Abstract

Curcumin, the yellow pigment in the spice turmeric, has potent chemopreventive activities that involve diverse molecular pathways. It is widely believed that curcumin pro-apoptotic properties are mediated by downregulation of NF kappa B (NFκB). The p65/RelA subunit of NFκB may influence cell death, in part by activation of NFκB anti-apoptotic target genes including X-linked inhibitor of apoptosis ( XIAP ), A20 , bcl-xL and inhibition of sustained activation of c-Jun N-terminal kinase (JNK). We have shown previously that curcumin inhibits NFκB, activates JNK and promotes apoptosis in HCT116 colorectal cancer cells. Here, we show that forced overexpression of p65 does not affect curcumin-induced JNK activation. Indeed, overexpression of p65 enhanced curcumin-mediated apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and poly(ADP-ribose) polymerase (PARP) cleavage. This potentiating effect of p65 upon curcumin-mediated apoptosis was reversed by transfection of cells with an IκB super-repressor (ΔNIκB). Curcumin treatment inhibited expression of NFκB anti-apoptotic target genes in mock-transfected and in p65-overexpressing HCT116 cells, although expression levels remained higher in the latter. Taken together, these results show that curcumin-mediated activation of JNK or induction of apoptosis does not require inhibition of p65. Furthermore, curcumin/p65 synergy in promotion of apoptosis cannot be attributed to active repression of NFκB anti-apoptotic genes.

Introduction

Curcumin (diferuloylmethane) is a naturally occurring polyphenolic pigment, isolated from the rhizomes of the plant Curcuma longa (Linn.), and is commonly used as a colouring and flavouring agent in food products. Curcumin has antioxidant ( 1 ) or anti-inflammatory properties ( 2 ) and inhibits tumourigenesis in various tissues in animal models ( 36 ). These anti-tumourigenic effects are associated with induction of apoptosis ( 3 , 79 ). Curcumin has diverse molecular effects ( 1014 ) including inhibition of nuclear factor kappa B (NFκB) in colon cancer and other cells ( 1517 ).

The NFκB family of transcription factors consists of five Rel-domain-containing proteins: p65/RelA, RelB, c-rel, p50/NFκB1 and p52/NFκB2. In most cells the predominant form of active NFκB consists of a p65/p50 heterodimer although other homo/heterodimers also form ( 18 ). NFκB has been shown to promote cell survival through induced expression of anti-apoptotic genes such as bcl-xL ( 19 ) and X-linked inhibitor of apoptosis ( XIAP ) ( 20 ). In physiological conditions, NFκB is sequestered in inactive form in the cytoplasm by inhibitory IκB proteins. However, constitutive activation of NFκB has been observed in several cancer cell types and may contribute to apoptosis resistance ( 21 , 22 ). The p65 subunit of NFκB may promote cell survival by induction of growth arrest and DNA damage-inducing gene beta ( GADD45β ) ( 23 ) or XIAP ( 24 ) leading to inhibition of c-jun N-terminal kinase (JNK) activity. When p65 is inhibited, cells show sustained activation of JNK in response to a stimulus such as tumour necrosis factor α (TNFα) and undergo apoptosis. Inhibition of NFκB may also upregulate GADD45α , which in turn activates JNK ( 25 ).

We have shown previously that curcumin induces sustained activation of JNK, which promotes apoptosis in human HCT116 colon cancer cells ( 26 ). As HCT116 cells exhibit constitutive NFκB activity, which in turn may be inhibited by curcumin ( 26 ), we hypothesized that sustained activation of JNK and induction of apoptosis by curcumin may occur in these cells as a result of inhibition of NFκB transcriptional activity. In order to test this hypothesis we sought to override the inhibitory effect of curcumin on constitutive NFκB activity by overexpressing p65 in HCT116 cells. While p65 may impede apoptosis, growing evidence suggests that it may also potentiate cell death depending upon the nature of the apoptotic stimulus ( 2729 ). Indeed, p65 can be both an activator and repressor of its target genes depending upon the manner in which it is induced ( 30 ). Here, we show that overexpression of p65 has no effect on curcumin-induced JNK activation but potentiates curcumin-induced apoptosis. Furthermore, enhancement of curcumin-induced apoptosis by p65 overexpression cannot be attributed to active repression of NFκB anti-apoptotic target genes, XIAP , bcl-xL or A20 .

Materials and methods

Materials

Curcumin was 80% pure (98% curcuminoid content) and was obtained from Sigma-Aldrich (Poole, Dorset, UK). All antibodies were obtained from New England Biolabs (Hitchin, Hertfordshire, UK), except for anti-β-actin, which was purchased from Sigma-Aldrich. All other reagents were widely available commercially.

Plasmids

The NFκB-responsive luciferase reporter construct, 3EnhConALuc ( 31 ), was kindly provided by Prof. Ron Hay, University of St Andrews. The p65 expression vector, pCMV-p65 ( 32 ), was a kind gift from Dr Warner Greene, University of California, San Francisco. The pCMV-ΔNIκBα construct, an N-terminal deletion mutant of IκBα ( 33 ), was kindly provided by Dr Dean Ballard, Vanderbilt University, Nashville.

