Enhancing the Anti-cancer Activity of Non-steroidal Anti-inflammatory Drug and Down-regulation of Cancer Stemness-related Markers in Human Cancer Cells by DAPT and MHY2245

DAPT 및 MHY2245의 비스테로이드소염제(NSAID)의 항암 활성 증강 및 종양줄기세포관련 표지자 발현 감소 활성에 대한 분자적 기전

Abstract

This study investigated the mechanisms underlying the anti-cancer effects of non-steroidal anti-inflammatory drugs (NSAIDs) in human cancer cells in combination with either N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor, or MHY2245, a new synthetic sirtuin 1 inhibitor. The results showed both DAPT and MHY2245 as novel chemosensitizers of human colon cancer KM12 and human hepatocellular carcinoma SNU475 cells to NSAIDs involving celecoxib and 2, 5-dimethyl celecoxib. The NSAID-induced cytotoxicity of these cells was significantly increased by DAPT and MHY2245 in a cyclooxygenase-2 independent manner. In addition, DAPT and MHY2245 reduced levels of p62, Notch1 intracellular domain, and multiple cancer stemness (CS)-related markers including Notch1, CD44, CD133, octamer-binding transcription factor 4, mutated p53 and c-Myc. However, the level of activating transcription factor 4 (ATF4) was enhanced, probably indicating the down-regulation of multiple CS-related markers by DAPT or MHY2245-mediated autophagy induction. Moreover, the NSAID-mediated reduction of p62/nuclear factor erythroid-derived 2-like 2 and CS-related marker proteins and the up-regulation of C/EBP homologous protein (CHOP)/ATF4 were accelerated by DAPT and MHY2245. As such, the combination of NSAID and either DAPT or MHY2245 resulted in higher cytotoxicity than NSAID alone by accelerating the down-regulation of multiple CS-related markers and PARP activation, indicating that both inhibitors promote NSAID-mediated autophagic cell death, possibly through the CHOP/ATF4 pathway. In conclusion, either combination strategy may be useful for the effective treatment of human cancer cells expressing CS-related markers.

비스테로이드소염제(NSAID)와 γ-secretase 저해제(DAPT) 또는 SIRT1저해제(MHY2245)의 병용 효과를 인간 대장암(KM12) 및 간암(SNU475) 세포를 대상으로 조사한 결과, celecoxib (CCB) 및 2, 5-dimethyl celecoxib (DMC)를 포함하는 NSAID는 DAPT 또는 MHY2245와의 병용에 의하여 COX-2활성과 상관없이 NSAID의 암세포 증식 억제능이 현저히 증강되었다. DAPT와 MHY2245는 p62단백질 감소와 동시에 Notch1, CD44, CD133, octamer- binding transcription factor 4 (Oct4) 등의 다수의 종양 줄기세포 표지자 및 NICD1 발현 양을 감소시켰지만, activating transcription factor 4 (ATF4) 발현은 증강시켰다. 또한 NSAID 단독처리 보다 NSAID/DAPT 및 NSAID/MHY2245 병용 처리에 의하여 오토파지가 촉진되므로서 종양 줄기세포 표지자의 발현 및 단백질양의 감소가 가속화되고, 이에 따라 PARP 활성화 및 세포사멸이 현저히 증강 되었다. 결론적으로 NSAID/DAPT 및 NSAID/MHY2245의 병용 투여는 종양 줄기세포 표지자를 발현하는 인간 암세포의 증식 억제 및 제거에 효과적인 처리방법으로, 임상에 적용시킬 수 있는 학문적 근거로서 제공 될 수 있다.

