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Blockade of Paclitaxel-Induced Thymidine Phosphorylase Expression Can Accelerate Apoptosis in Human Prostate Cancer Cells

Departments of ¹ Urology, ² Biochemistry and Molecular Medicine, and ³ Pathological Laboratories, Shimane University School of Medicine, Izumo, Japan

	ABSTRACT

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Recently, survival benefit by chemotherapy using paclitaxel (PTX) and the induction of thymidine phosphorylase (TP) by PTX have been reported in several solid tumors. On the other hand, TP confers antiapoptotic effect on tumor cells through inhibition of caspase-8 activation in vitro. On the basis of these previous observations, we hypothesized that (a) TP can be induced after PTX treatment in human prostate cancer (PC) and (b) blockade of PTX-induced TP expression can enhance the apoptotic processes in human PC cells. PTX was used to find TP expression in all eight hormone-refractory PC cases after chemotherapy; however, cleaved caspase-8 was not expressed after chemotherapy in the six hormone-refractory PC cases with strong TP expression. In PC cell lines (PC-3, DU 145, and LNCaP), TP expression after PTX treatment was clearly up-regulated in a dose-dependent manner. Cell viability of PC cell lines treated with PTX and TP antisense was significantly reduced in a time-dependent and dose-dependent manner compared with the PTX treatment alone. Likewise, apoptotic index of PC cells treated with PTX and TP antisense was significantly increased in comparison with PTX alone. After complete blockade of PTX-induced TP translation by TP antisense transfection, cleaved form of caspase-3 and poly(ADP-ribose) polymerase was increased, and this exaggeration of apoptosis also ran parallel with caspase-8 activation in a PTX dose-dependent manner. However, in PC cell lines treated with TP antisense alone, neither caspase-3 nor poly(ADP-ribose) polymerase was cleaved despite caspase-8 activation. These results indicate that PTX-induced TP up-regulation is associated with decreased caspase-8 activation. This study is the first report showing that blockade of PTX-induced TP expression could exaggerate the processing of apoptosis in PC cells treated with PTX. Our results provide preclinical evidence that TP could be a new molecular target for enhancing the potency of PTX-mediated apoptosis in PC cells.

	INTRODUCTION

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Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor and functions as chemotactic and angiogenic molecules (1) . TP reversibly catalyzes the phosphorylation of thymidine to thymine and 2-deoxyribose-1-phosphate. TP also promotes tumor growth and confers resistance on apoptosis independent of angiogenesis, playing a key role in the invasiveness and metastasis of TP-expressing solid tumors (2) . Recently, TP has been reported to inhibit Fas-induced caspase-8 activation, which in turn leads to the loss of mitochondrial membrane potential (3) , and subsequently to prevention of the cytochrome c release, caspase-3 activation, and finally to inhibition of apoptosis (3) . On the other hand, several lines of evidence show that TP is clearly induced in established human cancer cells by taxanes such as paclitaxel (PTX; ref. 4 ).

PTX, first isolated from the bark of the Western yew tree (Taxus brevifolia), is a complex diterpene that is distinct from other antimicrotubule agents in that it binds directly to polymerized tubulin, promoting microtubule assembly and stability (rather than instability) and preventing depolymerization (5) . In clinical trials in which PTX is used in chemotherapy, PTX is generating excitement for the treatment of numerous types of tumors, including several refractory tumors such as ovarian carcinoma, myeloblastic leukemia, and hormone-refractory prostate cancer (HRPC; ref. 6, 7, 8 ).

