Human DLD-1 CRC cells (Korean Cell Line Bank; Korean Cell Line Research Foundation, Seoul, Korea) were maintained at 37°C in 5% CO₂ in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Capricorn Scientific GmbH, Ebsdorfergrund, Germany).

The following antibodies were used in the present study: Anti-phosphorylated (p)AKT (1:2,000 dilution; catalog no. 9271; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-AKT (1:2,000 dilution; catalog no. 9272; Cell Signaling Technology, Inc.), anti-eukaryotic translation initiation factor 4E-binding protein 1 (1:2,000 dilution; 4EBP1; catalog no. 9452; Cell Signaling Technology, Inc.), anti-p4EBP1 (1:2,000 dilution; catalog no. 9459; Cell Signaling Technology, Inc.), anti-c-Myc (1:2,000 dilution; catalog no. ab32072; Abcam, Cambridge, UK), anti-β-actin (1:5,000 dilution; catalog no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and horseradish peroxidase (HRP)-conjugated anti-rabbit (1:10,000 dilution; catalog no. A120-101p; Bethyl Laboratories, Inc., Montgomery, TX, USA) and anti-mouse (1:10,000 dilution; catalog no. A90-116p-33; Bethyl Laboratories, Inc.) antibodies.

The following chemicals were used: Dimethylsulfoxide (DMSO) (catalog no. D1370.0100; Duchefa Biochemie, Haarlem, Netherlands), Forskolin (catalog no. BML-CN100; Enzo Life Sciences, Inc., Farmingdale, NY, USA), rolipram (catalog no. R1012; AG Scientific, Inc., San Diego, CA, USA), roflumilast (catalog no. 2675-50; BioVision, Inc., Milpitas, CA, USA), rapamycin (catalog no. sc3504; Santa Cruz Biotechnology, Inc.), JQ1 (catalog no. A1910; ApexBio Technology, Houston, TX, USA) and resveratrol (catalog no. 554325; Merck KGaA, Darmstadt, Germany).

DLD-1 cells (3×10⁵ cells/well) were seeded in 6-well plates. Following incubation overnight at 37°C in a 5% CO₂ incubator, the cells were treated with forskolin (20 or 40 µM) for 2 h at 37°C, following pre-incubation with rolipram (40 µM) for 6 h or resveratrol (20 µM) for 16 h at 37°C. The concentration of cAMP was determined using a Parameter cAMP assay kit (catalog no. KGE002B; R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocol.

DLD-1 (3×10⁵ cells/well) were seeded in 6-well dishes and treated with DMSO, forskolin (10 or 20 µM for 16 h), rolipram (20 or 40 µM for 16 h), roflumilast (40 µM for 16 h), JQ1 (0.5 or 1 µM for 24 h), rapamycin (25 or 50 nM for 24 h), and resveratrol (20 µM for ~48–72 h) at 37°C. Following treatment, cells were harvested by scraping and rinsed with PBS. The cells were lysed in radioimmunoprecipitation assay buffer (Elpis Biotech, Inc., Daejeon, Korea) mixed with sodium vanadate (1 mM; Sigma-Aldrich; Merck KGaA), β-glycerol phosphate (50 mM; Sigma-Aldrich; Merck KGaA), protease inhibitor (1X; G-Biosciences, St. Louis, MO, USA), EDTA (5 mM; G-Biosciences) and β-mercaptoethanol (142 mM; Bioworld Technology, Inc., St. Louis Park, MN, USA). The lysates were separated on 10 or 15% polyacrylamide gels, transferred onto polyvinylidene difluoride membranes and then blocked with 1% bovine serum albumin for 1 h at room temperature (MP Biomedicals, LLC, Santa Ana, CA, USA) dissolved in TBS and Tween-20. The membranes were probed with the aforementioned primary antibodies overnight at 4°C and HRP-conjugated secondary antibodies for 1 h at room temperature. Subsequently, the signals were visualized using enhanced chemiluminescent reagent (EzWestLumi plus; ATTO Corporation, Tokyo, Japan) and analysis of the relevant protein levels was performed using Luminograph II image analysis software (WSE-6100; ATTO Corporation).

To measure clonogenicity, 1×10³ DLD-1 cells/well were seeded in 6-well plates, incubated overnight at 37°C and then treated with DMSO (control), 20 µM forskolin and/or 40 µM rolipram/roflumilast for 7 days at 37°C. The visible colonies that developed were stained with 0.5% crystal violet solution in 25% methanol, and counted with the naked eye at room temperature.

