Gene expression profiling revealed novel mechanism of action of Taxotere and Furtulon in prostate cancer cells

Background Both Taxotere and Capecitabine have shown anti-cancer activity against various cancers including prostate cancer. In combination, Taxotere plus Capecitabine has demonstrated higher anti-cancer activity in advanced breast cancers. However, the molecular mechanisms of action of Taxotere and Capecitabine have not been fully elucidated in prostate cancer. Methods The total RNA from PC3 and LNCaP prostate cells untreated and treated with 2 nM Taxotere, 110 μM Furtulon (active metabolite of Capecitabine), or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 hours, was subjected to Affymetrix Human Genome U133A Array analysis. Real-time PCR and Western Blot analysis were conducted to confirm microarray data. Results Taxotere and Furtulon down-regulated some genes critical for cell proliferation, cell cycle progression, transcription factor, cell signaling, and oncogenesis, and up-regulated some genes related to the induction of apoptosis, cell cycle arrest, and differentiation in both cell lines. Taxotere and Furtulon also up-regulated some genes responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents. Conclusions Taxotere and Furtulon caused the alternation of a large number of genes, many of which may contribute to the molecular mechanisms by which Taxotere and Furtulon inhibit the growth of prostate cancer cells. This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against prostate cancer.


Background
Prostate cancer is the most common cancer and the second leading cause of cancer related deaths in men in the United States with an estimated 230,110 new cases and 29,500 deaths in 2004 [1]. Initial treatment for prostate cancer is usually androgen-ablative therapy, radiotherapy or radical prostatectomy and the patients respond to androgen-ablative therapy in the beginning of treatment. However, many patients eventually fail this therapy and die of recurrent androgen-independent prostate cancer and metastasis [2]. Up to 30% of men undergoing radical prostatectomy will also relapse, often as a result of micrometastatic cancer present at the time of surgery [3]. The efficacy of cytotoxic chemotherapy for treatment of hormone-refractory prostate cancer has been tested in clinical trials. In general, response rates of <10% were observed in single-agent studies [2]. Thus, there is a tremendous need for the development of mechanism-based targeted strategies for treatment of prostate cancer.
Taxotere, a member of taxane family, is semi-synthesized from an inactive taxoid precursor extracted from the needles of the European yew, Taxus baccata. Its known basic mechanism of action is that it binds to tubulin and deranges the equilibrium between microtubule assembly and disassembly during mitosis [4]. Stabilization of microtubules by Taxotere impairs mitosis and exerts an anticancer effect in tumors [4]. Taxotere has shown clinical activity in wide spectrum of solid tumors including breast, lung, ovarian, prostate cancers, etc [5,6]. In metastatic breast, lung, and ovarian cancer, randomized trials have shown that Taxotere-containing therapies are superior to or as effective as established standard chemotherapeutic regimens and are often associated with an improved safety profile [6]. Clinical trials have also found that weekly Taxotere in patients with metastatic hormonerefractory prostate cancer is associated with improvements in clinical benefit response and quality of life [7,8]. Thus, Taxotere is currently considered to be among the most important anticancer drugs in cancer chemotherapy. In addition to its effects on microtubules, Taxotere also induces apoptosis with down-regulation of bcl XL and bcl-2, and upregulation of p21 WAF1 and p53 [9,10]. We have previously reported that Taxotere down-regulates some genes for cell proliferation, mitotic spindle formation, transcription factors, and oncogenesis, and up-regulates some genes related to induction of apoptosis and cell cycle arrest in prostate cancer cells, suggesting the pleiotropic effects of Taxotere on prostate cancer cells [11].
Clinical trials showed that chemotherapeutic combination with Taxotere and Capecitabine resulted in improved objective response rate and overall survival without a significant increase in the treatment related adverse effects in metastatic breast cancer and advanced non-small cell lung carcinoma [18,19]. However, the molecular mechanism(s) of action of Taxotere and Capecitabine have not been fully elucidated. In this study, we utilized highthroughput gene chip, which contains 22,215 known genes, to determine the alternation of gene expression profiles of hormone insensitive (PC3) and sensitive (LNCaP) prostate cancer cells exposed to Taxotere and Furtulon. The purpose of this study was: 1) to identify novel genes that have key roles in cancer cell killing and resistance induced by Taxotere and/or Furtulon; 2) to test whether similar genes are altered by Taxotere and Furtulon; 3) to test whether combination therapy alters genes that may reflect better treatment outcome or may provide information whether combination therapy could be antagonistic; 4) finally to provide molecular information for further mechanistic investigation and future clinical application.

