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1 Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia; 2 Department of Pharmacology, Dartmouth Medical School, Hanover, New Hampshire; and 3 Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DR4 and DR5 more recently have attracted much more attention because their ligand TRAIL induces apoptosis preferentially in transformed or malignant cells and is different from the death ligands TNF and Fas, which, in addition to inducing apoptosis in cancer cells, cause an inflammatory response and liver damage, respectively, when administered systemically (1) . Therefore, TRAIL is considered to be a tumor-selective apoptosis-inducing cytokine and a promising new candidate for cancer treatment (2 , 3) , although there is a concern about its potential hepatotoxicity (4) , which may be avoided by appropriate preparation of recombinant human TRAIL (5 , 6) .
TRAIL also can bind to three decoy receptors, DcR1 (TRAIL-R3 or TRID), DcR2 (TRAIL-R4 or TRUNDD), and osteoprotegerin (OPG), that contain either no cytoplasmic death domain or a truncated death domain and can compete with DR4 and DR5 for ligand binding and thereby act as antagonists (1) . TRAIL and its receptors DR4 and DR5 are expressed widely in normal and malignant cells, whereas DcR1 and DcR2 are expressed preferentially in many normal tissues but in only a few transformed cells (1) . Therefore, it was initially suggested that TRAIL’s low toxicity toward normal tissues is caused by the expression of decoy receptors in normal tissues, which can protect normal cells from induction of apoptosis by TRAIL (1) .
Many studies have shown that TRAIL-induced apoptosis can be augmented by certain types of anticancer agents in a variety of cancer types in vitro and in vivo (7, 8, 9, 10) . The mechanism underlying the augmentation of TRAIL-induced apoptosis by these agents is largely related to their ability to up-regulate the expression level of TRAIL receptors (i.e., DR4 and DR5). Therefore, agents that up-regulate the expression of TRAIL receptors DR4 and/or DR5 may have the potential for clinical management of cancer in combination with TRAIL through augmentation of TRAIL-induced apoptosis. Because DR4 and DR5 can be regulated by either a p53-dependent or -independent mechanism (11, 12, 13, 14, 15, 16) , it is possible that augmentation of TRAIL-induced apoptosis by anticancer agents may be either p53 dependent or p53 independent.
Triterpenoids, biosynthesized in plants by the cyclization of squalene, are used for medicinal purpose in many Asian countries, and some of them were reported to have anticarcinogenic activity (17, 18, 19, 20) . Because of the relatively weak biological activities of the natural triterpenoids, new analogs of these molecules were synthesized in an attempt to identify more potent agents (21, 22, 23) . One of these analogs is methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me), which was found to induce apoptosis in human lung cancer cells and other types of cancer cells (24, 25, 26) . Because CDDO-Me has the potential as a cancer therapeutic agent, we examined the mechanism by which it induces apoptosis. We previously have shown that CDDO-Me induces apoptosis in human lung cancer cells by activating the cytochrome c/caspase-9–mediated signaling pathway (24) . Other reports indicate that CDDO-Me and its analog CDDO induce a caspase-8–dependent apoptosis (26, 27, 28, 29, 30) . Moreover, these agents also were reported to enhance apoptosis caused by death ligands, including TNF and TRAIL (30 , 31) . However, the mechanisms underlying induction of apoptosis and activation of caspase-8, as well as enhancement of TRAIL-induced apoptosis, by these novel triterpenoids are largely unclear.
In the present study, we, for the first time, showed that CDDO-Me activated the caspase-8 pathway in human lung cancer cells and that this effect is required for CDDO-Me–induced apoptosis. Furthermore, we found that CDDO-Me induced a p53-independent expression of DR5 and DR4 and enhanced TRAIL-induced apoptosis. Importantly, we showed that CDDO-Me activates the c-Jun NH2-terminal kinase (JNK) pathway and that this effect contributes to DR up-regulation, caspase-8 activation, and induction of apoptosis by CDDO-Me. Silencing of DR5 expression using siRNA attenuated CDDO-Me–induced apoptosis. Collectively, our results show a novel DR5-mediated mechanism underlying CDDO-Me–induced apoptosis in human lung cancer cells.
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MATERIALS AND METHODS |
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Cell Lines and Cell Cultures.
Human non–small-cell lung carcinoma cell lines H460, A549, and H1944, which possess wild-type p53, and H522, H157, and H1792, which express mutant or no p53 (32)
, were purchased from the American Type Culture Collection (Manassas, VA). These cells were grown in monolayer culture in a 1:1 (v/v) mixture of DMEM and Ham’s F12 medium supplemented with 5% FCS at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. Normal human bronchial epithelial (NHBE) cells also were purchased from Clonetics (San Diego, CA). These cells were grown in a BEGM BulletKit (Clonetics) at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air.
