypsin and other protease inhibitors may be important in regulating tumorigenic potential of breast cancer cell lines. It has been demonstrated that Bowman Birk inhibitor could be more effective as an anticarcinogenic agent when coupled to poly D-lysine (75).…”