Original Paper | Published:

Curcumin confers radiosensitizing effect in prostate cancer cell line PC-3

Oncogene volume 23, pages 15991607 (26 February 2004) | Download Citation

Subjects

Abstract

Curcumin (Diferuloylmethane) is a major chemical component of turmeric (curcuma longa) and is used as a spice to give a specific flavor and yellow color in Asian food. Curcumin exhibits growth inhibitory effects in a broad range of tumors as well as in TPA-induced skin tumors in mice. This study was undertaken to investigate the radiosensitizing effects of curcumin in p53 mutant prostate cancer cell line PC-3. Compared to cells that were irradiated alone (SF2=0.635; D0=231 cGy), curcumin at 2 and 4 μM concentrations in combination with radiation showed significant enhancement to radiation-induced clonogenic inhibition (SF2=0.224: D0=97 cGy and SF2=0.080: D0=38 cGy) and apoptosis. It has been reported that curcumin inhibits TNF-α-induced NFκB activity that is essential for Bcl-2 protein induction. In PC-3 cells, radiation upregulated TNF-α protein leading to an increase in NFκB activity resulting in the induction of Bcl-2 protein. However, curcumin in combination with radiation treated showed inhibition of TNF-α-mediated NFκB activity resulting in bcl-2 protein downregulation. Bax protein levels remained constant in these cells after radiation or curcumin plus radiation treatments. However, the downregulation of Bcl-2 and no changes in Bax protein levels in curcumin plus radiation-treated PC-3 cells, together, altered the Bcl2 : Bax ratio and this caused the enhanced radiosensitization effect. In addition, significant activation of cytochrome c and caspase-9 and -3 were observed in curcumin plus radiation treatments. Together, these mechanisms strongly suggest that the natural compound curcumin is a potent radiosesitizer, and it acts by overcoming the effects of radiation-induced prosurvival gene expression in prostate cancer.

Introduction

Prostate cancer is the cancer of second largest incidence among the male populations in the US, and the incidence has been increasing rapidly in the recent years (Greenlee et al., 2000). According to a WHO report, 36% of the prostate cancer patients of the world, as in 2000, belong to US population (Wilkinson et al., 2002). Prostate cancer cells are only modestly responsive or even unresponsive to the cytotoxic effects of chemotherapeutic agents or radiotherapy. Increased concentrations of cytotoxic drugs and higher dosages of irradiation fail to improve the response to therapy and it leads to resistance to apoptosis in prostate cancer cells. Thus, it is imperative to identify anticancer agents that are nontoxic and highly effective to induce cell death preferentially in tumor cells. Compounds occurring naturally in the human diet may be devoid of toxicity. Curcumin (Singh et al., 1996) is a major chemical component of turmeric (Bhaumik et al., 1999) and is used as a spice to give a specific flavor and yellow color in Asian food (Sharma, 1976). It is also used as a cosmetic as well as in some medical preparation. Curcumin has been reported to have several pharmacological effects including antitumor, anti-inflammatory and antioxidant properties (Sharma, 1976; Huang et al., 1991). An epidemiological study revealed that low incidence of bowl cancer in Indians can be part in attributed to the presence of natural additives like curcumin in Indian cookery (Mohandas and Desai, 1999). Curcumin is inhibitory to a broad range of tumors such as mammary tumor, duodenal and colon cancer and TPA-induced skin tumors in mice (Huang et al., 1998).

Curcumin has a potent role in inhibiting cellular migration and curcumin treatment of DU-145 cells suppressed the constitutive activation of both NF-κB and AP-1 (Mukhopoadhyay et al., 2001). The molecular mechanism of NFκB inhibition by curcumin is unclear, but involved inhibition of IκB degradation (Jobin et al., 1999; Mukhopadhyay et al., 2001). In addition, the antiproliferaive activity of curcumin may also relate to its ability to block activation of RAS protein by inhibiting farnesyl protein transferase (Jiang et al., 1996). Curcumin significantly inhibits prostate cancer growth (Dorai et al., 2001) and has the potential to prevent the progression of this cancer to its hormone-refractory state (Huang et al., 1998). Curcumin induced apoptosis in both androgen-dependent and androgen-independent prostate cancer cells, by interfering with the signal transduction pathways and prevent the progression of the tumor to the hormone-refractory state (Singh and Aggarwal, 1995).

