Abstract
Background/Aim: The anticancer potential of indomethacin and diclofenac has been reported against several types of cancer cells. In this study, we investigated the enhancement effect of a coumaric acid derivative found in some Piper plants, N-trans-p-coumaroyltyramine (TCT) on indomethacin and diclofenac cytotoxicity in breast cancer cells. Materials and Methods: MCF-7 and mitoxantrone-resistant MCF-7 (MCF-7/MX) cancer cells were treated with indomethacin or diclofenac in the presence of TCT for 48 h. Cell viability, apoptosis, mitochondrial function and signaling proteins were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Hoechst 33342, tetramethyl-rhodamine-ethyl-ester and western blot analysis, respectively. Results: Combination treatment resulted in significant reduction of cell viability and mitochondrial membrane potential, whereas the ratio of BCL2-associated X, apoptosis regulator to BCL2 apoptosis regulator, and apoptosis increased. The enhancing effect of TCT was related to reduced nuclear factor-erythroid factor 2-related factor 2/heme oxygenase-1 expression, and increased activation of the protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2 alpha/activating transcription factor 4/C/EBP homologous protein signaling pathways. Conclusion: TCT in combination with indomethacin or diclofenac promoted endoplasmic reticulum stress-dependent apoptosis in breast cancer cells.
Failure of cancer chemotherapy may stem from various factors, such as high expression levels of drug efflux transporters, change in cellular intrinsic sensitivity, and patients’ intolerability toward adverse events of cytotoxic drugs (1). In this regard, the addition of a chemo-sensitizing agent to treatment regimens might be a promising approach to augmenting therapeutic success (2). Through this approach, titrating up the dose of cytotoxic anticancer drugs may not be needed, and patients can consequently better tolerate chemotherapy. Several natural compounds, such as flavonoids, naphthoquinone, terpenes and alkaloids, have been reported to be able to enhance the cytotoxicity of anticancer agents through various mechanisms, including inhibition of efflux transporters, increasing cellular stress, and inducing apoptosis (3–5).
Indomethacin and diclofenac are non-steroidal anti-inflammatory drugs (NSAIDs) known mainly for pain relief via inhibition of cyclo-oxygenase. However, the anticancer potential of both NSAIDs has also been reported (6, 7). The underlying mechanisms of their cytotoxicity have been correlated to increased production of reactive oxygen species (ROS) and accumulation of unfolded proteins in endoplasmic reticulum (ER) stress (8, 9). Under stress conditions, misfolded proteins increase excessively in the ER and trigger an unfolded protein response. Activation of key unfolded protein response stress sensors i.e., PKR-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1a) and activating transcription factor-6 (ATF6) transduces signals to initiate the cascade of apoptotic cell death (10, 11). Moreover, using NSAIDs in combination with certain phytochemicals, such as resveratrol, curcumin, piperine or naringenin, was shown to increase cancer cell death effectively both in vitro and in vivo (12–14).
In addition to promoting stress-mediated cell death, the chemosensitizing effects of phytochemicals may be related to their interference with the activities of the ATP-binding cassette (ABC) drug efflux transporters, particularly P-glycoprotein (ABCB1), ABC subfamily C member 1 (ABCC1) and ABC subfamily C member 2 (ABCC2), and ABC subfamily G member 2 (ABCG2) (15, 16). Suppression of these drug efflux pumps can increase the accumulation of their cytotoxic drug substrates to effective levels within target cells. Examples of such natural chemosensitizers are alkaloids (e.g., stemofoline), flavonoids (e.g., naringenin), polyphenols (e.g., resveratrol), and naphthoquinone (e.g., rhinacanthin-C) (3, 17–19).
The stems and fruits of Piper wallichii (Miq.) Hand.-Mazz (family Piperaceae) have been used medicinally in the treatment of gastro-intestinal, inflammatory and circulatory diseases in China, India and Thailand (20, 21). We have isolated a sizable amount of N-trans-p-coumaroyltyramine (TCT; Figure 1), a coumaric acid derivative with an amide moiety, from the stem of this plant. Although this compound is also a constituent of several other dietary plants, its pharmacological activities have rarely been studied. TCT was reported to possess anti-trypanosomal and inhibitory activities against the enzymes acetylcholinesterase and α-glucosidase (22–24). Several amide alkaloids, including piperine and piperlongumine, from Piper plants were able to potentiate the cytotoxicity of anticancer agents such as oxaliplatin and placlitaxel via mechanism involving ROS-mediated ER stress and inhibition of drug efflux transporters (4, 25, 26). In view of this, we studied the chemo-enhancing potential of TCT on NSAID-mediated cytotoxicity in breast cancer models.
