Abstract
Background/Aim: Anti-inflammatory drugs, such as aspirin, have attracted attention as anticancer agents that can be applied to standard chemotherapy for pancreatic cancer. This study aimed to examine the antitumour effects and possible fundamental mechanisms of aspirin in pancreatic cancer cells. Materials and Methods: We appraised the antitumour effects of aspirin on cell proliferation and tumour growth, cell cycle distribution, apoptosis, signalling pathways, angiogenesis-related proteins, and phosphorylated receptor tyrosine kinases (p-RTKs) and identify miRNAs associated with its antitumour effects. Results: Aspirin inhibited cell proliferation in pancreatic cancer cell lines and induced G0/G1 cell cycle arrest by decreasing the expression of cyclin D1. Aspirin inactivated glycogen synthase kinase (GSK)-3β but had no effect on the p38 mitogen-activated protein kinase (MAPK) pathway, p-RTKs, or angiogenesis-related molecules. Aspirin treatment statistically increased the expression of 274 miRNAs in PANC-1 cells and 30 miRNAs in PK-8 cells and suppressed the expression of 294 miRNAs in PANC-1 cells and 13 miRNAs in PK-8 cells. Conclusion: Aspirin inhibited the proliferation of pancreatic cancer cells and induced cell cycle arrest. Aspirin also inactivated GSK-3β but not the p38 MAPK pathway. Thus, aspirin may be used in combination with chemotherapeutic agents for pancreatic cancer.
Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and high case fatality rate because of low curative resection rates caused by local invasion and distant metastases. PDAC and its related variants account for 85%-90% of all pancreatic neoplasms and are, therefore, referred to as pancreatic cancer. The overwhelming majority (80%-85%) of patients with PDAC present with locally advanced or distant metastases, and only a minority (15%-20%) are amenable to surgical resection (1, 2). As a risk factor for pancreatic cancer, chronic pancreatitis is a progressive inflammatory disease of the pancreas that results in destruction of acinar cells and significant pathological fibrosis (3, 4). The major aetiologies of chronic pancreatitis include alcoholism, heredity, and idiopathy with a strong genetic predisposition (5). The inflammatory pathways that contribute to the pathogenesis of pancreatic cancer are currently being elucidated; however, the development of anti-inflammatory molecules to prevent pancreatic cancer and elucidation of their mechanisms remain a challenge.
Aspirin is a classic non-steroidal anti-inflammatory drug (NSAID) that is universally used as an anti-inflammatory and antiplatelet agent to reduce the risk of cardiovascular disorders (6). Its mechanism of action is irreversible selective inhibition of cyclooxygenase, which is different from other NSAIDs. Aspirin is a candidate drug for cancer chemoprevention and has been reported to reduce the risk of various types of cancer, including pancreatic cancer (7-10). According to the latest clinical statistics, long-term aspirin use may reduce the risk of pancreatic cancer in a dose-dependent manner (11). Aspirin has been reported in population studies to inhibit cancer development, making it a useful repositioning drug (12).
Nuclear factor kappa B (NF-B) regulates proliferative and anti-apoptotic genes, such as cyclin D1 and BCL2, and it is believed that NF-
B plays a major role in promoting gemcitabine resistance (13). Aspirin has an inhibitory effect on NF-
B, which is a transcription factor that activates genes involved in inflammation and cell proliferation (14). It has also been suggested that aspirin inhibits cell proliferation by arresting the cell cycle. Furthermore, aspirin has been reported to inactivate key target molecules in various carcinomas via AMP-activated protein kinase/mammalian target of rapamycin (15), WNT/β-catenin (16), and p38 mitogen-activated protein kinase (MAPK) signalling pathways (17). In pancreatic cancer, glycogen synthase kinase (GSK)-3β intervenes in several cellular processes. This kinase is involved in the regulation of NF-
B, cell survival, and proliferation (18). Administration of NSAIDs appears to inhibit AKT protein kinase activation, which in turn increases GSK-3β expression; high expression of GSK-3β leads to β-catenin degradation, arrest of abnormal cell proliferation, and chemopreventive effects (19). Although there have been several reports on aspirin and its mechanisms as described above, the microRNA (miRNA) signatures of PDAC cells and tumours treated with aspirin are not well understood in vitro or in vivo. The purpose of this study was to investigate the functional effects of aspirin on the cell cycle and apoptosis in PDAC cell lines and xenograft mouse models and identify miRNAs associated with the antitumour effects of aspirin.
