비스테로이드소염제(Nonsteroidal Anti-inflammatory Drug, NSAID)에 의한 인간 암세포의 imatinib 및 TRAIL의 세포 독성 증강 기전 연구

Potentiation of the Cytotoxic Effects of Imatinib and TRAIL by Nonsteroidal Anti-inflammatory Drugs on Human Cancer Cells

Abstract

항암 요법의 실패의 주요 원인으로 암세포의 항암제에 대한 내성 획득이 잘 알려져 있다. 비스테로이드소염제(NSAID)는 항염증작용뿐만 아니라 항암제와의 병용요법으로 임상적인 암 치료 요법에 응용되고있다. 본 연구에서는 NSAIDs 인 celecoxib 및 이의 구조 유사체인 2,5-dimethyl celecoxib 그리고 ibuprofen의 인간 암세포에 대한 imatinib 및 TNF-related apoptosis inducing ligand (TRAIL) 세포 독성 변화에 미치는 영향을 조사하였다. NSAID는 TRAIL 및 imatinib에 각각 약제 내성을 나타내는 간암 세포와 백혈병 세포에서 이들 약물의 세포독성을 증강시키는 활성을 나타내었다. NSAID는 ATF4/CHOP의 발현 증강으로 소포체 스트레스 및 오토파지(Autophagy, 자가포식)를 유도하였다. 이로 인한 DR5 발현 증강과 함께 c-FLIP 발현 억제로 TRAIL의 세포독성을 증강시키는 기전을 나타내었다. NSAID로 유도되는 오토파지 활성은 imatinib-resistant CD44^highK562 백혈병세포의 imatinib 감수성을 증강시켰으며, NSAID는 이 세포에서 높은 발현을 나타내는 다양한 stemness-related marker 단백질의 발현 감소를 촉진시키는 활성으로 세포사멸을 유도하는 것을 알 수 있었다. 이러한 결과는 NSAID의 오토파지 유도 활성이 TRAIL과 imatinib의 세포 독성을 증강시키는 것으로서, NSAID와 이들 약물과 병용 처리방법은 인간 암세포의 TRAIL 및 imatinib 내성을 극복 시킴과 동시에 암세포에 이들 약물의 독성 부작용을 감소시킬 수 있는 낮은 농도의 처리를 가능하게 할 것으로 사료된다.

The resistance of cancer cells to anti-cancer drugs is the leading cause of chemotherapy failure. The clinical use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been gradually extended to cancer treatment through combination with anti-cancer drugs. In the current study, we investigated whether NSAIDs including celecoxib (CCB), 2,5-dimethyl celecoxib (DMC), and ibuprofen (IBU) could enhance the cytotoxic effects of imatinib and TNF-related apoptosis inducing ligand (TRAIL) on human cancer cells. We found that the NSAIDs potentiated TRAIL and imatinib cytotoxicity against human hepatocellular carcinoma (HCC) cell lines SNU-354, SNU-423, SNU-449, and SNU-475/TR and against leukemic K562 cells with high level of CD44 (CD44^highK562), respectively. More specifically, CCB induced endoplasmic reticulum stress via up-regulation of ATF4/CHOP which is associated with the induction of autophagy against HCC and CD44^high K562 cells. NSAID-induced autophagic activity accelerated TRAIL cytotoxicity of HCC cells through up- and down-regulation of DR5 and c-FLIP, respectively. The NSAIDs also potentiated imatinib-induced cytotoxicity and apoptosis through down-regulation of markers in CD44^highK562 cells that express a stemness phenotype. Our results suggest that the ability of NSAIDs to induce autophagy could enhance the cytotoxicity of TRAIL and imatinib, leading to a reverse resistance to these drugs in the cancer cells. In conclusion, NSAIDs in combination with low-dose TRAIL or imatinib may constitute a novel clinical strategy that maximizes therapeutic efficacy of each drug and effectively reduces the toxic side effects.