Chemical structure of N-trans-p-coumaroyltyramine.
Materials and Methods
Chemicals and reagents. Calcein acetoxymethyl (calcein-AM), 5(6)-carboxy-2’,7’-dichlorofluorescein diacetate (CDCFDA), 5(6)-carboxy-2’,7’-dichlorofluorescein (CDCF), cyclosporine A, diclofenac, 2’,7’-dichlorofluorescin diacetate (DCFH-DA), hydrogen peroxide, indomethacin, KO143, pheophorbide A, rotenone, tetramethylrhodamine-ethyl-ester (TMRE) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Fetal bovine serum, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), L-glutamine, and RPMI-1640 medium were purchased from Gibco Life Technologies (Grand Island, NY, USA). Mouse monoclonal anti-BCL2 associated X (BAX), anti-BCL2 apoptosis regulator (BCL2), anti-eukaryotic initiation factor 2 alpha (eIF2a), anti-heme oxygenase 1 (HO1), and anti-nuclear factor-erythroid factor 2-related factor 2 (NRF2) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Mouse monoclonal anti-activating transcription factor 4 (ATF4), anti-C/EBP homologous protein (CHOP), anti-IRE1a, anti-PERK, anti-phospho (p)-IRE1a, and rabbit monoclonal anti-p-eIF-2a, anti-c-Jun N-terminal kinase (JNK), anti-p-JNK, anti-p-NRF2, anti-p-PERK, and anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase and anti-mouse IgG HRP-conjugated antibodies were purchased from Calbiochem (San Diego, CA, USA). Super Signal West Pico chemiluminescent substrates was obtained from Pierce Biotechnology (Rockford, IL, USA).
Plant material. Dried P. wallichii stems (3 kg) were ground, macerated in MeOH (3×10 l for 3 days each) and the filtrate was evaporated under reduced pressure at 45°C to obtain the methanolic extract (100 g). The extract was fractionated on a silica-gel column (2 kg, 10×35 cm), eluted with n-hexane-acetone gradient (7:3 to 3:7) into six fractions (A-F). Repeated size-exclusion chromatography of fraction D (2.1 g) on Sephadex LH-20 columns, washed down with CH2Cl2-MeOH (1:1), yielded subfractions D1-D4. Subfraction D3 (0.66 g) was fractionated on a silica-gel column (30 g, 3×13 cm), using CH2Cl2-MeOH (9:1) as the eluent, into subfractions D31-D34. Purification of subfraction D32 (90 mg) with a Sephadex LH-20 column, eluted with CH2Cl2-MeOH (1:1), yielded N-trans-p-coumaroyltyramine (36.3 mg).
Cell culture. Human breast adenocarcinoma MCF-7 cells (American Type Culture Collection, Rockville, MD, USA) were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. Mitoxantrone-resistant MCF-7 cells (MCF-7/MX) were established by adding mitoxantrone in stepwise gradual increasing concentrations (0.01-0.7 μM) to RPMI-1640 complete medium (27). Finally, MCF-7/MX cells were maintained in RPMI-1640 complete medium containing 0.7 μM mitoxantrone. One week before experiments, MCF-7/MX cells were cultured in mitoxantrone-free RPMI-1640 complete medium.
Cell viability assay. The MTT reduction assay was used to assess cell viability. Cells (5×103 cells/well) were seeded onto 96-well plates overnight prior to treatment with non-toxic concentration of the test compounds [namely, indomethacin (10 μM), diclofenac (10 μM), mitoxantrone (2, 24 μM), tamoxifen (10 μM), vinblastine (1 nM), camptothecin (0.5 nM), thapsigargin (0.0125 μM), tunicamycin (0.3 μM), hydrogen peroxide (10 μM), and rotenone (1 μM) in the presence and absence of TCT (10, 100 μM) or piperine (10, 100 μM)]. After 48-h treatment, the cells were further incubated with MTT solution (0.5 mg/ml) in an incubator at 37°C with CO2 for 4 h. The formazan crystals were dissolved in 100 μl of dimethyl sulfoxide and then quantified spectrophotometrically at 570 nm, using a microplate reader.
