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. 2018 Jan;26(1):221-231.
doi: 10.1016/j.jfda.2017.03.009. Epub 2017 Apr 18.

The apple polyphenol phloretin inhibits breast cancer cell migration and proliferation via inhibition of signals by type 2 glucose transporter

Kuan-Hsun Wu  1   2   3 , Chi-Tang Ho  4 , Zhao-Feng Chen  5 , Li-Ching Chen  6   7   8   9 , Jacqueline Whang-Peng  6   9 , Teng-Nan Lin  10 , Yuan-Soon Ho  11   12   5   6
Affiliations

The apple polyphenol phloretin inhibits breast cancer cell migration and proliferation via inhibition of signals by type 2 glucose transporter

Kuan-Hsun Wu et al. J Food Drug Anal. 2018 Jan.

Abstract

Human triple-negative breast cancer (TNBC) is the most aggressive and poorly understood subclass of breast cancer. Glucose transporters (GLUTs) are required for glucose uptake in malignant cancer cells and are ideal targets for cancer therapy. To determine whether the inhibition of GLUTs could be used in TNBC cell therapy, the apple polyphenol phloretin (Ph) was used as a specific antagonist of GLUT2 protein function in human TNBC cells. Interestingly, we found that Ph (10-150 μM, for 24 h) inhibited cell growth and arrested the cell cycle in MDA-MB-231 cells in a p53 mutant-dependent manner, which was confirmed by pre-treatment of the cells with a p53-specific dominant-negative expression vector. We also found that Ph treatment (10-150 μM, for 24 h) significantly decreased the migratory activity of the MDA-MB-231 cells through the inhibition of paxillin/FAK, Src, and alpha smooth muscle actin (α-sMA) and through the activation of E-cadherin. Furthermore, the anti-tumorigenic effect of Ph (10, 50 mg/kg or DMSO twice a week for six weeks) was demonstrated in vivo using BALB/c nude mice bearing MDA-MB-231 tumor xenografts VSports手机版. A decrease in N-cadherin, vimentin and an increase in p53, p21 and E-cadherin were detected in the tumor tissues. In conclusion, inhibition of GLUT2 by the apple polyphenol Ph could potentially suppress TNBC tumor cell growth and metastasis. .

Keywords: Apple polyphenol; Breast cancer; Glucose transporter 2; Phloretin; Triple-negative breast cancer V体育安卓版. .

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Conflict of interest statement

Disclosure of potential conflicts of interest

The authors declare that no financial competing interests or financial relationships exist with other people or organizations involved in this study.

Figures (V体育ios版)

Fig. 1
Fig. 1
Ph-induced human TNBC cell growth. (A) Human breast TNBC cancer (MDA-MB-231) cells were treated with 25–150 μM Ph for 24 h. Some cells were also treated with 0.05% DMSO as a control. The gross morphology of the cells was determined, and the scale bar represents 200 μm. (B) The human breast TNBC cancer (MDA-MB-231) and normal (MCF-10A) cells were treated with 25–150 μM Ph for 24 h. Cells were also treated with 0.05% DMSO as a control. MTT assays were performed, and the results were observed for 1–5 days; *p < 0.05. (C) The Ph-induced cytotoxic effects were determined by trypan blue exclusion assay. The results are presented as percentage of the control; *p < 0.05. All results presented above were repeated at least three times.
Fig. 2
Fig. 2
Effects of Ph-induced G0/G1 phase cell cycle arrest in TNBC cancer cells. (A) Flow cytometry (FACS) analysis of DNA was conducted after MDA-MB-231 cells were synchronized by 0.04% FCS for 24 h and then switched to culture media supplemented with 10% FBS containing 0.05% DMSO (control) or Ph (10–150 μM in 0.05% DMSO) for an additional 16 h. The lower panel shows the flow cytometry chart of all these cells. The data were analyzed by nonparametric two-sided tests (Kruskal–Wallis and Mann–Whitney tests, *p = 0.05 for all comparisons). (B) The MDA-MB-231 cells were treated with or without Ph (10–150 μM) for 16 h. The protein levels of GLUT2, p21/Cip1, p27/Kip1, cyclin E1, cyclin D1, and p53 were determined by immunoblotting analysis. In each case, GADPH expression served as a control. (C) The MDA-MB-231 cells were pre-treated with a p53-specific dominant-negative expression vector (indicated as p53DN cells) for 24 h. Both the MDA-MB-231 and p53DN cells were treated with Ph (50–100 μM) for 16 h, and the protein was harvested for detection of p53 protein by immunoblotting analysis. Membranes were also probed with anti-GADPH antibodies to correct for differences in protein loading. All results presented above were repeated at least three times, and representative results are shown.
Fig. 3
Fig. 3
The mechanisms of Ph-induced TNBC cell migration. (A) The MDA-MB-231 tumor cells were treated with Ph (25–100 μM) for 24 h, and tumor cell growth and migration was calculated by the wound-healing method. The data were analyzed by paired t-tests. The mean value determined from the DMSO-treated cells was significantly different from that of Ph-treated cells (*p = 0.05 for all comparisons). (B) Total protein from the Ph-treated cells was harvested for detection of proteins related to cell migration by immunoblotting analysis. Membranes were also probed with anti-GADPH antibodies to correct for differences in protein loading. All results presented above were repeated at least three times, and representative results are shown.
Fig. 4
Fig. 4
The Ph-induced in vivo anti-tumor effects in MDA-MB-231-xenografted tumors. (A) The in vivo anti-tumor effect of Ph was evaluated by treating mice bearing MDA-MB-231 tumor xenografts. After transplantation, tumor size was measured using calipers, and tumor volume was estimated according to the following formula: tumor volume (mm3) = L × W2/2, where L is the length and W is the width. Once the tumors reached a mean volume of 200 mm3, the animals received intraperitoneal injections of DMSO or Ph at 10 mg/kg, or 50 mg/kg three times per week for 6 weeks. All animal studies were performed according to the local guidelines for animal care and protection. (B) After the mice were sacrificed, protein lysates were isolated from the MDA-MB-231 xenograft tumors, and proteins related to cell growth, arrest or migration were detected by immunoblotting analysis. Membranes were also probed with anti-GADPH antibodies to correct for differences in protein loading. All results presented above were repeated at least three times, and representative results are shown.
Fig. 5
Fig. 5
The molecular mechanisms underlying the Ph-induced anti-tumor effects in human MDA-MB-231 cancer cells. In response to Ph treatment, cell glucose uptake was suppressed through GLUT2. Subsequently, low concentrations of intracellular glucose transcriptionally activated HNF6 protein expression as described in our previous paper [5]. The overexpressed HNF6 was reported to be a transcription factor [56] that induced GLUT2 compensatory upregulation in response to glucose deprivation (shown in Fig. 2B). HNF6 also acts as a transcription factor [57] to induce p53-mediated signals and trigger G0/G1 cell cycle arrest, which then caused the in vivo anti-tumor effects in the MDA-MB-231 xenograft mice (right panel). In this study, we also found that Ph could inhibit Paxillin/FAK, N-cadherin, and α-SMA-mediated migratory signals in the tumor-bearing mice treated with Ph. Our results indicated that apple polyphenol Ph could be an important chemopreventive or therapeutic agent for TNBC breast cancer patients.
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References

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