It has been shown that curcumin inhibits NFκB activation that in turn downregulates endogenous bcl-2 and baxxL protein (Mukhopadhyay et al., 2001). These observations lead us to hypothesize that curcumin can potentially inhibit radiation-induced prosurvival factors such as NFκB activation and Bcl-2 expression. To ascertain this hypothesis, we performed the experiments using androgen-independent, p53-null PC-3 cells. Our results show that curcumin in combination with radiation inhibits TNF-α-mediated NFκB activity, resulting in Bcl-2 protein downregulation in PC-3 cells. Also, curcumin enhanced radiation-induced apoptosis by releasing cytochrome c and activated caspases in combinations with radiation in PC-3 cells.

Results

Radiation-induced resistance through upregulation of prosurvival factors in prostate cancer cells

Resistance to radiation or chemotherapy may be due to interference of apoptotic pathways in cancer treatment. NFκB activation is thought to exert antiapoptotic effects in most cancer cells. In some cell types, the antiapoptotic effects of TNF-α appears to be mediated by the upregulation of NFκB activity (Beg and Baltimore, 1996; Wang et al., 1996), resulting in induction of bcl-2 gene expression that causes resistance to treatments in cancer cells (Chendil et al., 2002; Inayat et al., 2002). Radiation caused an induction of TNF-α protein expression (Figure 1a), NFκB activity (Figure 1b) and Bcl-2 upregulation (Figure 1c) in PC-3 cells. The peak induction of TNF-α protein expression was observed at 48 h, and these results show that radiation induced prosurvival factors in p53-null prostate cancer cells.

Figure 1
Figure 1

Ionizing radiation induces TNF-α protein, NFκB activity and Bcl-2 protein in PC-3 cells. (a) TNF-α protein estimation in a medium of untreated and irradiated PC-3 cells by enzyme-linked immunosorbent assay (ELISA). Error bars represent s.e.m. of two separate experiments performed in triplicate. (b) The effect of radiation on DNA binding activity of NFκB in PC-3 cells. EMSAs of NFκB binding complexes from PC-3 cells with 5 Gy of radiation. Lanes 2–5, positive control nuclear was used for appropriate experimental controls. Arrows indicate the position of super shift, NFκB complex band shift nonspecific binding and free probe. (c) Western blot analysis of Bcl-2 protein induction in PC-3 cells. Whole cell protein extracts were prepared from PC-3 cells that were left untreated (UT) or exposed to a 5 Gy dose and incubated for the indicated time interval. The blot was probed with an antibody for Bcl-2 or β-actin

Anti-TNF-α neutralizing antibody inhibits radiation-induced NFκB activity leading to repression of Bcl-2 protein

The protective responses of cells against radiation are DNA repair, activation of prosurvival transcription factors and induction of antiapoptotic genes. One of the prosurvival transcription factor, NFκB, is activated by ionizing radiation (Brach et al., 1991). NFκB activates several downstream target genes such as Bcl-2 (Dixon et al., 1997; Tamatani et al., 1999), which is responsible for the protection of cells against radiation-induced apoptosis. The activation of NFκB by radiation also depends on radiation-induced TNF-α protein (Baldwin, 1996; Bierhaus et al., 1997). Moreover, NFκB is known to be a cell survival and antiapoptotic molecule (Beg and Baltimore, 1996; Van Antwerp et al., 1996). To test whether TNF-α induction by radiation is necessary for NFκB activation and Bcl-2 upregulation, we performed Western blot analysis of cells either left untreated or treated with anti-TNF-α neutralizing antibody plus radiation. As shown in Figure 2a, DNA binding activity of NFκB increased approximately sixfolds in these cells treated with recombinant TNF-α protein. Whereas, the radiation-induced NFκB was inhibited when PC-3 cells were exposed to anti-TNF-α neutralizing antibody (Figure 2a). Similarly, recombinant TNF-α protein induced Bcl-2 expression in these cells while anti-TNF-α neutralizing antibody repressed the radiation-induced Bcl-2 expression (Figure 2b). These results suggest that radiation-induced NFκB activity depends on radiation-induced expression of TNF-α. Altogether, these observations indicate that radiation induces TNF-α that in turn triggers NFκB activation and Bcl-2 induction in PC-3 cells.