Northern Blot Analysis.
Total cellular RNA was prepared, and Northern blot analysis was performed as described previously (33)
. Thirty micrograms of total RNA were loaded in each lane. Human DR5 cDNA was obtained from Dr. W. S. El-Deiry (University of Pennsylvania School of Medicine, Philadelphia, PA). Human DR4 cDNA was purchased from Alexis Biochemicals, and 18S rRNA cDNA was purchased from Ambion, Inc. (Austin, TX).
Western Blot Analysis.
Whole-cell lysates were prepared for Western blot analysis as described previously (34)
. Fifty micrograms of whole-cell lysates were electrophoresed through 7.5% to 12% denaturing polyacrylamide slab gels and transferred to a Hybond enhanced chemiluminescence membrane (Amersham, Piscataway, NJ) by electroblot analysis. The blots were probed or reprobed with the primary antibodies, and then second antibody binding was detected using the enhanced chemiluminescence system (Amersham) in accordance with the manufacturer’s protocol. Mouse monoclonal anti–caspase-3 and anti-DR4 and rabbit polyclonal anti-DR5 antibodies were purchased from IMGENEX (San Diego, CA). Rabbit polyclonal anti–caspase-9, anti–caspase-6, anti–poly(ADP-ribose) polymerase (PARP), anti–c-Jun, anti-ATF2, anti–phospho (p)-c-Jun (Ser63), and anti–phospho (p)-ATF2 (Thr71) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Mouse monoclonal anti–caspase-8 and rabbit polyclonal anti-DFF45 were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-Bid antibody was purchased from Trevigen (Gaithersburg, MD). Mouse monoclonal anti-RIP and anti–caspase-7 antibodies were purchased from PharMingen (San Diego, CA). Rabbit polyclonal anti–ß-actin antibody was purchased from Sigma Chemical Co. (St. Louis, MO).
Detection of Cell Surface DR4 and DR5 Using Flow Cytometry.
The procedure for direct antibody staining and subsequent flow cytometric analysis for cell surface protein was described previously (35)
. The mean fluorescence intensity that represents antigenic density on a per cell basis was used to represent the DR4 or DR5 expression. Phycoerythrin (PE)-conjugated mouse antihuman DR4 monoclonal antibody (DJR1), PE mouse antihuman DR5 monoclonal antibody (DJR2–4), and PE mouse IgG1 isotype control (MOPC-21/P3) were purchased from eBioscience (San Diego, CA).
Detection of Apoptosis.
Apoptosis was primarily evaluated by measuring cytoplasmic histone-associated DNA fragments (mononucleosome and oligonucleosomes) formed during apoptosis using a Cell Death Detection ELISAPlus kit (Roche Molecular Biochemicals, Basel, Switzerland) according to the manufacturer’s instructions. We also measured the sub-G1 population as another indicator of apoptosis as described previously (36)
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Silencing of DR5 Expression Using Small Interfering RNA.
The target sequence of DR5 small interfering RNA (siRNA) was 5'-AAGACCCTTGTGCTCGTTGTC-3', which was described previously (37)
. The target sequence of control (nonsilencing) siRNA was 5'-AATTCTCCGAACGTGTCACGT-3'. These siRNAs were ordered from Qiagen (Valencia, CA). The transfection of siRNA was conducted in a 24-well plate (1 µg/well) using RNAiFect transfect reagent purchased from Qiagen following the manufacturer’s instructions. The cells were either harvested for preparation of whole-cell lysates and Western blot analysis after 72 hours to detect DR5 expression or reseeded in a 96-well plate for additional treatment with CDDO-Me on the second day to detect apoptosis. After a given time, cells in the 96-well plate exposed to different treatments then were subjected to detection of apoptosis using a Cell Death Detection ELISAPlus kit as described previously.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Bid and RIP, two known substrates of caspase-8 (38, 39, 40)
, were activated or cleaved as indicated by the decreases in their expression levels (Fig. 1A)
during CDDO-Me treatment. Thus, it appears that CDDO-Me activates caspase-8 and its mediated downstream apoptotic events. Additional experiments showed that the activation of casapse-8 could be detected as early as 9 hours in H460 cells and at 3 hours in H157 cells, respectively, after exposure to CDDO-Me (Fig. 1B)
, indicating that caspase-8 activation is an early event in CDDO-Me–induced apoptosis. We also noted that the activation of caspase-8 by CDDO-Me was more apparent in H460 and H157 cells than in A549 cells (Fig. 1A)
, which corresponds to our previous finding that H460 and H157 cells were more sensitive than A549 cells to CDDO-Me–induced apoptosis (24)
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, indicating that z-IETD-fmk suppresses CDDO-Me–induced apoptosis. This result shows that caspase-8 activation is required for induction of apoptosis by CDDO-Me.
