Intra-islet glucagon signalling regulates beta-cell connectivity, first-phase insulin secretion and glucose homoeostasis.

OBJECTIVE: Type 2 diabetes (T2D) is characterised by the loss of first-phase insulin secretion. We studied mice with ß-cell selective loss of the glucagon receptor (Gcgrfl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor (GCGR) signalling on pan-islet [Ca2+]I activity and insulin secretion. METHODS: Metabolic profiling was conducted on Gcgrß-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for ß-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in ß-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. RESULTS: Gcgrß-cell-/- mice exhibited higher glucose levels following intraperitoneal glucose challenge (control 12.7 mmol/L ± 0.6 vs. Gcgrß-cell-/- 15.4 mmol/L ± 0.0 at 15 min, p = 0.002); fasting glycaemia was not different to controls. In vitro, Gcgrß-cell-/- islets showed profound loss of pan-islet [Ca2+]I waves in response to glucose which was only partially rescued in vivo. Diet induced obesity and hyperglycaemia also resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n = 8). CONCLUSION: These data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised [Ca2+]I waves and support a possible therapeutic role for glucagonergic agents to restore the insulin secretory capacity lost in T2D.

Isoenzymes of lactate dehydrogenase in human leukemic cells in culture treated with inducers of differentiation.

The human leukemic cell lines HL60 and K562, were induced to differentiate terminally by chemical agents. The isoenzyme patterns of lactate dehydrogenase (LD) in the cells before and after differentiation were determined electrophoretically on agarose gels. In general, treatment of the leukemic cells with inducers of differentiation resulted in a quantitative shift of the isoenzyme pattern towards anodic or cathodic forms. This was correlated with the conversion of the chemically treated cells to morphologically more normal cells, as verified by light microscopy and/or synthesis of hemoglobin. The LD isoenzyme patterns of the chemically differentiated cells were: (a) characteristic for the particular cell type obtained rather than for the nature of the inducer used; and (b) not similar to those of normally differentiated cells of the corresponding lineage, indicating that incomplete differentiation had occurred.

Simian sarcoma virus--transformed cells secrete a mitogen identical to platelet-derived growth factor.

Normal rat kidney (NRK) cells transformed by simian sarcoma virus (SSV) release into the culture medium a biologically active mitogen with properties identical to those of human platelet-derived growth factor (PDGF). Like PDGF, the growth factor derived from SSV-NRK cells was shown to be stable to heat and sensitive to reducing agents. It was capable of inhibiting binding of labeled PDGF to the receptor on human fibroblasts. It also stimulated the phosphorylation of the same membrane protein (185 kilodaltons) in isolated plasma membranes from human fibroblasts. Immunoprecipitation of metabolically labeled proteins released by SSV-NRK cells showed that a 34-kilodalton protein was specifically precipitated by antiserum to PDGF. Upon reduction, this protein had a molecular size of 17 kilodaltons. PDGF has been shown to consist of two 14- to 18-kilodalton proteins linked by disulfide bonds.

Expression of platelet-derived growth factor (PDGF)-related transcripts and synthesis of biologically active PDGF-like proteins by human malignant epithelial cell lines.

Human malignant epithelial cell lines were analyzed for expression of platelet-derived growth factor (PDGF) genes. Of the 12 cell lines tested, 9, derived from breast, lung, gastric, and ovarian carcinomas, were found to express both PDGF-1 and PDGF-2 genes. The levels of both PDGF-1 and PDGF-2 transcripts were superinduced when these cells were treated with cycloheximide, an inhibitor of protein synthesis. These cells also released an activity that in studies with BALB-c/3T3 cells, inhibited binding of 125I-labeled PDGF and stimulated incorporation of [3H]thymidine. This stimulating activity was inhibited after reduction of the conditioned media by mercaptoethanol or after preincubation with antibodies to PDGF. Moreover, this activity was not affected by heat treatment. Immunoprecipitation studies revealed that breast, lung, and gastric carcinoma cells produced PDGF-like proteins that migrated as 30- and 32-kD species under nonreducing conditions and as 15- and 16-kD species under reducing conditions. In contrast, malignant cells of ovarian origin produced 14-16-kD PDGF-like proteins that were unchanged in mobility after reduction. As PDGF receptors were not detected on these malignant epithelial cells, the production of PDGF-like proteins may affect other cells in the microenvironment by paracrine mechanisms and may contribute to excessive cell proliferation, inflammatory reactions, and connective tissue remodeling seen in certain carcinomas.

Phosphorylation of histones in cells treated with hypertonic and acidic media.