Figure 2
Figure 2

Neutralizing antibody to TNF-α or AD5-IκB super-repressor inhibits radiation-induced NFκB activity leading to repression of Bcl-2 protein in PC-3 cells. (a) EMSAs of NFκB binding complexes from PC-3 cells were left untreated or treated with recombinant TNF-α and/or 5 Gy of radiation with neutralizing anti-TNF-α antibody. Nuclear cell extracts (5 μg) from untreated or irradiated cells were incubated with 32p-labeled NFκB DNA probe, followed by analysis of DNA binding activities. Arrows indicated the position of NFκB complex bindings. (b) Whole cell protein extracts were prepared from PC-3 cells that were left untreated (UT) or treated with recombinant TNF-α and/or irradiated at 5 Gy and incubated for the indicated time interval. The blot was probed with an antibody for Bcl-2 or β-actin

Curcumin inhibits radiation-induced prosurvival factors in PC-3 cells

Previously, it has been shown that curcumin inhibits TNF-α-mediated NFκB activation. In addition, curcumin downregulates endogenous bcl-2 and baxxL protein level (Mukhopadhyay et al., 2001). In order to inhibit the radiation-induced antiapoptotic function in cells, we treated PC-3 cells either with curcumin or radiation alone or in combination. Interestingly, curcumin inhibited radiation-induced TNF-α protein expression (Figure 3a) and resulted in downregulation of NFκB activation (Figure 3b) and Bcl-2 protein expression (Figure 3c). Together, these data strongly suggest that curcumin is a potent inhibitor of radiation-induced prosurvival factors in PC-3 cells.

Figure 3
Figure 3

Curcumin downregulates radiation-induced NFκB activity and Bcl-2 protein expression. (a) TNF-α protein estimation in a medium of untreated/ftreated with curcumin (5 μM) or radiation (5 Gy) or in combination (curcumin 2 μM plus radiation (5 Gy)) of PC-3 cells by ELISA. Error bars represent s.e.m. of two separate experiments performed in triplicate. (b) The effect of Curcumin on radiation-induced NFκB activity in PC-3 cells. Arrows indicated the position of super shift, NFκB complex, nonspecific binding and free probes. (c) Western blot analysis of Bcl-2 expression on PC-3 treated with curcumin with or without radiation. Whole cell protein extracts were prepared from PC-3 cells that were left untreated (UT) or treated with or with out curcumin or exposed to a 5 Gy dose. The blot was probed with antibodies for Bcl-2, or β-actin

Curcumin enhance radiation-induced clonogenic inhibition in PC-3 cells

PC-3 cells conferred enhanced resistance to radiation since curcumin inhibits radiation-induced prosurvival factors such as NFκB and Bcl-2. Effects of curcumin either alone or in combination with radiation on cell survival studied with colony-forming assay. Curcumin enhanced significantly the radiation-induced clonogenic inhibition (SF2=0.224: D0=97 cGy and SF2=0.080:D0=38 cGy at 2 and 4 μM concentrations) compared to cells treated with curcumin alone (Figure 4a) or radiation alone (SF2=0.635; D0=231 cGy) (Figure 4b). Interestingly, a significant enhancement in the radiosensitizing effect of curcumin was observed at 2 and 4 μM concentrations. These results indicate that the natural compound curcumin inhibited the growth of PC-3 cells and significantly enhanced the effect of radiation (Table 1).

