In Silico Evaluation of the Binding Energies of Androgen Receptor Agonists in Wild-Type and Mutational Models.

Anabolic androgenic steroids (AAS) are substances with androgenic and anabolic characteristics. Among the many side effects of hormone therapy with AAS, the following stand out: heart problems, adrenal gland disorders, aggressive behavior, increased risk of prostate cancer, problems related to lack of libido and impotence. Such substances vary in the relationship between androgenic activity, and the activation of the androgen receptor (AR) is of fundamental importance for the singularity of the action of each AAS. In this sense, our study evaluates the aspects that comprise the interactions of testosterone agonists (TES), dihydrotestosterone (DHT) and tetrahydrogestrinone (THG) in complex with the AR. In addition, we also evaluated the impact of ligand-receptor affinity differences in a mutation model. We apply computational techniques based on density functional theory (DFT) and use, as methodology, Molecular Fractionation with Conjugate Caps (MFCC). The energetic specificities present in the interaction between the analyzed complexes attest that the highest affinity with the AR receptor is found for AR-THG, followed by AR-DHT, AR-TES and AR-T877A-DHT, respectively. Our results also show the differences and equivalences between the different agonists, in addition to evaluating the difference between the DHT ligand in complex with the wild-type and mutant receptor, presenting the main amino acid residues that involve the interaction with the ligands. The computational methodology used proves to be an operative and sophisticated choice to help in the search for pharmacological agents for various therapies that have androgen as a target.

Investigating a new C2-symmetric testosterone dimer and its dihydrotestosterone analog: Synthesis, antiproliferative activity on prostate cancer cell lines and interaction with CYP3A4.

The synthesis of a 17α-linked C2-symmetric testosterone dimer and its dihydrotestosterone analog is reported. The dimers were synthesized using a short five-step reaction sequence with 28% and 38% overall yield for the testosterone and dihydrotestosterone dimer, respectively. The dimerization reaction was achieved by an olefin metathesis reaction with 2nd generation Hoveyda-Grubbs catalyst. The dimers and their corresponding 17α-allyl precursors were tested for the antiproliferative activity on androgen-dependent (LNCaP) and androgen-independent (PC3) prostate cancer cell lines. The effects on cells were compared with that of the antiandrogen cyproterone acetate (CPA). The results showed that the dimers were active on both cell lines, with an increased activity towards androgen-dependent LNCaP cells. However, the testosterone dimer (11) was fivefold more active than the dihydrotestosterone dimer (15), with an IC50 of 11.7 µM vs. 60.9 µM against LNCaP cells, respectively, and more than threefold more active than the reference drug CPA (IC50 of 40.7 µM). Likewise, studies on the interaction of new compounds with drug-metabolizing cytochrome P450 3A4 (CYP3A4) showed that 11 was a fourfold stronger inhibitor than 15 (IC50 of 3 µM and 12 µM, respectively). This suggests that changes in the chemical structure of sterol moieties and the manner of their linkage could largely affect both the antiproliferative activity of androgen dimers and their crossreactivity with CYP3A4.

Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF.

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

Partial Agonistic Actions of Sex Hormone Steroids on TRPM3 Function.

Sex hormone steroidal drugs were reported to have modulating actions on the ion channel TRPM3. Pregnenolone sulphate (PS) presents the most potent known endogenous chemical agonist of TRPM3 and affects several gating modes of the channel. These includes a synergistic action of PS and high temperatures on channel opening and the PS-induced opening of a noncanonical pore in the presence of other TRPM3 modulators. Moreover, human TRPM3 variants associated with neurodevelopmental disease exhibit an increased sensitivity for PS. However, other steroidal sex hormones were reported to influence TRPM3 functions with activating or inhibiting capacity. Here, we aimed to answer how DHEAS, estradiol, progesterone and testosterone act on the various modes of TRPM3 function in the wild-type channel and two-channel variants associated with human disease. By means of calcium imaging and whole-cell patch clamp experiments, we revealed that all four drugs are weak TRPM3 agonists that share a common steroidal interaction site. Furthermore, they exhibit increased activity on TRPM3 at physiological temperatures and in channels that carry disease-associated mutations. Finally, all steroids are able to open the noncanonical pore in wild-type and DHEAS also in mutant TRPM3. Collectively, our data provide new valuable insights in TRPM3 gating, structure-function relationships and ligand sensitivity.

