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INTERNATIONAL JOURNAL OF ONCOLOGY 14: 713-719, 1999

Anti-tumor effects of PC-SPES an herbal formulation in prostate cancer

R.K. T'IWARI' , J. GELIEBTER , V.P.S. GARIKAPATY', S.P.K. YEDAVELLI', S. CHEN and A. MITTELMAN

Departments of 1 Microbiology and Immunology, 2 Medicine, Division of Oncology, New York Medical College, Valhalla, NY 10595, USA

Received January 7, 1999; Accepted February 8, 1999

Abstract:

Prostate cancer is the most common cancer amongst males in developed countries. Surgical removal of the prostate effectively cures the primary disease but the metastatic disease is refractory to most forms of chemotherapy. There is a clinical need to develop novel treatment strategies that exploit the mode of action of both conventional and alternative drugs/medicinal plants. We have been investigating the antiproliferative and anti-tumor effects of an herbal preparation termed PC-SPES (patent pending, US serial number 08/697, 920) which is a refined powder of eight different medicinal plants. PC-SPES administered as a food supplement caused a dramatic decrease in prostate specific antigen levels in some prostate cancer patients with advanced disease. These preliminary clinical findings laid the foundation for a program to examine the in vitro and in vivo effects of PC-SPES, and identify the active component in this mixture so that a standardized treatment regimen can be formulated. In this communication, we report the anti-tumor effects of PC-SPES incorporated in the diet utilizing a well studied Dunning R3327 rat prostate cancer model. Dietary PC-SPES at levels of 0.05% and 0.025% did not exhibit any toxicity and no significant difference in food intake was noted at the end of six weeks. Dose dependent inhibitory effect of dietary PC-SPES was observed on both tumor incidence (P=0.01) and rate of tumor growth when tumors were induced in syngeneic Copenhagen rats by intradermal injections of MAT-LyLu cells that are known to metastasize in the lung and lymph nodes. The number of pulmonary metastases in animals on PC-SPES that showed no primary tumor growth had no metastatic lesions in the lung, however, in animals that did not respond to PC-SPES, the number of pulmonary metastases was not significantly different from the nontreated controls. The significant anti-tumor effects of PC-SPES on MAT-LyLu induced tumorigenesis and metastasis in Copenhagen rats, in general refractory to most conventional therapy, suggests a therapeutic benefit of this herbal food supplement and may be a useful adjuvant to conventional therapeutic modalities. Correspondence to: Dr Raj K. Tiwari, Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA Key words: rat model, anti-tumor effects, PC-SPES, toxicity, pulmonary metastases

Introduction

Prostate cancer accounts for over 180,000 new cases with approximately 40,000 deaths among American men every year. Removal of the prostate is curative for the primary disease with a substantial decline in the quality of life but metastatic prostate cancer is refractory to most prevalent forms of radiation and chemotherapy (1,2). Furthermore, conventional treatments that employ physical or chemical hormone deprivation leads to a more aggressive, rapidly proliferating subset of prostate cancer cells that are hormone independent and presumably acquire a distinct set of growth factors that mediate androgen-independent proliferation (3,4). Specific therapy that targets the functional activity of the epidermal growth factor receptor (EGFR) and the oncogene product HER-2/neu are in clinical evaluation but a combination treatment schedule that can target all the subsets of prostate cancer cells is still elusive. Since regulation of cell cycle, induction of apoptosis and steroid hormone induced signal transduction pathways and invasion of the extracellular matrix by enhanced tumor vasculature are all involved in the growth, maintenance and spread of prostate cancer, it is imperative that successful treatment strategy needs to target all or several of the growth regulating pathways simultaneously.

A multi-targeted approach to prostate cancer can be promulgated in several ways. Combination chemotherapy with drugs that target the cell cycle and angiogenesis, or radiation and chemotherapy, or anti-hormone therapy with growth factor/signal transduction inhibitors have shown promise but the success of each novel approach depends on its toxicity, morbidity and nature of the prostate cancer (5-8). Since it is not always possible to examine the phenotypic characteristics of prostate cancer cells by non-invasive procedures, the treatment options are postponed until the appearance of overt clinical or pathological symptoms. At this point potential treatment benefits are significantly reduced. It is therefore important to focus research attention on the prevention of progression of benign disease utilizing a non-toxic, multi-targeted approach.

