Artesunate와 비스테로이드소염제 NSAID의 병용 처리에 의한 항암 활성 증강

Potentiation of Anticancer Effect of Artesunate by Combination with Nonsteroidal Anti-inflammatory Drugs on Human Cancer Cells

Abstract

본 연구에서는 다양한 인체 암포주를 대상으로 NSAID의 항암 효과를 증강시키는 artesunate (ART)의 역할과 이에 대한 분자적 기전을 연구하였다. 다양한 타입의 암세포주를 대상으로 암세포 성장 억제 활성을 조사한 결과, ART는 NSAID인 celecoxib (CCB) 또는 dimethyl-CCB (DMC)와의 병용 효과를 나타내었다. ART 처리로 ATF4/CHOP의 발현 증강과 함께 오토파지 유도 표식인 p62 감소의 결과로서, ATF4/CHOP 경로가 ART의 오토파지 유도 활성에 관여할 것으로 예상되었으며, ART의 오토파지 활성과 관련하여 NRF2 및 암 줄기 세포 관련 단백질인 CD44, CD133, ALDH1, Oct4, mutated p53 (mutp53) 및 c-Myc의 발현이 감소되었다. 또한 DMC 단독처리 보다 ART와 DMC의 병용으로 ATF4/CHOP의 발현 증강과 p62의 감소가 더욱 촉진되고, NRF2 및 암 줄기 세포 관련 단백질 발현 감소도 현저히 촉진되면서 궁극적으로 PARP 활성화에 의해 apoptosis가 유도됨을 알 수 있었다. 이러한 결과는 ART/DMC 병용 처리가 각 물질 단독 처리보다 암세포의 성장 억제 및 apoptosis 유도에 더욱 효과적이고, ART 및 DMC 의 오토파지 유도 활성은 암 줄기 세포 관련 단백질의 분해를 촉진함으로써, 암 줄기 세포가 제거될 수 있음을 시사하였다. 이와 같이 ART는 NSAID 뿐만 아니라 imatinib의 항암 효과를 증강시키는 활성으로, chemosensitizer로서 중요한 후보 물질이 될 수 있음을 밝혔다.

The purpose of present study is to investigate the role of artesunate (ART) in enhancing anticancer effect of nonsteroidal anti-inflammatory drug (NSAID) on human cancer cells, and we elucidate a possible molecular mechanism of this combination effect. We showed that the combined effect of ART with NSAID such as celecoxib (CCB) or dimethyl-CCB (DMC) in various type of human cancer cells. After ART treatment, the expression of p62, nuclear factor erythroid 2-like 2 (NRF2) and cancer stemness (CS)-related proteins including CD44, CD133, aldehyde dehydrogenase 1 (ALDH1), octamer-binding transcription factor 4 (Oct4), mutated p53 (mutp53) and c-Myc was down-regulated. ART induced autophagy as reduction of the autophagy receptor p62, which was associated with up-regulation of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and simultaneous down-regulation of NRF2 and CS-related proteins was occurred in the human cancer cells. These results indicate a possibility that ART activates autophagy through ATF4-CHOP cascade leading to down-regulation of CS-related proteins and subsequently eradicated cancer stem cells. In addition, co-treatment with ART and imatinib was more effective than either drug alone on growth inhibition and apoptosis induction of cancer cells. In conclusion, induction of autophagy-dependent cell death by ART might play a critical role in mediating the synergistic effect of drug combination (ART/NSAID and ART/imatinib). Therefore, ART could be a promising candidate as a chemosensitizer to enhance the anticancer effects of NSAID and imatinib.

Introduction

The use of natural compounds that exhibit remarkable anticancer effects without side effects has gained increasing interest in the development of anticancer agents. Artesunate (ART), a semi-synthetic derivative of artemisinin, is the active ingredient of the Chinese herb Artemisia annua and its derivatives have many advantages including low toxicity to normal tissue/cells [10], low cross-drug resistance [5] and synergistic effects with many traditional chemotherapeutic antitumor drugs [24]. Indeed, ART is being investigated currently in a number of clinical trials for cancer treatment and has been shown to be a high selective anti-tumor activity against various tumors via several mechanisms including induction of apoptosis and caspase-independent forms of non-apoptotic apoptosis such as autophagy, inhibition of tumor invasion and metastasis and tumor-related signal transduction pathways [6,37].