Apoptosis assay. Apoptotic cell death was determined by Hoechst 33342 staining assay. Cells (5×103 cells/well) were seeded onto 96-well plates overnight prior to treatment with non-toxic concentrations of the test compounds [namely, indomethacin (10 μM), diclofenac (10 μM), thapsigargin (0.0125 μM), tunicamycin (0.3 μM) and hydrogen peroxide (10 μM) in the presence and absence of TCT (10, 100 μM) or piperine (10, 100 μM)] for 48 h. After treatment, cells were stained with Hoechst 33342 (10 μM) and then fixed in 4% paraformaldehyde for 30 min. Cells were visualized under a fluorescence microscope at 350/461 nm (excitation/emission). The apoptotic cells were defined by the appearance of cell shrinkage, chromatin condensation or nuclei fragmentation.
Uptake assays. Activities of major ABC drug transporters ABCB1, ABCC1, ABCC2, and ABCG2 in MCF-7 and MCF-7/MX cells were demonstrated by substrate accumulation assay. Cells (2×105 cells/well) were seeded onto 24-well plates overnight prior to treatment with 50 μM cyclosporine A, 500 μM indomethacin, 10 μM KO143 or 100 μM TCT for 30 min. Then the specific substrate of each transporter [namely, 0.4 μM calcein-AM, 5.2 μM CDCF, 5 μM CDCFDA or 10 μM pheophorbide A, respectively] was added for another 30 min. At the end of this co-incubation period, the cells were lysed with 1% Triton X-100 and the fluorescent intensity of each substrate was measured with a microplate reader at 485/535 nm (excitation/emission) for calcein and CDCF, or at 488/675 nm (excitation/emission) for pheophorbide A.
Determination of ROS. To measure ROS production, cells (2×104 cells/well) were grown overnight in 96-well plates prior to DCFH-DA assay. Cells were incubated with a fluorescent probe DCFH-DA (100 μM) for 30 min, followed by addition of the test compounds [namely, indomethacin (10 and 500 μM), diclofenac (10 and 500 μM), rotenone (20 μM) in the presence and absence of TCT (100 μM)] for 2 h. At the end of this co-incubation period, the cells were lysed with 1% Triton X-100 and fluorescent intensity was measured with a microplate reader at 485/535 nm (excitation/emission).
Determination of mitochondrial function. Mitochondrial function was assessed by TMRE assay. Cells (5×103 cells/well) were seeded onto 96-well plates overnight, followed by incubation with non-toxic concentrations of the test compounds [namely, indomethacin (10 μM), diclofenac (10 μM), thapsigargin (0.0125 μM), tunicamycin (0.3 μM), and rotenone (1 μM) in the presence and absence of TCT (100 μM)] for 48 h. Then 1 μM TMRE was added for another 30 min, and the fluorescent intensity was read with a microplate reader at 549/575 nm (excitation/emission).
Western blotting analysis. After treatment, cell lysates were prepared in RIPA lysis buffer (50 mM Tris-HCl pH 6.8, 150 mM NaCl, 1% Tritron-X, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM, Na3VO4 and protease inhibitor cocktails). Equal amounts of proteins (40 μg) were separated by 7-12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and blotted on a polyvinylidine fluoride membrane. After blocking with 5% skim milk, the membranes were probed with primary antibodies at optimal dilutions (1:1,000-1:2,000), followed by incubation with the HRP-conjugated secondary antibodies (1:2,000). Protein bands were then developed using the Super signal West Pico chemiluminescent substrates. The immunoblots were visualized and quantified by a GE ImageQuant™ LAS 4000 (GE Healthcare Biosciences, Tokyo, Japan). Protein expression levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (an internal control).
Statistical analysis. Data are presented as mean±standard error of the mean from three individual experiments (n=3). The differences between groups were determined by one-way analysis of variance, followed by the Bonferroni post-hoc test. Statistical significance was considered at values of p<0.05.