Induction of apoptosis appears to be the main mechanism behind the antitumor effect of PTX (9 , 10) . Although several proteins involved in the PTX-mediated apoptosis have been identified, the molecular pathways underlying the apoptotic processes associated with PTX are not clearly defined (11, 12, 13) . Understanding how PTX induces apoptosis is crucial to the elucidation of clinical relevance of chemotherapy using PTX. Furthermore, there is no definite link between TP expression and PTX-mediated apoptosis in cases of prostate cancer (PC). This relationship should be addressed to investigate the anticancer effect of PTX. In the present study, we examined TP expression in relation to apoptosis-related protein expression by an immunohistochemical analysis using HRPC samples before and after PTX-based chemotherapy, and human PC cell lines (PC-3, DU 145, and LNCaP) in vitro were used to attempt to elucidate whether TP influences PTX-mediated apoptosis or has cytoprotective function against PTX.

	MATERIALS AND METHODS

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Tissue Samples.
Tissue samples from systematic sextant needle biopsy were collected in the same HRPC patients before and after PTX-based chemotherapy (14) . From a total of 32 patients, eight cases (median age, 73.5; range, 54–80 years; clinical staging, T₄N₀M₁ in five and T₄N₁M₁ in three cases), whose biopsy samples before and after chemotherapy equally contained viable cancer cells within the same target areas, were recruited. Written form of informed consent was obtained from all patients. Tosoh II tPSA assay (Tosoh Medics, South San Francisco, CA) was used to measure serum levels of prostate-specific antigen (PSA) within a week just before and after chemotherapy.

Cell Culture.
The human prostate cancer cell lines PC-3, DU145, and LNCaP were obtained from the American Type of Culture Collection (Manassas, VA) and incubated in F-12K and MEM-E supplemented with 10% fetal bovine serum and 2 mmol/L L-glutamine. The cells were maintained at 37°C in a humidified atmosphere of 5% CO₂/95% air. Culture media were changed every 48 hours.

Immunohistochemistry.
The tissue samples were fixed in 10% buffered formalin (pH 7.0) and processed for embedding in paraffin wax. The envision-peroxidase method (Dako, Carpinteria, CA) was used to perform immunohistochemical staining. Mouse monoclonal antibody against TP (1/100 dilution; Dako), rabbit polyclonal antibodies against cleaved caspase-8 and cleaved caspae-3 (1:1,000 dilution; Sigma, St. Louis, MO), and poly(ADP-ribose) polymerase (PARP) p85 fragment (1:100 dilution; Promega Corp., Madison, WI) were used in this study. The reaction products were visualized using diaminobenzidine (Dako). Normal mouse and rabbit IgG instead of monoclonal and polyclonal antibodies, respectively, served as a negative control. A pathologist not involved in the present study evaluated the immunostaining under an experimental blind condition. The immunohistochemical staining was graded on an arbitrary scale from 0 to 2+; 0 represents negative expression (0–20% positive cells), 1+ represents weakly positive expression (20–50% positive cells), and 2+ represents strongly positive expression (50–100% positive cells). The scale was determined according to the average rate of positive cells in ten random fields of all slides.

Morpholino Antisense Oligonucleotide Transfection.
A specific morpholino antisense oligonucleotide (5'-GGTCATCAAGGCTGCCATCGCTCCG-3') was designed complementary to the TP mRNA based on the entire cDNA sequence in the GenBank database (Genbank accession no. NM001953). Antisense containing 5 bp mismatch pairs (5'-GGTTATCAAAGCTACCGTCGCTTCG-3') was used as a control sense oligonucleotide. For the oligonucleotide treatment of each cell line, a special delivery system following the manufacturer’s protocol (Funakoshi, Tokyo, Japan) was used to transfect cells. The specificity (the degree of cross-hybridization with the entire sequence) was confirmed by Western blot analysis.

Growth Inhibition Assay.
Cells treated with or without TP antisense were cultured for 24 hours in 6-well plates at a density of 5 x 10⁵ cells/well in growth medium. Media were then changed to growth media containing PTX (1 x 10^–9 to 1 x 10^–7 mol/L). Cells were harvested with trypsin, and a hemocytometer was used to count them on days 2, 3, and 4. Experiments were done in quadruplicate for each time point.

