Guest guest Posted March 5, 2003 Report Share Posted March 5, 2003 Impression: The PC-SPES & analogues have an 'adaptogenic effect' This explains why in some cases testosterone is not completely suppressed (or suppressed at all for that matter). I have enclosed the text below with [ ***** ] to indicate where a mechanistic explanation of this complex activity is emerging. To fully appreciate the significance of this it is useful to read the recent Leibowitz paper on 'testosterone therapy' for PC. See http://www.paactusa.org/newsletters/2003.html or recent EPCEL messages. Comments welcome particularly from our colleagues with experience in TCM. Sammy. 27 February 2003, Volume 22, Number 8, Pages 1261-1272 Oncogenomics - Gene profiling and promoter reporter assays: Novel tools for comparing the biological effects of botanical extracts on human prostate cancer cells and understanding their mechanisms of action Dora Bigler1, Kay M Gulding1, Roger Dann2, Fayad Z Sheabar2, Mark R Conaway3 and Dan Theodorescu1 1Departments of Molecular Physiology and Biological Physics and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA; 2Kemin Foods, Proteins and Bioassays Development Group, Des Moines, IA, USA; 3Department of Health Evaluation Sciences (Biostatistics), University of Virginia Health Sciences Center, Charlottesville, VA, USA Correspondence to: D Theodorescu, Department of Molecular Physiology and Biological Physics - Box 422, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA. E-mail: dt9d Abstract The use of botanical mixtures is commonplace in patients with prostate cancer, yet the majority of these products have not been rigorously tested in clinical trials. Here we use PC-SPES, a combination of eight herbs that has been shown to be effective in clinical trials in patients with prostate cancer, as a model system to demonstrate 'proof of principle' as to how gene expression profiling coupled with promoter assays can evaluate the effect of herbal cocktails on human prostate cancer. In addition, we demonstrate how such approaches may be used for standardization of herbal extract activity by comparing the gene profile of PC-SPES with that of PC-CARE, a product with a similar herbal composition. Since prior studies have shown that PC-SPES contains estrogenic organic compounds, and such compounds are known to affect prostate cancer, an important issue is whether these are the primary drivers of the gene profile. Our data suggest that gene expression profiles of LNCaP human prostate cancer cells in response to PC-SPES are different from those found when diethylstilbestrol (DES), a synthetic estrogen, is used, suggesting that the estrogenic moieties within PC-SPES do not drive this expression signature. In contrast, the expression profile of PC-CARE was almost identical to that of DES, highlighting that mixtures containing similar herbal compositions do not necessarily result in similar biological activities. Interestingly, these three agents cause similar in vitro morphological changes and growth effects on LNCaP. To validate the expression profiling data, we evaluated the protein expression and promoter activity of prostate-specific antigen (PSA), a gene induced by PC-SPES but repressed by DES. [ ***** In order to gain a mechanistic understanding of how PC-SPES and DES affect PSA expression differently, LNCaP cells were transiently transfected with wild-type and mutagenized PSA promoter, ARE concatemers and appropriate controls. We provide evidence that androgen response elements (ARE) II and III within the promoter region are responsible for the suppressive effects of DES and stimulatory effects of PC-SPES. In addition, we show that the effects on PSA transcription are ARE specific in the case of DES while PC-SPES affects this promoter nonspecifically. ***** ] In conclusion, expression profiling coupled with mechanistic target validation yield valuable clues as to the mode of action of complex botanical mixtures and provides a new way to compare objectively mixtures with similar components either for effect or quality assurance prior to their use in clinical trials. Oncogene (2003) 22, 1261-1272. doi:10.1038/sj.onc.1206242 Keywords prostate neoplasms; gene profiling; androgen; estrogen; prostate-specific antigen/PSA; plant extracts Introduction Billions of dollars are spent by American consumers on complementary and alternative therapies for the treatment of cancer. Unfortunately, the majority of these products have not been rigorously tested in clinical trials. In stark contrast, one product stands out both for its clinically documented effects as well as its popularity among patients. PC-SPES (Botaniclab, Brea, CA, USA), a combination of eight herbs, has been shown to decrease prostate-specific antigen levels (PSA) >50% in many patients with androgen-independent prostate cancer (AIPC) (DiPaola et al., 1998; de la Taille et al., 2000; Small et al., 2000; Oh et al., 2001). This magnitude of PSA decrease has in turn been associated with improved survival in clinical trials (Bubley et al., 1999). Since