While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-κB (NF-κB) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-κB and AP-1 in the survival of human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our results show that the faster rate of proliferation of DU145 cells when compared to LNCaP cells correlated with the constitutive expression of activated NF-κB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-κB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative effects of tumor necrosis factor (TNF). TNF induced NF-κB activation but not AP-1 activation in LNCaP cells. In DU145 cells both c-Fos and c-Jun were expressed and treatment with TNF activated c-Jun NH2-terminal kinase (JNK), needed for AP-1 activation. In LNCaP cells, however, only low levels of c-Jun was expressed and treatment with TNF minimally activated JNK. Treatment of cells with curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LNCaP) NF-κB activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis in both cell types, which correlated with the downregulation of the expression of Bcl-2 and Bcl-xL and the activation of procaspase-3 and procaspase-8. Overall, our results suggest that NF-κB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms.
Prostate cancer is the most prevalent malignancy in men in the United States, and leads to the highest number of cancer-related deaths among them (Parker et al., 1997). Efforts to improve prognosis requires the understanding of the role of androgens in prostate cancer. Although androgens are required for the normal growth and development of prostate, they may also play a role in prostate carcinogenesis by acting either as initiators or promoters. Most patients respond initially to androgen-ablative therapies; the regression of the prostate tumors indicates that androgen functions as a survival factor. However, the disease generally recurs within 1 to 3 years of treatment, and the recurrent tumors no longer require androgen for growth or survival. Experimental evidence demonstrates a clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors (Craft et al., 1999a). In addition, these tumors tend to be highly resistant to conventional cytotoxic agents such as cisplatin. Presently available treatments for advanced hormone-resistant prostate cancer are marginally effective, and so newer agents are needed to selectively kill the cancer cells.
How prostate cancer cells develop resistance is not fully understood, but Bcl-2 (Raffo et al., 1995; Herrmann et al., 1997a; McConkey et al., 1996), peroxisome proliferator-activated receptor-γ (Kubota et al., 1998), altered expression of nuclear phosphoprotein pp32 (Kadkol et al., 1999), defective androgen receptor signaling (Craft et al., 1999b), ubiquitously secreted glycoprotein SGP2 (also called clusterin) (Sintich et al., 1999), mutated or nonfunctional p53 (van Brussel et al., 1999; Ewing et al., 1995), nonspecific activation of proteosome (Adams et al., 1999; Herrmann et al., 1998), expression of low-affinity insulin-like growth factor-binding protein (Degeorges et al., 1999), inactivation of tumor suppressor gene PTEN/MMAC (Davies et al., 1999), phosphotidylinositol 3′-kinase (Lin et al., 1999), and high constitutive levels of cyclooxygenase (COX)-2 expression (Liu et al., 1998) have been implicated as survival factors for prostate cancer. In contrast, mitogen-activated protein kinase kinase kinase-1, prostate apoptotic response-4 (Nalca et al., 1999) and a transcription factor, early growth response-1, have been shown to mediate apoptosis in prostate cancer cells (Abreu-Martin et al., 1999; Ahmed et al., 1997).
Within the last few years, several reports have suggested that nuclear transcription factors NF-κB and AP-1 can also act as survival factors and are required for the proliferation of a variety of different tumor cell types (Colotta et al., 1992; Karin et al., 1997; Miyamoto and Verma, 1995; Bargou et al., 1997). For instance, the proliferation of Hodgkin's disease is dependent on the constitutive activation of NF-κB (Bargou et al., 1997). There are some studies to suggest NF-κB activation may also play a role in the proliferation of prostate cancer cells (Herrmann et al., 1997b). Why some prostate cancer cells are sensitive to TNF-induced apoptosis while others are resistant is not understood. We hypothesized that this difference is related to the expression of NF-κB and AP-1 in prostate cancer cells. Therefore, in the present studies, we examined in detail the role of both constitutive and inducible NF-κB and AP-1 in TNF-induced apoptosis in androgen-dependent LNCaP and androgen-independent DU145 prostate cancer cells. Our results indicate that DU145 cells constitutively express the activated form of NF-κB and AP-1 and were resistant to TNF-induced apoptosis, whereas LNCaP cells did not express constitutive NF-κB and AP-1 and were sensitive to TNF-induced apoptosis. Curcumin, a chemopreventive agent, suppressed the constitutive NF-κB and AP-1 in DU145 cells and inducible NF-κB in LNCaP cells, which correlated with augmentation of the TNF-induced apoptosis.
The molecular changes that occur when androgen-dependent prostate tumors become androgen-independent is not very well understood. We used androgen-dependent LNCaP and androgen-independent DU145 cell lines to dissect the molecular differences.