Results
Enhancement of drug-induced cytotoxicity by TCT. The half-maximal inhibitory concentration (IC50) values for indomethacin, diclofenac and mitoxantrone cytotoxicity to MCF-7 and MCF-7/MX cells after 48-h exposure are shown in Table I. Notably, the resistant MCF-7/MX cells were approximately 2.3, 5.1, and 11.7 times less sensitive, respectively, to indomethacin-, diclofenac-, and mitoxantrone-mediated cytotoxicity than MCF-7 cells. At 10 μM, indomethacin and diclofenac did not induce toxicity in MCF-7 or MCF-7/MX cells (Figure 2), whereas at 100 μM, TCT and piperine significantly enhanced indomethacin- and diclofenac-mediated cytotoxicity in both cell types. The indomethacin- and diclofenac-mediated cytotoxicity, when used in combination with TCT, was 2.6 and 2.3 times higher, respectively, for MCF-7 cells, and was 2.3 and 1.5 times higher, respectively, for MCF-7/MX cells. In addition, both TCT and piperine (100 μM) were able to significantly increase the sensitivity of MCF-7 cells to MX (at the IC50 value of 2 μM) (Figure 2A). However, a sensitizing effect of both compounds was not observed in MCF-7/MX cells exposed to mitoxantrone (at the IC50 value of 24 μM) (Figure 2B). The combination of piperine and mitoxantrone did not significantly increase cytotoxicity to MCF-7/MX cells as compared to piperine alone. It is worth noting that, at 100 μM, TCT had no effect on cell viability, whereas piperine significantly reduced the viability of MCF-7 and MCF-7/MX cells to approximately 38% (Figure 2).
The half-maximal inhibitory concentration (IC50) values of indomethacin, diclofenac, mitoxantrone for MCF-7 and mitoxantrone-resistant MCF-7/MX cells.
Effects of N-trans-p-coumaroyltyramine (TCT) and piperine (PIP) at 100 μM on drug-induced cytotoxicity in MCF-7 (A) and mitoxantrone (MX)-resistant MCF-7/MX cells (B) after 48-h exposure to indomethacin (INDO), diclofenac (DIC), and MX. Each bar represents the mean±standard error of the mean (n=3). Significantly different at p<0.05 compared with: *drug-treated cells in the absence of the test compound or #untreated control.
We further evaluated the ability of TCT and piperine to increase the chemosensitivity of both MCF-7 and MCF-7/MX cells to different anticancer drugs and ER stressors (Figure 3). The cancer cells were exposed to each drug at its non-cytotoxic concentration in the presence of either TCT or piperine (100 μM) for 48 h. Our results showed that neither TCT nor piperine was able to increase the sensitivity of these cells to 10 μM tamoxifen, 1 nM vinblastine or 0.5 nM camptothecin after 48-h exposure (Figure 3). In contrast, the combination of 100 μM TCT with ER stressors [namely 10 μM hydrogen peroxide, 1 μM rotenone, 0.0125 μM thapsigargin, or 0.3 μM tunicamycin] significantly induced cell death in both wild type and resistant MCF-7 cells (Figure 3). Piperine-mediated sensitization to hydrogen peroxide was observed in both MCF-7 and MCF-7/MX cells. However, piperine did not significantly potentiate the cytotoxicity of rotenone and tunicamycin in resistant cells.
Effects of N-trans-p-coumaroyltyramine (TCT) and piperine (PIP) at 100 μM on the viability of MCF-7 (A) and mitoxantrone (MX)-resistant MCF-7/MX cells (B) after 48-h exposure either to cytotoxic anticancer agents [tamoxifen (TAM), vinblastine (VBL), and camptothecin (CPT)] or endoplasmic reticulum stressors [thapsigargin (TGN), tunicamycin (TUN), rotenone (ROT), and hydrogen peroxide (H2O2)]. Each bar represents the mean±standard error of the mean (n=3). Significantly different at p<0.05 compared with: *drug-treated cells in the absence of test compound or #untreated control.