We have been investigating the anti-tumor and antiproliferative effect Of PC SPES, a combination of eight different medicinal plants each having a distinct clinical effect (9-11). A dramatic drop in the prostate specific antigen (PSA) levels was observed in some advanced prostate cancer patients. PC-SPES is a refined powder containing Ganodermab lucidium Karst. Dendranthema morifolium Tzvel, Glycyrrhiza glabra L, Isatis indigotica, Panax pseudo-ginseng Wall, Rabdosia rubescens, Scutellaria baicalensis Georgi, and Serenoa repens ( 11 ,12). Several of these components exert significant biological effects for e.g. Ganoderma lucidium Karst exerts a strong immunomodulatory effects and the immunomodulatory protein LZ-8 has been identified (12); Baicalein a flavonoid derived from Scuttellaria baicalensis has anti-proliferative and lipooxygnesase-inhibitory activity (13); Serenoa repens is a potent phytoestrogen and its lipid extracts is an inhibitor of both type I and type 2 5a-reductase, the enzyme that converts testosterone to dihydrotestosterone, the active androgen in the prostate (14); Rg 1, a saponin derived from Panax ginseng has mitotic activity towards T cells similar to Concanavalin A (15,16) and ginseoside-Rb2 inhibits angiogenesis in a transplantable tumor model; extracts of Glycyrrhiza glabra has been shown to have anti-mutagenic activity (17,18). It is the combination of several such potent medicinal plants that presumably exert a synergistic anti-proliferative effects by acting on multiple targets simultaneously.

We examined the effect of PC-SPES in the R3327 syngeneic Copenhagen rat model using MAT-LyLu cells. The advantage of this model is that several cell lines have been developed from a spontaneous tumor that arose in the Copenhagen rats with varied properties. The parental G subcell line is androgen responsive and grows slowly and is not known to metastasize. A variant of this cell line termed as MAT-LyLu is rapidly proliferating, is androgen unresponsive and metastasizes to the lung and lymph nodes (19). Mat-LyLu cells have a doubling time of 8 to 10 h and have a 100% tumor take in syngeneic Copenhagen rats and are in general refractory to most conventional forms of therapy.

In the present study we examined the effect of dietary PC-SPES at concentrations of 0.025% and 0.05% on MATLyLu cell line induced tumorigenesis in Copenhagen rats. End-points of investigation included tumor incidence, rate of tumor growth, pulmonary metastasis, tumor histology and toxicity and palatability of PC-SPES containing diet. The concentration of PC-SPES have been computed based on the doses used in the clinic and doses that can be achieved by dietary supplementation. Our results suggest that PC-SPES affects tumor incidence, rate of tumor growth and pulmonary metastasis. Animals may be classified as responders and nonresponders with complete resistance to tumors in the responders. Animals that showed no tumor were free of metastasis suggesting that both processes of lodging at the primary site and at metastatic site are affected by PC-SPES treatment.

Materials and Methods

Growth Of MAT-LyLu cells in vitro and in vivo. MAT-LyLu cells (a gift from John Isaacs Laboratory, Johns Hopkins, Baltimore, MD) were grown in RPMI-1640 containing 10% fetal bovine serum (FBS) supplemented with penicillin (50 IU/ml), streptomycin (50 ug/ml) 2 mM L-glutamine and 2.5 mM dexamethasone. Cells were fed twice a week and were trypsinized with 0.05% trypsin-EDTA at 80 to 90% cell confluency. Since the cells are rapidly growing, care is taken that the cells do not reach 100% confluency. Cells injected for growth in Copenhagen rats were Generally taken from flasks that were between and 50 and 75% confluent. MATLyLu cells were washed twice with phosphate buffered saline (PBS) and then trypsinized and suspended in PBS at a concentration of 100,000 cells per ml. Each animal was injected with 0.1 ml of cell suspension with an effective dose of 10,000 live MAT-LyLu cells per rat. Cell viability was always determined by the trypan blue exclusion test and samples that exhibited less than 98% viability were discarded. Cells were injected into the right flank of the animal which has been shaved prior to the injection. All injections were intradermal (i.d.), using an insulin syringe. Cells were kept at 4'C at all times prior to injections. This method of injections with MAT-LyLu, in our experience, yields 100% tumor incidence. All experimental groups and the control animals were injected at the same time with the same batch of cells.