Introduction

Cancer stem (-like) cells (CSCs) exist in various tumors as a minor subpopulation and are tightly associated with metastasis and therapeutic resistance, and also CSCs have indefinite proliferative potential and are often responsible for tumor progression and recurrence. Therefore, eradicating CSCs is promising aspect for the cure of cancer. The stemness properties of CSCs such as long-term self-renewal, differentiation and proliferation abilities have been reported to be governed by stemness genes [29]. Notch signaling activation in CSCs promotes cell survival, self-renewal and metastasis and inhibits apoptosis, which mostly relies on the activity of the γ-secretase enzyme that cleaves the Notch receptors and releases the active intracellular domain (ICD). The inhibition of γ-secretase may provide an integrative targeting therapeutic strategy for the suppression of Notch signaling. CD44, IGF-1R and ErbB4 as well as Notch are proteolytically cleaved by γ-secretase/presenilin in membrane and form ICD of respective proteins [10]. CD44 also plays a role in the progression of tumors and is expressed in CSCs. CD44-positive cells promote tumorigenesis in breast and colorectal cancers, displaying stem cell properties such as self-renewal and differentiation [13]. The expression of CD44 is important to the maintenance and possible acceleration of tumorigenesis through the cleavage of its ICD that activates stemness factors such as Nanog, Sox2 and Oct4. Therefore, the cleavage of CD44ICD potentially regulates cancer stemness characteristics. Treatment with a γ-secretase inhibitor blocks the cleavage of CD44ICD and the nuclear translocation of cancer stemness factors [4]. In addition, MiR199-5p that targeted directly the expression and function of the deacetylase SIRT1 and inhibited SIRT1 expression, and miR-199a-5p overexpression resulted in effectively reduction of the CD44ICD release and stemness characteristics [25].

Autophagy, as a type II programmed cell death, plays a dynamic tumor-suppressive or tumor-promoting role in different types of cancer. The potential ability of autophagy to modulate cell death makes it a therapeutic target in cancer. The signaling adaptor p62 is an autophagy receptor and a selective substrate for autophagy. Since p62 can simultaneously bind to LC3, a key component of the autophagosome membrane, the binding of p62 to the ubiquitinated proteins will lead them to autophagosomes for subsequent delivery to lysosomes and degradation [23]. In addition, p62 is an important mediator of stem-like properties for cancer cells, and suppression of p62 diminishes the enrichment of genes that are regulated by c-Myc in breast cancer [37]. It has been shown that autophagy-induced Notch1 degradation could be a promising treatment strategy for preventing tumor progression, and p62 binds to multiple sites on Notch1 intracellular domain (NICD1) to promote its degradation. [38]. When Notch signaling is activated, the transcription machinery, including NICD1, increases NRF2 synthesis. It has been reported that NICD1 can be directly degraded by autophagy, and p62 binds to multiple sites on NICD1 to regulate its degradation, and multiple p62 binding sites on NICD1 independently but collectively promotes its degradation [1]. Therefore, autophagy-induced Notch1 degradation could be a promising treatment strategy for eradicating cancer stem cells. Indeed, it has been reported that inhibition of γ-secretase by NMK-T-057 or DAPT up-regulates autophagy genes such as LC3-II and Beclin and induces autophagy [6, 34]. SIRT1 inhibitor also markedly increases levels of LC3-II and ATG5 and induces autophagy [35]. It is therefore necessary to determine whether combination of autophagy inducer and g-secretase inhibitor as well as SIRT1 inhibitor requires an enhanced specificity to ICD of Notch1 and blockage of the nuclear translocation of stemness transcription factors, which could be useful for developing Notch1-targeting therapeutics.

Materials and Methods

Cell culture and reagents

The human colon cancer cell line KM12 was a kind gift from professor Isaiah J. Fidler (University of Texas MD Anderson cancer center, USA). Human hepatocellular carcinoma (HCC) SNU-475 cell line derived HCC tissues of patients purchased from the Korea Cell Line Bank. 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℃. Celecoxib (CCB) and 2, 5-dimethyl celecoxib (DMC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-Sphenylglycine t-butyl ester (DAPT) was purchased from Selleck (Houston, TX, USA). MHY2245 was kindly donated by professor Hyung Ryong Moon (college of pharmacy, Pusan National University, KOREA).

Cell proliferation (viability) assay

Assay for cell proliferation was measured by the 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium 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, DAPT and/or MHY2245 at 37℃. After 96 hr, the medium was removed using centrifugation, and MTT-formazan crystals solubilized in 100 ml dimethyl sulfoxide. 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 using a minigel apparatus (Bio-Rad, Hercules, CA, USA). Following electrophoresis, gels were transferred onto a nitrocellulose membrane (Hybond-ECL; GE Healthcare, Piscataway, NJ, USA). Each membrane was blocked with 5% skim milk in Tris-buffered saline plus 0.05% Tween-20. 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: Oct4, CHOP, CD44, CD133, ALDH1, NICD1 and Notch1 (Cell Signaling Technology), 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. b-actin antibody (Sigma-Aldrich), p62 (Novus Biologicals, Littleton CO, USA), c-Myc (Epitomics, CA, USA) were also used.

