CCT-327 enhances TRAIL-induced apoptosis through the induction of death receptors and downregulation of cell survival proteins in TRAIL-resistance human leukemia cells
Yan-Jin Liu1, Ying-Chao Lin2,3, Jang-Chang Lee4, Sheng-Chu Kuo1, Chi-Tang Ho5, Li-Jiau Huang1, Daih-Huang Kuo6**, Tzong-Der Way7,8,9*
1 Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University, Taichung, Taiwan
2Division of Neurosurgery, Buddhist Tzu Chi General Hospital, Taichung Branch, Taiwan
3School of Medicine, Tzu Chi University, Hualien, Taiwan
4Department of Pharmacy, China Medical University, Taichung, Taiwan
5Department of Food Science, Rutgers University, New Brunswick, New Jersey, USA 6Graduate Institute of Pharmaceutical Technology, College of Pharmacy & Health
Care, Tajen University, Pingtung, Taiwan
7Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan
8Institute of Biochemistry, College of Life Science, National Chung Hsing University, Taichung, Taiwan
9Department of Health and Nutrition Biotechnology, College of Health Science, Asia University, Taichung, Taiwan
*Correspondence author:
Tzong-Der Way, Ph.D.
Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan
No.91 Hsueh-Shih Road, Taichung, Taiwan 40402 Tel: +886-4-2205-3366 ext: 5209
Fax: +886-4-2203-1075
E-mail: [email protected] **Co-corresponding author: Li-Jiau Huang, Ph.D.
Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University, Taichung, Taiwan
No.91 Hsueh-Shih Road, Taichung, Taiwan 40402 Tel: (886)-4-2205-3366 ext.: 5609
Fax: (886)-4-22030760
E-mail: [email protected]
Abbreviations:
CCT327, 2-(5-Methylselenophen-2-yl)-methylenedioxyquinolin-4-one; 2PQs, 6,7-substituted 2-phenylquinolin-4-ones; TRAIL, tumor necrosis factor (TNF)-related apoptosis-inducing ligand; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide; PI, propidium iodide; NAC, N-acetylcysteine; DR, death receptor; DcR, decoy receptor; OPG, osteoprotegerin; FADD, fas-associated protein with death domain; DISC, death initiating signaling complex; PARP, poly ADP-ribose polymerase; ROS, reactive oxygen species; FLICE, FADD-like interleukin-1 beta converting enzyme; c-FLIP, FLICE inhibitory protein; MAPKs, mitogen-activated protein kinases; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide; ECL, enhanced chemiluminescence; PBS, phosphate-buffered saline; RPMI 1640, Roswell Park Memorial Institute (RPMI) 1640.
Abstract
Tumor necrosis-factor–related apoptosis-inducing ligand (TRAIL) has a potential therapy of cancer, and its ability is selectively to kill cancer cells without affecting normal cells. However, resistance to TRAIL cannot be avoided to develop. This study investigated the effects of CCT327, an analogue of quinolin-4-one, could sensitize cancer cells to TRAIL and potentiates TRAIL-induced apoptosis in TRAIL-resistance human leukemia cells (HL60-TR). We found that CCT327 could enhance TRAIL-induced apoptosis through up-regulation of death receptor (DR) 4 and DR5. In addition to up-regulating DRs, CCT327 suppressed the expression of decoy receptor (DcR) 1 and DcR2. CCT327 significantly down-regulated the expression of cFLIP and other antiapoptotic proteins. We also demonstrated that CCT327 could activate p38, and JNK. Moreover, CCT327-induced induction of DR5 and DR4 was mediated by reactive oxygen species (ROS), and N-acetylcysteine (NAC) blocked the induction of DRs with CCT327. Taken together, these results showed that CCT327 combined with TRAIL treatment may provide an effective therapeutic strategy.