Materials and Methods
Cell culture and reagents. Four pancreatic adenocarcinoma cell lines (PANC-1, PK-1, PK-8, and PK-9) were obtained from Biobank Japan (Tokyo, Japan). All cells were cultured using standard conditions (37°C and 5% CO2) in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan) comprising 10% foetal bovine serum (FUJIFILM Wako Pure Chemical Industries) and penicillin/streptomycin (100 mg/l; Gibco-Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Aspirin (FUJIFILM Wako Pure Chemical Industries) was resolved in DMSO, and the pH was adjusted to 7.2-7.5, which is a suitable range for cell growth.
Cell proliferation assay. Cell proliferation assays were performed as per the manufacturer’s protocol using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). According to the standard protocol, 5.0×103 cells were seeded in 96-well plates in triplicate. After 24 h, PANC-1, PK-1, PK-8, and PK-9 cells were treated with 0, 2.5, 5, or 10 mM aspirin for 24-72 h at 37°C in 5% CO2. The absorbance per well was measured at 450 nm using a microplate reader. The absorbance of the control samples (medium only) represented 100% viability, and the results for the aspirin-treated samples were expressed as a percentage of the control samples.
Cell cycle and apoptosis analyses. Cell cycle and apoptosis analyses were performed as described in our previous study (20). To evaluate cell cycle progression, cells were seeded in 60-mm plates and incubated for 24 h for exponential growth. PANC-1 and PK-8 cells were incubated with or without 2.5 or 5 mM aspirin for an additional 24-48 h.
The Annexin V-FITC Early Apoptosis Detection Kit (Cell Signaling Technology, Danvers, MA, USA) was used to measure apoptosis following the manufacturer’s protocol. PANC-1 and PK-8 cells were incubated with or without 2.5 mM aspirin for 24-48 h, collected, washed with phosphate-buffered saline (PBS), resuspended in annexin V binding buffer, and incubated with annexin V-FITC and propidium iodide. The following items were used for flow cytometric analysis: a Cell Cycle Phase Determination Kit (Cayman Chemical, Ann Arbor, MI, USA), a Cytomics FC500 (Beckman Coulter, Brea, CA, USA) and a CytoFLEX S (Beckman Coulter) both with an argon laser (488 nm), a Kaluza software v2.1 (Beckman Coulter), and CytExpert software (Beckman Coulter).
Western blot analysis. Western blotting was performed as previously described (20). The following antibodies were used: anti-cyclin D1, anti-retinoblastoma protein (RB), and anti-cyclin E (1:1,000, Thermo Fisher Scientific); anti-phosphorylated (p)RB (1:1,000, BD Pharmingen, San Jose, CA, USA); anti-cyclin-dependent kinase (CDK)6 (1:500) and anti-CDK2 (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA); anti-β-actin (1:5,000, Sigma-Aldrich, St. Louis, MO, USA); and anti-GSK-3β, anti-p-GSK-3β (Ser9), anti-AKT, anti-p-AKT (Ser473), anti-p38 MAPK, anti-p-p38 MAPK (Thr180/Tyr182), anti-mitogen-activated protein kinase kinase (MKK)3, and anti-p-MKK3 (Ser189)/MKK6 (Ser207) (1:1000, Cell Signaling Technology). The secondary antibodies included horse-radish peroxidase-conjugated anti-mouse and anti-rabbit IgG (#7076, #7074; Cell Signaling Technology). Band intensities were semi-quantified using ImageJ software v1.53k (National Institutes of Health, Bethesda, MD, USA) and normalized to β-actin.