Apoptosis assay

KM12 cells (1×105 cells/ml) were treated with DAPT in the presence or absence of DMC 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 for 15 min at room temperature. After incubation, 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 exhibited Annexin V-FITC+ /PI− staining patterns; whereas late apoptotic cells showed Annexin V-FITC+ /PI+ staining patterns due to a loss of plasma membrane integrity.

Calculation of Combination Index (CI)

The specific interaction between DAPT, MHY2245, CCB and DMC on KM12 and SNU475 cells 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<0.01, and p<0.001 were considered statistically significant in all experiments.

Results

Synergistic growth inhibition of human cancer cells expressing cancer stemness (CS)-related markers by NSAID in combination with either DAPT or MHY2245

Previously, we showed that human colon cancer KM12 cells exhibited elevated expression of many CS-related markers [21], and targeting CS-related markers could be useful for eradication of CSCs. Since treatment of γ-secretase inhibitor blocked the nuclear translocation of stemness factors [4], we investigated chemo-sensitization by γ-secretase inhibitor DAPT in KM12 cells expressing various CS-related markers. First, we examined whether DAPT could enhance the cytotoxicity of NSAID such as CCB, cyclooxygenase-2 (COX-2) inhibitor available for clinical use, and DMC, a derivative of CCB that lacks COX-2-inhibitory function. The sensitivity of KM12 cells to either CCB or DMC was significantly enhanced by DAPT (Fig. 1A, Fig. 1B). The CI values for co-treatment of KM12 cells with DAPT (1, 5 or 10 μM) and CCB (2 or 8 μM) ranged from 0.16 to 0.76 , indicating gave a CI value less than 1, a synergistic effect of the 2 drugs. Similar synergistic effect was observed in DAPT/ DMC combination treatment under same concentrations, with the CI values ranging from 0.11 to 0.71, and therefore DAPT/DMC combination was more effective than DAPT/ CCB combination. Our data suggest synergistic combination effect of γ-secretase inhibitor with NSAID. Previously, we reported that NSAID induced apoptotic and autophagic cell death, and high expression of CS-related markers in cancer cells were suppressed by SIRT1 inhibition [20, 26]. We therefore determined whether a new SIRT1 inhibitor MHY 2245 could enhance the cytotoxicity of NSAID in cancer cells expressing CS-related markers. When the combination effect of either MHY2245/CCB or MHY2245/DMC on cell growth inhibition of KM12 cells was evaluated by MTT assay, two types of combination (MHY2245/CCB and MHY2245/DMC) were more effective than either drug alone (Fig. 2A, Fig. 2B). When we calculated to analyze whether there is a synergistic effect between MHY2245 and CCB, the combination index (CI) value for combination of MHY2245 and CCB in KM12 cells was determined. The CI for co-treatment of KM12 cells with CCB (1 or 5 μM) and MHY2245 (0.2 or 2 μM) was found to be all below 1, indicating that the combination of CCB and MHY2245 interacts synergistically. Similarly, a combination of DMC (1 or 5 μM) and MHY2245 (0.2 or 2 μM) also produced CI values less than 1, and combined treatment of MHY2245 and DMC was more synergistic cytotoxicity than combined treatment of MHY2245 and CCB when compared with CI values of two type of combination in the cells, indicating that MHY2245 promotes NSAID-induced cytotoxicity in the cells in a COX-2 independent manner. Therefore, inhibition of COX-2 by CCB might not play a significant role in enhancing combination effect of MHY2245 and NSAID.

Fig. 1. Synergistic anticancer effect of a combination of DAPT with NSAID on viability in human colon cancer KM12 cells. The cells were treated with indicated doses of celecoxib (CCB; A) or 2, 5-dimethyl celecoxib (DMC; B) in the presence or absence of serial doses of DAPT. 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.

Fig. 2. Combination effect of MHY2245 with NSAID on viability in KM12 cells. The cells were treated with indicated doses of CCB (A) or DMC (B) in the presence or absence of MHY2245 (0.2- or 2 μM). 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.