MTT Assay.
Cells were grown at 5 x 10³ in the culture medium containing 100 µL of serum dispensed into 96-well microplates. After treatment with chemical agents, 10 µL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml; Sigma) was added to each well, and the plates were incubated at 37°C for 4 hours. A measurement wavelength of 450 nm and a reference wavelength of 655 nm was used to read absorbance on a microplate reader (Model 3550, Bio-Rad, Richmond, CA). IC₅₀ was defined as the PTX concentration that inhibited cell growth by 50% compared with control cells, according to the average cell cycle length. IC₅₀ values were calculated from a linear regression analysis of plotted values.

Evaluation of Apoptosis.
The APOPercentage Apoptosis Assay kit (Biocolor Ltd. United Kingdom) was used to examine alteration in the apoptosis membrane. Briefly, cells were seeded at 5 x 10³ in medium containing 100 µL of serum that was dispensed into 96-well microplates. After treatment with 1 x 10^–9 PTX for 48 hours, the culture medium was replaced with fresh medium containing APOPercentage Dye Label. The APOP% Dye Release Reagent was added to each well to aid cell lyses and the release of the bound dye from the apoptotic cells. A microplate colorimeter was used to measure cell-bound dye recovered in solution. A reference wavelength of 655 nm was used to estimate the apoptotic index at a measurement wavelength of 595 nm.

Preparation of Cell Lysates.
After drug treatment for 48 hours, cells were harvested with 0.02% trypsin, centrifuged, and the cell pellets immediately frozen at –80°C until use. Frozen tumor cells were homogenized in lysis buffer [1% Triton X-100; 20 mmol/L Tris-HCl (pH 7.6); 0.1% SDS; 1% sodium deoxycholate], lysates were centrifuged at 15,000 x g for 20 minutes at 4°C, and each supernatant was used in the immunoblotting analysis. Protein concentrations were determined by the Bradford method (15) .

Immunoblotting.
Samples were resolved by 11% SDS-PAGE. A Bio-Rad Transblot SD was used to electrophoretically transfer proteins to nitrocellulose membranes (Millipore Corp., Bedford, MA). The membranes were immersed in 5% skim milk in 0.02 mol/L Tris-HCl, 0.4 mol/L NaCl (pH 8.0), and 0.05% Tween 20 buffer (TTBS) for 1 hour and probed with monoclonal antibody against TP, caspase-8, and ß-actin or polyclonal rabbit antibody against caspase-3, PARP, and cleaved PARP. Blots were then labeled with antimouse or antirabbit antibody conjugated with peroxidase; an enhanced chemiluminescence and Western blotting was used for visualization. The software package NIH was used to quantify Image Signal intensities. The protein expression level was depicted as the relative yield to the ß-actin level.

Statistical Analysis.
The difference between two groups was statistically analyzed by Mann-Whitney U test, or the Stat View V statistical package (SAS Institute Inc., Cary, NC) was used for Student’s t test. The P value of <0.05 was regarded as statistically significant.

	RESULTS

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Immunostaining of TP and Apoptosis-Related Proteins in HRPC Specimens.
PTX on the expression of TP and apoptosis-related cleaved form of caspase-8, capase-3, and PARP was used to immunostain eight HRPC samples and assess the effect of chemotherapy. Typical immunostaining of TP, cleaved form of caspase-8, caspase-3, and PARP before and after chemotherapy was shown in Fig. 1 . PTX in four HRPC cases (cases 1, 3, 4, and 5) was used to increase TP expression after chemotherapy, whereas the remaining four cases (cases 2, 6, 7, and 8) showed no difference in TP expression before and after chemotherapy. In all eight HRPC samples, cleaved forms of caspase-3 and PARP were not detectable before chemotherapy, whereas they were detectable after chemotherapy. In six HRPC samples (cases 3–8), cleaved form of caspase-8 expression was not detectable either before or after chemotherapy. On the other hand, the remaining two HRPC samples (cases 1 and 2) showed increased expression of cleaved caspase-8 after chemotherapy. In these 2 HRPC samples, cleaved form of caspase-3 and PARP expression was scored as 2+ (strongly positive expression) after chemotherapy.