Cell culture

Human colon cancer cell line HCT116 was obtained from the European Collection of Cell Cultures (ECACC; Salisbury, UK) and grown as monolayers at 37°C in a humidified atmosphere with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) heat inactivated foetal calf serum, glutamine (2 mM), penicillin (50 U/ml) and streptomycin (50 µg/ml). Curcumin was dissolved in dimethyl sulfoxide (DMSO) and used in the previously determined optimal concentration of 35 µM ( 26 ). Controls were treated with 0.1% DMSO alone. All treatments were carried out on cells at 60–80% confluence.

Transient transfections and luciferase assay

Transfection was performed in 24-well plates using Lipofectin reagent (Invitrogen, Paisley, UK) according to the manufacturer's instructions. Briefly, cells were grown in 24-well plates and transfected with the appropriate vector (250 ng) the following day. After 5 h the transfection mix was removed and replaced with complete medium. Cell treatments were then carried out 24 h post-transfection as indicated. Luciferase activity was determined using the luciferase assay system with reporter lysis buffer from Promega, Southampton, UK. Results, which were expressed as relative luciferase activity, were corrected for differences in transfection efficiency by co-transfection with a pCMV-βGal construct followed by β-galactosidase assay (Promega).

Western blotting

Cell monolayers were washed with phosphate-buffered saline (PBS) and lysed directly into SDS–PAGE loading buffer. Soluble protein (30 µg) was resolved by SDS–PAGE and transferred to nitrocellulose membrane. For all experiments, equal protein loading was confirmed by staining the nitrocellulose with Ponceau S or by probing with an antibody to β-actin. Membranes were probed with the appropriate primary antibodies. Reactions were visualized with a suitable secondary antibody conjugated to horseradish peroxidase (HRP) (Dako, Ely, UK) using an enhanced chemiluminescence system (Santa Cruz, California, US).

Cell viability assay

Cells were seeded at 1 × 10 4 cells/well in 96-well plates and transfected as described above. Cells were treated with curcumin (35 µM) for 24 h and then 5 mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; 20 µl] was added and incubated for 1 h. Cells were then incubated overnight with 20% SDS and the formation of coloured formazan dye was assessed colorimetrically at 550 nm. Results are expressed as percentage loss of cell viability compared with control.

Semi-quantitative reverse transcriptase–polymerase chain reaction (RT–PCR)

Total RNA was prepared using Trizol reagent (Gibco, Paisley, Scotland). RT–PCR was carried using a One-Step RT–PCR kit (Qiagen, Crawley, UK) in a final volume of 12.5 µl according to the manufacturer's instructions. Primer sequences were as follows: bcl-xL forward, 5′-CCCAGAAAGATACAGCTGG; bcl-xL reverse, 5′-GCGATCCGACTCACCAATAC; XIAP forward, 5′-CAGATAGGCTTAACAAATGGAGCT; XIAP reverse, 5-ATAGTGTCCCGCCACTTGCATT; A20 forward, 5′-ACTCTTTGGGTTATTACTGTC; A20 reverse, 5′-ACAGGTTATTATTATTTAGCC. Reactions were normalized by evaluating the level of amplification of the glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) transcript using the following primers: GAPDH forward, 5′-AGTTCAACGGATTTGGTCGTA, reverse, 5′-AAATGAGCCCCAGCCTTCT. For semi-quantitative analysis, each sample was amplified within the exponential phase of the PCR ( bcl-xL and XIAP , 26 cycles; A20 , 28 cycles; GAPDH , 21 cycles). Amplified DNA was electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining.

Statistical analysis

Data are presented as mean ± SD. Comparisons between treatment groups were made with the Student's t -test. Values of P < 0.05 were considered statistically significant.

Results

Overexpression of p65 increases NFκB transcriptional activity in HCT116 cells

HCT116 cells express constitutive NFκB transcriptional activity, which is inhibited by curcumin ( 26 ). In order to confirm that overexpression of p65 resulted in an increase in NFκB activity, we co-transfected cells with pCMV-p65 expression construct and an NFκB-dependent luciferase reporter plasmid ( 31 ). p65 overexpression was confirmed by western blotting ( Figure 1A ). Overexpression of p65 resulted in a significant increase in constitutive NFκB transcriptional activity as measured after 6 and 18 h, respectively ( Figure 1B and C ). Curcumin (35 µM) treatment had no significant effect on NFκB transcriptional activity after 6 h in mock- or p65-transfected cells ( Figure 1B ) but resulted in a significant inhibition after 18 h ( Figure 1C ). NFκB transcriptional activity was significantly higher in p65-transfected cells compared with mock-transfected cells after 6 and 18 h, respectively, even after curcumin treatment ( Figure 1B and C ).

Fig. 1.

Overexpression of p65 increases NFκB transcriptional activity. ( A ) Cells were transfected with pCMV-p65 or empty vector and treated with curcumin (35 µM) for 6 h. Overexpression of p65 was confirmed by western blotting using an anti-p65 antibody using whole-cell lysates. ( B and C ) Cells were co-transfected with a p65 expression construct and an NFκB-responsive luciferase reporter plasmid 3EnhConALuc. Cells were treated with curcumin (35 µM) for 6 h (B), or 18 h (C). Luciferase activity of cell lysates was measured and normalized to β-galactosidase activity obtained by co-transfection with a pCMVβGal internal control plasmid. Results are expressed as the mean ± SD from three separate experiments. * , P < 0.001, significantly different from mock-transfected cells; * , P < 0.001, significantly different from curcumin-treated mock-transfected cells; * , P < 0.05, significantly different from untreated cells.

