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Tze-chen Hsieh^a, Sophie S. Chen^b, Xuhui Wang^b and Joseph M. Wu ^{a ,*}

^a Department of Biochemistry & Molecular Biology, New York Medical College,
Valhalla, N.Y 10595
^b International Medical Research, Inc., Brea, CA 92821

Summary. As part of the study on the potential use of natural product-based combination therapy for treating prostate cancer, we have investigated the effects of a "HPLC standardized" herbal preparation, PC-PES, on the prostate LNCaP cell line. Proliferation of the LNCaP cells was inhibited by a 4-6 day incubation with ethanolic extracts of PC-SPES. Decrease of cell growth was accompanied by a 60-70 % down-regulation of the proliferating cell nuclear antigen (PCNA) and level of secreted PSA. A smaller and more variable decrease (20-40%) in the level of intracellular PSA was also observed. The PC-SPES-modulated PSA changes occurred concurrently with the decrease of AR expression, based on Western blot analysis and binding to the radioactive ligand [³H]R1881. A 60% decrease in R1881 binding occurred after a 24 h incubation with PC-SPES. These results suggest that PC-SPES negatively affects cell growth in part through its ability to modulate changes in PCNA, and may decrease PSA levels indirectly by suppressing AR expression.

INTRODUCTION

Despite progress made over the last several decades in the field of surgery, radiation, and chemotherapy for cancer treatment, cancer continues to be a major cause of human morbidity and mortality in developed and developing countries. Lack of apparent success to control malignancy is probably caused by the constellation of changes seen in cancer cells, that in turn leads to increased mutations owing to inadequate and/or deficient DNA repair mechanisms, results in the decoupling of cell proliferation from differentiation, and promotes a loss or dysregulation of apoptosis. The combination of these modified events culminates in uncontrolled growth and spread of abnormal cells. Design of an encompassing strategy for cancer treatment is made difficult by virtue of the fact that each of these biological properties of malignant cells may involve multiple genetically distinct but functionally overlapping molecular pathways and participation of different protein moieties. Furthermore, many forms of cancer are known to actively undergo multi-stage in situ transitions prior to the clinical manifestation of infiltration and metastasis, due to the genetic plasticity of neoplastic cells and their ability to propagate along divergent pathways (1).

Before the advent of modern chemistry in the nineteenth century, minerals and extracts of living organisms (both animal and vegetable sources) have provided the principle source for the development of drugs (2,3). Subsequent advances in chemistry made it possible to isolate and synthesize chemically pure compounds that would give reproducible biological results, and provided guidelines for developing rational strategies in the synthesis and evaluation of modern age pharmacological agents. To date, about 25% of all the drugs on the pharmacist's shelf have been derived from study and analysis of less than 0.5% of the higher plants on this planet (4), suggesting that the vast majority of the globally diverse plant species has yet to be tested for agents with pharmaceutical potentials.

Current approaches to treat cancer have primarily involved use of single agents. In addition to being overly toxic at the effective dosage, presumably in part due to lack of balancing chemicals commonly found in the traditional remedy, single agents are not sufficiently broad-based in their mechanism of action as to effectively control all of the underlying complexities of malignant cells described. Accordingly, in recent years increasing efforts have been directed at studying "combination" or "sequential" approaches, wherein putatively effective agents, at suboptimal concentrations, are either presented as a group or are given in a particular sequence, to target cells (5,6). A. variation of the "combination" theme is to apply whole herbal preparations as "alternative" disease treatment modalities. An example of such an approach is the study of Suh et al. (7) in which ethyl acetate extracts of some 400 plants were tested for ability to induce HL-60 leukaemic cell differentiation, resulting in the demonstration of activity for 17 extracts. It should be noted that one limitation of herb therapies is poor to inadequate characterization of the active ingredients. Furthermore, even in cases where active ingredients have been identified, reconstituting individual components to the same potency as the crude preparation is uncertain.

The incidence of prostate cancer has witnessed a steep rise in the US in the last decade (8). The subclinical (latent) form of prostate carcinoma is estimated to be present in about 30-50% of men by age 75, and deaths from the disease have surpassed lung cancer to become the leading cause of cancer mortality in males. At present there is no known cure for advanced prostate cancer, which is partly due to insufficient understanding of the cellular, biochemical, molecular and genetic features of prostate carcinoma. This knowledge deficiency is probably attributed to many factors, among which are the limited number of prostate cell lines currently available for research, and the paucity of information concerning the transition of prostate cancer from latent to the highly aggressive, therapy-refractory terminal state.

