Characterization of Precursor-Dependent Steroidogenesis in Human Prostate Cancer Models.

Castration-resistant prostate tumors acquire the independent capacity to generate androgens by upregulating steroidogenic enzymes or using steroid precursors produced by the adrenal glands for continued growth and sustainability. The formation of steroids was measured by liquid chromatography-mass spectrometry in LNCaP and 22Rv1 prostate cancer cells, and in human prostate tissues, following incubation with steroid precursors (22-OH-cholesterol, pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone). Pregnenolone, progesterone, 17-OH-pregnenolone, and 17-OH-progesterone increased C21 steroid (5-pregnan-3,20-dione, 5-pregnan-3,17-diol-20-one, 5-pregnan-3-ol-20-one) formation in the backdoor pathway, and demonstrated a trend of stimulating dihydroepiandrosterone or its precursors in the backdoor pathway in LNCaP and 22Rv1 cells. The precursors differentially affected steroidogenic enzyme messenger RNA (mRNA) expressions in the cell lines. The steroidogenesis following incubation of human prostate tissue with 17-OH-pregnenolone and progesterone produced trends similar to those observed in cell lines. Interestingly, the formation of C21 steroids from classical pathway was not stimulated but backdoor pathway steroids (e.g., 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one) were elevated following incubations with prostate tissues. Overall, C21 steroids were predominantly formed in the classical as well as backdoor pathways, and steroid precursors induced a diversion of steroidogenesis to the backdoor pathway in both cell lines and human prostate tissue, and influenced adaptive steroidogenesis to form C21 steroids.

Next-generation steroidogenesis inhibitors, dutasteride and abiraterone, attenuate but still do not eliminate androgen biosynthesis in 22RV1 cells in vitro.

Castration resistant prostate cancer (CRPC) is often lethal and inevitably develops after androgen ablation therapy. However, in the majority of cases it remains androgen dependent. CRPC tumors have the ability to synthesize their own androgens from cholesterol by engaging in de novo steroidogenesis. We investigated the potential of 22RV1 prostate cancer cells to convert the supplemented steroid precursors within this pathway under the effects of current clinical steroidogenesis inhibitors such as abiraterone and dutasteride, either alone or in combination. Under steroid starved conditions, enzymes responsible for de novo steroidogenesis were upregulated. Testosterone and dihydrotestosterone (DHT) were formed by using both dehydroepiandrosterone (DHEA) and progesterone as substrates. Formation of testosterone and DHT was higher following incubation with DHEA compared to progesterone. Progesterone decreased the mRNA expression of enzymes responsible for steroidogenesis. Abiraterone treatment decreased testosterone production but increased several precursor steroids in both classical and backdoor pathways in the presence of progesterone. In contrast, the DHT levels were elevated following treatment with abiraterone when progesterone was absent. Dutasteride decreased the formation of testosterone, DHT and precursor steroids in the backdoor pathway but increased steroid precursors in the classical steroidogenesis pathway. The combination of abiraterone and dutasteride decreased testosterone and DHT in the presence of progesterone but increased DHT in the absence of progesterone. Abiraterone inhibited androgen receptor (AR) activation but not to the same extent as MDV3100. However, abiraterone and dutasteride treatment, either alone or in combination, were more effective in decreasing prostate specific antigen secretion into the media than MDV3100. Thus, while interventions with these drugs alone or in combination fail to completely inhibit steroidogenesis in the 22RV1 cells, the combined inhibition of androgen production and blockade of AR can exceed the effect of MDV3100. Further characterization of bypass mechanisms that may develop as a response to these inhibitors is necessary to achieve optimal suppression of testosterone and DHT synthesis as a part of therapeutic regimens for the treatment of CRPC.

Pomegranate extracts impact the androgen biosynthesis pathways in prostate cancer models in vitro and in vivo.

Castration-resistant prostate cancer (CRPC) remains largely dependent on androgen receptor (AR). Residual tissue androgens are consistently detected within CRPC tumors and play a critical role in facilitating AR-mediated signaling pathways which lead to disease progression. Testosterone and dihydrotestosterone (DHT) are the major androgens detected in tumors. They are produced through three biosynthesis pathways: Δ(4), Δ(5), and backdoor pathways. Both androgens bind to and stimulate AR activation. The current study investigates the effects of pomegranate extracts (POM) and their ability to inhibit androgen biosynthesis using PCa cell lines (22RV1 and LNCaP) in vitro as well as the PTEN knockout mouse model representing prostate cancer. Steroids were extracted using ethyl acetate or solid phase extraction, and then analyzed by UPLC/MS/MS. The results showed that POM (0-12µg/mL) reduced the production of testosterone, DHT, DHEA, androstenedione, androsterone, and pregnenolone in both cell lines. In addition our in vivo data supports this observation with a reduction in serum steroids determined after 20 weeks of POM treatment (0.17 g/L in drinking water). In accordance with these results, Western blotting of cell lysates and tPSA analysis determined that PSA was significantly decreased by the treatment of POM. Interestingly, AKR1C3 and AR levels were shown to be increased in both cell lines, perhaps as a negative feedback effect in response to steroid inhibition. Overall, these results provide mechanistic evidence to support the rationale for recent clinical reports describing efficacy of POM in CRPC patients.