Fig. 1.

Overexpression of p65 increases NFκB transcriptional activity. ( A ) Cells were transfected with pCMV-p65 or empty vector and treated with curcumin (35 µM) for 6 h. Overexpression of p65 was confirmed by western blotting using an anti-p65 antibody using whole-cell lysates. ( B and C ) Cells were co-transfected with a p65 expression construct and an NFκB-responsive luciferase reporter plasmid 3EnhConALuc. Cells were treated with curcumin (35 µM) for 6 h (B), or 18 h (C). Luciferase activity of cell lysates was measured and normalized to β-galactosidase activity obtained by co-transfection with a pCMVβGal internal control plasmid. Results are expressed as the mean ± SD from three separate experiments. * , P < 0.001, significantly different from mock-transfected cells; * , P < 0.001, significantly different from curcumin-treated mock-transfected cells; * , P < 0.05, significantly different from untreated cells.

Activation of JNK signalling by curcumin is unaffected by overexpression of p65

To determine the effects of overexpression of p65 on JNK activation, we transiently transfected HCT116 cells with p65 or empty vector and assessed whole-cell lysates from curcumin-treated cells by western blotting analysis using an antibody that specifically recognizes the phosphorylated form of JNK. As shown in Figure 2 , curcumin treatment resulted in sustained phosphorylation of both p46 and p54 isoforms of JNK. Western blotting with an antibody that recognizes JNK regardless of its phosphorylation status revealed that overall levels of JNK were unaffected by curcumin. The activation of JNK by curcumin was confirmed by western blotting using an antibody specific to the phosphorylated form of the JNK substrate c-jun. Overexpression of p65 had no significant effect on sustained curcumin-induced activation of JNK or c-jun ( Figure 2 ).

Fig. 2.

Activation of JNK signalling by curcumin is unaffected by overexpression of p65. ( A ) Cells were transfected with pCMV-p65 or empty vector and treated with curcumin (35 µM) for 24 h. JNK activity was determined by western blotting and by assessment of phosphorylated c-jun levels in whole-cell lysates. ( B ) Fold induction of JNK was calculated by densitometric analysis of phospho-JNK western blots. Results are expressed as the mean ± SD from three separate experiments.

Fig. 2.

Activation of JNK signalling by curcumin is unaffected by overexpression of p65. ( A ) Cells were transfected with pCMV-p65 or empty vector and treated with curcumin (35 µM) for 24 h. JNK activity was determined by western blotting and by assessment of phosphorylated c-jun levels in whole-cell lysates. ( B ) Fold induction of JNK was calculated by densitometric analysis of phospho-JNK western blots. Results are expressed as the mean ± SD from three separate experiments.

p65 overexpression enhances curcumin-induced apoptosis

We have previously shown that curcumin induces apoptosis in HCT116 cells ( 26 ). In order to assess the effects of overexpression of p65 on curcumin-induced apoptosis, we transfected cells with p65 or empty vector and then treated with 35 µM curcumin for 24 h. Cell death was assessed by MTT assay and Poly(ADP-ribose) polymerase (PARP) cleavage assay. Figure 3A shows that p65 overexpression resulted in a significant reduction in cell viability after curcumin treatment compared with cells transfected with empty vector. Similarly, p65-overexpressing cells showed increased PARP cleavage in comparison with mock-transfected controls after curcumin treatment ( Figure 3B ), indicating an increase in apoptosis. Basal levels of apoptosis in untreated cells were unaffected by p65 overexpression. Treatment with vehicle (DMSO) alone did not induce apoptosis in either mock- or p65-transfected cells ( Figure 3B ).

Fig. 3.

p65 overexpression enhances curcumin-induced cell death ( A ) and apoptosis ( B ). (A) Cells were transfected with pCMV-p65 or empty vector and treated with curcumin (35 µM) for 24 h. Cell death was assessed by MTT assay. Results, showing percentage cell viability compared with vehicle-treated cells, represent the mean ± SD of three separate experiments. * , P < 0.001, significantly different from mock-transfected cells. (B) Cells were treated as in (A) and apoptosis was assessed by western blotting for PARP cleavage using whole-cell lysates.

Fig. 3.

p65 overexpression enhances curcumin-induced cell death ( A ) and apoptosis ( B ). (A) Cells were transfected with pCMV-p65 or empty vector and treated with curcumin (35 µM) for 24 h. Cell death was assessed by MTT assay. Results, showing percentage cell viability compared with vehicle-treated cells, represent the mean ± SD of three separate experiments. * , P < 0.001, significantly different from mock-transfected cells. (B) Cells were treated as in (A) and apoptosis was assessed by western blotting for PARP cleavage using whole-cell lysates.

IkB reverses the potentiation of curcumin-induced apoptosis after p65 overexpression

In order to confirm that activation of NFκB transcriptional activity was involved in the increase in curcumin-induced apoptosis after overexpression of p65, we transfected cells with a super-repressor form of IκBα (ΔNIκBα) that has been shown to efficiently block the activation of NFκB ( 33 ). As shown in Figure 4A , co-expression of p65 and ΔNIκBα resulted in a potent reduction in NFκB transcriptional activity, in both untreated and curcumin-treated cells, compared with cells transfected with p65 only. MTT and PARP cleavage assays showed that inhibition of NFκB transcriptional activity in cells overexpressing p65 was associated with a decrease in curcumin-induced cell death ( Figure 4B and C ). Cells co-transfected with pCMV4 and ΔNIκBα showed reduced NFκB transcriptional activity compared with transfection with pCMV4 alone, but this did not result in a change in basal levels of apoptosis in untreated cells. Curcumin-induced apoptosis was also unaffected by transfection with pCMV4 and ΔNIκBα, compared with PCMV4 alone. Taken together, these results show that increased NFκB transcriptional activity resulting from overexpression of p65 results in a potentiation of curcumin-induced apoptosis.