Activities of drug efflux transporters in the presence of TCT. As shown in Figure 4A, both cell types expressed active ABCC1 and ABCC2. Co-incubation with indomethacin (an ABCC1/ABCC2 inhibitor) significantly increased intracellular accumulation of the ABCC1 substrate CDCF and the ABCC2 substrate CDCFDA. Moreover, MCF-7/MX cells also showed an appreciable level of ABCG2 activity (Figure 4A). The presence of ABCG2 inhibitor KO143 significantly increased accumulation of the ABCG2 substrate pheophorbide A in MCF-7/MX cells by approximately 2.4 times. Neither MCF-7 nor MCF-7/MX cells exhibited significantly increased levels of intracellular calcein in the presence of the ABCB1 inhibitor cyclosporine A, suggesting the absence of ABCB1 function. Furthermore, neither TCT nor piperine were able to increase significantly the accumulation of CDCF and pheophorbide A in MCF-7/MX cells, suggesting the lack of inhibitory action against ABCC1, ABCC2, and ABCG2 activities (Figure 4B).
Activities of drug efflux transporters in MCF-7 and mitoxantrone (MX)-resistant MCF-7/MX cells. Basal activities of ATP-binding cassette subfamily B member 1 (P-glycoprotein, ABCB1), ABCC1, ABCC2 and ABCG2 (A) and intracellular accumulation of specific fluorescent substrates of transporters 5(6)-carboxy-2’,7’-dichlorofluorescein (CDCF), 5(6)-carboxy-2’,7’-dichlorofluorescein diacetate (CDCFDA) and pheophorbide A (Phe A) (B) in the presence of N-trans-p-coumaroyltyramine (TCT) and piperine (PIP) at 100 μM. Each bar represents the mean±standard error of the mean (n=3). *Significantly different at p<0.05 compared with the control (without inhibitor).
Increased indomethacin- and diclofenac-mediated apoptotic cell death in the presence of TCT. In this study, we determined the apoptosis-inducing effect of 10 μM indomethacin and diclofenac, along with ER stressors (0.0125 μM thapsigargin, 0.3 μM tunicamycin, and 10 μM hydrogen peroxide), in the presence of 100 μM TCT after 48-h exposure, using Hoechst 33342 staining assay. As shown in Figure 5, both MCF-7 and MCF-7/MX cells treated with TCT in combination with indomethacin, diclofenac or ER stressors exhibited a higher number of apoptotic cells compared to those treated with single compounds. The percentage of drug-induced apoptotic MCF-7 and MCF-7/MX cells significantly increased, by approximately 4-fold, in the presence of TCT. At 100 μM, piperine alone increased apoptotic cell death by 4- to 5-fold compared to untreated MCF-7 and MCF-7/MX cells. Apparently, when piperine was used in combination with indomethacin, diclofenac or ER stressors, piperine itself was mainly responsible for the observed apoptotic cell death after 48-h treatment (Figure 5). It should be noted that the sensitivity of MCF-7 and MCF-7/MX cells to each treatment was comparable.
Fluorescent imaging of Hoechst 33342 staining in MCF-7 (A) and MCF-7/MX cells (B) after 48-h exposure to endoplasmic reticulum stressors indomethacin (INDO), diclofenac (DIC), thapsigargin (TGN), tunicamycin (TUN), and H2O2) in the presence and absence of N-trans-p-coumaroyltyramine (TCT) and piperine (PIP) at 100 μM (scale bar=500 μm). Apoptotic cells having condensed chromatin and fragmented nuclei were counted and expressed relative to total cells. Each bar represents the mean±standard error of the mean (n=3). Significantly different at p<0.05 compared with: *stressor-treated cells in the absence of test compound or #untreated control.
Effects of TCT on ROS production and mitochondrial function. In this study, we explored whether TCT is able to potentiate indomethacin- and diclofenac-mediated cytotoxicity via the increase of intracellular ROS levels. As shown in Figure 6, indomethacin and diclofenac significantly increased intracellular ROS by approximately 1.5-fold in both MCF-7 and MCF-7/MX cells. However, the combination of each NSAID with TCT did not increase ROS levels in either cell type. In addition, TCT did not enhance rotenone-mediated ROS generation.
Reactive oxygen species (ROS) production in MCF-7 (A) and mitoxantrone (MX)-resistant MCF-7/MX cells (B) after 2-h exposure to indomethacin (INDO), diclofenac (DIC) and rotenone (ROT) in the presence and absence of 100 μM N-trans-p-coumaroyltyramine (TCT). Each bar represents the mean±standard error of the mean (n=3). #Significantly different at p<0.05 compared with the untreated control.
