Carcinogenesis Advance Access originally published online on February 25, 2006
Carcinogenesis 2006 27(8):1636-1644; doi:10.1093/carcin/bgi371
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Curcumin mediates ceramide generation via the de novo pathway in colon cancer cells
1 The Jack Bell Research Centre 2660 Oak Street, Vancouver, BC, Canada
2 Department of Biochemistry and Molecular Biology, Faculty of Science, University of the Basque Country PO Box 644, 48080 Bilbao, Spain
3 Division of Gastroenterology, University of British Columbia 100-2647 Willow Street, Vancouver, BC, Canada V5Z 3P1
*To whom correspondence should be addressed. Tel: +1 604 875 5287; Fax: +1 604 875 5447; Email: bsalh{at}interchange.ubc.ca
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Abstract |
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A wealth of evidence supports the notion that curcumin, a phytochemical present in turmeric, is a potent chemopreventive agent for colon cancer. Its mechanism of action remains incompletely understood. Here we report that curcumin's apoptosis-inducing effects in colon cancer cell lines are accompanied by robust ceramide generation. This occurs through de novo synthesis as the increase in ceramide could be attenuated by pre-incubation of the cells with myriocin, and no changes were observed in sphingomyelin levels, or in either acidic or neutral sphingomyelinase activities. Furthermore, cell death could in part be reversed by myriocin, indicating, for the first time, that endogenous ceramide generation by this agent contributes towards its biological activity. We then investigated the role of reactive oxygen species (ROS) in this phenomenon and demonstrated that curcumin induced robust oxidant generation in the cell lines tested, and its reversal by N-acetylcysteine, completely attenuated apoptosis. We next confirmed that curcumin could activate c-jun N-terminal kinase (JNK) and that its modulation could reverse cell death; however, this intervention could not block ceramide generation, or ROS production. Conversely, however, the inhibition of ROS using N-acetylcysteine led to an inhibition of JNK activation. Hence, we conclude that curcumin induces apoptosis via a ROS-associated mechanism that converges on JNK activation, and to a lesser extent via a parallel ceramide-associated pathway.
Abbreviations: BSO, L-buthionine-[S,R]-sulfoximine; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; JNK, c-jun N-terminal kinase; NAC, N-acetylcysteine; PBS, phosphate-buffered saline; ROS, reactive oxygen species; TCL, thin layer chromatography.
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Introduction |
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Colon cancer is without doubt an important health concern globally, and consumes considerable resources in terms of disease management and screening strategies (1). Hence, increasing attention has been focused on ways to reduce its incidence. The roles of dietary fiber, folate, calcium and non-steroidal anti-inflammatory drugs are receiving considerable attention (2,3). Other dietary agents, such as curcumin (diferuloylmethane) [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], are believed to play a significant role in populations that have a significantly reduced incidence of this disorder (4).
Curcumin is a yellow pigment that has been isolated from the ground rhizome of Curcuma species. Besides its use as a coloring and flavoring agent, curcumin has anti-carcinogenic properties, induces apoptosis in numerous cancer cell lines, and inhibits carcinogen-induced tumorigenesis in rodent intestine, as well as at other sites (5). At a cellular level curcumin inhibits NF
B, AP-1, Cox-2, angiogenesis, MMP9, and suppresses cell proliferation by inhibiting protein kinase PKC and the epidermal growth factor receptor. Its apoptosis-inducing effect is mediated through caspase 8, Bid cleavage, Bax, reactive oxygen species (ROS) generation and c-jun N-terminal kinase (JNK) (6–9).
Ceramides are the lipid backbone of sphingomyelin and glycolipids, and are capable of influencing cell growth, viability and apoptosis. They are generated via two main pathways: the de novo and the sphingomyelinase pathways. Both of these have been implicated in the induction of apoptosis by several drugs, including camptothecin, irinotecan and gemcitabine (10–12). Additionally, ceramides activate stress-activated protein kinases (SAPK) such as JNK, which have also been implicated in the induction of apoptotic cell death (13). Ceramides have also been shown to act on mitochondria, promoting the release of ROS (generated by the respiratory chain), as well as promoting the release of cytochrome c, thereby inducing apoptosis.