Animal experiments.

Four- to five-week old Copenhagen rats were purchased from Harlan Sprague Dawley, Indianapolis, IN, and allowed to acclimate for one week, feeding on Purina 5001 rat chow. At the end of one week the rats were randomized into three groups of seven rats. Groups were fed either Purina 5001 rat chow (control) or Purina 5001 rat chow containing 0.05% or 0.025% PC-SPES. The rats were allowed to continue on the experimental diet for two weeks and their food intake and body weight monitored. Food intake was determined by giving pre-measured amount of food and monitoring consumption of food by weighing the remaining food every alternate day. Body weights of the animals were measured twice a week. Average food consumption per animal per day from each group was compared. This computation also took into account the food wastage of each animal in each of the groups that was in the range of 4.2-4.4 g per rat per day. Live MATLyLu (10,000 cells/rat) were injected in all animals on day 14 after randomization into groups. All animals were individually housed in hanging cages and had ad libitum access to food and drinking water and were kept on twelve hour diurnal cycle. All injections and tumor measurements were performed under light anesthesia (metofane inhalation). Experimental end point measurements included, dietary intake, body weight gain, tumor incidence, rate of tumor growth, number animals with pulmonary metastases and number of visible metastatic nodules per lung of the tumor bearing animal and histopathological examination of the tumors. Tumor diameter was measured using vernier caliper.

Dietary formulation.

PC-SPES was obtained from BotanicLab. (Brea, CA) and was incorporated into rodent chow 5001 by Purina Labs. (10). Three different concentrations 2.5 g (0.25%), 0.5 g (0.05%), 0.25 g (0.025%) of PC-SPES per kg, were formulated. The concentration of 0.05% corresponded to the supplementation to prostate cancer patients. In a separate experiment (not shown here) diets containing 0.25% PC-SPES was found to be non-palatable in the case of Copenhagen rats as 50% of the animals were found to be malnourished due to refusal of food. Taste was not an issue in the case of prostate cancer patients as they received PC-SPES in capsules. These experiments formed the rationale for the use of 0.05% and 0.025% of PC-SPES in our experimental group. Statistical analysis. Data was analyzed using a two-tailed Student's t-test and mean values differing from each other at P=0.05 was considered significant.

Results

Effect of PC-SPES on the body weight of Copenhagen rats. Four to five week old Copenhagen rats were maintained for two weeks on Purina 5001 rodent chow containing PC-SPES at 0.05% and 0.025%, for acclimatization. Change of mean body weight per group (control, 0.05% and 0.025%) is presented in Fig. 1. The mean food intake per group in grams in the different groups is also given in Fig. 2. It is clear that animals on PC-SPES had a decrease in the amount of food ingested both at 0.025% and 0.05% groups as compared with the control (P=0.002) in the first four weeks of the start of the dietary treatment. The lowered food intake is also reflected in the body weight of the animals that is lower in the PC-SPES treated group in a dose dependent manner. The animals on PC-SPES were found to exhibit normal activity with no hyperactive behavior when compared with the animals on normal diet that contained no PC-SPES. Animals on PC-SPES at 0.05% ingested doses of PC-SPES similar to prostate cancer patients on three 333 mg capsules three times a day. No reports of weight loss was reported in these patients (personal communication, manuscript in preparation), on the contrary some patients had either a significant weight gain or had maintained a steady body weight. This reflects the effect of PC-SPES on the general well being of the prostate cancer patients on this supplement. It should be mentioned that on a dose as high as 0.25% PC-SPES (unpublished observations and data not shown), the animals were malnourished as they refused the supplemented food. It is presumed that such a high concentration of PC-SPES interfered with the palatability of the chow to Copenhagen rats. The fact that 0.05% dose was well tolerated by these animals with no significant overt toxicities as determined by hair loss, loss of appetite, lack of locomotion suggests that supplementation of PC-SPES at this dose assuming similar metabolism in rats and humans should not produce any toxicities in humans. In fact supplementation Of PC-SPES at this dose in human patients is well tolerated by prostate cancer patients.