TNF inhibits the proliferation of LNCaP cells but not of DU145 cells
We first investigated the rate of proliferation of prostate cancer cell lines LNCaP and DU145 in the absence and presence of TNF. To determine this, cells were cultured for 2, 4, and 6 days in the absence or presence of 10 nM TNF and then examined for cell viability by the trypan blue exclusion method. As shown in Figure 1, in the absence of TNF the rate of proliferation of DU145 cells was about two-times faster than LNCaP cells (approximately 30 000 vs 17 000 on day 6). The presence of TNF inhibited the proliferation of LNCaP cells (a) but had little effect on the proliferation of DU145 cells (b). These results indicate that TNF inhibits the proliferation of androgen-dependent cells but not androgen-independent cells.
Several cell-survival proteins have been implicated which can induce TNF-resistance (Aggarwal, 2000). It is possible that DU145 cells synthesize the proteins that can induce resistance to TNF. Therefore, LNCaP and DU145 were exposed to TNF in the presence of cycloheximide, an inhibitor of protein synthesis. The results in Figure 1c show that TNF was now cytotoxic to DU145 cells and the level of cytotoxicity was similar to that observed with LNCaP cells. Furthermore, when examined for TNF-induced caspase activation by PARP cleavage, both cell types were found to be equally sensitive. Thus these results suggest that the resistance of DU145 cells to TNF is due to synthesis of antiapoptotic proteins. These results also indicate that TNF signaling machinery is intact in DU145 cells.
LNCaP and DU145 cells differ for NF-κB
Because NF-κB has been implicated as a cell-survival signal (for references see Aggarwal, 2000) and several recent reports suggest that NF-κB activation protects cells from TNF-induced apoptosis, we investigated the possibility that DU145 cells resist the antiproliferative effects of TNF because of their constitutive expression of NF-κB. Therefore, NF-κB activation was examined using EMSA in LNCaP and DU145 cells. As shown in Figure 2a, LNCaP cells did not express constitutive NF-κB but DU145 did. Thus, constitutive expression of NF-κB in DU145 cells may be responsible for the induction of resistance to TNF.
TNF is one of the most potent activators of NF-κB. Whether TNF could activate NF-κB in LNCaP cells was examined. For this, cells were treated with 0.1 nM of TNF for 30 min and then examined for NF-κB using EMSA. As shown in Figure 2a (right panel), TNF induced NF-κB in LNCaP cells. Interestingly, TNF induced NF-κB in the DU145 cells even more.
The NF-κB induced in prostate cell lines contained p65 (RelA) and p50 subunits, inasmuch as incubation of nuclear extracts with either anti-p65 (Figure 2b) or anti-p50 antibodies resulted in abrogation in the NF-κB/DNA complex, whereas preimmune sera had no effect. The specificity of the TNF-induced NF-κB/DNA complexes was further confirmed by demonstrating that the binding was abolished in the presence of 100-fold excess unlabeled κB-oligonucleotide, but not by mutant oligonucleotide (Figure 2b).
To investigate whether NF-κB in DU145 and LNCaP cells was transcriptionally active, NF-κB-dependent reporter activity assay was performed. As shown in Figure 2c (left panel) without any treatment about six-fold increase in NF-κB-dependent reporter activity over the vector control was observed in DU145 cells, which increased to 9.5-fold when cells were treated with TNF. On the other hand, LNCaP cells showed a 3.7-fold increase in NF-κB-dependent reporter activity only after treatment with TNF. These results suggest that both constitutive and TNF-inducible NF-κB in prostate cells were transcriptionally active.
LNCaP and DU145 cells differ for AP-1
The AP-1/c-jun gene product is a nuclear protein that forms a heterodimer with the c-fos gene product to stimulate transcription of genes that contain AP-1/c-jun response elements (Karin et al., 1997). Expression of these gene products have been implicated for the G0/G1 transition of the cell cycle, and they have been reported to protect cells from apoptotic death (Dixit et al., 1989). We therefore analysed nuclear extracts of DU145 and LNCaP for AP-1, and the results are shown in Figure 3a. DU145 prostate cells showed constitutive AP-1 activity, which was further enhanced by TNF treatment. The DNA-binding assay revealed the absence of both constitutive and inducible AP-1 in LNCaP cells (Figure 3a).
The lack of either constitutive or inducible AP-1 in LNCaP cells prompted us to examine the expression of two essential components of AP-1, c-Fos and c-Jun. For this, nuclear extracts prepared from both cell types were probed by Western blot using anti-c-Jun and anti-c-Fos antibodies. DU145 cells expressed a significant amount of both c-Jun and c-Fos proteins, but LNCaP cells expressed only small amounts of c-Jun and no c-Fos (Figure 3b). This may explain why EMSA results showed no constitutive or inducible AP-1 in LNCaP cells. This is the first report to indicate that LNCaP cells lack AP-1 and c-Fos expression.
