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Regulation of Prostate Growth in Culture with the Pollen Extract, Cernitin ™ T60™, and the Impact of the Drug on the EGF Tissue Profiles

F. K. Habib

Introduction

A major difference between the prostate and other accessory reproductive glands is the susceptibility of the prostate to hyperplasia in aging men. Indeed, benign hyperplasia of the prostate (BPH) affects most males over 60 years of age and causes enlargement of the inner gland. When the urethra becomes constricted, treatment is required to relieve the kidney and circulating system of the damaging effects of back pressure.

Surgery in the form of transurethral resection still remains the "gold standard" for the treatment of outflow tract obstruction (1) but recently attention has also focussed on alternative forms of therapy, namely hormonal (2, 16), 5(ax-reductase inhibitors (11), and (ax-adrenergic blockers (5). However, the longterm prognosis for medical treatment has been poor and many of the endocrine and pharmacological agents presently in use have side effects (4). This has prompted the medical and scientific community to consider new lines of treatment of BPH. One recent development was the sudden and unexpected interest in phytotherapy, which was in part instigated by the encouraging results and the undoubted beneficial effects of the pollen extract, Cernilton ®, in the symptomatic relief of BPH (1).

The mechanism by which the pollen induces its effect on the hyperplastic prostate is not yet clear even though extensive experimentation has been undertaken by many workers (8,9, 10). Notably however, the bulk of the earlier research was focussed on experiments with animal tissue, which constitutes an unsatisfactory model for the human gland. Additionally, the few studies on the human prostate were carried out either on whole organ homogenates or on prostate epithelial cell lines (8), both of which ignore the potential heterogeneity of the cellular activity within the gland and the importance of stroma / epithelial interactions. Furthermore, the immortal cell lines represent a highly selective cell population which might have undergone phenotypic changes and may therefore be distinctive from the cells of origin.

In an attempt to overcome these earlier limitations, efforts in our laboratory have been directed towards developing primary culture of the human prostate and the serial culture of epithelial and fibroblast cells from BPH employing defined media. Initially, progress was slow and attempts to find the optimal concentration of ingredients to permit the growth of the cells and increase their plating efficiency were repeatedly frustrated. However, thanks to our collaboration with Dr. D. Chaproniere, to whom much of the credit for the earlier work goes (3), combined with the perseverance of my chief tissue culturist, Mrs. Margaret Ross, we managed to overcome many of the initial obstacles and finally establish a reliable technique for the serial culture of both prostate stroma. and epithelial cells in serum-free medium (manuscript in preparation). This model was subsequently adapted to our Cernilton ® studies in which the experiments were confined to the water-soluble Cernitin ™ T60™ fraction; this fraction accounts for approximately 60 % of the pollen extract. Details of the procedures followed and a summary of our findings on the characterization of the cultured cells along with the impact of the Cernitin ™ T60™ are described within.

This chapter also includes some preliminary data on growth factor profiles in prostate tissue specimen and in expressed prostate secretions (EPS) obtained from a group of BPH patients receiving the pollen extract. The relevance of growth factor peptides and particularly epidermal growth factor (EGF) to the prostate stems from their ability to maintain and regulate prostate growth either by acting in tandem with androgens or possibly even by by-passing the steroid hormones and imprinting their own characteristics on the gland (7,12). Recent reports on the preferential accumulation of EGF in BPH when compared to normal prostate tissue (6,14) supports the belief that this peptide might be implicated in the pathogenesis of this condition. Since the action of Cernilton ® in the prostate has been found not to be mediated via the androgen delivery system of the cell (8), we are now looking at the possibility of an association between the expression of some of these growth factors and their response to Cernilton ® in patients receiving the drug.

Serial Culture of Prostate Epithelial and Fibroblast Cells

BPH specimens obtained by transurethral resection were transported under sterile conditions to the laboratory in transport medium. Acini and fibroblast cells were released from prostate tissue by collagenase digestion and primary and sub-cultures were grown by plating onto plastic culture flasks and incubating at 37oC in a 95% air-5% C02 humidified atmosphere. By using this system it was possible to establish and serially culture pure populations of both epithelial (Fig 1 a) and fibroblasts (Fig I b) cells in well-defined media. For epithelial cells the WAJC404 medium (3) was serum free and was supplemented with insulin (2.5µg/ml), EGF (10µg /ml), dexamethasone (1µM), and choleratoxin (10µg/ml); this medium selects against the growth of the fibroblast cells. Four days after inoculation of the epithelial cells onto T-75 flasks, the acini demonstrated good spread, and confluence was usually reached by day five. Fibroblast cells were maintained in RPM11640 supplemented with fetal calf serum (10%) and penicillin and streptomycin (10µg/ml each). Fibroblast cells were initially slow in growing and confluence was reached usually after ten days.