Effect Of PC-SPES on tumor incidence in Copenhagen rats. Results presented in Fig. 3 shows the dose dependent effect of PC-SPES on tumor incidence in Copenhagen rats, when tumor was induced by i.d. injections of 10,000 live MAT-LyLu cells. PC-SPES incorporated in the diet at 0.025% resulted in a 20% decrease in palpable tumors in animals, whereas, when the dose Of PC-SPES in the diet is increased to 0.05% tumor incidence was decreased by more than 40% (P=0.05). Similar observations were obtained earlier in another experiment with five animals per group. Thus the observations presented here are reproducible and seem to reflect the biological effect of ingested PC-SPES. Dose dependent effect of PC-SPES on tumor incidence suggests that there appears to be responders and non-responders even in inbred strain of experimental animals. Copenhagen rats on PC-SPES diet with no evidence of primary tumor showed no pulmonary metastasis. All the animals in the control showed primary tumor as well as pulmonary metastases. The effect on tumor induction as observed in reduction of tumor incidence at both 0.05% and 0.025% PC-SPES together with the observation that animals that did not have primary tumor lacked pulmonary metastases has significant implication in the preventive use of this herbal supplement provided long term use of PC-SPES does not have in vivo toxicity or any side effects. It is also clear from our experiments that doses below 0.025% PC-SPES may not have any significant impact on tumor incidence and hence pulmonary metastasis suggesting an optimal threshold cellular concentration prior to manifestation of its biological effect.

Effect of PC-SPES on the rate of tumor growth and pulmonary metastases in Copenhagen rats. Dose dependent decrease in the tumor burden was noted in animals that were administered PC-SPES. The reduction was noted three weeks after tumor injection with increasing difference in rate of tumor growth inhibition. The mean values were lower than the control as much as 50% [compare mean values of 0.05% treated animals 1.35 to 2.98 in control (Fig. 4)]. A wide range in the tumor sizes was observed in the experimentally treated animals and as such the values did not attain statistical significance. Nevertheless the wide difference in the mean values together with the observation that some animals were devoid of tumors suggests a differential action of this herb and in animals which are responders or partial responders an antitumor effect is observed. Animals that received 0.05% PCSPES showed a plateau in the growth rate, however animals that were scored positive for the primary tumor also showed lung metastasis. Statistically significant decrease (P=0.05) in pulmonary metastasis was observed in the PC-SPES treated (0.05%) when compared with the untreated controls (Fig. 5). It should be noted that 20% of the animals in 0.025% and 40% of the animals in the 0.05% PC-SPES treated animals did not exhibit any lung metastasis. Similar to the effect of PC-SPES on tumor growth the number of macroscopically visible lung metastases showed considerable variation, however, the mean values in the PC-SPES treated control were found to be statistically different from the control group of animals.

Discussion.

Effect of PC-SPES on the growth of Copenhagen rats. PC-SPES at doses of 500 and 250 parts per million was well tolerated by Copenhagen rats with no overt toxicity symptoms. A difference in the mean amount of food consumed can be attributed to peripheral rodent identifiable factors such as taste, odor or texture. These results are contrary to results obtained with PC-SPES supplementation in a capsular form in prostate cancer patients where either a weight gain or stabilization but no weight loss was observed. Since palatability and taste was not a factor in the human population, it is presumed that taste was the major determinant in lowered food intake.