Autophagy is basically a protective mechanism that sustains cell survival under adverse growth conditions. In established tumors, autophagy usually acts as a prosurvival pathway to favor tumor progression and mediates resistance to anticancer therapies in response to intracellular and environmental stress. However, when stress exceeds a certain level of autophagy, excessive and persistent autophagy can cause large-scale and irreversible destruction of cellular contents and leads to cell death ultimately [11,38]. Cancer therapy manipulating autophagic inhibition or activation has gained an increasing interest among clinicians and research- ers. Certain autophagic inhibitors would be promising antichemoresistance candidates, which can kill cancer cells through inhibiting autophagy. On the other hand, autophagy induction by certain reagents may trigger autophagic cell death or increase the interaction with other death programs [20]. Several approved and/or experimental drugs, together with natural compounds, have been reported to induce autophagy in different cancer types [35].

Nonsteroidal anti-inflammatory drugs (NSAIDs) have attracted increasing attention in recent years, and is considered as a potential anticancer drug due to its broad anticancer activity as observed in multiple cancer models [44]. NSAIDs such celecoxib (CCB), meloxicam and aspirin activate autophagy by blocking mTOR signaling, and low concentrations of NSAIDs can induce autophagy at early stages, whereas higher concentrations of NSAIDs can inhibit autophagy and induce apoptosis at later stages [43]. Imatinib is a specific tyrosine kinase inhibitor, effectively controlling chronic myeloid leukaemia (CML), gastrointestinal stromal tumors and other cancers. It has been demonstrated that imatinib can induce autophagy in BCR-ABL expressing cells, and autophagic receptor p62 mediates homoharringtonineinduced BCR-ABL autophagic degradation [7,25]. Proper drug combination is an effective strategy to improve anticancer activity since the use of a multiple drug regimen provides an effective mean to target multiple cellular abnormalities with a potential to have synergistic therapeutic effect, to overcome drug resistance and to possibly reduce toxic side effects due to dosage reduction for each individual drug in the combination [1,22].

The inability to efficiently eliminate highly tumorigenic and invasive cancer stem (-like) cells (CSCs) during cancer therapy may result in treatment failure due to cancer relapse and metastases [36]. Even if CSCs are emerging as a promising target for cancer therapies, the best way to treat many cancers would kill all malignant cells including CSCs and bulk tumor cells. Therefore, we hypothesize that combination therapy with CSCs-targeted agent and conventional cytotoxic drug will improve cancer treatment. A great attention in designing novel combination chemotherapy of autophagy inducers in cancer treatment acted as both autophagic cell death and apoptosis may provide enhanced efficacy and reduce toxicity. We identified that ART could be used to potentiate the therapeutic activity of NSAID as well as imatinib to kill both CSCs and bulk tumor cells for cancer treatment.

Materials and Methods

Cell culture and reagents

Hepatocellular carcinoma (HCC) cell line SNU-475 derived HCC tissues of patients purchased from the Korea Cell Line Bank. TRAIL-resistant SNU-475/TR cells isolated from parental SNU-475 cells by stepwise increases in concentrations of TRAIL [23]. Human CML K562 cell line was obtained from American Type Culture Collection (Manassas, VA, USA). The CD44-high K562 (CD44^highK562) cells were resistant to imatinib, and the cells were stable in complete medium without imatinib [28]. The poorly metastatic KM12 cell line was established from a primary colorectal carcinoma classified as Dukes B2. The highly metastatic cell line KM12L4A was derived from KM12. KM12SM cell line was derived from a rare liver metastasis produced by parental KM12 cells growing in the cecum wall of a nude mouse [18]. Cells were maintained in RPMI medium (Welgene, Gyeongsan, Korea) or Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) 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℃. Artesunate (ART), celecoxib (CCB), 2, 5-dimethyl celecoxib (DMC), and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell proliferation assay

Cell proliferation was measured by counting viable cells by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra- zolium 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 CCB, DMC, imatinib or/and ART 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. Lysates containing proteins were quantified using a Bradford protein assay kit (Pierce, Rockford, IL., USA). Protein samples were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) using a minigel 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: ALDH1, Oct4, CHOP, CD44 and CD133 (Cell Signaling Technology), PARP, NRF2, 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. β-actin (actin) antibody (Sigma-Aldrich), p62 (Novus Biologicals, Littleton CO, USA), c-Myc (Epitomics, CA, USA) were also used.

