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Abstract

Lung cancer is the main cause of cancer incidence and mortality around the world. Prucalopride is an agonist for the 5-hydroxytryptamine 4 receptor, but it was unknown whether prucalopride could be used to treat lung cancer. To investigate the biological effects of prucalopride on proliferation, apoptosis, invasion, and migration of lung cancer cells, and its underlying molecular mechanism in the progression of lung cancer, we performed this study. The Cell Counting Kit 8 assay was used to measure the proliferation of A549/A427 lung cancer cells treated with prucalopride. Transwell assay was applied to evaluate cell invasion and migration. Cell apoptosis was detected by flow cytometry and Western blot analyses. The expression levels of related proteins in the PI3K/AKT/mTor signaling pathway were analyzed by Western blotting. Prucalopride inhibited the proliferation, invasion, and migration of A549/A427 human lung cancer cells. It also induced autophagy and apoptosis and decreased the expression of the phosphorylated protein kinase B (AKT) and mammalian target of rapamycin (mTor) in these cells. This study implied an inhibitory role for prucalopride in the progression of human lung cancer.

Keywords

Prucalopride lung cancer proliferation invasion migration autophagy PI3K/AKT

Introduction

Lung cancer is the most common malignancy, and the morbidity and mortality of lung cancer patients were 11.6% and 18.4%, respectively.¹ Generally, lung cancer can be classified into two main histological groups: small cell lung cancer (SCLC), which accounts for 15% of all lung cancers, and non-SCLC (NSCLC), which accounts for 85%.² According to the statistics, the 5-year survival rate for lung cancer is less than 20%.³ Currently, chemotherapies are the major options of treatment for lung cancer. However, because they are usually limited by drug sensitivity, drug resistance, and toxic side effects, there is an urgent need to develop a broad spectrum of anticancer drugs with low toxicity and high efficiency.

Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine mainly known for its neurotransmission and vasoactive properties⁴ and has various receptors consisting of 5HTR₁-5HTR₇.⁵ Indeed, genome-wide association studies illustrated that the variations of gene encoding the 5-HT₄ receptor were associated with airway obstruction and chronic obstructive pulmonary disease, which was regarded as a risk factor in lung cancer.^6,7 A selective 5-HT₄ receptor agonist, mosapride, was reported to inhibit the proliferative activity of human umbilical vein endothelial cells, depending on cell cycle arrest.⁸ These results indicated that drugs that target the 5-HT₄ receptor might be effective treatments for lung cancer.

Prucalopride, a dihydrobenzofuran carboxamide compound, is a highly selective and high-affinity 5-HT4 receptor agonist with a motility-enhancing activity that stimulates gut motility associated with chronic constipation by normalizing bowel movements. It was approved for use by the European Medicines Agency in 2009.⁹ Prucalopride has been reported to improve the condition of digestive disease patients with negligible cardiac adverse effects.¹⁰ Liu et al. confirmed that prucalopride can inhibit cell behaviors in ovarian cancer.¹¹ However, it is unknown whether prucalopride could be used to treat lung cancer. Therefore, this study attempted to investigate the antitumor activity of prucalopride in lung cancer cells.

PI3K/AKT/mTor signaling pathway is well-known as an important pathway that is activated in multiple biological and pathological processes, thereby affecting tumorigenesis.^12
–14 Furthermore, autophagy and apoptosis of cancer cells were regulated by the PI3K/AKT/mTor pathway.¹⁵ Considering its significance in the progression of cancers, we detected the PI3K/AKT/mTor pathway-related proteins to identify whether the PI3K/AKT/mTor pathway was involved in the regulatory effects caused by prucalopride in lung cancer.

Materials and methods

Cell culture

The human normal lung cell line BEAS-2B and human lung cancer cell lines A549 and A427 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone Laboratories Inc., Logan, Utah, USA) including 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, Missouri, USA) and 10% fetal bovine serum (GIBCO® Cell Culture, Carlsbad, California, USA) at 37°C with 5% CO₂. Cells at the logarithmic growth phase were washed three times in phosphate-buffered saline (PBS) and then digested with 0.1% trypsin (Solarbio, Beijing, China). When the cells had become rounded, culture medium was used to terminate the digestion, and cells were resuspended into single-cell suspensions for further experiments.

Prucalopride (MedChemExpress, Monmouth Junction, New Jersey, USA) was added to cells after dilution with the concentration of 0, 0.1, 1, 10, 50, and 100 μM for 72 h to detect the cell viability and select the suitable concentration of prucalopride.