Introduction

Chemotherapy is an important therapeutic strategy of treatment for cancer patients. But a major problem to targeted cancer therapy is the inevitable emergence of drug resistance. In order to treat drug-resistant cancers, a combination of chemotherapeutics with different acting agents may lead to useful treatment strategies [2].The anti-cancer benefit of chemotherapy was thought to be the effect of direct cytotoxicity, which may then lead to cell death. Despite the differences between two processes of apoptosis (type I cell death) and autophagic cell death (type II cell death), their regulation is connected, and the same regulators can control both apoptosis and autophagy [16]. Autophagy can protect cells from apoptotic stimuli, including growth factor deprivation and endoplasmic reticulum (ER) stress but it may also induce cell death, which depends on the distinct cellular context. Therefore, autophagy and apoptotic signals cooperatively function to induce cell death [7]. Although it is widely accepted that anti-tumor treatment induces autophagy, it remains to be determined whether activation of autophagy promotes cell survival as a response to stress, or induces cell death under the condition of apoptotic defects. Autophagy plays an anti-tumor effect in suppression of the formation of hepatocellular carcinoma (HCC), while serving as a pro-survival mechanism to promote liver cancer development, and results in resistance to anti-HCC therapy. Therefore, targeting autophagy is a promising strategy for the treatment of HCC [23]. Autophagy is activated for cell survival after ER stress. The ER stress-induced autophagy can not only play a cytoprotective role but also can promote cell death. ER is also acting as a cell sensor to monitor and maintain cellular homeostasis. Both the ER stress and autophagy pathways interact with those of apoptosis and cell fate is determined by the most dominant response during the event of irreparable or prolonged damage [8]. Induction of ER stress enhanced expression of the pro-apoptotic CCAAT/enhancer-binding protein homologous protein (CHOP), which has been found to up-regulate DR5 expression in cancer cells [24]. ER stress has also been reported to down-regulate anti-apoptotic proteins, including c-FLIP [10]. TNF-related apoptosis-inducing ligand (TRAIL) is currently under clinical development as a cancer therapeutic because it can induce apoptosis selectively in cancer cells. TRAIL has been shown to induce autophagy as well as apoptosis [11], and the therapeutic relevance of ER stress inducers in cancer as a sensitizer to TRAIL-based therapies has been reported [24]. However, many hepatocellular carcinoma (HCC) cells show resistance to TRAIL-induced apoptosis [9].

In cases of failure towards treatment of imatinib, a small molecule Bcr-Abl tyrosine kinase inhibitor, leukemia patients that are resistant to imatinib therapy due to the low sensitivity of cancer cells to the drug even if first-line antineoplastic agent for both chronic myeloid leukemia (CML) and acute lymphoblastic leukemia [18]. In these cases, novel treatment strategies are urgently needed to overcome drug resistance of these antitumor agents. Therefore, the combination of two agents that are able to trigger aggravated ER stress might be enhanced tumor cell killing.

Celecoxib (CCB), a novel non-steroidal anti-inflammatory drug that directly targets cyclooxygenase-2 (COX-2) inhibitor, prevented the progression of a number of types of cancer and exhibited several potential antitumor mechanisms, including inhibition of proliferation, induction of apoptosis, antiangiogenic effect, and resensitization of anticancer drugs. Although CCB is a COX-2 inhibitor, it also exerts antitumor activity in tumor cells and tissues that lack the COX-2 enzyme [19], indicating that CCB can inhibit tumor growth independently of its COX-2-inhibitory activity. Therefore, NSAID including celecoxib could be a potential candidate for combination therapy through antitumor activity of its own and resensitization of other anticancer drugs [14]. Treatment with CCB induces the up-regulation of ER chaperones, and promotes tumor cell death in vitro and in vivo through the ER stress response [20]. Therefore, it is necessary to examine whether it is more efficacious to treat cancer with a combination of drugs, rather than NSAID alone. In the present study, we determined whether NSAID in combination with TRAIL or imatinib able to trigger ER stress might result in further aggravated ER stress, leading to significantly enhanced cell death of human HCC and chronic myelogenous leukemia (CML) cells.