Apoptosis assay

SNU475 cells (1×10⁵ cells/ml) were treated with ART in the presence or absence of DMC (or imatinib) for 24 hr, respectively. 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 phosphatidylserine 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

Calculation of Combination Index (CI)

The specific interaction between ART and CCB, DMC, or imatinib on various cancer cell lines was evaluated by the combination index (CI) assay, respectively. The CI values were calculated based on the results of MTT tests. CI method for drug combination studies was used to evaluate multiple drug-effect interactions with CalcuSyn software (Biosoft, Cambridge, UK). When CI values less than 1 indicate the effect is synergistic, and values above 1 indicate the effect is antagonistic. The CI is equal to 1 the effect is additive between two agents.

Statistical analysis

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

Growth inhibition effect of combination of ART with NSAID on different types of human cancer cells

In this study, we investigated the combination effect of ART and NSAID in human hepatocellular carcinoma (HCC) cell line SNU-475 and its TRAIL-resistant SNU475/TR cells. The cytotoxicities of ART, CCB and DMC alone and their combination (ART/CCB and ART/DMC) in these cells were determined (Fig. 1A and 1B). ART exhibited remarkably dose-dependent cytotoxicity in the micromolar range against SNU475 and SNU475/TR cells. Since ART has been used in combination with other agents, chosen based on target diversity, we next investigated whether combining ART/ CCB or ART/DMC was more effective than either treatment alone. The combination effect of ART with CCB, a cyclo-oxygenase-2 (COX-2) inhibitor available for clinical use, on cell growth inhibition of human HCC cells was evaluated by MTT assay. When we calculated to analyze whether there is a synergistic effect between ART and CCB, the combination index (CI) value for combination of ART and CCB in SNU475 cells was calculated. The CI for co-treatment of SNU475 cells with ART (12.5, 25 or 50 μM) and CCB (5 or 10 μM) was found to be all below 1 (the CI values ranged from 0.59 to 0.87), indicating that the combination of ART and CCB interacts synergistically. Similarly, the CI values for co-treatment of SNU475/TR cells with ART (12.5, 25 or 50 μM) and CCB (5 or 10 μM) ranged from 0.39 to 0.90, indicating that CCB enhanced the susceptibility of SNU475/ TR cells to ART. Moreover, the DMC, a derivative of CCB that lacks COX-2-inhibitory function, was as efficacious as CCB at enhancing ART toxicity in SNU475 cells. As expected, co-treatment of ART and DMC was significantly greater than that of single-agent treatment in SNU475/TR cells. These findings indicate that CCB promotes ART-in- duced cytotoxicity of the cells in a COX-2 independent fashion and therefore, inhibition of COX-2 by CCB might not play a significant role in enhancing cellular sensitivity to ART. We also examined whether the sensitivity of CCB could be potentiated by ART in SNU475 and SNU475/TR cells. The susceptibility of these cells to CCB was increased by ART, indicating mutually cooperative interaction between ART and CCB (Fig. 1C). Next, we evaluated the combinatory effect of ART and DMC against other cancer cells such as human leukemic cell line K562 and its CD44-high K562 (CD44^highK562) cells. We found that co-treatment of ART and DMC was significantly greater cytotoxicity than that of single-agent treatment in both cells (Fig. 2A). Similar results were observed in human colorectal carcinoma KM12 cells and their highly metastatic variants, KM12SM and KM12L4A cells. The CI values for co-treatment of KM12, KM12SM or KM12L4A cells with ART (12.5 or 25 μM) and CCB (1 or 5 μM) was found to range from 0.56 to 0.98, indicating combination of two drugs is synergistic (data not shown). In addition, the combination of ART and DMC is more effective in killing KM12 and KM12L4a cells than either drug alone (Fig. 2B). These results demonstrated that the combination of ART and NSAID significantly augmented the growth inhibition of cancer cells compared with single agent treatment. Importantly, the sensitivities of both K562 and CD44^highK562 cells to imatinib were significantly enhanced by ART (Fig. 3A). The CI values for co-treatment of K562 cells with multiple doses of imatinib and ART (20 μM) ranged from 0.33 to 0.63. Similar synergistic effect was observed in imatinib/ART combination treatment, with the CI values ranging from 0.46 to 0.74 on CD44^highK562 cells. We also found that ART enhanced the imatinib sensitivity against both SNU475 and SNU475/TR cells (Fig. 3B). The combination of ART with imatinib gave a CI value less than 1, indicating a synergistic effect of the 2 drugs on both cells. These results suggest that ART could synergistically enhance the sensitivity of imatinib as well as NSAIDs including CCB and DMC in multiple cancer cell lines of different origins, and thus the combination of ART with NSAID (or imatinib) might be a promising strategy for human cancer.