Anti-tumor effects of PC-SPES. Our studies using Copenhagen rats and the transplantable, rapidly proliferating, highly metastatic cell line, MAT-LyLu indicates that dietary supplementation with the Chinese herbal preparation of PC-SPES can modulate tumor incidence, rate of tumor growth and pulmonary metastases. These results are of special significance in the MAT-LyLu model as growth of these cells are in general refractory to most forms of treatment. Decrease in tumor incidence in a dose dependent manner suggests that some animals respond to the anti-tumor effect of PC-SPES better than others and that even in genetically identical inbred strains of rats we may be able to identify PC-SPES responders and non-responders. This is reminiscent of the human experience with PC-SPES where some patients showed dramatic response or were partial responders, while others were completely refractory (Mittelman A, et al, manuscript in preparation). Although the precise mechanism for such an action is elusive, one can speculate that since PC-SPES is a mixture of several different medicinal plants, each with its target of action, induction of a synergistic response may be host dependent (11). While published data on the biological effects of PC-SPES suggests that specific ethanol extracts of PC-SPES induces apoptosis and cell cycle deregulation predominantly a prolongation of the G1 phase of the cell cycle, immunological modulation by induction of tumor specific cytolytic T cells by specific component(s) Of PC-SPES in its in vivo effect cannot be ruled out (11).

The anti-tumor effect of a non-toxic dietary supplement of combination of medicinal plants suggests the relevance of the use of combination therapy that is capable of utilizing multiple targets. These multiple targets consist of biochemical intermediaries that mediate the cell cycle transition by ethanol extracts of PC-SPES (11). Components of PC-SPES can target specific biochemical targets e.g. Glycerrhiza can reverse mutations (20), Serenoa repens, a phytoestrogen lowers estrogen levels and affects androgen receptor binding with its ligand ( 14,21,22), Scuttelaria baicalensis that contains baicalin inhibits lipooxygenase and DNA topoisomerase activity (23,24), extracts of Ganoderma lucidum have potent immunomodulatory effects (12), Panax ginseng, probably the most studied herb, has anti-carcinogenic effects on spontaneous and carcinogen induced tumor model systems as well as can affect tumor metastases (11,25). Thus, while most of the active components individually have anti-tumor effects, several different herbs in a mixture in the Chinese and Japanese traditional medicine has been found to be more efficacious. The Generation and success of popular mixtures such as 'sho-saiko-to' and 'juzentaihoto' for hepatocellular diseases and enhancement of drug induced anti -neoplastic effects in China and Japan relates to the effectiveness of combination therapy. Each individual component, though potent in their individual effects cannot compare with the effect of the combination preparation (25,26). These anecdotal observations support the hypothesis that preparation that contain mixtures of individually potent drugs have enhanced efficacies that may be related to either synergism of the individual components or the simultaneous hit on multiple cellular and biochemical targets that not only eliminates existing malignant cells but can also prevent the clonal expansion of neoplasms and their further differentiation and thus prevention of secondary metastasis. A recent report f the use of PC-SPES examined the estrogenic effect of PCSPES in an animal model and in eight patients (27). While the substantial decrease in prostate specific antigen correlated with our observations, the doses used were 'too high' and thus the validity of the estrogenic effect was questioned.

Standardized treatment using PC-SPES. The long-term goal of our studies is to identify the active component(s) of herbal preparations that have shown anti-tumor effects. PC-SPES is one such preparation that has similarities with other Chinese and Japanese preparations from medicinal plants but is unique in other respects. Evaluation of the water soluble and ethanol extracts of PC-SPES in in vitro and in vivo studies, and separation of the active components using high performance liquid chromatography (HPLC) and examination of the cellular and molecular effects will help development of a standardized drug-like treatment procedures. These studies will help alternative medicinal plant based therapy to enter into the field of molecular medicine. Furthermore, identification of the cellular and molecular targets of the active component of PC-SPES will provide an intermediate biomarker that can presumably evaluate the efficacy of the treatment and or compliance to a treatment schedule.

Acknowledgements

These studies were supported by grants from AICR (94B66), USARMY (DAMD 17-98-1-8534) and Granoff Foundation to R.K.T., Zita Spiss Foundation to R.K.T. and J.G., AICR (97AO72) to J.G. and Zalmin A. Arlin Cancer Fund to A.M.

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