View larger version (47K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Representative immunostaining of TP (A, B), cleaved caspase-8 (C, D), cleaved caspase-3 (E, F), and cleaved PARP (G, H) in the prostate biopsy specimens having viable PC cells (x400) are shown. A, C, E, and G, before chemotherapy with PTX. PC cells in the tumor tissues that comprise small glandular structures are almost completely negative for TP (A), cleaved caspase-8 (C), cleaved caspase-3 (E), and cleaved PARP (G). All of these expressions were scored as 0. B, D, F and H, after chemotherapy with PTX. Small subsets of PC cells and stromal cells in the tumor tissues are weakly positive for TP (B) and cleaved caspase-8 (D) and are strongly positive for cleaved caspase-3 (F) and cleaved PARP (H). These expressions were scored as 1+, 1+, 2+ and 2+, respectively. I, summary of immunostaining data and PSA alteration before and after chemotherapy with PTX.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Relationship of TP expression with PSA value and PSA reduction rate before and after chemotherapy with PTX in eight HRPC cases are shown. A and B show the alteration of serum PSA levels before and after chemotherapy with PTX. Group 1 includes four patients with increased TP expression after chemotherapy with PTX, whereas group 2 comprises the other four patients with TP expression being unchanged before and after chemotherapy. The difference in PSA reduction rate between Groups 1 and 2 is shown in C. Statistical significance was analyzed with Mann-Whitney U test. The values are expressed as mean ± SE.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Cell viability of PC cell lines treated with PTX and/or antisense TP is shown. A, B, and C show the dose-response curves of PC-3, DU 145, and LNCaP, respectively, and cells treated () or untreated () with antisense oligonucleotide for TP mRNA (5'-GGTCATCAAGGCTGCCATCGCTCCG-3') after exposure of PTX at five different concentrations (1 x 10–9, 1 x 10–8, 5 x 10–8, 1 x 10–7, and 1 x 10–6 mol/L) for 24 hours. Viable cells were measured by MTT assay and expressed as a percentage of the controls. Asterisk in Fig. 3A–C indicates the statistical difference in cell viability between TP antisense (–) and TP antisense (+) treated cell lines (P < 0.05). D, E, and F show the time-response curves of PC-3, DU145, and LNCaP cell lines, respectively. Cell viability of control nontreated PC cell lines (), PC cell lines treated with TP antisense alone (), treated with 1 x 10–9 mol/L PTX alone (), and treated with a combination of both TP antisense and 1 x 10–9 mol/L PTX () for 24, 48, and 72 hours is shown. Asterisks, * and **, in Fig. 3D–F indicate the statistical difference in cell viability between control and PTX or between control and combination of PTX and TP antisense (P < 0.05and P < 0.01, respectively). # in Fig. 3D–F shows the statistical difference in cell viability between PTX and combination of PTX and TP antisense (P < 0.05). The values are expressed as mean ± SE at each point.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Alteration of apoptotic index in PC cell lines with different treatment modalities is shown. Apoptotic indices of each group are expressed as its relative proportion to the untreated control (mean ± SE). Statistical significance was evaluated by student’s t test. The difference in apoptotic index between PTX alone versus PTX + TP antisense, control versus PTX alone, and control versus PTX + TP antisense reached statistical significance (P < 0.05, P < 0.05, and P < 0.01, respectively).

