Combined Treatment of Nonsteroidal Anti-inflammatory Drugs and Genistein Synergistically Induces Apoptosis via Induction of NAG-1 in Human Lung Adenocarcinoma A549 Cells
Cho Hee Kim, Min Young Kim, Su Yeon Lee, Ji Young Moon, Song Iy Han
1, Hye Gyeong Park
2and Ho Sung Kang*
Department of Molecular Biology, College of Natural Sciences, and Research Institute of Genetic Engineering, Pusan National University, Pusan 609-735, Korea
1
Research Center for Resistant Cells, College of Medicine, Chosun University,Gwangju 501-759, Korea
2
Korea Nanobiotechnology Center, Pusan National University, Pusan 609-735, Korea
Received July 24, 2009 /Accepted July 31, 2009A number of studies have demonstrated that the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce the risks of colorectal, oesophageal and lung cancers. NSAIDs have been shown to exert their anti-cancer effects through inducing apoptosis in cancer cells. The susceptibility of tumor cells to anti-tumor drug-induced apoptosis appears to depend on the balance between pro-apoptotic and anti-apoptotic programs such as nuclear factor kB (NF-kB), phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) and MEK1/2-ERK1/2 pathways. We examined the effects of pro-survival PI3K and ERK1/2 signal pathways on cell cycle arrest and apoptosis in response to NSAIDs including sulindac sulfide and NS398. We show that simultaneous inhibition of the Akt/PKB and ERK1/2 signal cascades could synergistically enhance the potential pro-apoptotic activities of su- lindac sulfide and NS398. Similar enhancement was observed in cells treated with sulindac sulfide or NS398 and 100 μM genistein, an inhibitor of receptor tyrosine kinases (RTKs) that are upstream of PI3K and MEK1/2 signaling. We further demonstrate that NAG-1 is induced and plays a critical role(s) in apoptosis by NSAIDs-based combined treatment. In sum, our results show that combinato- rial treatment of sulindac sulfide or NS398 and genistein results in a highly synergistic induction of apoptotic cell death to increase the chemopreventive effects of the NSAIDs, sulindac sulfide and NS398.
Key words : Sulindac, NS398, genistein, apoptosis, PI3K, ERK1/2, NAG-1
*Corresponding author
*Tel：+82-51-510-2275, Fax：+82-51-513-9258
*E-mail : [email protected]
Introduction
NSAIDs including sulindac and NS398 exert anti-in- flammatory effects through inhibition of cyclooxygenase (COX, prostaglandin H synthase). COX-1 is constitutively expressed in many tissues, while COX-2 is up-regulated by growth factors and is highly expressed in different types of premalignant and malignant tumors, indicating its im- plication in tumor growth [31,33,34]. Several experimental studies showed that NSAIDs have an anti-neoplastic effect against many types of cancer cells; the regular use of NSAIDs can reduce the risk of colorectal, oesophageal and lung cancer [15,31,33,34]. A number of molecular mecha- nisms responsible for the chemopreventive effects of NSAIDs have been proposed. Anti-tumorigenic activity of
NSAIDs may be dependent on its inhibitory effects on COX activity, but other results suggest that the anti-cancer activity of NSAIDs may be independent of COX inhibition [31,33,34].
COX-independent mechanism is explained by NSAIDs-in- duced cell cycle arrest and apoptosis. Sulindac sulfide and sulindac sulfone are the two major metabolites of sulindac that is the most extensively investigated chemopreventive NSAID that reduces the number and size of tumors in hu- mans and animals [31,33,34]. Sulindac sulfide is COX se- lective, whereas sulindac sulfone is believed to lack COX-in- hibitory activity. Sulindac sulfide induces apoptosis through the mitochondrial pathway involving caspase 9 and Bax [39].