Figure 4
Figure 4

Curcumin enhance radiation-induced clonogenic inhibition in PC-3 cells: (a) Curcumin alone or (b) radiation or in combination with 2 or 4 μM concentration of curcumin induced clonogenic inhibition of PC-3 cells. Cell survival curve of PC-3 cells with treatments as assayed by colony-forming ability and analysed by Single Hit Multi-Target (SHMT) model curve fit. The data shown are representative of the combined mean of two independent experiments

Table 1: Radiation inactivation estimates of PC-3 cells treated with curcumin or radiation alone or in combination

Curcumin enhances radiation-induced apoptosis in PC-3 cells

Terminal transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining was performed with or without curcumin/radiation after 24 and 48 h of treatment to determine the induction of apoptosis. By TUNEL assay, the incidence of apoptosis after 24 and 48 h of radiation over the untreated population was 2.61 and 4.88% compared to curcumin alone treated cells (7.23 and 11.56%, respectively). The combination of curcumin and radiation significantly enhanced induction of apoptosis in these cells after 24 h (21.39%) and 48 h (27.57%) of treatment (Figure 5a). By flow cytometry assay, using MC-540 and Hoechst 342 staining, the incidence of apoptosis after 24 and 48 h of radiation over the untreated population was 3.81 and 6.25%, compared to curcumin alone treated cells (9.6 and 13.22%, respectively). However, with combination of radiation and curcumin the incidence of apoptosis after 24 and 48 h was 18.31 and 29.90%, respectively (Figure 5b). Thus, these results demonstrate that curcumin significantly enhanced the radiation-induced apoptosis in PC-3 cells.

Figure 5
Figure 5

Curcumin enhances radiation-induced apoptosis in PC-3 cells: (a) Quantification of apoptosis by TUNEL method. (b) Quantification of apoptosis in by Hoechst 33342 (Ho342) and merocyanin 540 (MC540) staining. The error bar represents s.e. and the data shown are representative of the combined mean from two independent experiments

Curcumin downregulates radiation-induced Bcl-2 protein expression in PC-3 cells

After confirming that curcumin inhibits radiation-induced prosurvival factors in PC-3 cells, we performed Western blot analysis for Bcl2 and Bax protein expression after treating PC-3 cells with curcumin or radiation or in combination. Bax and Bcl-2 are two discrete members of the gene family involved in the regulation of cellular apoptosis. Interestingly, no change in the level of bax protein was observed after the combined treatment (Figure 6). Since curcumin downregulates radiation-induced Bcl-2 and no changes in the bax protein levels were observed, Bcl2 : Bax ratio changed and it may have caused the induction of apoptosis in PC-3 cells.

Figure 6
Figure 6

Curcumin downregulated radiation-induced Bcl-2 protein in PC-3 cells: Whole cell protein extracts were prepared from PC-3 cells that were left untreated or treated with radiation or curcumin or in combination and incubated for the indicated time interval. The blot was probed with antibodies for Bcl-2, Bax and β-actin

Curcumin induced cytochrome c release and caspases activation in PC-3 cells

To confirm the involvement of the mitochondrial pathway of apoptosis, we analysed the activation of cytochrome c release from the mitochondria by Western blot analysis. It is known that cytochrome c releases from mitochondria into the cytosol and binds to the apoptotic protease activating factor (Apaf) complex and triggers the activation of procaspase-9 to the active caspase-9 (Reed, 1997). As shown in Figure 7, a marked fraction of the cytochrome c was released from the mitochondria, of curcumin and in combination with radiation-treated cells at 3 h, and the release was more pronounced at 6 h.