Co-treatment of testosterone and estrogen mitigates heat-induced testicular dysfunctions in a rat model.

The two gonadal steroid hormones, testosterone and estrogen, regulate spermatogenesis by proliferation, differentiation, and apoptosis of testicular cells. It has been reported that heat stress or increased scrotal temperature impairs spermatogenesis in many mammals. Moreover, testicular heat stress has also been shown to suppress testosterone and estrogen biosynthesis. Furthermore, it is well known that testosterone and estrogen are important for testicular activity. Therefore, we hypothesised that exogenous testosterone and estrogen, alone or in combination, might alleviate the testicular activity in a heat-stressed rat model. To the best of our knowledge, this will be the first report of the exogenous treatment of both testosterone and estrogen in the heat-stressed rat. Our results showed that a combined testosterone and estrogen treatment significantly increased sperm concentration. The histopathological analysis also exhibited a normal histoarchitecture in the combined treatment group along with decreased oxidative stress. The improved spermatogenesis in the combined treatment group was also supported by the increase in PCNA, GCNA, tubule diameter, germinal epithelium height, and Johnsen score in the combined treatment group. Furthermore, the combined treatment also increased the expression of Bcl2, pStat3, and active caspase-3 and decreased expression of Bax. Thus, increased proliferation, apoptotic and anti-apoptotic markers, along with improved histology in the combined treatment group suggest that estrogen and testosterone synergistically act to stimulate spermatogenesis by increasing proliferation and differentiation of germ cells and may also remove the heat-induced damaged germ cells by apoptosis. Overall, the final mechanism of testosterone- and estrogen-mediated improvement of testicular activity could be attributed to amelioration of oxidative stress.

Innovative C2-symmetric testosterone and androstenedione dimers: Design, synthesis, biological evaluation on prostate cancer cell lines and binding study to recombinant CYP3A4.

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17ß-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17ß-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17ß-OH (14α and 14ß) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14ß) with an average IC50 of 13.3 µM. In LNCaP cells, 14ß was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0 µM vs. 59.6 µM, respectively). At low concentrations (0.25-0.5 µM), 14α and 14ß were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.

11-Oxygenated Estrogens Are a Novel Class of Human Estrogens but Do not Contribute to the Circulating Estrogen Pool.

Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen both in men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we use steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in 3 different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11ß-hydroxy-17ß-estradiol binds and activates estrogen receptors α and ß and that 11ß-hydroxy-17ß-estradiol and the classic androgen pathway-derived active estrogen, 17ß-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data show that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.

Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa.

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection.

Synthesis of some steroidal mitocans of nanomolar cytotoxicity acting by apoptosis.

Several steroids (abiraterone, prednisone, testosterone, cholesterol) and the BCL-2 inhibitor bexarotene were used as starting materials to synthesize iperazinyl-spacered rhodamine B conjugates. The conjugates were screened for their cytotoxicity in SRB assays against several human tumor cell lines and found to be active in a low µM to nM range. The conjugate derived from testosterone held an EC50 = 59 nM against MCF-7 tumor cells and acted mainly by necrosis. The prednisone conjugate, however, was less cytotoxic but acted mainly by apoptosis and held a moderate selectivity against MCF-7 tumor cells.

Nickel and zinc complexes of testosterone N4-substituted thiosemicarbazone: Selective cytotoxicity towards human colorectal carcinoma cell line HCT 116 and their cell death mechanisms.

Two new Schiff base ligands (TE and TF) were prepared from conjugation of testosterone with 4-(4-ethylphenyl)-3-thiosemicarbazide and 4-(4-fluorophenyl)-3-thiosemicarbazide, respectively. Their nickel (NE and NF) and zinc (ZE and ZF) complexes were reported. X-ray crystallography revealed a distorted square planar geometry was adopted by NE. The compounds demonstrated excellent selectivity towards the colorectal carcinoma cell line HCT 116 despite their weak preferences towards the prostate cancer cell lines (PC-3 and LNCaP). Against HCT 116, all these compounds were able to arrest cell cycle at G0/G1 phase and induce apoptosis via mitochondria-dependent (TE, NE, and TF) and extrinsic apoptotic pathway (ZE, NF, and ZF). Moreover, only ZE was able to act as topoisomease I poison and halt its enzymatic reactions although all compounds presented excellent affinity towards DNA.

Progestins Related to Progesterone and Testosterone Elicit Divergent Human Endometrial Transcriptomes and Biofunctions.

