Prostate secretory protein (PSP94) suppresses the growth of androgen-independent prostate cancer cell line (PC3) and xenografts by inducing apoptosis.

BACKGROUND: PSP94 (prostate secretory protein of 94 aa; also called PIP), one of the predominant proteins secreted into the seminal fluid, was proposed as an independent diagnostic/prognostic marker for prostate cancers. It was also shown to inhibit rat prostate cancer growth. In this study, we investigated the effect of purified PSP94 on the growth of androgen-independent human prostate cancer cells (PC3) and its potential mechanism of action. METHODS AND RESULTS: PSP94, in a dose- and time-dependent manner, inhibited the growth of PC3 cells. The protein demonstrated a stronger inhibitory effect on the colony-forming ability of PC3 cells in soft agar. A daily injection of PSP94 at 5 microg/kg/body weight resulted in a 50-60% inhibition in the growth of PC3 xenografts in athymic mice. PC3 cell growth inhibition by PSP94 resulted from cell death characteristic of morphological apoptosis, which was confirmed by dual fluorescence microscopy, electron microscopy, and DNA fragmentation assays. Mechanistic studies indicated that PSP94 enhanced the expression of proapoptotic protein Bax without affecting Bcl-2 levels. CONCLUSIONS: This study suggests that PSP94 may represent a novel, apoptosis-based, antitumor agent applicable to the treatment of hormone-refractory human prostate cancers.

Hormone-refractory prostate cancer cells express functional follicle-stimulating hormone receptor (FSHR).

PURPOSE: Understanding growth regulation in hormone-refractory prostate cancer may provide avenues for novel treatment interventions. This study was conducted to characterize the expression of the receptor (FSHR) for follicle-stimulating hormone (FSH) in androgen-independent prostate cancer cell lines and in human malignant prostate tissues. MATERIALS AND METHODS: Western blotting, immunohistochemistry (IHC), and flow cytometric analysis were used to study the expression of FSHR. The effect of FSH on cell growth and clonogenicity was studied using proliferation and clonogenic assays. RESULTS: Immunohistochemistry revealed expression of FSH in PC3 and Du145 cells. FSHR was identified in PC3 and Du145 cells, as well as in human adenocarcinoma of the prostate. The specificity of the FSHR detected on prostate cancer tissues or cells by IHC and Western blotting was confirmed by preabsorbing the antibodies with the immunizing antigens. Stimulation of these hormone-refractory cells with FSH triggered a proliferative response in vitro, suggesting that the receptor is biologically active. CONCLUSION: Hormone-refractory prostate cancer cells express FSH and biologically active FSHR. Our results suggest that FSHR and its ligand may play a role in the regulation of the growth of hormone-refractory prostate cancers.

Identification of binding proteins for PSP94 in human prostate adenocarcinoma cell lines LNCaP and PC-3.

BACKGROUND: Prostatic secretory protein of 94 amino acids (PSP94) is one of the predominant proteins found in human seminal fluid. Limited information is available regarding a physiological function for PSP94. An important step in the elucidation of this function is the determination of the mechanism of interaction of PSP94 with potential cellular targets. METHODS: Equilibrium binding assay was employed to demonstrate specific binding of biotinylated-PSP94 to the LNCaP and PC-3 cell lines. Binding proteins were partially purified by PSP94 affinity-chromatography from LNCaP, PC-3 cells, and prostate tissues. RESULTS: Binding of biotinylated-PSP94 to LNCaP and PC-3 cells was saturable and time and temperature dependent. The binding could be specifically competitively inhibited by unlabelled PSP94. Two types of PSP94 binding sites with distinct affinity (Kd) and density (Bmax) were determined by Scatchard analysis for each of the two cell lines. For the LNCaP cells, these values were Kd 1 = 0.75 nM and Bmax1 = 300 fmol/mg protein and Kd 2 = 4.5 nM, Bmax2 = 780 fmol/mg protein, respectively. Similar affinity and density results were obtained for PC-3 cells: Kd 1 = 0.83 nM, Bmax1 = 250 fmol/mg protein, and Kd 2 = 5.0 nM, Bmax2 = 700 fmol/mg. The binding of biotinylated-PSP94 to the LNCaP cells was competitively inhibited by the partially purified proteins. Analysis of these proteins SDS-PAGE showed three main bands and the molecular weights of these three bands were approximately 180, 100 and 60 kD, respectively. CONCLUSIONS: The data showed the presence of specific binding proteins to the PSP94 in LNCaP, PC-3 cells, and prostate tissue.