Because CDDO-Me caused Bid cleavage (Fig. 1A)
, we speculated that caspase-8 activation resulted in caspase-9 activation via a Bid-mediated mechanism. Therefore, we further examined whether the caspase-8 inhibitor z-IETD-fmk blocked CDDO-Me–induced caspase-9 activation. As shown in Fig. 1D
, z-IETD-fmk, as expected, decreased level of the cleaved form of caspase-8 induced by CDDO-Me, indicating that it suppresses CDDO-Me–induced caspase-8 activation. Correspondingly, we detected the cleaved form of caspase-9 in cells treated with CDDO-Me alone but not in cells treated with CDDO-Me in the presence of z-IETD-fmk, indicating that z-IETD-fmk blocks CDDO-Me–induced caspase-9 activation. Collectively, these results show that CDDO-Me induces caspase-8 activation upstream of caspase-9 activation.
CDDO-Me Induces DR5 and DR4 Expression.
It is known that caspase-8 plays a central role in apoptosis mediated by death receptors, such as Fas, DR4, and DR5 (40, 41, 42, 43)
. Therefore, we wished to determine whether CDDO-Me could affect the expression of these death receptors. We first examined the effects of CDDO-Me on the expression of Fas, DR4, and DR5 in H460 and A549 cells, both of which have wild-type p53 (32)
. CDDO-Me increased Fas mRNA levels in the A549 cells but not in the H460 cells. This increase was weak at 6 hours and reached a peak at 18 hours (data not shown). When we expanded this examination to more lung cancer cell lines, including H157, H522, H292, and H1944 cells, we failed to see such an effect on Fas expression (data not shown). However, CDDO-Me induced DR5 and DR4 expression more rapidly in H460 and A549 cells, with peak levels detected at 12 hours (Fig. 2A)
. Considering a late and limited effect of CDDO-Me on Fas expression, we then focused our analyses on DR4 and DR5 expression in the subsequent experiments.
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. Collectively, these results suggest that CDDO-Me induced a p53-independent expression of DR4 and DR5 in human lung cancer cells.
It should be noted that DR5 induction generally was more apparent than DR4 up-regulation by CDDO-Me in all of the tested lung cancer cell lines (Fig. 2A)
. This suggests that DR5 may play a more important role than DR4 in mediating CDDO-Me–induced apoptosis if they are involved in CDDO-Me–induced apoptosis. By examining the expression of DR4 and DR5 at the protein level, we found that DR5 protein expression increased after treatment with 0.5 µmol/L and 1 µmol/L of CDDO-Me in all of the tested cell lines, albeit to various degrees. In contrast, DR4 protein was weakly elevated in one (H157) of six cell lines only when treated with 1 µmol/L CDDO-Me (Fig. 2B)
. Moreover, CDDO-Me increased cell surface DR5 by 68% (mean fluorescent intensity increased from 26.9 to 45.3) and 78% (mean fluorescent intensity increased from 27.1 to 48.3) in A549 and H1792 cell lines, respectively, compared with DMSO control, whereas it either did not increase (A549) or even decrease (H1792) cell surface DR4 (H1792; Fig. 2C
). These results further support a more important role for DR5 than DR4 in CDDO-Me–induced apoptosis.
CDDO-Me Augments TRAIL-Induced Apoptosis.
If CDDO-Me induces the expression of DR5 gene leading to increased amount of DR5 on the cell surface, the treatment of cells with CDDO-Me combined with exogenous TRAIL should result in augmented induction of apoptosis. TRAIL at concentrations ranging from 25 to 100 ng/mL (H1792 and H522) or 25 to 1000 ng/mL (A549 cells) weakly increased DNA fragmentation in A549, H1792, and H522 cells, whereas the addition of CDDO-Me augmented DNA fragmentation, which reached levels much greater than those induced by each agent alone (Fig. 3A)
. Similarly, the sub-G1 population induced by CDDO-Me and TRAIL combination (39%) was much greater than those induced by either CDDO-Me (17.5%) or TRAIL (13.6%) alone (Fig. 3B)
. These results indicate that the combination of CDDO-Me and TRAIL augments induction of apoptosis. Moreover, the augmentation of apoptosis by the combination of CDDO-Me and TRAIL was suppressed in the presence of recombinant human decoy receptors DcR2 or OPG (Fig. 3C)
. This shows that induced DR5 is functional (in terms of ligation with TRAIL), and CDDO-Me enhances TRAIL-induced apoptosis by up-regulating its receptors. Importantly, neither TRAIL alone nor the combination of CDDO-Me and TRAIL increased DNA fragmentation in NHBE cells (Fig. 3A)
, suggesting that the combination of CDDO-Me and TRAIL might not be toxic to normal cells.
