The "Cross Talk" between the Receptors of Insulin, Estrogen and Progesterone in Neutrophils in the Synthesis of Maspin through Nitric Oxide in Breast Cancer

Abstract

Purpose: The binding of either insulin, or estrogen or progesterone to their specific receptors on neutrophils has been reported to stimulate nitric oxide (NO) induced maspin synthesis in these cells. Experiments were carried out to determine the role of insulin receptor interaction in the nitric oxide induced maspin synthesis in neutrophils that was effected by estrogen or progesterone.

Methods: Estrogen receptor positive (ER+) and progesterone receptor positive (PR+) neutrophils were isolated from the blood cancer subjects. Maspin was determined by enzyme linked immunosorbent assay after in vitro translation of maspin mRNA. NO was determined by methemoglobin method.

Results: It was found that pre incubation of normal neutrophils with insulin to reach equilibrium binding decreased both ER and PR numbers by ≈50% without changing the dissociation constants of the steroids binding. The reduction of ER or PR numbers on neutrophils due to the pretreatment with insulin resulted in the decreased NO induced maspin synthesis from 2.383 ± 0.014 nM to 1.454 ± 0.004 nM in the case of estrogen and in the decrease of maspin synthesis from 2.329 ± 0.012 nM to 1.410 ± 0.002 nM in the case of progesterone. The incubation of ER+ neutrophils or PR+ neutrophils with insulin further decreased the maspin synthesis from 1.422 ± 0.029 nM to 0.790 ± 0.004 nM in the case of estrogen, and from 1.138 ± 0.024 nM to 0.555 ± 0.003 nM maspin in the case of progesterone respectively compared to normal control.

Conclusion: These results suggested that a "cross-talk" between the insulin receptors and the steroid receptors down regulated maspin synthesis in normal and in breast cancer neutrophils.

Keywords: breast-cancer; crosstalk; estrogen; insulin receptor; maspin; neutrophils; nitric oxide; progesterone; steroids receptors.

Figure 1

Scatchard plot of the equilibrium binding of insulin to normal neutrophils. The equilibrium binding of insulin to neutrophils was carried out by incubating purified porcine ¹²⁵I-insulin in the binding assay mixture as described in the Materials and Methods. After incubation for 2.5h at 23°C, the bound ligand was separated from the free insulin by using glass micro fibre filter (GF/C) in a Millipore manifold filtration unit as described (6).

Figure 2

Scatchard plot of estrogen binding to normal neutrophils pre incubated with or without insulin. A. Scatchard plot of equilibrium estrogen binding to normal neutrophils; B. Scatchard plot of equilibrium estrogen binding to the normal neutrophils pre incubated with insulin. Detail of the equilibrium binding of estrogen to neutrophils was carried out as described in Materials and Methods. The estrogen was quantitated by ELISA using polyclonal antibody raised in rabbits as described in the Materials and Methods. In the experiment the neutrophils were treated with 200 μunits of insulin for 2.5 h at 23°C before these cells were used for the study of binding of estrogen without removing insulin from the cell suspension.

Figure 3

Scatchard plot of the equilibrium binding of progesterone to normal neutrophils pre incubated with or without insulin. The equilibrium binding of progesterone to the normal neutrophils was carried out by incubating these cells with progesterone as described in the Materials and Methods. The progesterone binding was determined by ELISA using polyclonal antibody raised in rabbits. A, Scatchard plot of the equilibrium binding of progesterone to neutrophils incubated in the absence of insulin; B, Scatchard plot of the equilibrium binding of progesterone to the normal neutrophils preincubated with insulin. Neutrophils to suspension were incubated with 200 μU of insulin for 2.5 h at 23°C to reach equilibrium binding of insulin to these cells. These cells were next treated with progesterone to determine the binding of progesterone to neutrophils pre incubated with insulin without removing insulin from the binding assay mixture.

Figure 4

Effect of pre incubation of normal neutrophils with insulin on the estrogen induced NO and maspin synthesis. Normal neutrophils suspension was pre incubated with 200 μU of insulin for 2.5 h at 23°C to attain equilibrium binding of the hypoglycemic protein. The neutrophils pre incubated with insulin were next treated with different amounts of estrogen as indicated. After incubation for 4 h at 37°C the estrogen induced synthesis of both NO and maspin were subsequently determined. Solid square (▪) = effect of estrogen induced maspin synthesis; Hollow square (□) = effect of pre incubation of neutrophils with insulin on the estrogen induced maspin synthesis; Solid circle (●) = estrogen induced NO synthesis in neutrophils; Hollow circle (o) = estrogen induced NO synthesis in neutrophils pre incubated with insulin; Each point is mean ± S.D. of five different experiments each in triplicate using blood samples from 10 different donors.

Figure 5

Effect of pre incubation of normal neutrophils with insulin on the progesterone induced synthesis of NO and maspin. Normal neutrophils suspension was incubated with 200 μU of insulin to reach equilibrium binding of the protein hormone as describing under Figure 4. These cells were next treated with different amounts of progesterone as indicated and after incubation for 4 h at 37°C, the synthesis of both NO and maspin was determined. Solid square (▪) = progesterone induced maspin synthesis in neutrophils; Hollow square (□) = progesterone induced maspin synthesis in neutrophils pre incubated with insulin; Solid circle (●) = progesterone induced NO synthesis in neutrophils; Hollow circle (o) = progesterone induced NO synthesis in neutrophils previously incubated with insulin for 2 h at 23°C; Each point is mean±S.D.of five different experiments each in triplicate using blood samples from 10 different donors.