Purpose Estrogen, through its binding to nuclear estrogen receptor (ER), has been implicated in the development of human breast cancer. The presence or absence of ER in breast lesions has been used to classify breast cancer into ER+ or ER- type. Maspin, an anti-breast cancer protein produced in normal mammary cells, has also been reported to control the condition. Studies have been conducted to determine the role of ER+ and ER- status in neutrophils in the synthesis of maspin in human breast cancer. Methods Maspin presence was determined by enzyme linked immunosorbent assay, while nitric oxide (NO) level was determined using the methemoglobin method. Results Scatchard plots of the equilibrium binding of estrogen demonstrated the presence of 4.18×10 7 receptors per normal neutrophil and 2.46×10 7 receptors per ER+ neutrophil with a similar dissociation constant (0.926 nM). The ER- type showed nonspecific estrogen binding only. At 0.6 nM estrogen, NO synthesis was maximally increased to 1.829 and 0.887 µM NO/10 9 cells at 4 hours in normal and ER+ neutrophils respectively, with synthesis of 2.383 and 1.422 nM maspin in normal and ER+ neutrophils respectively. Estrogen failed to produce these effects in ER- neutrophils. Conclusion ER status in neutrophils determined maspin synthesis in breast cancer through the stimulation of NO synthesis. Neutrophils with ER- status which do not produce any maspin when treated with estrogen, might imply a worse prognostic outcome in ER- breast cancer due to the lack of anti-breast cancer protein synthesis.

Purpose: The binding of either progesterone or insulin 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 progesterone receptor interaction in the nitric oxide induced maspin synthesis in neutrophils that was preincubated with insulin. Methods: progesterone receptor positive (PR+), progesterone receptor negative (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: Immunohistochemical studies of progesterone receptor (PR) demonstrated the presence of progesterone receptor in the normal peripheral neutrophils and less in number in PR+ breast cancer neutrophils. In contrast, PR-breast cancer neutrophils lacked the progesterone receptor, suggesting pathophysiological defects in the synthesis of PR protein in peripheral PRneutrophils. It was also found that as a result of incubation of neutrophils with insulin the binding affinity for progesterone to its receptor in normal neutrophils remained essentially unchanged which demonstrated Kd = 47.619 nM compared to Kd of the binding of progesterone is 46.08 nM in the normal neutrophils that were not pretreated with insulin. The progesterone receptors which were 11.5×10 10 /cell in the untreated cells was found to be decreased to 8.2×10 10 /cell (p<0.005, n=6) after the same cell were treated with 200µU of insulin. The reduction of PR number on normal neutrophils due to the pretreatment with insulin resulted in the decreased NO induced maspin synthesis from 2.329 ± 0.012 nM to 1.410 ± 0.002 nM. Decreased PR number in PR+ breast cancer neutrophils due to disease condition and pretreatment with insulin reduced the maspin synthesis from 1.138 ± 0.024 nM to 0.555 ± 0.003 nM compared to normal control. Conclusion: These results suggested that insulin down regulated maspin synthesis in normal and in breast cancer neutrophils by decreasing the progesterone receptor number in both cases.

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These results suggest that estrogen and progesterone complement each other in NO-induced maspin synthesis, and do not necessarily antagonize in the synthesis of the anti-breast-cancer protein.