Fig. 4.

IκBα reverses the potentiation of curcumin-induced apoptosis after RelA overexpression. ( A ) Cells were co-transfected with pCMV4, p65 and ΔNIκBα expression vectors and an NFκB-responsive luciferase reporter plasmid 3EnhConALuc, as indicated. Cells were treated with curcumin (35 µM) for 18 h, and luciferase activity of cell lysates was measured and normalized to β-galactosidase activity obtained by co-transfection with a pCMVβGal internal control plasmid. Results represent the mean ± SD of three separate experiments. * , P < 0.001, significantly different compared with pCMV4-transfected cells; ** , P < 0.01, significantly different compared with p65-transfected cells, without ΔNIκB. ( B ) Cells were transfected as in (A) and treated with curcumin (35 µM) for 24 h. Cell death was assessed by MTT assay. Results, showing percentage cell viability compared with vehicle-treated cells, represent the mean ± SD of three separate experiments. * , P < 0.001, significantly different from pCMV4-transfected cells. ( C ) Cells were transfected as in (A) and treated with curcumin (35 µM) for 24 h. Apoptosis was assessed by western blotting for PARP cleavage using whole-cell lysates.

Fig. 4.

IκBα reverses the potentiation of curcumin-induced apoptosis after RelA overexpression. ( A ) Cells were co-transfected with pCMV4, p65 and ΔNIκBα expression vectors and an NFκB-responsive luciferase reporter plasmid 3EnhConALuc, as indicated. Cells were treated with curcumin (35 µM) for 18 h, and luciferase activity of cell lysates was measured and normalized to β-galactosidase activity obtained by co-transfection with a pCMVβGal internal control plasmid. Results represent the mean ± SD of three separate experiments. * , P < 0.001, significantly different compared with pCMV4-transfected cells; ** , P < 0.01, significantly different compared with p65-transfected cells, without ΔNIκB. ( B ) Cells were transfected as in (A) and treated with curcumin (35 µM) for 24 h. Cell death was assessed by MTT assay. Results, showing percentage cell viability compared with vehicle-treated cells, represent the mean ± SD of three separate experiments. * , P < 0.001, significantly different from pCMV4-transfected cells. ( C ) Cells were transfected as in (A) and treated with curcumin (35 µM) for 24 h. Apoptosis was assessed by western blotting for PARP cleavage using whole-cell lysates.

Curcumin downregulation of NFκB-responsive anti-apoptotic genes is not enhanced by overexpression of p65

Previous studies have shown that pro-apoptotic effects of p65 may be mediated by active repression of anti-apoptotic gene expression ( 30 ). To assess the expression of such genes in curcumin-treated cells overexpressing p65 we carried out semi-quantitative RT–PCR of XIAP , bcl-xL and A20 genes, which have been shown to be actively repressed by p65 in pro-apoptotic contexts, on RNA prepared from cells transfected with the p65 expression vector or empty vector and treated with curcumin. Figure 5 shows that expression of XIAP and bcl-xL was similar in mock- and p65-transfected cells, whereas A20 was significantly upregulated by p65 overexpression. Curcumin treatment resulted in a reduction in expression of XIAP and bcl-xL to a similar degree in both mock- and p65-transfected cells. A20 expression was reduced by curcumin in mock-transfected cells but was more highly expressed in p65-overexpressing cells at all time points.

Fig. 5.

Curcumin downregulation of the anti-apoptotic NFκB target genes bcl-xL , XIAP and A20 is not enhanced by overexpression of p65. ( A ) Semi-quantitative PCR was performed using primers specific to bcl-xL , XIAP , A20 or a GAPDH control on 100 ng total RNA prepared from cells transfected with pCMV-p65 or empty vector as indicated, and treated with curcumin (35 µM) for the times shown. ( B ) Band intensities were calculated by densitometric analysis and normalized to GAPDH levels. Results, shown as percentage intensity of untreated mock-transfected controls, are presented as the mean ± SD of three separate experiments (i–iii). * , P < 0.05, significantly different from untreated pCMV4-transfected cells, at timepoint zero (0 h). #, P < 0.05, significantly different from untreated p65-transfected cells, at timepoint zero (0 h). ‡, P < 0.05, significantly different from pCMV4-transfected cells at the same timepoint.

Fig. 5.

Curcumin downregulation of the anti-apoptotic NFκB target genes bcl-xL , XIAP and A20 is not enhanced by overexpression of p65. ( A ) Semi-quantitative PCR was performed using primers specific to bcl-xL , XIAP , A20 or a GAPDH control on 100 ng total RNA prepared from cells transfected with pCMV-p65 or empty vector as indicated, and treated with curcumin (35 µM) for the times shown. ( B ) Band intensities were calculated by densitometric analysis and normalized to GAPDH levels. Results, shown as percentage intensity of untreated mock-transfected controls, are presented as the mean ± SD of three separate experiments (i–iii). * , P < 0.05, significantly different from untreated pCMV4-transfected cells, at timepoint zero (0 h). #, P < 0.05, significantly different from untreated p65-transfected cells, at timepoint zero (0 h). ‡, P < 0.05, significantly different from pCMV4-transfected cells at the same timepoint.