Fig. 1 Phase-contrast micrograph of a primary culture of epithelium (1 a; x 100) and a serial culture of fibroblast (1 b; x 200) from a patient with benign prostatic hyperplasia.

a
b

Verification of the culture as prostatic fibroblast and epithelial cells is accomplished by immunocytochemical staining employing a variety of antibodies including those for vimentin, desmin, prostatic-specific antigen (PSA), prostatic acid phosphatase (PAP), and cytokeratin. Assessment of the staining patterns and their intensities was always undertaken by an independent pathologist. A typical pattern of the staining profiles obtained is illustrated in Table 1.

In addition to the immunostaining (Table 1), the cells were also examined by phase contrast microscopy Analysis of the photomicrographs (Figs.1a and 1b) suggest that the resultant epithelial monolayers contain very little or no contaminants - any residual fibroblasts will be totally destroyed by the epithelium growth medium. Furthermore, the bulk of the epithelial cells appear to be of a secretory nature since PAP and PSA are strongly expressed (Table 1). The epithelial cells also stain uniformly for cytokeratin and recognize the antibody for the epidermal growth factor receptor. This confirms our earlier findings on the presence of EGF-receptors in epithelial cells of human prostate tissue (14).

Tab. 1 Immunocytochemical Staining of Epithelial and Fibro-blast Cells in Culture.

Markers used Fibroblast cells Epithelial cells

Prostatic acid phosphatase - ++

Prostatic specific antigen - ++

Epidermal growth factor receptor -/+ ++

Cytokeratin -/+ ++

Vimentin ++ -/+

Desmin + -

HMFG (Human Milk Fat Globulin) -/+ ++

Intensity of staining: (++) strongly positive; (+) moderately positive; (-/+) patchy;
(-) negative.

In contrast, the fibroblast cells failed to stain for PAP and PSA but were positively labelled by antibodies for vimentin and desmin. Somewhat surprisingly, the fibroblast cells were also outlined by the antibodies for cytokeratin and for Human Milk Fat Globulin (HMFG), which are exclusively epithelial in nature. This raises the possibility that the fibroblast cells might contain small contaminants of a secondary cell. Closer examination of those fibroblastic cells by microscopy highlights the presence of small numbers of epithelial-like cells amongst the stromal monolayers. The secondary cells could be either non-secretory epithelial or endothelial cells which maintain an "epithelioid"-Iike appearance, but this needs to be confirmed. The presence of the fibroblast contaminants was also confirmed by flow cytometry and we are at present attempting to segregate the two cell populations employing a cell sorter. Interestingly, however, the "epitheloid"-Iike material appears not to multiply but remains constant throughout each passage and might merely act as a supportive matrix for the fibroblast.

Fig. 2 Effect of T60™ concentration on fibroblast cell survival. Fibroblast cells (2.5 x 10 4 cells/well) were plated overnight in 96 well plates. Increasing concentrations of T60™ were added for varying times. (3 H)-thymidine was then added for 24 hours and the cells were trypsinized in 10 % TCA. The cells were then harvested onto filter mats, dried and counted in scintillation fluid. The data is normalized relative to the untreated control (100 %).

The Effect of T60™ on Epithelial and Stromal Cell Growth in vitro

Dose response curves of Cernitin ™ T60™ treat ment were determined using the following method: triplicate determinations for each treatment were performed in 96 well culture plates; each well was seeded with 2.5x104 cells and incubated overnight at 37C in the medium under defined incubation conditions. The following day, the Cernitin ™ T60™ stock solution was serially diluted in the defined medium to yield a concentration varying from 0.05-1 mg/ml. Controlled cultures received culture medium alone. For the dose response curve studies, the cells were exposed to Cernitin ™ T60™ for a total period up to 4 days with changes of freshly diluted T60™ in medium every 2 days. For the time course study, cells were treated in the presence and absence of T60™ a total period of 7 days. After the incubation periods, the cells were pulselabelled with radiolabelled thymidine whilst remaining in the defined medium for a further 24 hours.

