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Keywords:

  • Prostate cancer;
  • S-1;
  • docetaxel;
  • thymidylate synthase;
  • castration resistant

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

S-1 is a recently developed dihydropyrimidine dehydrogenase inhibitor fluoropyrimidine and has demonstrated high maximum plasma 5-Fluorouracil (5-FU) levels with mild toxicity, and an oral formulation has resulted in an improvement in patient quality of life. The aims of the present study were to determine the efficacy of S-1 or S-1 combined with docetaxel (DOC) using castration resistant prostate cancer (CRPC) cells and to explore their clinical potential for treating CRPC patients. LNCaP cells, androgen dependent prostate cancer (ADPC) cells and C4-2 cells, which are a CRPC subline of LNCaP cells, were used. Specimens obtained from ADPC and CRPC patients were also evaluated. The CRPC specimens and C4-2 cells exhibited significantly lower thymidylate synthase (TS) expression, a target of 5-FU, than the ADPC specimens and LNCaP cells. In vitro, C4-2 cells exhibited higher sensitivity to 5-FU than LNCaP cells. In C4-2 xenograft model, S-1 monotherapy suppressed tumor growth and low-dose DOC enhanced the anti-tumor effect of S-1. In vitro, low-dose DOC, which did not induce G2/M arrest, increased p53 and p21 and resulted in down-regulation of TS in C4-2 cells, and down-regulation of TS is considered to be responsible for the synergistic effect of S-1 in vivo. The present findings indicate that CRPC patients with androgen ablation may be good candidates for 5-FU based chemotherapy, and these regimens have attractive therapeutic potential for clinical practice, and they may have a significant impact on therapeutic options.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

Prostate cancer is the most common cancer and the second leading cause of cancer death in men in the United States.1 Androgen ablation is the standard treatment for patients with advanced prostate cancer and controls the disease temporarily; however, many patients will ultimately become refractory to androgen ablation and suffer disease progression. Various methods to treat castration resistant prostate cancer (CRPC) have been attempted, including chemotherapy.

Chemotherapy was previously thought to have an insignificant role because of low response rates; however, recently a new novel role has emerged for systemic chemotherapy based on docetaxel (DOC) as reported in two landmark phase III studies, TAX3272 and SWOG9916.3 However, because toxicity at the dose of DOC (75 mg/m2), which is the usual dose for CRPC patients, is significant, DOC may not be the preferred option to treat elderly or far advanced patients. Furthermore, no standard therapy is available for patients who have disease progression after DOC treatment.

5-Fluorouracil (5-FU), an antitumor pyrimidine, has frequently been used clinically to treat patients with various types of cancers. 5-FU is converted to FdUMP and this inhibits thymidylate synthase (TS), which is the enzyme that catalyzes de novo synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP) from 2′-deoxyuridine-5′-monophosphate (dUMP), by forming a stable ternary complex with methylene tetrahydrofolate, and the ternary complex leads to the inhibition of DNA synthesis.4 Several studies in other types of cancer have shown that TS activity was greater in cancerous tissue specimens than in normal tissue samples and that the TS activity level was correlated with stage progression.5, 6 In general, high TS expression is a predictor of a low response to 5-FU cytotoxic therapy.7 Several studies have indicated that TS expression can help to predict the clinical outcome and the response to 5-FU cytotoxic therapy.8

The response-limiting factor for 5-FU is plasma 5-FU levels. Continuous infusion is necessary to obtain higher responses due to the rapid catalysis of 5-FU by dihydropyrimidine dehydrogenase (DPD) in the liver. DPD-inhibitory fluoropyrimidine (DIF) was developed to resolve the problem of rapid reduction of 5-FU by DPD. S-1 is a new oral formulation of DIF recently developed in Japan. S-1 consists of three drugs, tegafur (FT), 5-chloro-2,4-dihydroxypyrimidine (CDHP; gimeracil) and potassium oxonate (OXO; oteracil) at a molar ratio of 1: 0.4: 1. FT is a prodrug of 5-FU and is gradually converted to 5-FU by cytochrome p450 enzymes in liver. CDHP is a potent inhibitor of DPD. Gastrointestinal (GI) toxicities such as diarrhea and mucositis are dose-limiting factors associated with the use of 5-FU. OXO is a reversible competitive inhibitor of orotate phosphoribosy1 transferase (OPRT), an enzyme that is responsible for GI toxicity via its phosphorylation of 5-FU. Therefore, the oral administration of S-1 can achieve a more potent antitumor effect through an increased 5-FU concentration without any enhancement of GI toxicity. S-1 has recently been adopted as a front line chemotherapeutic regimen for the treatment of gastric cancer in Japan, based on the findings of several randomized trials.9–11 Furthermore, S-1 has been also identified as an effective agent for the treatment of colorectal,12 head and neck,13 breast,14 non-small-cell lung,15 biliary tract16 and pancreatic cancers.17 5-FU is not used in patients with prostate cancer because of its poor efficacy and severe side effects.18–20 The therapeutic effect of S-1 for treating prostate cancer is still not fully understood.

