Phloretin induces apoptosis of human esophageal cancer via a mitochondria-dependent pathway

Abstract

2,4,6-trihydroxy-3-(4-hydroxyphenyl)-propiophenone (phloretin) is found in apple tree leaves and the Manchurian apricot, and is a potent compound that exhibits anti-inflammatory, antioxidant and antitumor activities. However, the effect of phloretin on esophageal cancer cells is not well-defined. The present study aimed to examine whether and how phloretin induced apoptosis in human esophageal cancer cells. EC-109 cells were cultured in Dulbecco's modified Eagle's medium and incubated with 60, 70, 80, 90 and 100 µg/ml phloretin for 6, 12, 24 and 48 h. Cell proliferation was measured by an MTT assay. Cell apoptosis rate was measured using flow cytometric analysis subsequent to propidium iodide (PI) staining. The protein expression levels were determined by western blot analysis. It was found that phloretin significantly decreased viable cell numbers in a dose- and time-dependent manner and induced apoptosis in EC-109 cells. Additionally, phloretin exhibited potent anticancer activity in vitro, as evidenced by the downregulation of the anti-apoptosis-associated molecule B-cell lymphoma 2 (bcl-2) and an increase in the levels of the apoptosis-associated molecules bcl-2-like protein 4 and tumor protein p53. Phloretin treatment also affected the expression of apoptotic protease activating factor-1, the protein product of the direct binding of the inhibitor of apoptosis protein with low PI to the X-linked inhibitor of apoptosis protein. The present results indicated that phloretin may inhibit EC-109 cell growth by inducing apoptosis, which may be mediated through a mitochondria-dependent pathway.

Keywords: apoptosis; mitochondria-dependent pathway; phloretin.

Figure 1.

The effect of phloretin, a fruit tree extract, on cell viability. (A) Chemical structure of phloretin. (B) Survival rate of EC-109 cells when treated with phloretin at concentrations ranging from 60–100 µg/ml for 6, 12, 24 and 48 h. The cell viability was quantified using an MTT assay.

Figure 2.

Flow cytometry analysis and histograms. Cell cycle distribution profiles are included for (A) the Ct group and cells treated with phloretin at (B) 60, (C) 70 and (D) 80 µg/ml for 12 h. (E) A bar graph demonstrating the rate of apoptosis using flow cytometry analysis. Data represent the mean ± standard deviation of 3 independent experiments. *P<0.05 between indicated groups, unpaired Student's t-test. FITC-A, fluorescein isothiocyanate-A; PI-A, propidium iodide-A, Ct, control.

Figure 3.

The relative expression level of cells treated with phloretin, as determined by western blot and relative quantification of (A) Bax and (B) Bcl-2. Data represent the mean ± standard deviation of three independent experiments. *P<0.05 between indicated groups, unpaired Student's t-test. Ct, control; Bax, Bcl-2-associated X protein.

Figure 4.

The relative p53 expression level of cells treated with phloretin. (A) Western blotting was used to detect p53. (B) The western blot image was then used for relative quantification. *P<0.05 between indicated groups, unpaired Student's t-test. Ct, control.

Figure 5.

The relative expression level of cells treated with phloretin, as determined by western blot and relative quantification of (A) Smac/DIABLO, (B) APAF-1 and (C) XIAP. *P<0.05 between indicated groups, unpaired Student's t-test. Ct, control; DIABLO (Smac/DIABLO), direct binding protein with low PI; APAF-1, apoptotic protease activating factor 1; XIAP, X-linked inhibitor of apoptosis protein.