Factors in the extracellular environment, specifically hypertonic or acidic growth media, are shown to alter the modification of histones in several cell lines. For histone 2A, changes in modification were visible in the mass pattern and were found to be primarily changes in phosphorylation. The increased modification of the core histones was quickly reversed when cells were returned to normal medium.

Cif (Cytochrome c efflux-inducing factor) activity is regulated by Bcl-2 and caspases and correlates with the activation of Bid.

The cytosolic factor Cif (cytochrome c-efflux inducing factor) was activated by the apoptosis inducers staurosporine and anti-Fas antibodies and rapidly induced the efflux of cytochrome c from purified human mitochondria. HL-60 cells that stably overexpressed a bcl-2 cDNA transgene (Bcl-2:HL-60 cells) contained mitochondria and a cytosol that were resistant to exogenous Cif and that lacked detectable endogenous Cif activity, respectively. Therefore, Bcl-2 overexpression negated Cif activity and suggested that the requirement for Cif resides upstream of Bcl-2 on the apoptotic signal transduction pathway. The addition of purified caspase 3, caspase 7, or caspase 8 to the cytosolic extract from Bcl-2:HL-60 cells, however, restored Cif activity, demonstrating that the inhibition of Cif by Bcl-2 overexpression could be overcome by activated caspases. Moreover, the addition of purified caspases to cytosolic extracts prepared from parental HL-60 cells was also sufficient to cause Cif activation, suggesting that caspases might be required for Cif activation. Consistent with these observations, Fas-induced apoptosis in Jurkat cells resulted in caspase 8 activation and subsequently in activation of Cif. Finally, we demonstrate that the activation of Cif correlated with the activation of the Bcl-2 family member Bid by caspases and that Cif activity was selectively neutralized by anti-Bid antibodies. Taken together, these results indicate that Cif is identical to Bid and that it can be inhibited by Bcl-2 and activated by caspases. Thus, Cif (Bid) is an important biological regulator for the transduction of apoptotic signals.

Evidence for a G2 checkpoint in p53-independent apoptosis induction by X-irradiation.

The p53 tumor suppressor gene is thought to be required for the induction of programmed cell death (apoptosis) initiated by DNA damage. We show here, however, that the human promyelocytic leukemia cell line HL-60, which is known to be deficient in p53 because of large deletions in the p53 gene, can be induced to undergo apoptosis following X-irradiation. We demonstrate that the decision to undergo apoptosis in this cell line appears to be made at a G2 checkpoint. In addition, we characterize an HL-60 variant, HCW-2, which is radioresistant. HCW-2 cells display DNA damage induction and repair capabilities identical to those of the parental HL-60 cell line. Thus, the difference between the two cell lines appears to be that X-irradiation induces apoptosis in HL-60, but not in HCW-2, cells. Paradoxically, HCW-2 cells display high levels of expression of bax, which enhances apoptosis, and no longer express bcl-2, which blocks apoptosis. HCW-2 cells' resistance to apoptosis may be due to the acquisition of expression of bcl-XL, a bcl-2-related inhibitor of apoptosis. In summary, apoptosis can be induced in X-irradiated HL-60 cells by a p53-independent mechanism at a G2 checkpoint, despite the presence of endogenous bcl-2. The resistance shown by HCW-2 cells suggests that bcl-XL can block this process.

Morphogen transport in epithelia.

We present a general theoretical framework to discuss mechanisms of morphogen transport and gradient formation in a cell layer. Trafficking events on the cellular scale lead to transport on larger scales. We discuss in particular the case of transcytosis where morphogens undergo repeated rounds of internalization into cells and recycling. Based on a description on the cellular scale, we derive effective nonlinear transport equations in one and two dimensions which are valid on larger scales. We derive analytic expressions for the concentration dependence of the effective diffusion coefficient and the effective degradation rate. We discuss the effects of a directional bias on morphogen transport and those of the coupling of the morphogen and receptor kinetics. Furthermore, we discuss general properties of cellular transport processes such as the robustness of gradients and relate our results to recent experiments on the morphogen Decapentaplegic (Dpp) that acts in the wing disk of the fruit fly Drosophila.

Labd-14-ene-8,13-diol (sclareol) induces cell cycle arrest and apoptosis in human breast cancer cells and enhances the activity of anticancer drugs.