Combining different anticancer agents is a reasonable strategy with which to obtain a potent cytotoxic effect in cancer cells. In this respect, S-1 could offer an interesting option. For example, clinical trials involving a regimen of S-1 combined with low-dose DOC to treat gastric cancer,21 lung cancer and breast cancer are currently ongoing.

In our study, we investigated the efficacy of S-1 monotherapy and S-1 combined with a low dose of DOC, as well as the detailed mechanism by which combined S-1 and DOC could affect TS activity.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

Clinical samples and clinicopathological features

Archival paraffin-embedded sections were obtained from 11 patients who underwent retropubic radical prostatectomy for localized prostate cancer between January and December 2002. None of the patients had received hormonal treatment before the operation. The average age of the patients at the time of surgery was 66.4 ± 1.2 yr. The Gleason score was 7 in all patients. According to the TNM system, there were five patients in clinical stage II and six in stage III.

Patients with CRPC often show local progression and then suffer from urinary obstruction due to tumor growth. In these patients, palliative transurethral resection (TUR) of the tumor often helps to regain normal voiding. In our study, nine TUR prostate specimens were also selected from the archival paraffin-embedded sections of patients with CRPC who underwent TUR. The backgrounds of the patients differed depending on the transition of the trends of treatment; however, all patients received surgical or medical castration with or without antiandrogen, and PSA rose again during hormone treatment. The average age of these patients was 76.2 ± 2.3 yr. The Gleason score of all nine samples was more than 8.

Cell lines and agents

The androgen-dependent prostatic carcinoma cell line, LNCaP cells, an androgen-independent LNCaP subline, C4-2 cells and androgen-independent PC3 cells were used in our study.22 These cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Dainippon Pharmaceutical, Tokyo). To examine the androgen-ablated treatment, C4-2 cells were grown with 10% charcoal-stripped FBS.

5-FU was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Nocodazole, fascaplysin and monoclonal anti-β actin antibody were purchased from Sigma Aldrich Japan (Tokyo). Polyclonal anti-p16 and polyclonal anti-p53 antibody were obtained from Santa Crutz Biotechnology, Inc (CA, USA). Monoclonal anti-p21 and monoclonal anti-Ki67 antibody were obtained from Millipore Japan (Tokyo) and Dako Japan (Tokyo), respectively. S-1 and polyclonal anti-TS antibody were generously supplied by Taiho Pharmaceutical Co, Ltd (Tokyo) and DOC was provided by Sanofi-Aventis (Tokyo).

Immunohistochemistry

Immunohistochemistry was performed as previously described.23–25 Primary antibody (polyclonal anti-TS antibody and monoclonal anti-Ki67 antibody at a dilution of 1:250) was applied, and they were incubated with secondary antibodies conjugated to peroxidase-labeled dextran polymer. Visualization of the immunoreaction was performed with diaminobenzidine (DAB). Counterstaining was conducted with 10% hematoxylin.

Microscopic images of representative places were scanned with a digital microscope, and the staining intensities of cancer cells and background (tissue free area) for each image were quantified automatically using NIH Image J software. The staining intensities of TS were calculated by subtracting the background from the cancer cells. The mean staining intensity for TS was assessed in ten different microscopic fields of all sections from each group, and staining intensity was compared to the counterparts. A dark accumulation of DAB in the nuclei was judged to indicate a positive reaction for Ki67. The percentage of cancer cells with nuclei stained for Ki67, that is, the Ki67 index, was calculated for each section on the basis of over 1,000 cancer cell nuclei.