Materials and Methods

Cell culture and reagents

Hepatocellular carcinoma (HCC) cell lines (SNU-475, SNU423, SNU-353, and SNU-449) derived HCC tissues of patients purchased from the Korea Cell Line Bank [6]. SNU-475/TR cells isolated from parental SNU-475 cells by stepwise increases in concentrations of TRAIL. Human K562 CML cell line was obtained from American Type Culture Collection (Manassas, VA, USA). CD44^highK562 cells were established during isolation of imatinib-resistant K562 cells after treatment with increasing concentrations of imatinib, and were stable in complete medium without imatinib [5]. Cells were maintained in RPMI medium (Welgene, Gyeongsan, Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Welgene), 100 unit/ml penicillin and 100 μg/ml streptomycin in a 5% CO₂ humidified incubator at 37℃. Recombinant human soluble TRAIL was obtained from R&D systems (Minneapolis, MN, USA). Celecoxib (CCB), 2,5-dimethyl celecoxib (DMC), ibuprofen (IBU) cycloheximide (CHX), 3-methyladenine (3-MA) chloroquine (CQ), and 4- phenylbutyric acid (4-PBA) were purchased from SigmaAldrich (St. Louis, MO, USA). Imatinib was kindly donated by Dr. I. J. Fidler (University of Texas MD Anderson Cancer Center, Houston, TX).

Cell proliferation assay

Cell proliferation was measured by counting viable cells by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric dye-reduction method. Exponentially growing cells (1×10⁴ cells/well) were plated in a 96-well plate and incubated in growth medium treated with the indicated concentrations of TRAIL, imatinib, CCB, DMC or IBU at 37℃. After 96 hr, the medium was removed using centrifugation, and MTT-formazan crystals solubilized in 100 ml DMSO. The optical density of each sample at 570 nm was measured using ELISA reader. The optical density of the medium was proportional to the number of viable cells. Inhibition of proliferation was evaluated as a percentage of control growth (no drug in the medium). All experiments were carried out in triplicate.

Western blot analysis

Cells were washed with ice-cold phosphate buffer, lysed in lysis buffer consisting of 1% (w/v) sodium dodecyl sulfate (SDS), 1 mM sodium ortho-vanadate, and 10 mM Tris (pH 7.4), and sonicated for 5 sec. Lysate containing proteins were quantified using a Bradford protein assay kit (Pierce, Rockford, IL., USA). Protein samples were separated by 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE) using a mini gel apparatus (Bio-Rad, Hercules, CA, USA). Following electrophoresis, gels were transferred onto a nitrocellulose membranes (Hybond-ECL; GE Healthcare, Piscataway, NJ, USA). Each membrane was blocked with 5% skim milk in Tris-buffered saline plus 0.05% Tween-20 (TBST). Protein bands were probed with primary antibody followed by labeling with horseradish peroxidase-conjugated anti-mouse, anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA, USA). The antibodies were used: c-FLIP, β-tubulin, ALDH1, Nanog, Oct4, CHOP, DR5, CD44, caspase-8 and caspase-3 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP), ATF4 and p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The p53 antibody (DO-1) is a mouse monoclonal antibody raised against amino acids 11-25 of p53 of human origin (Santa Cruz Biotechnology), which was recommended for detection of wild and mutant p53 of human origin. b-actin antibody (Sigma-Aldrich), LC3B, and p62 (Novus Biologicals, Littleton CO, USA), c-Myc (Epitomics, CA, USA) were also used.

Flow cytometric analysis

Cell surface expression of DR5 on HCC cells (5×10⁵ cells/ well) treated with or without NSAID was determined by flow cytometry. Briefly, cells were washed once at the time of harvesting with PBS/0.1% sodium azide and aliquoted into polystyrene tubes. Cells were stained with FITC-labeled anti-DR5 mAb. Autofluorescence and isotype (IgG2b)-matched control Abs (BD Biosciences, San Jose, CA, USA) were included. Data were acquired on a CANTO II and Calibur (BD Biosciences) and analyzed by using FlowJo (ver. 10; Tree Star, Ashland, OR, USA).

