Pharmacological polyamine catabolism upregulation with methionine salvage pathway inhibition as an effective prostate cancer therapy.

Prostatic luminal epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen, sensitizing them to perturbations of connected metabolic pathways. Enhanced flux is driven by spermidine/spermine N1-acetyltransferase (SSAT) activity, which acetylates polyamines leading to their secretion and drives biosynthetic demand. The methionine salvage pathway recycles one-carbon units lost to polyamine biosynthesis to the methionine cycle to overcome stress. Prostate cancer (CaP) relies on methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme, to relieve strain. Here, we show that inhibition of MTAP alongside SSAT upregulation is synergistic in androgen sensitive and castration recurrent CaP models in vitro and in vivo. The combination treatment increases apoptosis in radical prostatectomy ex vivo explant samples. This unique high metabolic flux through polyamine biosynthesis and connected one carbon metabolism in CaP creates a metabolic dependency. Enhancing this flux while simultaneously targeting this dependency in prostate cancer results in an effective therapeutic approach potentially translatable to the clinic.

Antitumour activity and tolerability of an EphA2-targeted nanotherapeutic in multiple mouse models.

Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.

Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade.

Androgen deprivation therapy (ADT) is palliative and prostate cancer (CaP) recurs as lethal castration-recurrent/resistant CaP (CRPC). One mechanism that provides CaP resistance to ADT is primary backdoor androgen metabolism, which uses up to four 3α-oxidoreductases to convert 5α-androstane-3α,17ß-diol (DIOL) to dihydrotestosterone (DHT). The goal was to determine whether inhibition of 3α-oxidoreductase activity decreased conversion of DIOL to DHT. Protein sequence analysis showed that the four 3α-oxidoreductases have identical catalytic amino acid residues. Mass spectrometry data showed combined treatment using catalytically inactive 3α-oxidoreductase mutants and the 5α-reductase inhibitor, dutasteride, decreased DHT levels in CaP cells better than dutasteride alone. Combined blockade of frontdoor and backdoor pathways of DHT synthesis provides a therapeutic strategy to inhibit CRPC development and growth.

Serum-free complete medium, an alternative medium to mimic androgen deprivation in human prostate cancer cell line models.

BACKGROUND: Almost all men who present with advanced prostate cancer (CaP) and some men who fail therapy for clinically localized CaP are treated with androgen deprivation therapy (ADT). CaP cell lines are used to identify and characterize new agents for ADT or investigate mechanisms of ADT resistance. CaP cell lines are maintained in culture medium that contains fetal bovine serum, which contains testosterone (T). Androgen deprivation experiments are performed using media supplemented with androgen-free serum, such as charcoal stripped fetal bovine serum (CS-FBS). However, CS-FBS composition varies from batch-to-batch and variations may impact experimental reproducibility. Serum free media (SFM) may provide a better defined alternative to media supplemented with CS-FBS (CSM). METHODS: Cell growth of six human CaP cell lines was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Androgen levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: MTT assays showed 5 of 6 CaP cell lines grew after 6 days of culture in androgen- deprived SFM or CSM. LNCaP and VCaP growth was stimulated when cells were cultured in SFM or CSM supplemented with T. LNCaP, C4-2, LAPC-4, and VCaP cell growth was inhibited when cultured in SFM or CSM with T and bicalutamide. LC-MS/MS data showed LAPC-4 cells produced similar DHT levels when cultured in T-supplemented SFM or CSM. Dutasteride impaired T to DHT metabolism in LAPC-4. CONCLUSIONS: Media composition contributed to growth differences observed between CaP cells cultured in SFM or CSM. However, the differences in media composition did not impair CaP cell response to T-stimulated growth, bicalutamide growth inhibition, metabolism of T, or dutasteride efficiency. SFM can be used as a better defined alternative to CSM for androgen deprivation experiments.

Dietary folate levels alter the kinetics and molecular mechanism of prostate cancer recurrence in the CWR22 model.

Folate impacts the genome and epigenome by feeding into one-carbon metabolism to produce critical metabolites, deoxythymidine monophosphate and s-adenosylmethionine. The impact of folate exposure and intervention timing on cancer progression remains controversial. Due to polyamine metabolism's extraordinary biosynthetic flux in prostate cancer (CaP) we demonstrated androgen stimulated CaP is susceptible to dietary folate deficiency. We hypothesized dietary folate levels may also affect castration recurrent CaP. We used the CWR22 human xenograft model which recurs following androgen withdrawal. Engrafted mice were fed a folate depleted or supplemented diet beginning at androgen withdrawal, or prior to xenograft implantation. Both folate depletion and supplementation at the time of withdrawal significantly decreased recurrence incidence. Folate supplementation prior to xenograft implantation increased time to recurrence, suggesting a protective role. By contrast, folate depleted recurrent tumors exhibited transcriptional adaptive responses that maintained high polyamine levels at the expense of increased DNA damage and DNA methylation alterations. Mining of publically available data demonstrated folate related pathways are exceptionally dysregulated in human CaP, which correlated with decreased time to biochemical recurrence. These findings highlight the potential for novel therapeutic interventions that target these metabolic pathways in CaP and provide a rationale to apply such strategies alongside androgen withdrawal.