LNCaP and DU145 cells differ for JNK
The amino-terminal activation domain (Ser-63 and Ser-73) of c-jun is phosphorylated by a JNK, thereby increasing its transcriptional activity (Pulverer et al., 1991). To determine whether androgen-dependent and -independent cells differ in their activation of JNK (which if so might explain the difference in AP-1 activity), we prepared cell extracts from untreated and TNF-induced prostate cell lines and analysed them for JNK activity. As shown in Figure 3c, both cell lines showed some basal level of JNK activity. On treatment with TNF, a sixfold increase in kinase activity was observed in DU145 cells, whereas the activation in LNCaP cells was marginal (less than twofold).
Curcumin inhibits both constitutive and inducible NF-κB and AP-1
NF-κB has been implicated as an antiapoptotic transcription factor, which may override the effects of apoptosis pathways in some cells (Van Antwerp et al., 1996; Wang et al., 1996; Beg and Baltimore, 1996). Our laboratory and several others have shown that curcumin is a potent inhibitor of NF-κB (Singh and Aggarwal, 1995) and AP-1 (Huang et al., 1991). Whether curcumin can suppress constitutive NF-κB and AP-1 in DU145 cells and inducible NF-κB in LNCaP cells was investigated. The results in Figure 4a indicate that TNF induced NF-κB in LNCaP cells and curcumin completely suppressed TNF induced NF-κB activation in a dose-dependent manner, with maximum suppression occurring at around 25 μM. When examined for constitutive NF-κB in DU145 cells, curcumin completely suppressed it at around 75 μM (Figure 4b). The difference in the concentration of curcumin required may depend on the mode of activation of NF-κB, as it is inducible in LNCaP cells and constitutive in DU145 cells.
Curcumin also completely suppressed AP-1 in DU145 cells at around 50 μM (Figure 4c). While suppressing AP-1, curcumin induced another band below the AP-1 band (Figure 4c). Both the super-shift analysis and the cold-competition revealed that AP-1 band consisted of c-Jun and c-Fos (Figure 4d). The band below the AP-1 band, however could not be supershifted by antibodies against c-Jun and c-Fos, indicating that it is a non-specific band (NSB). These results indicate that curcumin suppresses both NF-κB and AP-1 in prostate cancer cells.
Curcumin downregulates the expression of antiapoptotic gene products and activates caspases
Whether suppression of NF-κB by curcumin leads to the suppression of NF-κB-regulated gene products was examined. It has been shown that the expression of Bcl-2 and Bcl-xL are regulated by NF-κB (Tamatani et al., 1999; Hinz et al., 1999). Therefore, the expression of these gene products after treating the cells with 50 μM curcumin was examined using Western blot analysis. Results indicate that curcumin downregulated the expression of both Bcl-2 and Bcl-xL in LNCaP and DU145 cells in a time-dependent manner (Figure 5a,b). The downregulation of the expression of both Bcl-2 and Bcl-xL was not a result of apoptosis of these cells, because the structural protein β-actin was unaltered under these conditions.
We also investigated whether curcumin could activate the proapoptotic procaspase-3 and procaspase-8 in prostate cancer cells. As shown in Figure 5, 100 uM curcumin activated both the procaspase-3 (c) and procaspase-8 (d) in LNCaP and in DU145 cells. When examined for the enzymatic activity of caspase-3 and caspase-8, curcumin (50 uM) was found to activate both the caspases (Figure 5e) in LNCaP cells (left panel) as well as in DU145 cells (right panel). These results thus suggest that curcumin alone can induce apoptosis.
Curcumin potentiates the anti-proliferative effect of TNF
Whether or not the suppression of NF-κB and NF-κB-regulated gene products by curcumin leads to the potentiation of the antiproliferative effects of TNF was investigated. As shown in Figure 6a, under the same conditions in which TNF inhibited the proliferation of LNCaP cells by only 25% in the absence of curcumin, the presence of 10 μM curcumin potentiated the inhibition by almost 75%, suggesting synergism between TNF and curcumin. In the case of DU145 cells, the potentiation was less dramatic, as the cytotoxicity was enhanced from 15% in the absence of curcumin to 35% in the presence of curcumin (Figure 6b).
The cytotoxic effects of TNF are mediated, in part, through the activation of caspases, which can be monitored through the cleavage of a PARP substrate. Whether curcumin potentiates TNF-induced caspase activation was examined. As shown in Figure 6c, where both TNF and curcumin by themselves failed to induce any significant PARP cleavage in LNCaP cells, the addition of the two together induced PARP cleavage, indicating synergistic effects. In spite of their ability to suppress cell growth, neither TNF, nor curcumin, nor the combination of the two had any effect on PARP cleavage in DU145 cells (Figure 6d).