Analysis of angiogenesis-related protein and phosphorylated receptor tyrosine kinase (pRTK) profiles using an antibody array. PANC-1 and PK-8 cells (1.0×106 cells/100 mm dish) were incubated with 2.5 or 5.0 mM aspirin for 48 h at 37°C, and proteins were extracted with PRO-PREP solution containing a protease inhibitor mixture (Intron Technology, Hong Kong, China). Human Angiogenesis and Human Phospho-RTK Antibody Arrays (RayBiotech Life, Inc., Norcross, GA, USA) were used in accordance with the manufacturer’s protocol; these arrays are dot-based assays that enable the detection and comparison of 20 angiogenesis-specific cytokines and 71 different phosphorylated receptor tyrosine kinases, respectively. Each membrane array was analysed using ImageQuant LAS 4010 (GE Healthcare, Boston, MA, USA).
MiRNA array. PANC-1 and PK-8 cells were treated with 2.5 mM aspirin for 48 h and total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Limburg, the Netherlands) following the manufacturer’s protocol. The samples were labelled using miRCURY Hy3 Power Labeling Kit (Exiqon A/S, Vedbaek, Denmark) after affirming the purity and quantity of each RNA using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA 6000 Nano Kit (Agilent Technologies). The labelled RNA was crossbred to a human miRNA Oligo chip (v.21; Toray Industries, Tokyo, Japan). Scanning was performed with a 3D-Gene Scanner 3000 (Toray Industries). The signal intensities of the images were calculated using 3D-Gene Extraction Software version 1.2 (Toray Industries). The obtained data were analysed using GeneSpring GX 10.0 software (Agilent Technologies) to evaluate changes in the miRNA expression between untreated and aspirin-treated samples. To remove background, the obtained data were quantile normalized. The false discovery rate was calculated using the Benjamini-Hochberg method. Heat maps were created with the relative expression intensity of each miRNA, and the bottom two log intensities in each column were the median centres.
Xenograft model analysis. Animal studies were approved and conducted in accordance with the guidelines established by the Kagawa University Experimental Animal Committee. Female nude mice (BALB/c-nu/nu; 4-weeks old) were purchased from SLC Japan (Shizuoka, Japan). Mice were maintained under specific pathogen-free conditions using laminar air flow racks, sterilized (γ-irradiated) food (CL-2; Clare Japan, Tokyo), and autoclaved water. A total of 1×107 PK-8 cells were injected subcutaneously into the right flank of each mouse. The mice were randomly assigned to two groups (aspirin and control) with 14 mice/group. The mice were treated three times/week by intraperitoneal injection with 60 mg/kg aspirin or PBS (control). Tumour growth was observed and tumour volume (V) mm3 was calculated as V=long tumour diameter (mm)×short tumour diameter (mm)×short tumour diameter (mm). All animals were sacrificed seven days after beginning treatment. None of the mice died during this period.
Immunohistochemistry. Tumours were fixed, embedded in paraffin, and sectioned. Tumour sections were dewaxed, soaked in ethanol, and then blocked with 0.5% hydrogen peroxidase. Antigen-specific immunoreactivity was blocked using diluted normal rabbit serum at room temperature. After washing with PBS, the sections were immunostained. Expression of cyclin D1 was determined by incubation with a mouse monoclonal antibody (1:200 dilution) overnight. Then, the sections were incubated with suitable peroxidase-conjugated secondary antibodies (PK-6102, PK-6101; Vectastain Elite ABC kit; Vector Laboratories, Inc, Burlingame, CA, USA) for 30 min, stained with diaminobenzidine solution, and counterstained with Mayer’s hematoxylin. Samples treated with PBS served as negative controls.
Statistical analysis. GraphPad Prism software version 6.0 (GraphPad Software, San Diego, CA, USA) was used for all analyses. Student t-tests were used to examine significant differences between the different groups. One-way or two-way ANOVA was used for multiple comparisons. A p-value of <0.05 was considered statistically significant.
Results
Aspirin inhibited the growth of four human PDAC cell lines. We examined the inhibitory effect of aspirin on the proliferation of four PDAC cell lines (PANC-1, PK-1, PK-8, and PK-9 cells) by performing the CCK-8 assay. The PDAC cells were treated with 0, 2.5, 5, or 10 mM of aspirin for 24 to 72 h. Aspirin inhibited the growth of each PDAC cell line in a concentration- and time-dependent manner (Figure 1A). Figure 1B shows the inhibition of cell proliferation in the four PDAC cell lines after 72 h of aspirin treatment.