Occurrence of FSH, inhibin and other hypothalamic-pituitary-intestinal hormones in normal fertility, subfertility, and tumors of human testes.

OBJECTIVE: To compare the distribution of peptide hormones in presumably normal human testicular tissues and specimens exhibiting any of five pathologies. METHODS: Biopsies from patients having testicular malfunctions were prepared as sections and specifically immunohistochemically stained for inhibin, FSH, serotonin, AUP, and oxytocin. RESULTS: Immunocytochemical studies revealed the presence of various hypophysial-pituitary-intestinal hormones, viz., FSH, inhibin, arginine vasopressin (AVP), calcitonin, serotonin, oxytocin, adrenocorticotropin (ACTH), gastrin, secretin, and somatostatin in human testicular biopsies exhibiting normal spermatogenesis, Sertoli-cell-only syndrome, spermatogenic arrest, Leydig cell hyperplasia, Leydig cell tumor, and seminoma. Intensity of immunostaining for all peptides except FSH was stronger in cases of subfertile as compared to normal testis. Intensity of immunostaining with inhibin was maximum in Leydig cell tumor. CONCLUSION: These regulatory peptides may be involved in the pathophysiology of the testes.

Expression of a 10.5 kDa inhibin in the endometrium of bonnet monkey on treatment with an antiprogestin ZK 98.299: an immunocytochemical study.

Immunocytochemical localization of inhibin was carried out in paraffin embedded tissue sections of the control and antiprogestin (ZK 98.299)-treated bonnet monkey endometrium using polyclonal antibodies generated against human seminal plasma inhibin (10.5 kDa). The study shows that administration of low doses (5 mg/week) of antiprogestin results in an increase in the expression of inhibin by the endometrium. Treatment with higher doses (20 mg/week) caused a decrease in the expression. Since treatment with higher doses also caused atropic changes in the endometrium, the decrease in inhibin could be the result of morphological damage to the endometrium rather than the effects of antiprogestin on the expression of inhibin. The potential involvement of endometrial inhibin in the process of nidation is speculated.

Prostate inhibin peptide (PIP) in prostate cancer: a comparative immunohistochemical study with prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP).

Prostate inhibin peptide (PIP) is a polypeptide synthesized by the prostate gland that is involved in prostatic growth and differentiation. The objective of this study was to evaluate PIP as an immunocytochemical marker for prostatic adenocarcinoma (PCA) by comparing it with PSA and PAP. A total of 71 cases of primary PCA and 5 cases of metastatic PCA were studied. Primary tumors were specially selected to include a disproportionate number of high-grade tumors. The distribution of cases by Gleason score was 2-5, 14 cases; 6-7, 24 cases; and 8-10, 33 cases. Four metastases were to bone (decalcified tissue) and one to soft tissue. All 71 cases of primary PCA stained positively for the three antibodies tested, with none demonstrating obvious superiority, although individual case variability was seen. In one bone metastasis, staining for PSA was negative, with both PAP and PIP giving positive results. All non-prostatic carcinomas tested were negative. These results indicate that PIP is as sensitive and specific an immunohistochemical marker as PSA and PAP in untreated prostate adenocarcinomas. Further, the androgen-independent nature of PIP may give it an advantage over PSA/PAP in tumors exposed to androgen ablating agents.

Occurrence and de novo biosynthesis of follicle stimulating hormone (FSH) in benign and malignant conditions of human breast.

