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Glut-1 antibodies induce growth arrest and apoptosis in human cancer cell lines
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Cancer Letters 257 (2007) 244–251 www.elsevier.com/locate/canlet
Glut-1 antibodies induce growth arrest and apoptosis in human cancer cell lines Shipra Rastogi a, Sarmistha Banerjee a,b, Srikumar Chellappan a, George R. Simon b,* a
b
Drug Discovery Program, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, MRC-4W, Tampa, FL 33612, United States Thoracic Oncology Program, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, MRC-4W, Tampa, FL 33612, United States Received 11 October 2006; received in revised form 24 July 2007; accepted 26 July 2007
Abstract Glucose transporters (Gluts) facilitate glucose uptake and tumors frequently over express the Gluts, especially Glut-1. Breast cancer and lung cancer (NSCLC) cell lines were incubated with anti-Glut-1 antibodies alone or with cisplatin, paclitaxel or gefitinib. Inhibition of proliferation and apoptosis was assessed. Antibodies to Glut-1 inhibited proliferation by 50% and 75% in the tested NSCLC and breast cancer cell lines, respectively. They also potentiate the anti-proliferative effects of cisplatin, paclitaxel and gefitinib. Our results indicate that anti-Glut-1 antibodies inhibit proliferation and induce apoptosis in the evaluated cell lines and provide preliminary evidence of their anti-tumor activity. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Anti-Glut antibodies; Lung cancer; Breast cancer; Glucose uptake; Glut-1
1. Introduction Many human cancers display a high rate of aerobic glycolysis [1]. 18-F-2-Deoxyglucose positron emission tomography (FDG-PET) is a noninvasive diagnostic and prognostic tool that measures tumor metabolism and makes imaging of primary and metastatic tumors possible by taking advantage of this phenomenon [2]. Up regulation of glycolytic metabolism occurs downstream of multiple oncogenic * Corresponding author. Tel.: +1 813 972 8372; fax: +1 813 903 6875. E-mail address: george.simon@moffitt.org (G.R. Simon).
pathways [3,4]. Metabolism of glucose via the glycolytic pathway not only provide ATPs to meet the tumor’s energy demands but may also provide precursors and reducing equivalents for the synthesis of macromolecules, such as nucleotides, proteins, and lipids. Hence, the acquisition of the glycolytic phenotype plays a pivotal role in tumor growth and survival and has been shown to correlate with increased tumor aggressiveness and poor patient prognosis in several tumor types [5]. Glucose utilization by cancer cells is therefore greatly enhanced when compared to normal or benign tissues. Glucose is taken up by cells and then phosphorylated to glucose-6-phosphate. Facilitative
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.07.021
S. Rastogi et al. / Cancer Letters 257 (2007) 244–251
glucose uptake is achieved by five transmembrane transporters, termed Glut1–5, which are protein products of their respective GLUT genes. The Glut transporters differ in their kinetics and are tailored to the requirements of the cell type they serve. Although more than one Glut may be expressed by a particular cell type, tumors frequently over express Glut-1, which is a high affinity glucose transporter [6]. The relationships between FDG uptake and the expression of facilitative glucose transporters have been previously evaluated. FDG uptake and the expression of five glucose transporters and the proportions of proliferating cell and macrophage populations were studied in paraffin sections from surgically resected primary non-small cell lung cancers (NSCLC) by immunohistochemistry (IHC). The patients were imaged with FDG-PET before surgery. Glut-1 expression was significantly higher than that of any other glucose transporters. All tumors tested (n = 23) were Glut-1-positive (70.8 ± 26.1% of tumor cell area was positive and staining intensity was 2.8 ± 1.2) and Glut-1 was the major glucose transporter expressed. Both FDG uptake and Glut-1 expression appear to be associated with increased tumor size [6]. Autoradiographic studies of excised tumors demonstrate increased FDG uptake in viable cells near necrotic portions of tumor. In conclusion, human tumor cell lines, in response to hypoxia, increase glucose uptake by up regulating membranous expression of the Glut-1 glucose transporter. The ability to survive periods of hypoxia confers tumors with an aggressive malignant phenotype enabling it to be resistant to both chemotherapy and radiotherapy and consequently poor overall survival. In several tumors including, NSCLC, colon cancer, bladder cancer, breast cancer and thyroid cancers, increased Glut-1 expression not only confers a malignant phenotype but also predicts for inferior overall survival [7–9]. We postulated that anti-Glut-1 antibodies would disrupt Glut-1 transporter function and consequently, not only lead to decreased tumor growth but also render it sensitive to chemotherapy and radiotherapy. This is presumably achieved by inhibiting intra-tumoral glucose uptake. In the experiments outlined below, we show that antibodies to Glut-1 decrease proliferation and induce apoptosis in NSCLC and breast cancer cell lines by decreasing glucose uptake. This work lays the foundation for future in vivo studies.
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2. Materials and methods 2.1. Cell culture Breast carcinoma cell lines MCF-7, T47D, p53 negative osteosarcoma cell line Saos-2 and NSCLC cell line H1299 were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). SV40-transformed human lung fibroblast WI-38-VA13 was obtained from ATCC and cultured in Minimal essential medium eagle with Earle’s salt and L-glutamine containing 10% FBS, sodium pyruvate, and non-essential amino acids. NSCLC cell lines A549, H358, H226, H1650 were grown in Ham’s F12K containing 10% FBS. Human microvascular endothelial cells of lung (HMEC-L) and human aortic endothelial cells (HAEC) were grown in EGM bullet kit medium containing 5% FBS from Clonetics. 2.2. Cell lysate preparation and Western blot Lysates from cells were prepared by NP40 lyses. Samples were boiled in equal volume of 2· SDS sample buffer, and separated on 8% polyacrylamide gels. After semi-dry transfer to supported nitrocellulose membranes, the blots were probed with monoclonal antibody to Glut-1 from R&D systems. The proteins were detected by using an enhanced chemiluminescence assay system from Amersham Biosciences. 2.3. Immunofluorescence Glut-1 monoclonal antibody was purchased from R&D Systems Inc (Minneapolis, MN). Cells were plated onto poly-D-lysine (Sigma) coated 8-well glass chamber slides (10,000 cells per well) for immunostaining. Cells were fixed in 3.5% paraformaldehyde for 25 min, permeabilized in 0.2% Triton X-100/PBS for 5 min, and blocked in 5% normal goat serum in PBS at room temperature for 1 h. Primary antibody incubation was performed overnight at 4 C. After washing, secondary antibody incubation was performed with goat anti-mouse IgG Alexa Fluor-488 for 30 min at room temperature. DAPI was detected using Vectashield Mounting Medium with DAPI (Vector Laboratories, Inc.). Control experiments demonstrated that there was no detectable staining by secondary antibodies only (data not shown). Slides were observed by fluorescence microscopy using Leica DM LB2 microscope (40·/0.75 numerical aperture) with a Qimaging Retiga1300 camera. 2.4. MTT assay MTT assays were performed by the following wellestablished method. In a 96 well tissue culture plate 10,000 cells were plated in each well. The cells were
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incubated in presence or absence of Glut-1 antibody for 18 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in PBS (10 mg/ml) and filter sterilized. Three hours before the end of the incubation 20 ll of MTT solution was added to each well containing cells in 96 well plates. The plate was incubated in an incubator at 37 C for 3 h. Media was aspirated gently and 200 ll of DMSO was added to each well to dissolve formazan crystals. The absorbance was measured at 550 nm. 2.5. Proliferation assay MCF-7, H1299 and H1650 cells were plated onto poly(Sigma) coated 8-well glass chamber slides (10,000 cells per well). The cells were incubated with 0.1 mg/ml Glut-1 monoclonal antibody for 18 h. The cells were fixed and stained using 5-Bromo-2 0 -deoxyuridine labeling and Detection kit from Roche according to manufacturer’s protocol. D-lysine
2.6. Apoptosis assay MCF-7, H1299 and H1650 cells were plated onto poly(Sigma) coated 8-well glass chamber slides (10,000 cells per well). The cells incubated with 0.1 mg/ ml Glut-1 monoclonal antibody for 18 h served as controls. The cooperative effect of drugs was evaluated by adding 5 lM of cisplatin or paclitaxel or 10 lM gefitinib. After 18 h of incubation cells were fixed and stained according to manufacturer’s instructions using Promega’s DeadEnd Colorimetric TUNEL system. D-lysine
2.7. Glucose uptake assay Cellular glucose uptake was measured by incubating cells in glucose-free RPMI 1640 with 0.2 Ci/mL [3H]2deoxyglucose (specific activity, 40 Ci/mmol) for 60 min. After the cells were washed with ice-cold PBS, the radioactivity in the cell pellets was quantified by liquid scintillation counting. 2.8. Statistical analyses Unless other wise specified experiments were done in triplicate. Error bars were generated based on the 95% confidence intervals obtained from these experiments. 3. Results 3.1. Expression levels of Glut-1 in transformed and primary cell lines
Fig. 1. Glut-1 expression in different cell lines. Cell lysates were prepared from various cell lines and 100 lg protein was electrophoresed and blotted on nitrocellulose membrane. The lysates were boiled in 20 ll of SDS sample buffer and separated on 8% polyacrylamide gel. After semi-dry transfer to supported nitrocellulose membranes, the blots were probed with the Glut-1 monoclonal antibody. The proteins were detected by using an enhanced chemiluminescence assay system from Amersham. The blot revealed increased expression of Glut-1 in H1299, H1650, MCF-7 and T47D.
and H1650 as well as primary cell line HMEC-L. MCF-7 and T47D breast cancer cell lines were also evaluated. Additionally we analyzed Jurkats, U937, Saos-2, WI38VA13 and HAEC for expression of Glut-1 protein by Western blot. As depicted in Fig. 1, H1299, H1650, MCF-7, T47D and Jurkats show comparatively higher expression of Glut-1. At the same time, Glut-1 levels appeared to be low in U937, H226 and A549 cells. Actin levels were comparable across the cell lines, suggesting that observed differences in the levels of Glut-1 are genuine. 3.2. Effect of Glut-1 antibody on cell proliferation Next we attempted to assess the ability of monoclonal Glut-1 antibody to affect cell proliferation. We therefore incubated H1299, H1650, T47D and MCF-7 cell lines in presence of different dilutions of the monoclonal antibody for 18 h. Similar dilutions of a non-specific IgG1 isotype antibody were used as the control. An MTT assay revealed that 0.1 mg/ml of the antibody was able to repress the proliferation of these cell lines by at least 50% (Data not shown). These results were further confirmed by BrdU proliferation assay. H1299, H1650, T47D and MCF-7 Cells were cultured on 8-well chamber slides and incubated with 0.1 mg/ml monoclonal Glut-1 antibody for 18 h. A concentration of 0.1 mg/ml IgG1 antibody was used as control. As shown in Fig. 2, the treatment with the Glut-1 antibody led to 45–70% decrease in the proliferation of all the four cell lines tested, suggesting that incubation with this antibody can inhibit cell proliferation. 3.3. Localization of Glut-1 receptor
Glut-1 is the most widely expressed isoform of the Glut family that provides cells with their basic glucose requirement. We examined levels of Glut-1 in various cell lines including NSCLC cell lines A549, H1299, H358, H226
An immunofluorescence experiment was performed to confirm the ability of antibody to bind the Glut-1 receptor. MCF-7 and H1650 cells were plated on chamber
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uptake may be enough to reduce proliferation and induce apoptosis in aggressive cancer cell lines who require high levels of glucose to meet their energy needs. Additionally, by immunostaining the predominantly membranous localization of the Glut-1 receptor is demonstrated in the breast cancer cell lines in comparison to the primary breast cell line MCF10A, which is particularly evident in the MCF-7 and T47D cell lines. Please see Fig. 4. 3.4. Glut-1 enhances the inhibition of cell proliferation by chemotherapeutic drugs
Fig. 2. H1299, H1650, T47D and MCF-7 cells were grown on poly-D-lysine coated chamber slide in presence or absence of 0.1 mg/ml Glut-1 antibody or IgG1 control isotype antibody and BrdU assay was performed. The results show 40–70% decrease in BrdU incorporation in these cell lines.
slides. After washing the cells were fixed and immunostained with the Glut-1 monoclonal antibody. The cells were then visualized with secondary antibody conjugated to Alexafluor-488. As shown in Fig. 3 staining for Glut-1 is visible in the cytoplasm and confocal microscopy confirmed the localization of the Glut-1 transporter to the cell membrane. This suggests that transporter is present on the cell surface and implies that anti-Glut-1 antibody prevents cell proliferation by altering Glut-1 transporter’s function, presumably by inducing conformational changes in Glut-1 transporter function. In Fig. 3, we show that the MDAMB231 breast cancer cell line demonstrate a 10–50% reduction in glucose uptake when incubated with the anti-Glut-1 antibody. This reduction of glucose
Experiments were done to evaluate whether anti-Glut1 antibody could synergize with standard chemotherapeutic agents to inhibit proliferation of H1299, H1650 and MCF-7. The cells were plated on chamber slides and incubated in presence of chemotherapeutic agents(5 lM cisplatin, 5 lM paclitaxel or 10 lM geftinib) in absence or presence of 0.1 mg/ml anti-Glut-1 antibody. The treatment with drugs led to significant decrease in proliferation of all the three cancer cell lines, however, addition of Glut-1 antibody led to a greater reduction in cell proliferation in NSCLC cell lines H1299 and H1650, and breast cancer cell line MCF-7 (Fig. 4). In the H1299 line, treatment with the anti-Glut-1 antibody alone inhibited proliferation by 62%; when added to cisplatin it enhanced cisplatin-induced inhibition of proliferation by 62%, paclitaxel by 74% and gefitinib by 42%. Similarly in the H1650 cell line treatment with anti-Glut-1 antibody alone inhibited proliferation by 55%; when added to cisplatin, it enhanced cisplatin induced inhibition of proliferation by 18%; paclitaxel by 23% and gefitinib by 46%. In the MCF-7 cell line, anti-Glut-1 antibody alone inhibited proliferation by 59%, when added to cisplatin it enhanced cisplatin-induced inhibition of proliferation by 40%, paclitaxel by 47% and gefitinib by 59%. The results are shown in Fig. 5.
65 60
3.5. Glut-1 enhances the apoptosis by chemotherpeutic drugs
Specific Activity
55 50 45 40 35 30 25 20 Control IgG
Glut1 IgG
Fig. 3. Results of the glucose uptake assay in MDA-MB 231 cells treated with an anti-Glut-1 antibody for 72 h. Error bars were generated from two independent experiments. Treatment with Glut-1 antibody demonstrates an inhibition of glucose uptake in comparison to a control IgG1 antibody.
Since the above drugs are known to be strong inducers of apoptosis, attempts were made to assess whether antiGlut-1 antibody synergizes with them to induce apoptosis. NSCLC cell lines, H1299 and H1650, and breast cancer cell line, MCF-7 were evaluated for apoptosis by the TUNEL assay, after treatment with 5 lM cisplatin, 5 lM paclitaxel or 10 lM geftinib alone, or with antiGlut-1 antibodies. Glut-1 antibodies enhanced the apoptotic effects of cisplatin, paclitaxel and gefitinib (Fig. 6) in H1650 cell line by 43%, 62% and 111%; in H1299 by 111%, 30% and 71% and in MCF-7 cell line by 37%, 91% and 133%, respectively. Induction of apoptosis was assessed by measuring PARP cleavage. PARP cleavage was assessed by Western
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Fig. 4. In the breast cancer cell lines MCF-7, T47D, and MDAMB231, Glut-1 is cytoplasmic and localized to the cell membrane. However, in the primary breast cell line MCF10A, the localization of Glut-1 appears to be diffusely cytoplasmic with no particular predilection to be membranous.
blotting of lysates from MCF-7 and H1650 cells treated with paclitaxel alone or after co-incubation with 0.1 mg/ ml of anti-Glut-1 antibody for 18 h. Results of these experiments demonstrate that the combined treatment of cells with paclitaxel and Glut-1 antibody lead to enhanced apoptosis in comparison to paclitaxel alone (Fig. 7). In summary, our results indicate that anti-Glut-1 antibodies inhibit proliferation and induce apoptosis in the evaluated NSCLC cell lines and breast cancer cell lines providing evidence that the use of antibodies to Glut-1 may be a viable but an as yet unexplored therapeutic strategy in tumors that over express Glut-1 and consequently demonstrate increased glucose uptake in FDG-PET.
4. Discussion We demonstrate here that tumors’ dependence on glucose can be exploited for therapeutic gain by using anti-Glut antibodies The proposed mechanism is the reduction of glucose uptake by altering the function of the high affinity glucose transporter Glut-1. Since tumors preferentially up regulate Glut-1 over other members of the Glut family of transporters, we used a monoclonal antibody to Glut-1 in the experiments detailed above. We demonstrate that anti-Glut-1 antibodies not only decrease proliferation in the breast cancer and
NSCLC cell lines tested but also enhance chemotherapy-induced apoptosis. Other investigators have also explored similar strategies. Aft et al. investigated the effects of the anti-metabolite 2-deoxy-D-glucose (2-DG) on breast cancer cells in vitro [10]. This compound has been shown to inhibit glucose metabolism. Treatment of human breast cancer cell lines with 2-DG resulted in a decrease of cell growth in a dose dependent manner. The cell death induced by 2-DG was due to apoptosis as demonstrated by induction of caspase 3 activity and PARP cleavage. In response to exposure to 2-DG, breast cancer cells expressed higher levels of Glut-1 transporter protein and consequently increased its glucose uptake when compared to non-treated breast cancer cells. These data suggest that breast cancer cells treated with 2-DG, owing to the perceived deprivation of glucose, respond by expressing higher levels of glucose transporter proteins, which allows for further increased uptake of 2-DG, and subsequent induction of cell death. Decreasing GLUT-1 expression as a means of abrogating tumor growth was investigated by Noguchi et al. [11,12]. Noguchi et al. transfected MKN45 cells with cDNA for anti-sense GLUT-1.
S. Rastogi et al. / Cancer Letters 257 (2007) 244–251
Fig. 5. Combined effect of chemotherapeutic drugs and Glut-1 antibody on cell proliferation. H1299, H1650, and MCF-7 cells were grown on chamber slides as described above and treated either with cisplatin, paclitaxel or iressa in presence or absence of Glut-1 antibody (0.1 mg) for 18 h. The proliferation was measured by BrdU incorporation. The results show that combination of chemotherapeutic drugs with the Glut-1 antibody further diminishes the cell proliferation.
Glucose transport was significantly decreased in cells with anti-sense GLUT-1 compared with wildtype cells or cells with vector alone. Suppression of GLUT-1 mRNA resulted in a decreased number of cells in the S phase. This was accompanied by over expression of p21 protein. Tumorigenicity in the nude mice injected with anti-sense GLUT-1 expressing cells was significantly slower than in those with wild-type MKN45 cells. These results corroborate our data. Differential expression of glucose transporters, particularly Glut-1, in normal and pathologic tissue
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Fig. 6. Combined effect of chemotherapeutic drugs and Glut-1 antibody on cell apoptosis. H1299, H1650, and MCF-7 cells were grown on chamber slides as described above and treated either with cisplatin, paclitaxel or iressa in presence or absence of Glut-1 antibody (0.1 mg) for 18 h. The apoptosis was measured by TUNEL assay. The results demonstrate that the combination of chemotherapeutic drugs with the Glut-1 antibody results in additive effect on cell apoptosis.
is critical to enable the potential therapeutic utility if anti-Glut-1 antibodies in clinic. Suganuma et al. examined 71 kidney surgical samples using reverse transcriptase-polymerase chain reaction (RT-PCR) for GLUT1–14 in normal and tumor tissues. The expression levels for GLUT1–5, 9, 10 and 12 were quantified by real-time quantitative PCR. The RTPCR results showed that normal kidney tissue expresses all the GLUT isoforms. In clear cell carcinoma of the kidney, GLUT-1 expression increased (p < 0.001) while GLUT4, 9 and 12 decreased (p < 0.001) [13]. In a similar study conducted in thyroid tissue, Matsuzu et al. examined the expression
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Fig. 7. MCF-7 and H1650 cells were treated with 20 lM paclitaxel in presence or absence of Glut-1 antibody for 18 h. Apoptosis was assessed using PARP cleavage. Combined treatment of cells with paclitaxel and Glut-1 antibody leads to more potent apoptotic stimuli in comparison to paclitaxel alone.
pattern and levels of GLUT genes in normal and pathologic thyroid tissues to evaluate the clinical significance of GLUT mRNA levels. One hundred fifty-two surgically resected thyroid tissue samples from 103 patients were evaluated. Samples included normal thyroid tissue (n = 58), benign thyroid disease (n = 61), and thyroid carcinoma (n = 33). Expression of the GLUT-1, GLUT-2, GLUT-3, GLUT-4, and GLUT-10 genes were examined by RT-PCR and mRNA levels were quantitated by real-time RT-PCR. All thyroid parenchymal cells expressed GLUT-1, GLUT-3, GLUT-4, and GLUT-10. GLUT-1 showed increased expression in carcinoma cases (p < 0.0001) and also in comparison with paired normal tissue samples from the same patient (p < 0.0001). Other GLUTs were statistically unchanged in pathologic tissues. The authors concluded that the up regulation of GLUT-1 during carcinogenesis plays a major role in enhanced glucose uptake in thyroid cancer cells [14]. Since normal cell express several of the Glut isoforms and tumors have increased dependence on Glut-1, we anticipate that the treatment with Glut-1 antibody would differentially affect tumors while relatively sparing normal cells. Indeed even minor alterations in Glut-1 function may have significant effects on the tumor. Membranous up regulation of glucose transporters may not just be essential for tumor survival but may also be integrally involved in the carcinogenic process. Healthy colonocytes derive 60–70% of their energy supply from short-chain fatty acids, particularly butyrate. Butyrate is transported across the luminal membrane of the colonic epithelium via a monocarboxylate transporter, MCT1. Analysis of healthy colonic tissues and carcinomas using IHC and Western blotting by Russo et al. revealed a sig-
nificant decline in the expression of MCT1 protein during transition from normality to malignancy [15]. This was reflected in a corresponding reduction in MCT1 mRNA expression, as measured by Northern analysis. Carcinoma samples displaying reduced levels of MCT1 were found to express the high affinity glucose transporter, GLUT-1, suggesting that there is a switch from butyrate to glucose as an energy source in colonic epithelia during transition to malignancy. To the best of our knowledge this is the first paper to show that anti-Glut-1 antibodies inhibit proliferation and induce apoptosis in NSCLC cell lines and breast cancer cell lines evaluated. Furthermore, they enhance apoptosis caused by chemotherapeutic agents like cisplatin and paclitaxel and targeted agents like gefitinib. These and our data clearly indicate that Glut-1 is a legitimate targets for anti-neoplastic drug development and are worthy of further rigorous pre-clinical investigation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2007.07.021. References [1] O. Warburg, On the origin of cancer cells, Science 123 (1956) 309–314. [2] S.S. Gambhir, Molecular imaging of cancer with positron emission tomography, Nat. Rev. Cancer 2 (2002) 683–693. [3] R.L. Elstrom, D.E. Bauer, M. Buzzai, et al., Akt stimulates aerobic glycolysis in cancer cells, Cancer Res. 64 (2004) 3892–3899. [4] C.V. Dang, G.L. Semenza, Oncogenic alterations of metabolism, Trends Biochem. Sci. 24 (1999) 68–72. [5] F.C. Detterbeck, S. Falen, M.P. Rivera, J.S. Halle, M.A. Socinski, Seeking a home for a PET. Part 2: defining the appropriate place for positron emission tomography imaging in the staging of patients with suspected lung cancer, Chest 125 (2004) 2300–2308. [6] A.C. Clavo, R.S. Brown, R.L. Wahl, Fluorodeoxyglucose uptake in human cancer cell lines is increased by hypoxia, J. Nucl. Med. 36 (1995) 1625–1632. [7] M. Younes, L.V. Lechago, J.R. Somoano, M. Mosharaf, J. Lechago, Wide expression of the human erythrocyte glucose transporter Glut-1 in human cancers, Cancer Res. 56 (1996) 1164–1167. [8] M. Younes, L.V. Lechago, J. Lechago, Overexpression of the human erythrocyte glucose transporter occurs as a late event in human colorectal carcinogenesis and is associated with an increased incidence of lymph node metastases, Clin. Cancer Res. 2 (1996) 1151–1154.
S. Rastogi et al. / Cancer Letters 257 (2007) 244–251 [9] M. Younes, R.W. Brown, D.R. Mody, L. Fernandez, R. Laucirica, GLUT1 expression in human breast carcinoma: correlation with known prognostic markers, Anticancer Res. 15 (1995) 2895–2898. [10] R.L. Aft, F.W. Zhang, D. Gius, Evaluation of 2-deoxy-Dglucose as a chemotherapeutic agent: mechanism of cell death, Br. J. Cancer 87 (2002) 805–812. [11] Y. Noguchi, T. Okamoto, D. Marat, et al., Expression of facilitative glucose transporter 1 mRNA in colon cancer was not regulated by k-ras, Cancer Lett. 154 (2000) 37–142. [12] Y. Noguchi, A. Saito, Y. Miyagi, et al., Suppression of facilitative glucose transporter 1 mRNA can suppress tumor growth, Cancer Lett. 154 (2000) 175–182.
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[13] N. Suganuma, F. Segade, K. Matsuzu, D.W. Bowden, Differential expression of facilitative glucose transporters in normal and tumour kidney tissues, BJU. Int. 99 (2007) 1143– 1149. [14] K. Matsuzu, F. Segade, U. Matsuzu, A. Carter, D.W. Bowden, N.D. Perrier, Differential expression of glucose transporters in normal and pathologic thyroid tissue, Thyroid 14 (2004) 806–812. [15] V.C. Russo, K. Kobayashi, S. Najdovska, N.L. Baker, G.A. Werther, Neuronal protection from glucose deprivation via modulation of glucose transport and inhibition of apoptosis: a role for the insulin-like growth factor system, Brain Res. 1009 (2004) 40–53.
Cancer Letters 257 (2007) 244–251 www.elsevier.com/locate/canlet
Glut-1 antibodies induce growth arrest and apoptosis in human cancer cell lines Shipra Rastogi a, Sarmistha Banerjee a,b, Srikumar Chellappan a, George R. Simon b,* a
b
Drug Discovery Program, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, MRC-4W, Tampa, FL 33612, United States Thoracic Oncology Program, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, MRC-4W, Tampa, FL 33612, United States Received 11 October 2006; received in revised form 24 July 2007; accepted 26 July 2007
Abstract Glucose transporters (Gluts) facilitate glucose uptake and tumors frequently over express the Gluts, especially Glut-1. Breast cancer and lung cancer (NSCLC) cell lines were incubated with anti-Glut-1 antibodies alone or with cisplatin, paclitaxel or gefitinib. Inhibition of proliferation and apoptosis was assessed. Antibodies to Glut-1 inhibited proliferation by 50% and 75% in the tested NSCLC and breast cancer cell lines, respectively. They also potentiate the anti-proliferative effects of cisplatin, paclitaxel and gefitinib. Our results indicate that anti-Glut-1 antibodies inhibit proliferation and induce apoptosis in the evaluated cell lines and provide preliminary evidence of their anti-tumor activity. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Anti-Glut antibodies; Lung cancer; Breast cancer; Glucose uptake; Glut-1
1. Introduction Many human cancers display a high rate of aerobic glycolysis [1]. 18-F-2-Deoxyglucose positron emission tomography (FDG-PET) is a noninvasive diagnostic and prognostic tool that measures tumor metabolism and makes imaging of primary and metastatic tumors possible by taking advantage of this phenomenon [2]. Up regulation of glycolytic metabolism occurs downstream of multiple oncogenic * Corresponding author. Tel.: +1 813 972 8372; fax: +1 813 903 6875. E-mail address: george.simon@moffitt.org (G.R. Simon).
pathways [3,4]. Metabolism of glucose via the glycolytic pathway not only provide ATPs to meet the tumor’s energy demands but may also provide precursors and reducing equivalents for the synthesis of macromolecules, such as nucleotides, proteins, and lipids. Hence, the acquisition of the glycolytic phenotype plays a pivotal role in tumor growth and survival and has been shown to correlate with increased tumor aggressiveness and poor patient prognosis in several tumor types [5]. Glucose utilization by cancer cells is therefore greatly enhanced when compared to normal or benign tissues. Glucose is taken up by cells and then phosphorylated to glucose-6-phosphate. Facilitative
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.07.021
S. Rastogi et al. / Cancer Letters 257 (2007) 244–251
glucose uptake is achieved by five transmembrane transporters, termed Glut1–5, which are protein products of their respective GLUT genes. The Glut transporters differ in their kinetics and are tailored to the requirements of the cell type they serve. Although more than one Glut may be expressed by a particular cell type, tumors frequently over express Glut-1, which is a high affinity glucose transporter [6]. The relationships between FDG uptake and the expression of facilitative glucose transporters have been previously evaluated. FDG uptake and the expression of five glucose transporters and the proportions of proliferating cell and macrophage populations were studied in paraffin sections from surgically resected primary non-small cell lung cancers (NSCLC) by immunohistochemistry (IHC). The patients were imaged with FDG-PET before surgery. Glut-1 expression was significantly higher than that of any other glucose transporters. All tumors tested (n = 23) were Glut-1-positive (70.8 ± 26.1% of tumor cell area was positive and staining intensity was 2.8 ± 1.2) and Glut-1 was the major glucose transporter expressed. Both FDG uptake and Glut-1 expression appear to be associated with increased tumor size [6]. Autoradiographic studies of excised tumors demonstrate increased FDG uptake in viable cells near necrotic portions of tumor. In conclusion, human tumor cell lines, in response to hypoxia, increase glucose uptake by up regulating membranous expression of the Glut-1 glucose transporter. The ability to survive periods of hypoxia confers tumors with an aggressive malignant phenotype enabling it to be resistant to both chemotherapy and radiotherapy and consequently poor overall survival. In several tumors including, NSCLC, colon cancer, bladder cancer, breast cancer and thyroid cancers, increased Glut-1 expression not only confers a malignant phenotype but also predicts for inferior overall survival [7–9]. We postulated that anti-Glut-1 antibodies would disrupt Glut-1 transporter function and consequently, not only lead to decreased tumor growth but also render it sensitive to chemotherapy and radiotherapy. This is presumably achieved by inhibiting intra-tumoral glucose uptake. In the experiments outlined below, we show that antibodies to Glut-1 decrease proliferation and induce apoptosis in NSCLC and breast cancer cell lines by decreasing glucose uptake. This work lays the foundation for future in vivo studies.
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2. Materials and methods 2.1. Cell culture Breast carcinoma cell lines MCF-7, T47D, p53 negative osteosarcoma cell line Saos-2 and NSCLC cell line H1299 were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). SV40-transformed human lung fibroblast WI-38-VA13 was obtained from ATCC and cultured in Minimal essential medium eagle with Earle’s salt and L-glutamine containing 10% FBS, sodium pyruvate, and non-essential amino acids. NSCLC cell lines A549, H358, H226, H1650 were grown in Ham’s F12K containing 10% FBS. Human microvascular endothelial cells of lung (HMEC-L) and human aortic endothelial cells (HAEC) were grown in EGM bullet kit medium containing 5% FBS from Clonetics. 2.2. Cell lysate preparation and Western blot Lysates from cells were prepared by NP40 lyses. Samples were boiled in equal volume of 2· SDS sample buffer, and separated on 8% polyacrylamide gels. After semi-dry transfer to supported nitrocellulose membranes, the blots were probed with monoclonal antibody to Glut-1 from R&D systems. The proteins were detected by using an enhanced chemiluminescence assay system from Amersham Biosciences. 2.3. Immunofluorescence Glut-1 monoclonal antibody was purchased from R&D Systems Inc (Minneapolis, MN). Cells were plated onto poly-D-lysine (Sigma) coated 8-well glass chamber slides (10,000 cells per well) for immunostaining. Cells were fixed in 3.5% paraformaldehyde for 25 min, permeabilized in 0.2% Triton X-100/PBS for 5 min, and blocked in 5% normal goat serum in PBS at room temperature for 1 h. Primary antibody incubation was performed overnight at 4 C. After washing, secondary antibody incubation was performed with goat anti-mouse IgG Alexa Fluor-488 for 30 min at room temperature. DAPI was detected using Vectashield Mounting Medium with DAPI (Vector Laboratories, Inc.). Control experiments demonstrated that there was no detectable staining by secondary antibodies only (data not shown). Slides were observed by fluorescence microscopy using Leica DM LB2 microscope (40·/0.75 numerical aperture) with a Qimaging Retiga1300 camera. 2.4. MTT assay MTT assays were performed by the following wellestablished method. In a 96 well tissue culture plate 10,000 cells were plated in each well. The cells were
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incubated in presence or absence of Glut-1 antibody for 18 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in PBS (10 mg/ml) and filter sterilized. Three hours before the end of the incubation 20 ll of MTT solution was added to each well containing cells in 96 well plates. The plate was incubated in an incubator at 37 C for 3 h. Media was aspirated gently and 200 ll of DMSO was added to each well to dissolve formazan crystals. The absorbance was measured at 550 nm. 2.5. Proliferation assay MCF-7, H1299 and H1650 cells were plated onto poly(Sigma) coated 8-well glass chamber slides (10,000 cells per well). The cells were incubated with 0.1 mg/ml Glut-1 monoclonal antibody for 18 h. The cells were fixed and stained using 5-Bromo-2 0 -deoxyuridine labeling and Detection kit from Roche according to manufacturer’s protocol. D-lysine
2.6. Apoptosis assay MCF-7, H1299 and H1650 cells were plated onto poly(Sigma) coated 8-well glass chamber slides (10,000 cells per well). The cells incubated with 0.1 mg/ ml Glut-1 monoclonal antibody for 18 h served as controls. The cooperative effect of drugs was evaluated by adding 5 lM of cisplatin or paclitaxel or 10 lM gefitinib. After 18 h of incubation cells were fixed and stained according to manufacturer’s instructions using Promega’s DeadEnd Colorimetric TUNEL system. D-lysine
2.7. Glucose uptake assay Cellular glucose uptake was measured by incubating cells in glucose-free RPMI 1640 with 0.2 Ci/mL [3H]2deoxyglucose (specific activity, 40 Ci/mmol) for 60 min. After the cells were washed with ice-cold PBS, the radioactivity in the cell pellets was quantified by liquid scintillation counting. 2.8. Statistical analyses Unless other wise specified experiments were done in triplicate. Error bars were generated based on the 95% confidence intervals obtained from these experiments. 3. Results 3.1. Expression levels of Glut-1 in transformed and primary cell lines
Fig. 1. Glut-1 expression in different cell lines. Cell lysates were prepared from various cell lines and 100 lg protein was electrophoresed and blotted on nitrocellulose membrane. The lysates were boiled in 20 ll of SDS sample buffer and separated on 8% polyacrylamide gel. After semi-dry transfer to supported nitrocellulose membranes, the blots were probed with the Glut-1 monoclonal antibody. The proteins were detected by using an enhanced chemiluminescence assay system from Amersham. The blot revealed increased expression of Glut-1 in H1299, H1650, MCF-7 and T47D.
and H1650 as well as primary cell line HMEC-L. MCF-7 and T47D breast cancer cell lines were also evaluated. Additionally we analyzed Jurkats, U937, Saos-2, WI38VA13 and HAEC for expression of Glut-1 protein by Western blot. As depicted in Fig. 1, H1299, H1650, MCF-7, T47D and Jurkats show comparatively higher expression of Glut-1. At the same time, Glut-1 levels appeared to be low in U937, H226 and A549 cells. Actin levels were comparable across the cell lines, suggesting that observed differences in the levels of Glut-1 are genuine. 3.2. Effect of Glut-1 antibody on cell proliferation Next we attempted to assess the ability of monoclonal Glut-1 antibody to affect cell proliferation. We therefore incubated H1299, H1650, T47D and MCF-7 cell lines in presence of different dilutions of the monoclonal antibody for 18 h. Similar dilutions of a non-specific IgG1 isotype antibody were used as the control. An MTT assay revealed that 0.1 mg/ml of the antibody was able to repress the proliferation of these cell lines by at least 50% (Data not shown). These results were further confirmed by BrdU proliferation assay. H1299, H1650, T47D and MCF-7 Cells were cultured on 8-well chamber slides and incubated with 0.1 mg/ml monoclonal Glut-1 antibody for 18 h. A concentration of 0.1 mg/ml IgG1 antibody was used as control. As shown in Fig. 2, the treatment with the Glut-1 antibody led to 45–70% decrease in the proliferation of all the four cell lines tested, suggesting that incubation with this antibody can inhibit cell proliferation. 3.3. Localization of Glut-1 receptor
Glut-1 is the most widely expressed isoform of the Glut family that provides cells with their basic glucose requirement. We examined levels of Glut-1 in various cell lines including NSCLC cell lines A549, H1299, H358, H226
An immunofluorescence experiment was performed to confirm the ability of antibody to bind the Glut-1 receptor. MCF-7 and H1650 cells were plated on chamber
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uptake may be enough to reduce proliferation and induce apoptosis in aggressive cancer cell lines who require high levels of glucose to meet their energy needs. Additionally, by immunostaining the predominantly membranous localization of the Glut-1 receptor is demonstrated in the breast cancer cell lines in comparison to the primary breast cell line MCF10A, which is particularly evident in the MCF-7 and T47D cell lines. Please see Fig. 4. 3.4. Glut-1 enhances the inhibition of cell proliferation by chemotherapeutic drugs
Fig. 2. H1299, H1650, T47D and MCF-7 cells were grown on poly-D-lysine coated chamber slide in presence or absence of 0.1 mg/ml Glut-1 antibody or IgG1 control isotype antibody and BrdU assay was performed. The results show 40–70% decrease in BrdU incorporation in these cell lines.
slides. After washing the cells were fixed and immunostained with the Glut-1 monoclonal antibody. The cells were then visualized with secondary antibody conjugated to Alexafluor-488. As shown in Fig. 3 staining for Glut-1 is visible in the cytoplasm and confocal microscopy confirmed the localization of the Glut-1 transporter to the cell membrane. This suggests that transporter is present on the cell surface and implies that anti-Glut-1 antibody prevents cell proliferation by altering Glut-1 transporter’s function, presumably by inducing conformational changes in Glut-1 transporter function. In Fig. 3, we show that the MDAMB231 breast cancer cell line demonstrate a 10–50% reduction in glucose uptake when incubated with the anti-Glut-1 antibody. This reduction of glucose
Experiments were done to evaluate whether anti-Glut1 antibody could synergize with standard chemotherapeutic agents to inhibit proliferation of H1299, H1650 and MCF-7. The cells were plated on chamber slides and incubated in presence of chemotherapeutic agents(5 lM cisplatin, 5 lM paclitaxel or 10 lM geftinib) in absence or presence of 0.1 mg/ml anti-Glut-1 antibody. The treatment with drugs led to significant decrease in proliferation of all the three cancer cell lines, however, addition of Glut-1 antibody led to a greater reduction in cell proliferation in NSCLC cell lines H1299 and H1650, and breast cancer cell line MCF-7 (Fig. 4). In the H1299 line, treatment with the anti-Glut-1 antibody alone inhibited proliferation by 62%; when added to cisplatin it enhanced cisplatin-induced inhibition of proliferation by 62%, paclitaxel by 74% and gefitinib by 42%. Similarly in the H1650 cell line treatment with anti-Glut-1 antibody alone inhibited proliferation by 55%; when added to cisplatin, it enhanced cisplatin induced inhibition of proliferation by 18%; paclitaxel by 23% and gefitinib by 46%. In the MCF-7 cell line, anti-Glut-1 antibody alone inhibited proliferation by 59%, when added to cisplatin it enhanced cisplatin-induced inhibition of proliferation by 40%, paclitaxel by 47% and gefitinib by 59%. The results are shown in Fig. 5.
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3.5. Glut-1 enhances the apoptosis by chemotherpeutic drugs
Specific Activity
55 50 45 40 35 30 25 20 Control IgG
Glut1 IgG
Fig. 3. Results of the glucose uptake assay in MDA-MB 231 cells treated with an anti-Glut-1 antibody for 72 h. Error bars were generated from two independent experiments. Treatment with Glut-1 antibody demonstrates an inhibition of glucose uptake in comparison to a control IgG1 antibody.
Since the above drugs are known to be strong inducers of apoptosis, attempts were made to assess whether antiGlut-1 antibody synergizes with them to induce apoptosis. NSCLC cell lines, H1299 and H1650, and breast cancer cell line, MCF-7 were evaluated for apoptosis by the TUNEL assay, after treatment with 5 lM cisplatin, 5 lM paclitaxel or 10 lM geftinib alone, or with antiGlut-1 antibodies. Glut-1 antibodies enhanced the apoptotic effects of cisplatin, paclitaxel and gefitinib (Fig. 6) in H1650 cell line by 43%, 62% and 111%; in H1299 by 111%, 30% and 71% and in MCF-7 cell line by 37%, 91% and 133%, respectively. Induction of apoptosis was assessed by measuring PARP cleavage. PARP cleavage was assessed by Western
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Fig. 4. In the breast cancer cell lines MCF-7, T47D, and MDAMB231, Glut-1 is cytoplasmic and localized to the cell membrane. However, in the primary breast cell line MCF10A, the localization of Glut-1 appears to be diffusely cytoplasmic with no particular predilection to be membranous.
blotting of lysates from MCF-7 and H1650 cells treated with paclitaxel alone or after co-incubation with 0.1 mg/ ml of anti-Glut-1 antibody for 18 h. Results of these experiments demonstrate that the combined treatment of cells with paclitaxel and Glut-1 antibody lead to enhanced apoptosis in comparison to paclitaxel alone (Fig. 7). In summary, our results indicate that anti-Glut-1 antibodies inhibit proliferation and induce apoptosis in the evaluated NSCLC cell lines and breast cancer cell lines providing evidence that the use of antibodies to Glut-1 may be a viable but an as yet unexplored therapeutic strategy in tumors that over express Glut-1 and consequently demonstrate increased glucose uptake in FDG-PET.
4. Discussion We demonstrate here that tumors’ dependence on glucose can be exploited for therapeutic gain by using anti-Glut antibodies The proposed mechanism is the reduction of glucose uptake by altering the function of the high affinity glucose transporter Glut-1. Since tumors preferentially up regulate Glut-1 over other members of the Glut family of transporters, we used a monoclonal antibody to Glut-1 in the experiments detailed above. We demonstrate that anti-Glut-1 antibodies not only decrease proliferation in the breast cancer and
NSCLC cell lines tested but also enhance chemotherapy-induced apoptosis. Other investigators have also explored similar strategies. Aft et al. investigated the effects of the anti-metabolite 2-deoxy-D-glucose (2-DG) on breast cancer cells in vitro [10]. This compound has been shown to inhibit glucose metabolism. Treatment of human breast cancer cell lines with 2-DG resulted in a decrease of cell growth in a dose dependent manner. The cell death induced by 2-DG was due to apoptosis as demonstrated by induction of caspase 3 activity and PARP cleavage. In response to exposure to 2-DG, breast cancer cells expressed higher levels of Glut-1 transporter protein and consequently increased its glucose uptake when compared to non-treated breast cancer cells. These data suggest that breast cancer cells treated with 2-DG, owing to the perceived deprivation of glucose, respond by expressing higher levels of glucose transporter proteins, which allows for further increased uptake of 2-DG, and subsequent induction of cell death. Decreasing GLUT-1 expression as a means of abrogating tumor growth was investigated by Noguchi et al. [11,12]. Noguchi et al. transfected MKN45 cells with cDNA for anti-sense GLUT-1.
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Fig. 5. Combined effect of chemotherapeutic drugs and Glut-1 antibody on cell proliferation. H1299, H1650, and MCF-7 cells were grown on chamber slides as described above and treated either with cisplatin, paclitaxel or iressa in presence or absence of Glut-1 antibody (0.1 mg) for 18 h. The proliferation was measured by BrdU incorporation. The results show that combination of chemotherapeutic drugs with the Glut-1 antibody further diminishes the cell proliferation.
Glucose transport was significantly decreased in cells with anti-sense GLUT-1 compared with wildtype cells or cells with vector alone. Suppression of GLUT-1 mRNA resulted in a decreased number of cells in the S phase. This was accompanied by over expression of p21 protein. Tumorigenicity in the nude mice injected with anti-sense GLUT-1 expressing cells was significantly slower than in those with wild-type MKN45 cells. These results corroborate our data. Differential expression of glucose transporters, particularly Glut-1, in normal and pathologic tissue
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Fig. 6. Combined effect of chemotherapeutic drugs and Glut-1 antibody on cell apoptosis. H1299, H1650, and MCF-7 cells were grown on chamber slides as described above and treated either with cisplatin, paclitaxel or iressa in presence or absence of Glut-1 antibody (0.1 mg) for 18 h. The apoptosis was measured by TUNEL assay. The results demonstrate that the combination of chemotherapeutic drugs with the Glut-1 antibody results in additive effect on cell apoptosis.
is critical to enable the potential therapeutic utility if anti-Glut-1 antibodies in clinic. Suganuma et al. examined 71 kidney surgical samples using reverse transcriptase-polymerase chain reaction (RT-PCR) for GLUT1–14 in normal and tumor tissues. The expression levels for GLUT1–5, 9, 10 and 12 were quantified by real-time quantitative PCR. The RTPCR results showed that normal kidney tissue expresses all the GLUT isoforms. In clear cell carcinoma of the kidney, GLUT-1 expression increased (p < 0.001) while GLUT4, 9 and 12 decreased (p < 0.001) [13]. In a similar study conducted in thyroid tissue, Matsuzu et al. examined the expression
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Fig. 7. MCF-7 and H1650 cells were treated with 20 lM paclitaxel in presence or absence of Glut-1 antibody for 18 h. Apoptosis was assessed using PARP cleavage. Combined treatment of cells with paclitaxel and Glut-1 antibody leads to more potent apoptotic stimuli in comparison to paclitaxel alone.
pattern and levels of GLUT genes in normal and pathologic thyroid tissues to evaluate the clinical significance of GLUT mRNA levels. One hundred fifty-two surgically resected thyroid tissue samples from 103 patients were evaluated. Samples included normal thyroid tissue (n = 58), benign thyroid disease (n = 61), and thyroid carcinoma (n = 33). Expression of the GLUT-1, GLUT-2, GLUT-3, GLUT-4, and GLUT-10 genes were examined by RT-PCR and mRNA levels were quantitated by real-time RT-PCR. All thyroid parenchymal cells expressed GLUT-1, GLUT-3, GLUT-4, and GLUT-10. GLUT-1 showed increased expression in carcinoma cases (p < 0.0001) and also in comparison with paired normal tissue samples from the same patient (p < 0.0001). Other GLUTs were statistically unchanged in pathologic tissues. The authors concluded that the up regulation of GLUT-1 during carcinogenesis plays a major role in enhanced glucose uptake in thyroid cancer cells [14]. Since normal cell express several of the Glut isoforms and tumors have increased dependence on Glut-1, we anticipate that the treatment with Glut-1 antibody would differentially affect tumors while relatively sparing normal cells. Indeed even minor alterations in Glut-1 function may have significant effects on the tumor. Membranous up regulation of glucose transporters may not just be essential for tumor survival but may also be integrally involved in the carcinogenic process. Healthy colonocytes derive 60–70% of their energy supply from short-chain fatty acids, particularly butyrate. Butyrate is transported across the luminal membrane of the colonic epithelium via a monocarboxylate transporter, MCT1. Analysis of healthy colonic tissues and carcinomas using IHC and Western blotting by Russo et al. revealed a sig-
nificant decline in the expression of MCT1 protein during transition from normality to malignancy [15]. This was reflected in a corresponding reduction in MCT1 mRNA expression, as measured by Northern analysis. Carcinoma samples displaying reduced levels of MCT1 were found to express the high affinity glucose transporter, GLUT-1, suggesting that there is a switch from butyrate to glucose as an energy source in colonic epithelia during transition to malignancy. To the best of our knowledge this is the first paper to show that anti-Glut-1 antibodies inhibit proliferation and induce apoptosis in NSCLC cell lines and breast cancer cell lines evaluated. Furthermore, they enhance apoptosis caused by chemotherapeutic agents like cisplatin and paclitaxel and targeted agents like gefitinib. These and our data clearly indicate that Glut-1 is a legitimate targets for anti-neoplastic drug development and are worthy of further rigorous pre-clinical investigation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2007.07.021. References [1] O. Warburg, On the origin of cancer cells, Science 123 (1956) 309–314. [2] S.S. Gambhir, Molecular imaging of cancer with positron emission tomography, Nat. Rev. Cancer 2 (2002) 683–693. [3] R.L. Elstrom, D.E. Bauer, M. Buzzai, et al., Akt stimulates aerobic glycolysis in cancer cells, Cancer Res. 64 (2004) 3892–3899. [4] C.V. Dang, G.L. Semenza, Oncogenic alterations of metabolism, Trends Biochem. Sci. 24 (1999) 68–72. [5] F.C. Detterbeck, S. Falen, M.P. Rivera, J.S. Halle, M.A. Socinski, Seeking a home for a PET. Part 2: defining the appropriate place for positron emission tomography imaging in the staging of patients with suspected lung cancer, Chest 125 (2004) 2300–2308. [6] A.C. Clavo, R.S. Brown, R.L. Wahl, Fluorodeoxyglucose uptake in human cancer cell lines is increased by hypoxia, J. Nucl. Med. 36 (1995) 1625–1632. [7] M. Younes, L.V. Lechago, J.R. Somoano, M. Mosharaf, J. Lechago, Wide expression of the human erythrocyte glucose transporter Glut-1 in human cancers, Cancer Res. 56 (1996) 1164–1167. [8] M. Younes, L.V. Lechago, J. Lechago, Overexpression of the human erythrocyte glucose transporter occurs as a late event in human colorectal carcinogenesis and is associated with an increased incidence of lymph node metastases, Clin. Cancer Res. 2 (1996) 1151–1154.
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