Introduction
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, as an attractive anticancer agent. The ability of TRAIL is selectively to kill a variety of cancer cells without affecting normal cells (1-4). TRAIL is the most promising experimental cancer therapeutic drug, therefore is currently being tested in clinical trials (5-8). TRAIL receptors have been identified five different receptors as death receptor (DR) 4 (TRAIL-R1) and DR5 (TRAIL-R2), decoy receptor (DcR) 1, DcR2, and osteoprotegerin (OPG) so far. DR4 and DR5 are able to transducing apoptotic signal, whereas the other three (DcR1, DcR2, and OPG) are decoy receptors to impede TRAIL-induced apoptosis, which play a dominant negative role by competing with DR4 and DR5 for interaction with TRAIL (9-12).
TRAIL binding to two closely related receptors, DR4 and DR5, leads to recruitment of the adaptor protein, which fas-associated protein with death domain (FADD) and initiator caspase-8 to form the death initiating signaling complex (DISC). The resulting leads to cleavage and activation of caspase-8, which in turn activates downstream caspase cascade, like caspase-9 and caspase-3 in the presence or absence of mitochondrial amplification machinery (13-18). However, human cancer cell lines and primary tumor cells are found to develop resistance to TRAIL through intrinsic or acquired resistance mechanisms. This resistance shows to be mediated through
deregulation of apoptotic-related signaling molecules, such as down-regulation of DR4, DR5, caspase-8, or Bax, and enhanced expression of antiapoptotic molecules such as survivin, or overexpression of the Bcl-2 family proteins (19-21). FLICE indicates activation of caspase-8, and FLICE-like inhibitors such as cFLIP, has been reported to bind to caspase-8 and impeded the activation of downstream incidents leading to apoptosis, including TRAIL-mediated apoptosis (22-25). Consequently, the relation between tumor and TRAIL was caused large interest in find out effector mechanisms and the search for novel compounds which can resensitize tumor cells to TRAIL-induced apoptosis.
Reactive oxygen species (ROS), such as superoxide, H2O2, and hydroxyl radicals, trigger a variety of cellular responses leading to cell growth, differentiation, or cell death (26–31). Mitogen activated protein kinases (MAPKs), such as stress activated protein kinase/c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 are principal mediators for ROS-induced signaling pathway (31-35). In addition, when MAPKs are activated to triggers diverse signaling cascades resulting in cell proliferation, differentiation, or cell death in various tumor cells (30, 34-39).
Several 6,7-substituted 2-phenylquinolin-4-ones (2PQs) were synthesized and identified them as novel antimitotic agents (40). Recent report, novel 2-selenophenyl quinolin-4-ones and their isosteric compounds were designed, synthesized, and
evaluated for in vitro anticancer activity. The most promising target compound, as 2-(5-Methylselenophen-2-yl)-6,7-methylenedioxyquinolin-4-one (CCT327) was represented highly selective and potent inhibition against to MDA-MB-435 melanoma. CCT327 regulates expression of mitotic phase- and apoptosis-associated proteins. CCT327 decrease expression of cyclin B1 and CDK1 proteins in a concentration-dependent manner in HL-60 cells. Next, CCT327 was found to activate caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage (41). Whether CCT327 can sensitize tumor cells to TRAIL-induced apoptosis is not known. In this study, we attempted to ascertain that CCT327 on TRAIL-induced apoptosis in TRAIL-resistance human leukemia cells. Our investigation showed that CCT327 can sensitize the TRAIL-induced apoptosis through the up-regulation of DR4 and DR5 expression and the down-regulation of cFLIP, and other antiapoptotic proteins. Furthermore, JNK and p38 regulated expression of DR4 and DR5 via ROS-mediated.