Murine xenograft prostate cancer model

SCID athymic mice (6 weeks old) were obtained from CLEA Japan, Inc (Tokyo). The mice were castrated by a scrotal incision under general anesthesia with pentobarbital and were used to create a xenograft model with C4-2 cells. C4-2 cells (5 × 106 cells), suspended in 100 μl of Matrigel (Becton Dickinson Labware, Lincoln Park, NJ) and were implanted subcutaneously in one flank of each mice. From 8 to 12 mice were used in each group to study the high and low-dose combinations and from 5 to 7 mice were used in each group for the immunohistochemical staining of TS. Tumor volume was calculated using the following formula: tumor volume (mm3) = length × width × height × 0.5. The treatments were started when the average tumor volume had reached over 300 mm3, and we converted the tumor volumes to a percentage relative to the starting volume in each individual. The mice were administered S-1 daily by gavage and/or DOC on the first day by intraperitoneal infusion. In clinical practice for the treatment of prostate cancer patients, DOC is administered intravenously. Shimada et al.26 compared the pharmacokinetic behavior of DOC after intravenous and intraperitoneal administration in mice.26 The values of the plasma area under concentration-time curves were similar for both administration routes. They concluded that intravenous and intraperitoneal administration were not the same, although the difference between them in mouse model was not remarkable. After a predetermined period of time, the animals were killed, and the subcutaneous tumors were harvested. All procedures were performed according to Japanese government guidelines, and the protocol was approved by the Animal Care Committee of Keio University.

Real-time quantitative PCR

Cell lysates and cDNAs were extracted using a TaqMan Fast Cells-to-CT Kit (Applied Biosystems, CA, USA), according to the manufacturer's protocol. The reaction mixture was then used as a template in a TaqMan Fast real-time quantitative PCR assay using a StepOne real time PCR System (Applied Biosystems). The primers and TaqMan probe sets (TaqMan Gene Expression Assays, Inventoried) for TS (Hs00426591_m1) and human β actin endogenous control (Hs99999903_m1) were purchased from Applied Biosystems (sequences not disclosed). The TS to β actin mRNA ratios were calculated for each sample to determine the relative mRNA expression.

Cell lysate preparation and Western blotting analysis

Whole protein was isolated using RIPA buffer (Cell Signaling Technology Japan, Tokyo). Western blot analysis was performed as previously described.27 Briefly, 50 μg of total protein was boiled for 5 min with SDS-PAGE sample buffer (50 mmol/L Tris-HCL, 2% SDS, 0.01% bromophenol blue, 10% glycerol and 5% 2-mercaptoethanol), and samples were subjected to electrophoresis on 12.5% SDS-polyacrylamide gels. Proteins were transferred onto a polyvinylidene difluoride membrane in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween 20). The primary antibodies for TS, p53, p21 and p16 at a dilution of 1:1000 were reacted. Anti-β actin antibody was used as an internal control. Then membrane was incubated with species-specific secondary antibodies and immunodetection was performed using an ECL Plus Western Blotting Detection System (GE Healthcare Japan, Tokyo).

Cell growth assay

Cells were seeded at a density of 5 × 103 cells per well into 96-well culture plates. Cell viability was determined using a Premix WST-1 Cell Proliferation Assay System (Takara Bio Inc, Shiga, Japan) to examine the cytotoxic effect of each agent. The absorbance value of each well was determined in a microplate spectrophotometer (Bio-Rad Laboratories, Inc, Tokyo).

Cell cycle analysis

Flow cytometric analysis was performed as previously described.28 C4-2 cells stained with FITC-labeled BrdU and propidium iodide were analyzed by flow cytometry (Beckman Coulter, Fullerton, CA, USA).

Statistical analysis

Statistical analyses were performed using the Mann-Whitney U test. p-values <0.05 were considered to be statistically significant. Combination index (CI) analysis provided a qualitative measure of the extent of drug interaction.29 CI values of less than 1, equal to 1 and greater than 1 indicate synergy, additive and antagonism, respectively.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

TS expression in clinical samples and prostate cancer cell lines

We confirmed the expression of TS in human samples by immunohistochemistry (Fig. 1a). Almost all cancer cells in surgical specimens from localized prostate cancer showed high levels of TS expression. In contrast with the surgical specimens, many cells with no or only low expression levels of TS expression were observed in the TUR specimens from patients with CRPC who received androgen ablation therapy. Overall, TUR specimens with CRPC showed significantly lower TS expression than surgical specimens (0.58 ± 0.15 compared to surgical specimens, p < 0.05, Fig. 1b).