Apoptosis assay

CD44^highK562 cells (2×10⁵ cells/ml) were treated with imatinib in the presence or absence of CCB (or DMC) for 24 hr. Apoptosis was measured by annexin V assay. The cells were centrifuged and resuspended in 500 μl of a staining solution containing annexin V fluorescein (FITC Apoptosis Detection Kit; BD Pharmingen, San Diego, CA, USA) and propidium iodide (PI) in PBS. After incubation at room temperature for 15 min, the cells were analyzed by flow cytometry. Annexin V binds to cells that express phosphati dylserine on the outer layer of their cell membrane, and PI stains the cellular DNA of cells with a compromised cell membrane. This allows for the discrimination of live cells from apoptotic cells and necrotic cells. Viable cells remained unstained (Annexin V-FITC⁻/PI⁻). Early apoptotic cells showed Annexin V-FITC⁺/PI⁻ staining patterns; whereas late apoptotic cells exhibited Annexin V-FITC⁺/PI⁺ staining patterns due to a loss of plasma membrane integrity

Statistical analysis

The results obtained were expressed as the mean ± SE of at least three independent experiments. The statistical significance of differences was assessed using the Student’s t-test. Values of p<0.05, p<0.01, and p<0.001 were considered statistically significant in all experiments.

Results

Enhancement of TRAIL-mediated cytotoxicity by NSAID in human cancer cells

We first compared the effect of NSAID with TRAIL on the growth of human HCC SNU-354 and leukemia K562 with high level of CD44 (CD44^highK562) cells. SNU-354 cells were treated with increasing concentrations of TRAIL in the presence or absence of celecoxib (CCB), one of NSAIDs, for 4 days, and then cell growth inhibition was determined by MTT assay. High susceptibility to TRAIL following combined treatment with CCB and TRAIL than TRAIL alone was observed in SNU-354 cells, indicating potentiation of TRAIL sensitivity in HCC cells by CCB. Moreover, 2,5-dimethyl-celecoxib (DMC), one of CCB derivative that lacks the ability to inhibit COX-2, also potentiated TRAIL-induced cytotoxicity of SNU-354 cells (Fig. 1), suggesting that NSAID exerts TRAIL-potentiating effects, regardless of COX-2 activity. Similar results were observed in CD44^highK562 cells. Both CCB and DMC significantly enhanced TRAIL-induced cytotoxicity of CD44^highK562 cells (Fig. 1B). Ibuprofen (IBU), another non-selective COX inhibitor, also significantly potentiated TRAIL cytotoxicity in four HCC cell lines including SNU-475/TR, SNU-423, SNU-449 and SNU-354 cells, leading to sensitization of sensitize HCC cells to TRAIL by IBU (Fig. 2). These results strongly suggest the possibility that NSAID could be a promising candidate for a new class of TRAIL sensitizer.

Fig. 1. Enhancement of TRAIL cytotoxicity by celecoxib or 2, 5-dimethyl celecoxib in HCC and leukemia cells. SNU-354 HCC cells (A) or CD44^highK562 cells (B) were treated with serial doses of TRAIL in the presence or absence of indicated doses of celecoxib (CCB) or 2, 5-dimethyl celecoxib (DMC). Percentage of cell survival was determined after 96 hr of incubation using MTT assay. Each bar represents the mean ± SD of triplicate experiments. *p<0.05, **p<0.01, ***p<0.001.

Fig. 2. Enhancement of TRAIL cytotoxicity by ibuprofen in various HCC cells. SNU-475/TR, SNU-423, SNU-449 and SNU-354 HCC cells were treated with serial doses of TRAIL in the presence or absence of indicated doses of ibuprofen (IBU). Percentage of cell survival was determined after 96 hr of incubation using MTT assay. Each bar represents the mean ± SD of triplicate experiments. *p<0.05, **p<0.01, ***p<0.001.

Down-regulation of c-FLIP and up-regulation of DR5 and caspases by NSAID through ER-dependent autophagy activation