Aspirin inhibits cell proliferation of pancreatic ductal adenocarcinoma cells (PANC-1, PK-1, PK-8, and PK-9 cells). (A) Cells were treated with four concentrations of aspirin (0, 2.5, 5, and 10 mM), and cell growth was determined using the CCK-8 assay at 24, 48, and 72 h. (B) PANC-1, PK-1, PK-8, and PK-9 cells were treated with 0, 2.5, 5, and 10 mM aspirin for 72 h. Data points represent the mean cell number from three independent cultures, and the error bars represent the standard deviation. Data are presented as means±SD; *p<0.05, **p<0.01 vs. control.
Aspirin induced cell cycle arrest at the G0/G1 to S phase transition in pancreatic cancer cells and inhibited cell cycle-related protein expression. To determine whether aspirin affected the cell cycle of PANC-1 and PK-8 cells, we examined cell cycle progression by flow cytometry and evaluated the expression of cell cycle-related proteins by western blot. Among the four PDAC cell lines, PANC-1 and PK-8 cells showed the greatest response to aspirin; therefore, these two cell lines were used for cell cycle analysis. PANC-1 and PK-8 cells were treated with 2.5 mM or 5 mM aspirin for 24 or 48 h, and untreated cells were used as controls. After 24 and 48 h of aspirin treatment, the number of cells in the G0/G1 phase was significantly increased, and the number of cells in S phase was significantly decreased (Figure 2A and B).
Effects of aspirin on the cell cycle in PANC-1 and PK-8 cells. (A) Cell cycle distributions in aspirin-treated PANC-1 and PK-8 cells. (B) Histograms showing the percentage of PANC-1 and PK-8 cells in G0/G1 S, and G2/M phases. (C) Western blot analysis of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, CDK6, retinoblastoma (RB), and p-RB in PDAC cells treated with 2.5 and 5 mM aspirin for 24 or 48 h. Data are presented as means±SD; *p<0.05, **p<0.01; n.s.: not significant vs. control.
Next, we confirmed by western blot that aspirin treatment significantly altered cell cycle-related proteins that induce progression from the G0/G1 to S phase. PANC-1 cells showed decreased expression of cyclin D1, CDK2, and CDK4 at 24 and 48 h after aspirin treatment, and PK-8 cells showed decreased expression of cyclin D1. In addition, expression of phosphorylated RB, CDK2, and CDK4 was inhibited in PK-8 cells after aspirin treatment for 24 and 48 h (Figure 2C).
Flow cytometry was used to examine the effect of aspirin on apoptosis; the quadripartite figure shows viable cells (lower left square), early apoptotic cells (lower right square), and late apoptotic cells (upper right square). At 24 and 48 h after aspirin administration, no significant increase in the percentage of early apoptosis was observed in the aspirin-treated group (PANC-1: 3.27%, PK-8: 9.86%) compared with that in the control group (PANC-1: 3.54%, PK-8: 13.21%; Figure 3A and B).
Effects of aspirin on apoptosis in PANC-1 and PK-8 cells. (A) Changes in early apoptosis induced by aspirin at 24 or 48 h were assessed by flow cytometry. Cells positive for annexin V and negative for propidium iodide (PI) were considered early apoptotic cells (lower right square). (B) Percent of annexin-V-positive cells (n.s.: not significant vs. control).
