ISI Impact Factor (2004): 1.096

Editor-in-Chief
Prof. Yi-Fei WANG,

Combination of genistein with ionizing radiation on androgen-independent prostate cancer cells

Sen-Xiang Yan¹, Yasuo Ejima², Ryohei Sasaki², Shu-Sen Zheng¹, Yusuke Demizu², Toshinori Soejima², Kazuro Sugimura²

Asian J Androl  2004 Dec; 6: 285-290            

Keywords: prostate cancer; genistein; ionizing radiation (IR); apoptosis; cell cycle

Abstract

Aim: To study the effect of the combined use of genistein and ionizing radiation (IR) on prostate DU145 cancer cells. Methods: DU145, an androgen-independent human prostate cancer cell line, was used in the experiment. Clonogenic assay was used to compare the survival of DU145 cells after treatments with genistein alone and in combination with graded IR. Apoptosis was assayed by DNA ladder and TUNEL stain. Cell cycle alterations were observed by flow cytometry and related protein expressions by immunoblotting. Results: Clonogenic assay demonstrated that genistein, even at low to medium concentrations, enhanced the radiosensitivity of DU145 cells. Twenty-four hours after treatment with IR and/or genistein, apoptosis was mainly seen with genistein at high concentrations and was minimally related to IR. At 72 h, apoptosis also occurred in treatment with lower concentration of genistein, especially when combined with IR. While both IR and genistein led to G₂/M cell cycle arrest, combination of them further increased the DU145 cells at G₂/M phase. This G2/M arrest was largely maintained at 72 h, accompanied by increasing apoptosis and hyperdiploid cell population. Cell-cycle related protein analysis disclosed biphasic changes in cyclin B1 and less dramatically cdc-2, but stably elevated p21^cip1 levels with increasing genistein concentrations. Conclusion: Genistein enhanced the radiosensitivity of DU145 prostate cancer cells. The mechanisms might be involved in the increased apoptosis, prolonged cell cycle arrest and impaired damage repair.

1 Introduction

Epidemiologically, prostate cancers are much more often found in Western than in Asian countries. One of the reasons is that the Asians consume more soybean products rich in isoflavonoids than the Western people. According to a recent survey, the mean serum concentrations of genistein, the main form of isoflavonoids found in soybean products, were approximately 15 times higher in Japanese than that in British people [1]. Apart from its seemingly prophylactic action, genistein has also been reported to directly inhibit cell growth, induce apoptosis and cell cycle arrest and lead to genetic and protein expression changes in vitro in a wide range of solid tumors.

As one of the established treatments for prostate cancer, radiotherapy can be used alone or in combination with chemo-, hormonal or surgical therapies. Combined therapy is believed to yield more satisfactory results, especially for androgen-independent prostate cancer. In this paper the combined use of ionizing radiation (IR) with genistein on DU145, an androgen-independent prostate cancer cell line, and the possible action mechanism were studied.

2 Materials and methods

The human prostate cancer line DU145 was obtained from the American Type Culture Collection (Rockville, MD, USA) and was maintained in RPMI-1640 culture medium (CM) supplemented with 10 % fetal bovine serum (Sigma, St. Louis, USA) in a 5 % CO₂ atmosphere at 37 ℃. DU145 demonstrated two mutations at codons 223 and 274, and proline to leucine/valine to phenylalanine of amino-acid changes [2].

Genistein was purchased from the Calbiochem Co. (USA) and was dissolved in DMSO (Sigma) as 100 mmol/L stock. Cells in genistein-free treatment were incubated using the vehicle (DMSO of similar concentration). IR was performed at room temperature in cell culture dishes or chamber-slides at a dose rate of 1.67 Gy/min with an X-ray apparatus (Toshiba, Japan). During IR, control dishes were also taken out of the incubator for mock IR.

The IC₅₀ of genistein on DU145 cells was determined through clonogenic assay. In the IR only group, DU145 cells reaching 70 % - 80 % confluent in 6 cm dishes were exposed with graded doses of IR (0, 1, 3 and 5 Gy). In the IR plus genistein groups, CM within the dishes was changed to contain genistein of different concentrations (5 and 15 µmol/L) 24 h before IR. Immediately after IR, cells were replaced to 6 cm dishes containing the same concentrations of genistein as before. After 10 days of incubation, cells in all dishes were fixed with 10 % formalin and stained with crystal violet. The number of colony (defined as an aggregation of more than 50 cells) was counted and survival fraction curves calculated using the single-hit multi-target (SHMT) model: S/S₀=1-(1-e^-D/D0)ⁿ, where S/S₀ is the survival fraction and D the dose (Gy). Experiments were repeated in triplicate with each treatment.

