Adding abiraterone or docetaxel to long-term hormone therapy for prostate cancer: directly randomised data from the STAMPEDE multi-arm, multi-stage platform protocol.

Background: Adding abiraterone acetate with prednisolone (AAP) or docetaxel with prednisolone (DocP) to standard-of-care (SOC) each improved survival in systemic therapy for advanced or metastatic prostate cancer: evaluation of drug efficacy: a multi-arm multi-stage platform randomised controlled protocol recruiting patients with high-risk locally advanced or metastatic PCa starting long-term androgen deprivation therapy (ADT). The protocol provides the only direct, randomised comparative data of SOC + AAP versus SOC + DocP. Method: Recruitment to SOC + DocP and SOC + AAP overlapped November 2011 to March 2013. SOC was long-term ADT or, for most non-metastatic cases, ADT for ≥2 years and RT to the primary tumour. Stratified randomisation allocated pts 2 : 1 : 2 to SOC; SOC + docetaxel 75 mg/m2 3-weekly×6 + prednisolone 10 mg daily; or SOC + abiraterone acetate 1000 mg + prednisolone 5 mg daily. AAP duration depended on stage and intent to give radical RT. The primary outcome measure was death from any cause. Analyses used Cox proportional hazards and flexible parametric models, adjusted for stratification factors. This was not a formally powered comparison. A hazard ratio (HR) <1 favours SOC + AAP, and HR > 1 favours SOC + DocP. Results: A total of 566 consenting patients were contemporaneously randomised: 189 SOC + DocP and 377 SOC + AAP. The patients, balanced by allocated treatment were: 342 (60%) M1; 429 (76%) Gleason 8-10; 449 (79%) WHO performance status 0; median age 66 years and median PSA 56 ng/ml. With median follow-up 4 years, 149 deaths were reported. For overall survival, HR = 1.16 (95% CI 0.82-1.65); failure-free survival HR = 0.51 (95% CI 0.39-0.67); progression-free survival HR = 0.65 (95% CI 0.48-0.88); metastasis-free survival HR = 0.77 (95% CI 0.57-1.03); prostate cancer-specific survival HR = 1.02 (0.70-1.49); and symptomatic skeletal events HR = 0.83 (95% CI 0.55-1.25). In the safety population, the proportion reporting ≥1 grade 3, 4 or 5 adverse events ever was 36%, 13% and 1% SOC + DocP, and 40%, 7% and 1% SOC + AAP; prevalence 11% at 1 and 2 years on both arms. Relapse treatment patterns varied by arm. Conclusions: This direct, randomised comparative analysis of two new treatment standards for hormone-naïve prostate cancer showed no evidence of a difference in overall or prostate cancer-specific survival, nor in other important outcomes such as symptomatic skeletal events. Worst toxicity grade over entire time on trial was similar but comprised different toxicities in line with the known properties of the drugs. Trial registration: Clinicaltrials.gov: NCT00268476.

Comparison of efficacy of letrozole and clomiphene citrate in ovulation induction in Indian women with polycystic ovarian syndrome.

OBJECTIVES: The objective of this study was to compare the efficacy of letrozole versus clomiphene citrate as an ovulation induction drug in polycystic ovarian syndrome (PCOS) patients of Indian origin. METHOD: One hundred and forty seven infertile PCOS patients were randomly given letrozole (2.5 mg) (n = 69) or clomiphene (100 mg) (n = 78) from day 3 to day 7 of menstrual cycle, followed-up with transvaginal serial folliculometry from day 9. 10,000 IU of human chorionic gonadotropin (hCG) was administered when at least 1 ovarian follicle was ≥ 18 mm in size. RESULTS: The pertinent results of the study are as follows: on the day of hCG injection, mean E2 level was significantly higher in the clomphene citrate group (817 ± 286.70 pg/ml) in comparison with letrozole group (444.03 ± 85.42 pg/ml). Mean endometrial development was 8.72 ± 1.41 mm in the letrozole and 8.78 ± 1.16 mm in the clomiphene group (P = 0.004). CONCLUSION: Letrozole has beneficial effect on endometrium, thereby potentially increasing pregnancy rates after successful ovulation induction in women with PCOS.