For the determination of the rate of the DNA synthesis the cells were trypsinized and 10 % ice cold trichloroacetic acid was added for 2 hours. The cells were subsequently harvested onto filter mats ' dried at 60oC for 30 minutes and each disc of filter paper containing the precipitable material was then counted in scintillation fluid. The results illustrated in Fig. 2 (fibroblast cells) and Fig. 3 (epithelial cells) are expressed as the percentage of 3H-thymidine incorporated relative to the untreated control. These demonstrate that the effect of Cernitin ™ T60™ on stroma and epithelial cells is biphasic: initially and at the low concentrations of T60™ (up to approximately 0.1 mg / ml) we detect significant stimulation, particularly in the fibroblast cells which show after 2 days of exposure an increase of approximately 75 % in DNA synthesis. However, exposure to higher concentrations of the T60™ inhibits the uptake of thymidine and after 3-4 days exposure we do find that the concentrations of T60™ (P> 0.25 mg / ml) almost totally inhibit the fibroblast growth.

Although the epithelial cells do also show an inhibition in cell growth which is timeand concentration-dependent, it appears that the epithelial cells are slightly more resistant to the pollen extract than the fibroblast cells. Though there is initially a minute stimulation in DNA synthesis of up to 25 % after 2 days of exposure (results not shown), this is rapidly reversed, and inhibition is observed at approximately the same concentrations of T60™ as those required to induce the same effect with the fibroblast but following longer periods of response to the Cernitin ™ T60™ (Fig. 3).

EGF Concentrations in Prostate Tissue and Prostate Secretions following Cernitin ™ Treatment

Fig. 3 Effect of T60™ concentration on epithelial cell survival. Details were identical to those followed for the fibroblast cells as detailed in legend to Fig. 2

Prostate tissue was obtained at the time of the transurethral resection from 19 patients with BPH; the patients had been entered into a Cernilton ® double-blind placebo-controlled study over a six-month period. The tissue was transported immediately to the laboratory in iced saline, dry blotted, snap-frozen in liquid nitrogen and stored at - 70oC until analysis. Matching expressed prostatic secretions (EPS) were collected by transrectal massage before the commencement of the trial and at approximately three-month intervals with the last specimen obtained immediately prior to transurethral resection whilst the patient was under either regional or general anesthesia. The fluid was collected into 1-ml insulin syringes, frozen without delay, and stored at -70oC until needed.

Studies on EPS Specimens

Pre- and post-treatment samples of EPS were obtained from 8 patients in the Cernilton ® treatment group and 5 patients in the placebo group; the mean length of treatment with Cernilton ® was 147 ± 42 days. A comparison of EGF concentrations in both groups before commencement of treatment revealed no significant difference (P > 0.5; Fig. 4). Similarly, comparison of the EGF concentrations in samples before and after treatment also showed no significant difference (P > 0.5); these data are illustrated in Fig. 4. In addition we have also examined the changes in EGF concentrations of consecutive samples of EPS from individual participants in the doubleblind placebo-controlled study; the patterns obtained are illustrated in Fig.5. Clearly, there are no consistent patterns of change which could be of use for monitoring response to treatment.

Studies on Prostate Tissue

Fig. 4 EGF concentrations in expressed prostate fluid (EPS). Aliquots of EPS were taken from a group of patients who had entered the doubleblind placebo trial of Cemilton, and EGF was measured in samples taken at the start and towards the end of the trial. The concentrations in the treated group were compared to those on placebo. Results are expressed as mean ± SEM for 8 patients in the treated group and 5 patients on placebo. Bars show SEM.

In addition to the measurements undertaken on EPS, we have also measured the EGF concentrations in prostate tissue obtained from 15 BPH patients undergoing prostatectomy. These were compared to the concentrations found in a parallel group of 7 patients who were taking Cernilton ® as part of the doubleblind placebo-controlled study. The data was expressed as ng EGF/mg protein in the tissue and the results obtained for the individual patients are outlined in Table 2. Although the levels of EGF in the treated group appeared to be considerably lower than those measured in the controlled group, the difference is not statistically different. However, it should be noted that the population receiving the Cernilton ® tablet is comparatively small and the results obtained might have been slightly biased by the fact that 2 out of 7 patients showed relatively high concentrations of EGF whereas the remainder of the population had levels considerably lower than those measured in any of the other individuals in the controlled group. We are at present extending this study to incorporate a further 20 patients on the drug in the hope that this might throw some light on the mechanism of action of Cernilton ® and whether the differences between the control and test groups are genuine and reflect actual changes in the metabolic pathways of the gland following treatment with the pollen extract.