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Figure 1. TS expressions in prostate cancer specimens, and TS expressions and chemosensitivities to 5-FU in prostate cancer cells. (a) Immunohistochemical staining of surgical specimens with ADPC and TUR specimens with CRPC. Magnification, x200. (b) Intensities of TS expression in human specimens by dot plot. Staining intensities of TS for each image were quantified with NIH Image J software. *p < 0.05 compared to ADPC. (c) Real time PCR analysis of prostate cancer cells. LNCaP cells were supplemented with 10% FBS, and C4-2 cells with 10% charcoal-stripped FBS.

(d) Western blotting analysis of prostate cancer cells. Both cells were cultured under the same conditions as described in c. (e) Effect of 5-FU on cytotoxicity in prostate cancer cells by WST-1 assay. Points/columns, mean from at least three individual experiments; bars, +SD. *: p < 0.05 compared to LNCaP.

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In vitro, the androgen-independent subline C4-2 with androgen-ablated treatment showed significantly lower TS mRNA (44.4 ± 2.2% in C4-2, compared to LNCaP) and protein expression than LNCaP supplemented with 10% FBS by real time PCR and Western blotting, respectively (Figs. 1c and 1d). This finding was consistent with human samples.

5-FU chemosensitivity in prostate cancer cell lines

LNCaP and C4-2 were incubated with various concentrations of 5-FU to examine the effects of 5-FU on sensitivity (Fig. 1e). After 48 hr of incubation, 5-FU inhibited cell growth in a dose-dependent manner in both cells, and the IC50 values at 48 hr of 5-FU were 68.5 and 17.7 μM in LNCaP and C4-2 cells, respectively. C4-2 with relatively low TS expression was more sensitive to 5-FU than LNCaP.

Effects of high dose of S-1, DOC, and combined S-1/DOC on C4-2 tumor growth in SCID mice

S-1 contains FT, CDHP and OXO. Based on their respective biological actions, experiments to evaluate the efficacy of S-1 should be evaluated in vivo not in vitro.

To investigate the effects of S-1 and DOC, we orally administered S-1 and intraperitoneal injections of DOC to SCID mice bearing a C4-2 tumor. At first, we started the treatment when the average tumor volume had reached about 100 mm3. With the high dose regimens (S-1: 5 mg/kg daily oral administration, DOC: 10 mg/kg intra-peritoneal injection on the first day, combination: S-1 plus DOC), the tumors had completely disappeared with DOC alone and combined S-1/DOC (data not shown), so we could not evaluate the synergistic interference on combination. Therefore, the treatment was started at a high dose again when the average tumor volume had reached over 300 mm3, followed 3 weeks later by inoculation of C4-2 cells (Fig. 2a). The tumor volume was 72.6 ± 14.0%, 53.0 ± 7.8%, and 22.9 ± 7.7% of control for S-1 monotherapy, DOC monotherapy and high dose combined S-1/DOC at 16 days, respectively (Fig. 2a). The combination therapy resulted in a dramatic decrease in the volume of subcutaneous tumors compared to the control as well as with the S-1 and DOC monotherapies (p < 0.05). S-1 monotherapy and DOC monotherapy were also significantly effective compared to control (p < 0.05).

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Figure 2. Effect of DOC and S-1 on tumor growth in a mouse xenograft model of C4-2 from the day tumor volume reached 300 mm3 and for 16 days. Mice (n = 8–12 in each group) were treated with DOC alone, S-1 alone, their combination, or vehicle control. (a), (b). Tumor volumes at high dose (a) and low dose (b). Points, mean tumor volume relative to the tumor volume of starting point (%); bars, +SE. *p < 0.05 compared to control group. †p < 0.05 compared to combination group. (c), (d). Photo panels of Ki67 immunostaining of xenograft tumors at high dose (c) and low dose (d). Magnification, x200. (e) Ki67 indexes of Ki67 immunostaining of xenograft tumors at high dose and low dose.

Columns, mean Ki67 index; bars, +SD. *p < 0.05 compared to control group. †p < 0.05 compared to combination group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Effects of low dose of S-1, DOC and combined S-1/DOC on C4-2 tumor growth in SCID mice

In light of the significant effect observed with a high dose of combined S-1/DOC, we examined whether the doses of S-1 and DOC could be reduced. The doses adjusted for humans were 3 mg/kg of S-1 daily and 2 mg/kg of DOC on the first day (Fig. 2b). The tumor volume was 76.1 ± 5.2%, 92.0 ± 4.2%, and 52.1 ± 2.4% of control for S-1 monotherapy, DOC monotherapy and combined S-1/DOC, respectively (Fig. 2b). The combination therapy exhibited marked suppression of tumor growth compared to control, S-1 monotherapy, and DOC monotherapy after 16 days of treatment (p < 0.05). Tumor volume with S-1 monotherapy was significantly different from control (p < 0.05). However, tumor volume was not significantly different between DOC monotherapy and control throughout the treatment period (p = 0.890). Both the high- and low-dose treatments were well tolerated, leading to no body weight loss compared to controls.