Cell proliferation assay

Cells were seeded into 96-well plates at a density of 2 × 10³ cells per well and treated with 10 μM prucalopride for 24, 48, or 72 h. Cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8; Solarbio) every 24 h. Before detection, 10 μL of CCK8 reagent was added to the cell supernatant and incubated at 37°C for 1.5 h. The optical density at 450 nm was measured and used to plot a proliferation curve. Six replicate wells were included for each sample, and the result was expressed as the mean value ± standard deviation (SD).

Invasion and migration assay

Transwell chambers (8.0-µm pore size; Millipore, Billerica, Massachusetts, USA) coated with Matrigel (BD Biosciences, San Jose, California, USA) were used to explore cell invasion. A suspension (100 μL) of 1 × 10⁵ cells was seeded into the upper chambers, and lower chambers were filled with 500 μL RPMI 1640 medium with 10% FBS. After 24 h incubation, cells remaining in the upper chamber were removed by cotton swabs. Subsequently, invasive cells were fixed in 4% paraformaldehyde for 30 min and stained=with 0.1% crystal violet for 20 min at room temperature. The number of stained cells was counted in five random fields. The detection of cell migration was similar to the invasion assay but seeded 5 × 10³ cells into the upper chambers and did not require the Transwell chamber to be precoated with Matrigel.

Cell apoptosis assay

The effects of prucalopride on cell apoptosis were detected by annexin V/propidium iodide (PI) double staining. Cells were treated with prucalopride for 24 h, then harvested and resuspended in precooled PBS. Cells were then carefully centrifuged, aspirated, and resuspended in 1× binding buffer to a density of 1 × 10⁶/mL to 5 × 10⁶/mL. Then, 100 μL of cell suspension was mixed with 5 μL annexin V/fluorescein isothiocyanate (Sizhengbo, Beijing, China) in a 5 mL flow tube, and the mixture was placed in the dark for 5 min. A total of 10 μL PI dye and 400 μL PBS were added before the measurement of optical density at wavelengths of 488 and 530 nm. Results were analyzed by FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA).

Western blot analyses

Total protein was isolated from cells using radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) supplemented with Protease Inhibitor Cocktail (Cwbio, Beijing, China). The bicinchoninic acid (BCA) method (Cwbio) was used to measure protein concentrations. Next, 20 μg of protein and loading buffer were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, California, USA) and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). Membranes were incubated for 1 h at room temperature with 5% skimmed milk. They were then blocked with primary antibodies at 4°C overnight, washed three times with tris-buffered saline and Tween 20, and then incubated with the secondary antibody for 1 h. All primary antibodies (Proteintech Group Inc., Chicago, Illinois, USA) were diluted 1:1000 except for anti-GAPDH, which was diluted 1:5000. An enhanced chemiluminescence kit (Proteintech Group Inc.) was used to detect proteins that were then quantified with Quantity One software (Version number 1708195; Bio-Rad).

Statistical analysis

Data were expressed as the mean ± SD and evaluated with SPSS 18.0 software (SPSS Inc., Chicago, Illinois, USA). The Student’s t-test and one-way analysis of variance with Dunnett’s post hoc test were used to analyze differences between experimental groups. The p value <0.05 was considered statistically significant.

Results

Prucalopride repressed lung cancer cells proliferation

We first assessed the cell viability under the stimulation of prucalopride with different concentrations (0, 0.1, 1, 10, 50, and 100 μM) and selected 10 μM as the experimental concentration because 10 μM of prucalopride significantly inhibited cell viability, but the cell viability still remained over 50% of normal levels while 50 and 100 μM were not (Figure 1(a), p < 0.05). Subsequently, to examine the effect of prucalopride on cell growth, we assessed cell viability using the CCK-8 assay. Figure 1(b) showed that the viability of A549 cells was declined after treatment with 10 μM of prucalopride, and this influence differed significantly from the normal control (NC) at 48 and 72 h (p < 0.05). The viability of A427 cells showed the same pattern (Figure 1(c), p < 0.05). Collectively, these findings demonstrated that prucalopride hindered proliferation in lung cancer cells.

Figure 1.

Prucalopride inhibited proliferation as shown by the CCK-8 assay. (a) Cell viability induced by prucalopride with different concentrations 0, 0.1, 1, 10, 50, and 100 μM, *p < 0.05 and **p < 0.01 compared with the BEAS-2B cell line. (b) Cell proliferation of A549 cells treated with 10 μM of prucalopride. (c) Cell proliferation of A427 cells treated with 10 μM of prucalopride. *p < 0.05 compared with the NC group. CCK: Cell Counting Kit; NC: negative control.