the current therapy of AIPC does not lead to cure, the results obtained with PC-SPES offer exciting possibilities for the development of novel therapies if the active tumoricidal compound could be identified. In the initial studies on this product, high-performance liquid chromatography (HPLC), gas chromatography and mass spectrometry showed that PC-SPES contains estrogenic organic compounds. Since estrogen treatment of hormone refractory prostate cancer has shown some benefit (Malkowicz, 2001; Takezawa et al., 2001), the possibility exists that the observed effects of PC-SPES in prostate cancer are due solely to its estrogenic properties. Arguing against this hypothesis is the observation that the responses obtained with PC-SPES exceed those obtained with diethylstilbestrol (DES), a synthetic estrogen. In a recent clinical trial, 17/38 patients (45%) on PC-SPES demonstrated a PSA response compared to 8/39 patients (21%) on DES (Small et al., 2002). Nevertheless, while unlikely, it is still conceivable that the estrogens in the PC-SPES are more potent than DES and this could explain the observed clinical results. Likely because of the successful marketing and documented clinical effects of PC-SPES, products with similar names (i.e. PC-CALM, PC-PLUS and PC-CARE) and herbal compositions are appearing on the market. However, none of these products appear to have been studied and reported in the peer-reviewed scientific literature (White, 2002), yet are likely consumed by many patients with prostate cancer. Based on these observations, we propose to test the hypothesis that PC-SPES affects a completely different gene expression repertoire than does DES when applied to human prostate cancer. The practical importance of such findings is as follows: (1) If the induced gene profile of this herbal extract were similar to DES, this would lend support to the notion that the clinical result (Small et al., 2002) was because of potency differences among the estrogens as speculated above, and there would be significantly less interest in undertaking the difficult biochemical fractionation and subsequent screening to identify the exact chemical compounds responsible for its inhibitory effect on prostate cancer. (2) If the induced gene profile of this mixture were different from that of DES, this would suggest that PC-SPES exerts its biological effects differently from DES, and would pave the way for the development of rational reporter assays used to evaluate fractions of this mixture with subsequent identification of the active compound that may lead to a significant therapeutic advance in prostate cancer. In addition, we seek to use this approach to compare and contrast two similar herbal mixtures and thus demonstrate 'proof of principle' of a new paradigm for the 'functional' standardization of similar botanical products marketed directly to consumers. [ ***** Here we use PC-SPES to demonstrate how gene expression profiling coupled with promoter assays can suggest whether this herbal cocktail exerts its biological effect on prostate cancer via its estrogenic components or via completely different pathways. We show that gene expression profiles of human prostate cancer cells in response to PC-SPES are different from those found when DES is used, suggesting that the estrogens in this mixture are not the primary effectors of this gene profile. In contrast, the profiles of PC-CARE are similar to those of DES indicating that despite similar starting components, botanical mixtures can have profoundly different biological effects. In addition, we use prostate-specific antigen (PSA) expression and promoter mutants of this gene in human prostate cancer cells to validate the expression profiling data and to suggest that PC-SPES regulates this gene differently than does DES. ***** ] Results and discussion PC-SPES inhibits tumor growth in vitro and in vivo and is independent of the androgen sensitivity of the prostate cancer cell lines Ethanol extracts of PC-SPES can significantly reduce LNCaP proliferation (Hsieh et al., 1997). Although androgen-independent cell lines have been evaluated for their sensitivity to PC-SPES, a definite conclusion as to the relation between androgen sensitivity and PC-SPES response cannot be made because of the differing origins of the various cell lines. To test the hypothesis that the antiproliferative response to PC-SPES is not a function of androgen resistance, C4-2, a unique lineage related but androgen-independent LNCaP derivative, was evaluated and compared to LNCaP. Examination of the cell morphology in response to PC-SPES, PC-CARE and DES reveals cellular rounding and increased prominence of nucleoli (Figure 1a). No difference existed between compounds used on either cell lineage except for PC-CARE at 1 l/ml, which was associated with no morphological change. Following incubation with 0, 1, 2 and 5 l/ml of PC-SPES extract for 1-2 days, a dose- and time-dependent growth inhibition was observed and no difference between LNCaP and C4-2 was found (Figure 1b). Interestingly, 10 M when