Ki67 immunostaining of xenograft tumors at high and low doses

The Ki67 index of the tumors at the high dose was significantly decreased in the S-1 (21.4 ± 5.5%, p < 0.05) and DOC (18.8 ± 4.5%, p < 0.05) monotherapies and the S-1/DOC combination therapy (10.2 ± 4.5%, p < 0.05) compared to the control (35.2 ± 6.9%, Figs. 2c and 2e). The Ki67 index of the combination therapy differed significantly from that of the S-1 and DOC monotherapies (p < 0.05). The Ki67 index of the tumors at the low doses was significantly decreased in the S-1 monotherapy (18.7 ± 3.0%, p < 0.05) and combination therapy (8.81 ± 3.2%, p < 0.05) compared to the control (26.8 ± 1.8%) but not in the DOC monotherapy (22.9 ± 3.6%, p = 0.06, Figs. 2d and 2e). The Ki67 index in the combination therapy differed significantly from that in the S-1 monotherapy and DOC monotherapy (p < 0.05). The Ki67 indexes were closely correlated with tumor volume at both doses.

Combination of 5-FU and DOC in C4-2 cells in vitro

We next evaluated the combined effect of 5-FU and DOC on cell viability in C4-2 cells. Figure 3a shows the viability of cells exposed to the drugs, which indicates the maximal synergistic effect in the various combination series used in the experiments. The mean cell viability values at 48 hr of treatment with 5 μM 5-FU alone, 2 nM DOC alone and combined 5-FU/DOC were 85.9 ± 6.3%, 78.5 ± 5.8% and 58.9 ± 4.3%, respectively. The CI values of the combined treatment were 0.702 ± 0.045 (IC20, 30, 40 and 50 of DOC in C4-2 were 1.95, 2.54, 3.06 and 3.58 nM, respectively). Because the CI values were less than 1, the combination of 5-FU and DOC had a synergistic effect in vitro. We also evaluated the effect of combined therapy in other cell lines. In LNCaP cells, the mean cell viability values at 48 hr of treatment with 10 μM 5-FU alone, 5 nM DOC alone and combined 5-FU/DOC were 72.6 ± 6.1%, 69.2 ± 7.8% and 45.3 ± 7.3%, respectively (Fig. 3b). In PC3 cells, the mean cell viability values at 48 hr of treatment with 2.5 μM 5-FU alone, 10 nM DOC alone, and combined 5-FU/DOC were 84.1 ± 6.4%, 78.0 ± 6.3% and 65.0 ± 5.9%, respectively (Fig. 3c). The CI values were 0.677 ± 0.086 and 0.622 ± 0.030 in LNCaP and PC3, respectively (The IC50 of 5-FU was 19.5 μM in PC3 cells, and the IC50s of DOC were 11.6 nM and 68.0 nM in LNCaP and PC3 cells, respectively); therefore, a synergistic effect of combined therapy was observed in other prostate cancer cell lines.

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Figure 3. (a), (b), (c) Cytotoxic effects of combination therapy on C4-2 (a), LNCaP (b) and PC3 (c) by WST-1 assay. Columns, mean from at least three individual experiments; bars, +SD.*p < 0.05 compared to control group. †p < 0.05 compared to combination group. (d), (e), (f). Alteration of TS protein expression of combination therapy in C4-2 (d), LNCaP (e) and PC3 (f) by Western blotting analysis.

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Modulation of TS expression by 5-FU and DOC in vitro