Aspirin suppressed GSK-3β activation via activation of AKT signalling in PDAC cells. To evaluate the mechanism of aspirin-induced cell cycle arrest, we focused on GSK-3β signalling. As shown in Figure 4A, we observed a dose-dependent increase in the phosphorylation of GSK-3β after treatment with aspirin using western blot analysis of whole cell proteins, while no effect was observed for the total levels of GSK-3β. To further elucidate the signalling pathways that inactivated GSK-3β, we examined the effect of aspirin on AKT, which is implicated in the regulation of GSK-3β activity (21). After 24 h of treatment with aspirin, an increase in the levels of phosphorylated AKT was observed (Figure 4A). Next, we examined the effects of aspirin on p38 MAPK signalling in PDAC cells. Western blot analysis using phosphorylation-specific antibodies revealed no dose- or time-dependent phosphorylation of p38 MAPK after aspirin administration (Figure 4B). Therefore, aspirin suppressed the activation of GSK-3β in PDAC cells, but the p38 MAPK pathway was not involved in the inactivation. Furthermore, aspirin treatment did not induce cyclin D1 phosphorylation, suggesting that cyclin D1 degradation was independent of GSK-3β and the p38 MAPK signalling pathway.
Investigation of the pathway by which aspirin causes G0/G1 arrest. (A) Inactivation of GSK-3β by aspirin. Cells were treated with 2.5 and 5 mM aspirin for 24 and 48 h. Western blot analysis of GSK-3β, p-GSK-3β, AKT, and p-AKT in PANC-1 and PK-8 cells. (B) Western blot analysis of p38 MAPK, p-p38 MAPK, MKK3, p-MKK3/6, and p-cyclin D1 in PANC-1 and PK-8 cells. β-actin was used to normalized total protein.
Comprehensive analysis of angiogenesis-related cytokines, receptor tyrosine kinase phosphorylation, and altered miRNAs in aspirin-treated PDAC cells. We investigated the effects of aspirin on receptor tyrosine kinase phosphorylation and angiogenesis-related cytokines in PANC-1 and PK-8 cells using the Human RTK Phosphorylation Antibody Array Kit and Human Angiogenesis Antibody Array Kit, respectively. There was no significant difference between the untreated group and the 2.5 and 5 mM aspirin-treated groups in either of the cell lines (Figure 5A and B). The microarray platform was also used to analyse the expression of 2,555 miRNAs in aspirin-treated and control PANC-1 and PK-8 cells. Aspirin treatment statistically increased the expression of 274 miRNAs in PANC-1 cells and 30 miRNAs in PK-8 cells (Figure 5C). Aspirin treatment suppressed the expression of 294 miRNAs in PANC-1 cells and 13 miRNAs in PK-8 cells (Figure 5C, Table I and Table II). Aspirin treatment increased the levels of five common miRNAs in both cell lines (Table III).
Effects of aspirin on receptor tyrosine kinase phosphorylation, angiogenesis-related proteins, and miRNA levels. (A) Human RTK Phosphorylation Antibody Array and (B) Human Angiogenesis Antibody Array were used to detect protein expression changes in PANC-1 and PK-8 cells. There was no significant difference between the control and aspirin-treated groups for either of the cell lines. (C) MiRNA assays were performed using PANC-1 and PK-8 control cells (untreated) and after treatment with aspirin for 48 h. When a false discovery rate < 0.05 was applied, 274 and 30 miRNAs were up-regulated in PANC-1 and PK-8, respectively, and 294 and 13 miRNAs were down-regulated in PANC-1 and PK-8, respectively.
Statistical results of the miRNA assay of PANC-1 cells with or without aspirin treatment. The fold change was >3.0 and <0.5, and the false discovery rate (FDR) was <0.03 (miR-365a-5p, miR-204-3p, miR-663a, and miR-365b-5p are exceptions).
Statistical results of the miRNA assay of PK-8 cells with or without aspirin treatment. The fold change was >1.5 and <0.7, and the false discovery rate (FDR) was <0.03.
The common microRNAs found in both cell lines that were altered by aspirin treatment. The fold change was >1.5 and <0.7, and the false discovery rate (FDR) was <0.03.
Aspirin inhibited tumour growth in vivo. Nude mice were subcutaneously implanted with PK-8 cells and then treated intraperitoneally with aspirin (60 mg/kg) or PBS alone. The tumour sizes in aspirin-treated mice were significantly smaller than those in the control mice (Figure 6A and B). To further investigate whether aspirin also affected cell cycle regulatory proteins in vivo, we analysed protein expression by immunohistochemistry. Aspirin treatment decreased the expression levels of cyclin D1 (Figure 6C and D) compared with those in the control group.