To further confirm these results, we examined the cytotoxicity of other human cancer cells by NSAID in combination with either DAPT or MHY2245. Since hepatocellular carcinoma (HCC) is also a highly lethal cancer that is caused by the existence of CSCs, we therefore evaluated the combinatory effect of NSAID and DAPT against human HCC SNU 475 cells. The combination of either DAPT/CCB or DAPT/ DMC significantly augmented the cell growth inhibition compared with each drug alone (Fig. 3A, Fig. 3B). The sensitivity of SNU475 cells to either CCB or DMC was significantly enhanced by DAPT. The CI values for co-treatment of SNU 475 cells with DAPT (5 or 10 μM) and CCB (1 or 5 μM) ranged from 0.40 to 0.70. Similar synergistic effect was observed in DAPT/DMC combination treatment under same concentrations with the CI values ranging from 0.19 to 0.49, and therefore combination treatment with DAPT and DMC was more effective than combination treatment with DAPT and CCB. When SNU475 cells were co-treated with CCB and MHY2245, the CI values for co-treatment of the cells with CCB and MHY2245 was strictly less than 1, indicating combination of two drugs is synergistic (Fig. 3C). The co-treatment of SNU475 cells with DMC and MHY2245 resulted in strong synergistic combination effect (Fig. 3D), and the combined effect of MHY2245/DMC is higher than that of MHY2245/CCB in SNU475 cells. These results suggest synergistic combination effect of SIRT1 inhibitor with NSAID on human HCC cells.

Fig. 3. Enhancement of NSAID cytotoxicity by DAPT and MHY 2245 in human HCC SNU475 cells. The cells were treated with indicated doses of CCB or DMC in the presence or absence of DAPT (A, B) or MHY2245 (C, D). 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<0.001.

Down-regulation of p62 and CS-related markers by DAPT and MHY2245 possibly through ER-dependent autophagy, and acceleration of NSAID-mediated apoptotic effect by DAPT

The p62 protein acts as a cargo receptor for autophagic degradation of ubiquitinated targets and is up-regulated by various stressors. Autophagy promotes the degradation of Noch1, and p62 binds to multiple sites on NICD1, active form of Notch1, to promote its degradation [38], and treatment of γ-secretase inhibitor blocked the cleavage of CD44ICD and the nuclear translocation of stemness factors such as Nanog, Sox2, and Oct4, resulting in reduction of stemness factors [4]. Therefore, we determined whether the levels of p62 and CS-related markers in KM12 cells could be modulated by DAPT (Fig. 4A). When the changed level of p62 in KM12 cells was evaluated in the presence of DAPT, the cells showed that DAPT treatment resulted in down-regulation of p62 through up-regulation of ATF4 in a dose dependent manner, indicating autophagy-inducing activity of DAPT. Indeed, it has been reported that DAPT induces autophagy through up-regulation of autophagy-related genes [6, 34]. Since endoplasmic reticulum (ER) stress is a potent inducer of autophagy and ATF4 is important in the activation of autophagy, our data showed that DAPT-induced autophagy could be caused by activation of ATF4. Moreover, we showed that DAPT-induced autophagy resulted in down-regulation/degradation of Notch1, NICD1 and other CS-related marker proteins. The levels of Notch1, CD44, CD133, Oct4, mutated p53 (mutp53) and c-Myc as well as NICD1 by DAPT were dose-dependently reduced in KM12 cells. These results suggest that DAPT-induced autophagy may contribute to degradation/down-regulation of CS-related marker proteins, possibly contributing to eradicate CSCs. Since it has been reported that MHY2245 can also induce autophagy [35], we next determined whether MHY2245 could down-regulate p62 and subsequent NICD1 and CS-related markers in an ATF4-dependent manner (Fig. 4B). KM12 cells treated with MHY2245 showed down-regulation of p62 and subsequent NICD1, Notch1 and other CS-related marker proteins through possibly ATF4-dependent autophagy. These results indicated the possibility that down-regulation of CS-related markers might be associated with autophagy induction by either DAPT or MHY2245, eventually leading to kill cancer cells expressing CS-related markers. Therefore, we next examined the effect of DAPT and DMC, singly and in combination, on apoptosis induction in KM12 cells (Fig. 5A, Fig. 5B). Apoptotic cell death was monitored with flow cytometry using PI and annexin V. The total percentage of apoptotic cell populations is expressed as the sum of percentages in the second and fourth quadrants that are considered to be the early and late apoptotic cells, respectively. When the cells were treated with DMC in the presence or absence of DAPT, an increase in apoptosis after the combination treatment of DMC and DAPT was observed in KM12 cells. DAPT significantly accelerated DMC-mediated apoptosis compared with each drug alone. Indeed, p62 functions as a selective autophagy receptor for degradation of ubiquitinated substrates. In the p62-Kelch-like ECH-associated protein 1 (Keap1)-NRF2 axis, p62 functions as a modulator of NRF2 activation, and p62 was positively correlated with NRF2 and negatively correlated with Keap1 [16]. Indeed, NRF2 activation was associated with up-regulation of p62 in cancer stem like cells [17], and activation of NRF2 found in many types of tumors, which can be responsible for cancer resistance to therapies and poor patient prognosis. We found that DAPT accelerated DMC-mediated autophagy caused by down-regulation of p62/NRF2 and up-regulation of ATF4/CHOP, which promoted down-regulation of multiple CS-related marker such as CD44, CD133, Oct4 and c-Myc and consequently PARP activation in KM12 cells (Fig. 5C). These results suggest that DAPT accelerates DMC-mediated autophagic ability, which in turn causes autophagic degradation of multiple CS-related marker proteins. We also determined whether MHY2245 could enhance NSAID-mediated autophagy and accelerate reduction of multiple CS-related maker proteins, leading to PARP activation. MHY2245 also enhanced CCB-mediated down-regulation of p62/NRF2 through up-regulation of ATF4/CHOP and accelerated CCB-mediated reduction of Notch1/NICD1 and multiple CS-related marker proteins such as CD44, CD133, Oct4, c-Myc and ALDH1, eventually leading to PARP activation in KM12 cells (Fig. 6A). MHY2245 also enhanced DMC-mediated down- regulation of p62/NRF2 and up-regulation of ATF4/CHOP, indicating that DMC-mediated autophagy possibly through ATF4/CHOP signaling. In addition, MHY 2245 enhanced DMC-mediated down-regulation of Notch1/ NICD1 and concurrent multiple CS-related marker proteins, causing potent activation of PARP in KM12 cells (Fig. 6B). These results suggest that activation of ATF4/CHOP-mediated autophagy by DAPT and MHY2245 might be contributed to degradation/down-regulation of CS-related markers and consequently enhanced NSAID- mediated cell death, ultimately resulting in sensitization of cancer cells expressing CS-related markers to NSAID.