Figure 7
Figure 7

Curcumin induced cytochrome c release in PC-3 cells: Cytochrome c was assessed in untreated, treated with radiation or curcumin or in combination after 3 and 6 h in PC-3 cells, and cytosol and mitochondria extracts were extracted. Western blot analysis was used to determine the cytochrome c expression in both cytosol and mitochondria extracts. Loading control β-actin antibody was used for cytosol extracts

Caspases play a pivotal role in the execution of programmed cell death (Janicke et al., 1998; Juo et al., 1998; Kuida et al., 1998; Earnshaw et al., 1999), and in particular, we evaluated caspase-9 activity because it represents the apical caspase of the mitochondrial (intrinsic) pathway (Kuida et al., 1998). Caspase-3 activation has been shown to be one of the most important cell executioners for apoptosis (Janicke et al., 1998). A marked time-dependent increase in the activities of caspase-9 and -3 was observed in cells treated with 5 μM curcumin or in combination of curcumin (2 μM) with radiation. However, the sequential pattern of activation of these caspases was markedly different. There was significant activation of caspase-9 as early as 3 h of treatment, and the activity continued to increase till 24 h of the assay (Figure 8a). On the other hand, significant activation of caspase-3 activity was seen at 12 h after treatment (Figure 8b). The increase in the release of cytochrome c was in agreement with the data showing the continued increase in caspase-9 activity after 3 h treatment. The close association of the release of cytochrome c from mitochondria with the concurrent increase in caspase-9 provide evidence that curcumin induces apoptosis in PC-3 cells through the mitochondrial pathway.

Figure 8
Figure 8

Curcumin induce caspase-3 and caspase-9 activation in PC-3 cells: PC-3 cells were left untreated or treated with radiation or curcumin or in combination and and cell lysates prepared for time interval indicated. The enzymatic activity of cell lysates towards tetrapeptide chromogenic substrates, (a) DEVD-pNA (for caspase 3-like) and (b) LEHD-pNA (for caspase 9) was determined. Caspase activities are expressed as fold change of control and presented as mean±SE of two samples

Curcumin radiosensitizes PC-3 cells by inducing G2/M block of cell cycle

To understand the mechanism that curcumin causes enhanced radiation-induced clonogenic inhibition, we performed flow cytometry to analyse the cell cycle changes induced by radiation in PC-3 cells. Curcumin caused a strong G2/M block, which is an important phase sensitive to radiation. Curcumin-treated cells showed 32.55% of G2/M block at 12 h, 48.10% at 24 h, 42.75% at 48 h and 35.89% at 72 h(Table 2). Thus, the G2 block in curcumin-treated cells when combined with radiation caused enhanced radiosensitization, whereas radiation- or curcumin-treated cells showed no significant G2/M block.

Table 2: Effects of curcumin or ionizing radiation or in combination with curcumin and radiation on cell cycle distribution

Discussion

Our studies showed that radiation induces prosurvival factors such as increased NFκB activity and Bcl-2 upregulation in PC-3 cells. Ionizing radiation induce NFκB activation (Hallahan et al., 1989; Van Antwerp et al., 1996) and it plays an important role in inhibiting TNF-α or chemotherapy-induced apoptosis (Wang et al., 1996; Plummer et al., 1999). TNF-α is also a potent inducer of NFκB activity (Hallahan et al., 1989; Van Antwerp et al., 1996). The multiplicity of mechanisms of NFκB activation and its role in inhibition of antiapoptotic function is more complex. The antiapoptotic target genes for NFκB includes Bcl-2 (Tamatani et al., 1999), Bcl-xL (Dixon et al., 1997; Tamatani et al., 1999) and Bcl-2 homologue A1/Bfl-1(Wang et al., 1999). We reported that ectopic overexpression of Bcl-2 in prostate cancer cells showed enhanced radiation resistance and inhibition of apoptosis in prostate cancer cells and other tumor cell types (Hockenbery et al., 1990; Sentman et al., 1991). Induction of prosurvival and antiapoptotic genes strongly suggests that PC-3 cells harbor a tight regulatory loop that inhibits the cell killing effects of ionizing radiation.