The cyanogen bromide fragment I of asialoorosomucoid is transported more efficiently than asialoorosomucoid in rat hepatocytes.

Cultured rat hepatocytes internalized and degraded 125I-labeled asialoorosomucoid (125I-ASOR) through asialoglycoprotein receptor at rates about half that of its cyanogen bromide fragment I (125I-ASCNBr-I). Reduction and carboxymethylation of the fragment resulted in decreased rates of internalization and degradation which were still greater than those of 125I-ASOR. In the presence of 5 microM colchicine, degradation of all three ligands was inhibited. However, the intracellular level of 125I-ASOR at steady state remained unchanged, while those of the fragments increased continuously. Study of the binding of these ligands to hepatocytes at 4 degrees C indicated that there was no significant difference in binding parameters between ASOR, ASCNBr-I and RC-ASCNBr-I (reduced and carboxymet ASCNBr-I). Studies of the fate of these ligands preloaded in the cell at 37 degrees C indicated that a higher fraction of the internalized ASOR than of the fragments was released by diacytosis. In contrast to ASOR, diacytosis of the fragments was not enhanced by colchicine. Studies of the distribution of intracellular ligands by Percoll density gradient centrifugation indicated that they were internalized initially into two early endosomal compartments of d = 1.037 g/ml and d = 1.045 g/ml. In the presence of colchicine, accumulation of the ligands in a third endosomal compartment of d = 1.08-1.095 g/ml was revealed, while in the presence of leupeptin accumulation of the ligands in lysosomes was observed. The results of a kinetic analysis indicated that both cyanogen bromide fragments were transported to all these compartments more rapidly than was ASOR. It appears that they are internalized and degraded more rapidly than ASOR due to a more efficient sorting of the internalized ligand into the pathway of lysosomal degradation.

Synergistic activation of yeast-expressed rat androgen receptor by modulators of protein kinase-A.

We have employed a yeast (Saccharomyces cerevisiae) based rat androgen receptor expression system to examine the cross-talk between different signalling pathways. We report here the synergistic modulation of androgen regulated transcriptional activation of beta-galactosidase reporter activity by the activators of protein kinase-A, like forskolin and 8-bromo-cyclic AMP. A similar ligand-dependent enhancement of reporter activity compared to a DHT treated control has been noticed with okadaic acid, which is a potent inhibitor of protein phosphatase. The activation could be blocked by protein kinase-A/C inhibitor, H7. Forskolin treatment neither altered levels of receptor mRNA nor [3H]R1881 binding to the receptor. Although it promotes binding of receptor to an androgen response element, forskolin was unable to activate subsequent interaction with the transcription machinery in the absence of androgen. Additionally, the synergistic actions of these activators were independent of the degree of androgen response element occupancy. Anti-androgens, cyproterone acetate and flutamide, which failed to exhibit antagonistic behaviour with yeast expressed receptor, were able to antagonize only the forskolin mediated augmentation of reporter activity. Finally, analyses of mutants established the role of DNA and steroid binding domains of receptor for this synergism.

Localization of the vicinal dithiols involved in steroid binding to the rat glucocorticoid receptor.