Significantly, dietary sphingolipids (including ceramides) have recently been shown to reduce both the number of aberrant colonic crypt foci and number of tumors present in mice (14,15). Furthermore, modulation of this pathway has been reported to induce apoptosis in human colonic tumor xenografts as well as in metastatic colonic cancer (16,17).
In this study we demonstrate that curcumin induces ceramide generation in colonic cancer cells, and have examined its relationship with ROS generation and JNK activation; these factors having recently been shown to play a significant role in curcumin-induced apoptosis in Caki and HCT116 cells, respectively (7,8). The data indicate that both ROS generation and JNK activation are important mechanisms in curcumin-mediated apoptosis in colon cancer cell lines. Additional findings are supportive of ROS, but not ceramide, being upstream of curcumin-induced JNK activation.
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Materials and methods |
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Cell culture
HCT116 colon carcinoma cells were cultured using 10% fetal bovine serum (FBS) supplemented McCoy's 5A (Gibco) media containing penicillin and streptomycin. These cells were a gift from B. Vogelstein. DLD1 cells were cultured in 10% FBS containing RPMI1640 media (Fisher), and HT29 cells were cultured in M199 media (Fisher, Nepean, ON, Canada) containing 10% FBS and supplemented with penicillin and streptomycin. DLD1 and HT29 cells were obtained from American Type Culture Collection.
Cell death assay (MTS assay)
HCT116 cells were plated into 96-well plates at a density of 1 x 104 cells/well. Cells were grown for 24 h before being placed in 1% FBS containing media for 3 h. Cells were then stimulated with dose-escalating concentrations of compounds for 24 h. MTS [3-(4,5-dimethylthiaol-2-yl) 5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, (250 µg/ml) inner salt] (Promega, Nepean, ON, Canada) plus 20 µg of PMS (Gibco, Burlington, ON, Canada) solutions were mixed together and added to each well for 1.5 h. Colorimetric analysis was carried out using an ELISA plate reader at an absorbance of 490 nm. Each condition was plated in quintuplicate.
Fluorescence-activated cell sorter (FACS) analysis for sub G1 cell population determination
Cells were seeded in 12-well plates and grown to 80% confluence. After incubation in 1% FBS containing media for 3 h, cells were then exposed to curcumin (Sigma-Aldrich, Sigma-Aldrich, Oakville, ON, Canada), for a duration of 4 h. Cells were then mechanically lifted by repeated pipetting and washed twice with ice-cold phosphate-buffered saline (PBS) (Sigma). After centrifugation at 1200 r.p.m., cell pellets were re-suspended in ice-cold 70% ethanol for 1 h. Cells were then washed twice with ice-cold PBS and re-suspended in 50 µg/ml propidium iodide (Gibco) and 25 µg/ml RNAse (Fisher). The sub-diploid (sub G1, apoptotic) cell population was measured using FACS (Epics XL-MCL; Beckman Coulter, Fullerton, CA). At least 104 cellular events were counted.
Hoechst staining
Colon carcinoma cells were seeded on sterile cover slips in six-well plates. Upon reaching 80% confluence, cells were placed in 1% FBS containing media for 3 h, and then incubated with 50–100 µM curcumin for a 4 h period. Media were then removed and the cells were washed twice with PBS. Cells were fixed using 1 ml of 100% ice-cold ethanol for 1 h. One milliliter of diluted Hoechst 33258 (1 µg/ml in PBS) was added in each well for 1 h. The cover slips were then placed on glass slides. Pictures were taken from at least three different fields of view.
