Luteolin suppresses gastric cancer progression by reversing epithelial-mesenchymal transition via suppression of the Notch signaling pathway

Abstract

Background: Gastric cancer (GC) is one of the most malignant tumors and the second leading cause of cancer-related deaths in the world. Luteolin, a flavonoid present in many fruits and green plants, suppresses cancer progression. The effects of luteolin on GC cells and their underlying mechanisms remain unclear.

Methods: Effects of luteolin on cell proliferation, migration, invasion, and apoptosis were examined in vitro and in vivo by cell counting kit-8 (CCK-8), transwell assays, and flow cytometry, respectively. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blots were performed to evaluate Notch1 signaling and activation of epithelial-mesenchymal transition (EMT) in GC cells treated with or without luteolin. Immunohistochemistry was performed to examine proliferation and Notch1 expression in xenograft tumors.

Results: Luteolin significantly inhibited cell proliferation, invasion, and migration in a dose-dependent and time-dependent manner and promoted cell apoptosis. Luteolin reversed EMT by shrinking the cytoskeleton and by inducing the expression of epithelial biomarker E-cadherin and downregulating the mesenchymal biomarkers N-cadherin, vimentin and Snail. Furthermore, Notch1 signaling was inhibited by luteolin, and downregulation of Notch1 had similar effects as luteolin treatment on cell proliferation, migration, and apoptosis. In addition, luteolin suppressed tumor growth in vivo. A higher expression of Notch1 correlated with a poor overall survival and a poor time to first progression. Furthermore, co-immunoprecipitation analysis revealed that activated Notch1 and β-catenin formed a complex and regulated cell proliferation, migration, and invasion.

Conclusions: In this study, GC progression was inhibited by luteolin through suppressing Notch1 signaling and reversing EMT, suggesting that luteolin may serve as an effective anti-tumor drug in GC treatment.

Keywords: Apoptosis; EMT; Gastric cancer; Luteolin; Notch1; β-Catenin.

Fig. 1

Effects of luteolin on proliferation and colony formation ability in GC cells. a The proliferation of Hs-746T GC cells was inhibited upon luteolin treatment compared with the control group. Cell proliferation curves indicated that luteolin suppressed the growth of GC cells in a dose- and time-dependent manner. The significant inhibited effect on cell growth by luteolin was observed at 4th and 5th day after luteolin treatment. The results of 4th and 5th day were compared to that in their control groups using the Student’s t test. There was no statistical significance at 4th day after 10 μM lueolin treatment compared with 0 μM luteolin, but a statistical significance at 5th day. Both 20 and 30 μM luteolin resulted in a statistical significance at 4th and 5th day. b The proliferation of MKN28 GC cells was inhibited upon luteolin treatment compared with the control group. The results of 4th and 5th day were compared using the Student’s t test. There was a statistical significance at 4th and 5th day after lueolin treatment compared with 0 μM luteolin. c Luteolin significantly reduced the colony formation ability of GC cells. d Number of colonies in control and luteolin-treated groups in two GC cell lines. Results are the means of three independent experiments. *P < 0.05, **P < 0.01

Fig. 2

Effect of luteolin on cell apoptosis in GC. a, b The apoptosis of GC cells was increased upon luteolin treatment compared with the control groups. The percentages of both early and late apoptotic cells in 10 and 30 μM luteolin-treated groups were higher than the control groups. c, d The histograms show the percentage of cell apoptosis in GC cells. e Phosphorylation of Akt (Ser-473) was inhibited by luteolin, as observed by Western blot analysis. Results are the means of three independent experiments. *P < 0.05, **P < 0.01

Fig. 3

Effects of luteolin on cytoskeleton and motility in GC cells. NCI-N87 GC cells were treated with or without luteolin (30 μM) for 24 h, and analyzed by F-actin staining (Red F-actin, Blue DAPI, 200×). a Control NCI-N87 GC cells showed a spindle and fusiform shape, which indicates higher motility. b Luteolin treatment (30 μM) for 24 h caused shrinking in NCI-N87 GC cells and a decrease in the number of pseudopodia on the cell surface. c, d The cell motility was assessed by transwell assays (200×). e, f The number of migrating and invading cells is quantified. Results are the means of three independent experiments. *P < 0.05, **P < 0.01

Fig. 4

Effects of luteolin on EMT and Notch signaling in GC cells. a The chemical structure of luteolin. b The protein levels of the EMT markers were assessed by Western blot analysis in GC cells treated with different concentrations of luteolin. Luteolin increased E-cadherin levels and significantly decreased N-cadherin, β-catenin, vimentin, and Snail levels. c Immunofluorescence analysis showed that β-catenin was decreased in GC cells upon luteolin treatment (Green β-catenin, Blue DAPI, 200×). d The expression of Notch1, cyclin-D1, and Hes-1 was examined by Western blot analysis in GC cells after treatment with luteolin. e Gray scale ratio of Notch signaling markers in GC cells. f The mRNA levels of Notch targets were evaluated by RT-PCR. Results are the means of three independent experiments. *P < 0.05, **P < 0.01

Fig. 5

Effects of Notch1 on cell proliferation and EMT in GC cells. a The targets of Notch1 signaling were examined by Western blot assay after Notch1 downregulation using a shRNA. b Suppression of Notch1 caused inhibition of proliferation in GC cells. c The migration ability of GC cells was reduced in Notch1-silenced cells. d Suppression of Notch1 induced cell apoptosis. e The expression of E-cadherin was increased in Notch1 knocked down GC cells, while in contrast, N-cadherin, vimentin, and Snail expression levels were decreased. f Overexpression of Notch1 decreased E-cadherin expression following luteolin treatment in Hs-746T cells, while increased vimentin and pAKT expression. g The inhibiting effect of luteolin on cell migration was reversed subsequent to Notch1 overexpressing in Hs-746T cells. h Co-IP of β-catenin and NICD in GC cells. The interaction between NICD and β-catenin was abrogated with luteolin treatment in vitro and in vivo. i Proposed molecular model for Notch and β-catenin crosstalk. Results are the means of three independent experiments. *P < 0.05, **P < 0.01

Fig. 6

Effect of luteolin on tumor growth in vivo and effect of Notch1 on prognosis. a Images of MKN-28 xenograft tumors treated with PBS or luteolin. b Tumor volumes were measured every week (*P < 0.05, **P < 0.01). c Average weights of xenograft tumors in nude mice (*P < 0.05). d Expression of β-catenin, Notch1, and Ki-67 in xenograft tumors by IHC (200×). e TUNEL staining of xenograft tumors (200×). f, g Higher expression of Notch1 was correlated to a poor overall survival (OS) (P = 0.00022) and poor time to first progression (FP) (P = 0.00062)

Fig. 7

Proposed mechanisms of Notch and Wnt/β-catenin signaling in GC progression. When ligands bind to the Notch receptors, activated NICD translocates into the nucleus and forms a complex with activated β-catenin. The complex formation results in the regulation of target genes to induce cell proliferation and metastasis and inhibits apoptosis. Luteolin blocks the complex formation and inhibits cell proliferation and metastasis, and increases cell apoptosis, suggesting an anti-tumorigenic effect. Luteolin may be a drug for GC treatment