Aspirin inhibits PK-8 cell xenograft growth in nude mice. (A) Tumours in aspirin-treated mice were significantly smaller than those in vehicle-treated mice. (B) Tumour growth curves of aspirin-treated and control groups. Each point represents the mean±standard error of 14 mice. (C) Immunohistochemical staining of cyclin D1 in cancerous tissues from aspirin-treated and control groups of xenografted mice. (D) The number of cyclin D1-positive cells per 1 mm2 area in aspirin-treated mice was reduced compared with that in the untreated mice.
Discussion
In the present study, we demonstrated that aspirin inhibited the growth of PDAC cells, which was correlated with a decrease in cyclin D1 levels and concomitant G1 cell cycle arrest. Aspirin alone was found to inhibit the growth of human PDAC cells by interfering with cell cycle progression, and these effects of aspirin may be related to the inhibition of GSK-3β activity and down-regulation of cyclin D1. Furthermore, aspirin stimulated AKT activation via protein phosphorylation at Ser43, while p38 MAPK and its upstream MKK3/6 pathway were not affected. These findings add to our understanding of the function of aspirin in PDAC as well as the molecular mechanisms.
Aspirin inhibited cell cycle progression from G0/G1 phase to S phase, which was demonstrated by flow cytometric analysis. Previous reports have shown that the effect of aspirin on the cell cycle varied depending on the cell type. Qu et al. (22) reported that aspirin induced cell cycle arrest in the G0/G1 phase, and Gao et al. (23) reported that aspirin induced cell cycle arrest in the G2/M phase. Cell cycle regulatory proteins play an important role in the regulation of cell cycle progression (24). We examined the expression of cell cycle regulatory proteins involved in aspirin-induced G1 arrest. Western blot analysis showed that aspirin decreased the levels of cyclin D1, which regulates the G0/G1 to S phase transition. Furthermore, the expression pattern of the cyclin D1 partner CDK4 in PANC-1 and PK-8 cells was consistent with the aspirin-induced reduction of cyclin D1. Cyclin D1 is required for progression through the G1 phase of the cell cycle (25). Down-regulation of cyclin D1 after aspirin treatment in pancreatic cancer cell lines is consistent with cell cycle arrest in the G0/G1 phase. While previous studies have suggested that aspirin can induce apoptosis in pancreatic cancer cells (26), it was unable to induce apoptosis in gemcitabine-resistant pancreatic cancer cell lines (22). In our study, aspirin did not induce apoptosis in PANC-1 and PK-8 cells. High concentrations of aspirin (1-5 mM) can be achieved in vivo by oral administration of 4-10 g/d aspirin during the treatment of rheumatic diseases and arthritis (27). Therefore, we used an upper limit of 5 mM aspirin for the present study. The marked inhibition of pancreatic cancer cell growth by aspirin was mainly due to growth inhibition and induction of cell cycle arrest.
Next, we focused on GSK-3β as an upstream mechanism involved in the down-regulation of cyclin D1 expression. We found that aspirin suppressed the activation of GSK-3β. Cyclin D1 degradation is mediated by phosphorylation at Thr286 by various kinases, including GSK3β, p38 MAPK, and DYRK1 (17). The mechanism by which aspirin exerts the above biological activity is thought to involve suppression of the activity of GSK-3β. In our study, aspirin inhibited GSK-3β activation and induced AKT phosphorylation in PANC-1 and PK-8 cells. Furthermore, aspirin did not induce cyclin D1 phosphorylation, suggesting that cyclin D1 degradation is independent of GSK-3β and the p38 MAPK signalling pathway. Ou et al. reported that aspirin suppressed the activity of GSK-3β and decreased cyclin D1 and BCL-2 expression in PANC-1 cells, a gemcitabine-resistant pancreatic cancer cell line, which is consistent with our findings (22). The activity of GSK-3β is regulated not only by an activating phosphorylation at Tyr216 but also by an inhibitory phosphorylation at Ser9 (28). Ougolkov et al. suggested that pancreatic cancer cells have a pool of active GSK-3β and depletion or inhibition of this pool reduces cell viability (29). Shimasaki et al. suggested that ARA014418, a GSK-3β inhibitor, sensitizes pancreatic cancer cells to gemcitabine in vitro and in the PANC-1 mouse xenograft model. Untreated PANC-1 cells showed increased expression of GSK-3β, phosphorylation of Tyr216, and activity. Treatment with pharmacological doses of ARA014418 inhibited the proliferation of these cells, and this effect appeared to be synergistic with gemcitabine (27). Therefore, the combination of aspirin, which inactivates GSK-3β, and anticancer drugs may be effective for pancreatic cancer treatment.