In addition, it also involves the membrane DR pathway in-
volving DR5 and proximal caspase 8 [18] and suppression
of 14-3-3epsilon to induce apoptosis [22]. NS398, a COX-2
specific inhibitor that has chemopreventive activities against
many epithelial malignancies, could induce apoptosis
through a mechanism that is independent of its COX-2 in-
hibitory activity. NS398-induced apoptosis is known to be mediated by the release of cytochrome c from mitochondria and consequently, by the activation of caspase-9 and cas- pase-3 and by the cleavage of poly (ADP-ribose) polymerase [21,23,28]. Furthermore, NS398 may suppress the growth of human lung cancer cells by up-regulating the expression of p27Kip1, a typical CDKI via inhibition of protein degrada- tion [19]. In addition, the pro-apoptotic activity of NSAIDs is closely correlated to an increased expression of the non-steroidal anti-inflammatory drug activated gene-1 (NAG-1, also known as macrophage inhibitory cytokine-1 (MIC-1) and growth and differentiation factor-15 (GDF-15)), a member of the TGF-β superfamily that mediates apoptosis by the agents to prevent tumor formation and development such as several NSAIDs, resveratrol, and genistein [2,3,4,29, 35]. Clinical trials of the combination of NSAIDs with radio- therapy, chemotherapy or both have been shown to improve long-term survival rates in patients with a number of cancers. COX-2 inhibitors overcome apoptosis resistance and selectively sensitize tumor cells to the extrinsic death re- ceptor-induced apoptotic pathway independently of COX-2 [32]. Currently, several groups are conducting clinical trials in cervix cancer, lung cancer, and brain tumors, using in- hibitors of COX-2 in combination with chemotherapy and radiation therapy [1,5,30].
The susceptibility of tumor cells to apoptosis induced by cancer drugs depends on the balance between their intrinsic pro-apoptotic and anti-apoptotic programs. The PI3K-Akt/
PKB and Ras-MEK1/2-ERK1/2 signaling cascades is the best-characterized pro-survival protein kinase and is con- stitutively activated in many cancer cells including non- small cell lung cancer (NSCLC) cell lines [6,7,10,11,17,36].
Thus, the inhibition of the PI3K-Akt/PKB and MEK1/
2-ERK1/2 pathways by pharmacological or genetic ap- proaches is usually an effective approach to enhance apopto- sis by anti-cancer drugs [9,16,25,27].
In this study, the effects of inhibition of PI3K-Akt/PKB and MEK1/2-ERK1/2 on apoptotic activities of non-steroidal anti-inflammatory drugs (NSAIDs) including sulindac sul- fide and NS398 were examined using various human cancer cells. Here we show that simultaneous inhibition of the Akt/PKB and ERK1/2 signal cascades by specific inhibitors or genistein (an inhibitor of RTKs that are upstream of PI3K and MEK1/2 signaling) could synergistically enhance the potential pro-apoptotic activities of sulindac sulfide and NS398 and NAG-1 plays a key role in apoptosis by NSAIDs-
based combined treatment.
Materials and Methods Cell culture and drug treatment
Human lung adenocarcinoma A549 cells were obtained American Type Culture Collection (ATCC) and were grown in RPMI 1640 medium (Invitrogen) supplemented with 10%
(v/v) heat-inactivated FBS and 1% (v/v) penicillin- streptomycin (Invitrogen). Human colorectal carcinoma HCT116 and human colorectal adenocarcinoma HT29 cells were obtained American Type Culture Collection (ATCC) and were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin (Invitrogen). A549, HCT116 and HT29 cells were cultured at 37
oC humidified incubator with 5% CO
2. NS398 (EI-261, Biomol), Sulindac sulfide (AP-200, Biomol) was dissolved in DMSO at a concentration of 100mM. For NS398 or sulindac sulfide treatment, cells were incubated in the fresh media containing NS398 or sulindac sulfide at different concen- trations for 48 hr as described in Figure legends. For the studies concerning the effects of inhibitors, A549 cells were pretreated with LY294002 (20 μM), wortmannin (0.2 or 20 μ M), U0126 (20 μM), PD98059 (30 μM), genistein (100 μM, from Calbiochem), AG1478 (10 μM), AG1024 (10 μM). These inhibitors were pretreated for 1 hr followed by treatment with sulindc sulfide and NS398 in the presence of the inhibitors.