Methods
Cell culture and growth inhibition PC3 (ATCC, Manassas, VA) and LNCaP (ATCC) human prostate cancer cells were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO 2 atmosphere at 37°C. Taxotere (Aventis Pharmaceuticals, Bridgewater, NJ) was dissolved in DMSO to make 4 µM stock solution. Furtulon (Roche, Palo Alto, CA) was dissolved in PBS to make 100 mM stock solution. For growth inhibition, PC3 and LNCaP cells were treated with Taxotere (1, 2, and 4 nM), Furtulon (50, 100, and 200 µM), or 1 nM Taxotere plus 50 µM Furtulon for one to three days. Control PC3 and LNCaP cells received 0.01% DMSO or 0.1% PBS for same time points. After treatment, PC3 and LNCaP cells were incubated with MTT (0.5 mg/ ml, Sigma, St. Louis, MO) at 37°C for two hours and then with isopropyl alcohol at room temperature for one hour. The spectrophotometric absorbance of the samples was determined by using ULTRA Multifunctional Microplate Reader (TECAN, Durham, NC) at 595 nm. The concentrations of Taxotere and Furtulon used for our in vitro studies are easily achievable in humans, suggesting that our experimental results are relevant for human applications. The experiment was repeated three times and a t test was performed to verify the significance of cell growth inhibition after treatment. PC3 and LNCaP cells were treated with 2 nM Taxotere, 110 µM Furtulon, or 1 nM Taxotere plus 50 µM Furtulon for 6, 36, and 72 h. Total RNA from each sample was isolated by Trizol (Invitrogen, Carlsbad, CA) and purified by RNeasy Mini Kit and RNase-free DNase Set (QIAGEN, Valencia, CA) according to the manufacturer's protocols. cDNA for each sample was synthesized by Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) using the T7-(dT) 24 primer instead of the oligo(dT) provided in the kit. Then, the biotin-labeled cRNA was transcripted in vitro from cDNA by using BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY), and purified by RNeasy Mini Kit. The purified cRNA was fragmented by incubation in fragmentation buffer (40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc) at 95°C for 35 min and chilled on ice. The fragmented labeled cRNA was applied to Human Genome U133A Array (Affymetrix, Santa Clara, CA), which contains 22,215 human gene probes, and hybridized to the probes in the array. After washing and staining, the arrays were scanned. Two independent experiments were performed to verify the reproducibility of results.

Microarray data normalization and analysis
The gene expression levels of samples were normalized and analyzed by using Microarray Suite, MicroDB™, and Data Mining Tool software (Affymetrix, Santa Clara, CA). The absolute call (present, marginal, absent) and average difference of 22,215 gene expressions in a sample, and the absolute call difference, fold change, average difference of gene expressions between two or several samples were normalized and identified using these software. Statistical analysis of the mean expression average difference of genes, which show >2 fold change, was performed using a t test between treated and untreated samples. Clustering and annotation of the gene expression were analyzed by using Cluster and TreeView [20], Onto-Express [21], and GenMAPP [22]. Genes that were not annotated or not easily classified were excluded from the functional clustering analysis.

Real-time RT-PCR analysis for gene expression
To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes (Table 1) with varying expression profiles for real-time RT-PCR analysis. Two micrograms of total RNA from each sample were subjected to reverse transcription using the Superscript first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Real-time PCR reactions were then carried out in a total of 25 µL reaction mixture (2 µl of cDNA, 12.5 µl of 2X SYBR Green PCR Master Mix, 1.5 µl of each 5 µM forward and reverse primers, and 7.5 µl of H 2 O) in SmartCycler II (Cepheid, Sunnyvale, CA). The PCR pro-gram was initiated by 10 min at 95°C before 40 thermal cycles, each of 15 s at 95°C and 1 min at 60°C. Data were analyzed according to the comparative Ct method and were normalized by actin expression in each sample. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product.