Fig. 1. Combination effect of ART with NSAID on human HCC cell proliferation and viability. SNU475 (A) or SNU475/TR cells (B) were treated with serial doses of artesunate (ART) in the presence or absence of indicated doses of celecoxib (CCB) or 2, 5-dimethyl celecoxib (DMC). Both SNU475 and SNU475/TR cells were treated with serial doses of CCB in the presence or absence of 10 μM ART (C). Percentage of cell survival (left) and relative cell survival (right) 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. Synergistic anticancer effect of a combination of ART and DMC against human leukemic and colon carcinoma cells. K562 and CD44^highK562 cells (A) and KM12 and KM12L4A cells (B) were treated with serial doses of ART in the presence or absence of 5 μM DMC. Percentage of relative 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. 3. Enhancement of imatinib cytotoxicity by ART against multiple cancer cells. K562 and CD44^highK562 cells (A) and SNU475 and SNU475/TR cells (B) were treated with serial doses of imatinib in the presence or absence of indicated doses of ART. Percentage of relative cell survival was determined after 96 hr of incubation using MTT assay. Each bar represents the mean ± SD of triplicate experiments. **p<0.01, ***p<0.001.

Down-regulation of p62/NRF2 and cancer stemness (CS)-related proteins by ART possibly through ER- dependent autophagy

It has been reported that p62 serves as a selective autophagy adaptor protein that links ubiquitinated proteins to autophagy machinery for degradation, and the reduced level of p62 can be used to monitor autophagic flux [3, 16, 27]. The hyper-activation of nuclear factor erythroid 2-like 2 (NRF2), frequently found in many tumor types, can be responsible for cancer resistance to therapies and poor patient prognosis, and NRF2 activation is associated with up-regulation of autophagy-associated p62 in CSCs [17]. To investigate whether ART could induce autophagy and down-regulate NRF2 and CS-related proteins, we examined whether ART could modulate the expression of p62/NRF2 and subsequently change the levels of CS-related proteins in multiple cancer cells (Fig. 4). When the level of p62 in KM12L4A cells was evaluated in the presence or absence of ART, the cells showed that the treatment of ART resulted in dose-dependent reduction in NRF2 and concurrent p62. Since endoplasmic reticulum (ER) stress-inducible activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) signaling has been shown to play significant role in the induction of autophagy [30], we also determined whether ART-induced autophagy could be caused by modulation of ATF4/CHOP signaling axis. ART induced up-regulation of ATF4 and CHOP, and it caused ER stress response, suggesting induction of autophagy possibly through ATF4/ CHOP pathway by ART in KM12L4A cells. We further examined whether ART-induced autophagy caused a decrease in CS-related proteins in KM12L4A cells. After ART treatment, the levels of CS-related proteins involving CD44, CD133, aldehyde dehydrogenase 1 (ALDH1), octamer-binding transcription factor 4 (Oct4), mutated p53 (mutp53) and c-Myc were significantly reduced in the cells (Fig. 4A, left). Similarly, ART-treated KM12SM cells also showed down regulation of p62/NRF2 and up-regulation of ATF4/CHOP, which resulted in reduction of multiple CS-related proteins (Fig. 4A, right). Since KM12L4A and KM12SM cells with high metastatic potential exhibited increased CS-related protein levels [18], ART would be very useful in reducing the expression of CS-related proteins in cancer cells with high metastatic potential. Both SNU475 and SNU475/TR cells also exhibited down-regulation of NRF2/p62 and up-regulation of ATF4/CHOP, leading to reduce the levels of multiple CS-related proteins (Fig. 4B). These results suggest that ART-induced autophagy through ATF4/CHOP pathway may possibly contribute to degradation/down-regulation of CS-related proteins. Moreover, we checked whether the autophagy inhibitor chloroquine (CQ) could prevent ART-induced degradation/down-regulation of p62 and subsequent CS-related proteins (Fig. 5). CQ prevented ART-induced degradation/down-regulation of p62 and CS-related proteins including CD133, CD44 and mutp53 in SNU475 cells. The ART-induced ATF4 expression was significantly decreased by CQ but down-regulation of ATF4 was attenuated by ART, indicating that inhibition of autophagy suppresses induction of ATF4 (Fig. 5A). When both KM12L4A and KM12SM cells were treated with ART in the presence of CQ, ART‑induced reduction of p62 level and subsequent down regulation of CD133, CD44 and mutp53 were prevented by CQ in these cells (Fig. 5B). These results suggest the possibility that ART-induce autophagy through ATF4/CHOP signaling pathway may be associated with degradation/down- regulation of CS-related proteins and subsequent growth inhibition in CSCs.