Cell viability assay
For cell viability assay, A549 cells (1×10
4) were seeded in a 96-well tissue culture plate and incubated for 24 hr.
After treatment, 5 μl of MTT solution (5 mg/ml) was added to each well. After incubation for 3 hr at 37
oC, formazan crystals in viable cells were solubilized with 150 μl of DMSO.
The absorbance of each well was then read at 570 nm using microplate reader (Molecular devices, Palo Alto, CA).
SDS-PAGE and Western blot analysis
Cells were prepared by washing with cold-phosphate-buf- fered saline (PBS) and lysed by adding lysis buffer (0.1%
SDS, 1.0% Triton X-100 and 1.0% deoxycholate in PBS con-
taining 1 mM DTT, 1% protease inhibitor cocktail (Sigma),
1 mM PMSF, 1 mM Na
3VO
4, 1 mM NaF and 1 mM glycer-
ophosphate). After incubation on ice for 30 min, the resulting
extracts were centrifuged at 13,000 rpm for 20 min. The pro-
tein concentration was determined using the Bio-Rad protein assay kit. Equal amount of proteins was loaded, and sepa- rated by SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked for 1 hr with blocking buffer (5% skim milk and 0.1% Tween 20 in PBS) at room temperature on a shaker. The membranes were then incubated for overnight at 4
oC with primary antibody (in PBS with 0.1% Tween 20) including anti-PARP, anti-cas- pase-3, and anti-p27 (Santa Cruz, CA). Nitrocellulose mem- branes were washed three times in wash buffer (PBS with 0.1% Tween 20). Primary antibody was detected using 1:1,000 diluted HRP-conjugated goat anti-mouse or goat an- ti-rabbit IgG antibodies and visualized with the enhanced chemiluminescence detection system (Amersham-Pharmacia Biotech, Buckinghamshire, England). Western blot experi- ments were performed at least three times.
Morphological detection of apoptosis by Hoechst 33342 staining [20]
Percentage of apoptosis was calculated by counting the condensed and fragmented nuclei stained with Hoechst 33342 (Molecular Probe). Cells were grown on coverslips in multiwell culture plates. The attached cells were fixed by adding 3.7% formaldehyde in PBS for 30 min at 4
oC.
Detached cells were centrifuged at 5,000 rpm for 3 min at 4
oC, resuspended with 3.7% formaldehyde in PBS, and then attached to slide glass by using CytoSpin. After washing with cold-PBS three times, the cells were stained with Hoechst 33342 (10 μg/ml in D.W) for 15 min and then ob- served by fluorescence microscopy. Three fields of each con- dition were randomly selected to photograph, and frag- mented and condensed nuclei were counted. The ratio of apoptotic cells to total cells was calculated, and the average and error (±SD) of data for each field were calculated and presented into graph.
RNA extraction and reverse transcription- olymerase chain reaction (RT-PCR)
One microgram of each DNA-free total RNA sample, ex- tracted by RNAwiz (Ambion), was converted into cDNA with M-MLV reverse transcriptase and oligo (dT)
15(Bioneer Co., Korea) according to the manufacture's instruction.
Equal amounts of cDNA were subsequently amplified by PCR in a 20 μl reaction volume containing 10 mM Tris-HCl (pH 8.3), 200 μM dNTPs, 50 mM KCl, 1.5 mM MgCl
2, 2.5 U of Taq polymerase (Promega Co., USA) The primers were
: NAG-1, sense-TCTCAGATGCTCCTGGTGTT, antisense- AATCTGG GTCTTCGGAGTG; GAPDH, sense- TTC ACC ACC ATG GAG AAG GCT, antisense- A GCC TTG GCA GCA CCA GT. PCR-products were subjected to electro- phoresis on 1.5 % agarose gels and visualized by ethidium bromide staining.