DLD-1 cells (5×10³ cells/well) were mixed with 0.2% top agar (catalog no. A9414-5G; Sigma-Aldrich; Merck KGaA) and seeded on 0.6% bottom agar in 12-well plates to visualize their anchorage-independent growth. The treatment conditions (DMSO, 20 µM forskolin and 40 µM rolipram/roflumilast) were maintained for 21 days at 37°C with 5% CO₂. The colonies were then stained with 0.005% crystal violet solution for 1 h at room temperature. The colonies were counted using a light microscope (Olympus CKX41; Olympus Corporation, Tokyo, Japan) at ×20 magnification.

DLD-1 cells were seeded on day 0 at a density of 1×10⁵ cells/well into 6-well plates and cultured with DMSO (control), 20 µM forskolin and 40 µM rolipram/roflumilast for 6 days at 37°C. The cells were detached by trypsinization at 37°C and washed with PBS for counting with a hemocytometer at ×100 magnification every day for 6 days.

To quantify mRNA levels of PDE4A, 4B, 4C and 4D, RT-qPCR was performed and calculated using the 2⁻ΔΔ^Cq method (20). TATA-box binding protein (TBP) was used as an internal control. First, 1×10⁶ DLD-1 cells/well were seeded in 6-well plates overnight at 37°C and subsequently treated with DMSO, 40 µM forskolin for 2 h, 40 µM rolipram for 6 h, or a combination of forskolin and rolipram at 37°C. Finally, DLD-1 cells were harvested by scraping. Total RNA from DLD-1 CRC cells (1×10⁶ cells per sample) was extracted using Tri-RNA Reagent (Favorgen Biotech Cooperation, Kaohsiung, Taiwan). cDNA was synthesized using PrimeScript™ RT reagent kit with gDNA Erase (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. qPCR was then performed using a CFX connect™ Realtime system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and TOPreal™ qPCR 2X PreMIX (SYBR^® Green with low ROX; Enzynomics, Inc., Daejeon, Korea), according to the manufacturer's protocols. The primer sequences for PDE4A, 4B and 4D were as described previously (19,23,24). The primer sequences for PDE4C and TBP were as follows: PDE4C forward, 5′-AGTCCCATGTGTGACAAGCA-3′, and reverse, 5′-TCTGGTTGTCGAGGGGTAAG-3′; and TBP forward, 5′-TATAATCCCAAGCGGTTTGCTGCG-3′, and reverse, 5′-AATTGTTGGTGGGTGAGCACAAGG-3′. The following PCR amplification cycles were used: 95°C for 15 min, and 40 cycles of 95°C for 15 sec, 59°C for 15 sec and 27°C for 30 sec.

A non-parametric Mann-Whitney U test was performed using Excel 1810 software 2018 (Microsoft Corporation, Redmond, WA, USA) and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was carried out using One-way ANOVA with post-hoc Tukey HSD test calculator (http://www.astatsa.com). P<0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± standard deviation. All experiments were independently repeated a minimum of three times.

Considering that previous studies demonstrated a potential role of cAMP signaling in the survival of CRC cells (22,25), the present study investigated how intracellular cAMP expression levels are affected by forskolin, a chemical activator of adenylyl cyclase. Forskolin alone demonstrated an insignificant effect (Fig. 1A), which indicates that cAMP-degrading PDEs are present. It was identified that treatment with rolipram, a selective PDE4 inhibitor, in combination with forskolin, was associated with a significant increase in cAMP levels, which indicates that PDE4 is a major hydrolyzer of cAMP. Notably, treatment with rolipram alone did not increase cAMP expression levels, which may be due to low basal activities of adenylyl cyclase. Subsequently, RT-qPCR was performed, which identified that the PDE4D mRNA level was significantly increased, compared with PDE4A, 4B or 4C, which indicates that PDE4D is primarily responsible for cAMP degradation in DLD-1 CRC cells (Fig. 1B). However, other cAMP PDEs, including PDE3 and PDE7, may serve a role.

A previous study characterized the cAMP response elements (CREs) in the PDE4D promoter region using human airway smooth muscle cells (26). To investigate whether the expression of PDE4D is responsive to cAMP in DLD-1 cells, the present study exposed cells to forskolin, rolipram or a combination of forskolin and rolipram to modulate intracellular cAMP levels. The administration of single agents exhibited no significant effect on PDE4D expression; however, co-treatment with forskolin and rolipram significantly increased the expression level of PDE4D by >3-fold (Fig. 1C). This data is associated with cAMP concentrations upon treatment with these drugs, with forskolin/rolipram combination, but not forskolin or rolipram treatment alone, resulting in increased cAMP levels, indicating that PDE4D expression is regulated by cAMP in DLD-1 cells. The expression levels of other PDE4 isoforms, including PDE4A, 4B and 4C, were significantly reduced compared with PDE4D; however, their expression levels in response to forskolin, rolipram or a combination were also examined. As demonstrated in Fig. 1C, PDE4B mRNA levels significantly increased upon exposure to forskolin or forskolin/rolipram. This result agrees with a previous study, which demonstrated that CREs exist in the promoter region of PDE4B, but not PDE4A (27). To the best of our knowledge, the presence or absence of CREs in the PDE4C promoter region remains to be characterized.