We next determined whether NSAID could modulate cell surface expression of death receptor 5 (DR5) in human HCC cell lines since DR5 induction is one mechanism accounting for the sensitization of TRAIL-induced apoptosis by NSAID. Cell surface expression level of DR5 was quantified by flow cytometry. The cell surface expression of DR5 in HCC cells was significantly increased by NSAID treatment. In SNU-423 cells, the cell surface expression of DR5 was increased by CCB or DMC treatment (Fig. 3A, Fig. 3B). Similarly, up-regulation of DR5 surface expression was observed in IBU-treated SNU-475/TR cells (Fig. 3C). These results suggest that NSAID-induced up-regulation of DR5 surface expression could be involved in sensitization of TRAIL-resistant HCC cells to TRAIL. It has been reported that c-FLIP as well as DR5 are key components in the TRAIL/death receptor-medi ated apoptotic pathway, and down-regulation of c-FLIP plays an important role in overcoming TRAIL resistance and is caused by facilitating autophagy-mediated c-FLIP degradation, leading to apoptotic cell death [12]. We therefore determined whether CCB treatment could induce autophagy and subsequently modulate c-FLIP and DR5 expression in SNU-449 cells. Since LC3 is widely used to monitor autophagy. The amount of LC3 conversion (LC3-I to LC3-II) is clearly correlated with the number of autophagosomes, and an increase in endogenous LC3-II may be regarded as a marker for autophagy and another autophagy marker p62, a scaffolding protein that is degraded by autophagy [3]. When SNU-449 cells were treated with increasing doses of CCB for different times, we found an increase in conversion of LC3-I to LC3-II and a decrease in p62 protein level in the cells, indicating autophagy inducing ability of CCB. Decrease of c-FLIP_L level and concurrent increase of DR5 level were induced after treatment with above 20 μM CCB for 24 hr (Fig. 4A). Moreover, down-regulation of c-FLIP_L and up-regulation of DR5 were occurred between 12 and 36 hr at 30μM CCB (Fig. 4B). To determine whether the ability of CCB to induce autophagy in SNU-449 cells could be linked to the degradation/down-regulation of c-FLIP_L and concurrent up-regulation of DR5 through ER stress-mediated autoph agy, the cells were treated with CCB in the presence of chloroquine (CQ) that blocks an autophagy inhibitor that blocks late stage autophagy, and changed levels of CHOP/ATF4, c-FLIP_L DR5 and cancer stemness-related marker CD44 were evaluated (Fig. 4C). We first determined whether CCB could up-regulate CHOP and ATF4, markers of ER stress. Our data showed that treatment of SNU449 cells with CCB enhanced expression of CHOP and ATF4, which was prevented by CQ, and also CCB down-regulated c-FLIP_L/CD44 and upregulated DR5, which were prevented by CQ. These results indicate that NSAID induces ER stress-mediated autophagy through ATF4/CHOP pathway, which leads to autophagymediated degradation of c-FLIP_L and CD44 and simultaneous up-regulation of CHOP-dependent DR5 in HCC cells. Next, we investigated NSAID’s cooperative effect with TRAIL on the induction of apoptosis in SNU-449 cells. When the cells were co-treated with IBU and TRAIL, IBU exerted enhanced effects on the TRAIL-mediated activation of caspases and PARP through caspase-8, -3 and PARP cleavage (Fig. 4D), suggesting that NSAID cooperates with TRAIL to augment the induction of apoptosis in SNU-449 cells. Similar results were obtained in another HCC cells. SNU-475 cells showed that CCB-induced up-regulation of CHOP/ATF4 and DR5 and down-regulation of CD44 was significantly blocked by treatment of early stage autophagy inhibitor 3- methyladenine (3-MA). CQ also prevented CCB-induced upregulation of CHOP/DR5 and down-regulation of c-FLIP_L/ CD44 (Fig. 5A, Fig. B), indicating that ER stress-mediated autophagy induction by NSAID contributes to TRAIL-mediated autophagic cell death in HCC cells. For further confirming CCB-induced c-FLIP_L degradation, the changed level of c-FLIP_L in SNU-475 cells was determined in the presence of protein synthesis inhibitor cycloheximide (CHX) after treatment with CCB (Fig. 5C). When the half-life of c-FLIP_L protein in SNU-475 cells treated with or without CCB by performing a CHX chase assay, reduction of c-FLIP protein in CCB-treated cells was significantly accelerated in the presence of CHX compared with that in CCB-untreated cells, indicating reduction of c-FLIP_L level by CCB in HCC cells possibly through autophagic degradation. Since the above results indicate that up-regulation of DR5 and down-regulation of c-FLIP_L/CD44 by CCB occurred through ER stress ATF4/CHOP pathway, we therefore examined whether ER stress inhibitor 4-phenylbutyric acid (4-PBA) could affect the CCB-induced expression of DR5, c-FLIP_L and CD44 in SNU475 cells (Fig. 5D). Our data showed that inhibition of ER stress by 4-PBA blocked CCB-induced up-regulation of CHOP/DR5 and down-regulation of c-FLIP_L/CD44. These results suggest that CCB triggers ER stress ATF4/CHOP, which leads to up-regulation of DR5 and down-regulation of c-FLIP_L/CD44.