We report the occurrence as well as biosynthesis of a pituitary hormone, follicle stimulating hormone (FSH) in human breast. Using immunoperoxidase localization technique, both FSH and beta-FSH were localized in cytoplasm of epithelial cells but not in stromal cells. Immunostaining was more intense in benign and malignant specimens as compared to normal. In vitro radiolabelled precursor experiments with breast tissue explants indicate de novo synthesis of FSH. Human milk had higher concentrations of FSH as compared to serum. In gonads, FSH is involved in the cellular growth, differentiation and function. The presence of higher levels of FSH in benign mammary tumors and breast cancer when compared to normal breast supports the suggestion that FSH might have a role in the process of breast malignant transformation.

Effect of synthetic nonapeptide (Thr-Cys-Ser-Val-Ser-Glu-Trp-Gly-Ile) on ovarian follicular development in the bull frog Rana tigrina (Daud).

Effect of synthetic nonapeptide (Thr-Cys-Ser-Val-Ser-Glu-Trp-Gly-Ile) representing the amino acid sequence 86-94 of human seminal plasma was studied on the ovarian follicular growth in the bullfrog R. tigrina during preparatory phase of reproductive cycle. Daily (except on Sundays) injections of 10 micrograms nonapeptide for one month caused a significant increase in ovarian weight and number of second growth phase (SGP) or vitellogenic oocytes. The results suggest that the nonapeptide is biologically active in amphibians also.

Immunoperoxidase localization and denovo biosynthesis of a 10.5-kDa inhibin in benign and malignant conditions of human breast.

Using immunocytochemistry, we report the occurrence of a 10.5-kDa inhibin in human breast tissue specimens obtained from normal, fibroadenoma and adenocarcinoma cases. The immunostaining for inhibin was confined to the cytoplasm of the epithelium and myoepithelium cells. Expression of inhibin increased in following order: normal (1+); adenocarcinoma and lobular carcinoma in situ (2+) and fibroadenoma (4+). Breast explants has the ability of denovo biosynthesis of inhibin in vitro. In view of the growth modulating regulatory properties of 10.5 kDa inhibin, our findings are suggestive of the potential role of inhibin in breast pathology.

A comparative study on expression of prostatic inhibin peptide, prostate acid phosphatase and prostate specific antigen in androgen independent human and rat prostate carcinoma cell lines.

Prostatic inhibin peptide (PIP), consisting of 94 amino-acid residues is synthesized and secreted by the prostate gland. Previous studies on immunohistochemical localization of PIP in primary prostatic tumor and their metastasis, have documented the value of this peptide as a tumor marker for diagnosis of prostate cancer (PCa). The present study was undertaken to compare the expression of PIP with that of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) in androgen independent human PCa cell lines (PC-3, DU-145 and TSU-Prl) by immunoperoxidase technique. The results of the study indicated that the staining for PIP was more intense than that of PSA and PAP. The PSA staining was either weakly positive (PC-3) or totally absent (TSU-Prl and DU-145) while PAP staining was intense in PC-3 and moderate in the other two human cell lines. The intense staining observed for PIP in all of the androgen independent cell lines suggests that the synthesis and secretion of PIP is not primarily dependent on androgens. Furthermore, expression of these markers in Dunning rat cultured adenocarcinoma cell lines and tumors were studied. Positive staining for all three human tumor associated antigens (PIP, PSA and PAP) cross-reacting with the Dunning rat PCa cell lines and the tumors, suggest the suitability of this model for preclinical screening of various therapeutic agents.

Patterns of inhibin and FSH localization in endometrium of baboon (Papio anubis) during menstrual cycle and early pregnancy.

Immunoreactive 10.5 KDa moiety of inhibin and hFSH was present in the baboon endometrium during menstrual cycle, early pregnancy and in castrated animals treated with steroid hormones, estrogen and/or progesterone. Endometrial differences during the menstrual cycle altered the intensity of immunostaining of inhibin and FSH. Maximum staining was observed in late luteal phase for both the hormones. In early pregnancy (35th day), the conceptus increased the staining for inhibin in the adjoining endometrial glands. Treatment of castrated animals with steroids for 14 days caused increased staining for inhibin. Maximum staining was observed when treated with estradiol or progesterone, whereas combination of estrogen and progesterone treatment decreased the staining reaction. In conclusion, both inhibin and FSH were localized in baboon endometrium and were under the influence of estrogen and progesterone.