Our previous studies with the thiol-specific reagent methyl methanethiolsulfonate (MMTS) and the vicinal dithiol-specific reagent sodium arsenite have established that 2 spatially close thiols (i.e. vicinal dithiols) are involved in steroid binding to the intact 98 K rat glucocorticoid receptor. These 2 thiols form an intramolecular disulfide after treatment with low concentrations of MMTS. One of these thiols was proposed to by Cys-656. In an effort to identify both thiols, we have examined the effects of MMTS and arsenite on proteolytic fragments of the receptor, which contain progressively fewer cysteines. MMTS and arsenite are now found to cause the same dithiothreitol-reversible inhibition of steroid binding and affinity labeling of both the 42 K chymotrypsin fragment and the 16 K steroid-binding core fragment of the receptor as was seen for the intact receptor. Characteristic responses include a bimodal inhibition curve for steroid binding after preincubation with MMTS and an inhibition of binding by very low concentrations of arsenite. Low concentrations of MMTS could block steroid binding by forming a disulfide bond between the receptor and a tightly associated, nonreceptor protein. However, no evidence for such cross-linking was observed when intact 98 K receptors, 42 K chymotrypsin fragments, or 16 K trypsin fragments were treated with various concentrations of MMTS, separated on nonreducing sodium dodecyl sulfate-polyacrylamide gels, and visualized by Western blotting with antiheat shock protein 90 or antireceptor antibodies. One of the antireceptor antibodies (aP1) that had been raised against the rat receptor sequence 440-795 was now found to recognize at least 1 epitope in the 16 K core fragment. We conclude that the vicinal dithiols involved in steroid binding are 2 of the 3 cysteines in the sequence of Thr537-Arg673.

Activation of rat androgen receptor by androgenic ligands is unaffected by antiandrogens in Saccharomyces cerevisiae.

The E. coli lacZ has been utilized as a reporter to evaluate ligand-mediated activation of the rat androgen receptor (AR) in Saccharomyces cerevisiae strain YCR1. beta-galactosidase activity was androgen-specific and was found to be inducible approximately 260-fold by dihydrotestosterone (DHT), testosterone and R1881. None of the antiandrogens tested was able to antagonize the DHT-dependent induction of beta-galactosidase activity. In the gel retardation assay, exposure of the receptor to DHT in vitro led to the formation of a protein-DNA complex that was not detected in yeast extracts unexposed to hormone. However, activation of AR by a steroidal (cyproterone acetate) and a non-steroidal antiandrogen (flutamide) either alone or in combination with DHT also results in a similar migration pattern. Additionally, LEM1, the ABC transporter that selectively modulates the biological potency of steroids in yeast, although operative in YCR1, was not responsible for antiandrogen resistance. These results thus indicate the involvement of other non-receptor factor(s) in mediating the effect of antiandrogens in yeast.

Identification of an ABC transporter gene that exhibits mRNA level overexpression in fluoroquinolone-resistant Mycobacterium smegmatis.

We describe here the PCR amplification of a DNA fragment (mtp1) from Mycobacterium smegmatis using primers derived from consensus sequences of the ABC family of transporters. The fragment encodes amino acid sequences that exhibited significant homology with different ABC transporters. Amino acid sequence alignment of the full length gene with other transporters identified the ABC protein as the B-subunit of the phosphate specific transporter. Strikingly, a M. smegmatis colony which exhibited a high level of ciprofloxacin resistance showed mRNA level overexpression of mtp1. Thus this is the first report in any prokaryote indicating differential expression of an ABC transporter in a fluoroquinolone resistant colony.

Role of an ABC importer in mycobacterial drug resistance.

Phosphate specific transporter (Pst) in bacteria is involved in phosphate transport. Pst is a multisubunit system which belongs to the ABC family of transporters. The import function of this transporter is known to be operative at media phosphate concentrations below the millimolar range. However, we found amplification of this transporter in a laboratory generated ciprofloxacin resistant Mycobacterium smegmatis colony (CIPr) which was grown in a condition when phosphate scavenging function of this operon was inoperative. Our results therefore argue the role of this ABC importer in conferring high level of fluoroquinolone resistance in CIPr.

Recovery of fertility 14 years following radiotherapy and chemotherapy for testicular tumour.