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. A similar analysis of effector caspases revealed that combined CDDO-Me and TRAIL enhanced the cleavage of caspase-3, caspase-6, and caspase-7 and the subsequent cleavage of their substrates PARP and DFF45 (Fig. 4A and B)
. Furthermore, the enhanced induction of apoptosis by CDDO-Me and TRAIL combination was suppressed by the pan-caspase inhibitor z-VAD-fmk, the caspase-3 inhibitor z-DEVD-fmk, the caspase-8 inhibitor z-IETD-fmk, and the caspase-9 inhibitor z-LEHD-fmk, respectively (Fig. 4C)
. Collectively, these results show that the activation of caspase cascades, including initiator caspases, caspase-8 and caspase-9, and effector caspases, caspase-3, caspase-6, and caspase-7, mediates the augmented apoptosis induced by the CDDO-Me and TRAIL combination.
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. As a related gene, DR4 expression was not altered by the DR5 siRNA (Fig. 5A)
. The cells with silenced DR5 expression (transfected with the DR5 siRNA) had a much lower level of DNA fragments than those expressing normal level of DR5 (transfected with control siRNA) when exposed to CDDO-Me (Fig. 5B)
, indicating that DR5 silencing causes cell resistance to CDDO-Me–induced apoptosis. Therefore, we conclude that DR5 up-regulation is required for CDDO-Me–induced apoptosis. Moreover, CDDO-Me cooperated with TRAIL to augment apoptosis in control siRNA-transfected cells but not in DR5 siRNA-transfected cells (Fig. 5C)
, indicating that DR5 up-regulation also is required for enhancement of TRAIL-induced apoptosis by CDDO-Me.
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B (NFB; ref. 15
). Considering that CDDO-Me induces a p53-independent induction of DR5 in this study and that its analog CDDO inhibits NFB signaling (30)
but induces JNK activation (26)
in leukemia cells, we examined the possibility that CDDO-Me activates JNK pathway and, if so, whether JNK activation contributes to CDDO-induced DR5 induction and apoptosis in human lung cancer cells. As shown in Fig. 6A
, CDDO-Me increased the levels of p-c-Jun and p-ATF2, two well-known JNK substrates, in a time-dependent manner in H157 cells. The increases in p-c-Jun and p-ATF2 occurred rapidly at 30 minutes or 50 minutes after treatment with CDDO-Me. It transiently and weakly increased the total ATF2 level but enhanced the total c-Jun level in a similar fashion as it increased the p-c-Jun level because phosphorylation of c-Jun regulates its transcriptional activity (45)
. These results indicate that CDDO-Me activates JNK pathway.
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), caspase-8 activation (3 to 9 hours; Fig. 1B
), and apoptosis (after 12 hours; ref. 24
) can be induced after cells are exposed to CDDO-Me, thus representing a early event. The question was raised as to whether JNK activation plays any role in mediating CDDO-Me–induced death receptor up-regulation and apoptosis. To address this issue, we examined the effects of CDDO-Me on death receptor expression, caspase activation, and apoptosis in the presence of the JNK-specific inhibitor SP600125 (46)
. SP600125 at concentrations of 10 to 30 µmol/L, as expected, completely abolished not only CDDO-Me–induced increase of p-c-Jun but also the basal level of p-c-Jun in the H157 cell line. Correspondingly, the elevated c-Jun level induced by CDDO-Me, as well as the basal level of c-Jun, was suppressed by SP600125 (Fig. 6B)
. These results confirm that SP60012 is a JNK inhibitor in our cell system.
We next examined the effect of SP600125 on DR5 and DR4 induction by CDDO-Me in the H157 cell line. As shown in Fig. 6C
, CDDO-Me increased DR5 and DR4 expression. However, SP600125 at concentrations of 10 to 30 µmol/L, which are effective in inhibiting JNK activation, blocked CDDO-Me–induced up-regulation of DR5 and DR4 in a concentration-dependent fashion. This result indicates that CDDO-Me induces DR expression via a JNK-mediated mechanism.