Curcumin has been reported to be a potent antiproliferative agent for many tumor types (Rao et al., 1995; Sikora et al., 1997) and it acts as a proapoptotic agent in a variety of cancer cell lines (Kuo et al., 1996; Khar et al., 1999). Exposure of PC-3 cells to curcumin inhibited radiation-induced Bcl-2 expression, indicating that radiation-induced TNF-α is necessary to activate NFκB, which is required for the induction of Bcl-2 protein. This is the first report showing that curcumin inhibits endogenous TNF-α as well as radiation-induced TNF-α protein expression in PC-3 cells. Inhibitory effects of curcumin on NFκB activation have been documented in prostate cancer cells (Mukhopadhyay et al., 2001), mouse fibroblast cells (Huang et al., 1991), human leukemia cells (Singh and Aggarwal, 1995) and human colon epithelial cells (Plummer et al., 1999). Curcumin inhibits NFκB activation by inhibiting IκBα phosphorylation that is necessary to export NFκB from cytosol to nucleus and to activate its target genes. However, this is the first documented report demonstrating that curcumin is a potent radiosenstizer in prostate cancer cells and this sensitization is conferred by the inhibition of radiation-induced prosurvival factors such as NFκB and Bcl-2. Hence, the downregulation of endogenous and radiation-induced bcl-2 protein expressions in PC-3 cells will have significant therapeutic benefits in a majority of prostate cancer patients, since bcl-2 protein is overexpressed in these patients. Curcumin also inhibits cell proliferation induced by growth factors. Correlation between inhibition of cell proliferation and different phases of cell cycle by curcumin has been reported in the literature (Chen and Huang, 1998; Chen et al., 1999). Curcumin induces cell cycle arrest in G2/M phase in breast cancer cells. These findings well correlated with our results since curcumin-treated cells showed G2/M arrest of cell cycle, which is sensitive to radiation and therefore this leads to enhanced radiosensitization.

Activated protein (AP-1) is a transcription factor activated by UV radiation, phorbol ester and asbestosis (Shaulian and Karin, 2002). AP-1 promotes several cellular genes that are responsible for cell proliferation and also transformation of preneoplastic to neoplastic state (Dong et al., 1995). Several reports show that curcumin suppresses AP-1 activation; in our study also curcumin inhibits endogenous and radiation-induced AP-1 in PC-3 cells (data not shown).

We found that curcumin induced apoptosis by the activation of the downstream caspase-9, which has been shown to play an important role in apoptosis induced by several conditions (Ohta et al., 1997; Mow et al., 2001). In this study, caspase-9 activation was preceded by the activation of caspase-3, the apical caspase of the intrinsic mitochondrial pathway of apoptosis. Similarly, curcumin induced the release of cytochrome c into the cytosol after 3 h, and this release markedly increased after 6 h of treatment. Our results show curcumin downregulates radiation-induced bcl-2 protein expression that suggests that this protein is involved in the release of cytochrome c from mitochondria.

In recent clinical trails, curcumin was given a dosage of 8000 mg/day, and the peak serum concentration of 1.77±1.87 μM after 2 h of intake of curcumin has been reported (Cheng et al., 2001). In our results, a significant enhancement of radiosensitizing effect was observed at 2 and 4 μM concentrations of curcumin by colony-forming assays. Hence, it is possible to achieve 2 μM concentration of curcumin in the serum concentration by consumption of 8000 mg/day of curcumin and this dose of curcumin in the serum will enhance the radiation effect in prostate cancer patients.

Apoptotic assays indicate that radiation caused significantly enhanced apoptosis in curcumin-treated cells. These results indicate that the natural compound, curcumin, at nontoxic doses inhibited the growth and induced apoptosis in PC3 cells and significantly enhanced the effect of radiation. It has been shown that curcumin induce apoptosis either by mitochondrial-dependent or mitochondrial-independent mechanism depending on the cell types. Curcumin-induced mitochondrial-independent apoptosis has been shown in breast cancer cell lines (Mehta et al., 1997), basal cell carcinomas (Jee et al., 1998) and T-Jurkat cells (Piwocka et al., 1999).