We report a patient who developed a left-sided malignant teratoma with ipsilateral para-aortic nodes at the age of 20 years. Following treatment with left orchidectomy, abdominal and pelvic radiotherapy and chemotherapy, he was found to be azoospermic. More than 14 years after treatment, he regained his fertility. Similar prolonged iatrogenic depression of spermatogenesis has been reported in younger lymphoma patients; however, in post-pubertal patients with testicular tumour this has not been frequently reported.

Mycoplasma detection in cell cultures: a comparison of four methods.

Mycoplasma is a common contaminant of tissue culture samples. Infection is persistent, difficult to detect and diagnose, and very difficult to cure. The concentration of mycoplasma in infected cultures can be as high as 10(7) colony-forming units per mL, and their presence can change many of the cell reactions, including altering cell growth rate, inducing morphological changes or cell transformation, and mimicking virus infection. Therefore, it should be assumed that a mycoplasma-contaminated cell line may be significantly influenced in every respect, and, thus, experimental data derived from such a cell line is likely to be invalid. Contamination is not obvious, either macroscopically or microscopically; thus, routine mycoplasma testing is essential for any cell culture laboratory. Many of the testing procedures developed so far are time-consuming, expensive, inconclusive and unsuitable for screening large numbers of test specimens. This study compares DNA staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and PCR ELISA, to determine which is the best procedure for routine assessment of cell cultures. All four methods gave reproducible results with both infected and non-infected cell lines. Both ELISA methods were easy to perform, reproducible and easily interpreted.

In vitro testing of platinum-based drugs on a panel of human ovarian tumour cell lines.

Much improvement in the treatment of ovarian cancer has been achieved since the introduction of platinum compounds in the 1980s, with the result that single-agent platinum-based therapy following primary surgery is now the standard treatment for advanced ovarian cancer. The main therapeutic effect of chemotherapy is based on the sensitivity of the patient's tumour to the drug. However, testing a new chemical compound on humans requires much care, time and resources, whereas prior testing of drugs on cancer cell lines may indicate those drugs particularly suited to treatment of a specific disease. This study investigates the actions of two established platinum-based chemotherapeutic agents (cisplatin and carboplatin) on a panel of 10 human ovarian cancer cell lines. Each cell line was plated onto 96-well tissue culture plates, incubated for 72 hours with the drug, formalin-fixed and then assessed using the methylene blue colorimetric microassay to detect viable cells. The IC50 values for each cell line were calculated in order to assess the toxicity of each drug, and a wide range of responses were observed across the 10 cell lines investigated. This suggests that the panel reflected the heterogeneous nature of ovarian cancer, a malignancy in which a huge range of drug sensitivities can be seen even among tumours of the same histological type. The results indicate that the panel could be of use either as a primary screen to test new drugs against ovarian cancer or to investigate the drug resistance that is so common in this disease.

Effect of mercuric chloride and cadmium chloride on gonadal function and its regulation in sexually mature common carp Cyprinus carpio.

Gonadal function in fish, Cyprinus carpio was significantly affected by sublethal doses of mercuric chloride (HgCl2) and cadmium chloride (CdCl2) in chronic (45 days) exposure. Parameters investigated were nonesterified (NE) and esterified (E) cholesterol of ovary, liver and serum and ovarian 3 beta-Hydroxysteroid and 17 beta-Hydroxysteroid dehydrogenase enzyme activity and serum and pituitary gonadotropin (GtH) levels. Both the pollutants were able to reduce the hypothalamic extract (HE) or gonadotropin releasing hormone (GnRH) induced pituitary GtH release in vitro. Short term (96h) exposure of the fish to the pollutants had no significant effect on the gonadal function. In addition to the deleterious effect of pollutants on the gonadal steroidogenesis and pituitary gonadotropin release, using [4-14C] cholesterol as a tracer it was found that for 45 days exposure, HgCl2 had an adverse effect on the transport of cholesterol from circulation to ovary.

