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Abstract

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

Recently, we reported that combined ingestion of soy isoflavones and curcumin significantly decreased the serum level of prostate-specific antigen based on a randomized placebo-controlled double-blind clinical study. We investigated whether these polyphenols inhibited the proliferation of prostate cancer cells by activating a DNA damage response. The effects of isoflavones and curcumin on the expression and phosphorylation of ataxia-telangiectasia-mutated kinase (ATM), histone H2AX variant (H2AX) and checkpoint kinase2 (Chk2) were examined in LNCaP cells. The induction of apoptosis in LNCaP cells was evaluated by poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, the effects of a testosterone supplement on modulation of the DNA damage response were examined. Combined treatment of isoflavones and curcumin additively suppressed cellular proliferation and induced phosphorylation of ATM, histone H2AX, Chk2 and p53. Testosterone augmented the activation of the DNA damage response and PARP cleavage induced by curcumin. Our results indicate that activation of the DNA damage response by polyphenols might suppress the malignant transformation of prostate cancer. In addition, testosterone, when combined with curcumin, may have suppressive effects on the progression of prostate cancer. (Cancer Sci 2011; 102: 468–471)

While prostate cancer is the most common neoplasm in Caucasian men, the incidence in Asians has been relatively low. Observational studies have suggested that diet is one of the most important factors contributing to the lower observed incidence and mortality of prostate cancers in Asia.(1) Asian diets are rich in polyphenols, which have been suggested to prevent cancers.(2,3) Indeed, epidemiological studies have shown that soy intake is one of the major factors in the prevention of prostate cancer.(1) Polyphenols such as soy isoflavones and curcumin are common compounds in Asian diets, and both have anti-inflammatory and antioxidant properties. Previous studies show that curcumin and isoflavones induce apoptosis and cell cycle arrest in both androgen-dependent and androgen-independent prostate cancer cells.(4–6) Recently, we reported that a combined treatment of soy isoflavones and curcumin decreased serum levels of prostate-specific antigen (PSA) in subjects with a baseline PSA of more than 10 ng/mL in a randomized placebo-controlled clinical trial.(7) Furthermore, a combined treatment of soy isoflavones and curcumin inhibited the production of PSA and expression of the androgen receptor in cultured prostate cancer cells.(7)

The DNA damage response (DDR) emerges as an oncogene-inducible biological barrier against progression of cancer beyond its early stages. Recent evidence from cell culture and animal models and analyses of clinical specimens show that the early precursor lesions commonly express markers of an activated DDR.(8,9) These markers include phosphorylated kinases (ataxia-telangiectasia-mutated kinase [ATM] and checkpoint kinase2 [Chk2]), phosphorylated histone H2AX and phosphorylated p53. Such activation of the DDR network leads to senescence or death of oncogene-transformed cells that delays or prevents cancer progression. The DDR can also be induced by chemotherapeutic agents, UV and oxidative stress.(10) However, little is known about the effects of polyphenols, such as curcumin and isoflavones, on the induction of the DDR.

Androgens drive both the proliferation and differentiation of developing prostate epithelial cells. The maintenance of prostate epithelium requires continuous physiological levels of androgens, which inhibits apoptosis.(11,12) However, serum and tissue testosterone levels decrease as individuals age. Recently, several lines of evidence suggest that decreases in testosterone level accelerate the progression of prostate cancer. Indeed, low testosterone levels have been shown to be associated with an advanced tumor stage at presentation, positive surgical margins, high Gleason scores and worse overall survival.(13–17) Intraprostatic dihydrotestosterone (DHT) levels were significantly reduced in males with high-grade (Gleason scores 7–10) carcinomas compared with males with low grade carcinomas (Gleason scores ≤ 6).(18) These observations suggested that testosterone might suppress the malignant progression of prostate cancer. However, the mechanisms underlying the inhibition of malignant transformation in prostate cells by testosterone have not been fully elucidated.

The purpose of this study was to determine whether curcumin and isoflavone induced a DDR in prostate cancer cells as a potential mechanism of cancer prevention. Furthermore, we examined whether testosterone modulated the DDR in prostate cancer cells to elucidate the role of testosterone in cancer progression.