For DNA ladder, cells were dissolved and DNA fragments extracted. The supernatant containing DNA fragments was obtained and incubated with 2  µL of 20 mg/mL RNase A for 1 h at 37 ℃. After further incubation with 2 µL of 20 mg/mL proteinase K for 1 h at 37 ℃, samples were added 20 µL of 5 mol/L NaCl and 120 µL of isopropanol and stocked at -20 ℃ overnight. Before minigel electrophoresis, samples were centrifuged at 16 000 rpm for 15 min to remove isopropanol. Then 20 µL of Tris-EDTA (TE) buffer (containing 10 mmol/L tris-HCl and 1 mmol/L EDTA) was added and 12 µL of samples was loaded to each well along with the DNA molecular marker. After electrophoresis, minigels were exposed to ultraviolet and films taken with a Polaroid CRT camera.

TUNEL was performed using ApopTag in situ apoptosis detection kit (Intergen Co.). This method detects the nucleosome-sized DNA fragments by tailing the 3¹-OH ends of fragments with digoxigenin-nucleotide using terminal deoxynucleotidyl transferase.

70 % - 80 % confluent and asynchronous cells in 6 cm dishes were divided into the control, IR (6 Gy) and/or genistein (5, 15, 30 and 100 µmol/L) groups. Samples were taken 24 h after respective treatments. In the 30 mmol/L genistein plus IR treated group, further observation of cell cycle perturbations was made at 48 and 72 h. Just before loading the samples to the flow cytometer (Becton Dickinson, Franklin lakes, USA), stock samples were centrifuged at 4 000 rpm for 2 minutes and pellets re-suspended in 500 µL of cell cycle buffer (containing 400 µL PBS, 100 µL 5 mg/mL RNase A and 2.5 µL 20 % Triton X). Cell suspensions were transferred to Falcon 2052 tubes filtered through a nylon mesh. Then samples were incubated with 10 µL 5 mg/mL PI (prodidium iodide) in the dark for 10 min. Ten thousand events were acquired and the proportions of cells in each cycle phase were calculated using ModFit software. Each experiment was performed at least 3 times.

Immunoblotting was performed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fifty µg protein in each well was loaded onto 12 % SDS-PAGE gels for electrophoresis. Gels were then electroblotted to a sheet of Hypond-P polyvinylidane difluoride membrane (Amersham, Buckinghamshire, UK). Non-specific binding sites in the membrane were blocked by immersing in 3 % bovine serum albumin in Tris-buffered saline (100 mmol/L Tris-Cl, pH 7.5, and 140 mmol/L NaCl) supplemented with 0.1 % Tween 20 (TBS-T) for 1 h at room temperature. After that, the membrane was incubated with the first antibody diluted at 1:500-1000 for 2 h at room temperature or overnight at 4 ℃ and then a horseradish peroxidase-labeled second antibody diluted at 1:1 000 for 2 h at room temperature. Fluorescence was detected and exposed on an X-ray film using an enhanced chemiluminescence (ECL; Amersham) detection system.

2.7 Statistical analysis

One-way ANOVA was employed to assess statistical significance between treatments.

Genistein at low (5 µmol/L) to medium (15 µmol/L) concentrations significantly decreased the survival fractions of DU145 cells after graded IR (Figure 1). Survival fractions in the combined treatments of IR with genistein were calculated by adjusting the cytotoxicity of genistein alone. Morphologically, cells with combined treatments of IR and genistein exhibited various degrees of cytoplasmic swelling, vacuolization, nuclear condensation and apoptotic signs (data not shown).

Figure 1. Survival fractions of DU145 cells following graded IR with or without genistein. 0: Irradiation (IR only); u: IR plus 5 µmol/L genistein (IR+5mmol/L); O: IR plus 15 mmol/L genistein (IR+15 µmol/L). A. Survival fraction curves; B. Survival curve parameters, where a represents linear and b quadratic parts of the linear-quadratic cell killing model, D0 inverse of the slope of the survival curve, SF2 and SF4 survival fractions at doses 2Gy and 4Gy, respectively.

DNA ladder assay showed that 24 h after each treatment, laddering was seen in 100 µmol/L genistein alone or plus IR treatment. With longer observation time, laddering was also seen in treatment with a lower genistein concentration (30 µmol/L); combined use of IR further enhanced this phenomenon (Figure 2). TUNEL provided morphological information of apoptosis after treatment (Figure 3).