Fig. 4. Effect of ART on expression of cancer stemness (CS)-related proteins through ER-dependent autophagy. KM12L4A and KM12SM (A) and SNU475 and SNU475/TR (B) cells were treated with serial doses of ART for 48 hr, and the changed levels of the indicted molecules in these cells were determined by Western blot analysis. Actin was used as a loading control.

Fig. 5. Effect of ART on expression of autophagic degradation of CS-related proteins in multiple cancer cells. SNU-475 (A) and KM12L4A and KM12SM (B) cells were treated with 5 μM chloroquine (CQ) and 50 μM ART for 48 hr. Subsequently, altered levels of the indicted molecules in these cells were determined by Western blot analysis. Actin was used as a loading control.

Acceleration of NSAID-mediated down-regulation of CS-related proteins and potentiation of NSAID-in- duced apoptosis by ART

When SNU475 cells were co-treated with DMC and ART, ART significantly accelerated DMC-mediated reduction in p62/NRF2 levels and up-regulation of ATF4/CHOP, indicating that ART significantly augmented DMC-mediated autophagic ability. Concomitantly, ART accelerated DMC-mediated reduction of multiple CS-related proteins, leading to enhance PARP cleavage/activation in SNU475 cells (Fig. 6, left). Similar results were observed in SNU475/TR cells (Fig. 6, right). These results suggest that DMC-mediated elimination of CS-related proteins could be accelerated by ART through autophagic degradation of these proteins. We next examined the effect of ART and DMC, singly and in combination, on apoptosis induction in SNU475 cells. Induction of apoptosis was monitored with flow cytometry using PI and annexin V as determined by summing percentages in the second and fourth quadrants that are considered to be the percentage of early and late apoptotic cells. An increase in the induction of apoptosis after co-treatment with ART and imatinib compared to the individual drug treatments in SNU475 cells (Fig. 7A). When the cells were treated with ART in the presence or absence of imatinib, ART enhanced imatinib-induced apoptosis in the cells, indicating acceleration of imatinib‑mediated cell death by ART. An increase in apoptosis after the combination treatment of ART and DMC was observed in SNU475 cells (Fig. 7B). These results suggest that ART-mediated autophagic ability causes to down-regulation of multiple CS-related proteins, and consequently enhances apoptotic cell death, resulting in sensitization of SNU475 cells to NSAID or imatinib. Our data demonstrate that combination of ART and NSAID (or imatinib) will be provide promising therapeutic approach for cancer therapy.