To further study the mechanisms of the synergistic effects of 5-FU and DOC, we examined the possible changes in the expression levels of TS. C4-2 cells were treated with 2 nM DOC or 5 μM 5-FU alone and both in combination for 48 hr. Western blotting is capable of identifying both free TS and ternary complex using anti-TS antibody.30, 31 It was identified as a single band of 37 kDa adjusted for the estimated size for free TS in control, and 5-FU treatment showed an additional band in C4-2 cells (Fig. 3d). The shifted band was considered to represent the formation of a stable ternary complex of TS protein, 5-FU and folate, as described earlier.32 DOC treatment decreased free TS to 61.9% of control, while 5-FU treatment increased total TS (free plus ternary complex) to 223.4% of control. Combined 5-FU and DOC treatment decreased free TS to 69.2% and total TS to 37.3% compared to 5-FU alone. Briefly, 5-FU enhanced TS expression, DOC suppressed the expression of TS and combined 5-FU/DOC suppressed the TS (total or free), which was enhanced by 5-FU. These findings indicated that treatment with DOC resulted in down-regulation of TS with or without 5-FU in C4-2 cells. We also evaluated the modulation of TS expression by DOC, 5-FU and this combination in LNCaP and PC3 and observed the same tendency in both cells (Figs. 3e and 3f).

Effects of low-dose DOC on TS expression

DOC is an antimicrotubule disassembly agent, and this effect leads to an arrest in the G2/M phase of the cell cycle and eventually to apoptotic cell death. We next examined whether DOC regulates TS with or without cell cycle distribution.

We analyzed the cell cycle of C4-2 treated with 10–50 nM DOC for 24–48 hr by flow cytometry. Cells accumulated in the G2/M phase of the cell cycle and severe cytotoxicity was observed (data not shown). It is commonly believed that TS is a G1 to S phase dependent enzyme. To confirm whether G2/M arrest induced the redistribution of cells and resulted in down-regulation of TS, we examined the cell cycle effect on TS protein level using nocodazole, which interferes with the polymerization of microtubules and induces G2/M arrest. A total of 25 or 50 ng/ml nocodazole for 18 hr significantly induced G2/M arrest (Fig. 4a), but under the same conditions, nocodazole did not alter TS mRNA (data not shown) or protein levels (Fig. 4b). These data indicated that TS protein level is not controlled by cell cycle modulation by DOC in C4-2 cells.

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Figure 4. Efficacy of G2/M cell cycle arrest by nocodazole on TS expression. (a) Flow cytometric analysis of 25 or 50 ng/ml nocodazole for 18 hr in C4-2 cells. (b) Western blotting of TS on 25 or 50 ng/ml nocodazole for 18 hr in C4-2 cells.

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We next evaluated the doses of DOC, which do not cause cells to accumulate in G2/M phase. The low dose of DOC was confirmed by flow cytometry (1–2 nM of DOC for 48 hr, Fig. 5a). The estimated cytotoxicities of 1 nM and 2 nM DOC were 2.2% and 20.5%, respectively. The mRNA levels of TS were evaluated in C4-2 cells at low doses of DOC (1 and 2 nM), and these were down regulated in a dose-dependent manner (p < 0.05, Fig. 5b). A total of 1 nM and 2 nM DOC down regulated TS mRNA to 77.2 ± 2.2% and 58.4 ± 2.2%, respectively, of control in C4-2 cells. TS protein levels were also decreased at the same doses (Fig. 5c).

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Figure 5. Efficacy of low-dose DOC treatment on TS expression and cell cycle related proteins in C4-2 cells. (a) Flow cytometric analysis of 1 or 2 nM DOC for 48 hr. (b) Real time PCR of TS in 1 or 2 nM DOC for 48 hr. Columns, mean from at least three individual experiments; bars, +SD. *p < 0.05 compared to control. (c) Western blotting of TS in 1 or 2 nM DOC for 48 hr. (d) Western blotting of cell cycle related proteins in 1 or 2 nM DOC for 48 hr. (e) Western blotting of TS in fascaplysin for 24 hr.

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We determined the expression levels of cell cycle-related proteins to ascertain whether down-regulation of TS occurs. In a previous report, the activity of TS increased acutely as a cell passed from the late G1 phase to the early S phase of the cell cycle; therefore, we focused on three cell cycle proteins involved in the G1 to S phases, p53, p21 and p16 (Fig. 5d). p53 and p21 were significantly increased, but p16 was not affected by low-dose DOC. p21 is an inhibitor of the cyclin/Cdk family, and binds and inhibits the activity of cyclin-CDK2 and cyclin-CDK4 complexes and the up regulation of p21 leads to the inhibition of these CDKs. We also evaluated the effect of CDK4 inhibition on TS expression. Fascaplysin, a CDK4 specific inhibitor, significantly decreased TS expression in C4-2 cells in a dose-dependent manner (Fig. 5e).