Sclareol is a labdane-type diterpene that has demonstrated a significant cytotoxic activity against human leukemic cell lines. Here, we report the effect of sclareol against the human breast cancer cell lines MN1 and MDD2 derived from the parental cell line, MCF7. MN1 cells express functional p53, whereas MDD2 cells do not express p53. Flow cytometry analysis of the cell cycle indicated that sclareol was able to inhibit DNA synthesis induce arrest at the G(0/1) phase of the cycle apoptosis independent of p53. Sclareol-induced apoptosis was further assessed by detection of fragmented DNA in the cells. Furthermore, sclareol enhanced the activity of known anticancer drugs, doxorubicin, etoposide and cisplatinum, against MDD2 breast cancer cell line.

Constitutive production of platelet-derived growth factor-like proteins by human prostate carcinoma cell lines.

Prostate carcinoma cell lines DU-145 and PC-3 express both platelet derived growth factor (PDGF)-1 and PDGF-2/sis genes. Concomitantly, these cells synthesize and secrete PDGF-like proteins, as judged by indirect immunofluorescence and by direct immunoprecipitation with specific PDGF antiserum. Conditioned media derived from DU-145 and PC-3 cells stimulated the incorporation of [3H]thymidine by 3T3 cells and competed with 125I-labeled PDGF for its binding to cell surface receptors of 3T3 cells. The biological activity was stable to heating at 100 degrees C for 10 min, sensitive to reducing agents, and neutralized by the IgG fraction of PDGF antiserum, properties similar to those of authentic PDGF. Both DU-145 and PC-3 cell lines appear to lack receptors for PDGF as indicated by their inability to mitogenically respond to PDGF and receptor binding of 125I-labeled PDGF. Production of PDGF-like proteins by human prostate carcinoma cells may play an important role in a paracrine mode in the organization of the extracellular matrix of the malignant tissue.

Camptothecin and its derivatives induce expression of the c-jun protooncogene in human myeloid leukemia cells.

We have recently demonstrated that certain camptothecin derivatives are effective agents in the treatment of human tumor xenografts in nude mice. While camptothecin and its derivatives are recognized as inhibitors of topoisomerase I, little is known about the effects of these agents on specific gene expression, particularly genes involved in growth control. The c-jun early response gene codes for a leucine zipper transcription factor. The present studies demonstrate that 20(S)-camptothecin, 9-amino-20(S)-camptothecin, and 9-nitro-20(S)-camptothecin inhibit the growth of human U-937 myeloid leukemia cells and induce expression of the c-jun gene. c-jun transcripts were increased at 3 h and reached a maximum at 6 h of drug exposure. We also demonstrate that the induction of c-jun gene expression by these agents occurs at the transcriptional level. H7, a nonselective inhibitor of protein kinase C, completely blocked c-jun expression in 20(S)-camptothecin-treated cells, while another protein kinase inhibitor, HA1004, had no detectable effect. Similar findings were obtained for other leucine zipper encoding genes, including jun-B. These results suggest that 20(S)-camptothecin, 9-amino-20(S)-camptothecin, and 9-nitro-20(S)-camptothecin activate a cellular response involving the induction of early response genes. Finally, we demonstrate that induction of c-jun expression occurs in association with internucleosomal DNA fragmentation, a characteristic of programmed cell death.

Cytotoxic efficacy of 9-nitrocamptothecin in the treatment of human malignant melanoma cells in vitro.

In a recent study, we showed that the plant alkaloid camptothecin (CPT) and its derivatives 9-nitro-CPT (9NC) and 9-amino-CPT (9AC) inhibit growth of both human melanocytes (MEL cells) and their malignant counterparts, malignant melanoma (BRO) cells in vitro. This growth inhibition was accompanied by an increase in the size of BRO cells followed by death, whereas cell size increase and death were not observed in MEL cells. In this study, we have extended those investigations to identify parameters and factors that can modulate the cytotoxic action of 9NC against BRO cells in culture. MEL cells treated with 9NC accumulate at the S/G2 boundary of the cell cycle and remain there for a prolonged period of time with only a small number of cells dying by apoptosis. The extent of accumulation correlates with the length of 9NC treatment and/or 9NC concentration in the cell culture. Furthermore, treatment with low 9NC concentrations for a prolonged time or treatment with high drug concentrations results in a fraction of MEL cells with hyperdiploidy. In contrast, 9NC-treated BRO cells are arrested in the S phase before they die by apoptosis. Interestingly, lower 9NC concentrations are more effective than higher concentrations in inducing apoptosis. Once 9NC initiates the process of apoptosis in BRO cells, these cells are irrevocably committed to it and continue to die even after removal of the drug from the culture. The drug effectiveness to induce apoptosis correlates with the stage of the S phase, in which it affects DNA replication, with late stages resulting in higher numbers of apoptotic cells. Finally, although various 9NC concentrations result in inhibition of BRO cell proliferation, higher 9NC concentrations produce more enlarged BRO cells as assessed by microscopy. Taken together, these observations provide useful information for clinical application of 9NC as a chemotherapeutic agent against malignant melanoma.

