Aloe-emodin suppresses esophageal cancer cell TE1 proliferation by inhibiting AKT and ERK phosphorylation

Abstract

Aberrant AKT and extracellular signal-regulated kinase (ERK) activation is often observed in various human cancers. Both AKT and ERK are important in the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase kinase/ERK signaling pathways, which play vital roles in cell proliferation, differentiation and survival. Compounds that are able to block these pathways have therefore a promising use in cancer treatment and prevention. The present study revealed that AKT and ERK are activated in esophageal cancer TE1 cells. Aloe-emodin, an anthraquinone present in aloe latex, can suppress TE1 cell proliferation and anchor-independent cell growth. Aloe-emodin can also reduce the number of TE1 cells in S phase. Protein analysis indicated that aloe-emodin inhibits the phosphorylation of AKT and ERK in a dose-dependent manner. Overall, the present data indicate that aloe-emodin can suppress TE1 cell growth by inhibiting AKT and ERK phosphorylation, and suggest its clinical use for cancer therapy.

Keywords: aloe-emodin; esophageal cancer; signal transduction pathway.

Figure 1.

AKT and ERK were activated in EC cell lines. Four human EC cell lines were cultured, and cell lysates were harvested. A total of 50 µg of cell lysates of each cell line were subjected to western blot analysis. The phosphorylation of AKT and ERK was evaluated. Representative data of three independent experiments are shown. ERK, extracellular-signal regulated kinase; EC, esophageal cancer; p-, phosphorylated.

Figure 2.

(A) Chemical structure of aloe-emodin. (B) Toxicity of aloe-emodin in TE1 cells. TE1 cells (2×10⁴) were seeded into 96-well plates in 100 µl of 10% fetal bovine serum-Dulbecco's modified Eagle medium, and incubated in a 37°C, 5% CO₂ incubator overnight. The cells were treated with increasing doses of aloe-emodin (1, 5, 10, 25 and 50 µM), and cytotoxicity was analyzed at the indicated times using a Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol.

Figure 3.

AE suppresses TE1 cell proliferation and anchor-independent cell growth. TE1 cells (5×10³) were treated with different concentrations of AE. (A) AE significantly inhibited cell proliferation. Absorbance was measured at 24, 48, 72 and 96 h by Cell Counting Kit-8, as described in Materials and methods. Data are shown as means ± SD (*P<0.05 vs. untreated control, n=3). (B) AE significantly suppressed anchor-independent cell growth of TE1 cells. Colonies were counted, and data are shown as means ± SD (*P<0.05 vs. untreated control, n=3). DMSO, dimethyl sulfoxide; AE, aloe-emodin; SD, standard deviation.

Figure 4.

Aloe-emodin inhibits (A) AKT-glycogen synthase kinase 3β and (B) extracellular-signal regulated kinase-ribosomal S6 kinase activity. Western blot analysis of TE1 cells exposed to increasing concentrations of aloe-emodin was performed. Representative data of three independent experiments are shown. DMSO, dimethyl sulfoxide; ERK, extracellular-signal regulated kinase; p-, phosphorylated; CREB, cyclic adenosine monophosphate response element-binding protein; RSK, ribosomal S6 kinase; GSK3β, glycogen synthase kinase.

Figure 5.

Aloe-emodin effects on the cell cycle. (A) Aloe-emodin significantly decreased the number of TE1 cells in S phase (*P<0.05 vs. untreated, n=3). (B) Aloe-emodin significantly inhibited cyclin D1 transcription activity in TE1 cells in a dose-dependent manner (*P<0.05 vs. untreated, n=3). DMSO, dimethyl sulfoxide; Luc, luciferase.