A decrease in mitochondrial activity was observed in both MCF-7 and MCF-7/MX cells that were exposed to 10 μM NSAIDs in the presence of TCT for 48 h (Figure 7). At this concentration, neither indomethacin nor diclofenac given alone for 48 h had significant effect on mitochondrial membrane potential (MMP) and cytotoxicity. Similar results were also observed when the cells were challenged with ER stressors thapsigargin, tunicamycin, and rotenone at their non-cytotoxic concentrations (Figure 7). The reduction of MMP in cells exposed to either NSAIDs or ER stressors in the presence of TCT was well correlated with the increment of apoptotic BAX/BCL2 proteins and apoptotic cell death. As shown in Figure 8, combining TCT with NSAIDs or ER stressors resulted in higher pro-apoptotic BAX levels along with lower anti-apoptotic BCL2 expression, compared to groups without TCT.
Effect of N-trans-p-coumaroyltyramine (TCT) at 100 μM on mitochondrial membrane potential (MMP) in MCF-7 (A) and mitoxantrone (MX)-resistant MCF-7/MX cells (B) after 48-h exposure to endoplasmic reticulum stressors indomethacin (INDO), diclofenac (DIC), thapsigargin (TGN), tunicamycin (TUN), and rotenone (ROT). Each bar represents the mean±standard error of the mean (n=3). *Significantly different at p<0.05 compared with stressor-treated cells without TCT.
Immunoblots and their densitometrical analysis of BCL2-associated X (BAX) and BCL2 apoptosis regulator (BCL2) proteins in MCF-7 (A) and mitoxantrone (MX)-resistant MCF-7/MX cells (B) after 48-h exposure to endoplasmic reticulum stressors indomethacin (INDO), diclofenac (DIC), and tunicamycin (TUN) in the presence and absence of N-trans-p-coumaroyltyramine (TCT) at 100 μM. Data from densitometrical analysis are shown in the lower panels. Each bar represents the mean±standard error of the mean (n=3). *Significantly different at p<0.05 compared with stressor-treated cells without TCT.
Involvement of ER stress signaling pathways in the sensitizing effect of TCT on indomethacin- and diclofenac-mediated cytotoxicity. PERK/eIF2a/ATF4/CHOP and NRF2/HO1 signaling pathways: Activation of PERK proteins results in the stimulation of both eIF2a/ATF4/CHOP and NRF2/HO1 signaling cascades (28–31). At a non-cytotoxic concentration (10 μM), indomethacin increased the phosphorylation of PERK in MCF-7 and MCF-7/MX cells by approximately 1.43- and 1.5-fold, respectively, whereas diclofenac increased it by about 1.7- and 1.52-fold, respectively, compared to the untreated groups. Similarly, tunicamycin increased the phosphorylation of PERK in MCF-7 cells by 1.7-fold and in MCF-7/MX cells by 1.63-fold, without observable cytotoxicity. As shown in Figure 9, the increase in phosphorylated PERK were well correlated with increasing phosphorylated eIF-2a and ATF4 and higher CHOP expression levels in both cell types. The presence of 100 μM TCT in combination with indomethacin, diclofenac or tunicamycin led to significantly higher expression (about twice) of p-eIF2a/ATF4/CHOP proteins compared to those without TCT. Increased PERK/NRF2/HO1 activity was observed in both MCF-7 and MCF-7/MX cells exposed to indomethacin, diclofenac or tunicamycin for 48 h (Figure 10). The presence of TCT significantly increased the phosphorylated form of PERK and NRF2 in these cells but the expression level of HO1 decreased significantly.
Expression of PKR-like ER kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF-2α)/activating transcription factor-4 (ATF4)/C/EBP homologous transcription factor (CHOP) proteins and their phosphorylated forms in MCF-7 (A) and mitoxantrone (MX)-resistant MCF-7/MX cells (B) after 48-h exposure to endoplasmic reticulum stressors indomethacin (INDO), diclofenac (DIC), and tunicamycin (TUN) in the presence and absence of N-trans-p-coumaroyltyramine (TCT) 100 μM. Data from densitometrical analysis are shown in the lower panels. Each bar represents the mean±standard error of the mean (n=3). Significantly different at p<0.05 compared with: *stressor-treated group without TCT or #untreated control.