Curcumin alone induces apoptosis in both types of prostate cancer cells
Previously we have shown that curcumin inhibits the growth of breast tumor cell lines (Mehta et al., 1997). Because curcumin downregulates NF-κB and NF-κB-dependent gene products and activates the caspases in prostate cancer cells, we investigated whether curcumin could inhibit the proliferation of these cells. As shown in Figure 7a, while 10 μM alone had no significant effect on the proliferation of either LNCaP or DU145 cells, 50 μM completely inhibited the proliferation of both cell types. Curcumin also induced caspase-dependent cleavage of PARP in a time-dependent manner in both cell types (Figure 7b). The apoptosis of prostate cells by curcumin was further examined by DNA staining with propidium iodide. The kinetics of apoptosis in LNCaP and DU145 prostate carcinoma cells after exposure to curcumin are shown in Figure 8. Curcumin-induced apoptosis in LNCaP cells could be observed as early as 24 h (about 56% PI-positive cells), which reached maximum at 48 h (more than 80%). The kinetics of apoptosis by curcumin in DU145 cells, however, was slower with only 28 and 69% cells converted to PI-positive at 48 and 72 h, respectively (Figure 8). Microscopic observation revealed plasma membrane blebbing, cytoplasmic vacuoles, and chromatin condensation in curcumin treated LNCaP and DU145 prostate carcinoma cells (data not shown).
The mechanism by which prostate cancer cells develop resistance to various treatments is not well understood. We investigated the role of NF-κB and AP-1 in the induction of resistance to TNF in human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our results show that constitutively active NF-κB and AP-1 in DU-145 but not in LnCaP cells correlates with a faster proliferation of DU145 cells than LNCaP cells. It also correlated with the sensitivity of LNCaP cells and the resistance of DU145 cells to the antiproliferative effects of TNF. TNF induced NF-κB activation but not AP-1 in LNCaP cells. While both c-Fos and c-Jun were expressed in DU145 cells, LNCaP cells expressed only c-Jun. Also, TNF activated JNK needed for AP-1 activation in DU145 cells but not in LnCaP cells. Curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LnCaP) NF-κB and constitutive (DU145) AP-1 activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis that correlated with the activation of caspase-3 and caspase-8 and the downregulation of the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL.
Our results indicate that there are several molecular differences between LNCaP and DU145 cells. The first major difference is due to the constitutive NF-κB in DU145 cells but not in LNCaP cells. Why NF-κB is constitutively active in DU145 cells is not clear. It is possible that these cells either have a faster rate of IκBα (an inhibitor of NF-κB) degradation as seen in B cells (Miyamoto et al., 1994) or these cells produce TNF, a potent activator of NF-κB, as seen in T cells (Giri and Aggarwal, 1998). Because constitutive active NF-κB mediates the proliferation of various tumor cells (Bargou et al., 1997; Giri and Aggarwal, 1998; Aggarwal, 2000), this might explain why DU145 cells grow at a faster rate than LNCaP cells. The second difference between the two cell types, is the constitutive AP-1 activity in DU145 cells but not in LNCaP cells. Why DU145 cells express constitutive AP-1 is also not clear. LNCaP cells, however, do not have constitutive AP-1, or it is inducible with TNF. As AP-1 consists primarily of c-Fos and c-Jun, c-Jun was marginally expressed in LNCaP cells whereas c-Fos was not at all expressed. Why LNCaP cells do not express c-Fos is not clear. It has been reported that the repression of c-Fos causes a decline in AP-1 activity by altering the ratio of AP-1 (positive growth factor) to a protein known as QM or Jif (negative growth factor). The protein QM interacts with c-Jun and finally suppresses AP-1 activity (Colotta et al., 1992; Karin et al., 1997). Because AP-1 mediates the proliferation of various tumor cells (Dixit et al., 1989), this may also explain why DU145 cells grow at a faster rate than LNCaP cells.
A third major difference noted between androgen-dependent LNCaP cells and androgen-independent DU145 cells was their ability to undergo apoptosis in response to TNF. LNCaP cells were quite sensitive to TNF whereas DU145 cells were quite resistant. This difference may also be linked to the constitutively active NF-κB and AP-1 in DU145 cells and not in LNCaP cells. That NF-κB activation could downregulate TNF-induced apoptosis has been demonstrated by several groups in a variety of tumors (Van Antwerp et al., 1996; Wang et al., 1996; Beg and Baltimore, 1996; Giri and Aggarwal, 1998). The role of NF-κB in preventing TNF-induced apoptosis in prostate cancer cells has also been reported (Sumitomo et al., 1999). Similarly, the role of AP-1 in the induction of resistance to apoptosis has been documented (Karin et al., 1997; Dixit et al., 1989).