Furthermore, we studied whether JNK activation is required for CDDO-Me–induced caspase-8 activation and apoptosis. In the same cell line, we analyzed the effects of CDDO-Me on activation of caspase-8 and caspase-3, DNA fragmentation, and sub-G1 population formation in the absence and presence of SP600125. It is well known that activation of caspase-3 (a downstream effector caspase of caspase-8) and cleavage of its substrate PARP are hallmarks of apoptotic cell death. In the absence of SP600125, CDDO-Me appeared to activate caspase-8 and caspase-3 and to increase cleavage of PARP as indicated by the appearance of their cleaved forms detected by Western blot analysis. In the presence of SP600125, CDDO-Me failed to do so (Fig. 7A)
. These results show that CDDO-Me–mediated caspase-8 and caspase-3 activation and PARP cleavage are JNK dependent. In a similar manner, CDDO-Me increased the amount of DNA fragments (Fig. 7B)
and sub-G1 population (Fig. 7C)
, respectively, in the absence of SP600125. However, these effects were suppressed in the presence of SP600125. These results indicate that JNK activation also is required for CDDO-Me–induced DNA fragmentation and sub-G1 formation. Collectively, these results show that JNK activation is required for CDDO-Me–induced apoptosis in human lung cancer cells.
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DISCUSSION |
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It recently was reported that novel triterpenoids, including CDDO and CDDO-Me, activate the JNK pathway, which is required for induction of apoptosis by these triterpenoids in human leukemia cells (26)
. However, it has not been elucidated how JNK activation results in induction of apoptosis. In agreement with this report, we found that CDDO-Me rapidly increased the levels of p-c-Jun and p-ATF2, two substrates of JNK, revealing that CDDO-Me rapidly activates JNK pathway in human lung cancer cells. Moreover, the JNK inhibitor SP600125, which blocked CDDO-Me–induced JNK activation, also attenuated CDDO-Me–induced apoptosis including caspase-3 activation, PARP cleavage, and DNA fragmentation, showing that JNK activation is required for CDDO-Me–induced apoptosis in human lung cancer cells. For the first time, we have shown that JNK activation mediates DR5 and DR4 up-regulation and caspase-8 activation induced by CDDO-Me because SP600125 effectively suppressed CDDO-Me–induced death receptor up-regulation and caspase-8 activation. Together with the result that silencing of DR5 expression using siRNA decreased cell sensitivity to CDDO-Me–induced apoptosis, we conclude that CDDO-Me induces apoptosis through a JNK-mediated up-regulation of DR5 and DR4 (in some cell lines) in human lung cancer cells (Fig. 8)
.
It is known that DR5 and DR4 can be regulated in p53-independent mechanisms (13
, 15, 16, 17)
. However, it is largely unknown how p53-independent mechanisms regulate DR5 and DR4 expression. In the current study, we, for the first time, have shown that JNK activation up-regulates DR5 and DR4 expression. The question is how JNK activation regulates DR5 and DR4 expression. It is known that c-Jun/AP-1 is a major target of the JNK signaling pathway by phosphorylating the transactivation domain of c-Jun, leading to an increase in its transactivation potential (45)
. Thus, by increasing c-Jun phosphorylation, JNK promotes c-Jun expression transcriptionally, leading to an increase in AP-1 activity. We have shown that DR4 can be regulated by AP-1 activation via a functional AP-1 binding site in its promoter region (16)
. We also recently identified a functional AP-1 binding site in the DR5 promoter region and thus showed that DR5 can be up-regulated through an AP-1–mediated mechanism.4
In the current study, CDDO-Me increased not only p-c-Jun level but also total c-Jun level. Therefore, we suggest that CDDO-Me activates JNK, which in turn enhances AP-1 activity via increasing c-Jun expression, thus leading to up-regulation of DR5 and DR4 expression (Fig. 8)
.