Changes in the allergenicity during different preparations of Pomfret, Hilsa, Bhetki and mackerel fish as illustrated by enzyme-linked immunosorbent assay and immunoblotting.

BACKGROUND: Although the identification and characterization of several fish allergens have already been reported, there is almost no data on Indian fish allergens and the effect of thermal processing on their allergenicity. This study aimed at the evaluation of the changes in the level of allergenicity of 4 highly consumed Indian fishes, i.e. pomfret, hilsa, bhetki and mackerel, that occurred after boiling and frying. METHODS: In this study 110 patients with fish hypersensitivity as evidenced by clinical history and symptoms were recruited based on their positive skin prick test results. The raw, boiled and fried muscle extracts of the 4 fishes were prepared, and each extract was tested by ELISA and immunoblotting with patients' sera. RESULTS: ELISA and immunoblotting studies demonstrated that the raw muscle extracts of pomfret, hilsa, bhetki and mackerel were allergenic. While the allergenicity of boiled and fried extracts of pomfret and hilsa was considerably reduced, maximum allergenicity of bhetki was demonstrated in the fried extract. The degree of allergenicity of bhetki was demonstrated in the order fried>boiled>raw while that of mackerel followed the order raw>boiled approximately fried. CONCLUSION: The specific IgE-binding activity and immunoblot profile clearly showed that pomfret and hilsa fish allergens are heat-labile, while allergens of bhetki and mackerel maintained strong reactivity even after thermal treatment.

Bovine TSH-stimulation of fish thyroid peroxidase activity and role of thyroxine thereon.

bTSH augmented the fish thyroid peroxidase activity in a dose-response manner. Thyroxine could not modulate the effect of exogenous bTSH, but it decreased the peroxidase activity in a control system when administered alone. The data therefore suggest similar negative feedback control system for TSH-regulation as operative in the case of mammals.

Influence of gonadotropins and gonadal hormones on climbing perch thyroid nucleic acids.

Climbing perch thyroidal RNA content fluctuated in different phases of the reproductive cycle, highest at spawning (36.08 +/- 3.69 micrograms/mg tissue) and lowest at postspawning (6.88 +/- 0.76 microgram/mg tissue) whereas DNA remained unaltered. Treatment of intact perch with salmon gonadotropin (SG-G100) or ovine LH for 15 days significantly stimulated thyroidal RNA content. Stimulatory effect of SG-G100 was greater (p less than 0.001) than LH (p less than 0.005). FSH had no such effect. Gonadotropin (GtH) treatment could not alter thyroidal DNA. Ovarian steroids, 17 beta-estradiol (E2) and estrone (E1) remarkably elicited RNA content. Ovariectomy of perch caused striking depletion of RNA. Administration of GtH to ovariectomized perch had no effect on thyroid RNA but E1 and E2 supplementation resulted in significant stimulation in comparison to ovariectomized control. Findings indicate that GtH mediated its stimulatory effect on perch thyroidal RNA via the release of ovarian steroids.

Effects of prolactin and fish pituitary extract on plasma calcium levels in common carp, Cyprinus carpio.

The effects of prolactin (PRL) from both mammalian and piscine sources on plasma calcium levels in common carp, Cyprinus carpio, were investigated. Injections of ovine prolactin (oPRL) or homologous fish pituitary extract or partially purified prolactin of murrel, Channa punctatus pituitary (mPRL), caused significant increases in total and ultrafiltrable plasma calcium. Larger hypercalcemic responses were observed in fish kept in high-calcium fresh water and normal tap water than in fish in low-calcium fresh water. Injections of oPRL caused dose-dependent increases in plasma calcium level. The highest dose (1 mg/kg body wt) of oPRL had a reduced hypercalcemic effect. Administration of EGTA (200 microM/kg body wt) reduced the stimulatory effects of daily injections of oPRL or mPRL on plasma calcium in freshwater common carp under laboratory conditions. These results indicate that PRL may be involved in regulating plasma calcium levels in fish.