Western blot analysis
We also conducted Western Blot analysis to verify the alterations of genes at the level of translation for selected genes with varying expression profiles. The PC3 and LNCaP cells were treated with 1 and 2 nM Taxotere or 50 and 110 µM Furtulon for 24, 48, and 72 hours. After treatment, the cells were lysed in 62.5 mM Tris-HCl and 2% SDS, and protein concentration was measured using BCA protein assay (PIERCE, Rockford, IL). The proteins were subjected to 10% or 14% SDS-PAGE, and electrophoretically transferred to nitrocellulose membrane. The membranes were incubated with anti-cathepsin C (1:200, Santa Cruz, Santa Cruz, CA), anti-p16 (1:200, Santa Cruz,

Cell growth inhibition
MTT assay showed that the treatment of PC3 and LNCaP prostate cancer cells with Taxotere, Furtulon, or lower concentration of Taxotere plus Furtulon resulted in dose and time-dependent inhibition of cell proliferation (Figure 1), demonstrating the inhibitory effect of Taxotere and Furtulon on the growth of PC3 and LNCaP prostate cancer cells.

Regulation of mRNA expression by Taxotere and Furtulon treatment
Microarray analysis showed that the alterations of gene expression were occurred as early as 6 hours of Taxotere and/or Furtulon treatment, and were more evident with longer treatment (Table 2 and 3).
Clustering analysis based on gene function showed downregulation of some genes for cell proliferation and cell cycle progression (cyclin A, cyclin F, CDC2, CDK2, etc), transcription factors (transcription factor A, ATF5, TAF1131L, FOXM1, etc), and oncogenesis (GRO onco- Combination treatment with Taxotere and Furtulon also altered expression of some genes (CDC27, CDK9, p18, IKKα, etc) that showed no change in mono-treatment (Table 4 and 5), suggesting the synergic effects of combination treatment on some genes.
Taxotere and Furtulon also up-regulated some genes (S-100P, ALDH1A3, casein kinase, annexin, etc) responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents (Table 2 and 3). Taxotere and Furtulon also showed differential effects on PC3 cells with alteration of metastasis-related genes and on LNCaP cells with down-regulation of survivin, cyclin B & E, CDC2, CDC25, and specifically AR by Furtulon, suggesting their effects mediated by both AR-independent and dependent pathways ( Table 2 and 3).

Target verification by real-time RT-PCR and western blot
To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes with varying expression profiles for realtime RT-PCR and Western Blot analysis. The results of real-time RT-PCR for these selected genes were in direct agreement with the microarray data ( Figure 2). The same alternations of gene expression were observed by realtime RT-PCR analysis, although the fold change in the expression level was not exactly same between these two different analytical methods. The results of Western Blot analysis were also in direct agreement with the microarray and real-time RT-PCR data ( Figure 3 and our earlier report [11]). These results support the findings obtained from microarray experiments.