Fig. 6. Acceleration of DMC-mediated down-regulation of NRF2/p62 and CS-related proteins and PARP activity by ART in HCC cells. SNU-475 and SNU-475/TR cells treated with DMC (1 or 5 μM) or 50 μM ART for 48 hr. The changed levels of the indicted molecules in these cells co-treated with DMC and ART were determined by Western blot analysis. Actin was used as a loading control.

Fig. 7. Potentiation of imatinib or DMC-induced apoptosis by ART in HCC cells. SNU475 cells were treated with 10 μM imatinib or 10 μM DMC in the absence or presence of 15 μM ART for 24 hr, and the percentage of apoptotic cells was quantified using FACS (A). The upper right quadrants contain late apoptotic cells (positive for both PI and annexin V), and the lower right quadrants represent early apoptotic cells (annexin V + and PI). The percentages of viable, early apoptosis and late apoptosis/ necrosis cells were assessed by quantitative analysis (B).

Discussion

Artesunate (ART), a well-known anti-malarial drug with low toxicity, exhibits highly selective anti-cancer actions against various tumors, and ART, artemisinin and additional derivatives have many advantages including low toxicity to normal tissue/cells [10], low cross-drug resistance [5] and synergistic effects with traditional chemotherapeutic anticancer drugs [24]. However, the therapeutic efficacy and mode of action of ART in human HCC cells are poorly understood. We found that ART potentiated the anti-cancer effects of CCB (or DMC) and imatinib in human HCC cell lines. ART synergistically enhanced the both NSAID and imatinib cytotoxicity in multiple human cancer cell lines. It means that combination of ART and NSAID (or imatinib) leads to a dose-dependent enhancement of NSAID (or im- atinib)-induced growth inhibition by ART in human cells. Notably, the combination index values between ART and NSAID (or imatinib) in these cancer cells are all less than 1, indicating a strong synergy between the two drugs. In this study, DMC was acting in a COX2-independent manner when combined with ART to promote cancer cell death, indicating that ART and NSAID killed multiple cancer cells in COX2-independent fashion. Interestingly, we showed that ART inhibited cell growth and potentially sensitized cancer cells to NSAID and imatinib through the activation of autophagy. Indeed, ART activates lysosomal function and induces autophagy in cancer cells, which is attributed to the suppression of mTOR activity [39,42]. It has been reported that ART induces autophagy in chondrocytes through the suppression of the PI3K‐AKT‐mTOR pathway [8]. The endoplasmic reticulum (ER) is involved in the quality control of secreted protein via promoting the correct folding of nascent protein and mediating the degradation of unfolded or misfolded protein, namely ER-associated degradation, and ART activates the ER stress through ATF4-CHOP-CHAC₁ pathway [40]. We found that ART-induced ATF4/CHOP upregulation caused activation/induction of autophagy in multiple cancer cells, indicating a possibility that induction of autophagy by ART can occur possibly through ATF4/CHOP pathway. Previously, we reported that CCB and DMC also induced autophagy through the ATF/CHOP signaling axis in HCC cells [23]. Indeed, the non-coxib derivative of cele- coxib, OSU-03012, killed tumor cells through the induction of ER stress and autophagy [4].