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use.

Oleanolic Acid Ameliorates Benign Prostatic Hyperplasia by Regulating PCNA-Dependent Cell Cycle Progression In Vivo and In Vitro.

Oleanolic acid (OA) is a natural, biologically active pentacyclic triterpenoid found in Cornus officinalis. Although C. officinalis and OA have antiproliferative actions, the effects and mechanisms of OA in benign prostatic hyperplasia (BPH) are unclear. We examined the effect of OA in an animal model of testosterone-induced BPH. Male rats were injected with testosterone propionate with or without OA. The inhibitory effect of OA on BPH-1 cells was determined in vitro. Rats with BPH exhibited outstanding BPH symptoms, including prostatic enlargement, upregulated dihydrotestosterone and 5α-reductase 2 levels, and histological changes. Compared with the BPH group, the OA group showed fewer pathological alterations and regular androgen events. OA inhibited prostate cell proliferation by downregulating the expression of proliferating cell nuclear antigen (PCNA) and cell cycle markers in BPH-induced animals. This indicated that OA has superior therapeutic effect in the BPH animal model than finasteride. In vitro studies demonstrated upregulation of PCNA and cell cycle proteins, whereas OA clearly reduced this upregulation. Thus, OA may inhibit the development of BPH by targeting cell cycle progression markers. These suggest that OA is a potential agent for BPH treatment.

Perinatal concentrations of 17ß-estradiol and testosterone in the toe claws of female and male dogs from birth until 60 days of age.

This study was conducted to assess concentrations of 17ß-estradiol (E2) and testosterone (T) in toe claws of puppies collected at birth, at 30 and at 60 days of age, evaluating changes relating to age and effect of puppy sex, Apgar score, bodyweight at birth, "litter effect", litter size, and maternal age. Puppies (n = 89), 46 males and 43 females, with normal weight and without malformations, were assigned for the study. Within 12 h of birth tips of toe claws were clipped, and the re-growth tissue of the claws was collected at 30 and 60 days of age. Steroid quantifications occurred using a radioimmunoassay. The results indicated there were lesser concentrations (P < 0.001) of both hormones at 30 and 60 days of age than at birth and that concentrations were similar at 30-60 days of age. There were greater (P < 0.001) T concentrations in males than females, with there being an interaction between sex and sampling time (P < 0.01). The Apgar score was positively (P < 0.001) related to T concentrations in toe claws at birth. The bodyweight was positively correlated (P < 0.05) with T concentrations, with an interaction among puppy sex, bodyweight and sampling time (P < 0.05). Results of the present study confirmed the usefulness of toe claws as a matrix for study of hormonal changes in perinatology of dogs. Results of the study also indicate there are greater E2 and T concentrations at birth compared with 30 and 60 days of age that could be the result of these prenatal steroids affecting fetal development.

Enzymatic Synthesis of Anabolic Steroid Glycosides by Glucosyltransferase from Terribacillus sp. PAMC 23288.

The application of steroids has steadily increased thanks to their therapeutic effects. However, alternatives are required due their severe side effects; thus, studies on the activities of steroid derivatives are underway. Sugar derivatives of nandrolone, which is used to treat breast cancer, as well as cortisone and prednisone, which reduce inflammation, pain, and edema, are unknown. We linked O-glucose to nandrolone and testosterone using UDP-glucosyltransferase (UGT-1) and, then, tested their bioactivities in vitro. Analysis by NMR showed that the derivatives were 17ß-nandrolone ß-D-glucose and 17ß-testosterone ß-D-glucose, respectively. The viability was higher and cytotoxicity was evident in PC12 cells incubated with rotenone and, testosterone derivatives, compared to the controls. SH-SY5Y cells incubated with H2O2 and nandrolone derivatives remained viable and cytotoxicity was attenuated. Both derivatives enhanced neuronal protective effects and increased the amounts of cellular ATP.

Molecular interactions between sex hormone-binding globulin and nonsteroidal ligands that enhance androgen activity.