NAG-1 RNA interference
The NAG-1 small interference RNA (siRNA) vector (pSuper-neo-gfp-Si NAG1) was constructed using a pSuper-neo-gfp and a synthetic oligonucleotide targeting 5’-ACATGCACGCG CAGATCAA-3’ corresponding to posi- tions 780-798 on NAG-1 mRNA. A549 cells at 50% con- fluence were transfected with pSuper-neo-gfp NAG-1 siRNA (1 μg/well) for 3 hr using GeneJuice (Novagen) according to manufacturer’s protocol. After incubation for 48 hr at 37
oC (5% CO2), the transfected cells were selected by using 1 mg/ml G418, and were maintained in the presence of 0.2 mg/ml G418 (Invitrogen). G418-resistant A549 cells were pooled and used for RT-PCR analysis and for apoptosis analysis.
Results and Discussion
Sulindac sulfide induces apoptosis, whereas NS398 induce cell cycle arrests in A549 lung cancer cells
Several NSAIDs have been reported to induce cell cycle arrest or apoptosis depending on cell types [31,33,34]. In this study, the effects of sulindac sulfide and NS398 in hu- man cancer cells including A549 lung cancer cells and HCT-116 and HT-29 colon cancer cells were examined.
Apoptosis was assessed with fluorescence microscopic anal- ysis for nuclear condensation/fragmentation and by Western blot analysis of PARP degradation that is in- dicative of caspase 3 activation. Apoptotic cells undergo nu- clear condensation, degradation of DNA to ladder form of oligonucleotides and formation of apoptotic bodies, which are accompanied biochemically with activation of intra- cellular caspases [12,24]. As shown in Fig. 1, HCT-116 cells were more sensitive to sulindac sulfide, a COX-1/2 in- hibitor, than A549 and HT29 cells; 50 μM sulindac sulfide could induce apoptosis in HCT-116 colon cancer cells, whereas it induced apoptosis A549 lung cancer cells at con- centrations higher than 100 μM. In contrast, HT-29 cells were resistant to sulindac sulfide up to 100 μM (Fig. 1).
NS398 (a COX-2 specific inhibitors) up to 100 μM appeared
Fig. 1. Sulindac sulfide and NS398 induces cell cycle arrest or apoptosis in cancer cells. (A) A549 cells were treated with 50, 100 and 200 μM sulindac sulfide for 48 hr and were photographed under a microscope (X200). (B) A549, HCT-116 and HT-29 cells were treated with 50 and 100 μM sulindac sulfide for 48 hr. For morphological apoptosis analysis, the cells were stained with Hoechst 33342. (C) A549 cells were treated with 50, 100 and 200 μM sulindac sulfide for the indicated times and analyzed by SDS-PAGE and Western blotting with antibodies to PARP and pro-caspase-3. (D) A549 cells were treated with 50, 100, 200 μM NS398 for 48 hr and were photo- graphed under a microscope (X200). (E) A549, HCT-116 and HT-29 cells were treated with 50 and 100 μM NS398 for 48 hr. For morphological apoptosis analysis, the cells were stained with Hoechst 33342. (F) A549 cells were treated with 50, 100 and 200 μM NS398 for the indicated times and analyzed by SDS-PAGE and Western blotting with antibodies to PARP and p27.
to suppress the cell proliferation but did not induce apopto- sis in A549, HCT-116, and HT-29 cells (Fig. 1). NS398 has been shown to induce apoptosis in other cancer cells, in- dicating differential sensitivities to NS398 depending on cell types. As demonstrated previously[19], NS398 increased the intracellular level of p27Kip1, a typical CKI that could in- duce cell cycle arrest, in A549 cells (Fig. 1).