Seminal inhibin--prospects for immunocontraception.

Passive immunization of adult rats, hamsters and marmosets with rabbit anti-seminal inhibin resulted in complete or partial block of fertility. The antiserum treatment presumably neutralized endogenous inhibin resulting in an unopposed rise in circulating FSH. This probably led to a refractoriness of the testes to FSH resulting in complete spermatogenic arrest. Nevertheless, there was no change in the mating behaviour of the animals. The antibodies also affected the epididymal spermatozoa by causing large scale agglutination.

Antifertility effects in rats actively immunized with 80 kDa human semen glycoprotein.

A 80 kDa human sperm antigen has been identified using the serum of an infertile woman having circulating antisperm antibodies. The antigen was then purified to homogeneity by gel permeation chromatography using HPLC (protein PAK-125 column) system and on FPLC (superose-12 column) system. The antigen was found to be a glycoprotein. The antigen was mainly localized in the postacrosomal region of the human sperm, while it was localized in the head region of the rat sperm as demonstrated by immunofluorescent staining. The presence of this antigen was also demonstrated in the human prostate and endometrium and in the rat testis; epididymis and the prostate by immunocytochemical staining. The purified protein upon active immunization in female rats caused infertility in 100 percent animals. While in male rats it caused infertility in 90 percent animals. On morphometric analysis of testicular tissue it was observed that there was no significant change in spermatogonia and spermatocytes, but significant decrease in spermatids and sperm number as well as daily sperm production in the immunized male rats. The epididymal spermatozoa were markedly reduced in number and were largely found to be agglutinated. The results suggest that 80 kDa human sperm antigen appears to be a suitable candidate for immunocontraception both in male and female.

Immunocytochemical localization of bioregulatory peptides in marmoset testes.

Immunocytochemical localization of neuropeptides (beta-endorphin, substance P, arginine vasopressin, oxytocin), pituitary hormones (adrenocorticotropin, prolactin, growth hormone, follicle stimulating hormone (FSH), gonadal inhibin, gastrin, and human chorionic gonadotrophin (hCG)) was carried out in marmoset testis during development. Both intensity of immunostaining and distribution of these peptides in testicular compartments viz. seminiferous tubules and Leydig cells changed dramatically during development. In vitro biosynthesis of inhibin and FSH was increased by hCG, whereas prolactin (5 micrograms) and prostatic inhibin peptide suppressed the synthesis of these hormones.

Inhibin: unity in diversity.

Historically, inhibin was thought to be a testicular hormone involved in the regulation of pituitary FSH by a negative feedback control. The ability of inhibin to preferentially suppress FSH without affecting LH triggered extensive research for its possible use as a male contraceptive, suggesting a plurality of molecular forms and a multiplicity of biological actions of this putative hormone. It also became evident that inhibin is not unique to the testis, as presumed earlier, and can even be obtained from the ovary. This has necessitated a fundamental revision of the original concept of inhibin. Unfortunately, not many perceive inhibin as a loose conglomerate of structurally dissimilar, FSH-suppressing proteins and insist on singling out a 32-kDa protein derived from ovarian follicular fluid to be designated as inhibin. This article highlights features common to two distinctly different types of inhibin: seminal inhibin and ovarian inhibin. Evidence is also provided to indicate that the term inhibin need not be specific to the ovarian protein, but encompasses proteins hitherto dismissed as inhibin-like or inhibin-related proteins.

Passive immunization against prostatic 'inhibin' peptide as a male contraceptive.