Discussion

Pro-apoptotic effects of curcumin are central to its chemopreventive efficacy. Multiple intracellular signalling pathways including NF-kappaB, JNK and caspases interact in the regulation of programmed cell death ( 29 , 30 ). NFκB is an ubiquitous transcriptional control factor, involved in diverse cellular processes. To dissect any NFκB/JNK signalling interactions implicated in curcumin-mediated cell death, the current study overexpressed p65, the major transactivating subunit of NFκB, in HCT116 cells and assessed effects on curcumin-induced JNK activation, NFκB transcriptional activity and apoptosis.

In this study, overexpression of p65 in HCT116 cells resulted in increased p65 protein expression, enhanced NFκB transcriptional activation and upregulation of the NFκB responsive gene, A20 . Curcumin treatment, at 35 µM, which optimally induces apoptosis in HCT116 cells ( 26 ), inhibited NFκB transcriptional activity and expression of NFκB target genes in both mock transfectants and p65-overexpressing cells. Curcumin suppression of NFκB was incomplete in p65 transfectants since activity remained at least 3-fold higher than in mock-transfected cells. Curcumin induced phosphorylation of JNK and c-jun as shown previously ( 26 ) and was unaffected by p65 overexpression. While the precise molecular mechanisms involved in curcumin-mediated activation of JNK remain unclear, upstream signalling pathways that could potentially be implicated include MKK4 and MKK7 kinases ( 34 ), apoptosis-signal-regulating kinase ( 35 ), PKC-δ (protein-regulating kinase-δ) ( 36 ), mixed lineage kinases ( 37 ) and reactive oxygen species ( 38 ). Taken together, these results show that curcumin-induced activation of JNK occurs independently of its inhibition of NFκB transcriptional activity, in HCT116 cells.

Previous studies have shown that curcumin inhibition of NFκB is accompanied by induction of apoptosis and it is widely considered that this mechanism is causal ( 16 , 39 , 40 ). Indeed, overexpression of p65 in L929 mouse fibrosarcoma cells induced resistance to curcumin-induced apoptosis ( 41 ). The present study shows that curcumin inhibits p65 expression, NFκB-dependent transcriptional activity and expression of NFκB-dependent anti-apoptotic genes. While these pathways have a well-defined role in apoptosis in various cell types ( 42 ), the present study indicates that they may be superfluous to curcumin-mediated apoptosis in HCT116 colorectal cancer cells. Indeed, overexpression of p65 sensitized the cells to curcumin-mediated apoptosis, as assessed by PARP cleavage and MTT assay. Differences between our work and the previous study ( 41 ) may be attributed to species or cell-type-specific differences in expression of unknown curcumin target genes or other signalling pathways implicated in cell death. Cell-type-specific effects of curcumin have been shown previously. For example, curcumin-induced apoptosis is p53-dependent in MCF-7 cells ( 43 ) but independent of p53 in melanoma cells ( 44 ).

Overexpression of p65 has been shown to have pro- or anti-tumourigenic properties in various tissues. For example, prostatic intraepithelial neoplasia shows overexpression of p65 compared with benign prostatic epithelium ( 45 ). Increased expression of p65 correlates with colorectal tumourigenesis ( 46 ). Furthermore, p65 overexpression in thyroid carcinoma cell lines increases proliferation rate and colony formation in soft agar ( 47 ). Conversely, overexpression of p65 in MCF7ADR cells reduces their tumourigenic ability in nude mice ( 48 ). In breast cancer cells, overexpression of p65 inhibits TRAIL-induced apoptosis through inhibition of caspase 8 and DR4 and DR5 expression ( 49 ), whereas in a pro-B cell line it induces G 1 arrest and apoptosis ( 50 ). Clearly, the role of p65 in the carcinogenic process is complex and may involve interactions with multiple signalling pathways in a context-specific manner. It is suggested that p65 may function as an inhibitor or activator of apoptosis, depending upon the nature of the stimulus ( 2729 ). For example, ultraviolet light and daunorubicin induce p65–DNA binding but inhibit NFκB transcriptional activity and suppress NFκB anti-apoptotic target genes. Indeed, p65 can be both an activator and repressor of its target genes ( 30 ). However, pro-apoptotic effects of p65 overexpression in combination with curcumin cannot be explained on this basis since we found that (i) NFκB transcriptional activity in the presence of curcumin was increased in p65-transfected cells and (ii) downregulation of bcl-xL , XIAP and A20 mRNA expression by curcumin was not enhanced in p65-transfected cells compared with mock-transfected cells. In this study, p65 pro-apoptotic effects only occurred in synergy with curcumin treatment since cell death was unchanged in p65-overexpressing cells that were untreated by curcumin. Our results suggest that the pro-apoptotic effect of p65 in concert with curcumin could result from activation of one or more unknown pro-apoptotic target genes. NFκB has been shown to induce pro-apoptotic genes such as fas ligand ( 51 ) and fas ( 52 ) in response to arsenic trioxide and etoposide, respectively. Stabilization of p53 ( 53 ) and activation of polo-like kinase 3 ( 54 ) have also been implicated in apoptosis induction by NFκB. Conceivably, one or more of these pathways may be involved in the synergistic effect of curcumin and p65 observed in this study. This theme will be explored in further work.