Fig. 4. Effect of DAPT and MHY2245 on modulation of cancer stemness (CS)-related markers. KM12 cells were treated with serial doses of DAPT (A) or MHY2245 (B) for 48 hr, and the changed levels of the indicted molecules in KM12 cells were determined by Western blot analysis. Actin was used as a loading control.

Fig. 5. Potentiation of DMC-induced apoptosis and acceleration of autophagy-induced down-regulation of CS-related markers by DAPT. KM12 cells were treated with 10 μM DMC in the absence or presence of 50- or 100 μM DAPT for 24 hr, and the percentage of apoptotic cells was quantified using FACS (A). The upper right quadrants contain late apoptotic cells and the lower right quadrants represent early apoptotic cells. The percentages of early apoptosis, late apoptosis and necrosis cells were assessed by quantitative analysis (B). KM12 cells were treated with 100 μM DAPT in the absence or presence of DMC (1 or 5 μM) for 48 hr, and the changed levels of the indicted molecules in KM12 cells were determined by Western blot analysis (C).

Fig. 6. Acceleration of NSAID-induced down-regulation of CS-related markers and PARP activation by MHY2245. KM12 cells were treated with CCB or DMC (1- or 5 μM) in the absence or presence of 2 μM MHY2245 for 48 hr, and the changed levels of the indicted molecules in these cells were determined by Western blot analysis.

Discussion

Autophagy-mediated cell death is a new strategy for anticancer drug development. Drug-induced autophagy could be used to kill cancer cells and decrease the resistance of CSCs to anticancer therapy [8]. Apoptosis and autophagy are two different physiological processes but these two events might be interdependent or mutually independent [3]. Autophagy can regulate Notch signaling pathway that plays important roles in angiogenesis, cancer immunity and the maintenance of CSCs and directly modulates Notch1 degradation. Therefore, the loss of autophagy leads to the activation of the Notch signal [36]. Indeed, Notch is involved in migration and invasion of tumor cells, and elevated expression of Notch is associated with metastasis of human malignancies and poor outcome [31]. The interaction between the Notch1 receptor and the ligand leads to cleavage of Notch1 by γ-secretase, thus releasing the NICD1. NICD1 translocates to the nucleus where it binds to its partner proteins to activate stemness-related gene transcription program, and thus overexpression of NICD1 leads to acquisition of CSC characteristics.