Mitochondrial isolation for cytochrome c immunoblotting
Cells were seeded in six-well plates. After incubation in 1% FBS containing media for 3 h, the cells were treated with curcumin for 4 h. Cells were harvested by centrifugation at 1000 g for 5 min at 4°C. Cells were washed with ice-cold PBS, and then collected using mitochondrial buffer (20 mM HEPES–KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 250 mM sucrose, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 1 µg/ml aprotinin and 10 µg/ml pepstatin). After a 15 min incubation of cells on ice, they were lysed by repeatedly passaging through a 25-gauge needle 10–15 times. After a further 15 min incubation on ice, the homogenates were centrifuged for 5 min at 1000 g (4°C) and the supernatants were then centrifuged for 5 min at 10 000 g (4°C). The supernatant containing the cytosolic fraction was collected and the pellet was re-suspended in 50 µl mitochondrial buffer, thus yielding the mitochondrial fraction. Western blot analysis was carried out to detect cytochrome c (Transduction Laboratories, Mississauga, ON, Canada) released from mitochondria into the cytosol, using both fractions.
Western immunoblotting
Cell lysates containing equivalent amounts of protein were resolved using 12% SDS–PAGE and transferred on to nitrocellulose membrane using Biorad transblot apparatus at 300 mA for 90 min. The nitrocellulose membrane (Gibco) was then blocked in 5% skimmed milk in Tris-buffered saline/Tween-20 (TBST; 20 mM Tris–HCl, pH 7.4, 250 mM NaCl and 0.05% Tween-20) for 1 h. The primary antibody was applied to the membrane overnight (1 : 1000 dilution) and the latter then washed with TBST. The secondary antibody (conjugated to horseradish peroxidase) was added at a dilution of 1 : 5000 for 1 h and the membrane washed three times in TBST. Enhanced chemiluminescence was utilized to visualize the blots.
Measurement of ceramide generation
Cells were labeled with 5 µCi [3H]palmitate (Mandel Scientific, Guelph, ON, Canada) overnight for the determination of sphingomyelin. The radioactive medium was removed and the cells were washed with non-radioactive medium. For the determination of ceramide generation the cells were left in [3H]palmitate-containing medium (18). After 3 h of starvation in 1% FBS containing media, curcumin at increasing concentrations was added. The lipids were then extracted with chloroform/methanol. Cells were scraped into 1 ml of ice-cold methanol. One milliliter of chloroform and 0.9 ml of 2 M KCl + 0.2 M H3PO4 was added to each aliquot and the chloroform phases were dried under nitrogen. Ceramides were separated by thin layer chromatography (TLC) utilizing Silica Gel 60-coated glass plates (Fisher). Fifty percent of the lengths of these TLC plates were developed in chloroform/methanol/acetic acid (9 : 1 : 1) and then dried. The plates were re-developed in petroleum ether/diethylether/acetic acid (60 : 40 : 1) and then dried and stained with iodide vapor. The identity of the ceramide was standardized by the addition of authentic ceramide standards. Radioactive ceramides were than quantified by scraping from the TLC plates followed by liquid scintillation counting.
ROS determination
Cells were seeded on 12-well plates and grown to 
80% confluence. After 3 h incubation in 1% FBS containing media, 2 µM DCFH-DA (Molecular probes) was added. In the presence of ROS this dye was oxidized and the change is detected by using flow cytometry. After 1 h incubation with the dye, cells were then treated with curcumin with or without antioxidants for 4 h. The cells were than manually lifted by repeated pipetting, and spun down to remove the media at 1200 r.p.m. for 1 min. The pellets were then re-suspended in 1 ml PBS (after washing in PBS) and fluorescence measured. Where inhibitor studies were performed, ROS scavengers were added after the cells had been labeled with 2 µM DCHF-DA dye for 1 h.
Statistical analysis
This was performed using the Student's t-test (two-tailed).