We found that curcumin treatment suppressed both constitutive NF-κB and AP-1 activity in DU145 cells. Curcumin also abolished the TNF-inducible NF-κB activity in LNCaP cells. These results are in agreement with previous reports on other tumor cell types (Singh and Aggarwal, 1995; Huang et al., 1991; Kumar et al., 1998). How curcumin downregulates NF-κB is not clear, but the suppression of IKK activity, the kinase required for IκBα phosphorylation and degradation, has been demonstrated (Jobin et al., 1999). Curcumin is also known to inhibit the activation of JNK (Chen and Tan, 1998), the kinase required for AP-1 activation, which may explain how curcumin blocked constitutive AP-1 activity in DU145 cells. Alternatively, curcumin could also act through an antioxidant mechanism because antioxidants have been shown to block androgen-induced AP-1 and NF-κB activity in prostate carcinoma cells (Ripple et al., 1999). We found that suppression of NF-κB and AP-1 in prostate cancer cells enhanced TNF-induced apoptosis. These results are consistent with reports that suggest that NF-κB and AP-1 have an antiapoptotic role.
Our results indicate that the suppression of NF-κB activation by curcumin correlates with the downregulation of expression of Bcl-2 and Bcl-xL in both prostate cancer cell lines. The expression of Bcl-2 and Bcl-xL have been shown to be regulated by NF-κB (Tamatani et al., 1999; Hinz et al., 1999). Both Bcl-2 and Bcl-xl have been reported to inhibit apoptosis by suppressing the activation of caspases (Raffo et al., 1995; Herrmann et al., 1997a). The downregulation of Bcl-2 and Bcl-xL by curcumin also coincided with the activation of both caspase-8 and caspase-3 acting at upstream and downstream sites, respectively, in the pathway leading to apoptosis. Curcumin alone at high concentration caused the suppression of proliferation in both cell types and the caspase-dependent cleavage of PARP substrate, suggesting that curcumin can induce apoptosis. Because curcumin is known to inhibit cyclooxygenase-2 (COX-2) expression in colon cancer cells (Plummer et al., 1999) and suppression of COX-2 activity in LNCaP cells by NS398, a COX-2-selective inhibitor, causes apoptosis (Liu et al., 1998), it is possible that the apoptotic effects of curcumin observed in our system are mediated through the downregulation of COX-2. Morphological evidence (not shown), and DNA staining with propidium iodide further confirmed that curcumin is a potent inducer of apoptosis in both androgen-dependent as well as androgen -independent prostate carcinoma cells. It is quite likely that this apoptosis is mediated through the downregulation of NF-κB and AP-1, leading to the suppression of Bcl-2 and Bcl-xL, and resulting in the activation of caspases and apoptosis. Alternatively, it is also possible that curcumin-induced apoptosis is not mediated through the downregulation of NF-κB and AP-1, as LNCaP do not constitutively express these transcription factors. Because downregulation of Bcl-2 and Bcl-xL in both cell types precede apoptosis, suggests that curcumin may induce apoptosis through modulation of antiapoptotic proteins.
Curcumin, a major active component of turmeric, has been reported to induce growth inhibition or apoptosis in many cancerous cells (Mehta et al., 1997; Kuo et al., 1996; Jiang et al., 1996; Chen and Huang, 1998), by a mechanism that is not fully understood. Our results suggest a mechanism by which curcumin may mediate its effects. The chemopreventive activity of curcumin is well documented (Kawamori et al., 1999). The fact that curcumin can be used to downregulate the cell survival mechanisms in prostate cancer and other cell types is very appealing. Lack of any known pharmacological toxicity (Li et al., 1993 and references therein) prompts future investigation into the downregulation of tumor cell survival mechanisms.
Materials and methods
Human prostate cell lines (DU145 and LNCaP) were originally obtained from the American Type Culture Collection (Rockville, MD, USA). The rabbit polyclonal antibodies anti-IκBα, anti-p50, anti-p65, anti-JNK1, anti-c-jun, and anti-procaspases-8 and -3, and the anti-c-Fos and c-Jun antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-PARP rabbit polyclonal antibody was purchased from New England Bio Labs (Beverly, MA, USA). Curcumin and MTT were purchased from Sigma Chemicals (St. Louis, MO, USA). Bacteria-derived recombinant human TNF, purified to homogeneity with a specific activity of 5×107 units/mg, was kindly provided by Genentech, Inc. (South San Francisco, CA, USA). RPMI-1640, MEM, and FBS were procured from Life Technologies, Inc. (Grand Island, NY, USA). Protein A/G-sepharose beads were obtained from Pierce (Rockford, IL, USA).
A 20 mM of stock solution of curcumin was prepared in dimethyl sulfoxide and then further diluted in cell culture medium.