TRAIL binds to DR4 and/or DR5, leading to a tumor-selective induction of apoptosis, and thus is considered to be a promising candidate for cancer treatment (2 , 3) . However, human cancer cells exhibit a heterogeneous response to TRAIL-induced apoptosis (2 , 7 , 8 , 50) . Some cancer cells, including lung cancer cells, are resistant to TRAIL. Fortunately, many therapeutic agents can enhance cancer cell response or sensitize cancer cells to TRAIL treatment to overcome this resistance (2 , 7 , 8) . Our finding that CDDO-Me up-regulates the expression of DR5, and possibly DR4 in some cell lines, independently of p53 has important clinical implications because CDDO-Me combined with TRAIL or other TRAIL-inducing agents will augment induction of apoptosis by TRAIL–death receptor interaction. We found that combined CDDO-Me and TRAIL exhibited a more than additive induction of apoptosis in several lung cancer cell lines, regardless of p53 status. Because silencing of DR5 expression using siRNA abrogated the apoptosis induced by the combination of CDDO-Me and TRAIL, we conclude that CDDO-Me enhances or augments TRAIL-induced apoptosis in a p53-independent fashion by up-regulating DR5 expression. In A549 cells, TRAIL alone even at 1 µg/mL weakly increased DNA fragmentation, indicating that they are not sensitive to TRAIL-induced apoptosis. However, in the presence of CDDO-Me, TRAIL at 25 ng/mL was sufficient to induce high level of DNA fragmentation. Therefore, it appears that TRAIL combined with CDDO-Me can overcome cell resistance to TRAIL-induced apoptosis.
The ability of CDDO and CDDO-Me to enhance apoptosis induced by death ligands recently has been shown in several other types of cancer cell lines. Stadheim et al. (30) reported that CDDO potently enhanced TNF-induced apoptosis in human leukemia cells by inhibiting TNF-mediated expression of antiapoptotic genes. In another study that is more relevant to our report, Kim et al. (31) have shown that CDDO-Me and CDDO augmented TRAIL-induced apoptosis in a panel of human cancer cell lines, including prostate, ovarian, colon, and cervical cancer cells. Importantly, this combination was not cytotoxic to normal cells, including primary cultures of hepatocytes, endothelial cells, peripheral blood leukocytes, and bone marrow. In agreement with their findings, we also found that the combination of CDDO-Me and TRAIL did not show enhanced apoptosis in NHBE cells. However, they did not find that CDDO up-regulated DR4 or DR5 expression using Western blot analysis in these cancer cell lines. They showed that the enhanced apoptosis induction by this combination is mediated by the ability of CDDO or CDDO-Me to decrease FLIP protein level through ubiquitination and proteasome-dependent degradation. A more recent study from the same group (51) showed that CDDO or CDDO-Me induced a caspase-8–dependent apoptosis and enhanced TRAIL-induced apoptosis in leukemia cell lines involving down-regulation of FLIP. They also found that CDDO induced a slight increase in DR4 and DR5 protein levels in U937 cells (51) ; however, they did not examine the effects of CDDO-Me on the expression of DR4 and DR5 (31 , 51) . Our results clearly showed that CDDO-Me activated caspase-9 (24) and caspase-8 (current study) in human lung cancer cells, whereas the report by Suh et al. (51) showed that CDDO activated caspase-8 but not caspase-9 in leukemia cells. Therefore, it is possible that CDDO-Me may have some distinct effect on apoptosis from CDDO or that these triterpenoids may have cell-type specific effects.
In summary, the current study has shown that CDDO-Me induces a JNK-dependent up-regulation of DR5 expression, leading to activation of caspase-8 and induction of apoptosis in human lung cancer cells. Thus, our findings in this study reveal a novel JNK-dependent, DR5-mediated mechanism by which CDDO-Me, as well as possibly other triterpenoids, induces apoptosis in human cancer cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Notes: S-Y. Sun and F. R. Khuri are the Georgia Cancer Coalition Distinguished Cancer Scholars. M. B. Sporn is the Oscar M. Cohn Professor.
Requests for reprints: Shi-Yong Sun, Winship Cancer Institute, Emory University School of Medicine, 1365-C Clifton Road, Clinical Building C3088, Atlanta, GA 30322. Phone: 404-778-2170; Fax: 404-778-5520; E-mail: shi-yong_sun{at}emoryhealthcare.org
4 Zou and Sun, unpublished data. 
Received 4/ 7/04. Revised 7/ 2/04. Accepted 8/12/04.
