Alisertib Induces Cell Cycle Arrest, Apoptosis, Autophagy and Suppresses EMT in HT29 and Caco-2 Cells

Abstract

Colorectal cancer (CRC) is one of the most common malignancies worldwide with substantial mortality and morbidity. Alisertib (ALS) is a selective Aurora kinase A (AURKA) inhibitor with unclear effect and molecular interactome on CRC. This study aimed to evaluate the molecular interactome and anticancer effect of ALS and explore the underlying mechanisms in HT29 and Caco-2 cells. ALS markedly arrested cells in G₂/M phase in both cell lines, accompanied by remarkable alterations in the expression level of key cell cycle regulators. ALS induced apoptosis in HT29 and Caco-2 cells through mitochondrial and death receptor pathways. ALS also induced autophagy in HT29 and Caco-2 cells, with the suppression of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), but activation of 5' AMP-activated protein kinase (AMPK) signaling pathways. There was a differential modulating effect of ALS on p38 MAPK signaling pathway in both cell lines. Moreover, induction or inhibition of autophagy modulated basal and ALS-induced apoptosis in both cell lines. ALS potently suppressed epithelial to mesenchymal transition (EMT) in HT29 and Caco-2 cells. Collectively, it suggests that induction of cell cycle arrest, promotion of apoptosis and autophagy, and suppression of EMT involving mitochondrial, death receptor, PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways contribute to the cancer cell killing effect of ALS on CRC cells.

Keywords: EMT; alisertib; cell cycle; colorectal cancer; programmed cell death.

Figure 1

Alisertib (ALS) inhibits the phosphorylation of Aurora kinase A (AURKA) in HT29 and Caco-2 cells. HT29 and Caco-2 cells were exposed to ALS at 0.1, 1, and 5 μM for 48 h and protein samples were subject to Western blotting assay. (A) Representative blots of p-AURKA and total AURKA examined by Western blotting assay; (B) Bar graphs showing the level of p-AURKA and AURKA in HT29 and Caco-2 cells. β-Actin was used as the internal control. Data are shown as the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01 by one-way analysis of variance (ANOVA).

Figure 2

ALS induces cell cycle arrest in G2/M phase in HT29 and Caco-2 cells. (A) HT29 and Caco-2 cells were treated with ALS at 0.1, 1, and 5 μM for 24 h and then subjected to flow cytometric analysis. Representative DNA fluorescence histograms of propidium iodide (PI)-stained HT29 and Caco-2 cells showing the cell cycle distribution; (B) Time course of ALS-induced cell cycle change over 72 h in HT29 cells. Flow cytometric histograms showing the cell cycle distribution when HT29 cells were incubated with ALS at 1 μM for 4, 8, 12, 24, 48, and 72 h; (C) Time course of ALS-induced cell cycle change over 72 h in Caco-2 cells. Flow cytometric histograms show the cell cycle distribution when Caco-2 cells were incubated with ALS at 1 μM for 4, 8, 12, 24, 48, and 72 h. Cells were stained with PI and subjected to flow cytometric analysis that collected 15,000 events. Data represent the mean ± SD of three independent experiments. Dip: diploid; Ani: aneuploid.

Figure 2

ALS induces cell cycle arrest in G2/M phase in HT29 and Caco-2 cells. (A) HT29 and Caco-2 cells were treated with ALS at 0.1, 1, and 5 μM for 24 h and then subjected to flow cytometric analysis. Representative DNA fluorescence histograms of propidium iodide (PI)-stained HT29 and Caco-2 cells showing the cell cycle distribution; (B) Time course of ALS-induced cell cycle change over 72 h in HT29 cells. Flow cytometric histograms showing the cell cycle distribution when HT29 cells were incubated with ALS at 1 μM for 4, 8, 12, 24, 48, and 72 h; (C) Time course of ALS-induced cell cycle change over 72 h in Caco-2 cells. Flow cytometric histograms show the cell cycle distribution when Caco-2 cells were incubated with ALS at 1 μM for 4, 8, 12, 24, 48, and 72 h. Cells were stained with PI and subjected to flow cytometric analysis that collected 15,000 events. Data represent the mean ± SD of three independent experiments. Dip: diploid; Ani: aneuploid.

Figure 3

Effect of ALS on the expression level of key regulators of cell cycle in HT29 and Caco-2 cells. (A) Representative blots showing the expression of CDK1/CDC2, cyclin B1, p21 Waf1/Cip1, and p53 when HT29 cells were treated with ALS at 0.1, 1, and 5 μM for 48 h; (B) Representative blots showing the level of PLK1, CDK1/CDC2, p-CDC2 (Tyr15), cyclin B1, p-cyclin B1 (Ser133), and p-CDC25C (Ser216) in Caco-2 cells.