Isolation and characterization of an apoptosis-resistant variant of human leukemia HL-60 cells that has switched expression from Bcl-2 to Bcl-xL.

Human promyelocytic leukemia HL-60 cells treated with 8-chloroadenosine-3',5'-cyclic monophosphate (8-Cl-cAMP) undergo growth arrest and subsequently die by apoptosis. We describe here the isolation of a variant of HL-60 cells, HCW-2, which was resistant to the cytotoxic effects of 8-Cl-cAMP, but still underwent growth arrest. Thus, HCW-2 cells appeared to be altered in their ability to undergo apoptosis. HCW-2 cells were also completely refractory to the apoptotic action of cycloheximide and staurosporine, two compounds which were very potent inducers of apoptosis in the parental HL-60 cells, suggesting that the resistance to apoptosis was not unique to 8-Cl-cAMP. Western blot analysis demonstrated that the parental HL-60 cells expressed both Bcl-2 and Bax, two factors known to be intimately involved in the control of apoptosis. Surprisingly, HCW-2 cells no longer expressed Bcl-2 protein and paradoxically contained Bax protein at a level that was approximately 50-fold higher than in HL-60 cells. However, Northern and Western analyses indicated that the apoptotic suppressor gene, bcl-xL, which is not expressed in the parental HL-60 cells, was expressed in HCW-2 cells. Thus, the Bcl-2-independent resistance of HCW-2 cells to apoptotic induction is discussed in terms of the expression of bcl-xL.

Regression of human breast carcinoma tumors in immunodeficient mice treated with 9-nitrocamptothecin: differential response of nontumorigenic and tumorigenic human breast cells in vitro.

We have shown recently that the plant alkaloid camptothecin and its derivatives inhibited growth of human carcinoma and melanoma cells in vitro and induced regression of advanced human malignant melanoma tumors growing in immunodeficient (nude) mice. Here, we have extended these studies to show that the camptothecin derivative 9-nitro-20(S)- camptothecin (9NC) induces complete regression of advanced breast carcinoma tumors growing in nude mice. We also report that 9NC inhibits growth of nontumorigenic and tumorigenic breast cells in vitro. However, flow cytometry studies show that 9NC elicits differential effects on the cell cycle of nontumorigenic and tumorigenic cells. In general, 9NC-treated nontumorigenic cells accumulate slowly at the G2 phase of the cell cycle with no cell death. In contrast, 9NC-treated tumorigenic cells transverse rapidly from G1 to S phase followed by cell death. Removal of 9NC from the cell cultures resulted in most nontumorigenic cells dividing, whereas tumorigenic cells continued to die after removal of 9NC. Taken together, the findings indicate a different response of nontumorigenic and tumorigenic breast cells to 9NC.

Complete inhibition of growth followed by death of human malignant melanoma cells in vitro and regression of human melanoma xenografts in immunodeficient mice induced by camptothecins.

The plant alkaloid camptothecin and three camptothecin derivatives were used to study responses of human malignant melanoma (BRO) cells xenografted in immunodeficient (nude) mice. Camptothecin and its derivatives 9-nitro-20(S)-camptothecin and 9-amino-20(S)-camptothecin inhibited growth of tumors and caused regression in all tumor-bearing mice. Tumor regression was accompanied by degenerative changes in the tumor cells, as assessed by microscopic observations of histological sections prepared from the tumors. No toxic effects were observed in the drug-treated mice, with or without xenografts. In parallel experiments, camptothecin, 9-nitro-20(S)-camptothecin, and 9-amino-20(S)-camptothecin inhibited proliferation of BRO cells in vitro and resulted in dramatic morphological cellular changes comparable to those observed in the sections of solid tumors. The derivative 12-nitro-20(S)-camptothecin had no effect on BRO tumors or cell cultures. The difference between 9-nitro-20(S)-camptothecin and 12-nitro-20(S)-camptothecin is the position at which the NO2 group is attached to the camptothecin molecule. In contrast to BRO melanoma cells, none of the camptothecin derivatives had any effect on cultured human melanocytes, the normal counterparts of melanoma cells. Taken together, the findings indicate that camptothecin and derivatives exert different effects on the growth and morphology of normal and malignant (BRO) melanocytes.

Characterization of a novel topoisomerase I mutation from a camptothecin-resistant human prostate cancer cell line.

In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.