Fig. 3. Enhanced cell surface expression of DR5 by NSAID in HCC cells. SNU-423 (A, B) and SNU-475/TR (C) cells treated with indicated dose of CCB, DMC or IBU for 24 hr. The cells were stained with control IgG or anti-DR5 antibody and subsequently labeled with PE-conjugated secondary antibodies to determine the surface expression of DR5, and the DR5 surface expression was measured by a flow cytometer.

Fig. 4. Down-regulation of c-FLIP and up-regulation of DR5, and subsequent activation of caspases by NSAID through autophagy induction. SNU-449 cells were treated with serial doses of CCB for 24 hr (A) or 30 μM CCB for indicated times (B). The cells were pretreated with 5 μM chloroquine (CQ) for 3 hr, and followed with treatment of 40 μM CCB for 24 hr (C). The cells treated with indicated doses of TRAIL (ng/ml) in the presence or absence of 500 μM IBU for 24 hr (D). The changed levels of LC3-I/II, p62, c-FLIP, DR5 (mature form), CHOP, ATF4, CD44, caspase-8 and -3 (Casp8 and Casp3), their cleavage (Cl. Casp8 and Cl. Casp3), and cleaved PARP (Cl. PARP) were determined by Western blot analysis. Actin or tubulin was used as a loading control.

Fig. 5. Effect of NSAID on expression of CHOP/DR5 and c-FLIP through ER-dependent autophagy. SNU-475 cells were pretreated with 10 mM 3-MA (A) or 5 μM CQ (B) for 3 hr and followed with treatment of 40 μM CCB for 24 hr. (C) The cells were treated with or without 50 μM CCB for 24 hr, and were collected at 0, 3 and 6 hr after following treatment with 20 μg/ml cycloheximide (CHX). (D) The cells were pretreated with or without 1.5 mM 4-phenylbutyric acid (4-PBA) for 24 hr before treatment of 40 μM CCB for 24 hr (D), and the changed levels of indicated molecules were determined by western blot analysis. Actin was used as a loading control.

Potentiation of imatinib-induced cell death and acceleration of down-regulation of stemness-related markers by CCB

We investigated whether NSAIDs are capable of inducing sensitization of imatinib-resistant CD44^highK562 cells to imatinib through enhancement of imatinb-mediated cytotoxicity since NSAIDs potentiated TRAIL-induced cytotoxicity of CD44^highK562 cells in Fig. 1B. Our data showed that CCB treatment resulted in a dose-dependent reduction in cell viability against imatinb-treated CD44^highK562 cells (Fig. 6A). To evaluate if the combinatorial effect of imatinb and NSAID observed in these cells were correlated with enhancement of imatinib-mediated apoptosis, CD44^highK562 cells were treated with imatinib in combination with either CCB or DMC, which were stained with annexin V/PI, and FACS analysis was performed (Fig. 6B). When compared with CD44^highK562 cells only treated by imatinib, the cells co-treated with imatinib and CCB (or DMC) displayed a significant increase in Annexin V positivity, indicating that NSAID exerted enhanced effects on the induction of im atinib-mediated apoptosis. Therefore, our findings suggest that NSAID potentiates imatinib-mediated cytotoxicity and apoptosis, which could lead to reverse drug resistance to imatinib in leukemic cells. We previously reported that CD44^highK562 cells exhibited high expression of various stemness-related markers and resistance to imatinib [5]. To examine whether imatinib has the capability to induce ER-mediated autophagy and it causes down-regulation of stemness-related marker proteins, CD44^highK562 cells were treated with various concentration of imatinib. Treatment of CD44^highK562 cells with imatinib resulted in up-regulation of ATF4 and CHOP and reduction of p62 level, indicating ER-stress mediated autophagy induction by imatinib, in parallel with down-regulation of stemness-related marker proteins such as CD44, Oct4, c-Myc and mutated p53 (mutp53) at high concentration of CCB (Fig. 7A). Next, we determined whether autophagy inhibition could block CCB-induced autophagy, leading to attenuation of down- regulation of stemness-related markers (Fig. 7B). CD44^high K562 cells co-treated with imatinib and CQ showed that the existence of CQ markedly sealed the autophagy inducing effect of imatinib and subsequent down-regulation of CD44, Oct4 and c-Myc. Since imatinib-mediated cytotoxicity and apoptosis were enhanced by autophagy-inducing ability of NSAID, we determined whether CCB could accelerate imatinib-mediated autophagy and modulation of CD44 and Oct4 in CD44^highK562 cells (Fig. 7C). As expected, CCB accelerated imatinib-induced up-regulation of ATF4 and CHOP, indicat ing ER-mediated autophagy, in parallel with the acceleration of enhancement of LC3-II and subsequent down-regulation of CD44 and Oct4 in CD44^highK562 cells. In addition, CCB accelerated imatinib-mediated reduced level of p62 and subsequent of c-Myc and mutp53, which generated the typical cleavage products of PARP-1, the main substrate of caspase-3, thus verifying functional activation of the apoptotic caspase cascade in CD44^highK562 cells (Fig. 7D). These results suggest that enhanced autophagic capability of imatinib by NSAID is associated with autophagic cell death, and it potentiated imatinib-mediated cell death of CD44^high K562 cells.