View larger version (77K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Typical results of Western blotting of TP and apoptosis-related proteins in PC cell lines treated with different concentrations of PTX and/or TP antisense are shown. Protein levels of TP, procaspase-8, -3, cleaved caspase-8, -3, PARP, cleaved PARP and ß-actin in PC-3, DU 145, and LNCaP cells untreated (A) or treated (B) with antisense oligonucleotide for TP mRNA. These cells were exposed to various concentrations of PTX (1 x 10–9 to 1 x 10–7 mol/L) for 48 hours. Different antibodies were used to apply total protein (100 µg) to Western blotting. The software package NIH Image was used to quantify signal intensities. Protein expression levels were depicted as their relative yield to the ß-actin level.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. TP expression after treatment with PTX (A), and cleaved caspase-8 expression after combined treatment with PTX and TP antisense (B) in PC cell lines are shown.

	DISCUSSION

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Next, to verify the mechanism underlying PTX-induced apoptosis in relation to simultaneous TP expression, we focused on the molecular pathway involved in the apoptotic process. Two major apoptotic pathways are known in mammalian cells. One is the Fas-induced caspase-8 activation pathway, and the other is the mitochondrial pathway. Although these two apoptotic pathways operate independently, they converge at the level of caspase-3 activation (17) . PTX-induced apoptosis has also been implicated in caspase-8 activation in breast and colon cancer cell lines (18 , 19) . TP has been reported to inhibit Fas-induced caspase-8 cleavage followed by the release of cytochrome c, the activation of caspase-3, and the apoptosis (3) . However, no reports have shown a positive link between apoptotic pathways involved in PTX-induced TP expression and caspase-8 activation in PC cells. In breast and colon cancer cell lines, PTX induces proapoptotic effect on cancer cells through caspase-8 activation in addition to the activation of mitochondrial membrane potential (18 , 19) . However, as shown in Fig. 5 , PTX-induced apoptosis in PC cell lines seems to be independent of caspase-8 activation. Thus, the mechanism underlying the antitumor effect of PTX on cancer cells appears to be potentially varied among cancer cells of different origins. On the basis of the present finding of dose-dependent TP induction by PTX treatment in PC cell lines as well as the previous report of potential inhibitory effect of TP on caspase-8 activation (3) , we hypothesized that blockade of inhibitory effect of TP on caspase-8 activation could enhance the PTX-induced apoptosis in PC cells. In all three PC cell lines, after complete blockade of TP translation by TP antisense transfection, proapoptotic events such as cleaved form of caspase-3 and PARP were enhanced in a PTX dose-dependent manner. In addition, this exaggeration of apoptosis also ran parallel with the caspase-8 cleavage in a PTX dose-dependent manner. On the other hand, in all of three PC cell lines TP blockade itself did not confer any effects on the acceleration of apoptosis despite caspase-8 activation. These results suggest that cross-talk between caspase-3 activation through mitochondrial membrane potential and direct effect of caspase-8 activation pathway on cytochrome c release can modulate proapoptotic effect of PTX as a chemotherapeutic agent on PC cells. In turn, we can expect more antitumor apoptotic effect of PTX on PC cells with an inhibition of "adverse" effect of PTX-induced TP overexpression.

To our knowledge, this study is the first report to investigate the induction of TP expression by PTX and to present the possibility that overexpressed TP might be related to the potential decrease in caspase-8 cleavage in PC cell lines. Our results support the hypothesis that TP could be a new molecular target for enhancing the potency of PTX-mediated apoptosis in PC cells. Therefore, it is necessary to perform the clinical trial after treatment with a combination of TP antisense and PTX in the future.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. M. Fukushima (Institute for Applied Oncology, Taiho Pharmaceutical Co., Ltd., Saitama, Japan) for encouraging us throughout this study.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Mikio Igawa, MD, PhD., Professor and Chairman, Department of Urology, Shimane University School of Medicine, 89–1 Enya-cho, Izumo 693-8501. Phone: 81-853-20-2251; Fax: 81-853-20-2250; E-mail: igawam{at}med.shimane-u.ac.jp

Received 4/15/04. Revised 7/11/04. Accepted 8/16/04.

	REFERENCES

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