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 S. Chen, X. Liu, P. Yue, A. H. Schonthal, F. R. Khuri, and S.-Y. Sun CCAAT/Enhancer Binding Protein Homologous Protein-Dependent Death Receptor 5 Induction and Ubiquitin/Proteasome-Mediated Cellular FLICE-Inhibitory Protein Down-Regulation Contribute to Enhancement of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis by Dimethyl-Celecoxib in Human Non Small-Cell Lung Cancer Cells Mol. Pharmacol., November 1, 2007; 72(5): 1269 - 1279. [Abstract] [Full Text] [PDF]
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 H. Malhi, F. J Barreyro, H. Isomoto, S. F Bronk, and G. J Gores Free fatty acids sensitise hepatocytes to TRAIL mediated cytotoxicity Gut, August 1, 2007; 56(8): 1124 - 1131. [Abstract] [Full Text] [PDF]
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 S.-Y. Sun, X. Liu, W. Zou, P. Yue, A. I. Marcus, and F. R. Khuri The Farnesyltransferase Inhibitor Lonafarnib Induces CCAAT/Enhancer-binding Protein Homologous Protein-dependent Expression of Death Receptor 5, Leading to Induction of Apoptosis in Human Cancer Cells J. Biol. Chem., June 29, 2007; 282(26): 18800 - 18809. [Abstract] [Full Text] [PDF]
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X. Liu, P. Yue, S. Chen, L. Hu, S. Lonial, F. R. Khuri, and S.-Y. Sun The Proteasome Inhibitor PS-341 (Bortezomib) Up-Regulates DR5 Expression Leading to Induction of Apoptosis and Enhancement of TRAIL-Induced Apoptosis Despite Up-Regulation of c-FLIP and Survivin Expression in Human NSCLC Cells Cancer Res., May 15, 2007; 67(10): 4981 - 4988. [Abstract] [Full Text] [PDF]
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Y. Dai, P. Khanna, S. Chen, X.-Y. Pei, P. Dent, and S. Grant Statins synergistically potentiate 7-hydroxystaurosporine (UCN-01) lethality in human leukemia and myeloma cells by disrupting Ras farnesylation and activation Blood, May 15, 2007; 109(10): 4415 - 4423. [Abstract] [Full Text] [PDF]
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X. Ling, M. Konopleva, Z. Zeng, V. Ruvolo, L. C. Stephens, W. Schober, T. McQueen, M. Dietrich, T. L. Madden, and M. Andreeff The Novel Triterpenoid C-28 Methyl Ester of 2-Cyano-3, 12-Dioxoolen-1, 9-Dien-28-Oic Acid Inhibits Metastatic Murine Breast Tumor Growth through Inactivation of STAT3 Signaling Cancer Res., May 1, 2007; 67(9): 4210 - 4218. [Abstract] [Full Text] [PDF]
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R.-Y. Su, Y. Chao, T.-Y. Chen, D.-Y. Huang, and W.-W. Lin 5-Aminoimidazole-4-carboxamide riboside sensitizes TRAIL- and TNF{alpha}-induced cytotoxicity in colon cancer cells through AMP-activated protein kinase signaling Mol. Cancer Ther., May 1, 2007; 6(5): 1562 - 1571. [Abstract] [Full Text] [PDF]
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 H. Sasaki, N. Ohara, Q. Xu, J. Wang, D. A. DeManno, K. Chwalisz, S. Yoshida, and T. Maruo A Novel Selective Progesterone Receptor Modulator Asoprisnil Activates Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Mediated Signaling Pathway in Cultured Human Uterine Leiomyoma Cells in the Absence of Comparable Effects on Myometrial Cells J. Clin. Endocrinol. Metab., February 1, 2007; 92(2): 616 - 623. [Abstract] [Full Text] [PDF]
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S. Chintharlapalli, S. Papineni, and S. Safe 1,1-Bis(3'-Indolyl)-1-(p-substitutedphenyl)methanes Inhibit Growth, Induce Apoptosis, and Decrease the Androgen Receptor in LNCaP Prostate Cancer Cells through Peroxisome Proliferator-Activated Receptor {gamma}-Independent Pathways Mol. Pharmacol., February 1, 2007; 71(2): 558 - 569. [Abstract] [Full Text] [PDF]
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M. S. Yates, M. Tauchi, F. Katsuoka, K. C. Flanders, K. T. Liby, T. Honda, G. W. Gribble, D. A. Johnson, J. A. Johnson, N. C. Burton, et al. Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes Mol. Cancer Ther., January 1, 2007; 6(1): 154 - 162. [Abstract] [Full Text] [PDF]
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D. Chen, R. Chan, S. Waxman, and Y. Jing Buthionine Sulfoximine Enhancement of Arsenic Trioxide-Induced Apoptosis in Leukemia and Lymphoma Cells Is Mediated via Activation of c-Jun NH2-Terminal Kinase and Up-regulation of Death Receptors Cancer Res., December 1, 2006; 66(23): 11416 - 11423. [Abstract] [Full Text] [PDF]