Fig. 6. Potentiation of imatinib-induced cytotoxicity and apoptosis by CCB. (A) CD44^highK562 cells were treated with serial doses of imatinib in the presence or absence of CCB (1 or 5 μM). Percentage of cell survival was determined after 96 hr of incubation using MTT assay. Each bar represents the mean ± SD of triplicate experiments. *p<0.05, **p<0.01, ***p<0.001. (B) CD44^highK562 cells were treated with imatinib (2 or 10 μM) in the absence or presence of 10 μM CCB or DMC for 24 hr, and the percentage of apoptotic cells was quantified using FACS. The upper right quadrants contain late apoptotic cells, and the lower right quadrants represent early apoptotic cells. Images shown are representative of three independent experiments.

Fig. 7. Acceleration of imatinb-mediated autophagy and down-regulation of stemness-related markers by CCB. CD44^highK562 cells were treated with serial doses of CCB for 24 hr (A) or the cells were pretreated with 5μM CQ, and followed with treatment of 25 μM CCB for 24 hr (B). The cells were treated with 10 μM imatinib (C) or 5 μM imatinib (D) in the absence or presence of 10 μM CCB for 24 hr. The changed levels of LC3B-1/II, p62, mutated p53 (mutp53), c-Myc, CD44, Oct4, PARP and cleaved PARP (Cl. PARP) were determined by Western blot analysis. Actin was used as a loading control.

Discussion

Autophagy is involved in the initiation of HCC, and autophagy modulation could provide new prospects in antiHCC therapy [23]. Therefore, it is necessary to define how to regulate autophagy (inhibition or activation) in order to ensure maximum therapeutic advantage. We found that a novel combination of TRAIL and NSAID (CCB, DMC or IBU) was able to enhance TRAIL-mediated cell death in human HCC and leukemic CD44^highK562 cells through induction of ER stress-mediated autophagy. NSAID also sensitized imatinibresistant CD44^highK562 cells to imatinib-mediated cell death through a similar mechanism.