In summary, the present study shows that inhibition of NFκB by curcumin is not essential for its anti-apoptotic effects in HCT116 cells and suggests that other unidentified regulators of cell death may be implicated.

We thank the following: Prof. Ron Hay, University of St Andrews, for providing the 3EnhConALuc construct; Dr Warner Greene, University of California, San Francisco, for providing the pCMV-p65 construct; and Dr Dean Ballard, Vanderbilt University, Nashville, for providing the pCMV–ΔNIκB construct. This work was supported by the Research and Development Office, Department of Health, Social Services and Public Safety Northern Ireland.

Conflict of Interest Statement : None declared.

References

1.
Oyama,Y., Masuda,T., Nakata,M., Chikahisa,L., Yamazaki,Y., Miura,K. and Okagawa,M. (
1998
) Protective actions of 5′- n -alkylated curcumins on living cells suffering from oxidative stress.
Eur. J. Pharmacol.
  ,
360
,
65
–71.
2.
Abe,Y., Hashimoto,S. and Horie,T. (
1999
) Curcumin inhibition of inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages.
Pharmacol. Res.
  ,
39
,
41
–47.
3.
Collett,G.P, Robson,C.N., Mathers,J.C. and Campbell,F.C. (
2001
) Curcumin modifies Apc min apoptosis resistance and inhibits 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced tumour formation in Apc min mice.
Carcinogenesis
  ,
22
,
821
–825.
4.
Kawamori,T., Lubet,R., Steele,V.E., Kelloff,G.J., Kaskey,R.B., Rao,C.V. and Reddy,B.S. (
1999
) Chemopreventive effect of curcumin, a naturally occurring anti-inflammatory agent, during the promotion/progression stages of colon cancer.
Cancer Res.
  ,
59
,
597
–601.
5.
Limtrakul,P., Lipigorngoson,S., Namwong,O., Apisariyakul,A. and Dunn,F.W. (
1997
) Inhibitory effect of dietary curcumin on skin carcinogenesis in mice.
Cancer Lett.
  ,
116
,
197
–203.
6.
Ikezaki,S., Nishikawa,A., Furukawa,F., Kudo,K., Nakamura,H., Tamura,K. and Mori,H. (
2001
) Chemopreventive effects of curcumin on glandular stomach carcinogenesis induced by N -methyl- N ′-nitro- N -nitrooguanidine and sodium chloride in rats.
Anticancer Res.
  ,
21
,
3407
–3411.
7.
Samaha,H.S., Kelloff,G.J., Steele,V., Rao,C.V. and Reddy,B.S. (
1997
) Modulation of apoptosis by sulindac, curcumin, phenylethyl-3-methylcaffeate and 6-phenylhexyl isothiocyanate: apoptotic index as a biomarker in colon cancer chemoprevention and promotion.
Cancer Res.
  ,
57
,
1301
–1305.
8.
Kuo,M.L., Huang,T.S. and Lin,J.K. (
1996
) Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells.
Biochim. Biophys. Acta
  ,
1317
,
95
–100.
9.
Jiang,M.C., Yang-Yen,H.F., Yen,J.J. and Lin,J.K. (
1996
) Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines.
Nutr. Cancer
  ,
26
,
111
–120.
10.
Mukhopadhyay,A., Banerjee,S., Stafford,L.J., Xia,C.Z., Liu,M.Y. and Aggarwal,B.B. (
2002
) Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation.
Oncogene
  ,
21
,
8852
–8861.
11.
Batth,B.K., Tripathi,R. and Srinivas,U.K. (
2001
) Curcumin-induced differentiation of mouse embryonal carcinoma PCC4 cells.
Differentiation
  ,
68
,
133
–140.
12.
Aggarwal,B.B., Kumar,A. and Bharti,A. (
2003
) Anticancer potential of curcumin: preclinical and clinical studies.
Anticancer Res.
  ,
23
,
363
–398.
13.
Chen,H.W., Yu,S.L., Chen,J.J.W. et al . (
2004
) Anti-invasive gene expression profile of curcumin in lung adenocarcinoma based on a high throughput microarray analysis.
Mol. Pharmacol.
  ,
65
,
99
–110.
14.
Campbell,F.C. and Collett,G.P. (
2005
) Chemopreventive properties of curcumin.
Future Oncology
  ,
1
,
405
–414.
15.
Plummer,S.M., Holloway,K.A., Manson,M.M., Munks,R.J.L., Kaptein,A., Farrow,S. and Howells,L. (
1999
) Inhibition of cyclo-oxygenase 2 expression in colon cells by the chemopreventive agent curcumin involves inhibition of NF-kappa B activation via the NIK/IKK signalling complex.
Oncogene
  ,
18
,
6013
–6020.
16.
Bharti,A.C., Donato,N., Singh,S. and Aggarwal,B.B. (
2003
) Curcumin (diferuloylmethane) down-regulates the constitutive activation of nuclear factor-kappa B and I kappa B alpha kinase in human multiple myeloma cells, leading to suppression of proliferation and induction of apoptosis.