Passive immunization of adult male hamsters for 12 weeks against peptide (As-PIP), a sperm coating antigen, resulted in selective elevation of the blood levels of FSH, impairment of spermatogenesis, and complete infertility when males were mated with normal cycling females. Passive immunization of male marmosets with As-PIP for 8 weeks was also effective and was reversible, without causing any obvious change in mating behaviour. These preliminary studies in hamsters and marmosets indicate that antibodies to a prostatic 'inhibin' peptide represent a promising new approach to male contraception.

Hormonal modulation of biosynthesis of prostatic specific antigen, prostate specific acid phosphatase and prostatic inhibin peptide.

Hormonal modulation of in vitro biosynthesis of three prostatic secretory proteins, prostate specific acid phosphatase (PSAP), prostate specific antigen (PSA) and prostatic inhibin peptide (PIP) by human benign hyperplasia (BPH) tissue was studied. LH and inhibins caused increase in the synthesis of all three proteins whereas FSH enhanced the synthesis of PIP and PSA only but decreased PSAP synthesis. Prolactin and thyroid releasing hormone decreased synthesis of PIP and PSAP. However, PSA synthesis was enhanced by TRH and was decreased by prolactin. Estradiol caused significant increase in PSA and PSAP but no discernible changes in PIP synthesis were noticed. Testosterone caused an increase in PIP, PSA and PSAP. These data indicate that biosynthesis of PIP, PSA and PSAP by BPH tissue is under multihormonal regulation.

Endometrium--an extragonadal source of inhibin.

Using polyclonal antibodies generated against human seminal plasma inhibin (10.5 KDa), immunocytochemical localization was carried out in paraffin embedded tissue sections of human endometrial biopsies obtained at various phases of the menstrual cycle. A positive reaction which indicated the presence of inhibin was characterized by the presence of golden yellow or brown colour in the cytoplasm of epithelial cells that formed the glands as well as the luminal lining. The stromal cells however, showed negative staining. In early proliferative phase, the endometrial glands exhibited weak positive staining for inhibin which gradually increased and was intense in late follicular and early secretory phases. The intensity of the staining although was not diminished in the glandular epithelium in the mid as well as late secretory phases, the number of cells showing positive staining appeared to be reduced. Incubation of endometrial biopsies in vitro with labelled amino acid and immunoprecipitation of newly synthesized protein with specific antibodies to inhibin indicated that endometrium is capable of de novo synthesizing inhibin. The above results suggest that endometrium is an extra ovarian source of inhibin and the possible role of endometrial peptide in sperm fertilizing capabilities as well as in pre and post implantation events is suggested.

FSH in testes of marmosets during development: immunocytochemical localization and de novo biosynthesis.

Immunocytochemical localization of FSH was carried out in various cell types of marmoset testes during development using antisera generated against intact as well as beta-subunit of human FSH. Significant differences in the intensity as well as distribution of FSH in various cell types were observed in neonatal, pubertal, and adult marmosets. Intensity of staining in Leydig cells was maximum at day 1 and in adults (1-3 years), whereas it was minimum at 3 months. In seminiferous tubules (Sertoli cells), FSH was present in trace amount until puberty and subsequently increased at maturity. Further studies demonstrate de novo biosynthesis of FSH-like moiety in vitro by testicular tissue, which was age dependent.

De novo biosynthesis and localization of inhibin in marmoset (Callithrix jacchus) and rat epididymis.

Using specific polyclonal antibodies generated against a 13 Kd human testicular inhibin, immunocytochemical localization was carried out in epididymis of intact and castrated marmoset monkey and rat epididymis. Inhibin was found to be present in the cytoplasm of epithelial cells of caput, corpus and cauda epididymis. The intensity of staining and pattern of distribution did not change following castration. Further, the in vitro biosynthesis of inhibin studied by incorporating 3H-leucine and precipitating it with specific antibody indicated maximum biosynthesis in the corpus epididymis in case of marmosets and cauda in case of rats. Following castration in rats, the epididymal tissue still retained the capacity to biosynthesize inhibin. These studies indicate that marmoset and rat epididymis are capable of biosynthesizing/absorbing inhibin and whose synthesis does not depend on androgens.