we evaluated the effect of 5 l/ml PC-CARE and DES on these two cell lines, a similar degree of suppression of proliferation to that of PC-SPES was seen in both cell lines (data not shown). Since the effects of PC-SPES on androgen-dependent LNCaP cells have never been evaluated in vivo, we used this herbal mixture in a xenograft model. Feeding male mice PC-SPES resulted in a significant depression of growth for the whole duration of treatment when compared to vehicle-treated controls. In addition, growth stabilization of the tumors occurred between 10 and 30 days with a resumption of growth following this period of time, indicating the possible emergence of a resistant tumor phenotype (Figure 1c). It is important to note that this is the only currently published animal data with PC-SPES in which the lot used is known to be free of exogenous DES (Sovak et al., 2002), yet has a profound effect on xenograft growth. PC-SPES has different HPLC fingerprints when compared to known estrogenic compounds Because of recent concerns that some lots of PC-SPES contain small amounts of exogenous estrogens (Small et al., 2002), we tested for the presence of estrogens and other isoflavones in our lot of PC-SPES. HPLC profile of the PC-SPES extracted in a fashion similar to that used for in vitro and in vivo assays was compared to that of various standards. In addition, the spectrum of various eluting peaks was determined as well. PC-SPES shows a major peak at 1.6 min (Figure 2a). A minor peak that elutes at 2.6 min appears to partially coelute with diadzein. When the spectrum under each peak is compared (Figure 2b), differences are seen between PC-SPES and both standards, diadzein and 17-estradiol. The primary PC-SPES peak may be related to diadzein, but is probably not identical. No DES was found on our lot of PC-SPES confirming previous reports (Sovak et al., 2002). ***** Surely good evidence to suggest previous samples were tampered with to 'prove' they were contaminated with conventional drugs and thus explain-away the success of PC-SPES. Remember the FDA labs found no DES contamination either - DES was found in samples where independent auditing of procurement and storage was not made. Moral of the story: " Don't go down 'Skid Row' to sample a good whiskey " ;-/ ***** Prostate cancer cells exposed to PC-SPES exhibit a different gene profile than cells exposed to PC-CARE and DES Taken together, the data presented above support the notion that PC-SPES and DES are equally effective at reducing prostate cancer proliferation irrespective of the cell's androgen-sensitivity status and this is associated with similar cellular morphological changes. Superficially, these data could be interpreted as supporting the notion that PC-SPES and DES affect cell growth via similar mechanisms. However, this notion is challenged by the findings that PC-SPES shows more favorable results in patients with prostate cancer than DES (Small et al., 2002). We sought to test the hypothesis that PC-SPES and DES affect the expression of a similar repertoire of genes by carrying out gene expression profiling. By analysing duplicate samples, a stringent statistical analysis aimed at determining the genes that are differentially expressed in the two groups was carried out. It is important to note that cellular incubation conditions and timing for this analysis was carried out in an identical fashion to that used to obtain the morphological changes shown in Figure 1a. Therefore, any differences in gene expression in this setting are not because of gross differences in cellular appearance in response to either PC-SPES or DES. The genes that were statistically significantly induced or suppressed by PC-SPES or DES are shown in Table 1. Examination of these data suggests a strikingly different expression profile between the two tested agents. Interestingly, PSA expression is significantly changed by both agents but in the opposite direction. Whereas PC-SPES induces PSA, DES suppresses the expression of this gene indicating that these compounds have dramatically different effects on this androgen-responsive gene in human prostate cancer. In addition, irrespective of the statistical significance of their induction or suppression by either agent, all the genes with names containing the text search term 'prostate' found on the chip were listed in Table 2. It is interesting to note that with the exception of PSA, none of these are significantly affected by PC-SPES or DES. We have validated the Affymetrix results using quantitative RT-PCR technology using five genes randomly picked from those shown in Table 1. As can be seen in Table 3, there is excellent agreement between Affymetrix 'fold' expression of these five genes and the 'fold' expression obtained by RT-PCR. In addition to highlighting differences between the gene expression repertoires in response to various pharmacologic products, gene expression profiling has two other benefits. First, it can lead to clues as to the mechanism of action of PC-SPES by identifying the genes, which are induced by