Downregulation of aquaporins and PI3K/AKT and upregulation of PTEN expression induced by the flavone scutellarein in human colon cancer cell lines

Abstract

Scutellarein has an anticancer potential, but the pathway of its action has not been elucidated. This study investigated scutellarein efficacy against human colorectal cancer (CRC) and explored the possible pathway of its action. MTT assay was employed to detect scutellarein effect on HT-29, SW-480, and HCT116 cells viability. Scutellarein impact on programmed cell death was studied by Annexin V-FITC/PI and its role on migration and invasion was detected by wound healing and transwell chambers. Aquaporin (AQP) 1, 3, and 5 expression before and after scutellarein treatment was approached by quantitative polymerase chain reaction (RT‒qPCR) and immunostaining while Western blotting was used to explore scutellarein effect on PI3K/AKT pathway. Scutellarein induced apoptosis and necrosis in CRC cells, thus inhibiting proliferation, migration, and invasion. Colon cancer cells exhibited positive staining for AQP 1, 3, and 5 which was downregulated by scutellarein. PI3K/AKT pathway mediating cell proliferation and growth was also modulated by scutellarein; phosphatase and tensin (PTEN) was upregulated, whereas PI3K, AKT, and p-AKT expressions were downregulated, and the downstream mTOR and phosphorylated mTOR were also suppressed at the protein level. Data indicated that scutellarein suppressed growth, migration as well as invasion of these CRC cells by downregulating the expression of AQP 1, 3, and 5 and upregulating PTEN where the latter inhibited the genes and the proteins involved in PI3K/AKT pathway. The data indicate that scutellarein is a promising therapeutic agent that inhibits growth, migration, and invasion of CRC cells by down-regulating the expression of AQP 1, 3, and 5 and by PTEN up-regulation, thus inhibiting PI3K/AKT. These molecular alterations represent potential prognostic biomarkers for the metastasis of colon cancer, where the down-regulation of AQPs enhances patient survival.

Keywords: Apoptosis; Aquaporins; Colorectal cancer; Metastasis; PI3K/AKT signaling; PTEN; Scutellarein.

Fig. 1

Scutellarein inhibitory effects on the viability of the indicated cell lines. After 72 h of incubation with the specified concentrations of scutellarein, viability was assessed using the MTT assay. Data were evaluated using one-way ANOVA (F = 27.9541; P = 0.1313) and represent the means and standard deviations of three separate tests. The percentage of viability in comparison to untreated cells is used to express the values.

Fig. 2

Apoptosis induced by scutellarein in HT-29 (A, B), SW-480 (C, D), and HCT-116 (E, F) cells. A yellow arrow indicates chromatin condensation, a black arrow ruptured plasma membrane, a brown arrow apoptotic bodies, a white arrow cell swelling, a red arrow nuclear fragmentation, a violet arrow vacuolated cytoplasm, and an orange arrow membrane blebbing. Magnification was 400X; scale bars of 50 μm.

Fig. 3

Representative flow cytometry plots after 72 h (right panel). HT-29 (A), SW-480 (B) and HCT-116 (C) cells were treated with the specified concentrations of scutellarein for 24, 48, or 72 h (left panel). Flow cytometry plots after incubation of the three cell types for 24 and 48 h are shown in Supplementary Fig. 1 (Fig. S1).

Fig. 4

The inhibitory effect of scutellarein on the wound healing process in (A) HT-29, (B) SW-480, and (C) HC-T116 cell lines. The analysis involved subjecting the assay to various concentrations of scutellarein (0 μM, 3 μM, 10 μM, 30 μM, 100 μM, and 300 μM) for a duration of 72 h. The data obtained from both the control group and the treated groups were subjected to a one-way ANOVA, followed by Dunnett's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.002 and ∗∗∗p < 0.0006.

Fig. 5

Effect of scutellarein on HT-29, SW-480, and HCT-116 cell migration (A) and invasion (B) after 72 h of incubation. Transwell chamber assays were used to measure migration, while Matrigel chamber assays were used to measure invasion. For the control and treated groups, two-way ANOVA analysis was done, then Sidak's multiple comparisons test. Magnification was a 40X. ∗∗∗P˂ 0.003 and ∗∗∗∗P˂ 0.0001.