In this study, we propose γ-secretase inhibitor DAPT as a new sensitizer of NSAID involving CCB and DMC against human hepatocellular carcinoma SNU475 and colon cancer KM12 cells. Moreover, a new SIRT1 inhibitor MHY2245 also enhanced susceptibility of NSAID in these cancer cells. Both DAPT and MHY2245 significantly potentiated the sensitivity of these cancer cells to NSAID in a dose-dependent manner, possibly through induction of autophagic cell death. We demonstrate that the combination of NSAID and either DAPT or MHY2245 targeting Notch signaling exhibited a synergistic effect on growth inhibition in SNU475 and KM12 cells expressing multiple CS-related markers. DAPT and MHY2245 induced ATF4-mediated autophagy, leading to down-regulation/degradation of multiple CS-related markers in these cells, and therefore growth inhibitory activity of DAPT and MHY2245 might be associated with induction of autophagic cell death as well as apoptotic cell death. Moreover, DAPT and MHY2245 significantly accelerated NSAID-mediated autophagy caused by down-regulation of p62/NRF2, which caused a potent reduction of multiple CS-related marker proteins such as CD44, CD133, Oct4 and c-Myc and subsequent PARP activation in KM12 cells. It means that DAPT and MHY2245 commonly can enhance NSAID-mediated autophagic ability, which in turn causes autophagic degradation of multiple CS-related marker proteins. Our data provide the molecular mechanism underlying enhancement of apoptosis-induced cell death by DAPT and MHY2245-induced autophagic cell death, and it contributes to the synergistic antitumor effect of NSAID in combination with either DAPT or MHY2245 in human cancer cells. These two types of combination (NSAID/DAPT and NSAID/ MHY2245) than NSAID alone resulted in greater cytotoxicity by acceleration of NSAID-induced down-regulation/degradation of multiple CS-related makers likely through accelerated autophagy, ultimately leading to potentiation of NSAID-induced cytotoxicity in KM12 and SNU475 cells. Therefore, we propose that the use of NSAID in combination with either γ-secretase inhibitor or SIRT1 inhibitor may be effective in human CSCs.

It is well known that CSCs are resistant to chemotherapeutic agents, radiation, and cell death. CSC subpopulations from HCC cells play a critical role in cancer initiation, metastasis, recurrence and therapeutic resistance. CD133, CD44, ALDH1 and epithelial cell adhesion molecules (EpCAM) are widely recognized as liver CSC surface markers [24]. NRF2 signaling is activated in CD44high cancer-cell population, and high NRF2 expression in cancer cells resulted in growth enhancement and chemoresistance, and p62 is involved in CD44-mediated NRF2 activation [33], indicating that autophagy induction by down-regulation of p62 leads to degradation of CD44. Indeed, inhibition of autophagy blocked the degradation of CD44 in cancer cells [30]. Autophagy also caused degradation of CD133 [11]. In addition, autophagy-dependent degradation of mutp53 was attenuated by autophagic inhibition [5]. The NICD1 as the effector of Notch1 signaling pathway could form a complex with LC3/p62 and be degraded by autophagy [14]. It has also been reported that autophagy-induced decrease in NICD 1level is a consequence of Notch1 degradation rather than autophagic clearance of NICD1 [36].

In present study, we showed that DAPT induced autophagy as an early event and down-regulated/reduced the expression of Notch1 and NICD1 in a dose-dependent manner, which caused concurrent down-regulation of multiple CS-related marker proteins such as CD44, CD133, Oct4, mutp53 and c-Myc, possibly leading to autophagy-mediated cell death in KM12 and SNU475 cells. Therefore, we propose that DAPT can be a good drug candidate to abolish CSCs in human cancer. Indeed, treatment of DAPT decreased NICD, HESI, Oct4, and Sox2 protein expression in ovarian CSCs [15]. It has been also reported that γ-secretase inhibitor NMK-T-057 induces autophagy-dependent apoptosis by inhibiting γ-secretase-mediated activation of Notch signaling and inhibits stemness in breast cancer cells [6], indicating that activation of autophagy may be associated with inhibition of Notch signaling, and down-regulation of Notch1 may lead to trigger autophagy-mediated cell death in CSCs.