In conclusion, curcumin, a major active component of turmeric, has been reported to induce growth inhibition and induce apoptosis in many cancer cell types. In this study we, for the first time, report that curcumin is a potent radiosensitizer that inhibits growth of human prostate PC-3 cancer cells and downregulates radiation-induced prosurvival factors and enhance radiation-induced sensitivity in PC-3 cells.

Materials and methods

Cell line

Human prostate cancer cells (PC-3 cells) were obtained from the American Type Culture Collection and maintained as adherent monolayer cultures in RPMI-1640 medium supplemented with 10% fetal bovine serum.

Curcumin

Curcumin (99% purity) (E,E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) was purchased from Sigma Chemical Co. (St Louis, MO, USA) and stored as 100 mM stock solution in DMSO, protected from light at −20°C. At 24 h after plating the cells, the medium was removed and replaced with fresh medium containing DMSO or medium containing different concentrations of curcumin in RPMI medium. For treatments, cells were left untreated or treated with radiation (5 Gy) alone or curcumin (5 μM) alone or in combination of curcumin with radiation. For combination treatment, curcumin (2 μM) was added to the cultures 2 h prior to radiation (5 Gy).

Irradiation

A 100 kV industrial X-ray machine (Phillips, Netherlands) was used to irradiate the cultures at room temperature. The dose rate with a 2 mm Al plus 1 mm Be filter was 2.64 Gy/min at a focus-surface distance of 10 cm.

Western blot analysis

Total protein extracts from untreated cells or cells treated with curcumin alone or irradiated alone or in combination (curcumin plus radiation) at various time intervals were subjected to Western blot analysis as described (Chendil et al., 2002) using Bcl-2 monoclonal antibody (sc-509) (Santa Cruz, CA, USA), Bax monoclonal antibody (sc-493) (Santa Cruz, CA, USA), cytochrom c monoclonal antibody (Biovision, Inc., CA, USA), or for loading control the β-actin antibody (Sigma Chemical Co, St Louis, MO, USA) were detected using the chemiluminescence method.

Colony-forming assay

For clonogenic cell survival studies, two different cell concentrations in quadruplet sets were used for each treatment. Cell lines were left untreated or exposed to 0.5–6 Gy dose of radiation or treated with various concentration of curcumin or for combinatation treatment curcumin (2 μM or 4 μM) was added to the cultures 2 h prior to radiation (5 Gy). After incubation for 10 or more days, each flask was stained with crystal violet and the colonies containing more than 50 cells were counted. The surviving fraction (SF) was calculated as a ratio of the number of colonies formed and the product of the number of cells plated and the plating efficiency. The curve was plotted using XY log scatter (Delta Graph®4.0), and by using the formula of the SHMT model, the D0 was calculated. D0 is the dose required for reducing the fraction of cells to 37%, indicative of single-event killing. SF2 is the survival fraction of exponentially growing cells that were irradiated at the clinically relevant dose of 2 Gy.

Flow cytometry

Flow cytometry was performed as described earlier (Chendil et al., 2002). Untreated and treated cells (1 × 106) were washed in phosphate-buffered saline (PBS) and fixed in ice-cold ethanol. Fixed cells were pelleted and resuspended in 500 μl of PBS. RNA was eliminated by treating cells with RNAse A (Sigma, St. Louis, MO, USA). Then, the cells were stained by propidium iodide in PBS and analysed for cell cycle phases by flow cytometry, a FACStar calibur (Becton Dickinson) cell sorter.