Expression of nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxygenase 1 (HO1) proteins in MCF-7 (A) and mitoxantrone (MX)-resistant MCF-7/MX cells (B) after 48-h exposure to endoplasmic reticulum stressors indomethacin (INDO), diclofenac (DIC), and tunicamycin (TUN) in the presence and absence of N-trans-p-coumaroyltyramine (TCT) at 100 μM. Data from densitometrical analysis are shown in the lower panels. Each bar represents the mean±standard error of the mean (n=3). Significantly different at p<0.05 compared with: *stressor-treated cells without TCT or #untreated control.
IRE1a/JNK1/2 signaling pathway: Activation of IRE1α/JNK1/2 signaling can lead to an increase of the BAX/BCL2 ratio and apoptotic cell death. As shown in Figure 11, at their non-cytotoxic concentrations, indomethacin, but not diclofenac or tunicamycin, increased the phosphorylated form of IRE1a in both MCF-7 and MCF-7/MX cells by approximately 1.7-fold compared to untreated cells. However, indomethacin exhibited an insignificant effect on the phosphorylation of JNK1/2. TCT, at 100 μM, was able to significantly enhance the effect of indomethacin on IRE1a phosphorylation. The combination of TCT and indomethacin resulted in the buildup of more phosphorylated IRE1a proteins in both types of cancer cells by approximately 2.6-to 3-fold compared to the non-TCT groups (Figure 11).
Expression of inositol-requiring enzymes-1α (IRE1α)/c-Jun N-terminal kinases1/2 (JNK1/2) and their phosphorylated forms in MCF-7 (A) and mitoxantrone (MX)-resistant MCF-7/MX cells (B) after 48-h exposure to endoplasmic reticulum stressors indomethacin (INDO), diclofenac (DIC), and tunicamycin (TUN) in the presence and absence of N-trans-p-coumaroyltyramine (TCT) at 100 μM. Data from densitometrical analysis are shown in the lower paneIs. Each bar represents the mean±SEM (n=3). Significantly different at p<0.05 compared with: *stressor-treated cells without TCT or #untreated control.
Discussion
The combination of either indomethacin or diclofenac with selected phytochemicals has been reported to be effective in enhancing cancer cell death through several mechanisms, such as the inhibition of efflux transporters, increased cellular stress and induction of apoptosis in both in vitro and in vivo models (3, 4, 12–14, 32). In this study, we demonstrated that TCT, a natural coumaric acid derivative, potentiated the cytotoxicity of NSAIDs in breast cancer cells by activating ER stress-mediated apoptosis. In addition, the mechanism of potentiation was independent of drug efflux transporter.
High expression levels of the ABC efflux transporters, particularly ABCB1, ABCC1, ABCC2, and ABCG2, can lead to the failure of several cytotoxic anticancer drugs such as mitoxantrone and doxorubicin in breast cancer treatment (1). It should be noted that MCF-7/MX and MCF-7 cells in our study expressed ABCC1 and ABCC2, but not ABCB1. However, only the resistant subline MCF-7/MX cells exhibited high activity of ABCG2 efflux transporter. Both indomethacin and diclofenac are substrates of ABCC1, ABCC2, and ABCG2 efflux transporter (28). Treatment of the two MCF-7 cell types with combinations of NSAIDs and TCT at non-cytotoxic concentrations significantly increased cell death. Our results also revealed that the combinatorial effects were similar in both cell lines. In addition to TCT, we also tested the effects of combining piperine, a major bioactive compound in several Piper plants, with these two NSAIDs. Piperine has been reported to be able to potentiate the effect of cytotoxic drugs such as doxorubicin in normal and drug-resistant cancer cells (33). In this study, the combination of piperine with NSAIDs displayed no significant effect on cell death of both MCF-7 and MCF-7/MX cells when compared to piperine alone. Furthermore, TCT and piperine did not increase the cytotoxicity of mitoxantrone, tamoxifen, vinblastine and camptothecin, which are known substrates of efflux transporters. Neither TCT nor piperine elicited inhibitory effects on the activities of ABCC1, ABCC2 and ABCG2 in either cell type. Hence, TCT and piperine exert their potential chemo-sensitizing effect on indomethacin and diclofenac via mechanisms independent of drug efflux transporters.