SIRT1, an NAD-dependent deacetylase, plays important roles in human cancers by serving as either tumor suppressor or promoter depending on its targets in specific signaling pathways or in specific cancers [7]. Salermide, a SIRT1/2-specifc inhibitor also induced autophagy in human NSCLC cells [28], and a new SIRT1 inhibitor MHY2245 also induced autophagy in human ovarian cancer cells [35]. In present study, we showed that MHY2245 down-regulated Notch1/NICD1 and multiple CS-markers involving CD44 likely through ATP/CHOP signal-mediated autophagy in KM12 and SNU475 cells. The levels of CD44, CD133, Oct4, mutp53 and c-Myc were dose-dependently reduced in KM12 cells by MHY2245, indicating the possibility that down-regulation/degradation of CS-related markers by MHY2245 may contribute to eradicate CSCs. Previously, it has been shown that SIRT1 plays a key regulation role in the CD44-mediated signal transduction, and SIRT1 knockdown represses CD44 cleavage and nuclear translocation of CD44ICD since it is likely that CD44ICD nuclear translocation is associated with its deacetylation function, and knockdown of SIRT1 inhibits CSCs stemness [24]. Indeed, the molecular expression of cancer stemness markers such as Sox2, Nanog and Oct4 was induced by CD44ICD overexpression [4]. The up-regulated CS-related molecules in CSCs such as CD44high K562 cells are suppressed by SIRT1 inhibition [19]. Herein, we showed that level of CD44 was dose-dependently reduced in KM12 and SNU475 cells by MHY2245 as well as DAPT, indicating that the induction of autophagy by DAPT and MHY2245 is commonly associated with degradation of Notch1/NICD1 and concurrent inhibition of CD44 via prevention of the release of CD44ICD, finally leading to the down-regulation of other CS-related markers and subsequent cell death.

NSAIDs involving CCB and DMC exert anticancer activity via COX-2-independent pathways and display anticarcinogenic and chemopreventive properties through the regulation of autophagy in certain types of cancer [9]. The antitumor effect of NSAID is associated, at least in part, with their autophagy-modulating effect and an increase in autophagic flux, ultimately leading to cell death [2]. Previously, we reported that autophagic responses triggered by CCB activated the ATF/CHOP signaling axis in HCC cells, and CCB-induced autophagy occurred via AMPK activation and inhibition of Akt/mTOR/p70S6K/4EBP1 signaling pathway [22]. CCB-mediated autophagy down-regulated CS-markers including CD44 and Oct4 [26]. Therefore, we propose that the use of NSAID in combination with other autophagy inducer would be highly efficacious for the treatment of various type of tumors. Herein, we found that MHY2245 significantly accelerated NSAID-mediated reduction in levels of p62/NRF2 and concurrent down-regulation/degradation of multiple CS-related marker proteins, possibly through ATF4/CHOP signaling-dependent autophagy. KM12 cells showed that DAPT as well as MHY2245 significantly enhanced NSAID-mediated autophagic ability via ATF4/CHOP signaling pathway, which accelerated NSAID-induced downregulation of multiple CS-related markers. It means that NSAID in combined with either DAPT or MHY2245 induces a great increase in autophagic degradation of multiple CS-related marker proteins compared to each drug alone.

The multifunctional protein p62 integrates both survival and death signaling by regulating the ubiquitination of key cell signaling molecules that control survival and death [27, 32]. The elevation of p62 is a common molecular event in different types of CSC systems, and p62 knockdown reduces both the mRNA and the protein levels of Nanog, Sox2 and Oct4, whereas p62 up-regulation promotes the expression of these stemness markers [37]. NRF2 signaling is engaged in CSC-like properties of several types of cancer cells, and activation of NRF2-induced pathway in cancer is be critical for chemotherapeutic resistance and therefore NRF2 is emerging as a promising target to overcome cancer chemoresistance [17]. Overexpression of p62 and activation of NRF2 are critical for anchorage-independent growth observed in HCC cells [12]. NRF2 activation in ALDH-high CSC-like cells is associated with high p62 level [18], and p62 is also involved in CD44s-mediated NRF2 activation [33].

In the present study, we show that combine treatment of MHY2245/NSAID as well as DAPT/NSAID can provide a potential combination strategy for suppressing viability of CSCs probably owing to acceleration of NSAID-induced down-regulation/degradation of CS-related markers. Taken together, our findings highlight a novel mechanism underlying the synergistic anti-cancer effect of NSAID in combination with either DAPT or MHY2245 in human cancer cells, and thus Both DAPT and MHY2245 may have potential as novel chemosensitizers to NSAIDs for effective treatment of human cancer expressing multiple CS-related markers.