Inhibition of the PI3K-Akt/PKB and ERK1/2 pathways enhances pro-apoptotic activities of sulindac sulfide and NS398 in A549 lung cancer cells
The PI3K-Akt/PKB and MEK1/2-ERK1/2 signaling path-
ways, the best-characterized pro-survival program in cells, are known to be constitutively activated in many cancer cells including (NSCLC) cell lines [6,7,10,11,36]. To examine the effects of pro-survival PI3K and ERK1/2 signal pathways on NSAID-induced cell cycle arrest and apoptosis, A549 cells, one of NSCLC, were treated with the PI3K inhibitors and/or MEK1/2 inhibitors in different combinations.
Inhibition of both PI3K-Akt and ERK1/2 significantly stimu- lated 50 μM sulindac sulfide-potentiated cell death program, while inhibition of either PI3K-Akt or ERK1/2 did not affect the death program; combined treatment of 50 μM sulindac sulfide with PI3K inhibitor and ERK1/2 inhibitor induced apoptosis by 40 % at 48 hr (Fig. 2). In addition, combined treatment of 100 μM NS398 with the PI3K inhibitor and ERK1/2 inhibitor synergistically induced apoptosis at 48 hr (Fig. 2). The synergistic effect between NSAIDs and the PI3K inhibitors/MEK1/2 inhibitors was more prominent when NSAIDs+LY294002+U012 were used. Thus, inhibition of PI3K-Akt/PKB and ERK1/2 signaling pathways switched
A
B
Fig. 2. Inhibition of PI3K and MEK1/2 enhances apoptosis in- duced by sulindac sulfide, while it switches NS398- induced cell cycle arrest to apoptosis in A549 cells. (A) A549 cells were pretreated with PI3K inhibitors (LY294002 (LY or L, 20 μM) and wortmannin (W, 0.2 μM)) or MEK inhibitors (PD98059 (PD or P, 30 μM) and U0126 (U, 20 μM)) in different combinations for 1 h and then treated with 50 μM sulindac sulfide or 100 μM NS398 for 48 h. For morphological apoptosis analysis, the cells were stained with Hoechst 33342 and observed under a fluorescence microscope (A) and apoptotic cells were scored (B). Results (500-800 cells in each group) are expressed as the means +/- S.E.M. from three in- dependent experiments.
NS398-induced cell cycle arrest to apoptosis. These findings indicate that interruption of the cell protective signal path- way by the PI3K inhibitors and MEK1/2 inhibitors results in a highly synergistic induction of apoptosis, thereby in- creasing the chemopreventive effects of NSAIDs against hu- man cancer.
Genistein, a receptor tyrosine kinase inhibitor, enhances NSAIDs-induced apoptosis in A549 lung cancer cell line
In most cases, the PI3K-Akt/PKB and ERK1/2 signal pathways are activated via receptor tyrosine kinases (RTKs).
Thus, the effects of genistein (a general RTK inhibitor) and specific RTK inhibitors (AG1478, an ERBB 1 inhibitor;
AG1024, an insulin-like growth factor-1 receptor inhibitor) on NSAIDs-induced apoptosis were examined. Combined treatment of 100 μM genistein with either 50 μM sulindac sulfide or 100 uM NS398 for 48 hr in A549 cells resulted in a synergistic increase in apoptosis, while no prominent apoptosis was detected in cells treated with 100 μM genistein alone (Fig. 3). In contrast, no significant apoptosis was de- tected in A549 cells that were incubated under treatment of NSAIDs combined with either AG1478 or AG1024. Genistein is commonly used to examine intracellular signaling in con- centrations of 25-100 μM. Genistein at concentration of 100 μM could induce apoptosis of MCF-7 cells in 24 hr effectively. However, we could not detect a significant apop- totic induction in genistein-treated A549 cells. In general, NSCLC is known to be not a chemosensitive tumor, al- though the mechanism of resistance to the relevant anti- cancer drugs has not been fully elucidated. Interestingly, apoptosis by combination of genistein and NS398 was atyp- ical, when compared to that induced by other combinations.