Fig. 6

Scutellarein reduced AQP 1, 3, and 5 staining in HT-29 cells. Immunofluorescence images and quantification of AQP1 (A), AQP3 (B), and AQP5 (C) were obtained before (control) or 72-h incubation with scutellarein IC₅₀ (30.2 μM). ImageJ was used to determine the mean adjusted total cell fluorescence for AQP1, AQP3, and AQP5 (http://rsb.info.nih.gov/ij/(NIH), accessed December 23, 2022). Green fluorescence and blue nuclei indicate AQP-positive staining. Analyses used paired t tests. Magnification was 63X with 10 μm scale bars. ∗P < 0.05; n.s., non-significant (P = 0.15 for AQP3 and P = 0.17 for AQP5).

Fig. 7

Scutellarein reduced SW-480 cell AQP 1, 3, and 5 staining intensity. Immunofluorescence images and quantification of AQP1 (A), AQP3 (B), and AQP5 (C) were observed before (control) or 72-h incubation with scutellarein IC₅₀ (99.2 μM). ImageJ was used to determine the mean adjusted total cell fluorescence for AQP1, AQP3, and AQP5 (http://rsb.info.nih.gov/ij/(NIH), accessed December 23, 2022). Green fluorescence and blue nuclei indicate AQP-positive staining. Analyses used paired t tests. Magnification was 63X with 10 μm scale bars. ∗P value < 0.05; n.s., not significant (P = 0.0501 for AQP1 and P = 0.14 for AQP3).

Fig. 8

Scutellarein reduced HCT-116 AQP 1, 3, and 5 staining intensity. Immunofluorescence pictures and quantification of AQP1 (A), AQP3 (B), and AQP5 (C) were taken before (control) and after 72-h incubation with scutellarein (IC₅₀: 76.9 μM). ImageJ was used to determine the mean adjusted total cell fluorescence for AQP1, AQP3, and AQP5. Green fluorescence and blue nuclei indicate AQP-positive staining. Analyses used paired t tests. At 63× magnification and 10 μm scale bars, the P value was not significant (P = 0.13 for AQP1 and P = 0.24 for AQP3 and AQP5).

Fig. 9

Scutellarein increased PTEN (upper panel) but reduced PI3K (lower panel) mRNA expression levels in HT-29 (A, D), SW-480 (B, E) and HCT-116 (C, F) cells. Cells were incubated with the indicated concentrations of scutellarein for 24, 48, and 72 h. Data represent mean ± SD from two independent experiments, assessed using two-way ANOVA for control and treatment groups and Dunnett's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 compared to the control.

Fig. 10

Scutellarein reduced the mRNA expression levels of AQP1, AQP3, and AQP5 in HT-29 (A, D, G), SW-480 (B, E, H) and HCT-116 (C, F, I) cells. Data represent mean ± SD from two independent experiments, assessed using two-way ANOVA for control and treatment groups and Dunnett's multiple comparisons test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P˂ 0.001 when compared to the untreated control.

Fig. 11

Western blot demonstrating scutellarein effect on CRC cell line PTEN, AKT, p-AKT, mTOR, and p-mTOR protein expression. (A) HT-29, (B) SW-480, (C) HCT-116. Cells were incubated with scutellarein (0, 10, 30, 100 μM) for 24, 48, and 72 h. Data represent mean ± SD from two independent experiments, assessed using two-way ANOVA for control and treatment groups and Dunnett's multiple comparisons test. ∗P < 0.05 and ∗∗∗P˂ 0.001 when compared to the untreated control. Original blots for the proteins from the 3 cell lines are provided as supplementary material (Fig. S2; Original Western Blots Scutellarein).

Fig. 12

Hypothetical mechanism of action of scutellarein. (A) In the canonical form, epidermal growth factor receptor (EGFR), located in lipid rafts, dimerizes in the presence of epidermal growth factor (EGF) and initiates a signaling cascade that allosterically activates PI3K, making the pathway active to produce AKT and downstream targets, thus stimulating the expression of AQPs, which facilitate cell migration. (B) In the presence of scutellarein, the PI3K/AKT/mTOR pathway is disrupted to upregulate tumor suppressor phosphatase and tensin (PTEN). Upregulation of PTEN results in downregulation of AQP expression and inhibition of cell proliferation, invasion, and metastasis.