Characterization of Prostate Cancer in a Functional Eunuch.

BACKGROUND: Eunuchs rarely, if ever, develop prostate cancer (CaP). This article reports on a 62-year-old functional eunuch from prepubertal mumps orchitis who developed clinically localized CaP. METHODS: Serum and CaP and benign prostate tissue androgen levels were measured using a validated liquid chromatography-tandem mass spectrometry assay. The assay measures testosterone; dihydrotestosterone (DHT); the adrenal androgens, androstenedione and dehydroepiandrosterone; and the androgen metabolites, androsterone and androstanedione. Gene and protein expression levels of androgen metabolism enzymes, and androgen receptor and androgen-regulated genes were measured using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry, respectively. RESULTS: Intracrine androgen metabolism produced tissue DHT when serum and tissue testosterone levels were castrate and undetectable, respectively. Androgen receptor, androgen-regulated, and androgen metabolism enzyme genes were expressed but at lower levels in CaP than benign tissues. CONCLUSIONS: DHT was synthesized using the primary backdoor androgen metabolism pathway and not using androstenedione or dehydroepiandrosterone via the frontdoor or secondary backdoor pathways.

Sequential use of the androgen synthesis inhibitors ketoconazole and abiraterone acetate in castration-resistant prostate cancer and the predictive value of circulating androgens.

PURPOSE: Patients previously treated with ketoconazole were excluded from phase III trials of abiraterone acetate due to potential overlapping mechanism of action. The purpose of this study was to determine the clinical utility of abiraterone and its impact on circulating androgens following ketoconazole. EXPERIMENTAL DESIGN: Chemotherapy-naïve patients with progressive metastatic castration-resistant prostate cancer (mCRPC) and prior ketoconazole therapy ≥28 days received abiraterone acetate 1,000 mg daily and prednisone 5 mg twice daily. The primary endpoint was the proportion of patients with PSA response, defined as ≥30% PSA decline at 12 weeks. H0 = 0.30 versus H1 = 0.50 (α = 0.05, power = 0.83). Circulating androgen levels were measured using liquid chromatography tandem mass spectrometry. RESULTS: Thirty-nine patients were included in the final analysis. Twenty (51%; 95% confidence interval, 36%-66%) patients had ≥30% PSA decline; the null hypothesis was rejected. Sixteen (41%) had ≥50% PSA decline. Median PFS (progression-free survival) was 16 weeks; median radiographic PFS (rPFS) was 36 weeks. Samples for measurement of baseline androgens were available in 37 patients. The PSA response proportion was 59% in 29 patients with DHEA ≥ limit of quantitation (LOQ), compared with 13% in 8 patients with DHEA < LOQ (P = 0.042). Median PFS was 6 and 16 weeks in DHEA < LOQ and DHEA ≥ LOQ patients, respectively (P = 0.017); median rPFS was 14 and 36 weeks in DHEA < LOQ and DHEA ≥ LOQ patients, respectively (P < 0.001). CONCLUSIONS: Abiraterone demonstrates modest clinical efficacy in mCRPC patients previously treated with ketoconazole. Patients with DHEA ≥ LOQ were more likely to demonstrate PSA responses and longer PFS. Analysis of circulating androgens merits further investigation as a biomarker for response to androgen synthesis inhibitor therapy.

Androgenic biomarker prof|ling in human matrices and cell culture samples using high throughput, electrospray tandem mass spectrometry.

UNLABELLED: BACKGROUND. A high throughput, high pressure liquid chromatographic (HPLC) method with triple quadrupole mass spectral detection (LC/MS/MS) was validated for the measurement of 5 endogenous androgens in human plasma and serum and applied to various in vivo and in vitro study samples to pursue a better understanding of the interrelationship of the androgen axis, intracrine metabolism, and castration-recurrent prostate cancer (CaP). METHODS: A Shimadzu HPLC system interfaced with a Sciex QTRAP 5500 mass spectrometer with electrospray ionization was used with in line column-switching. Samples were liquid/liquid extracted and chromatographed on a Luna C18(2) column at 60°C with a biphasic gradient using a 15-min run time. RESULTS: The method was validated for five androgens in human plasma and serum, and applied to four sets of samples. Plasma (n=188) and bone marrow aspirate (n=129) samples from patients with CaP, who received abiraterone acetate plus prednisone for up to 945 days(135 weeks), had undetectable androgens after 8 weeks of treatment. Plasma dehydroepiandrosterone(DHEA) concentrations were higher in African Americans than Caucasian Americans with newly diagnosed CaP. Analysis of prostate tumor tissue homogenates demonstrated reproducible testosterone (T) and dihydrotestosterone (DHT) concentrations with a minimal sample size of 1.0­2.0 mg of tissue. Finally, cell pellet and media samples from the LNCaP C4-2 cell line showed conversion of T to DHT. CONCLUSION: The proposed LC/MS/MS method was validated for quantitation of five endogenous androgens in human plasma and serum, and effectively profiles androgens in clinical specimens and cell culture samples.