Materials and methods
Chemicals and reagents. CCT327 was synthesized in our laboratory (Fig. 1A). Recombinant soluble human TRAIL was purchased from PeproTech (Rocky Hill, NJ, USA). Primary antibodies of caspase-3, caspase-8, caspase-9, PARP, Bcl-2, survivin, JNK, phospho-JNK, ERK1/2, phospho-ERK1/2, p38 and phospho-p38 were purchased from Cell Signaling Technology (Danvers, MA, USA). DcR1 and DcR2 antibodies were purchased from ProSci Inc. (Poway, California, USA). Antibodies against Bax, FLIPs/L and Bid were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary DR4 and DR5 were purchased from Abcam Inc. (Cambridge, MA, USA) and Novus Biologicals (Littleton, CO, USA), respectively. Secondary antibodies, HRP-conjugated Goat anti-Mouse IgG and Goat anti-Rabbit IgG, were obtained from Millipore (Billerica, MA, USA). Cell culture materials were obtained from Corp. (Carlsbad, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI) and antibodies for β-actin were purchased form Sigma (St. Louis, MO, USA). PD98059, SB203580, and SP600125 were obtained from Calbiochem (San Diego, CA, USA).
Cell lines and cell cultures. The human leukemia cancer cell lines used in this study was HL60 (CCL 240) for parental obtained from the American Type Culture Collection (Manassas, VA, USA). HL60 cells derived from a human acute
promyelocytic leukemia and are usually sensitive to chemotherapeutic drugs and TRAIL. TRAIL-resistant HL60 cells (HL60-TR) were selected by exposure of HL60 cells to escalating doses of TRAIL (10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, 500 ng/mL, 1 μg/mL, 5 μg/mL, and 10 μg/mL) for 2 to 3 days. After each exposure, surviving cells were recovered and cultured in fresh medium for 3 days and then treated with the subsequent dose (42). HL60-TR was routinely maintained in RPMI 1640 (Invitrogen Corporation, Carlsbad, CA, USA). Medium supplemented with 2 mM L-glutamine, 100 μg streptomycin, 100 U penicillin and 10% fetal bovine serum (FBS) (Invitrogen Corporation, Carlsbad, CA, USA). Cells were grown in a humidified incubator at 37°C under 5% CO2 in air.
Cytotoxicity assay. In brief, cells were seeded on the 24-well plate (1 × 104 cells/well) overnight and then treated with different concentrations of CCT327 and TRAIL as indicated in the figure captions and incubated for 48 h. Following treatments, 80 μL of MTT (stock concentration 2 mg/mL) was added to each well, and incubated for 2 h under 5% CO2 and 37 °C. The cell viability was measured by MTT, which is converted by succinate dehydrogenase in mitochondria of viable cells to form a purple formazan dye by metabolically viable cells. The formazan dye was dissolved in dimethyl sulfoxide (DMSO). To measure absorbance was used by an enzyme-linked immunosorbent assay (ELISA) reader at O.D. 570 nm.
Flow cytometry analysis. To determine the effect of CCT327 combine TRAIL on the cell cycle, treated and untreated cells were stained with PI as mentioned earlier. Briefly, 5 × 105 cells were treated with CCT327 combine TRAIL for 48 h at 37°C and subjected to PI staining. Cells were collected by trypsinization, fixed with 70% (v/v) ethanol at 4°C for 30 min and washed with PBS. After centrifugation, cells were resuspended in 500 μL of PI solution comprising Triton X-100 (0.1%, v/v), RNase (100 mg/mL), and PI (80 mg/mL), and then analyzed with FACScan and the Cell Quest software (Becton Dickinson; Mountain View, CA, USA) (43).
Western blotting. HL60-TR cells on 100-mm culture dishes (1 × 106 cells/dish) were treated with various agents as indicated in figure legends, and then incubated for 48 h. Cells were harvested and protein fraction was extracted by adding 50 μL of gold lysis buffer (50 mM Tris-HCl, pH 7.4; 1 mM phenylmethylsulfonyl floride; 1 mM NaF; 1% NP-40; 150 mM NaCl; 1 mM EGTA; and 10 mg/mL leupeptin) to the cell pellets. Lysate protein was measured by the Lowry protein assay (Bio-Rad Laboratories, Berkeley, CA, USA). Proteins between 50 and 100 μg were used by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the PVDF membrane (polyvinylidene fluoride transfer membrane) (BioTrace, U.K.). The blotted membrane were blocked with 5% skim milk for 1 h at room temperature and probed with primary antibody overnight at 4°C. Finally, HRP-conjugated appropriated
secondary antibodies were used for 1h.