this agent. An examination of Table 1 indicates that several genes involved in the cell cycle are affected by PC-SPES. Never in mitosis gene a (NIMA)-related kinase 2 (Helps et al., 2000) is induced by PC-SPES. Induction of this gene in prostate cancer may be associated with growth arrest and thus may underlie the mechanism of action of PC-SPES. Another protein, S100P is a member of the S100 family of proteins containing two EF-hand calcium-binding motifs. S100 proteins are localized in the cytoplasm and/or nucleus of a wide range of cells, and are involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. Interestingly, the expression of S100P has previously shown to be downregulated in LNCaP cells following androgen deprivation (Averboukh et al., 1996). We are currently evaluating these and several other genes for their functional roles in prostate growth and progression. After submission of this manuscript, a study by Bonham et al. (2002) was published profiling 3000 prostate-derived genes in response to PC-SPES and DES. Interestingly, apart from the general conclusion that the expression profile of PC-SPES is not similar to that of DES, this manuscript reveals striking differences from our own, not only in the genes altered by PC-SPES and DES but also in the direction (up or down) of their regulation. For example, PSA is downregulated by DES and PC-SPES in their study while it is only downregulated by DES in our work. In addition, cdc-20, cdc-25B, cyclin A2, cyclin B2 and MAD2 were upregulated in our study while they were downregulated in theirs. Several genes including Cyclin E2, cdc-6, tubulin 1 and 2 were downregulated in both studies. The basis for these discrepancies may be because of several factors including: (1) one of the lots of PC-SPES used by Bonham et al. contained DES (lot 5431106 (Sovak et al., 2002), which would explain the PSA expression result among others; (2) the LNCaP cell line used was sourced differently from our own and phenotypic differences in this cell line have been reported by various laboratories. The second benefit of such profiling is in the determination of gene clusters, which are most specifically affected by either PC-SPES (Figure 3a) or DES (Figure 3b). These data are complementary to that shown in Table 1 and can be used in future studies to construct custom arrays for several applications such as quality control assessment of natural products. Surprisingly, the expression profiling of PC-CARE, which purportedly has the same starting components as PC-SPES is strikingly different from that of PC-SPES (Figure 3 and Table 1). This is even more interesting in view of the fact that the unsupervised cluster analysis and significantly altered genes shown in Figure 3 and Table 1, respectively, strongly indicate that the expression profiling of PC-CARE is more closely related to that of DES rather than that of PC-SPES. This finding has profound implications since it potentially offers an avenue for the molecular 'fingerprinting' of complex botanical extracts, which are claimed to be made from the same natural sources. Application of these approaches in preclinical studies may allow for both the standardization of the botanical mixtures allowing for rational interpretation of any discrepant human clinical data. In addition, companies producing botanical products can use this technology in quality assurance of their products, which may have biological relevance superior to conventional approaches such as HPLC. PC-SPES stimulates PSA secretion and PSA promoter activity in the presence or absence of androgen [ ***** - The chip data above indicate that PSA expression is regulated differently in response to PC-SPES as compared to DES. PC-SPES induces PSA expression while DES suppresses this gene indicating that these compounds may have fundamentally different effects on androgen-responsive genes in prostate cancer cells. Therefore, we chose PSA as a prototypical model for the study of androgen-responsive genes in prostate cancer. The availability of well-defined culture conditions in which androgen stimulation and deprivation can be evaluated combined with well-characterized promoter constructs makes this gene ideal for such studies. It is important to note that in order to fully characterize the effect of PC-SPES and DES on PSA transcription in both the presence and absence of androgen, we used standard culture conditions for this specific purpose that are somewhat different from those used to generate data on the morphological changes and microarray expression profiles in response to these two agents. Therefore, although qualitatively similar, results from the microarray data in regard to PSA cannot be quantitatively compared to those of the promoter reporter studies described below. Secreted endogenous PSA levels were evaluated in response to synthetic androgen R1881 stimulation and addition of PC-SPES for 3 days, following a 24 h incubation in serum-free, phenol red-free RPMI medium complemented with 5% charcoal-stripped FBS. This in vitro condition is analogous to