Cancer stem (-like) cells (CSCs) or tumor-initiating cells represent a small subpopulation of cancer cells within the tumor bulk tissue that retain the capacity for self-renewal, disease propagation, and metastasis. The presence of CSCs is decisive for tumor recurrences and therapeutic resistance [31], and autophagy modulates stemness of CSCs and development during cancer progression. Autophagy and apoptosis can function together in order to induce cell death, and autophagy can assist in cell death by activating apoptosis [30]. Since dysregulation of autophagy is associated with therapeutic resistance of CSCs, targeting autophagy could potentially represent a promising therapeutic target for CSCs. Therefore, modulation of autophagy could be re-sensitized CSCs to drugs by enhancing the efficacy of the drugs and reducing their toxicity and disease relapse. We previously showed that NSAID-induced autophagy was associated with degradation/down-regulation of cancer stemness (CS)-related proteins including CD44, ALDH1 and Nanog [23]. Indeed, it has been reported that rottlerin induces au- tophagy, and it causes apoptotic cell death in breast cancer stem cells [21], and nigericin-induced autophagy suppresses CSC properties in glioma cells [13], indicating that induction of autophagy consequently can suppress stemness and lead to cell death in CSCs. In human HCC cells, CD44 and CD133 markers were the most commonly used markers, and particularly CD44 expression was reported to be correlated with high HCC histologic grades, vascular invasion, and reduced survival outcomes [32]. Another potential CS-related protein CD133, a pentaspan membrane glycoprotein, is involved in maintenance and survival of HCC, and targeting CD133 in HCC and other types of cancer may be better approach for elimination of CSCs through inhibition of CD133-linked signaling pathway [2]. It was reported that the expression of CD44, CD133 and EpCAM in primary KM12 cells was higher than those in matastatic KM12L4A and KM12SM cells, and the mRNA levels of SOX-2, NANOG and Oct4 were essential for maintaining self-renewal in KM12L4A and KM12SM cells compared with those in KM12 cells [18], indicating that highly metastatic colon cancer cell populations may contain a greater proportion of CSCs. Previously, we reported that the levels of CS-related proteins including Oct4, CD34, β-catenin, c-Myc, mutp53, BCRP and P-gp in CD44^highK562 cells were higher than those in parental K562 cells [19]. In this study, we found that ART also induced degradation of CS-related proteins including reduction of CD44 level possibly through activation of ER stress-dependent autophagy, and treatment of autophagy inhibitor CQ blocked ART-in- duced reduction/degradation of CD44 in multiple cancer cells. Similarly, ART-induced autophagic degradation/re- duction of CD133 and mutp53 levels was prevented by CQ in the cells, strongly suggesting ART-induced autophagic degradation of CS-related proteins. It has been known that up-regulation of ATF4 and its downstream target gene CHOP may be involved in ER-associated degradation of proteins [30]. The present study showed that ART induced autophagy and subsequently degradation of CS-related proteins possibly through ATF4/CHOP signaling pathway. The lysosomal/autophagy mediated-degradation of CD44 in cancer cells was blocked by autophagy inhibition [12,29], and also autophagy caused degradation of CD133 and mutp53 [9,14]. We found that ART-induced reduction of p62 and NRF2 resulted in a concurrent decrease in the levels of ALDH1, Oct4 and c-Myc in multiple cancer cells. Indeed, ALDH1^high cells were found to demonstrate higher resistance to common chemotherapeutic reagents, and induction of autophagy can lead to degradation of ALDH1 family ALDH1A3 protein [41], c-Myc [26] and Oct4 [34]. It has been shown that p62 exerts a fundamental role in both autophagy and apoptosis regulation due to its ability to interact with key factors regulating these processes and signaling protein that accumulate in many types of tumors [15]. The elevation of p62 was responsible for NRF2 accumulation in spheroid CSCs and was identified as a molecular link between CD44 and NRF2 activation in breast cancer stem cells [33]. We found that the treatment of cancer cells with ART exhibited inhibition of p62-NRF2 signaling and subsequent reduction of CS-related protein levels, and therefore the suppressing p62-NRF2 signaling by ART, leading to cell death in CSCs. We previous reported degradation of CD44 caused by CCB (or DMC)-induced autophagy in HCC cell lines through ATF-CHOP signaling axis [23]. Our study showed that co treatment of DMC and ART significantly accelerated both DMC-mediated reduction of p62 and NRF2 and up-regulation of CHOP and ATF4, indicating augment of DMC-mediated autophagic ability by ART through potent down regulation of p62/NRF2. ART accelerated DMC-mediated reduction/degradation of CS-related proteins including CD44, CD133, c-Myc, mutp53 and ALDH1 and consequently caused to PARP cleavage/activation in HCC cells, which resulted in synergistic growth inhibitory effect by combination ART and NSAID. We also showed that the effect of autophagy regulation by combination of ART and NSAID (or imatinib) potently induced apoptotic cell death in SNU475 cells when compared with each agent alone, suggesting ART- mediated autophagic ability causes to down-regulation/deg- radation of multiple CS-related proteins, resulting in potentiation of CSCs to NSAID or imatinib by ART. In con- clusion, our data strongly suggest that combined treatment of ART and NSAID can offer a promising new therapeutic approach for eliminating CSCs.