Figure 2. Analysis of apoptosis with DNA ladder A: 24 h after each treatment. Lanes 1-11: DNA marker, Control, 5, 15, 30 and 100 µmol/L genistein, IR (6 Gy), IR+5, IR+15, IR+30 and IR+100 µmol/L genistein; B: Treatments with 30 µmol/L genistein alone or plus 6 Gy IR (lanes 1-7: DNA marker, 30 mmol/L of genistein at 24, 48 and 72 h, IR+30 mmol/L genistein at 24 , 48 and 72 h).

Figure 3. TUNEL analysis of apoptosis (A, B). A: 24 h after each treatment; B: 48 - 72 h after treatments with 30 µmol/L of genistein alone or plus IR. Scale bar = 20 µm.

At 24 h after treatment, genistein alone concentration-dependently increased cells at G₂/M phase accompanied with a corresponding decrease of cells at either G₀/G₁ or S phases, which was most obvious in 100 µmol/L genistein treatment. IR (6 Gy) also led to a mild G₂/M arrest. IR plus genistein at 30 µmol/L or lower concentrations further increased cells at G₂/M phase as compared to genistein or IR alone. This phenomenon was most clearly exhibited in 30 µmol/L plus IR treated group, where cells at G₂/M phase reached 89.2 % against 27.7 % in genistein alone or 23.7 % in IR alone. On the contrary, in 100 µmol/L genistein plus IR treatment showed a decrease in cells at G₂/M phase (74.6 % against 84.7 %), but an increase in S phase cells (24 % against 12.8 %) as compared to genistein alone. At 48 and 72 h after treatment, G₂/M arrest was largely maintained and 2 extra cell populations of apoptosis and hyperdiploid appeared in IR plus 30 µmol/L genistein treated group (Figure 4).

Figure 4. Representative flow cytometry plots and histograms of cell cycle alterations. A: 24 h after each treatment. Graphs showed averages of 3 replications. Control (Control cells), 5, 15, 30 and 100 祄ol/L genistein, IR (6Gy), IR+5 µmol/L, IR+15 µmol/L, IR+30 µmol/L and IR+100 µmol/L of genistein (^bP <0.05; ^cP <0.01). B: 24-72 h after 30 µmol/L genistein plus IR (6 Gy), where A and H represented apoptosis and hyperdiploid, respectively. Proportions of apoptosis and hyperdiploid cells were also shown. Note G2/M arrest was largely maintained at 72 h.

Tweenty-four h after genistein alone treatment, cyclin B1 exhibited a biphasic change depending on the concentrations of genistein. Specifically, cyclin B1 protein increased with higher concentrations of genistein until reaching its peak at about 30 µmol/L. Then at 100 µmol/L, cyclin B1 sharply decreased as compared to what was seen at 30 µmol/L genistein treatment. IR (6 Gy) alone also significantly raised the cyclin B1 protein level. Combination of IR with genistein followed the pattern of genistein alone treatment. The protein levels of cdc-2 showed similar but far less dramatic changes than cyclin B1 (Figure 5A).

p21^cip1 protein increased concentration-dependently with genistein treatment (Figure 5B) and this increase was not dependent on the level of p53 expression (data not shown). IR alone also mildly increased p21^cip1 protein. Combination of IR with genistein showed the similar pattern as with genistein alone.

Figure 5. Immunoblotting results of cyclin B1, cdc-2 and p21cip1 protein expressions 24 h after each treatment.

4 Discussion

Most prostate cancers are relatively androgen-dependent at their early stages and respond well with androgen deprivation therapy. However, they will eventually become androgen-independent within several years and refractory to most available therapies. DU145 is a well characterized androgen-independent human prostate cancer cell line. As shown from our result of clonogenic assay, genistein synergistically enhanced the IR effect on DU145 cells at low to medium concentrations of 5 and 15 µmol/L, which were not far above the upper limit of physiological serum concentrations in heavy soy product consumers [1]. The mechanisms of genistein on the inhibition of prostate cancer cells have not been clearly elucidated. Some hypotheses attributed this effect to its inhibition of receptor tyrosine kinases, which participate in transmembrane signaling and their enhancement can lead to persistent stimulation by autocrine growth factors that in turn lead to diseases and even induce cancers [3]. Genistein might have enhanced the radiosensitivity of DU145 cells in our experiment partly by inhibition of these so-called survival signals, which are either intrinsically over-expressed in tumor cells or further induced by IR.