PC-SPES treatment of prostate cancer patients, which have not yet had androgen ablation and serves to evaluate whether this agent can antagonize the effects of androgen. Data presented in Figure 4a indicate that PC-SPES counteracts PSA protein secretion by R1881 and this is dose dependent. However, these data also indicate that in the absence of androgen, lower doses of PC-SPES can stimulate PSA secretion. LNCaP cells were transfected with PSA-driven luciferase reporter plasmids (Figure 4b). After serum starving the cells for 24 h, PSA expression was induced by adding 1 nM R1881. At the same time, the cells were treated with various doses of PC-SPES extract or DES and incubated for 3 days. PSA promoter induction with R1881 was inhibited by DES in a dose-dependent manner (Figure 4c). In contrast, in uninduced cells (no R1881) PC-SPES itself seems to have an inducing effect at the 1.5 l/ml dose, which was still manifest in the context of R1881 addition. At higher doses the stimulating effect of R1881 on the PSA promoter was reduced by PC-SPES. When the levels of secreted PSA and those of PSA promoter activity are compared (Figure 4a and c), it becomes apparent that in the context of androgen, PC-SPES may diminish PSA secretion while having the opposite effect on PSA promoter activity suggesting that PC-SPES may function by inhibiting post-transcriptional events of the PSA gene in certain situations and at certain doses. PC-SPES and DES have opposite effects on PSA expression and this is mediated by ARE-II and III DES and PC-SPES have strikingly different dose-dependent effects on the whole PSA promoter both in the absence and presence of R1881 (Figure 4c). In contrast to PC-SPES, DES shows a definitive inhibition of androgen stimulated PSA expression at all doses (Figure 4c). While DES has been shown not to modulate mRNA expression of two androgen-responsive genes, C3 and SGP-2, in the prostate (Turner et al., 2001), it does not appear to bind to the androgen receptor directly (Montgomery et al., 1992) and thus its exact mechanism of action is unclear. Upon binding to androgen, the androgen receptor translocates into the nucleus and binds to three androgen-response elements (AREs) on the PSA promoter, where it interacts with other transcription factors and activates PSA gene transcription. In cells transfected with a construct containing three concatemerized copies of the PSA ARE I (Cleutjens et al., 1997), PC-SPES appears to have a nonspecific stimulatory effect as indicated by its similar pattern of induction of both the ARE-I and control contructs. The presence of androgen marginally increased the nonspecific stimulation (Figure 4d). In contrast, DES shows an ARE-I-specific inhibition of R1881-stimulated reporter activity similar in pattern to that observed with the whole PSA promoter (Figure 4c), suggesting that DES exerts its effect on PSA specifically via this ARE while PC-SPES does this via nonspecific effects on ARE I. To further investigate the role of the individual AREs in the overall and androgen-induced transcriptional responsiveness of the 6-kb PSA promoter in the context of PC-SPES and DES, we used knockout mutations introduced for each individual ARE in the full-length PSA promoter (Figure 4b). Cleutjens et al. (1996,1997) identified three consensus AREs within the 5.8-kb PSA promoter. ARE I and II are located within the proximal region of the promoter, whereas ARE III is contained within a 440-bp strong enhancer element core (AREc) located at -4.2 kb on the promoter. Transient transfection of LNCaP cells with the resulting mutated PSA promoter-LUC constructs showed that ARE II and III contributed to both the stimulatory and inhibitory effects of PC-SPES and DES, respectively (Figure 4e). These findings indicate that ARE-II and III are key elements in PSA promoter regulation by PC-SPES and by DES in the context of androgen stimulation. In addition, the reduction of PSA transcriptional activity seen with the wild-type promoter (Figure 4c) at higher PC-SPES doses is lost with the mutated full-length constructs (Figure 4e) and the ARE I concatemer (Figure 4d) indicating the presence of complex yet undefined ARE-dependent regulatory events. Materials and methods Cell lines, in vitro morphology, growth curves and PSA secretion Human prostate cancer cell lines LNCaP and C4-2 were obtained from Dr L Chung, Department of Urology, Emory University. C4-2 is an androgen-independent cell line derived from androgen-sensitive LNCaP cells (Wu et al., 1994). Unless otherwise specified, cells were grown and all experiments were carried out in T-media, containing 5% fetal calf serum (FCS). Both cell lines are tumorigenic and produce prostate-specific antigen (PSA) (Wu et al., 1994). Morphology of cells was assessed microscopically after 24 h of treatment with either 1 l/ml PC-SPES, 1 or 5 l/ml PC-CARE, 10 M DES or with 5 l/ml 70% EtOH as control. Growth curves were generated by the evaluation of cell numbers at various time intervals by the SyberGold Assay (Molecular Probe, Eugene, OR, USA) performed