Statistical analysis. One-way analysis of variance (ANOVA) was used for the comparison of more than two mean values. Results represent at least two to three independent experiments and are shown as averages ± S.E.M. Results with a P value less than 0.05 were considered statistically significant. (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Results
CCT327 sensitized HL60-TR cells to TRAIL-mediated apoptosis. We examined first the sensitivity of HL60 and HL60-TR cells to TRAIL. HL60 and HL60-TR cells were treated with increasing doses of recombinant TRAIL and then assessed for cell viability using the MTT method. The dose-response of HL60 and HL60-TR cells to TRAIL is shown in Fig. 1B. The HL60 cells were found to be highly sensitive to TRAIL whereas HL60-TR cells were completely resistant (Fig. 1B). Therefore, a detailed investigation of overcome the resistance mechanisms was used HL60-TR cells to carry out and identify the mechanisms.
We next determined whether CCT327 enhances TRAIL-induced apoptosis in HL60-TR cells. HL60-TR cells were treated with CCT327 (0-600 nM) and then exposed to TRAIL (0-200 ng/mL) for 48 h. Treatment with TRAIL had no effect on cell viability. However, combination treatment with CCT327 and TRAIL significantly enhanced TRAIL-induced cytotoxicity (Fig. 1C). To further confirm the effect of CCT327 on TRAIL-induced apoptosis, we also investigated the distribution of cells by PI staining. The percentage of apoptotic cells in the sub-G1 peak as evidenced by the increase in subdiploid fraction of treated cells was measured by flow cytometry. We found that TRAIL-induced apoptosis was increased from 9.7 to 50.8% in HL60-TR cells (Fig. 1D). Thus our results indicated that CCT327 converted the TRAIL-resistant
HL60-TR cells to TRAIL-sensitive cells.
Activation of caspases is an important hallmark of apoptosis induced by most agents. Next, we investigated whether the effect of CCT327 on TRAIL-induced cell death through activation of caspase-8, -9, and -3 and PARP cleavage. Treatment with 300 nM CCT327 alone had little effect on cleavage of procaspase-3, -8, -9 and PARP (Fig. 1E). Moreover, TRAIL alone did not induce processing of any caspases. Co-treatment with CCT327 and TRAIL effectively induced activation of all three caspases, thus leading to enhanced PARP cleavage. These results suggest that CCT327 can enhance TRAIL-induced apoptosis. CCT327 activated caspases that were essential involved in the stimulation of TRAIL-mediated apoptosis.
CCT327 inhibited the expression of antiapoptotic proteins. Several of antiapoptotic proteins are known to suppress TRAIL-induced apoptosis. How CCT327 enhances TRAIL-induced apoptosis was investigated next. HL60-TR cells were exposed to 300 nM CCT327 for different times and then examined for the expression of cFLIP (long and short), Bcl-2, Bcl-xL, and survivin. CCT327 suppressed expression of the antiapoptotic proteins such as Bcl-2 and both the short and long forms of cFLIP (Fig. 2A). It had no effect on expression of survivin. Expression of Bcl-xL was shown not distinct. Our results suggest that downregulation of antiapoptotic proteins is another mechanism by which CCT327 could sensitize TRAIL-induced apoptosis.
CCT327 regulated the expression of proapoptotic proteins. Whether CCT327 affects the expression of proapoptotic proteins, was examined. CCT327 caused the cleavage of bid protein, enhanced the expression of proapoptotic protein bax (Fig. 2B). Induction of bid and bax by CCT327 suggests that these proteins may disrupt mitochondrial homeostasis, which further would contribute to enhance the apoptotic effects of TRAIL.