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R. Ahmad, D. Raina, C. Meyer, S. Kharbanda, and D. Kufe Triterpenoid CDDO-Me Blocks the NF-{kappa}B Pathway by Direct Inhibition of IKKbeta on Cys-179 J. Biol. Chem., November 24, 2006; 281(47): 35764 - 35769. [Abstract] [Full Text] [PDF]
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P. Lei, M. Abdelrahim, and S. Safe 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit ovarian cancer cell growth through peroxisome proliferator-activated receptor-dependent and independent pathways. Mol. Cancer Ther., September 1, 2006; 5(9): 2324 - 2336. [Abstract] [Full Text] [PDF]
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S. Frese, M. Frese-Schaper, A.-C. Andres, D. Miescher, B. Zumkehr, and R. A. Schmid Cardiac Glycosides Initiate Apo2L/TRAIL-Induced Apoptosis in Non-Small Cell Lung Cancer Cells by Up-regulation of Death Receptors 4 and 5 Cancer Res., June 1, 2006; 66(11): 5867 - 5874. [Abstract] [Full Text] [PDF]
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A. Basu, V. P. Castle, M. Bouziane, K. Bhalla, and S. Haldar Crosstalk between Extrinsic and Intrinsic Cell Death Pathways in Pancreatic Cancer: Synergistic Action of Estrogen Metabolite and Ligands of Death Receptor Family. Cancer Res., April 15, 2006; 66(8): 4309 - 4318. [Abstract] [Full Text] [PDF]
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M. Ammirante, R. Di Giacomo, L. De Martino, A. Rosati, M. Festa, A. Gentilella, M. C. Pascale, M. A. Belisario, A. Leone, M. Caterina Turco, et al. 1-Methoxy-Canthin-6-One Induces c-Jun NH2-Terminal Kinase-Dependent Apoptosis and Synergizes with Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Activity in Human Neoplastic Cells of Hematopoietic or Endodermal Origin. Cancer Res., April 15, 2006; 66(8): 4385 - 4393. [Abstract] [Full Text] [PDF]
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 M. Abdelrahim, K. Newman, K. Vanderlaag, I. Samudio, and S. Safe 3,3'-Diindolylmethane (DIM) and its derivatives induce apoptosis in pancreatic cancer cells through endoplasmic reticulum stress-dependent upregulation of DR5 Carcinogenesis, April 1, 2006; 27(4): 717 - 728. [Abstract] [Full Text] [PDF]
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 M. S. Ricci and W.-X. Zong Chemotherapeutic approaches for targeting cell death pathways. Oncologist, April 1, 2006; 11(4): 342 - 357. [Abstract] [Full Text] [PDF]
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 S. Shishodia, G. Sethi, M. Konopleva, M. Andreeff, and B. B. Aggarwal A Synthetic Triterpenoid, CDDO-Me, Inhibits I{kappa}B{alpha} Kinase and Enhances Apoptosis Induced by TNF and Chemotherapeutic Agents through Down-Regulation of Expression of Nuclear Factor {kappa}B-Regulated Gene Products in Human Leukemic Cells Clin. Cancer Res., March 15, 2006; 12(6): 1828 - 1838. [Abstract] [Full Text] [PDF]
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H. Kim, E. H. Kim, Y. W. Eom, W.-H. Kim, T. K. Kwon, S. J. Lee, and K. S. Choi Sulforaphane Sensitizes Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Resistant Hepatoma Cells to TRAIL-Induced Apoptosis through Reactive Oxygen Species-Mediated Up-regulation of DR5 Cancer Res., February 1, 2006; 66(3): 1740 - 1750. [Abstract] [Full Text] [PDF]
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H. Hasegawa, Y. Yamada, K. Komiyama, M. Hayashi, M. Ishibashi, T. Yoshida, T. Sakai, T. Koyano, T.-S. Kam, K. Murata, et al. Dihydroflavonol BB-1, an extract of natural plant Blumea balsamifera, abrogates TRAIL resistance in leukemia cells Blood, January 15, 2006; 107(2): 679 - 688. [Abstract] [Full Text] [PDF]
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I. Samudio, M. Konopleva, N. Hail Jr., Y.-X. Shi, T. McQueen, T. Hsu, R. Evans, T. Honda, G. W. Gribble, M. Sporn, et al. 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) Directly Targets Mitochondrial Glutathione to Induce Apoptosis in Pancreatic Cancer J. Biol. Chem., October 28, 2005; 280(43): 36273 - 36282. [Abstract] [Full Text] [PDF]
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S. Chintharlapalli, S. Papineni, M. Konopleva, M. Andreef, I. Samudio, and S. Safe 2-Cyano-3,12-dioxoolean-1,9-dien-28-oic Acid and Related Compounds Inhibit Growth of Colon Cancer Cells through Peroxisome Proliferator-Activated Receptor {gamma}-Dependent and -Independent Pathways Mol. Pharmacol., July 1, 2005; 68(1): 119 - 128. [Abstract] [Full Text] [PDF]
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