Figure 4

ALS induces apoptotic death in HT29 and Caco-2 cells. (A) HT29 and Caco-2 cells were exposed to ALS at 0.1, 1, and 5 μM for 24 h and then subject to flow cytometric analysis. Flow cytometric dot plots of specific cell populations (live, early apoptosis and late apoptosis) in HT29 and Caco-2 cells; (B) HT29 and Caco-2 cells were treated with ALS at 1 μM for 4, 8, 12, 24, 48, and 72 h and then subject to flow cytometric analysis. Flow cytometric dot plots showing specific cell populations (live, early apoptosis, and late apoptosis) in HT29 and Caco-2 cells. The apoptotic cells were detected by an annexin-V:PE (phycoerythrin) and 7-Aminoactinomycin D (7-AAD) double staining assay. The flow cytometer collected 15,000 events. Q1 indicated necrotic cells due to the machanical damage. Early apoptotic cells are located in the lower right corner (annexin V:PE positive only). Late apoptotic cells are located in the upper right corner (double positive with annexin V:PE and 7-AAD). The viable cells are located in the lower left corner (double negative with annexin V:PE and 7-AAD staining).

Figure 4

ALS induces apoptotic death in HT29 and Caco-2 cells. (A) HT29 and Caco-2 cells were exposed to ALS at 0.1, 1, and 5 μM for 24 h and then subject to flow cytometric analysis. Flow cytometric dot plots of specific cell populations (live, early apoptosis and late apoptosis) in HT29 and Caco-2 cells; (B) HT29 and Caco-2 cells were treated with ALS at 1 μM for 4, 8, 12, 24, 48, and 72 h and then subject to flow cytometric analysis. Flow cytometric dot plots showing specific cell populations (live, early apoptosis, and late apoptosis) in HT29 and Caco-2 cells. The apoptotic cells were detected by an annexin-V:PE (phycoerythrin) and 7-Aminoactinomycin D (7-AAD) double staining assay. The flow cytometer collected 15,000 events. Q1 indicated necrotic cells due to the machanical damage. Early apoptotic cells are located in the lower right corner (annexin V:PE positive only). Late apoptotic cells are located in the upper right corner (double positive with annexin V:PE and 7-AAD). The viable cells are located in the lower left corner (double negative with annexin V:PE and 7-AAD staining).

Figure 5

Effect of ALS on the expression level of key proapoptotic and antiapoptotic molecules in HT29 and Caco-2 cells. (A) Effect of ALS on the expression level of Bcl-xl, Bax, Bcl-2, PUMA, cytochrome c, cleaved caspase 3, cleaved caspase 9, and cleaved PARP in HT29 cells; (B) Effect of ALS on the expression level of Bcl-xl, Bax, Bcl-2, p-FADD (Ser194), FADD, RIP, cytochrome c, cleaved caspase 3, and cleaved PARP in Caco-2 cells.

Figure 6

ALS induces autophagic cell death in HT29 and Caco-2 cells. (A) Cells were treated with ALS at concentrations of 0.1, 1, and 5 µM for 24 h and cell samples were subject to flow cytometry analysis. Flow cytometric dot plots showing autophagic HT29 and Caco-2 cells stained by Cyto-ID^®; (B) HT29 and Caco-2 cells were treated with ALS at 1 µM for 4, 8, 12, 24, 48, and 72 h and then subject to flow cytometry analysis. Flow cytometric dot plots showing autophagic HT29 and Caco-2 cells stained by Cyto-ID^®; (C) HT29 and Caco-2 Cells were treated with ALS at 0.1, 1, and 5 µM for 24 h. Cells were stained with green fluorescent Cyto-ID^® and subjected to confocal microscopy to detect autophagy. Confocal microscopic images showing autophagy in HT29 and Caco-2 cells. The box indicates the events that were counted. FL: fluoresence. Maganification: 40×.

Figure 6

ALS induces autophagic cell death in HT29 and Caco-2 cells. (A) Cells were treated with ALS at concentrations of 0.1, 1, and 5 µM for 24 h and cell samples were subject to flow cytometry analysis. Flow cytometric dot plots showing autophagic HT29 and Caco-2 cells stained by Cyto-ID^®; (B) HT29 and Caco-2 cells were treated with ALS at 1 µM for 4, 8, 12, 24, 48, and 72 h and then subject to flow cytometry analysis. Flow cytometric dot plots showing autophagic HT29 and Caco-2 cells stained by Cyto-ID^®; (C) HT29 and Caco-2 Cells were treated with ALS at 0.1, 1, and 5 µM for 24 h. Cells were stained with green fluorescent Cyto-ID^® and subjected to confocal microscopy to detect autophagy. Confocal microscopic images showing autophagy in HT29 and Caco-2 cells. The box indicates the events that were counted. FL: fluoresence. Maganification: 40×.