Conclusion

The precise mode of action of Cernilton ® in BPH is not clearly understood even though many studies have been undertaken to elucidate the mechanism by which this pollen extract promotes symptomatic relief in patients with BPH. The earlier studies concentrated mainly on animal models and as reported by Ito et al. (9), Cernilton ® produced in mature Wistar rats a significant reduction in the size of the prostate as well as in PAP concentrations whilst also inducing a parallel increase in blood and tissue zinc concentrations. Additionally, Cernitin ™ T60™ produced relaxation of the smooth muscle of the mouse and increased the contraction of the bladder muscle in a concentration-dependent manner (10).

Fig. 5 EGF concentrations in consecutive EPS specimens taken from patients receiving Cernilton ®. For each patient EPS specimens were obtained before commencement of treatment and then at approximately 3 and 6 months into the trial.

In view, however, of the specie differences in prostate anatomy and function, a fundamental distinction must be made between animal studies and experiments on human tissue. The attentions of this laboratory were therefore focussed initially on the immortal human prostate cell lines which demonstrated an inhibitory response following treatment with Cernitin ™ T60™ (7). Interestingly, the inhibitory effect was far more marked in the hormone unresponsive cell line when compared to the androgen-sensitive human prostate cells. Human cell lines derived from non-prostatic tissue failed to exhibit a similar sensitivity to the pollen extract (7).

Although the usage of immortal cell lines in our earlier studies was most helpful in identifying the specificity and selectivity of the drug, their use is somewhat limited because of: (a) the cancer nature of the continuous cells whilst Cernilton ® is prescribed purely for BPH; (b) immortal cells are identical clones and do not therefore take account of the morphological heterogeneity of the prostate; and (c) continuous cell lines may undergo phenotypic changes and this might render them distinctive from the cells of origin. In view of these limitations we have decided to continue our work on Cernitin ™ T60™ employing the well-established cultures of epithelial and fibroblast cells from human hyperplastic prostates (3,15). Those studies were facilitated by our ability to establish and serially culture pure populations of epithelial and fibroblast cells in a well-defined serum-free medium. By using this system the specific characteristics of Cernitin ™ T60™ could be assessed in a cohesive and systematic fashion.

Clearly, the data outlined in this report indicates that Cernitin ™ T60™ is a powerful mitogenic inhibitor of fibroblastic and epithelial proliferation. Although the mechanism involved is not as yet understood, we have evidence derived from our earlier studies (8) to indicate that these responses are not mediated via the androgenic pathways. We have therefore decided to look at the impact of Cernitin ™ T60™ on the expression of growth factors which have been implicated in the growth of the prostate cells. Though the results on the prostate fluid indicate little difference in EGF concentrations between the control and test groups, the evidence derived in this report suggests that there might be some impact on the epidermal growth factor concentration of the tissue.

EGF is a well-established secretory product of the prostate and is retained in large concentrations by BPH when compared to the normal gland (6). This retention might be associated with the high concentrations of the EGF receptors found in BPH which must sequester the growth factor for internal use (14). We are not too clear on the mechanism responsible for this build-up of EGF receptors and whether it is a causal factor or merely a result of the development of hyperplasia. We are also not certain whether there is an association between these abnormal growth factor concentrations and the dihydrotestosterone levels which have previously been linked to the growth of the gland (17). Significantly however, our most recent studies reveal no correlation between EGF receptors and the endocrine status of the gland, suggesting that androgens do not modulate EGF-receptor expression in the prostate (13). Since the action of Cernilton ® on the prostate seems also to be independent of the endocrine functions of the gland, the impact of the pollen extract on the tissue EGF concentrations might be of significant importance, not only in controlling the abnormal growth of the gland but also in pinpointing new pathways relating to the pathogenesis of BPH.

Tab. 2 hEGF Concentrations in Prostate Tissue following Treatment with Cernilton ®.

Control Group EGF concentration Cernilton ® Group EGF concentration

J. W. 1.50 W. F. 3.45

J. G. 1.35 C. F. 2.43

J. N. 2.09 C. S. 1.51

D. D. 1.61 K. B. 0.45

G. T. 2.07 A. S. 0.31

H. H. 1.20 T. S. 1.10

R. H. 2.84 R. H. 0.89

A. C. 3.98

W. T. 3.67

W. B. 4.38

W. H. 1.50

K. H. 3.40

H. J. 2.57

K. N. 3.00

T. S. 1.76

hEGF-Concentrations (mg/ EGF/mg Protein)

1.45 ± 1.31

Mean ± S.D. 2.39 ± 0.85

References

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