Because of the aforedescribed complexities of prostate cancer, a single drug approach may be expected to have limited efficacy as treatment modalities. As an alternative strategy, we have been interested in exploring the potentials of natural product-based combination approaches. In preliminary studies, a herbal preparation containing a complex mixture of eight herbs (herein referred to as PC-SPES), given as a dietary supplement to enhance the overall immune status and well-being of terminally ill cancer patients, was found to drastically reduce the level of serum PSA. This suggests that PC-SPES, as an example of herbal "neutraceuticals" (4), may affect the growth and gene expression of prostate cells. Accordingly we investigate changing levels of PSA in relationship to control of cellular proliferation by the ethanolic extract of PCSPES in the androgen-dependent LNCaP cells. Our results show that PC-SPES suppresses LNCaP prostate cell growth and results in drastic down-regulation of the levels of secreted and the intracellular PSA. Furthermore PSA change is concomitant with the suppressed expression of the androgen receptor, AR.

MATERIALS AND METHODS

PC-SPES was provided by BotanicLab, Inc. (Brea, CA). It contains one American and seven Chinese herbs: Isatis indigotica Fort, Panax pseudo-ginseng Wall, Ganoderma ludidium Karst, Scutellaria baicalensis Georgi, Dendranthema morifolium Tzvel, Glycyrrhiza glabra L, Robdosia rubescens, Serenoa repens. The herbal preparation was "standardized" by HPLC analysis of the 70% ethanol extract, which was prepared by stirring the content of one capsule (320 mg) of PC-PES with one ml of 70% ethanol at 150 rpm for one h at room temperature. Insoluble material was removed by centrifigation. The ethanolic extract was filtered through a 0.22 or 0.45 µM Millipore filter and stored in small aliquots at -20^oC. For HPLC analysis, 0.5 ml of the ethanolic extract was injected into an Alltech C18 reverse phase preparatory column (250 mm x 22 mm) and eluted at a flow rate of 4-8 ml/min, using a linear gradient generated by a Shimazu SC 6A two solvent pump system, with solvent A containing water and solvent B containing acetonitrile. Separation was monitored by absorbency at 254 nm. For tissue culture experiments, 100% ethanolic extracts were used.

Cell culture and treatment with PC-SPES. Culture of LNCaP cells and treatment with ethanolic extracts of PC-PES were identical to previous publications from this laboratory (9-11). At different times, cells were harvested by trypsinization. Morphological changes of control and treated cells were assessed by photomicroscopy (10,11).

Measurement of intracellular and secreted PSA. Levels of PSA were measured based on the cleavage of p-nitrophenyl phosphate substrate by the IgG-tagged alkaline phosphatase. The coloured products were quantified by measuring absorbency at 405- and 450-nm (10).

Western blot analysis. Postmitochondrial extracts were prepared from control and treated cells using buffers supplemented with multiple protease inhibitors as previously described (9-11). AR and PSA antibodies were obtained from commercial sources. Extracts from control and treated cells were separated on 10% SDS-PAGE, followed by transfer onto nitrocellulose membranes, and incubated with the respective primary and secondary antibodies. Specific immunoreactive bands were visualized with enhanced chemiluminescence system (ECL) or by color reaction, as described (9-11). Reprobing of blots with different antibodies was done after stripping with a buffer containing 62.5 mM Tris-HCI, pH 6.7, 100 nM 2-mercaptoethanol, 2% SDS, at 50^oC for 30 min.

Measurement of AR expression by binding to [³H]R1881. Cells were seeded in 6-well plates at a density of 1 x 10⁵ cells/ml and treated with PC-SPES (5 µl/ml). Specific binding to labelled R1881 was assayed as described (10,11) using 1 and 2 day PC-SPES-treated cells.

RESULTS

Preparation of PC-SPES. Since levels of chemicals in a given species of medicinal herb may vary considerably, both with the genetics and environment of the plant, and temporally and spatially within the plant (12), ethanolic extracts of PC-SPES were "standardized" by high performance liquid chromatography. Figure 1 shows a typical HPLC profile of 70% ethanolic extract of PC-SPES. "Standardization" of 3 preparations of PC-SPES was based on the consistent, relative appearance of six UV₂₅₄-absorbing peaks, identified as A-F. Components in A-F are presently being investigated using NMR and mass spectroscopy. Preliminary analysis suggest that they comprise mostly of small molecular weight entities, belonging to the group of alkaloids and polyphenols.

