In vitro modulation of multidrug resistance by pregnane steroids and in vivo inhibition of tumour development by 7α-OBz-11α(R)-OTHP-5ß-pregnanedione in K562/R7 and H295R cell xenografts.

Synthetic progesterone and 5α/ß-pregnane-3,20-dione derivatives were evaluated as in vitro and in vivo modulators of multidrug-resistance (MDR) using two P-gp-expressing human cell lines, the non-steroidogenic K562/R7 erythroleukaemia cells and the steroidogenic NCI-H295R adrenocortical carcinoma cells, both resistant to doxorubicin. The maximal effect in both cell lines was observed for 7α-O-benzoyloxy,11α(R)-O-tetrahydropyranyloxy-5ß-pregnane-3,20-dione 4. This modulator co-injected with doxorubicin significantly decreased the tumour size and increased the survival time of immunodeficient mice xenografted with NCI-H295R or K562/R7 cells.

Synthesis of new steroidal inhibitors of P-glycoprotein-mediated multidrug resistance and biological evaluation on K562/R7 erythroleukemia cells.

A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/ß-pregnane-3,20-dione or 5ß-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/ß-pregnane-3,20-dione, among which the 5ß-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 µM versus 1.2 and 10.6 µM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5ß-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 µM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

Mitotane has an estrogenic effect on sex hormone-binding globulin and corticosteroid-binding globulin in humans.

CONTEXT: Side effects of mitotane (o,p'-DDD) have suggested estrogenic effects. OBJECTIVE: The objective of the study was to explore o,p'-DDD potential estrogenic effect on SHBG and corticosteroid-binding globulin (CBG). DESIGN: Human hepatoma cell lines (HepG2), lacking estrogen receptor (ER)-alpha, and Hep89, stably transfected by ERalpha, were used. SETTING: The study was conducted at an academic research laboratory and medical center. PATIENTS AND OTHER PARTICIPANTS: The study included 10 male patients with recurrent adrenal carcinoma, receiving mitotane (4-6.5 g daily) for more than 6 months. MAIN OUTCOME MEASURES: The main outcome measures were SHBG/CBG mRNA levels measured by real-time PCR, culture medium SHBG/CBG concentrations measured by specific immunoassays, and transient transfection experiments with human SHBG proximal promoter reporter constructs. RESULTS: Increased serum SHBG and CBG concentrations, which exceeded normal male limits, were observed in most mitotane-treated patients. In the HepG2 cell line, 17beta-estradiol (E2) or o,p'-DDD treatment had no effect on mRNA or SHBG/CBG concentrations. In contrast, in the Hep89 cell line, E2 increased concentrations of SHBG (r = 0.44, P < 0.0001) and CBG (r = 0.585, P < 0.0001) secreted into culture media in a dose-dependent manner. o,p'-DDD significantly increased SHBG (150% vs. control, P < 0.05) and CBG (184% vs. control, P < 0.05) production by Hep89 cells, at a concentration of 2 x 10(-5) m. Transient transfection experiments in Hep89 cells showed that E2 or o,p'-DDD treatment did not increase the transcriptional activity of the minimal proximal promoter of human SHBG gene. CONCLUSIONS: Mitotane increased SHBG/CBG gene expression and liver production by mechanisms requiring the presence of ERalpha.

Corticosteroid binding globulin: a new target for cortisol-driven obesity.

We present data suggesting that corticosteroid-binding globulin (CBG) may be the causal gene of a previously identified quantitative trait locus (QTL) associated with cortisol levels, fat, and muscle content in a pig intercross. Because Cbg in human and mouse maps in the region orthologous to the pig region containing this QTL, we considered Cbg as an interesting positional candidate gene because CBG plays a major role in cortisol bioavailability. Firstly, we cloned pig Cbg from a bacterial artificial chromosome library and showed by fluorescent in situ hybridization and radiation hybrid mapping that it maps on 7q26 at the peak of the QTL interval. Secondly, we detected in a subset of the pig intercross progeny a highly significant genetic linkage between CBG plasma binding capacity values and the chromosome 7 markers flanking the cortisol-associated QTL. In this population, CBG capacity is correlated positively to fat and negatively to muscle content. Thirdly, CBG capacity was three times higher in Meishan compared with Large White parental breeds and a 7-fold difference was found in Cbg mRNA expression between the two breeds. Overall, the data accumulated in this study point to Cbg gene as a key regulator of cortisol levels and obesity susceptibility.

Influence of SHBG gene pentanucleotide TAAAA repeat and D327N polymorphism on serum sex hormone-binding globulin concentration in hirsute women.

SHBG is the specific plasma transport protein for sex steroid hormones in humans. Plasma SHBG concentration follows a gender dimorphism but varies with nutritional and hormonal status in both sexes. In addition, a genetic influence on SHBG in humans has recently been suggested by family studies. We investigated the relationship between a point mutation (D327N) in SHBG gene exon 8 that delays human SHBG half-life and a pentanucleotide repeat polymorphism [PNRP (TAAAA)(n)] in the SHBG gene 5' untranslated region that influences transcription in vitro, on the one hand, and SHBG levels on the other, in a population of 303 women referred for hirsutism. Of these patients, 154 (51%) met the criteria for polycystic ovary syndrome (PCOS) and 124 (41%) were overweight [body mass index (BMI) > or = 25 kg/m(2)]. The two SHBG gene alleles for D327N substitution, wild-type (W) and variant (v), were identified by restriction fragment length polymorphism in the whole population, and the GeneScan method was used to identify PNRP alleles in 245 subjects. Six alleles of the pentanucleotide motif with six to 11 repeats were present in our population. Plasma SHBG concentration was related to PCOS status, non-SHBG-bound testosterone, BMI, fasting blood glucose level, fasting insulinemia, and D327N allele v. The v allele was associated with higher SHBG levels [36.9 +/- 15.9 nmol/liter for W/v (n = 52) and 43.5 +/- 3.5 nmol/liter for v/v (n = 2)] than was the wild-type W allele [31.1 +/- 16.1 nmol/liter (n = 249); P = 0.039]. Multivariate analysis showed that BMI, PCOS status, and D327N polymorphism influenced plasma SHBG concentrations, each of these parameters contributing independently of the others. Investigating the role of each allele of the TAAAA repeat polymorphism on SHBG levels was more complex because of the number of different genotypes (as many as 18 in our population) and the low frequency of some of them. Moreover, a strong disequilibrium linkage was found between D327N allele v and the eight-TAAAA repeat allele (P < 0.0001). This could mask the effect of the TAAAA repeat polymorphism on SHBG concentration in vivo. Nevertheless, SHBG levels in patients who were homozygous for six repeats (34.9 +/- 16.2 nmol/liter; n = 21) were significantly (P = 0.043) higher than in nine-repeat homozygous patients (21.5 +/- 13.0 nmol/liter; n = 8), and lay between the two for eight-repeat homozygous patients (28.5 +/- 15.8 nmol/liter; n = 44). Delineating the precise role of this PNRP polymorphism will need further investigation in a large healthy population. In summary, although BMI and PCOS status have a major influence on circulating SHBG levels in hirsute women, the present results support the notion that polymorphism(s) within the coding sequence and, potentially, in the regulatory sequence of the SHBG gene are associated with circulating SHBG levels and may represent part of the genetic background of sex steroid hormone activity in humans.