When used in combination with indomethacin and diclofenac, TCT enhanced the chemosensitivity of MCF-7 and MCF-7/MX cells to ER stressors (i.e., hydrogen peroxide, rotenone, thapsigargin, and tunicamycin). These findings suggest that the enhancement of cytotoxicity by TCT may be attributed to ER stress-related mechanisms. Indomethacin and diclofenac have been demonstrated to cause apoptotic cell death by inhibition of mitochondria complex I, increasing the intracellular ROS level and production of ER stress in several types of cancer cells (12, 30, 34). Our results showed that the combination of TCT with NSAIDs did not increase ROS production in the NSAID-treated cells. However, loss of MMP and increased BAX/BCL2 expression ratio were observed in both cell types after exposure to TCT in combination with either indomethacin, diclofenac, or ER stressors. The loss of MMP was well correlated to increased BAX/BCL2 ratio and higher permeability of the outer mitochondrial membrane, leading to disruption of the mitochondrial function, accumulation of unfolded proteins in ER and apoptotic cell death (35, 36). Moreover, combining TCT with NSAIDs significantly increased the activation of ER stress PERK/eIF-2α/ATF4/CHOP signaling pathways. CHOP is a nuclear transcription factor that regulates the expression of proteins involved in controlling cell homeostasis and stress responses such as NRF2 signaling and apoptotic BCL2 protein (37). Increased CHOP expression could trigger cell death machinery and promote apoptosis through changes in the expression of pro-apoptotic BAX and anti-apoptotic BCL2 proteins.
Activation of PERK also results in the stimulation of cell survival mechanisms, particularly the NRF2/HO1 pathway (13, 28, 30). Interestingly, the combination of TCT with either indomethacin, diclofenac or tunicamycin increased the expression of p-PERK and p-NRF2 proteins, whereas that of HO1 protein was reduced. Hence, the potentiation by TCT of ER stress-mediated apoptosis in NSAID-treated cells, in part, stemmed from its suppression of HO-1 expression. HO-1, encoded by HMOX1 gene, is a cytoprotective molecule against stress-induced apoptosis (38). Phosphorylation of NRF2 leads to its dissociation from Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1, allowing its translocation into the nucleus in order to initiate HO1 transcription (39, 40). As such, TCT might interfere with the transport of NRF2 into the nucleus, causing the reduction of HO1 expression.
Indomethacin has been reported to activate IRE1α/JNK1/2 signaling, leading to increased BAX/BCL2 ratio and apoptotic cell death in various cancer cell lines (13, 34). In this study, indomethacin increased phosphorylation of IRE1α but not of JNK1/2 in MCF-7 and MCF-7/MX cells. Moreover, the enhancement of cytotoxicity by TCT was not mediated by JNK1/2 signaling. It has been proposed that IRE1α may also be linked to X box-binding protein 1 (XBP1) mRNA splicing (41, 42). IRE1α activity-dependent XBP1 mRNA splicing leads to increased CHOP expression and changes in BAX and BCL2 expression, resulting in mitochondrial apoptotic cell death in various cancer cell lines (37, 43). Thus, TCT might potentiate indomethacin-mediated apoptosis in both MCF-7 and MCF-7/MX cells via the activation of IRE1a/XBP1 signaling cascade. The role of XBP1 in enhancing the effects of TCT on indomethacin toxicity in breast cancer cells should be investigated further. It is notable that diclofenac cytotoxicity in the presence of TCT was not mediated through IRE1α signaling.
In conclusion, the present study was the first to demonstrate that exposure of breast cancer cells to combinations of TCT and NSAIDs (indomethacin, diclofenac) at non-toxic concentrations increase their apoptosis. TCT may, at least in part, exert its potentiating effect via the promotion of ER stress PERK/eIF2α/ATF4/CHOP signaling pathways.
Acknowledgements
This work was supported by the CU Graduate School Grant of Chulalongkorn University.
Footnotes
Authors’ Contributions
Angkana Wongsakul: Conceptualization, methodology, investigation, formal analysis, data interpretation and writing - original draft; Nonthalert Lertnitikul: resources; Rutt Suttisri: resources, writing - review and editing; Suree Jianmongkol: conceptualization, investigation, data interpretation, supervision, writing - review and editing, project administration.
Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received January 27, 2022.
- Revision received February 19, 2022.
- Accepted February 21, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.