CCT327 induced the expression of DR4 and DR5 in HL60-TR cells. To understand how CCT327 enhances TRAIL-induced apoptosis, we investigated its effect on DR4 and DR5 in HL60-TR cells. Treatment with 300 nM CCT327 induced both expression of DR4 and DR5 in a time-dependent manner (Fig. 3A, top). HL60-TR cells were treated with different concentrations of CCT327 for 48 h, and examined for expression of DR4 and DR5 proteins. CCT327 induced both DR4 and DR5 in a dose-dependent manner (Fig. 3A, bottom). Fig. 3B showed that treatment with TRAIL alone had no effect on DR4 and DR5. However, combination treatment with CCT327 (300 nM) and TRAIL (100 ng/mL) significantly enhanced the expression of DR4 and DR5. These data show that CCT327 regulated DR4 and DR5 that both play a major role in TRAIL-induced apoptosis. It is another mechanism which CCT327 enhanced the proapoptotic effects of TRAIL in HL60-TR cells.
receptors for ligand binding and thereby inhibit ligand-induced apoptosis (9-12). Therefore, we next examined whether CCT327 modulated the expression of DcRs. We found that indeed CCT327 decreased the expression of DcR1, but it did not influence the level of DcR2 (Fig. 3C). So, we know that CCT327 may potentiated TRAIL-induced apoptosis by inhibition of DcR1.
CCT327-induced up-regulation of TRAIL receptors is dependent on ROS. There has been a report that TRAIL-induced apoptosis is regulated by ROS (47-51). We attempted to determine whether CCT327-induced TRAIL receptors are also regulated by ROS. Our data showed that pretreating HL60-TR cells with the ROS scavenger N-acetylcysteine (NAC) reduced the CCT327-induced up-regulation of both DR5 and DR4 expression in a dose-dependent manner (Fig. 4A). This suggests that ROS was involved in the induction of TRAIL receptors by CCT327. Next, we examined whether ROS is needed for potentiation of TRAIL-induced apoptosis by CCT327. As shown in Fig. 4B, we found that pretreatment with NAC abolished the effect of CCT327 on TRAIL-induced cleavage of PARP. These results show that CCT327 potentated TRAIL-induced apoptosis through ROS.
CCT327 induced upregulation of TRAIL receptors is mediated activation of MAPKs. MAPKs, including ERK1/2, p38, and JNK, have been reported to mediate induction of TRAIL receptors (47, 49). For this, we determined whether CCT327 could activate
ERK1/2, p38, and JNK. Cells were pretreated with CCT327 for different times and then examined for the phosphorylated ERK, JNK and p38. We found that CCT327 activated ERK1/2 in a time-dependent manner (Fig. 5A, top). No activation of JNK was found. In addition, CCT327 activated p38 was also observed (Fig. 5A, bottom). Our result showed that induction of TRAIL receptors by CCT327 required ERK1/2 and p38. Next, we also determined whether these MAPKs have any role in CCT327-induced TRAIL receptors. Cells were pretreated with 20 μM ERK1/2 inhibitor (PD98059), 20 μM JNK inhibitor (SP600125) and 10 μM p38 inhibitor (SB202190), respectively (52). ERK1/2 inhibitor (Fig.5B) and p38 inhibitor (Fig. 5C) both suppressed the CCT327-induced upregulation of DR4 and DR5. No effect of the JNK inhibitor was observed on CCT327-induced DR4 and DR5 expression. Up-regulation of TRAIL receptors by CCT327 is reversed by inhibitors of ERK1/2 and p38. Thus, the activation of ERK1/2 and p38 is consistent with the results obtained with the effect of their inhibitors on the CCT327-induced expression of TRAIL receptors.