Prucalopride inhibited invasion and migration of lung cancer cells

We further tested whether prucalopride inhibited cancer cell invasion and migration. Transwell assay showed that prucalopride significantly inhibited the migration of A549 and A427 cell lines compared with the control group after 24 h treatment (31 ± 2 vs. 96 ± 4, p < 0.01, Figure 2). Similarly, prucalopride significantly inhibited cell invasion in A549/A427 cells compared with control cells (21 ± 2 vs. 72 ± 2, p < 0.01, Figure 2). These findings indicated that prucalopride significantly inhibited the invasion and migration of lung cancer cells.

Figure 2.

Prucalopride suppressed cell invasion and migration. (a) Effect of prucalopride administration on A549 cells and cell number was quantified in (c). (b) Effect of prucalopride administration on A427 cells and quantification of cell numbers from (d). Images were captured using an inverted microscope with ×100 magnification. **p < 0.01 compared with the NC group. NC: negative control.

Prucalopride induced cell apoptosis

To determine whether the impact of prucalopride on cell proliferation was associated with cell apoptosis, annexin-V and PI double-staining flow cytometry were performed using prucalopride-treated A549 cells (Figure 3(a)). Additionally, expression of the antiapoptosis protein B cell lymphoma 2 (Bcl-2) and the apoptosis-related proteins active caspase-3 and Bcl-2-associated X protein (Bax) was detected in prucalopride-treated A549 and A427 cells. Figure 3(a) revealed that prucalopride induced apoptosis in late (annexin V+/PI+) stages, resulting in higher apoptosis levels (4.2%) compared with the NC group (2.35%). The expression of both Bax and active caspase-3 was promoted, while Bcl-2 was significantly inhibited following treatment with prucalopride in both A549 and A427 cells (Figure 3(b) and (c), p < 0.05). Overall, these results demonstrated that prucalopride induced apoptosis in lung cancer cells.

Figure 3.

Prucalopride induced cell apoptosis. (a) A549 cells treated with prucalopride and labeled with annexin V/fluorescein isothiocyanate and PI were assessed by flow cytometric analysis. (b) Western blot analysis of A549 cells treated with prucalopride and relative protein levels of apoptosis-related protein. (c) Western blot analysis of A427 cells treated with prucalopride and relative protein levels of apoptosis-related protein. Results were normalized to the GAPDH loading control. *p < 0.05 compared with the control. Data are expressed as the mean ± SD (n = 3). SD: standard deviation. PI: propidium iodide.

Prucalopride repressed the PI3K/AKT signaling pathway in A549 cells

We next investigated the effect of prucalopride on the PI3K/AKT signaling pathway in A549 cells. Treatment with prucalopride for 24 h caused a significant reduction in the protein expression levels of phosphorylated-AKT (p-AKT) and p-mTor (Figure 4, p < 0.05), suggesting that prucalopride suppressed the PI3K/AKT signaling pathway in human lung cancer cells.

Figure 4.

Effects of prucalopride on the PI3K/AKT/mTor signaling pathway in A549 cells. (a) Expression levels of AKT, p-AKT, mTor, and p-mTor were measured in A549 cells treated with prucalopride using Western blot analysis. (b) Relative protein levels of AKT, p-AKT, mTor, and p-mTor compared with the NC group. *p < 0.05 compared with the control. Data were expressed as the mean ± SD (n = 3). SD: standard deviation; NC: negative control.

Prucalopride triggered autophagy in lung cancer cells

Considering the importance of AKT and mTor on autophagy and the abovementioned results, we used Western blotting to evaluate the roles of prucalopride in autophagy. We found that the LC3-II/LC3-I ratio and Beclin 1 levels were significantly upregulated, while p62 expression was significantly decreased (Figure 5, p < 0.05). These data suggested that prucalopride-induced autophagy might be involved in its anticancer role in A549 cells.

Figure 5.

Effects of prucalopride on autophagy in A549 cells. (a) Expression levels of LC3-I, LC3-II, Beclin 1, and p62 were measured in A549 cells treated with prucalopride using Western blot analysis. (b) Relative protein levels of LC3-I to LC3-II conversion, Beclin 1, and p62 compared with the NC group. *p < 0.05 compared with the control; **p < 0.01 compared with the control. Data were expressed as the mean ± SD (n = 3). SD: standard deviation; NC: negative control.