Blood
  ,
101
,
1053
–1062.
17.
Aggarwal,S., Takada,Y., Singh,S., Myers,J.N. and Aggarwal,B.B. (
2004
) Inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-kappa B signalling.
Int. J. Cancer
  ,
111
,
679
–692.
18.
Sun,Z.W. and Andersson,R. (
2002
) NF-kappa b activation and inhibition: a review.
Shock
  ,
18
,
99
–106.
19.
Chen,C.L., Edelstein,L.C. and Gelinas,C. (
2000
) The Rel/NF-kappa B family directly activates expression of the apoptosis inhibitor Bcl-x(L).
Mol. Cell. Biol.
  ,
20
,
2687
–2695.
20.
Lin,M.T., Chang,C.C., Chen,S.T., Chang,H.L., Su,J.L., Chau,Y.P. and Kuo,M.L. (
2004
) Cyr61 expression confers resistance to apoptosis in breast cancer MCF-7 cells by a mechanism of NF-kappa B-dependent XIAP up-regulation.
J. Biol. Chem.
  ,
279
,
24015
–24023.
21.
Shukla,S. and Gupta,S. (
2004
) Suppression of constitutive and tumor necrosis factor alpha-induced nuclear factor (NF)-kappa B activation and induction of apoptosis by apigenin in human prostate carcinoma PC-3 cells: correlation with down-regulation of NF-kappa B-responsive genes.
Clin. Cancer Res.
  ,
10
,
3169
–3178.
22.
Pham,L.V., Tamayo,A.T., Yoshimura,L.C., Lo,P. and Ford,R.J. (
2003
) Inhibition of constitutive NF-kappa B activation in mantle cell lymphoma B cells leads to induction of cell cycle arrest and apoptosis.
J. Immunol.
  ,
171
,
88
–95.
23.
De Smaele,E., Zazzeroni,F., Papa,S., Nguyen,D.U., Jin,R., Jones,J., Cong,R. and Franzoso,G. (
2001
) Induction of gadd45β by NFκB downregulates pro-apoptotic JNK signalling.
Nature
  ,
414
,
308
–313.
24.
Tang,G., Minemoto,Y., Dibling,B., Purcell,N., Li,Z., Karin,M. and Lin,A. (
2001
) Inhibition of JNK activation through NFκB target genes.
Nature
  ,
414
,
313
–317.
25.
Zerbini,L.F., Wang,Y., Czibere,A. et al . (
2004
) NF-κB-mediated repression of growth arrest- and DNA-damage-inducible proteins 45α and γ is essential for cancer cell survival.
Proc. Natl Acad. Sci. USA
  ,
101
,
13618
–13623.
26.
Collett,G.P. and Campbell,F.C. (
2004
) Curcumin induces c-jun N-terminal kinase-dependent apoptosis in HCT116 human colon cancer cells.
Carcinogenesis
  ,
25
,
2183
–2189.
27.
Kaltschmidt,B., Kaltschmidt,C., Hofmann,T.G., Hehner,S.P., Droge,W. and Schmitz,M.L. (
2000
) The pro- or anti-apoptotic function of NF-κB is determined by the nature of the apoptotic stimulus.
Eur. J. Biochem.
  ,
267
,
3828
–3835.
28.
Wang,S.W., Kotamraju,S., Konorev,E., Kalivendi,S., Joseph,J. and Kalyanaraman,B. (
2002
) Activation of nuclear factor-kappa B during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide.
Biochem. J.
  ,
367
,
729
–740.
29.
Ryan,K.M., Ernst,M.K., Rice,N.R. and Vousden,K.H. (
2000
) Role of NF-κB in p53-mediated programmed cell death.
Nature
  ,
404
,
892
–897.
30.
Campbell,K.J., Rocha,S. and Perkins,N.D. (
2004
) Active repression of antiapoptotic gene expression by RelA(p65) NFκB.
Mol. Cell
  ,
13
,
853
–865.
31.
Rodriguez,M.S., Wright,J., Thompson,J., Thomas,D., Baleux,F., Virelizier,J.L., Hay,R.T. and Arenzana-Seisdedos,F. (
1996
) Identification of lysine residues required for signal-induced ubiquitination and degradation of IκBα in vivo .
Oncogene
  ,
12
,
2425
–2435.
32.
Sun,S.C., Elwood,J., Beraud,C. and Greene,W.C. (
1994
) Human T-cell leukemia-virus type-I tax activation of NF-kappa-B/Rel involves phosphorylation and degradation of I-kappa-B-alpha and relA (p65)-mediated induction of the c-rel gene.
Mol. Cell. Biol.
  ,
14
,
7377
–7384.
33.
Brockman,J.A., Scherer,D.C., McKinsey,T.A., Hall,S.M., Qi,X., Lee,W.Y. and Ballard,D.W. (
1995
) Coupling of a signal response domain in IκBα to multiple pathways for NF-κB activation.
Mol. Cell. Biol.
  ,
15
,
2809
–2818.
34.
Fleming,Y., Armstrong,C.G., Morrice,N., Paterson,A., Goedert,M. and Cohen,P. (
2000
) Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7.
Biochem. J.
  ,
352
,
145
–154.
35.
Kanamoto,T., Mota,M., Takeda,K., Rubin,L.L., Miyazono,K., Ichijo,H. and Bazenet,C.E. (
2000
) Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons.