DU145 and LNCaP cells were maintained in RPMI-1640 that contained 10% FBS and a 1× antibiotic-antimycotic. Each cell line was split regularly before it attained 70–80% confluence.
NF-κB activation was analysed using EMSA as described previously (Chaturvedi et al., 2000). In brief, 8 μg of nuclear extracts prepared from the TNF-treated/untreated cells were incubated with 32P-end-labeled 45-mer double-stranded NF-κB oligonucleotide (16 fmoles DNA) from the HIV-LTR, 5′-TTGTTACAAGGGACTTTCCGCTG GGGACTTTCCAGGGA GGCGT GG-3′ (underlined indicates NF-κB binding sites) for 15 min at 37°C, and the DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5′-TTGTTACAACTCACTTTCCGCTGCT CACTTTCCAGGGAGG CGTGG-3′, was used to examine the specificity of binding of the NF-κB to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. The composition and specificity of binding was further determined by supershift of DNA-protein complex using specific antibodies and preimmune sera. The dried gels were visualized and radioactive bands quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA).
AP-1 activation was analysed in the same way as that for NF-κB except that the nuclear extracts were incubated with 32P-end-labeled double-stranded oligonucleotide of AP-1 (Manna et al., 1998). The AP-1/DNA complex was analysed on a 5% native polyacrylamide gel.
NF-κB-dependent reporter assay
The NF-κB-SEAP reporter gene expression assay was based on our earlier report (Darnay et al., 1999). In brief, 1.2×106 DU145 or LNCaP cells/1.5 ml were plated in 6-well plates and incubated for 16 h. Cells were then transiently transfected for 18 h with the expression vector (2 μg pCMVFlag1) or pNF-κB-SEAP2 (0.5 μg) by the FuGENE 6 transfection reagent. After transfection, cells were washed and incubated with medium in absence or in presence of 1 nM TNF for 24 h. The culture supernatant was removed and assayed for SEAP activity. The culture supernatant (25 μl) was mixed with 30 μl of 5×buffer (500 mM Tris Cl, pH 9, and 0.5% bovine serum albumin) in a total volume of 100 ul in a 96-well plate, and the heat-labile endogenous alkaline phosphatase deactivated by heating the mixture at 65°C for 30 min. The plate was chilled on ice for 2 min, 50 μl of 1 mM 4 methylumbelliferyl phosphate was added to each well, the plates incubated at 37°C for 2 h, and fluorescence read on a 96-well fluorescent plate reader (Fluoroscan II, Lab Systems, Needham, Heights, MA, USA) with excitation set at 360 nm and emission at 460 nm. The average (±s.e.m.) number of relative fluorescent light units for each transfection was determined.
Western blot analysis
For IκBα, 30 μg of cytoplasmic protein extracts, prepared as reported earlier (Chaturvedi et al., 2000), was resolved on 9% sodium dodecyl sulfate-polyacrylamide gel. Resolved samples were electrotransferred to a nitrocellulose membrane, then blocked with 5% nonfat milk protein for 1 h and incubated with anti-IκBα polyclonal antibodies (1 : 3000) for 1 h. The membrane was washed and treated with HRP-conjugated secondary antibodies, and finally detected by chemiluminescence (ECL, Amersham Pharmacia Biotech., Arlington Heights, IL, USA). To examine c-Jun and c-Fos expression, cells were induced with 0.1 nM TNF for 30 min, prepared the nuclear extracts and 60 μg of nuclear protein was resolved on 9% sodium dodecyl sulfate-polyacrylamide gel, and probed with appropriate antibodies. To probe for procaspases, Bcl-xl, and Bcl-2 proteins, 70 μg of whole cell proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel before being probed with the appropriate antibodies.
The JNK assay was performed using a modified method as described earlier (Kumar and Aggarwal, 1999). In brief, 100 μg of cytoplasmic extracts were incubated with anti-JNK1 antibodies, and the immune-complex that formed was precipitated with protein A/G-sepharose beads. The kinase assay was performed using washed beads as the source of enzyme and glutathione S-transferase-Jun (1–79) as the substrate (2 μg/sample) in the presence of 10 μCi [32P] ATP per sample. The kinase reaction was carried out by incubating the above mixture at 30°C in the kinase assay buffer for 15 min. The reaction was stopped by adding sodium dodecyl sulfate sample buffer and boiling the samples. Finally, protein was resolved on a 10% reducing gel. The radioactive bands of the dried gel were visualized and quantitated using a PhosphorImager as mentioned previously.
The cytotoxic effects of TNF alone, curcumin alone or the combination on DU145 and LNCaP cells were determined using the MTT method as described earlier (Haridas et al., 1998). The cells were plated in triplicate in 96-well plates and allowed to attach for overnight. Thereafter, the medium was replaced that contained TNF or curcumin or in combination. After 24 h or 72 h incubation at 37°C, the cell viability was examined by the MTT method.