all the apoptosis inducing cytokines. The unique property of triggering apoptosis in a variety of human cancer cells while sparing normal cells makes TRAIL a highly promising cancer therapeutic agent (2, 3). Both TRAIL and the agonistic antibodies against the receptor are currently in phase II clinical trial (53). TRAIL induces apoptosis through recognizing and binding to its cognate death receptors, DR4 and DR5 (also named as TRAIL-R1 and TRAIL-R2), on the cell surface. Upon ligand stimulation, DRs (Fas or death receptor 4/5, DR4/5) recruit FADD and the initiator caspases, caspase-8 or caspase-10, resulting in formation of the DISC, thereby inducing death signaling and the apoptosis pathway (9,10). However, TRAIL resistance is a major limitation in its clinical application as a cancer therapeutic agent. Nevertheless, a recent study has demonstrated that resistance of cancer cells to TRAIL is one of the major roadblocks to the development of this therapy (54). Thus, efforts to identify agents that activate DRs or block antiapoptotic effectors may improve therapeutic design.
In the present report, we describe a novel compound, CCT327, which has been shown to induce apoptosis in human leukemia cancer cells (41). Numerous literatures have shown convincing data that the up-regulation of DR4 or/and DR5 could sensitize TRAIL-resistant cells to TRAIL-induced cell death (49-52). We show that CCT327 can sensitize TRAIL-induced apoptosis through modulation of death receptors. Our
results also supported that DR4 and DR5 have important role to involve in CCT327 overcome TRAIL-resistance.
Many reports have showed that resistance to TRAIL can be due to several mechanisms, including overexpression of antiapoptotic proteins and decoy receptors (54). CCT327 decreased the expression of DcR1, but it did not influence the level of DcR2. Besides the induction of DcR 1, we also found that CCT327 downregulated expression antiapoptotic proteins including cFLIP (long and short), Bcl-2, Bcl-xL, and survivin. The effect was most pronounced on cFLIPs and Bcl-2. Recently, c-FLIP were shown to be correlated with TRAIL resistance in some tumor types, and c-FLIP downregulation has been implicated in chemotherapy sensitized TRAIL-induced apoptosis (56, 57). Several studies have shown that Bcl-2 blocks apoptosis by keeping mitochondrial function (58). Taken together, our results indicate that c-FLIP and Bcl-2 downregulation contributes to CCT327-facilitated TRAIL-mediated apoptosis.
ROS trigger a variety of cellular responses leading to cell growth, differentiation, or cell death (55). ROS generation has been proposed to be involved in death receptors up-regulation by cancer chemopreventive agents (51, 52). In the present study, we found that induction of ROS is critical for the sensitization of cells to TRAIL by CCT327. Our data show the mechanism by which CCT327 induces DRs up-regulation is through production of ROS. The antioxidant NAC abolished the upregulation of DR
by CCT327. The effect of CCT327 on TRAIL-induced apoptosis was also neutralized by the antioxidants. This reversal was apparently due to inhibition of induction of TRAIL receptors. An important downstream mediator of ROS-induced signaling is the MAPKs (31, 34). MAPKs, including ERK1/2, p38, and JNK, have been reported to mediate induction of TRAIL receptors (47, 49). Recent studies have been shown that activation of ERK, JNK or p38 are also associated with TRIAL-induced-apoptosis via up-regulation of DR4/5 (49, 50). CCT327 activated ERK1/2 p38 in a time-dependent manner. We questioned whether the activation of p38 and ERK1/2 was the cause or a downstream effect of up-regulation of the TRAIL receptors. ERK1/2 inhibitor (Fig.5B) and p38 inhibitor (Fig. 5C) both suppressed the CCT327-induced upregulation of DR4 and DR5. Interesting, the presence of JNK inhibitor had no effect on CCT327-induced DR4 and DR5 expression. CCT327 induced the expression of TRAIL receptors dependent of MAPK, especially ERK1/2 and p38.
Overall, we demonstrate that CCT327 can sensitize TRAIL-induced through the upregulation of DRs through JNK and p38-mediatedand the downregulation of cFLIP and other antiapoptotic proteins. CCT327 have an implication in treatment of cancer by TRAIL, especially tumors that develop resistance to TRAIL.