Furthermore, we evaluated whether low doses of DOC, S-1, and their combination could affect TS expression in vivo as they did in vitro. Based on our previous in vivo study, we studied the effects of a low dose of DOC (2 mg/kg) administered on the first day, a low dose of S-1 (3 mg/kg) given daily and their combination. TS expression levels in xenografts for 2 and 5 days were evaluated by immunohistochemistry (Fig. 6a). TS expression was significantly decreased to 65.7 ± 11.0% and 72.0 ± 3.3% of control after DOC treatment (p < 0.05) and significantly increased to 140.7 ± 6.7% and 132.9 ± 12.2% of control after S-1 treatment (p < 0.05) at 2 and 5 days, respectively. In the combined DOC and S-1 treatment, TS expression was also decreased to 59.7 ± 8.7% and 55.5 ± 12.7% that of S-1 at 2 and 5 days, respectively (p < 0.05).

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Figure 6. Immunohistochemical study of TS expression in C4-2 xenograft tumors at low dose. Xenograft tumors were harvested under 2 types of setting, 2 days and 5 days after initiation of administration (n = 5–7 in each group). (a). Photo panels of TS immunostaining of xenograft tumors. Magnification, x200. (b). Intensities of TS expression in xenograft tumors by dot plot. Staining intensities of TS for each image were quantified with NIH Image J software. *p < 0.05 compared to control group.†p < 0.05 compared to S-1 group.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

In our study, we examined the effects of changes in TS expression levels on the progression and disease process of prostate cancer. We then focused on S-1, clarified whether S-1 monotherapy or combination therapy consisting of S-1 with low-dose DOC is effective for the treatment for CRPC in vivo and investigated the mechanism of the synergistic effect in vitro and in vivo.

S-1 was recently developed as an oral DIF. CDHP inhibits 5-FU degradation approximately 180 times more effectively than uracil in vitro. S-1 has been administered in an adjuvant setting as a first line treatment in East Asian patients who have undergone a D2 dissection for locally advanced gastric cancer.10 Furthermore, S-1 plus cisplatin was reported to be a more effective regimen for locally advanced gastric cancer patients compared to S-1 monotherapy.11 CDDP inhibits the elimination of folate outside cells, resulting in promotion of the formation of a ternary complex, and enhanced 5-FU cytotoxicity. The effect of biochemical modulation by CDDP indicates that modulation of the working status of 5-FU could enhance the cytotoxicity of S-1 itself. Therefore, in our study, we focused on S-1 for the treatment of prostate cancer and evaluated the efficacy of S-1 or S-1 combined with DOC and expected to observe biochemical modulation of the functional status of 5-FU.

The four deoxyribonucleotides are directly synthesized from the corresponding ribonucleotides, but only dTMP needs an additional step. dUTP must be catalyzed by TS to complete dTMP synthesis. 5-FU is converted to an active form and exerts its cytotoxicity through the formation of a ternary complex with TS and folate and inhibition of TS results in blockade of the DNA synthetic process. Considering the sensitivity to 5-FU, TS plays key roles in the resistance to 5-FU in carcinoma cells and over-expression of TS is one mechanism by which tumors may develop resistance to 5-FU.8 In prostate cancer, TS was shown to be expressed at greater levels in cancer specimens than in normal prostatic tissue specimens.33 The roles of TS expression with or without hormone sensitivity and with or without androgen ablation have not been previously studied. In our study, immunohistochemistry showed that TS expression levels were low in CRPC tissues with androgen ablation therapy compared to tissues of localized prostate cancer without androgen ablation therapy. Consistent with the findings in human samples, TS expression levels were lower in C4-2 cells in media supplemented with 10% charcoal-stripped FBS than in LNCaP cells with normal 10% FBS. Furthermore, C4-2 cells were more sensitive to 5-FU than LNCaP cells. These results suggested that hormone insensitivity and/or receiving androgen ablation treatment may be related to low TS expression, resulting in high sensitivity to 5-FU. Therefore, we speculated that CRPC with androgen ablation therapy may be a good candidate for 5-FU based treatment.

The in vitro study results showed that TS expression was increased by 5-FU treatment alone, but TS was decreased in combination with DOC in C4-2, LNCaP and PC3 cells. Co-treatment with 5-FU and DOC resulted in a synergistic increase in cytotoxicity in these cells (The CIs were 0.702 ± 0.043, 0.677 ± 0.086 and 0.622 ± 0.030 in C4-2, LNCaP and PC3 cells, respectively) compared to 5-FU or DOC alone. Based on these results, a decrease in TS levels in prostate cancer cells by DOC treatment improved the chemosensitivity of 5-FU.