Although chromatin fragmentation was observed, the nu- clear membrane was intact. Combinatorial treatment with genistein and NS398 apoptotic nuclei appeared as nuclear membrane intact and condensed bodies of chromatin were also of small size. In contrast, apoptotic nuclei by other com- binations appeared as brightly staining heterogeneous mass- es of chromatin and nuclear membrane was disrupted.
Combined treatment with genistein and sulindac sulfide/NS398 synergistically induces apoptosis in A549 cells
NAG-1 was previously identified as a gene induced by several anti-tumorigenic compounds including NSAIDs and
Fig. 3. Combined treatment with genistein and sulindac sul- fide/NS398 synergistically induces apoptosis in A549 cells. A549 cells were exposed to genistein (100 μM, a general RTK inhibitor), AG1478 (10 μM, an ERBB 1 in- hibitor), or A1024 (10 μM, an insulin-like growth factor-1 receptor inhibitor) for 1 hr prior to treatment with 50 μM sulindac sulfide or 100 μM NS398 for 24 or 48 hr.
For morphological apoptosis analysis, the cells were stained with Hoechst 33342 and observed under a fluo- rescence microscope (A) and apoptotic cells were scored (B). Results (500-800 cells in each group) are expressed as the means +/- S.E.M. from three independent experiments.
many different compounds and has been shown to exhibit anti-tumorigenic and/or pro-apoptotic activities in vivo and in vitro [2,3,4,29,35]. Combined treatment of genistein with either 50 μM sulindac sulfide or 100 μM NS398 synergisti- cally induced NAG-1 (Fig. 4). To determine whether apopto- sis by NSAIDs-based combined treatment is regulated by NAG-1, we examined the effects of NAG-1 siRNA on apop- tosis by treatment of NSAIDs combined with genistein. As shown in Fig. 4 D, NAG-1 siRNA reduced apoptosis in re- sponse to combined treatment, indicating an important role(s) of NAG-1 in apoptosis by the combined treatment.
Genistein is an isoflavenoid found in soy that has potential
chemopreventive properties and anti-tumorigenic activities
with a wide variety of pharmacological effects in animal
cells, and is pharmacologically safe [13,14,26,38]. Recent re-
search has suggested that this plant polyphenol might be
A
B
Fig. 4. Combined treatment with genistein and sulindac sul- fide/NS398 synergistically enhances the expression of NAG-1 in A549 cells. (A-B) A549 cells were pre-treated with 100 μM genistein and then exposed to 50 μM su- lindac sulfide (A) or 100 μM NS398 (B) for 48 hr and analyzed by RT-PCR using specific probes for NAG-1 and GAPDH. 1 μg of RNA was subjected to reverse tran- scription as detailed in Materials and Methods. PCR- products were subjected to electrophoresis on 1.5 % agarose gels and visualized by ethidium bromide staining. (C) A549 cells pre-treated with NAG-1 siRNA were treated with genistein and sulindac sulfide and NS398 for 24 and 48 hr. For morphological apoptosis analysis, the cells were stained with Hoechst 33342 and the apoptotic cells with condensed/fragmented nuclei were scored by fluorescence microscopy.
used to sensitize tumor cells to chemotherapeutic agents and radiation therapy by inhibiting pathways that lead to treat- ment resistance and has also been found to be protective from therapy-associated toxicities [14]. Our results demon- strate that combinatorial treatment of genistein and NSAIDs at relatively low concentrations effectively induces apoptosis in A549 cells may provides a new approache for future effec- tive molecular cancer therapeutics using NSAIDs.
Acknowledgement
This work was supported for two years by Pusan National University Research Grant.
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