The ER acts as a cell sensor to monitor and maintain cellular homeostasis, and autophagy could be an adaptive mechanism against increased ER stress to eliminate misfolded proteins. ER stress has been found to be associated with the induction of both autophagy and apoptosis [22]. In the early stage of the ER stress response, unfolded protein response helps to promote cell survival, but the over-activation of ER stress can increase cell damage and even cause cell death. Therefore, ER stress inducer may be a potential target for pharmacological intervention targeted to autophagy to enhance damage of tumor cells induced by antitumor drugs. The antitumor properties of DMC, a non-COX-2 inhibitory analog of celecoxib, have been associated with their abilities to induce ER stress, which in turn inhibits protein translation and induce apoptosis [15]. IBU is also a non-selective COX inhibitor NASID that has antipyretic, analgesic and anti-inflammatory effects [17] and induces autophagy [13]. We provide evidence that DMC and IBU that lack COX-2 inhibitory activity retains CCB’s ability to enhance TRAIL or imatinib-induced cytotoxicity and no COX-2 inhibitory function appears to be required to enhance the anti tumor properties of TRAIL and imatinib. We also showed that COX-2-independent autophagy inducing effects occurred in cancer cell lines, whether or not they displayed drug-resistant phenotypes. Our results clearly demonstrate that DMC is able to potently mimic CCB with regard to enhancing growth inhibitory of anticancer drugs through induction of autophagy, and thereby DMC and CCB lead to sensitize in drug-resistant HCC and K562 cells to TRAIL and imatinib, respectively. DMC might have an additional advantage: because it does not inhibit COX-2, its use in human patients might not cause the serious cardiovascular side effects that recently emerged with the prolonged use of coxibs such as CCB, rofecoxib, and valdecoxib [21]. The expression of ATF4 and CHOP, one of ATF4 target genes, can be elevated by ER [4], which ultimately activates caspase 3 to induce apoptosis [26], and thus CHOP is a typical protein associated with ER stress-induced apoptosis. NSAID markedly increased CHOP-dependent up-regulation of DR5 and also reduced the level of c-FLIP, a key inhibitor of death receptormediated apoptosis by facilitating c-FLIP degradation through ER-dependent autophagic pathway. Both DR5 up-regulation and c-FLIP reduction contribute to cooperative induction of cell death by the combination of CCB and TRAIL. These results suggest that NSAID sensitizes human HCC cells to TRAIL-induced cell death via induction of DR5 and downregulation of c-FLIP. NSAID-induced autophagy also contributed to enhance imatinib-mediated cell death. We found that CCB or DMC induced ER stress and was able to increase the expression of ER stress markers including ATF4 and CHOP in CD44^highK562 cells, indicating that NSAID enhances the cytotoxicity of imatinib on leukemic cells caused by the induction of autophagy-related cell death through ER stress ATF4/CHOP-mediated autophagy activation. Therefore, the growth inhibitory property of NSAID might be due to ER stress-induced autophagic cell death as well as induction of apoptosis.

Cancer stem cell (CSC) population, a subgroup of cancer cells, is responsible for the chemoresistance and cancer relapse, as it has ability to self-renew and to differentiate into the heterogeneous lineages of cancer cells in response to chemotherapeutic agents [27]. Treatment of CD44^highK562 cells expressing a CSC-like phenotype with NSAID resulted in down-regulation of stemness-related marker proteins including CD44, Oct 4, c-Myc and mutated p53 through at least partially mediated by autophagic degradation. Indeed, it has been reported that IBU reduces the stemness features of gastric cancer cells by reducing CD44 and Oct3 and 4 transcript levels [1]. Our results suggest that NSAID can serve as attractive candidate for novel anticancer agents by eliminating cancer stem cells. Multiple signaling pathways have been demonstrated to play a significant role in the regulation of autophagy [25]. Among these pathways, the PI3K/Akt-mTOR pathway is inhibitor of the autophagy. We found that treatment of HCC or CD44^highK562 cells with autophagy inhibitor such as 3-MA, an inhibitor of class III PI3K inhibitor or CQ, an inhibitor of dual PI3K/mTOR resulted in blocking of NSAID-induced CHOP/ATF4-dependent autophagy in HCC and CD44^highK562 cells.

In summary, there is cooperation between CCB and imatinib on activation of autophagy associated proteins that accelerated autophagic degradation of c-FLIP and stemnessrelated proteins, suggesting that NSAID enhances TRAIL or imatinib-mediated apoptotic cell death by the induction of autophagic cell death. Although our present study provides the necessary basic studies of molecular and cellular enhancing cytotoxic effect of imatinib and TRAIL in human cancer cell lines by NSAID, Our findings are expected to be of clinical significance in terms of potential application of NSAIDs with TRAIL or imatinib in cancer treatment.