Figure 7

Effect of ALS on the expression or phospharylation levels of key autophagy-regulating molecules in HT29 and Caco-2 cells. Cells were treated with ALS at 0.1, 1, and 5 µM for 48 h. The phosphorylation level of PI3K, AMPK, Akt, p38 MAPK, and mTOR, and the total level of mTOR, beclin 1, PTEN, LC3-I, and LC3-II in HT29 and Caco-2 cells determined by Western blotting assay. (A) Representative blots of p-PI3K, PI3K, p-AMPK, AMPK, p-Akt, Akt, p-p38 MAPK, p38 MAPK, p-mTOR, mTOR, beclin 1, LC3-I, and LC3-II in HT29 and Caco-2 cells treated with ALS for 48 h; and (B) Bar graphs showing the ratio of p-PI3K/PI3K, p-AMPK/AMPK, p-Akt/Akt, p-p38 MAPK/p38 MAPK, p-mTOR/mTOR, LC3-II/LC3-I, and the expression levels of beclin 1 and PTEN in HT29 and Caco-2 cells treated with ALS for 48 h. β-Actin was used as the internal control. Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 by one-way ANOVA.

Figure 7

Effect of ALS on the expression or phospharylation levels of key autophagy-regulating molecules in HT29 and Caco-2 cells. Cells were treated with ALS at 0.1, 1, and 5 µM for 48 h. The phosphorylation level of PI3K, AMPK, Akt, p38 MAPK, and mTOR, and the total level of mTOR, beclin 1, PTEN, LC3-I, and LC3-II in HT29 and Caco-2 cells determined by Western blotting assay. (A) Representative blots of p-PI3K, PI3K, p-AMPK, AMPK, p-Akt, Akt, p-p38 MAPK, p38 MAPK, p-mTOR, mTOR, beclin 1, LC3-I, and LC3-II in HT29 and Caco-2 cells treated with ALS for 48 h; and (B) Bar graphs showing the ratio of p-PI3K/PI3K, p-AMPK/AMPK, p-Akt/Akt, p-p38 MAPK/p38 MAPK, p-mTOR/mTOR, LC3-II/LC3-I, and the expression levels of beclin 1 and PTEN in HT29 and Caco-2 cells treated with ALS for 48 h. β-Actin was used as the internal control. Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 by one-way ANOVA.

Figure 8

Effect of various inducers and inhibitors on the apoptosis and autophagy induced by ALS in HT29 and Caco-2 cells. The cells were pretreated with each of the compounds for 1 h before ALS was added and incubated for a further 24 h. Cells were double stained with Annexin V:PE (phycoerythrin) and 7-Aminoactinomycin D (7-AAD) to detect cellular apoptosis, after the cells were treated with ALS for 24 h. The autophagy was detected using the Cyto-ID^® green fluorescent dye to stain autophagy-associated vacuoles. (A) Flow cytometric dot plots showing the effects of a series of compounds on basal and ALS-induced apoptosis in HT29 and Caco-2 cells; (B) Flow cytometric dot plots showing the effects of the compounds on basal and ALS-induced autophagy in HT29 and Caco-2 cells. The box indicates the events that were counted. DMSO: dimethyl sulfoxide.

Figure 9

Effect of ALS on expression levels of epithelial to mesenchymal transition (EMT) associated markers in HT29 and Caco-2 cells. Cells were treated with ALS at concentrations of 0.1, 1, and 5 µM for 48 h and then protein samples were subject to Western blotting assay. (A) Representative blots showing the expression level of E-cadherin, N-cadherin, slug, TCF-8/ZEB1, and ZO-1 in HT29 and Caco-2 cells determined by Western blotting assay; (B) Bar graphs showing the expression level of E-cadherin, N-cadherin, slug, TCF-8/ZEB1, and ZO-1, and the ratio of E-cadherin over N-cadherin in HT29 and Caco-2 cells. β-Actin was used as the internal control. Data represent as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 by one-way ANOVA.

Figure 10

Schematic mechanism underlies the cancer cell killing effect of ALS in HT29 and Caco-2 cells.