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Curcumin induces apoptosis in colon cancer cell lines
The MTS assay was utilized to assess the effect of curcumin on the viability of colon cancer cells. Three cell lines HCT116, DLD1 and HT29 were exposed to increasing concentrations of curcumin for 24 h, which is shown to be capable of reducing cell viability in a dose-dependent manner (Figure 1B). Subsequently, HCT116 cells were treated with increasing concentrations of curcumin for 4 h, and then stained with propidium iodide. Using FACS, HCT116-treated cells exhibited a dose-dependent increase in the sub G1 cell population (Figure 1C). The effects of exposure of the HCT116 cells to curcumin on PARP and procaspase 3 were then examined. In accordance with the previous observations we observed changes compatible with an apoptosis-inducing effect (Figure 1D), as indicated by the presence of the cleaved form of PARP and disappearance of procaspase 3. Next, confirmation that curcumin leads to mitochondrial release of cytochrome c was then obtained in DLD1 cells (Figure 1E). Figure 1F illustrates chromatin fragmentation, another marker of apoptosis, in Hoechst 33258 stained HT29 cells (similar results were obtained using HCT116 cells).
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Curcumin exposure leads to ceramide generation
In order to assess the role of curcumin on ceramide generation HCT116 cells were incubated with tritiated palmitate. Labeled cells were then treated with increasing concentrations of curcumin. Cellular lipids were isolated and separated using TLC and the amount of radioactivity was analyzed on a scintillation counter. Here for the first time we have demonstrated that the treatment of HCT116 cells with curcumin leads to an increase in relative ceramide generation both dose- and time-dependently (Figure 2A and B). Over a 5-fold increase in ceramide generation was observed with curcumin used at a concentration of 100 µM, while peak induction appeared to occur after 12 h of incubation. Interestingly, the amplitude of ceramide generated appeared to peak at a curcumin concentration associated with the induction of apoptosis (50 µM).
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Ceramides are generated via two different pathways, the de novo pathway and the sphingomyelin hydrolysis pathway. In order to delineate which pathway curcumin utilizes to increase relative ceramide generation, we initially performed pre-labeling experiments and determined changes in sphingomyelin content of curcumin-treated cells. No changes were found in this parameter. To further establish that apoptosis did not rely on this pathway the activities of acidic and neutral sphingomyelinases were determined. No changes were found in these parameters either (data not shown). Subsequently, the specific de novo pathway inhibitor, myriocin (Figure 3A) was used to determine the role of this pathway. HCT116 cells were employed in continuous labeling experiments with tritiated palmitate. The data indicate that pre-treatment of HCT116 cells with myriocin leads to an impressive attenuation of curcumin-induced ceramide generation. Collectively, these findings provide the first evidence that curcumin effects robust ceramide generation through the de novo pathway.
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Inhibition of curcumin-induced ceramide generation leads to the attenuation of cell death
As ceramide generation has been linked with apoptotic cell death, the role of curcumin-induced ceramide generation in the induction of cell death was analyzed. Utilizing FACS, sub-diploid (apoptotic) populations were determined in HCT116 colon cancer cells, following exposure to curcumin, plus myriocin pre-treatment (25 nM). Figure 3B indicates that in curcumin-treated cells, pre-treated with myriocin, apoptosis is partially attenuated. Specifically there is a reduction in the sub G1 fraction from 23.9 to 17.4%. As this represented only a 50% reversal of cell death, it implied that other mechanisms likely operate concurrently, and are also important contributors to curcumin-induced apoptosis.
Treatment of HCT116 cells with curcumin leads to ROS generation
ROS have been implicated in cell death induced under a variety of circumstances. Previous work has also indicated a role for ROS in curcumin-induced cell death in other cell systems (7,19). Hence, we were keen to explore whether the same mechanism contributed towards curcumin's ability to effect cell death in our system. HCT116 cells were pre-labeled with DCHF-DA (a free radical recognizing dye). After the treatment of cells with increasing concentrations of curcumin, cells were analyzed using flow cytometry. The data indicate that curcumin leads to an impressive increase in ROS production (Figure 4A and B).