Propidium iodide staining
LNCaP and DU145 cells were exposed to curcumin for 0 h, 12 h, 24 h, 48 h, and 72 h. Thereafter cells were trypsinized, washed twice with PBS containing 2% BSA and resuspended in 500 μl PBS. One-hundred microliter of cell suspension was treated with 3 μl of PI (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 20 min in dark. Twenty-five microliter of cell suspension was applied to a glass slide and covered with a cover-slip. Number of PI-negative cells (in a normal light source) and PI-positive cells (in a fluorescence source) were counted from three representative fields and percentage PI positive cells was determined.
Assay of caspase-3 and -8
The caspase assay was essentially based on the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA). In brief, 100 μg cell lysate was incubated at 37°C for 4 h with 50 μl of reaction buffer and 5 μl of colometric substrate for either caspase-3 (DEVD-pNA) or caspase-8 (IETD-pNA). The optical density of the reaction mixture was read on a microplate reader at 405 nm wavelength.
PARP degradation by activated caspases
Apoptosis was examined by measuring the proteolytic cleavage of PARP (Haridas et al., 1998). LNCaP cells were left untreated or were cultured in the presence of 10 μM curcumin, 3 nM TNF, or a mixture of 3 μM TNF and 10 μM curcumin for 48 h. Whole-cell extracts were prepared, and 60 μg of protein was resolved in 7.5% sodium dodecyl sulfate-polyacrylamide electrophores gel, which was then electrotransferred to a nitrocellulose membrane, blotted with anti-PARP antibodies (1 : 2000), and detected using chemiluminescence. Apoptosis was represented by the cleavage of 116-kDa PARP into an 85-kDa degraded product.
electrophoretic mobility shift assay
fetal bovine serum
nuclear transcription factor-κB
inhibitory subunit of NF-κB
c-jun N-terminal kinase
poly (ADP-ribose) polymerase
tumor necrosis factor, SEAP, secretory alkaline phosphatase
Abreu-Martin MT, Chari A, Palladino AA, Craft NA, Sawyers CL . 1999 Mol. Cell. Biol. 19: 5143–5154
Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, Elliott P . 1999 J. Cancer Res. 59: 2615–2622
Aggarwal BB . 2000 Biochem. Pharmacol. 60: 1033–1039
Ahmed MM, Sells SF, Venkatasubbarao K, Fruitwala SM, Muthukkumar S, Harp C, Mohiuddin M, Rangnekar VM . 1997 J. Biol. Chem. 272: 33056–33061
Bargou RC, Emmerich F, Krappmann D, Bommert K, Mapara MY, Arnold W, Royer HD, Grinstein E, Greiner A, Scheidereit C, Dorken B . 1997 J. Clin. Invest. 100: 2961–2969
Beg AA, Baltimore D . 1996 Science 274: 782–784
Chaturvedi MM, Mukhopadhyay A, Aggarwal BB . 2000 Methods Enzymol. 319: 585–602
Chen HW, Huang HC . 1998 Br. J. Pharmacol. 124: 1029–1040
Chen YR, Tan TH . 1998 Oncogene 17: 173–178
Colotta F, Polentarutti N, Sironi M, Mantovani A . 1992 J. Biol. Chem. 267: 18278–18283
Craft N, Chhor C, Tran C, Belldegrun A, DeKernion J, Witte ON, Said J, Reiter RE, Sawyers CL . 1999a Cancer Res. 59: 5030–5036
Craft N, Shostak Y, Carey M, Sawyers CL . 1999b Nat. Med. 5: 280–285
Darnay B, Ni J, Moore PA, Aggarwal . 1999 J. Biol. Chem. 274: 7724–7731
Davies MA, Koul D, Dhesi H, Berman R, McDonnell TJ, McConkey D, Yung WK, Steck PA . 1999 Cancer Res. 59: 2551–2556
Degeorges A, Wang F, Frierson Jr HF, Seth A, Chung LW, Sikes RA . 1999 Cancer Res. 59: 2787–2790
Dixit VM, Marks RM, Sarma V, Prochownik EV . 1989 J. Biol. Chem. 264: 16905–16909
Ewing CM, Ru N, Morton RA, Robinson JC, Wheelock MJ, Johnson KR, Barrett JC, Isaacs WB . 1995 Cancer Res. 55: 4813–4817
Giri DK, Aggarwal BB . 1998 J. Biol. Chem. 273: 14008–14014