The aforementioned data demonstrated that only a combination of forskolin with rolipram significantly increased cAMP concentrations, whereas either agent alone exhibited no significant effect (Fig. 1). Subsequently, the present study examined whether the increase in cAMP levels by combinatory treatment affects the phenotype in DLD-1 cells. Clonogenic and soft agar assays demonstrated that treatment with forskolin/rolipram significantly inhibited colony formation, cell proliferation and anchorage-independent growth, which indicates that cAMP exhibits an inhibitory effect on the malignant properties of these cells (Fig. 2A-C). Roflumilast, an FDA-approved PDE4 inhibitor (28), when combined with forskolin, exhibited the same effect as rolipram (Fig. 2D-F). In summary, the data demonstrated that high cAMP concentrations can suppress the oncogenic properties of DLD-1 cells, which indicates that these cells may typically exhibit low cAMP levels.

Subsequently, the present study aimed to identify the signaling pathways that mediate the tumor-suppressive activities of cAMP. It has previously been demonstrated that the AKT/mTOR pathway and Myc, a well-characterized downstream target, regulate the survival of CRC cells (29–31). Additionally, the activities of AKT/mTOR signaling and the expression of Myc have been revealed to be regulated by cAMP in different types of cells, including human diffuse large B cell lymphoma cell lines (19,32,33). The present study hypothesized that a downregulation of the AKT/mTOR/Myc axis by increased cAMP levels may inhibit the survival of DLD-1 CRC cells. To test this, DLD-1 CRC cells were treated with forskolin, rolipram or forskolin/rolipram, and the expression levels of pAKT, p4EBP1 and Myc were measured by western blot analysis. Treatment with either agent alone exhibited no significant effect on pAKT, p4EBP1 and Myc expression levels, which were markedly downregulated by simultaneous administration of forskolin and rolipram (Fig. 3A-C). These results support the aforementioned observed intracellular cAMP levels upon the addition of these agents both when used separately and in combination. Specifically, co-treatment with forskolin and rolipram resulted in a significant increase in cAMP levels while either agent alone exhibited no significant effect (Fig. 1A), which indicates that cAMP inhibits the AKT/mTOR/Myc signaling pathway, which is associated with the regulation of the malignant properties of DLD-1 CRC cells. To confirm cAMP's negative impact on this signaling pathway, western blot analysis was also performed using another PDE4 inhibitor roflumilast, which demonstrated a similar effect on pAKT, p4EBP1 and Myc expression levels, compared with rolipram (Fig. 3D). Treatment with forskolin/roflumilast decreased the expression levels of pAKT, p4EBP1 and Myc. In summary, these data indicate that the oncogenic phenotype of DLD-1 cells is predominantly suppressed by cAMP through inhibition of the AKT/mTOR/Myc pathway; however, the possibility that cAMP modulates the survival of DLD-1 cells via other signaling pathways cannot be excluded.

To further characterize the mediators that regulate the oncogenic properties of DLD-1 cells, the present study used rapamycin and JQ1, chemical inhibitors of mTOR and bromo- and extra-terminal domain (BET) proteins (34), respectively. Treatment with rapamycin decreased the expression levels of Myc and p4EBP1, which indicates that Myc is a downstream target of mTOR signaling (Fig. 4A). Additionally, the inhibition of the mTOR/Myc axis by rapamycin was associated with a significant decrease in the number of colonies and the number of cells in proliferation assays (Fig. 4B-D). As expected, JQ1 also decreased the expression level of Myc and demonstrated a similar effect on colony number and cell number, compared with rapamycin (Fig. 4), which indicates that mTOR and Myc are critical mediators of the antitumor effect of cAMP in DLD-1 cells.

Based on previous studies that demonstrated that resveratrol, a type of natural phenol, inhibits PDE4 activities (35,36), the present study investigated whether resveratrol could exhibit similar effects to the selective chemical inhibitors rolipram and roflumilast. Following treatment with resveratrol and/or forskolin, the intracellular levels of cAMP were measured. Combined treatment with resveratrol and forskolin significantly increased the expression level of cAMP in DLD-1 cells, compared with the control; however, the addition of either agent alone did not exhibit any significant effect (Fig. 5A). This confirms that resveratrol exhibits PDE4-inhibitor activities. Consistent with the antitumor effect of cAMP, an increase in cAMP levels following the administration of resveratrol and forskolin was associated with decreased levels of p4EBP1 and Myc (Fig. 5B). This in turn was associated with suppression of cell proliferation and colony formation in DLD-1 cells (Fig. 5C and D). These data indicate that resveratrol may be an effective therapeutic agent for CRC with high PDE4 activity.