The present results also showed that the concentration of genistein predominantly affected early-stage apoptosis of DU145 cells. The mechanisms regarding the radioresistance of tumors have not been fully elucidated and might involve the increase in survival gene expression. There were reports that genistein inhibited NF-kappaB activation in prostate cancer cells [4], which might partly explain the dose-dependent induction of apoptosis in DU145 cells by genistein. Our prolonged observation revealed that apoptosis did also occur with genistein at lower concentration, especially when combined with IR. This might be explained by the fact that parts of these apoptotic cells might have derived from the sustained G₂/M arrest or hyperdiploid cells, as disclosed in our cell cycle analysis.

We found that the combination of genistein (at 5-30 µmol/L) with IR (6 Gy) caused more DU145 cells arrested at G₂/M phase than with genistein alone. Interestingly, 100 µmol/L of genistein plus IR did not further increase, but rather decreased the cells at G₂/M phase. This might be explained by the fact that a part of the G₂/M arrested cells have undergone apoptosis, as evidenced by our results of TUNEL/DNA ladder analyses. It was also likely that an increase of cells at S phase in 100 µmol/L of genistein plus IR treatment might have partly reflected the phenomenon of pre-G₂ apoptosis derived from G₂/M arrested cells [5-6]. Also, when we prolonged the observation time to 48 and 72 h with 30 µmol/L of genistein plus IR treatment, a time-dependent increase in two additional cell populations corresponding to apoptosis and hyperdiploid was detected. Hyperdiploid phenomenon can occur in various cancer cells as a result of treatment with DNA damaging agents like IR or anti-cancer drugs. Our results showed a notable appearance of hyperdiploid in DU145 cells following combined treatment of IR with genistein at 48 and 72 h. Hyperdiploid cells might be further sensitized to the effects of genistein because of impaired damage repair [7], which might also in part explain the significant decrease in the survival fraction seen with continuous exposure to genistein at moderate concentrations. Hyperdiploid cells reflected abnormal DNA endo-reduplications and were thought to be a result of deregulations or interactions of cell-cycle related proteins including p53, p21^cip1, pRb and cyclins [8-9]. The end result of hyperdiploidy has been postulated to be cell death including apoptosis, although some findings revealed that a small proportion of it would eventually survive and remain reproductively active [10].

Cyclin B1/cdc-2 complex was known to be a key regulator of the transition from G₂ to M phase of the cell cycle. p53 dependent or independent p21^cip1 over-expression accompanied with inhibition of cyclin B1/cdc-2 complex has been linked to the G₂/M arrest in several cancer lines by the action of genistein [10-11]. Our study also revealed that genistein (alone or plus IR) led to a concentration-dependent over-expression of p21^cip1, which was not dependent on p53 levels. Genistein had a biphasic effect on protein levels of cyclin B1 and less dramatically cdc-2. This phenomenon could not be sufficiently explained by association of the elevated cyclin B1 levels to an accumulation of cells at the G₂/M phase, since G₂/M arrested cells with genistein at 100 µmol/L were more than those at 30 µmol/L. Also, IR alone led to a mild degree of G₂/M arrest and yet an incompatibly significant increase in cyclin B1 level. A similar biphasic phenomenon of genistein on cyclin B1 protein levels was also seen in human breast cancer cells [13]. In fact, many conflicting results have been obtained regarding the relationship between cyclin B1 protein levels and mRNA expression after treatment with DNA damaging agents. For example, after a-irradiation cyclin B1 mRNA in Hela cells underwent a dose-dependent decrease, but cyclin B1 protein showed either oppositely increased with low doses (< 5 Gy) or correspondingly decreased with higher doses (> 5 Gy) [14]. The contradictory co-existence of augmented cyclin B1 protein and G₂/M arrest might have partly reflected the decreased ubiquitination of cyclin B1[15]. Deregulation in ubiquitination pathway per se has also been linked to accumulation of cyclin B1 and cell cycle arrest [16].

In conclusion, combined treatment of IR with genistein synergistically increased the radiosensitivity of DU145 prostate cancer cells. While early-stage apoptosis was mainly induced by genistein at high concentration; prolonged observation revealed that significant apoptosis also occurred with genistein at lower concentration, especially when combined with IR. Genistein plus IR could lead to more cell arrested at G₂/M phase and a time-dependent increase in hyperdiploid cells, which might have also contributed to the enhancement of radiosensitivity by genistein on DU145 cells.

The study was supported in part by the Japan-China Sasakawa Medical Fellowship.

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Correspondence to: Ryohei Sasaki, MD, Ph.D., Division of Radiology, Kobe Graduate School of Medicine, Hyogo 650-0017, Japan.
Tel.:+81-78-382 6104, Fax: +81-78-382 6129
E-mail: yuunasasaki@hotmail.com
Received 2004-05-08   Accepted 2004-11-03