Prostate cancer cells differ in testosterone accumulation, dihydrotestosterone conversion, and androgen receptor signaling response to steroid 5α-reductase inhibitors.

BACKGROUND: Blocking 5α-reductase-mediated testosterone conversion to dihydrotestosterone (DHT) with finasteride or dutasteride is the driving hypothesis behind two prostate cancer prevention trials. Factors affecting intracellular androgen levels and the androgen receptor (AR) signaling axis need to be examined systematically in order to fully understand the outcome of interventions using these drugs. METHODS: The expression of three 5α-reductase isozymes, as determined by immunohistochemistry and qRT-PCR, was studied in five human prostate cancer cell lines. Intracellular testosterone and DHT were analyzed using mass spectrometry. A luciferase reporter assay and AR-regulated genes were used to evaluate the modulation of AR activity. RESULTS: Prostate cancer cells were capable of accumulating testosterone to a level 15-50 times higher than that in the medium. The profile and expression of 5α-reductase isozymes did not predict the capacity to convert testosterone to DHT. Finasteride and dutasteride were able to depress testosterone uptake in addition to lowering intracellular DHT. The inhibition of AR activity following drug treatment often exceeded the expected response due to reduced availability of DHT. The ability to maintain high intracellular testosterone might compensate for the shortage of DHT. CONCLUSIONS: The biological effect of finasteride or dutasteride appears to be complex and may depend on the interplay of several factors, which include testosterone turnover, enzymology of DHT production, ability to use testosterone and DHT interchangeably, and propensity of cells for off-target AR inhibitory effect.

Covalently linked tandem lesions in DNA.

Reactive oxygen species (ROS) generate a type of DNA damage called tandem lesions, two adjacent nucleotides both modified. A subcategory of tandem lesions consists of adjacent nucleotides linked by a covalent bond. Covalently linked tandem lesions generate highly characteristic liquid chromotography-tandem mass spectrometry (LC-MS/MS) elution profiles. We have used this property to comprehensively survey X-irradiated DNA for covalently linked tandem lesions. A total of 15 tandem lesions were detected in DNA irradiated in deoxygenated aqueous solution, five tandem lesions were detected in DNA that was irradiated in oxygenated solution.

Lack of bioequivalence of ciprofloxacin when administered with calcium-fortified orange juice: a new twist on an old interaction.

Fluoroquinolones are known to interact with drugs containing multivalent ions. Current Food and Drug Administration (FDA) labeling states that ciprofloxacin and most other fluoroquinolones are safe to be given with food and dietary calcium but not calcium supplements. Although many of the currently marketed calcium fortified foods have calcium contents that usually exceed those in dietary calcium sources, it is unclear whether they represent a risk for less than optimal absorption of fluoroquinolones, which may result in subsequent clinical failures due to lack of bacterial eradication and antibiotic resistance. The purpose of this three-way, randomized, crossover study was to characterize and compare the bioequivalence of single doses of oral ciprofloxacin in 15 healthy volunteers when administered with water, concurrently with orange juice, and concurrently with calcium-fortified orange juice. Compared to the control arm, the Cmax of ciprofloxacin significantly decreased when it was given with orange juice (23%, p = 0.001) and with calcium-fortified orange juice (41%, p < 0.001). Twenty-four-hour ciprofloxacin AUCs were also decreased for both forms of the orange juice (22% [p < 0.001] and 38% [p < 0.001], respectively). When compared to each other, neither of the orange juice regimens were bioequivalent to each other, with the Cmax and AUC for the fortified form being 22% (p = 0.005) and 21% (p = 0.015) lower than those of the nonfortified form. By FDA standards, although ciprofloxacin is marginally bioequivalent when administered with orange juice, it is not when it is administered with calcium-fortified orange juice. The changes in Cmax and AUC have the potential to significantly decrease clinical efficacy and promote antibiotic resistance. Not warning patients about potential food-drug interactions with fortified foods may be a major unrealized and unstudied inadvertent source of clinical failures and resistance trends with fluoroquinolones.