Sex hormone-binding globulin (SHBG) determines the equilibrium between free and protein-bound androgens and estrogens in the blood and regulates their access to target tissues. Using crystallographic approaches and radiolabeled competitive binding-capacity assays, we report here how two nonsteroidal compounds bind to human SHBG, and how they influence androgen activity in cell culture. We found that one of these compounds, (-)3,4-divanillyltetrahydrofuran (DVT), present in stinging nettle root extracts and used as a nutraceutical, binds SHBG with relatively low affinity. By contrast, a synthetic compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI), bound SHBG with an affinity similar to that of testosterone and estradiol. Crystal structures of SHBG in complex with DVT or IPI at 1.71-1.80 Šresolutions revealed their unique orientations in the SHBG ligand-binding pocket and suggested opportunities for the design of other nonsteroidal ligands of SHBG. As observed for estradiol but not testosterone, IPI binding to SHBG was reduced by ∼20-fold in the presence of zinc, whereas DVT binding was almost completely lost. Estradiol-dependent fibulin-2 interactions with SHBG similarly occurred for IPI-bound SHBG, but not with DVT-bound SHBG. Both DVT and IPI increased the activity of testosterone in a cell culture androgen reporter system by competitively displacing testosterone from SHBG. These findings indicate how nonsteroidal ligands of SHBG maybe designed to modulate the bioavailability of sex steroids.

Oxime-based 19-nortestosterone-pheophorbide a conjugate: bimodal controlled release concept for PDT.

Photodynamic therapy has become a feasible direction for the treatment of both malignant and non-malignant diseases. It has been in the spotlight since FDA regulatory approval was granted to several photosensitizers worldwide. Nevertheless, there are still strong limitations in the targeting specificity that is vital to prevent systemic toxicity. Here, we report the synthesis and biological evaluation of a novel bimodal oxime conjugate composed of a photosensitizing drug, red-emitting pheophorbide a, and nandrolone (NT), a steroid specifically binding the androgen receptor (AR) commonly overexpressed in various tumors. We characterized the physico-chemical properties of the NT-pheophorbide a conjugate (NT-Pba) and singlet oxygen generation. Because light-triggered therapies have the potential to provide important advances in the treatment of hormone-sensitive cancer, the biological potential of this novel specifically-targeted photosensitizer was assessed in prostatic cancer cell lines in vitro using an AR-positive (LNCaP) and an AR-negative/positive cell line (PC-3). U-2 OS cells, both with and without stable AR expression, were used as a second cell line model. Interestingly, we found that the NT-Pba conjugate was not only photodynamically active and AR-specific, but also that its phototoxic effect was more pronounced compared to pristine pheophorbide a. We also examined the intracellular localization of NT-Pba. Live-cell fluorescence microscopy provided clear evidence that the NT-Pba conjugate localized in the endoplasmic reticulum and mitochondria. Moreover, we performed a competitive localization study with the excess of nonfluorescent NT, which was able to displace fluorescent NT-Pba from the cell interior, thereby further confirming the binding specificity. The oxime ether bond degradation was assayed in living cells by both real-time microscopy and a steroid receptor reporter assay using AR U-2 OS cells. Thus, NT-Pba is a promising candidate for both the selective targeting and eradication of AR-positive malignant cells by photodynamic therapy.

Comparison of Steroid Hormone Hydroxylations by and Docking to Human Cytochromes P450 3A4 and 3A5.

PURPOSE: Hydroxylation activity at the 6ß-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (P450 or CYP) 3A4 and CYP3A5 and their molecular docking energy values were compared to understand the catalytic properties of the major forms of human CYP3A, namely, CYP3A4 and CYP3A5. METHODS: Testosterone, progesterone, and cortisol 6ß-hydroxylation activities of recombinant CYP3A4 and CYP3A5 were determined by liquid chromatography. Docking simulations of these substrates to the heme moiety of reported crystal structures of CYP3A4 (Protein Data Bank code ITQN) and CYP3A5 (6MJM) were conducted. RESULTS: Michaelis constants (Km) for CYP3A5- mediated 6ß-hydroxylation of testosterone and progesterone were approximately twice those for CYP3A4, whereas the value for cortisol 6ß-hydroxylation mediated by CYP3A5 was similar to the value for that by CYP3A4. Maximal velocities (Vmax) of the three steroid hormones 6ß-hydroxylation catalyzed by CYP3A5 were 30%-63% of those by CYP3A4. Thus, Vmax/ Km values of these hormones for CYP3A5 resulted in 22%- 31% of those for CYP3A4. The differences in the docking energies between CYP3A4 and CYP3A5 for steroid hormones were slightly correlated to the logarithm of CYP3A5/CYP3A4 ratios for Km values (substrate affinity). CONCLUSIONS: The Vmax, rather than Km values, for CYP3A5-mediated 6ß-hydroxylation of three steroid hormones were different from those for CYP3A4. Molecular docking simulations could partially explain the differences in the accessibility of substrates to the heme moiety of human CYP3A molecules, resulting in the enzymatic affinity of CYP3A4 and CYP3A5.