The determination of urinary free and conjugated cortisol using UPLC-MS/MS.

BACKGROUND: To develop an UPLC-MS/MS method to replace the in-house immunoassay for the analysis of urinary cortisol. RESULTS: Cortisol was extracted from human urine by ethyl acetate and analyzed on a Waters ACQUITY TQD system using a BEH C18 column. Linear calibration curves were generated over the range of 27.6 to 1380 nmol/l and exhibited consistent linearity and reproducibility with a correlation coefficient greater than 0.9950. Intra-day coefficients of variation were between 3.74 and 5.10% and inter-day coefficients of variations were between 4.22 and 6.73%. The extraction recovery of cortisol was greater than 83%. CONCLUSION: An accurate, rapid and robust UPLC-MS/MS method for the determination of urinary cortisol has been developed and validated. With a lower flow rate (0.4 ml/min), a shorter running time per sample and a simple and cost-effective sample preparation, this method is a desirable option for clinical laboratories.

A rapid and accurate UPLC/MS/MS method for the determination of benzodiazepines in human urine.

A rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) assay method for simultaneous determination of 13 benzodiazepine compounds in human urine was developed and validated. Aliquots of 0.5 mL of urine specimens were used for the analysis and the benzodiazepines were extracted by single step methanol (containing 0.2% formic acid) precipitation and then separated on a BEH C18 (50 mm × 2.1 mm, 1.7 µm) analytical column with the temperature maintained at 45°C. The mobile phases consisted of methanol and water (both containing 0.2% formic acid) and the flow rate was 0.4 mL/min. The TQ detector, equipped with an electrospray ionization ion source, was set up with a positive mode. The acquisitions were performed in multiple-reaction monitoring (MRM) and the limit of quantification was 20 ng/mL for all of the 13 compounds. The low limits of detections (LODs) of the benzodiazepines in this method were between 0.5 and 2 ng/mL. The chromatographic separation time was 4 min and calibration curves in human urine were generated over the range of 20-2000 ng/mL. The method validation parameters such as accuracy, precision, carryover, recovery, stability, and specificity for all of the 13 compounds were within the acceptable range. This method is suitable for the high throughput screening of benzodiazepines in clinical laboratories.

Therapeutic drug monitoring of clozapine and norclozapine in human serum using ultra-performance liquid chromatography- tandem mass spectrometry.

A rapid, sensitive, and specific method was developed and validated using ultra-performance liquid chromatography- tandem mass spectrometry (UPLC-MS-MS) for simultaneous determination of clozapine and its major metabolite norclozapine in human serum. The compounds were extracted from serum by a single step protein precipitation and analyzed using a UPLC-triple-quadrupole detection (TQD) system. Separation of compounds was achieved on a BEH C18 (50 mm x 2.1 mm, 1.7 microm) analytical column using methanol and water (both containing 0.2% ammonium hydroxide) as the mobile phase at a flow rate of 0.40 mL/min. The compounds were ionized in the electrospray ionization ion source of the TQD and were detected in the multiple reaction monitoring (MRM) mode. The MRM transitions m/z 327 --> 270 and m/z 313 --> 192 for clozapine and norclozapine, respectively, were used for the quantification ions. Clozapine transition 327 --> 192 and norclozapine transition 313 --> 270 were used as confirmation ions. Linear calibration curves in human serum were generated over the range of 10-2000 ng/mL for both clozapine and norclozapine with a correlation coefficient (r(2)) > 0.9970. Calibration curves exhibited consistent linearity and reproducibility. Interassay coefficients of variation (CV) (n = 20) were 3.04-4.94% for clozapine and 2.84-6.07% for norclozapine. Intra-assay CVs (n = 6, 20 days) were 0.61-1.26% and 1.62-2.21% for clozapine and norclozapine, respectively. The extraction recoveries were larger than 95% for both clozapine and norclozapine. The method was applied to the quantification of clozapine and norclozapine in the sera of schizophrenic patients, and the data revealed that the concentrations of two compounds varied significantly in the patients treated with clozapine.

The structure and absolute configuration of Shuangkangsu: a novel natural cyclic peroxide from Lonicera japonica (Thunb.).