Haridas V, Darnay BG, Natarajan K, Heller R, Aggarwal BB . 1998 J Immunol. 160: 3152–3162
Herrmann JL, Menter DG, Beham A, von Eschenbach A, McDonnell TJ . 1997a Exp. Cell. Res. 234: 442–451
Herrmann JL, Beham AW, Sarkiss M, Chiao PJ, Rands MT, Bruckheimer EM, Brisbay S, McDonnell TJ . 1997b Exp. Cell. Res. 237: 101–109
Herrmann JL, Briones Jr F, Brisbay S, Logothetis CJ, McDonnell TJ . 1998 Oncogene 17: 2889–2899
Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, Strauss M . 1999 Mol. Cell. Biol. 19: 2690–2698
Huang TS, Lee SC, Lin JK . 1991 Proc. Natl. Acad. Sci. USA 88: 5292–5296
Jiang MC, Yang-Yen HF, Yen JJ, Lin JK . 1996 Nutr. Cancer 26: 111–120
Jobin C, Bradham CA, Russo MP, Juma B, Narula AS, Brenner DA, Sartor RB . 1999 J. Immunol. 163: 3474–3483
Kadkol SS, Brody JR, Pevsner J, Bai J, Pasternack GR . 1999 Nat. Med. 5: 275–279
Karin M, Liu Z, Zandi E . 1997 Curr. Opin. Cell. Biol. 9: 240–246
Kawamori T, Lubet R, Steele VE, Kelloff GJ, Kaskey RB, Rao CV, Reddy BS . 1999 Cancer Res. 59: 597–601
Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, Miyoshi I, Koeffler HP . 1998 Cancer Res. 58: 3344–3352
Kumar A, Dhawan S, Hardegen NJ, Aggarwal BB . 1998 Biochem. Pharmacol. 55: 775–783
Kumar A, Aggarwal BB . 1999 Meth. Enzymol. 300: 339–345
Kuo ML, Huang TS, Lin JK . 1996 Biochim. Biophys. Acta. 1317: 95–100
Li CJ, Zhang LJ, Dezube BJ, Crumpacker CS, Pardee AB . 1993 Proc. Natl. Acad. Sci. USA 90: 1839–1842
Lin J, Adam RM, Santiestevan E, Freeman MR . 1999 Cancer Res. 59: 2891–2897
Liu XH, Yao S, Kirschenbaum A, Levine AC . 1998 Cancer Res. 58: 4245–4249
McConkey DJ, Greene G, Pettaway CA . 1996 Cancer Res. 56: 5594–5599
Manna SK, Zhang HJ, Yan T, Oberley LW, Aggarwal BB . 1998 J. Biol. Chem. 273: 13245–13254
Mehta K, Pantazis P, McQueen T, Aggarwal BB . 1997 Anticancer Drugs 8: 470–481
Miyamoto S, Verma IM . 1995 Adv. Cancer Res. 66: 255–292
Miyamoto S, Chiao PJ, Verma IM . 1994 Mol. Cell. Biol. 14: 3276–3282
Nalca A, Qiu SG, El-Guendy N, Krishnan S, Rangnekar VM . 1999 J. Biol. Chem. 274: 29976–29983
Parker SL TT, Bolden S, Wingo PA . 1997 J. Clin. 46: 5–27
Plummer SM, Holloway KA, Manson MM, Munks RJ, Kaptein A, Farrow S, Howells L . 1999 Oncogene 18: 6013–6020
Pulverer BJ, Kyriakis JM, Avruch J, Nikolakaki E, Woodgett JR . 1991 Nature 353: 670–674
Raffo AJ, Perlman H, Chen MW, Day ML, Streitman JS, Buttyan R . 1995 O. Cancer Res. 55: 4438–4445
Ripple MO, Henry WF, Schwarze SR, Wilding G, Weindruch R . 1999 J Natl. Cancer Inst. 91: 1227–1232
Singh S, Aggarwal BB . 1995 J. Biol. Chem. 270: 24995–25000
Sintich SM, Steinberg J, Kozlowski JM, Lee C, Pruden S, Sayeed S, Sensibar JA . 1999 Prostate 39: 87–93
Sumitomo M, Tachibana M, Nakashima J, Murai M, Miyajima A, Kimura F, Hayakawa M, Nakamura H . 1999 J. Urol. 161: 674–679
Tamatani M, Che YH, Matsuzaki H, Ogawa S, Okado H, Miyake S, Mizuno T, Tohyama M . 1999 J. Biol. Chem. 274: 8531–8538
Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM . 1996 Science 274: 787–789
van Brussel JP, van Steenbrugge GJ, Romijn JC, Schroder FH, Mickisch GH . 1999 Eur. J. Cancer 35: 664–671
Wang CY, Mayo MW, Baldwin Jr AS . 1996 Science 274: 784–787
This research was conducted with support from The Clayton Foundation for Research.
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Mukhopadhyay, A., Bueso-Ramos, C., Chatterjee, D. et al. Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines. Oncogene 20, 7597–7609 (2001). https://doi.org/10.1038/sj.onc.1204997
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