Effects of PC-SPES on LNCaP cell growth and PCNA expression. To test the effects of PCSPES on growth of prostate carcinoma cells, the LNCaP were incubated with various amounts of PC-SPES and the growth of cells over time were determined. Ethanolic extracts of PC-SPES reduced cell growth in a time- and concentration-dependent mariner. Suppression of cellular proliferation was correlated with a 50-65 % reduction in expression of PCNA (Table 1), which is commonly used as a mitotic index to evaluate proliferative activity in mammalian cells (10).

Modulation of intracellular and secreted PSA by PC-PES. PSA is a 34 kDa-glycoprotein which has been used as a serum marker for screening and staging of prostate cancer (13-15). Since preliminary studies show PC-SPES, given to terminally ill prostate cancer patients, drastically reduced the circulating level of PSA, we checked for its ability to regulate PSA gene expression. Both intracellular and secreted PSA were suppressed following a minimum of 2 days of treatment with PC-SPES (Figure 2). However, whereas the level of secreted PSA decreased by 60-70% after a 4-6 day treatment with PC-SPES (Figure 2, panel B), a smaller and more variable decrease (20-40%) in the level of intracellular PSA was observed, suggesting that PCSPES could exert a complex and dynamic effect on the biosynthesis, processing and secretion of PSA in prostate cells.

Regulation of AR by PC-SPES. Since PSA gene responds to androgens, presumably via interaction with androgen receptors, changes in AR were also measured by Western blot analysis in both control and treated cell extracts. Results in Figure 3 show that intracellular AR and PSA expression was drastically down-regulated after treatment with PC-SPES. The PSA decrease was proportional to the amount of extract or media added to the assay mixture (data not shown). As an independent assessment of AR expression, the binding of the control and treated cells to [³H]R1881 was also studied. Results in Table 2 show that a one day treatment with PC-SPES decreased the binding of radioactive R1881 by 2.5-fold, which was in close agreement with the Western blot data.

DISCUSSION

It is becoming increasing clear that extracts of natural products from different sources will be an important available and excellent source for drug development. Because herbal medicine has been practiced in China for at least 2,000 years, a vast clinical data base has been accumulated, lending support to their claimed actions. Predictably for some diseases Chinese herbs may greatly increase the effectiveness of modern drug treatments, reduce their side-effects, and under the best circumstances replace them completely.

In preliminary studies, PC-SPES, given as a dietary supplement to end-stage cancer patients, was observed to unexpectedly reduce the circulating level of PSA. This prompted us to investigate its mechanism of action in LNCaP cells. Results of experiments reported in this communication show that PC-S PES suppressed LNCaP prostate cell growth and down-regulated the secreted and the intracellular pool of PSA, which was concomitant with the suppressed expression of the androgen receptor, AR. Since PC-SPES is a crude extract, a number of explanations may be offered for these observations. For example, both AR and PSA may be regulated by the same active ingredients present in PC-SPES. A second possibility is that AR and PSA are controlled by different chemicals present in PC-SPES. Alternatively, the regulation of AR and PSA may require defined ratios of several active components to be present simultaneously. Experiments are underway to compare efficacies of extracts prepared using different concentrations of ethanol, as well as various fractions derived from HPLC-generated UV-absorbing peaks, to further elucidate the manner in which AR and PSA are regulated by PC-SPES. Our current hypothesis on the pleiotropic effects of PC-SPES is as follows: it reduces prostate cell growth by intercepting the androgen receptor (AR) and down regulates PSA subsequent to reduced AR expression. With regard to the growth inhibitory effects of PC-SPES, we propose that, in addition to down-regulating PCNA, PC-SPES also modulates other cell cycle regulatory protein molecules such as pRB, and may additionally induce apoptosis. These and other possible mechanism of action of PC-SPES are currently being investigated.

ACKNOWLEDGEMENTS

The authors are grateful to Drs. Darzynkiewicz and Traganos of the Cancer Research Institute for their interest and advice. Appreciation is also extended to Dr. Mittleman for encouragement.

* Reprint requests should be sent to Dr. Joseph M. Wu at the Department of Biochemistry & Molecular Biology, New York Medical College, Valhalla, NY 10595.

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Accepted for Publication: BIOCHEMISTRY & MOLECULAR BIOLOGY INTERNATIONAL July 1997