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Figure legends
Figure 1. CCT327-potentiated TRAIL induced apoptosis of HL60-TR cells. (A) Chemical structure of CCT327. (B) Dose-response curves for HL60 and HL60-TR cells treated with TRAIL. Cells (1 × 104) were treated with TRAIL (0, 25, 50, 100, 200 ng/mL) for 48 h and cell viability was quantitated by MTT assay. In comparison with HL60 cells and HL60-TR were resistant to induction of apoptosis by TRAIL. Data are expressed as mean ± S.E.M. *, P < 0.05 and **, P < 0.01 significant when compared to HL60-TR. (C) HL60-TR cells (1 × 104 per well) were plated in 24-well plates for 48 h, treated various concentrations of with CCT327 (0, 75, 150, 300 or 600 nM) and TRAIL (0, 50, 100, or 200 ng/mL). Cell viability was determined by MTT assay, as described in Materials and Methods. Data are expressed as mean ± S.E.M. (D) HL60-TR cells (5 × 105) were treated with TRAIL (100 ng/mL) and with or without CCT327 (300 nM) for 48 h. Cells were stained with PI, and the sub-G1 fraction was analyzed using flow cytometry. Data are expressed as mean ± S.E.M. **, P < 0.01 significant when compared to CCT327; ###, P < 0.001 significant when compared to TRAIL. (E)
ng/mL) for 48 h. Whole-cell extracts were prepared and analyzed by Western blotting using antibodies against pro-caspase-3, pro-caspase-8, pro-caspase-9, and PARP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.
Figure 2. Effects of CCT327 on antiapoptotic and proapoptotic proteins expression. HL60-TR cells were treated with 300 nM CCT327 for indicated times. Whole-cell extracts were prepared and analyzed by Western blotting using the antibodies against antiapoptotic (A) and proapoptotic (B) proteins. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.
Figure 3. CCT327 induced DR5 and DR4 expression and suppressed decoy receptors. (A) HL60-TR cells (1 × 106 cells) were treated with indicated doses of CCT327 for indicated times. (B) HL60-TR cells were treated with TRAIL (100 ng/mL) and with or without CCT327 (300 nM) for 48 h. Whole-cell extracts were prepared and analyzed for DR4 and DR5 expression by Western blotting. (C) HL60-TR cells were treated with CCT327 (300 nM) for indicated times. Whole-cell extracts were prepared and analyzed for DcR1 and DcR2 expression by Western blotting. Western blotting data presented are representative of those obtained in at least 3 separate experiments. β-actin was used as a loading control.
HL60-TR cells (1 × 106 cells) were pretreated with various concentrations of NAC for 1 h. Cells were treated with 300 nM CCT327 for 48 h. Whole-cell extracts were prepared and analyzed for DR4 and DR5 expression by Western blotting. (B) NAC inhibited PARP cleavage induced by combination of TRAIL and CCT327. HL60-TR cells (1 × 106 cells) were pretreated with 10 mM NAC for 1 h, and then treated with 300 nM CCT327 or 100 ng/mL TRAIL for 48 h as indicated above. Whole-cell extracts were prepared and analyzed for PARP and Cleaved-PARP expression by Western blotting. Equal protein loading was evaluated by β-actin.
Figure 5. Upregulation of death receptors were ERK1/2, and p38 dependent. (A) HL60-TR cells (1 × 106 cells) were treated with 300 nM CCT327 as indicated above and whole-cell extracts were subjected to Western blotting for phosphorylated ERK1/2, p38, and JNK. The same blots were stripped and reprobed with ERK1/2, p38, and JNK to ensure equal loading. HL60-TR cells were pretreated with various concentrations of 20 μM ERK1/2 inhibitor, PD98059 (B); 20 μM JNK inhibitor, SP600125 (C); and 10 μM p38 inhibitor, SB202190 (D) for 1 h and then treated with 300 nM CCT327 for 48 h as indicated above. Whole-cell extracts were prepared and analyzed by Western blotting using DR4 and DR5 antibodies. β-actin was used as a loading control.