Induction of apoptosis in 9-nitrocamptothecin-treated DU145 human prostate carcinoma cells correlates with de novo synthesis of CD95 and CD95 ligand and down-regulation of c-FLIP(short).

Stimulation of CD95 leads to oligomerization of this receptor and the recruitment of the Fas-associated death domain (FADD) and procaspase-8 to form the death-inducing signaling complex (DISC). Subsequent proteolytic activation of caspase-8 at the DISC leads to the activation of downstream caspases and execution of apoptosis. The anticancer drug 9-nitrocamptothecin (9NC) inhibits the nuclear enzyme topoisomerase I (Top1), an event followed by apoptosis of cancer cells. We investigated whether other mechanisms downstream of the DNA-Top1-9NC complexing step regulate the apoptotic ability of 9NC in DU145 cells. We demonstrate that induction of apoptosis in DU145 cells, upon exposure to 9NC, is associated with de novo expression of CD95 and CD95L, suggesting that 9NC-induced apoptosis is mediated by the CD95 system. In this line, we observed early activation of procaspase-3, -7, and -8, but not -1, -9, and -10. Moreover, 9NC treatment resulted in the dramatic down-regulation of c-FLIP(short) expression, but not that of c-FLIP(long) or FADD. Furthermore, incubation of DU145 cells with a neutralizing antibody (NOK-1) to CD95L or transient transfection of a c-FLIP(short) expression vector into DU145 cells partially abrogated 9NC-triggered apoptosis. We propose that 9NC triggers apoptosis by driving DU145 cells from a nonapoptotic status (c-FLIP(short)(high), CD95(low), CD95L(low)) toward a proapoptotic status (c-FLIP(short)(low), CD95(high), CD95L(high)). These findings indicate that in addition to a Top1-mediated effect, 9NC can additionally activate a CD95/CD95L-dependent apoptotic pathway.

Mechanisms of resistance in a human cell line exposed to sequential topoisomerase poisoning.

Camptothecins are a new class of anticancer drugs that target DNA topoisomerase I; current efforts are directed toward elucidating optimal combinations of these drugs with other antineoplastic agents. A rationale for the use of sequential therapy involving the combination of camptothecins with topoisomerase II-targeting drugs, such as etoposide, has arisen from observations of increased topoisomerase II protein levels in cell lines resistant to camptothecin. In an effort to understand potential mechanisms of resistance to this strategy, we developed a U-937 cell subline, denoted RERC, that is capable of surviving exposure to sequential topoisomerase poisoning. The RERC cells are 200-fold resistant to camptothecin, 8-fold resistant to etoposide, and 10-fold hypersensitive to cisplatin compared to the parental U-937 cells. Biochemical analyses indicate that the resistant phenotype involves alterations in both topoisomerase I and topoisomerase IIalpha. Topoisomerase I catalytic activity in the resistant cells is similar to that of the parental line but is resistant to camptothecin. Moreover, the resistant cells express a single mRNA species of topoisomerase I that codes for a mutation in codon 533. In addition, topoisomerase IIalpha protein levels are decreased 10-fold in the resistant line, coincident with a two-fold decrease in the expression of topoisomerase IIalpha mRNA. Collectively, these results indicate that resistance to sequential topoisomerase poisoning may involve a reduction in total cellular topoisomerase activity.

Single neuron morphology in vivo with confined primed conversion.

Unraveling the structural organization of neurons can provide fundamental insights into brain function. However, visualizing neurite morphology in vivo remains difficult due to the high density and complexity of neural packing in the nervous system. Detailed analysis of neural morphology requires distinction of closely neighboring, highly intricate cellular structures such as neurites with high contrast. Green-to-red photoconvertible fluorescent proteins have become powerful tools to optically highlight molecular and cellular structures for developmental and cell biological studies. Yet, selective labeling of single cells of interest in vivo has been precluded due to inefficient photoconversion when using high intensity, pulsed, near-infrared laser sources that are commonly applied for achieving axially confined two-photon (2P) fluorescence excitation. Here we describe a novel optical mechanism, "confined primed conversion," which employs continuous dual-wave illumination to achieve confined green-to-red photoconversion of single cells in live zebrafish embryos. Confined primed conversion exhibits wide applicability and this chapter specifically elaborates on employing this imaging modality to analyze neural morphology of optically targeted single neurons in the developing zebrafish brain.

Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines.

While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-kappaB (NF-kappaB) 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-kappaB 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-kappaB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-kappaB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative effects of tumor necrosis factor (TNF). TNF induced NF-kappaB 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-kappaB 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-kappaB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms.