Studies on the Mechanisms of Apple Polyphenol (Phloretin)-Induced Apoptosis in Human Breast Cancer Cells

Malignant cells are known to have an accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is the first rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose transport in malignant cells has been associated with increased and deregulated expression of glucose transporter proteins with overexpression of GLUT in breast cancer cells, a characteristic feature. The experiments in this study evaluate a novel therapeutic stratagem using a glucose-transporter inhibitor, which blocks the uptake of glucose in breast cancer cells. To test whether our hypothesis was valid, 10 cases of human breast cancer and normal tissues were separately dissected by Laser Capture Microdissection (LCM), and the expression level of type 2 GLUT (GLUT-2) in human normal and tumor tissues were determined by real-time PCR analysis. Our results revealed that the expression of GLUT-2 was higher in tumor tissues (~98.8-fold) than in normal tissues (Fig. 1). Animal studies also demonstrated that inhibition of glucose uptake could be detected in vivo by micro PET technique and the results show that 18F-FDG absorption was nearly completely inhibited in GLUT-2 inhibitor (Phloretin, Ph)-treated tumors (Fig. 6,7). Our preliminary studies indicate that GLUT can act as a “molecular target” for designing novel anticancer agents. Year-1 proposal: Characterization of the GLUT-2 phenotypes and its biological roles in human breast cancer tissues in Taiwan Aim 1: Quantitative analysis of GLUT-2 expression levels by the LCM/real time PCR technique in human breast cancer tissues: LCM and real-time PCR analysis for GLUT-2 expression in human breast cancer tissues have been performed for more than 10 cases. Additional cases will be accumulated to more than 250 cases in which the clinical relevance of the GLUT-2 expression and its role in human breast cancer will be clearly investigated. Two breast cancer cell lines including MDAMB231 (ER -/-) and MCF-7 (ER +/+) were selected to see the factors of the estrogen receptor and its role involved in GLUT-2-mediated cancer cell proliferation. GLUT-2 knock-down (by SiRNA) and overexpressing (by tet-Off adenovirus) cell lines were also established in our laboratory to investigate their biological roles of GLUT-2 in cancer cell proliferation and its role in response to its specific inhibitors. Aim 2: Evaluations for the antitumor activities of a GLUT-2 specific inhibitor in a human breast cancer cell-xenografted tumor model: The human breast cancer cell-xenografted tumor model was established in immunodeficient SCID mice (Fig. 6). Our results revealed that a GLUT-2 specific inhibitor (Ph) has significant antitumor effects in SCID mice through inhibition of the glucose uptake as evidenced by micro PET (18F-FDG) absorption analysis (Fig. 7). Such results imply that GLUT-2 may have some significant role in human breast cancer growth in vivo. In this proposal, the GLUT-2 specific inhibitor was adapted as a molecular targeting agent. However, the best protocols for drug administration are still being established. The toxicity of the GLUT-2 inhibitors also needs to be carefully investigated. Year-2 proposal: Studies on the molecular mechanisms of GLUT-2 inhibitor-mediated antitumor effects Aim 1: Studies on the mechanisms of Ph-induced cell cycle regulation in human breast cancer 1. Establishment of GLUT-2 knock-down (SiRNA) and overexpressing (tet-Off) cell lines: all of these cell lines were established in our preliminary results (see the text of proposal). 2. In vitro studies of the cell cycle regulatory proteins treated by Ph in human breast cancer cells: Our preliminary results demonstrated that MCF-7 cells were more sensitive to Ph in the absence of glucose in culture medium (Fig. 3A). However, Ph-induced apoptosis in MCF-7 cells was completely attenuated when additional glucose was added (Fig. 3B). G2/M cell cycle arrest instead of apoptosis was observed when the Ph-treated cells were rescued from high concentrations of glucose (Fig. 3B). The G2/M phase cell cycle regulatory mechanisms will be investigated to study Ph-induced cell growth arrest effects. The roles of GLUT-2 involved in cell growth signaling pathways will also be studied by using the GLUT-2 knock-down and overexpressing MC-7 cells. 3. In vivo studies of the cell cycle regulatory proteins in Ph-treated tumors: The GLUT-2 knock-down and overexpressing MCF-7-xenografted tumors dissected from Ph-treated SCID mice will be isolated for analysis of the cell cycle regulatory proteins. Such results will unveil the significance of GLUT-2-mediated cell survival signals and the in vivo mechanisms of GLUT-2 inhibitor-mediated antitumor effects. Aim 2: Studies on the mechanisms of Ph-induced cell apoptosis mechanisms in human breast cancer cells 1. Studies on the mechanisms of Ph-induced apoptosis in breast cancer cells: Our preliminary results have revealed that GLUT-2 inhibitors could induce breast cell apoptosis. However, glucose presence in the cultured medium protected cells from the apoptosis-inducing effects (Fig. 2~3). Such results revealed that the GLUT-2 receptor may act as a molecular target to be applied in cancer chemotherapy. However, the molecular mechanisms of the GLUT-2 inhibitor-mediated apoptosis are still uncertain. In addition, how glucose links the survival signals through GLUT-2, which eventually results in cell survival, will also be illustrated. The GLUT-2 knock-down and overexpressing cell lines may be used to define the role of GLUT-2 in response to Ph-induced apoptosis. 2. In vivo observations of the apoptosis-signaling proteins in Ph-treated tumors: As described above, the GLUT-2 knock-down and overexpressing breast cancer cell-xenografted tumors will be isolated for analysis of the apoptosis regulatory proteins. Such results will unveil the significance of GLUT-2-mediated cell death signals and the in vivo mechanisms of GLUT-2 inhibitor-mediated antitumor effects. Year-3 proposal: Clinical application of GLUT-2 inhibitors for antitumor activity in human breast cancer Aim 1: Evaluation of the antitumor efficacy by combined treatment of Ph with clinically used anticancer agents: Our results revealed that combined treatment with a lower concentration of Ph (50 μM) plus Paclitaxel (10 nM) significantly potentiates apoptosis induction in MCF-7 cells (Fig. 8). In vivo tumor models further support such observations (Fig. 10). Such results imply that the combination of GLUT-2 inhibitors with clinically used anticancer agents may have significant benefits for breast cancer therapy. Our proposal requires further investigation of administration protocols for combination therapy and improved safety in an in vivo experimental model. Aim 2: Evaluation of GLUT-2 inhibitors used for cancer chemopreventive purposes: Our preliminary studies have revealed that Ph can be used as a candidate for anticancer activity through specific inhibition of the GLUT-2 function in human breast cancer. To our knowledge, the Ph has been isolated from apple juice and exists in other fruits and vegetables as a natural product. Such results promote us to ask whether GLUT-2 or other Glu-1, 4 inhibitors occur in natural products (such as Genestein exist in fruits), which could be used as chemopreventive agents for cancer. We have demonstrated that combined treatment of two GLUT-2 inhibitors potentiate apoptosis induction in MCF-7 cells (Fig. 5). In Year 3 of this proposal, in vitro and in vivo experiment models will be utilized to investigate whether combination of two other types of GLUT-2 inhibitors (such as Genestein, T-1095，Phloridzin，SB203580…) could potentiate the antitumor effects against a human breast cancer.