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To determine whether the underlying mechanism leading to this ROS generation could be modulated, several agents were employed. These included L-buthionine-[S,R]-sulfoximine (BSO), an agent depleting glutathione, N-acetylcysteine (NAC), an antioxidant and a glutathione (GSH) precursor and catalase (CAT). The data demonstrate that BSO (25 µM), NAC (5 mM) and catalase (1000 U) were all able to quench curcumin-generated ROS (Figure 4C).
Inhibition of ROS leads to the attenuation of cell death
We were interested in whether the observed increase in ROS had any relevance for curcumin-induced cell death. Using FACS analysis we demonstrate that NAC and BSO were both able to almost completely attenuate apoptosis in curcumin-treated HCT116 cells (Figure 5). These data indicate that the curcumin-induced ROS observed in our system is comparable with the previous findings in other systems where a direct link with apoptosis has been established.
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Curcumin-activated JNK is not upstream of ROS or ceramide pathways
In a previous work, curcumin has been shown to induce apoptosis by activating the JNK pathway in HCT116 cells. We first confirmed that JNK is activated in response to curcumin as early as 1 h post-exposure (Figure 6A). It is interesting to note that there appears to be a reduction in the JNK protein concomitant with the activation especially of the p54 isoform(s). Next we demonstrated that a specific JNK inhibitor, SP600125 (Anthra [1,9-cd] pyrazol-6 (2H)-one1, 9-pyrazoloanthrone), was capable of significantly preventing curcumin-induced apoptosis in both HCT116 and DLD cells (Figure 6B and 6C, respectively).
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In order to determine the sequence of events initiated by curcumin we performed additional experiments, which were directed at determining whether or not JNK was an upstream regulator of either ROS or ceramide generation. First, HCT116 cells were pre-labeled with DCHF-DA for 1 h and treated with 50 µM curcumin along with 20 µM SP600125. ROS generation was measured using flow cytometry. The data indicate that JNK inhibition does not influence the curcumin-induced change in ROS production (Figure 6D). Second, the effect of SP600125 on ceramide generation was investigated. There was no change in the level of relative ceramide generation in either of the two cell lines examined, consequent upon the inhibition of JNK (data not shown).
Collectively, these observations indicated that JNK activation is downstream of ROS generation, and indeed, NAC is able to attenuate the curcumin-induced JNK activation (Figure 7A). This figure shows a correlation between the generation of ROS and JNK activity, with re-activation of JNK occurring when the NAC concentration was reduced to below 1 mM. The data also indicate that a certain threshold of ROS may be required for JNK activation to occur as concentrations of NAC between 1 and 2.5 mM only partially attenuate ROS generation but there is no corresponding JNK activation.
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When the role of ceramide generation upon JNK activation was examined (Figure 7B), there appeared to be no obvious role for ceramide in generating ROS or JNK activation at the concentration of myriocin used throughout this study. However, a potential role for ceramide-mediated JNK activation (independently of ROS generation) cannot be completely excluded as some inhibition of the p45 JNK isoform may be seen at a myriocin concentration of 50 nM. Overall, the reported findings are compatible with the notion that JNK activation is predominantly downstream of ROS generation.
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Discussion |
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Chemopreventative agents, such as micronutrients and synthetic pharmacological compounds, lead to apoptotic cell death in pre-malignant cells. Curcumin has emerged as an important compound in this arena. Previous studies have shown that its administration to cells leads to apoptotic cell death, characterized by caspase 8 activation, ROS generation and JNK activation. Other important players include Bax and Hsp 70 (20). Our results extend these findings, by demonstrating that it leads to apoptosis in colon cancer cells via a mechanism that includes ceramide generation for the first time. In keeping with mechanisms utilized by some other drugs we have confirmed that curcumin utilizes the de novo pathway. Activation of sphingomyelinases has been thought to be the main pathway leading to the generation of ceramides participating in apoptosis, following stress stimuli. However, this appears not to be the case with curcumin as indicated by the following observations. First, there was no change in sphingomyelin levels in colon cancer cells following curcumin exposure. Second, by employing desipramine, a widely used sphingomyelinase inhibitor, levels of ceramide generation in HCT116 cells treated with curcumin were determined. The results demonstrated no significant difference in curcumin-induced ceramide generation in HCT116 cells, which were pre-treated with desipramine compared with those that were not (data not shown). Finally, direct evaluation of sphingomyelinase activity failed to demonstrate any change consequent upon curcumin exposure. Collectively, these results argue against acidic and neutral sphingomyelinases being involved in curcumin-induced apoptosis.