Quantification of apoptosis

Apoptosis was quantified by TUNEL staining and flow cytometry. The ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD, USA), which detects DNA strand breaks by TUNEL, was used as described (Ahmed et al., 1996). Briefly, cells were seeded in chamber slides and the next day the cells were left untreated or treated with curcumin or radiation or in combination. After 24 and 48 h, the DNA was tailed with digoxigenin-dUTP and conjugated with an anti-digoxigenin fluorescein. The specimen was counterstained with propidium iodide and antifade. The stained specimen was observed in triple band-pass filter using Nikon-microphot epifluorescence microscope. To determine the percentage of cells showing apoptosis, four experiments in total were performed, and approximately 1000 cells were counted in each experiment. For flow cytometry, cells were lifted by using nonenzymatic cell dissociation medium (Sigma) and washed with PBS and stained with Hoechst (Ho342) and merocyanine (MC540) and analysed by flow cytometry using a FACStar Plus cell sorter as described (Ahmed et al., 1996).

Electrophoretic mobility shift assay (EMSA)

Nuclear extracts were prepared from untreated and treated cells and EMSA was preformed as described previously (Chendil et al., 2002) with some modification. Briefly, cells were scraped and washed with cold PBS and repelleted. The pellet was suspended in 1 ml of Icecold buffer A (10 mM HEPES pH 7.8, 2 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 10 μg/ml of aprotinin, 0.5 μg/ml leupeptin, 3 mM PMSF and 3 mM dithiothreitol) for 5 min on ice. The crude nuclei were pelleted by centrifugation for 5 min. The crude nuclei pellet was suspended in 50 μl of buffer B (10 mM HEPES, pH 7.8, 2 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 10 μg/ml of aprotinin, 0.5 μg/ml leupeptin, 3 mM PMSF, 3 mM dithiothreitol and 10% NP-40). After 20 min of incubation on ice, the suspension was centrifuged, and supernatant was collected. Equal volume of cold buffer C (50 mM HEPES, pH 7.4, 300 mM NaCl, 50 mM KCl, 0.1 mM EDTA, 3 mM PMSF, 3 mM DTT and 10% (v/v) glycerol) was added to the supernatant and incubated on ice for 5 min with intermittent vortexing. The extracts were then centrifuged for 10 min and the supernatant was divided into aliquots and frozen at –80°C.

Analysis of DNA binding by EMSA was performed using 2 mg of poly(dI-dC) (Sigma Chemical Co, St Louis, MI, USA) as nonspecific competitor DNA. The binding reactions contained 10 000 c.p.m. of 32P-labeled double-stranded oligonucleotide probe with a high affinity for NFκB binding (Promega, Madison, WI, USA). For supershift experiments, anti-p65 antibody was incubated with binding buffer and nuclear extract for 1 h prior to adding the oligo probe. Binding reactions were electrophoresed on a 4% PAGE in 0.5 × TBE buffer to separate the bound and unbound probe.

Caspase activity

To measure the activity of caspases-3 and -9 in PC-3 cells, a fluorimetric assay was used according to the instruction of the manufacturer (Biovision, CA, USA). Briefly, cells were left untreated and treated either with curcumin or radiation or in combination. Cells were collected and resuspended in cold lysis buffer and incubated for 10 min on ice. In all, 50 μl of 2 × reaction buffer wad added and incubated for 2 h at 37°C with fluorogenic substrates, DEVD-AFC (caspase-3) and LEHD-AFC (caspase-9) in a reaction buffer. The release of fluorocrome AFC was measured at 400 nm excitation and 505 nm emissions using a fluorescence spectrophotometer.

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Acknowledgements

This work is supported by DOD PC020620 to DC.

Author information

Affiliations

  1. Department of Clinical Science, University of Kentucky, Lexington, KY 40536, USA

    • Damodaran Chendil
    • , Rama S Ranga
    •  & David Meigooni
  2. Department of Radiation Medicine, University of Kentucky, Lexington, KY 40536, USA

    • Sabapathi Sathishkumar
    •  & Mansoor M Ahmed

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Correspondence to Damodaran Chendil.

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https://doi.org/10.1038/sj.onc.1207284

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