Materials and Methods

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

Cell culture and reagents.  A human prostate cancer cell line, LNCaP, was obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were routinely maintained in RPMI 1640 supplemented with 10% FCS, 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were cultured at 37°C in a humidified incubator with 5% CO2. Curcumin (Medi Herb Inc., Bangalore, India) was dissolved in ethanol at a concentration of 10 mM and stored at −20°C. Isoflavones (Nichimo Co., Ltd, Tokyo, Japan) were dissolved in DMSO at a concentration of 20 mg/mL and stored at −20°C in the dark. For the androgen stimulation analysis, LNCaP cells were cultured in a serum-free medium for 2 days prior to the addition of various concentrations of DHT (Wako, Tokyo, Japan) or synthetic androgen R1881 (New England Nuclear, Boston, MA, USA). To block androgen signaling, LNCaP cells were cultured with Flutamide (Sigma-Aldrich Co., St Louis, MO, USA).

Cell proliferation assay.  LNCaP cells were seeded in poly-d-lysine-coated 96-well plates (Nalge Nunc, Rochester, NY, USA) at a density of 1 × 104 cells/well in 100 μL tissue culture medium. Cells were allowed to attach for 24 h and then treated with either increasing concentrations of curcumin or isoflavones. Cell proliferation was assessed using a colorimetric proliferation assay using tetrazolium3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) using the CellTiter 96 Aqueous One Solution Proliferation Assay System (Promega, Tokyo, Japan) according to the manufacturer’s instructions. After 48 h, MTS was added to the culture wells and the mixture was incubated for 60 min at 37°C. The absorbance at 490 nm was measured on a microplate reader.

Immunoblotting.  Subconfluent LNCaP cells were treated with curcumin, isoflavones or a combination of the two agents. Cells were washed twice with cold PBS and then lysed in radio immuno precipitation assay (RIPA) buffer on ice for 30 min. The cell lysate was centrifuged at 18500g for 30 min at 4°C and the supernatant was collected. Protein concentrations were measured using a BCA protein assay kit (Pierce Inc., Rockford, IL, USA) according to the manufacturer’s instructions. Protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore Inc., Tokyo, Japan). Immunoblotting was performed using rabbit anti-androgen receptor antibody (1:2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), mouse anti-prostate-specific antigen antibody (1:1000; DakoCytomation, Kyoto, Japan), rabbit anti-phospho-ATM (Ser1981) antibody (1:2000; Rockland Immunochemicals Inc., Gilbertsville, PA, USA), rabbit anti-phospho-Chk2 (Thr68) antibody (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), rabbit anti-phospho-p53 (Ser15) antibody (1:1000; Cell Signaling Technology), mouse anti-p53 (DO7) antibody (1:1000; DakoCytomation), rabbit anti-phospho-H2AX (Ser139) antibody (1:1000 dilution; Upstate Inc., Temecula, CA, USA), rabbit anti-PARP antibody (1:1000 dilution; Cell Signal Technology) or mouse anti-human β-actin antibody (1:10 000 dilution; Sigma–Aldrich Co.) as an internal loading control. Immunoreactive proteins were visualized with ECL detection reagents (GE Healthcare Biosciences, Tokyo, Japan). The results of western blotting were quantified by densitometric analysis using a LAS 3000 Densitometer and Multi Gauge v3.1 (Fujifilm, Tokyo, Japan). The results were normalized to β-actin values.

Results

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

Combined inhibitory effects of isoflavones and curcumin on prostate cancer cell proliferation.  We examined the effects of isoflavones and curcumin on the proliferation of LNCaP prostate cancer cells that express androgen receptors. Both isoflavones and curcumin inhibited the cell growth of LNCaP cells in a dose-dependent manner. Figure 1A shows that the combination of isoflavones and curcumin had a more potent inhibitory effect on cell proliferation than isoflavones alone. The cell viability in LNCaP cultures decreased by 42.5% when 20 μM curcumin and 20 μg/mL isoflavones were added to the culture medium. Figure 1B shows that 30 μM curcumin has an inhibitory effect on the cell growth of LNCaP in the presence of 100 nM DHT.

image

Figure 1.  (A) Effects of isoflavones and curcumin on the growth of LNCaP cells. Cells were treated with either increasing concentrations of curcumin and isoflavones. Varying concentrations of isoflavones, 0 μg/mL (▮), 10 μg/mL (bsl00066) and 20 μg/mL (♦), were added to the culture medium. After culturing for 2 days, cell viability was examined by MTS assay. Each point represents mean ± SD for six wells. (B) Curcumin has an inhibitory effect on cell viability of LNCaP even if the culture medium contained dihydrotestosterone (DHT). None (bsl00001), curcumin 30 μM (♦), DHT 100 nM (bsl00066), curcumin 30 μM + DHT 100 nM (•).

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Activation of the DDR by isoflavones and curcumin.  To determine the mechanism by which isoflavones and curcumin inhibit cell proliferation, we examined the phosphorylation of ATM, Chk2, histone H2AX and p53 by immunoblotting in cultures treated with polyphenols. The LNCaP cells were treated with 10 μg/mL isoflavones, 25 μM curcumin and a combination of these polyphenols for 48 h each. Phospho-specific antibodies that recognized each protein were used for this analysis. Isoflavones at a concentration of 10 μg/mL inhibited the expression of androgen receptors but did not induce phosphorylation of ATM, Chk2, histone H2AX or p53 in the LNCaP cells. Treatment with 25 μM of curcumin induced phosphorylation of these proteins. Moreover, a combination of the two compounds induced higher levels of ATM and Chk2 phosphorylation than curcumin only (Fig. 2). Densitometric analysis showed that combined treatment of 10 μg/mL isoflavones and 25 μM curcumin induced the phosphrylation of ATM (fivefold), Chk2 (1.4-fold), p53 (63-fold) and histone H2AX (60-fold), respectively, when compared with the control.

image

Figure 2.  Isoflavones and curcumin synergistically induce phosphorylation of ataxia-telangiectasia-mutated kinase (ATM), checkpoint kinase2 (Chk2), p53 (Ser15) and H2AX in the LNCaP cells. Protein expression was detected by immunoblotting after 48 h treatment. Immunoblots were probed for mouse anti-human β-actin antibody as an internal control.

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Testosterone induced activation of the DDR in curcumin-treated cells.  We showed that the proliferation effects of DHT were completely blocked by curcumin. Therefore, the effect of curcumin on the proliferation of LNCaP cells might be enhanced by testosterone through a DDR; this possibility was examined by western blotting analysis. For the androgen stimulation analysis, LNCaP cells were cultured in a serum-free medium for 2 days prior to the addition of androgens. Figure 3 shows that the phosphorylation of CHK, H2AX and p53 was induced by 1 nM R1881 in LNCaP cells treated with 25 μM curcumin, which express the androgen receptor. Isoflavones did not induce testosterone-mediated activation of the DDR. The expression of androgen receptor and PSA was increased by 1 nM R1881 as reported previously.(19,20) Curcumin suppressed the expression of PSA even in cells treated with 1 nM R1881 (Fig. 3).

image

Figure 3.  Testosterone induced the activation of the DNA damage response under the treatment of 25 μM curcumin. AR, androgen receptor; p-Chk2, phosphorylated-Chk2; PSA, prostate-specific antigen.