Second-generation testosterone-platinum(II) hybrids for site-specific treatment of androgen receptor positive prostate cancer: Design, synthesis and antiproliferative activity.

Prostate cancer is the most diagnosed type of cancer in men in Canada. One out of eight men will be stricken with this disease during the course of his life. It is noteworthy that, at initial diagnoses 80-90% of cancers are androgen dependent. Hence, the androgen receptor is a viable biological target to be considered for drug targeting. We have developed a new generation of testosterone-Pt(II) hybrids for site-specific treatment of hormone-dependent prostate cancer. The hybrid molecules are made from testosterone using an eight-step reaction sequence with about 7% overall yield. They are linked with a stronger tether chain between the testosterone moiety and the Pt(II) moiety in comparison to our first generation hybrids. The new hybrids were tested on hormone-dependent and -independent prostate cancer cell lines. The hybrid 3a presents the best antiproliferative activity and was selective on hormone-dependent prostate cancer with IC50 of 2.2 µM on LNCaP (AR+) in comparison to 13.3 µM on PC3 (AR-) and 8.8 µM on DU145 (AR-) prostate cancer cells. On the same cell lines, CDDP displayed IC50 of 2.1 µM, 0.5 µM and 1.0 µM, respectively. Remarkably, hybrid 3a was inactive on both colon carcinoma (HT-29) and normal human adult keratinocyte cells (HaCat) with an IC50 of >25 µM. This is not the case for CDDP showing IC50 of 1.3 µM and 5.1 µM on HT-29 and HaCat cells, respectively. The potential for selective activity on androgen-receptor positive prostate cancer cells is confirmed with hybrid 3a giving new hope for an efficient and less toxic platinum-based treatment of prostate cancer patients.

Concentrations of melatonin, thyroxine, 17ß-estradiol and 11-ketotestosterone in round goby (Neogobius melanostomus) in different phases of the reproductive cycle.

The aim of this study was to determine changes in concentrations of melatonin (Mel) and thyroxine (T4) in plasma, and 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) in plasma and gonads of female and male round gobies (Neogobius melanostomus) from the Southern Baltic Sea in four phases of the reproductive cycle classified as pre-spawning, spawning, late spawning and non-spawning periods. The concentrations of Mel, T4 and E2 were determined by radioimmunoassay (RIA) whereas 11-KT was quantified using an enzyme immunoassay (EIA). The maturity stage of gonads was determined using histological analyses. The pattern of changes in Mel concentrations of females and males was similar with the greatest concentrations in the spawning and non-spawning phases. In both sexes, there was a similar tendency of change in concentrations of T4 and E2 with the increase being in the pre-spawning and non-spawning phases. The greatest concentrations of 11-KT were observed in the plasma and gonads of males during the spawning phase. In females, there were no changes in 11-KT concentrations either in plasma or gonads during all phases where quantifications occurred. This is the first study for determination of the pattern of changes in Mel and T4 concentrations as well as gonadal steroids E2 and 11-KT, supported by histological analysis of gonads, in batch spawning fish during the reproductive cycle.

Location of multiple binding sites for testo and testo-Pt(II) with tRNA.

We report the binding of testo and testo-Pt(II) complexes (testosterone derivatives) with tRNA in aqueous solution at physiological pH. Thermodynamic parameter ΔH0 -8 to -3 (kJ mol-1), ΔS0 35 to 18 (J mol-1K-1) and ΔG0 -14 to -13 (kJ mol-1) and other spectroscopic results showed drug-tRNA binding occurs via ionic contacts with testo-Pt(II) forming more stable tRNA complexes in comparison to testo: Ktesto-Pt(II)-tRNA= 3.2 (± 0.9) × 105 M-1 > Ktesto-tRNA= 2.1 (± 0.7) × 105 M-1. Molecular modeling showed multiple binding sites for testo and testo-Pt(II) on tRNA molecule. Some of the useful molecular descriptors are calculated. Major structural changes were observed for biopolymers upon drug complexation, while tRNA remains in the A-family structures.