A novel cyclic peroxide, shuangkangsu (1), has been obtained from the water extract of the buds of Lonicera japonica (Thunb.). The structure was elucidated as 5,8-divinyl-1,4-dihydro-1,4-di-O-beta-d-glucopyranosyl-2,3-dioxane (1) on the basis of the spectral data. Its absolute stereochemistry was determined to be (1S, 4S) by comparison of its CD curves with those of its optically pure analogs 2 and 3, which were synthesized from the phthalaldehyde. The absolute configurations of 2 and 3 were determined to be (1R, 4R) and (1S, 4S) by X-ray analysis and CD spectra, respectively. Compound 1 showed significant antiviral activities against influenza virus in chicken embryo and respiratory syncytial virus in cells.

Bioactivity-guided isolation of the active compounds from Rosa nutkana and quantitative analysis of ascorbic acid by HPLC.

Rosa nutkana Presl. (Rosaceae) is distributed abundantly throughout central and southern areas of British Columbia, Canada. Aboriginal people in the Pacific Northwest have traditionally used R. nutkana as a food, medicine, and source of cultural material. The methanolic extract of the fruits of R. nutkana was previously found to have inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA). In our study, bioactivity-guided fractionation of the methanol extract from R. nutkana led to the isolation of the following 10 compounds: (i) tormentic acid, (ii) euscaphic acid, (iii) ursolic acid, (iv) maslinic acid, (v) quercetin, (vi) catechin gallate, (vii) quercetin-3-O-glucoside, (viii) 1,2,3,4,6-penta-O-galloyl-beta-D-glucoside, (ix) L-ascorbic acid (vitamin C), and (x) 1,6-digalloyl-beta-D-glucoside. Structures were elucidated by ultraviolet, infrared, mass spectrometry, and nuclear magnetic resonance data, as well as by comparison with those of the literature. The compounds quercetin, catechin gallate, quercetin-3-O-glucoside, 1,2,3,4,6-penta-O-galloyl-beta-D-glucoside, and 1,6-digalloyl-beta-D-glucoside exhibited weak antibacterial activity against MRSA. Our research demonstrates the value of traditional knowledge held by Aboriginal people in the Pacific Northwest with respect to uses of R. nutkana. Some described uses in the ethnobotanical literature correspond to activities observed under laboratory conditions. Further work on British Columbia Rosa spp. may contribute to identifying other potential therapeutic uses.

Bioactive compounds from Rhodiola rosea (Crassulaceae).

The methanol extract of the underground part of Rhodiola rosea was found to show inhibitory activity against Staphylococcus aureus. Bioactivity-guided fractionation of a 95% ethanol extract from the stems of R. rosea led to the isolation of five compounds: gossypetin-7-O-L-rhamnopyranoside (1), rhodioflavonoside (2), gallic acid (3), trans-p-hydroxycinnamic acid (4) and p-tyrosol (5). Their structures were elucidated by UV, IR, MS and NMR data, as well as by comparison with those of the literature. Compounds 1 and 2 were evaluated for their antibacterial and antiprostate cancer cell activities. Compounds 1 and 2 exhibited activity against Staphylococcus aureus with minimum inhibitory concentrations of 50 microg/mL and 100 microg/mL, respectively. Cytotoxicity studies of 1 and 2 also displayed activity against the prostate cancer cell line with IC(50) values of 50 microg/mL and 80 microg/mL, respectively.

Bioactive constituents from Iryanthera megistophylla.

Activity-guided fractionation of the 95% ethanol extract from the stem bark of Iryanthera megistophylla led to the isolation of two new compounds, named megislignan [2,3-dimethyl-4-(4-methoxyphenyl)-6-hydroxynaphthalene] (1) and megislactone [(2R,3R,4R)-3-hydroxy-4-methyl-2-(hexacos-17-enyl)butanolide] (2), along with seven known compounds, grandinolide (3), iryantherin K (4), iryantherin L (5), cinchonain I b (6), cinchonain I a (7), procyanidin B-2 (8), and cinchonain IIa (9). The structures of the new compounds were elucidated by spectral data interpretation. Isolates were evaluated for their antibacterial, antifungal, antiviral, and antiacetylcholinesterase activities.

A new compound from Geum rivale L.

A new compound, 1-O-methyl-6-O-caffeoyl-beta-D-glucopyranose (1), has been isolated from the aerial part of G. rivale, together with five known compounds, cecropiacic acid (2), niga-ichgoside (3), gallic acid (4), 1-o-protocatechuoylglucose (5), and sucrose (6). Their structures were elucidated by spectral methods and chemical reactions.

Antifungal activity of benzoic acid derivatives from Piper lanceaefolium.

Bioactivity-guided fractionation of a methanol extract from the leaves of Piper lanceaefolium resulted in the isolation of four new benzoic acid derivatives (1-4), together with taboganic acid, pinocembrin, and pinocembrin chalcone. Lanceaefolic acid methyl ester (3) and pinocembrin chalcone displayed activity against Candida albicans with a minimal inhibitory concentration value of 100 microg/mL in both cases.