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PARP cleavage was induced by testosterone with curcumin.  To verify the mechanism by which testosterone enhanced the DDR with curcumin, the expression of apoptosis-related proteins was monitored by western blot analysis (Fig. 4). Occurrence of PARP cleavage, a marker of apoptosis,(21) occurred in curcumin-treated cells. We could not observe the induction of Chk2 and PARP cleavage by 1 μM DHT when the cells were treated with 25 μM curcumin. However, PARP cleavage was induced by 1 μM DHT in combination with a higher concentration, 50 μM curcumin. Densitometric analysis showed that 50 μM curcumin with 1 μM DHT induced the phosphorylation of Chk2 (4.4-fold) and PARP cleavage (2.4-fold), respectively, when compared with 50 μM curcumin without 1 μM DHT. This testosterone-induced PARP cleavage was decreased by the addition of 10 μM fultamide, which is an inhibitor of the androgen receptor. Densitometric analysis showed that the treatment of flutamide reduced the phosphorylation of Chk2 (8.2-fold) and PARP cleavage (16.9-fold), respectively, when compared with 50 μM curcumin with 1 μM DHT. These results suggest that DHT enhance apoptosis through the DDR when combined with a higher concentration of curcumin, which is dependent on signaling mediated by the androgen receptor.

image

Figure 4.  Testosterone induced poly (ADP-ribose) polymerase (PARP) cleavage with activation of Chk2. Immunoblots were probed for mouse anti-human β-actin antibody as an internal control. DHT, dihydrotestosterone; p-Chk2, phosphorylated-Chk2.

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Discussion

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

Curcumin is extensively used as a spice or pigment in the Asian diet, especially in curry. An epidemiological study showed that a low incidence of large bowel cancer in Indians can be attributed to the intake of curcumin.(22) Animal studies revealed that dietary administration of curcumin inhibited the incidence of cancers.(23) Curcumin is under clinical trials mainly for various cancers and related diseases.(23) In in vivo analyses, curcumin has been shown to cause apoptosis and cell cycle arrest with inhibited cell growth, activation of signal transduction, and transforming activities in both androgen-dependent and independent prostate cancer cells.(5,6) Curcumin has anti-inflammatory and antioxidant properties through inhibiting the activation of NF-κB, reducing COX-2 and AKT, decreasing the production of IL-6.(24–27) Recent evidence suggests that genistein induces the DDR through ATM.(28) There is a report that curcumin induces the phosphorylation of ATM in pancreatic cancer cells.(29) To our knowledge, this is the first report that isoflavones and curcumin activate the DDR, such as the phosphorylation of ATM, histone H2AX, Chk2 and p53 proteins, in LNCaP cells. DNA damage checkpoints are activated in the early stages of human tumorigenesis, leading to cell-cycle blockade or apoptosis and thereby inhibiting tumour progression.(8) Thus, polyphenols such as isoflavones and curcumin might contribute to the prevention of prostate cancer by activating a DDR. The reduced incidence of prostate cancers in several Asian populations may be due to the additive effects of several compounds including curcumin and isoflavones commonly found in Asian foods.

Another finding of the present study was that testosterone enhanced apoptosis of LNCaP cells through a curcumin-induced DDR that was dependent on the androgen receptor. Several lines of study have shown that testosterone has a dual role of promoting both proliferation and differentiation in androgen-dependent prostate cancer cells.(30,31) A previous study has shown that testosterone mediates cellular proliferation signals in the prostate through increased expression of Skp2, the ubiquitin ligase that targets p27 for degradation,(32) while testosterone induces the cyclin-dependent kinase inhibitor p21Cip1.(33)

Our data demonstrated that in combination with curcumin testosterone lost its proliferative effect on LNCaP cells and contributed to a DDR. Currently, androgen deprivation is the gold standard for the treatment of metastatic prostate cancer. However, this treatment has the limitation that many of these cancers eventually become androgen independent, and the deprivation of androgen may cause serious morbidities, including obesity, hypertension and osteoporosis.(34) Therefore, our findings might have clinical implications for the development of new strategies for the prevention and treatment of prostate cancer that do not depend on androgen deprivation. The mechanism underlying this curcumin-induced testosterone-enhanced DDR warrants further study.

The present study also showed that certain combinations of polyphenols worked additively. It is both reasonable and practical to combine such components in daily diets rather than take single constituents daily to prevent the onset of cancer.

In conclusion, a combined treatment of soy isoflavones and curcumin additively induced a DDR in cultured prostate cancer cells. In addition, testosterone enhanced signals of DDR markers induced by curcumin treatment. Our findings suggest a potential protective effect of curcumin treatment against prostate carcinogenesis.

References

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