Discussion
It has been known that Taxotere binds to microtubules while Capecitabine is incorporated into DNA, inhibiting the bioprocess in cancer cells [4,13]. However, the precise molecular mechanisms for inhibiting cancer cell growth by Taxotere and/or Capecitabine have not been fully elucidated. From gene expression profiles of Taxotere and/or Capecitabine treated prostate cancer cells, we found that these chemotherapeutic agents caused alterations in the expression of many genes related to the control of cell proliferation, apoptosis, transcription, translation, cell signaling, oncogenesis, and angiogenesis (Figure 4), although the cellular target of Taxotere or Capecitabine appears to be different.
It has been well known that CDCs regulate the molecules related to the cell cycle initiation and progression and that cyclins associate with cyclin-dependent protein kinases (CDKs) and CDCs to control the process of cell cycle [23,24]. The CDK inhibitors including p21 WAF1 , p16 INK4A , and p18 INK4C have been demonstrated to arrest the cell cycle and inhibit the growth of cancer cells [23,24]. Our results showed that Cyclins (cyclin A2, cyclin E2, cyclin F, cyclin B1), CDK2, CDC2, and other cell growth promotion genes (pescadillo, spermidine synthase, mitotin) [25][26][27] were down-regulated in Taxotere and/or Furtulon treated prostate cancer cells, while CDK inhibitor p21 WAF1 and other growth inhibitor genes (BTG2, VDUP1, antiproliferative B-cell translocation gene 1) [28,29] were upregulated, suggesting that Taxotere and/or Furtulon inhibited the growth of prostate cancer cells through the arrest of cell cycle and the inhibition of cell proliferation ( Figure  4). The down-regulation of CDC27, CDK9, EGF, and FGF12B, and up-regulation of p16 INK4A and p18 INK4C were also observed in combination treatment but not in monotreatment, suggesting the synergic effect of combination treatment. These observations are novel in Taxotere and/ or Furtulon treated prostate cancer cells.
Induction of apoptosis by chemotherapeutic agents also leads to the inhibition of cancer cell growth. It has been reported that Taxotere is able to induce apoptosis by caspase-3 dependent or independent cell death mechanism [30]. Capecitabine may induce apoptosis through Fas/ FasL or Bax/Bcl-2 pathway [31,32]. From gene expression profile, we found that Taxotere and/or Furtulon increased level of growth arrest and DNA-damage-inducible alpha (GADD45A), GADD45B, p53 regulated PA26 nuclear protein (PA26), and p53-induced protein 11 (PIG11), all of which are related to the induction of apoptotic processes. GADD45A and GADD45B have been known to promote apoptosis and regulate G2/M arrest [33]. PA26 is a target of the p53 tumor suppressor and a member of the GADD family with the properties of inducing apoptosis [34]. PIG11 as a downstream target of p53 is also involved in the apoptotic processes [35]. The combination treatment also showed down-regulation of negative regulator of programmed cell death ICH-1S and Bcl-2-associated transcription factor, which was not occurred in monotreatment. The induction of apoptosis mediated by GADD45A, GADD45B, PA25, and PIG11 could be another molecular mechanism by which Taxotere and/or Furtulon inhibit the growth of prostate cancer cells. We also found that Taxotere and/or Furtulon inhibited the expression of transcription factors (FOXM1, ATF5, TFAM, TAFII31L), translation factors (EIF1A, EIF5A), oncogene (GRO1, GRO3, BRCA1-associated protein, tumor-associated nuclear protein p120), and heat shock protein, and up-regulated the genes for differentiation (prostate differentiation factor). These results are novel, and suggest the beneficial effects of Taxotere and/or Furtulon on the inhibition of cancer cell growth and oncogenesis.
It is important to note that Taxotere and/or Furtulon also up-regulated the expression of some genes which are known to induce cell resistance to chemotherapeutic agents and to favor cell survival. Among these genes, calcium-binding protein S100P has been found to be highly expressed in cells which develop acquired resistance to anti-tumor agents [36]. The overexpression of aldehyde dehydrogenase 1 (ALDH1) has also been detected solely in classical multidrug resistance cancer cells [37,38]. It has been reported that Annexin-I, casein kinase 1, and cispla-tin-resistance associated protein expressions modulate drug resistance in tumor cells [39,40]. The up-regulation of these molecules by Taxotere and/or Furtulon could induce cell resistance to chemotherapeutic agents. Also, Taxotere and/or Furtulon were found to up-regulate the expression of Notch 3, angiopoietin, activating transcription factor 3, which could favor cell survival [41][42][43]. Further in depth mechanistic studies are needed to address these issues. The investigation on overcoming these unbeneficial effects with other agents must be devised, which is ongoing in our laboratory.
Taxotere showed no effect on AR expression while Furtulon down-regulated AR expression in LNCaP cells, suggesting that the combination could be superior in ARpositive cells. The genes altered by Taxotere and/or Furtulon with respect to the control of cell growth, apoptosis, transcription, oncogenesis, and metastasis in androgen insensitive PC3 cells are different from that in androgen sensitive LNCaP cells, suggesting that the effects of Taxotere and Furtulon may be mediated by both ARdependent and independent signaling pathways. We observed up-regulation of tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2, and protease inhibitor 3 in Taxotere and/or Furtulon treated PC3 cells, suggesting that Taxotere and/or Furtulon may exert anti-metastatic effect. However, we also observed increase in the expression of MMP1, MMP9, cathepsin B, uPA, and tPA in Taxotere and Furtulon treated PC3 cells, therefore, more experimental studies are needed to reveal the overall effect of Taxotere and Furtulon on metastatic processes. These results were not observed in androgen sensitive LNCaP cells, suggesting difference in effects that could be mediated through different cell signal transduction pathways.

Conclusions
In conclusion, Taxotere and/or Furtulon directly and indirectly caused changes in the expression of many genes that are critically involved in the control of cell proliferation, apoptosis, transcription, translation, oncogenesis, angiogenesis, metastasis, and drug resistance (Figure 4). These findings could provide molecular information for further investigation on the mechanisms by which Taxotere and Furtulon exerts their pleiotropic effects on prostate cancer cells. These results could also be important in devising mechanism-based targeted therapeutic strategies for prostate cancer, especially in devising combination therapy for drug resistant prostate cancers. However, further in-depth investigations are needed in order to establish cause and effect relationships between these altered genes and therapeutic response in prostate cancer cells.

Competing interests
The author(s) declare that they have no competing interests.
Western Blot analysis showing the altered expression of spe-cific genes in Taxotere and Furtulon treated PC-3 or LNCaP cells