Combination therapy with different chemotherapeutic agents may sometimes increase the risks of side effects. After observing a marked decrease in the rate of tumor growth with high dose combination therapy in vivo, we decided to determine whether the doses of the chemotherapeutic agents could be reduced while at the same time maintaining the cytotoxic efficacy. We next investigated the growth inhibitory effects of combined low doses of DOC and S-1, adjusted for administration to humans. A significant decrease was observed in mice treated with a combination of these drugs, compared to S-1 monotherapy. Low-dose DOC alone did not influence tumor growth, but low-dose DOC enhanced the efficacy of S-1, providing strong evidence for an in vivo synergistic effect between S-1 and low-dose DOC. In addition, we also confirmed in vivo that low-dose S-1 enhanced TS expression, low-dose DOC suppressed the expression of TS, and combined S-1/DOC suppressed the TS, which was enhanced by S-1, and this tendency was also observed for at least 2–5 days. These experiments were started when the size of the tumor exceeded 300 mm3, representing an advanced stage of prostate cancer, although S-1 monotherapy, even if adjusted for the human dosage, could induce tumor growth suppression, and the high and low-dose combination therapies showed marked efficacy against tumor growth. It is hoped S-1 monotherapy and this combination of chemotherapy will show promise in clinical situations.

The usual dosage of DOC induces G2/M arrest and leads to cell death, and TS expression is induced in G1 to S phase. Even low-dose DOC has the potential to down-regulate TS without cell cycle modulation, suggesting there was another mechanism involved in the down-regulation of TS. We demonstrated that low-dose DOC decreased not only TS protein but also TS mRNA. This suggests that low-dose DOC regulated the transcription of TS. Le François et al.34 reported that over expression of p16INK4A or CDK4 inhibition was associated with decreased levels of TS protein in colon cancer cells. On the other hand, Panno et al.35 reported that “low dose” paclitaxel treatment is able to activate p21 promoter via p53 in breast cancer cells. p21, an inhibitor of the cyclin/Cdk family, binds to and inhibits the activity of cyclin-CDK2 and -CDK4 complexes. Based on these findings, we focused on cell cycle proteins involved in the G1 phase to S phase. p53 and p21 were significantly increased by low-dose DOC exposure (Fig. 5d), and the inhibition of CDK4, which is located downstream of p21, resulted in the down regulation of TS.

S-1 based chemotherapy is expected to have other advantages in addition to its effectiveness. Elderly patients or those with advanced disease may not be able to tolerate intensive chemotherapy with severe toxicity, often resulting in a decrease in compliance of adjuvant therapy. DIF such as S-1 whose profiles are characterized by mild toxicity, good effects, and oral administration may be superior as “standard” adjuvant chemotherapy regimens in terms of compliance.

Combining different anticancer agents is a reasonable strategy with which to obtain a potent cytotoxic effect in cancer cells, and a high PSA response rate was observed in a phase II trial of combination therapy consisting taxane and 5-FU in CRPC patients.36 Additionally, capecitabine, an oral fluoropyrimidine, is an interesting example that can be considered when discussing combination therapy involving docetaxel and fluoropyrimidine. Although capecitabine monotherapy showed only limited activity in CRPC patients (PSA response rate was 12%)37, the combination of weekly docetaxel (35–36 mg/m2) and capecitabine yielded extremely high PSA response rates of 73 and 68.2% in 2 phase II clinical trials.38, 39 Although direct comparisons are not feasible, these response rates were better than those in the TAX327 study (45 and 48%).2 According to a phase II clinical trial on S-1 monotherapy in CRPC patients, Akaza et al.40 reported that a PSA response was observed in 8 of 35 patients (22.9%). S-1 showed a higher PSA response rate than capecitabine, and S-1 could be a good candidate for combination therapy with docetaxel. Although there have been no clinical trials on the combination of S-1 and docetaxel in CRPC patients, our experimental data strongly support the potential clinical applicability of this combination in the future.

In conclusion, the present results demonstrate that S-1 monotherapy was effective in C4-2 bearing mice and that S-1 combined with low-dose DOC has a synergistic effect through the down-regulation of TS. We also believe that these regimens may have a significant impact on the management of CRPC patients and may also be effective in patients who experience disease progression during DOC treatment. It should be easy to transfer the results to the treatment of prostate cancer patients because S-1 has already demonstrated its usefulness as a front line chemotherapeutic agent for various cancers in Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References
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