Discussion

We investigated the potential anticancer effects of prucalopride on lung cancer and found that prucalopride significantly inhibited the proliferation, invasion, and migration of human lung cancer cells. Furthermore, our results indicated that prucalopride induced autophagy and apoptosis in lung cancer cells, possibly through regulating the PI3K/AKT/mTor signaling pathway.

In this present study performed by us, A549/A427 cells were stimulated with prucalopride. Analysis of CCK-8 revealed that prucalopride significantly suppressed proliferative ability of lung cancer cells. Furthermore, the invasion and migration of lung cancer cells were also inhibited after prucalopride treatment using Transwell assays. Prior evidence demonstrated that 5-HT has a two-sided effect on different cancers. On one hand, 5-HT can promote the growth of tumors. 5-HT and 5-HT antagonists were previously shown to significantly inhibit growth and induce apoptosis in PC3 prostate cancer cells,¹⁶ while Jiang et al. reported that increased levels of 5-HT and its receptor HTR2B promoted the growth of pancreatic and xenograft tumors.¹⁷ On the other hand, 5-HT inhibits cell proliferation in some tumors. For example, human melanoma cell proliferation has been reported to be impaired by serotonin,¹⁸ while Yoshikawa et al. suggested that 5-HT₄ agonist mosapride inhibited the activity and migration of human umbilical vein endothelial cells.¹⁹ In lung cancer, increased concentrations of 5-HT, even at 1 μM, stimulated cell proliferation.²⁰ In this present study, we demonstrated that the selective 5-HT4 receptor agonist prucalopride inhibited lung cancer cell proliferation, invasion, and migration.

Autophagy is important in the survival of cancer cells as an intracellular degradation process²¹ but is a double-edged sword in tumor cell survival and tumorigenesis because it both induces cancer cell apoptosis and maintains metabolically active cells. Bhatelia et al. showed that mediation of interferon regulatory factor 3 (IRF3) activation modulated the autophagy flux to promote breast cancer cell death, such that autophagy inhibited cancer progression.²² Conversely, autophagy can play a copromoter role in cancer progression, as seen in gastric cancer, whereas decreasing autophagy inhibited tumor growth.^22,23 To assess the role of prucalopride on apoptosis in the present study, we measured levels of hallmark proteins for autophagy and apoptosis. We observed increased numbers of annexin V and PI-positive cells, increased cleavage of caspase-3, promoted levels of Bax, and reduced levels of Bcl-2, which were indicative of enhanced apoptosis. Beclin 1 expression and conversion of LC3-I to LC3-II as typical markers of autophagy in A549 cells treated with prucalopride were increased.²⁴ P62 expression was reported to be negatively correlated with autophagy,²⁵ and P62 protein levels slightly reduced after prucalopride treatment, suggesting that autophagy was enhanced by the induction of prucalopride in lung cancer cells. To summarize, our results were consistent with the above-mentioned research, indicating that prucalopride is closely associated with autophagy.

PI3K/AKT/mTor is an important intracellular signaling pathway in the control of cell survival, apoptosis, and death,²⁶ It is also the critical signaling pathway in tumorigenesis.^27,28 PI3K typically refers to class I PI3K, which activates AKT and phosphorylates mTor, thereby inhibiting autophagy.²⁸ Qiao et al.²⁹ showed that prucalopride administration induced autophagy by inhibiting p-AKT and p-mTor expression. Other work revealed that autophagy was stimulated following the phosphorylation of AKT, the activation of mTor phosphorylation, and the repression of p70S6K, while prucalopride was suggested to hinder the initial step.^11,30 Consistent with this, we found that the inhibition of the PI3K/AKT/mTor signaling pathway contributed to autophagy. Specifically, we observed that the downregulation of p-AKT and p-mTor was accompanied by Beclin 1 reduction and the conversion of LC3.

In conclusion, our findings suggested that prucalopride inhibited human lung cancer cell proliferation, invasion, and migration by enhancing autophagy and apoptosis. These findings indicated that prucalopride might act as a potentially effective drug for lung cancer treatment.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Y-N Zhang

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Prucalopride inhibits lung cancer cell proliferation,invasion,and migration through blocking of the PI3K/AKT/mTor signaling pathway

Abstract

Keywords

Introduction

Materials and methods

Cell culture

Cell proliferation assay

Invasion and migration assay

Cell apoptosis assay

Western blot analyses

Statistical analysis

Results

Prucalopride repressed lung cancer cells proliferation

Prucalopride inhibited invasion and migration of lung cancer cells

Prucalopride induced cell apoptosis

Prucalopride repressed the PI3K/AKT signaling pathway in A549 cells

Prucalopride triggered autophagy in lung cancer cells

Discussion

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

References