Mol. Cell. Biol.
  ,
20
,
196
–204.
36.
Ham,Y.-M., Choi,J.-S., Chun,K.-H., Joo,S.-H. and Lee,S.-K. (
2003
) The c-jun N-terminal kinase 1 activity is differentially regulated by specific mechanisms during apoptosis.
J. Biol. Chem.
  ,
278
,
50330
–50337.
37.
Hirai,S., Katoh,M., Terada,M., Kyriakis,J.M., Zon,L.I., Rana,A., Avruch,J. and Ohno,S. (
1997
) MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates SEK1, an activator of c-Jun N-terminal kinase/stress-activated protein kinase.
J. Biol. Chem.
  ,
272
,
15167
–15173.
38.
Sakon,S., Xue,X., Takekawa,M. et al . (
2003
) NF-kappa B inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death.
EMBO J.
  ,
22
,
3898
–3909.
39.
Zheng,M.Z., Ekmekcioglu,S., Walch,E.T., Tang,C.H. and Grimm,E.A. (
2004
) Inhibition of nuclear factor-kappa B and nitric oxide by curcumin induces G(2)/M cell cycle arrest and apoptosis in human melanoma cells.
Melanoma Res.
  ,
14
,
165
–171.
40.
Han,S.S., Chung,S.T., Robertson,D.A., Ranjan,D. and Bondada,S. (
1999
) Curcumin causes the growth arrest and apoptosis of B cell lymphoma by downregulation of egr-1, C-myc, Bcl-X-L, NF-kappa B, and p53.
Clin. Immunol.
  ,
93
,
152
–161.
41.
Anto,R.J., Maliekal,T.T. and Karunagaran,D. (
2000
) L-929 cells harboring ectopically expressed RelA resist curcumin-induced apoptosis.
J. Biol. Chem.
  ,
275
,
15601
–15604.
42.
Orlowski,R.Z. and Baldwin,A.S.,Jr (
2002
) NFκB as a therapeutic target in cancer.
Trends Mol. Med.
  ,
8
,
385
–389.
43.
Choudhuri,T., Pal,S., Agwarwal,M.L., Das,T. and Sa,G. (
2002
) Curcumin induces apoptosis in human breast cancer cells through p53-dependent bax induction.
FEBS Lett.
  ,
512
,
334
–340.
44.
Bush,J.A., Cheung,K.-J.J.,Jr and Li,G. (
2001
) Curcumin induces apoptosis in human melanoma cells through a fas receptor/caspase-8 pathway independent of p53.
Exp. Cell Res.
  ,
271
,
305
–314.
45.
Sweeney,C., Li,L., Shanmugam,R., Bhat-Nakshatri,P., Jayaprakasan,V., Baldridge,L.A., Gardner,T., Smith,M., Nakshatri,H. and Cheng,L. (
2004
) Nuclear factor-kappaB is constitutively activated in prostate cancer in vitro and is overexpressed in prostatic intraepithelial neoplasia and adenocarcinoma of the prostate.
Clin. Cancer Res.
  ,
10
,
5501
–5507.
46.
Yu,H.G., Yu,L.L., Yang,Y., Luo,H.S., Yu,J.P., Meier,J.J., Schrader,H., Bastian,A., Schmidt,W.E. and Schmitz,F. (
2003
) Increased expression of RelA/nuclear factor-κB protein correlates with colorectal tumorigenesis.
Oncology
  ,
65
,
37
–45.
47.
Visconti,R., Cerutti,J., Battista,S. et al . (
1997
) Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression.
Oncogene
  ,
15
,
1987
–1984.
48.
Ricca,A., Biroccio,A., Trisciuoglio,D., Cippitelli,M., Zupi,G. and Del Bufalo,D. (
2001
) RelA over-expression reduces tumorigenicity and activates apoptosis in human cancer cells.
Br. J. Cancer
  ,
85
,
1914
–1921.
49.
Chen,X., Kandasamy,K. and Srivastava,R.K. (
2003
) Differential roles of RelA (p65) and c-Rel subunits of nuclear factor κB in tumor necrosis factor-related apoptosis-inducing ligand signaling.
Cancer Res.
  ,
63
,
1059
–1066.
50.
Sheehy,A.M. and Schlissel,M.S. (
1999
) Overexpression of RelA causes G 1 arrest and apoptosis in a pro-B cell line.
J. Biol. Chem.
  ,
274
,
8708
–8716.
51.
Kasibhatla,S., Brunner,T., Genestier,L., Echeverri,F., Mahboubi,A. and Green,D.R. (
1998
) DNA damaging agents induce expression of fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-κB and AP-1.
Mol. Cell
  ,
1
,
543
–551.
52.
Woo,S.H., Park,I.C., Park,M.J. et al . (
2004
) Arsenic trioxide sensitizes CD95/Fas-induced apoptosis through ROS-mediated upregulation of CD95/Fas by NF-kappa B activation.
Int. J. Cancer
  ,
112
,
596
–606.
53.
Fujioka,S., Schmidt,C., Sclabas,G.M. et al . (
2004
) Stabilization of p53 is a novel mechanism for proapoptotic function of NF-kappaB.
J. Biol. Chem.
  ,
279
,
27549
–27559.
54.
Li,Z.K., Niu,J.G., Uwagawa,T., Peng,B.L. and Chiao,P.J. (
2005
) Function of polo-like kinase 3 in NF-kappa B-mediated proapoptotic response.
J. Biol. Chem.
  ,
280
,
16843
–16850.