Several important findings have implicated disturbances in ceramide metabolism as being important in colon cancer. First, ceramide levels have been reported to be decreased in human colon cancer; treatment using ceramide analogs and ceramidase inhibitors led to rapid cell death in SW403 cells. Furthermore, the ceramidase inhibitor B13 prevented the growth of metastatic lesions (16). Sphingomyelin supplementation has not only been shown to reduce colonic aberrant colonic crypts and adenocarcinomas in 1, 2-dimethylhydrazine (DMH)-treated CF1 mice (14,21) but also enhances 5FU efficacy in colonic tumor xenografts (17). Thus, the potential of combining agents that increase sphingomyelinase activity and elevate intracellular ceramide through de novo generation will warrant further attention in future work.
A number of studies have established a link between ceramide elevation and ROS generation. Other reports have established the role of ROS in the induction of apoptosis. Since curcumin leads to apoptotic cell death utilizing the mitochondrial apoptotic pathway (i.e. cytochrome c release), we investigated the effect of curcumin on ROS generation. In accordance with the recent studies (19), we have demonstrated that the exposure of HCT116 colon cancer cells to curcumin leads to an increase in ROS generation. Furthermore, we confirm findings in other cancer cell systems, specifically that pre-treatment with NAC in curcumin-treated human breast epithelial cells MCF10A, and human renal Caki cells, attenuates apoptosis.
Notably, it was observed that the activity of curcumin could be attenuated by BSO as well as NAC. This seems somewhat counterintuitive since BSO inhibits 
-glutamylcysteinyl synthetase and would affect the redox status of the cell toward a climate favoring apoptosis. However, curcumin has been shown to affect the same enzyme also (22) as well as modulating glutathione levels and forming glutathionylated products; hence, we cannot exclude an alternative mode of action for BSO in this study. One possibility includes that of glutathione acting as a possible cofactor for mediating curcumin's oxidant effect. Hence the depletion of glutathione may reduce both the extent of ROS generation and thus apoptosis. In this respect (albeit without a demonstrable oxidant effect) BSO has been shown to attenuate methylseleninic-acid-induced apoptosis in Hep G2 cells (23). While NAC elevates glutathione levels it is likely that its antioxidant activity is more important in the context of our study, hence explaining its ability to reduce curcumin-induced apoptosis.
Previously, it has been demonstrated that the stimulation of HCT116 cells with exogenous C2-ceramide does not lead to JNK activation (24). Our data indicate that JNK activation via curcumin is likely to be independent of endogenous ceramide generation as well. The picture is rendered more complex by the fact that ROS are robustly generated by curcumin and that these species could potentially saturate JNK signaling, and thus making the pathway refractory to other inputs. Indeed, the possible involvement of JNK-mediated signaling being responsible for the small attenuation of curcumin-induced apoptosis by myriocin cannot be completely excluded by the findings reported in this study.
In conclusion, we have shown that curcumin's apoptosis-inducing effects on colon cancer cells incorporate a mechanism that is predominantly dependent upon ROS production and downstream JNK activation, and to a lesser degree by ceramide generation (summarized in Figure 8).
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Acknowledgments |
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This work was supported by funds from the Canadian Society of Intestinal Research, and in part from the Crohn's and Colitis Foundation of Canada.
Conflict of Interest Statement: None declared.
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