according to the manufacturer's protocol. For secreted, endogenous PSA levels LNCaP cells were grown for 24 h in phenol red-free RPMI medium complemented with 5% charcoal-stripped FBS before induction with R1881 and addition of PC-SPES. At 3 days after induction, the medium was evaluated for PSA by the University of Virginia Medical Laboratories using the Olympus AU640 analyzer and reagents (Olympus Diagnostics Systems). Preparation of PC-SPES and PC-CARE extracts PC-SPES was obtained from BotanicLab Inc. (Brea, CA, USA) via direct purchase from their website (www.botaniclab.com) in April 2001. The PC-SPES (lot 5431219) used for these experiments does not contain any exogenous DES (Sovak et al., 2002). PC-Care (Natrol, Chatsworth, CA, USA) was obtained on June 2001 via direct web site purchase (www.drugstore.com). Stock solutions were prepared by suspending the content of one capsule in 1 ml of 70% EtOH, stirring the suspension for 1 h at room temperature and removing the insoluble material by centrifugation as previously described (Darzynkiewicz et al., 2000). The clear 70% EtOH extract was filtered through a 0.22 m Millipore filter and stored in small aliquots at -20?C. Synthetic androgen R1881(Cleutjens et al., 1997) and estrogen diethylstilbestrol (DES) were purchased from Sigma. HPLC and spectroscopy In view of the known estrogenic compounds found in PC-SPES (DiPaola et al., 1998), two standards were used for the HPLC: 17-estradiol and diadzein. Two standards were prepared by dissolving <0.5 mg in 50:50 H2O:ACN (1 ml). Various amounts were chromatographed by HPLC (Hewlett-Packard 1100 series) equipped with a Diod Array Detector on a C18 column, 4.6?250 mm 100 A pore size, 5 M particles (Varian Inc.) at 25?C and an isocratic gradient of 50:50 H2O:ACN at a flow rate of 1 ml/min. PC-SPES samples were prepared by suspending the content of two capsule in EtOH followed by vigorous mixing and centrifugation at 6000 r.p.m. The supernatant was dried under nitrogen flow and redissolved in 50:50 H2O:ACN (1.5 ml mobile phase, and diluted 1:1). In vivo tumor growth and statistical analysis Nude male mice, 6 week old, were obtained from Taconic farms and maintained strictly according to the NIH and institutional guidelines at the University of Virginia animal facility. A total of 5?106 LNCaP cells in 0.1 ml Matrigel were injected subcutaneously in each animal. At 4 weeks after injection, the mice were randomly divided into a group of 20 mice that received PC-SPES and a group of 20 mice that received the carrier only. PC-SPES was administered orally (gavage) at a concentration of 250 mg/kg/day as previously described (Kubota et al., 2000). Mice were weighed every week and tumor sizes were measured twice a week as described (Seraj et al., 2000). Mice were euthanized 40 days postinjection. Two-sample t-tests were used to compare average tumor volumes between groups on each day. More sophisticated analyses using repeated measures models yielded nearly identical results, and we present the results from the simpler analyses based on t-tests. PSA reporter constructs and transient transfections All plasmid DNA used for transfection was prepared by the 'EndoFree Plasmid Maxi kit' from Qiagen (Valencia, CA, USA) following the manufacturer's protocol. The various PSA promoter constructs were a gift from Dr Michael Weber (University of Virginia). The 6 kb promoter fragment (whole PSA promoter) in pGL3 basic, 3ARE-I-TATA (from PSA and TK promoters, respectively), ARE minimal sequence - TATA and TATA only control constructs were previously described (Cleutjens et al., 1996,1997). The ARE I-1, ARE II-1 and ARE III-1 mutant constructs are point mutants in the three different ARE motifs at position -170 bp (ARE I), -394 bp (ARE II) and -4200 kb (ARE III), respectively, in the context of the whole PSA promoter and have been previously described (Cleutjens et al., 1996,1997). DNA transfection was performed using the lipofectin method (Invitrogen, Palo Alto, CA, USA) as described by the manufacturer. Cells were plated at a density of 1.5?105 cells/ml in six-well plates and fed the next day. Transfection was performed the day after and the cells were allowed to recover in T-media containing 5% FBS. After 24 h, the medium was switched to serum-free, phenol red-free RPMI medium complemented with 5% charcoal-stripped FBS for another 24 h before PSA-driven reporter expression was induced by adding the synthetic androgen R1881 at a final concentration of 1 nM. Where indicated, PC-SPES or DES was added at the time of induction. After 72 h cells were harvested, washed in PBS and lysed in 400 l Reporter Lysis Buffer (Promega, Madison, WI, USA). Lysed samples were analysed in a Turner Designs Luminometer by adding 20 l cell lysate to 100 l Luciferase Assay Reagent. Luciferase activity was corrected for cell number, determined with the SyberGold assay mentioned above. Affymetrix chip processing and analysis LNCaP cells were treated with 1 l/ml PC-SPES, 1 and 5 l/ml PC-CARE, 10 M DES or with 5 l/ml 70% EtOH as control and grown for 24 h. This experiment was repeated with completely different cells one week after the initial experiment. RNA was extracted at the end of the 24 h incubation period with the Qiagen RNeasy kit (Qiagen, San Diego, CA, USA). Microarray analysis was performed as described in the manufacturer's instructions (Affymetrix, Santa Clara, CA, USA). Briefly, cRNA was prepared from 8 g of total RNA, hybridized to HG-U95AV2 Affymetrix oligonucleotide arrays, which contain approximately 12 000 human genes or ESTs. After washing in a fluidic station, the arrays were scanned with a 2.5 m resolution HP Microarray Scanner (Hewlett Packard). Scanned images were first examined for visible defects and then checked for the fitness of the gritting. When passed, the image file was analysed to generate a raw data file named cell files. From this point on, a coordination of two paths of analysis was carried on using the Affymetrix Microarray Analysis Suite 5.0 (MAS 5.0, Affymetrix) and the Dchip software (Schadt et al., 2000,2001; Li and Wong, 2001). The detection of a particular gene, present, absent or marginal, was made using the MAS 5.0, those detection calls were later imported into and utilized by the Dchip program. Scatter plots were also generated using this software to inspect the reproducibility of the duplicates or the degree of changes among samples. Quantitation of the genes was obtained using the Dchip, which applied the model-based approach to derive the probe sensitivity index and expression index. The two indices were combined to quantify a particular gene. When certain probes or transcrips deviated from the model to a set extent, they were excluded from the quantitation process. Normalization of the arrays was performed using the invariant set approach. Comparative analysis of the samples using Dchip generated fold changes and paired sample t-test P-values. Regularly, we consider a P0.05 and a fold change 1.2 in combination of a % Present 50 as an indication of significant change of gene expression for upregulation or downregulation. A Spearman correlation coefficient was generated for all the possible pairs involved using the Dchip quantitation results for quality control. Filtering of the genes was performed to select the genes for hierarchical clustering using the build-in module in Dchip. The following criteria were applied for the filtering process: 0.5s.d./mean 10 AND Present call 25% among all samples AND Signal intensity 20 in 50% of all samples. Before clustering, expression values for a specific gene are standardized (linearly scaled) in all samples to have mean 0 and s.d. 1, and these standardized values are used to calculate correlations between genes and samples and serve as the basis for merging nodes. The distance of two neighboring genes is calculated using the Pearson's correlation model and is expressed as 1-|r|, which would be the length of the stem connecting the two branches. Validation of Affymetrix chip results In order to validate the Affymetrix chip expression data, we have carried out real-time quantitative RT-PCR for five genes that showed expression differences in the three experimental groups (Table 1). Suitable probes were ordered from the Applied Biosystems Assays-on-DemandO Gene Expression products library, a comprehensive collection of predesigned and functionally tested assays. Each assay consisted of two unlabeled PCR primers and a FAMO dye-labeled TaqManO MGB probe. All components are QC tested and formulated into a single 20x mix. TaqManO Universal PCR Master Mix was added to the cDNA sample to generate quantitative gene expression data on an Applied Biosystems ABI PRISMO 7900HT Sequence Detection System. 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Schadt EE, Li C, Ellis B and Wong WH. (2001). J. Cell Biochem. Suppl., (Suppl. 37), 120-125. Schadt EE, Li C, Su C and Wong WH. (2000). J. Cell Biochem., 80, 192-202. Article PubMed Seraj MJ, Harding MA, Gildea JJ, Welch DR and Theodorescu D. (2000). Clin. Exp. Metast., 18, 519-525. Small EJ, Frohlich MW, Bok R, Shinohara K, Grossfeld G, Rozenblat Z, Kelly WK, Corry M and Reese DM. (2000). J. Clin. Oncol., 18, 3595-3603. PubMed Small EJ, Kantoff P, Weinberg VK, Nguyen S, Smith MR, Bubley GJ and Oh WK. (2002). J. Clin. Oncol., 21, Abstract # 709. Sovak M, Seligson AL, Konas M, Hajduch M, Dolezal M, Machala M and Nagourney R. (2002). J. Natl. Cancer Inst., 94, 1275-1281. Takezawa Y, Nakata S, Kobayashi M, Kosaku N, Fukabori Y and Yamanaka H. (2001). Scand. J. Urol. Nephrol., 35, 283-287. Turner KJ, Morley M, MacPherson S, Millar MR, Wilson JA, Sharpe RM and Saunders PT. (2001). Mol. Cell Endocrinol., 178, 73-87. White J. (2002). J. Natl. Cancer Inst., 94, 1261-1263. Wu HC, Hsieh JT, Gleave ME, Brown NM, Pathak S and Chung LW. (1994). Int. J. Cancer, 57, 406-412. PubMed Figures Figure 1 PC-SPES effect on LNCaP and C4-2 prostate cell morphology and growth. (a) Morphology: cells were treated with either 1 l/ml PC-SPES, 5 l/ml PC-CARE, 10 M DES or with 5 l/ml 70% EtOH as control. Pictures were taken after 24 h incubation. (b) In vitro growth: cells were treated with either 1, 2 or 5 l/ml PC-SPES or with 5 l/ml EtOH as control and incubated for 1 and 2 days. The cells were lysed and the relative number of cells determined by a SyberGold assay, as described in materials and methods. Error bars represent the s.e. from the mean of quadruplicate sample. Data are representative for two independent experiments with similar results. © In vivo growth: mean changes in tumor volume by day and treatment group. The errors bars represent 1 s.e. The values along the axis are P-values for comparing treatment means at each day Figure 2 Reverse phase HPLC chromatograms of PC-SPEC sample and standards and their corresponding spectra. PC-SPES was extracted with ethanol and separated by RP-HPLC. (a) Retention time of various peaks and (b) their spectra were compared to those of various estrogens or isoflavones Figure 3 Cluster analysis of PC-SPES, PC-CARE and DES-treated LNCaP cells. Unsupervised cluster analysis displaying differential gene expression of the duplicate samples. Hierarchical clustering was performed with the D-Chip software after filtering the genes with the defined criteria as described in the materials and methods section. Genes coded in red color are deemed to have been upregulated and those in blue color downregulated, while those showing no significant changes are white. The brighter colors signify higher magnitude in the scale of the changes in either direction. The color bar in front of a gene name indicates the classification in gene ontology (www.geneontology.org). Panel (a) shows genes preferentially clustered with PC-SPES; Panel (b) shows genes preferentially clustered with PC-CARE and DES. Abbreviations: S: PC-SPES (1 l/ml); C1: PC-CARE (1 l/ml); C5: PC-CARE (5 l/ml); D: DES (10 M) Figure 4 The effect of PC-SPES and DES on PSA secretion and expression in LNCaP cells. (a) Untransfected LNCaP cells were treated as indicated, adding 1 nM R1881 for induction of PSA and at the same time either 0, 0.5 or 1.5 l/ml PC-SPES. The cells were pelleted and the media analysed for secreted PSA level. (b) Diagrammatic representation of the location of three consensus AREs (and resultant mutant constructs used in (e)) within the PSA promoter (Cleutjens et al., 1996,1997). © PSA expression with whole PSA promoter constructs: LNCaP cells were transiently transfected with whole PSA promoter pGL3 reporter construct and treated as indicated: adding 1 nM R1881 for induction of PSA and at the same time either 0, 0.5, 1.5 l/ml PC-SPES, or 0, 10, 20 M DES. The cells were harvested after 3 days and assayed for luciferase activity. (d) PSA expression with ARE-TATA constructs: LNCaP cells were transiently transfected with three ARE-I-TATA or TATA only PSA pGL3 reporter construct and treated as indicated in (a). The suffix 'Ind'. indicates samples exposed to R1881 while 'Unind'. are samples not exposed to this agent. (e) PSA expression with ARE mutant constructs: LNCaP cells were transiently transfected with either AREmin, ARE I, ARE II or ARE III pGL3 reporter construct and treated as indicated in (a). All samples in this experiment were treated with R1881. Error bars represent the s.e. from the mean of duplicate samples. Data are representative for two or more independent experiments with similar results Tables Table 1 Genes on HG-U95AV2 Affymetrix oligonucleotide arrays, which had a significantly altered expression in PC-SPES, PC-CARE and DES Table 2 All prostate-related genes on HG-U95AV2 Affymetrix oligonucleotide arrays Table 3 Validation of expression of 5 genes from Table 1 using real-time quantitative RT-PCR Received 22 July 2002; revised 8 November 2002; accepted 12 November 2002 27 February 2003, Volume 22, Number 8, Pages 1261-1272 Table of contents Previous Article Next PDF Privacy PolicyC 2003 Nature Publishing Group Quote Link to comment Share on other sites More sharing options...
Guest guest Posted March 5, 2003 Report Share Posted March 5, 2003 In a message dated 3/5/03 4:21:36 AM Eastern Standard Time, ga.bates writes: > PC-SPES, a combination of eight herbs that > has been shown to be effective in clinical trials in patients with prostate > cancer, I believe that product has been discontinued. I believe the FDA and something to do with that. I do not wish to say any more except look at the herbs that were in the product. Stan Quote Link to comment Share on other sites More sharing options...
Guest guest Posted March 5, 2003 Report Share Posted March 5, 2003 ga.bates wrote: > > Impression: The PC-SPES & analogues have an 'adaptogenic effect' This Can't quite remember exactly what the problem with PC-SPES was, but it was ultimately taken off the market, I believe it had been laced with pharmaceuticals to cause the prostate to shrink. Do a search for this product, you'll see that it is no longer available. -al. Al Stone L.Ac. <AlStone http://www.BeyondWellBeing.com Pain is inevitable, suffering is optional. Quote Link to comment Share on other sites More sharing options...
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