Józef Krzysiek

Regulation of Msx2 gene expression by steroid hormones in human nonmalignant and malignant breast cancer explants cultured in vitro

Muscle segment homeobox genes, which regulate developmental programs and are expressed in embryonic and adult tissue, play a role in development of some malignancies. There are no reports on the expression of these families of genes in breast cancer tissue. The aim of this study was to compare expression of Msx2 gene in breast cancer of different genotypes as well as in surrounding nonmalignant tissues. Explants obtained during surgery were divided according to their sex steroid receptor status determined by immunocytochemistry. Four explants obtained from malignant and nonmalignant tissue of each individual patient were incubated in a control medium or with the addition of progesterone (10− 7 M) alone, estradiol 17 β (10− 5 M) or both. The relative level of Msx2 transcripts was evaluated by a semiquantitative RT-PCR and cell proliferation by Alamar Blue test. Results of RT-PCR analysis showed that the relative expression of Msx2 gene depended on the presence of ER/PR receptors both in nonmalignant and malignant tissues Relative amount of Msx2 mRNA was the highest in surrounding nonmalignant ER + /PR − and ER − /PR + tissue, whereas in ER − /PR − and ER + /PR + tissue it was 1.4–1.6-fold lower. Tumorigenesis led to about a twofold decrease in the relative amount of Msx2 mRNA except for ER + /PR + immunophenotype, where no changes were observed. Addition of estradiol or progesterone to the culture of ER − /PR − type tissue explants did not change significantly the relative amount of Msx2 gene mRNA. An opposite effect was observed in ER + /PR − type of tissue. Addition of estradiol alone, or estradiol and progesterone together to tissue culture explants decreased two to three fold the relative amount of Msx2 gene mRNA in both, malignant and surrounding tissues. Progesterone alone had no effect on Msx2 gene expression in this type of tissue. The most complicated regulation was observed in ER + /PR + type of tissue. Culture of tissue explants supplemented with estradiol significantly increased the relative amount of Msx2 gene mRNA in the surrounding tissue. Progesterone enhanced the stimulatory effect of estradiol in surrounding tissues but not in the malignant tissue. Increased expression of Msx2 correlated with an increased proliferation in ER − /PR − and ER + /PR + types, but not in ER + /PR − type of tissues. In conclusion, obtained results provide evidence that estrogen affects Msx2 gene expression. Significant changes in the relative amount of Msx2 gene mRNA and lack of canonical ERE element in 5′-upstream sequence of this gene suggest that regulation takes place indirectly probably by protein-protein interaction. The decrease in the relative amount of Msx2 gene mRNA in ER + /PR − type tumor suggests that progesterone also affects Msx2 gene expression by an indirect mechanism(s).

Effect of hormone therapy on the enteroinsular axis.

Objective: Menopause is associated with a decline in insulin response to glucose and with insulin resistance. It has been proven that hormone therapy (HT) improves carbohydrate metabolism in postmenopausal women. However, it is known that gastrointestinal hormones play a key role in the coordination of digestion and absorption of ingested nutrients and in the regulation of pancreatic endocrine function. Therefore, the aim of this study was to investigate the effect of HT on gastrointestinal hormones and carbohydrate metabolism in postmenopausal women. Design: The prospective study was performed in 90 healthy postmenopausal women (mean age 54.5 years, standard deviation 3.34 years), of whom 49 completed the study. They were randomized and treated either with continuous transdermal HT (0.05 mg 17β-estradiol every 24 hours) combined with 5 mg oral dydrogesterone daily (group A, n = 25), or with oral HT (2 mg 17β-estradiol semihydrate every 24 hours) combined with 10 mg dydrogesterone as a continuous therapy (group B, n = 8). The control group (group C, n = 16) received no HT. Both basal and meal-stimulated plasma concentrations of glucose, insulin, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), and cholecystokinin (CCK), as well as basal estrogen levels, were measured before HT and after 6 and 12 months of treatment. At the same time intervals, all the studied parameters were measured for group C. Results: After 12 months of the transdermal HT, a decrease in both fasting (P < 0.002) and postprandial (P < 0.05) plasma glucose levels was observed. Oral HT reduced only the fasting plasma glucose level in the 12th month of treatment (P < 0.05). Regardless of the route of administration, HT reduced postprandial plasma levels of insulin (oral HT: P < 0.05; transdermal HT: P < 0.02). Fasting plasma levels of GIP were reduced after 6 and 12 months of transdermal HT (P < 0.002 and P < 0.001, respectively). Moreover, levels of postprandial GIP were reduced after 6 and 12 months of transdermal HT (P < 0.002 in both cases). Fasting and postprandial GLP-1 levels were reduced by transdermal HT after 12 months of supplementation. Oral HT also decreased these levels, but not significantly. The observed differences may, however, be related not only to the route of administration, but also to the difference in the dose of estradiol. Regardless of the route of administration, HT did not influence plasma levels of CCK. Conclusions: Hormone therapy significantly influences the enteroinsular axis in postmenopausal women and contributes to the normalization of plasma glucose levels.

Reduction in the level of antibodies against heat shock proteins 60 during different hormonal protocols in postmenopausal women

Introduction In current literature, the immune-inflammatory theory of atherosclerosis is widely discussed. The role of how heat shock proteins 60 (HSP60) lead to the development of the atheromatous plaque is especially underlined. The aim of the study is to estimate the influence of three hormonal protocols on behavior of antibodies against HSP60. It determines the state of endothelium in postmenopausal women. Material and methods The study was carried out on 90 women between 2007 and 2012. All the women were in their menopausal age (51 ± 3 years), from the south region of Poland, with a follicle stimulating hormone (FSH) level above 25 mIU/ml, and with menopausal symptoms disturbing their normal daily activity. The study was done for a period of 6 months. Three groups of 30 randomized patients were formed. In the first group we used transdermal estrogen therapy in a 37.5 µg/24 h dose combined with a 10 mg dose of dydrogesterone. In the second group we applied transdermal estrogen therapy in a 50 µg/24 h dose with 2.5 mg of oral medroxyprogesterone. In both these groups, gestagens were administered continuously. In the third group, we prescribed continuous, oral, low-dose combined estrogen-gestagen therapy with 1 mg of ethinyl estradiol and 0.5 mg of norethisterone acetate. The control group consisted of 30 volunteers who were also from the south region of Poland, in good health, with menopausal symptoms, no menstrual period for the last 12 months, selected considering their age and weight, with an FSH level above 25 mIU/ml and with normal levels of thyroid stimulating hormone (TSH) and prolactin. All patients treated and in the control group were seronegative to Chlamydia pneumonia for the entire duration of the study. In the analysis conducted, nonparametric tests were used (Mann-Whitney U test, Wilcoxon test, Kruskal-Wallis test – ANOVA). Results After 6 months of hormonal therapy, we found that all schemes of treatment promote a significant reduction in antibodies against HSP60 in all treated groups vs. the control group. Conclusions All of the investigated estrogen protocols have a favorable impact on the blood level of HSP60 antibodies in early postmenopausal women who have no cardiovascular risk factors. It triggers a better condition of endothelium.