Previously, aspirin was reported to induce apoptosis in colorectal cancer because of its effects on p38 MAPK, cyclin D1/CDK4, and NF-B pathways (17). We focused on p38 MAPK and the upstream MKK3/6 pathway in PDAC cells. However, p38 MAPK and MKK3/6 were not altered by aspirin treatment in PANC-1 or PK-8 cells. These results suggest that aspirin-induced degradation of cyclin D1 in PDAC cells may be mediated by an alternative pathway other than the p38 MAPK pathway. We used membrane-based antibody arrays for p-RTK and angiogenesis to search for pathways upstream of GSK-3β. However, no differences in molecules involved in the upstream pathway were detected between the control and aspirin-treated group in either of the cell lines. In summary, GSK-3β was significantly inactivated by aspirin treatment, but no correlation between p38 MAPK and cyclin D1 degradation was observed.
Multiple studies have reported on the relationship between gemcitabine, a typical anticancer drug, and miRNAs in pancreatic adenocarcinoma cells (30); however, there are few studies on aspirin and miRNAs (31). Therefore, in this study, we performed a cluster analysis of miRNAs that were altered by aspirin treatment in PDAC cells. The miRNAs miR-204, miR-663a, and miR-365 have been reported to be involved in gemcitabine resistance, and these miRNAs showed conflicting actions against gemcitabine resistance after aspirin administration. MiR-301a is known to act as a pivotal regulator in hypoxia-induced gemcitabine resistance through down-regulation of TAp63 (32). Furthermore, miR-365 can enhance gemcitabine resistance by reducing SHC1 and BAX, which are apoptosis-promoting molecules in pancreatic cancer (33). Thus, these miRNAs may be strong candidates for amelioration of gemcitabine drug resistance by concomitant aspirin administration. However, the lack of experiments to analyse these miRNAs and their functions in this study indicates that further experiments will be required to elucidate the mechanism of gemcitabine drug resistance.
The high metastatic rate of pancreatic cancer also contributes to its poor prognosis, and it has been reported that tumor progression and metastasis may be more active in the perioperative period (34). Malsy et al. reported that ropivacaine and sufentanil use in the perioperative period decreases the migratory ability of pancreatic cancer cells (35). Aspirin, an antipyretic analgesic, has also been reported to decrease tumor cell migration in prostate cancer cells (36), and similar results in pancreatic cancer cells would provide further insight into the antitumor effects of aspirin.
In conclusion, aspirin arrests PDAC cells at the G0/G1 phase of the cell cycle by down-regulating cyclin D1 expression. Aspirin treatment also deactivated GSK-3β and phosphorylated AKT but did not affect the p38 MAPK pathway. Thus, aspirin, a traditional drug, may be used in combination with existing chemotherapeutic agents for pancreatic cancer.
Acknowledgements
The Authors thank Kayo Hirose, Megumi Okamura, Mari Yamada, Fuyuko Kokado, and Keiko Fujikawa for skilful technical assistance. The Authors also thank Susan Zunino, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
Footnotes
Authors’ Contributions
RN and TM designed the experiments. SF, HI, SH, TM, MH, TK, DN, NF, HY, KK, HK, HKa, and HKo conducted the experiments, analysed the data, and drafted and wrote the final manuscript. SF contributed to drafting the manuscript. TM was involved in the research design and drafting of the final manuscript. All Authors have read and approved the final version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received May 20, 2022.
- Revision received June 6, 2022.
- Accepted June 7, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.