GLUT 5 Is Not Over-Expressed in Breast Cancer Cells and Patient Breast Cancer Tissues

F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues

The p53 binding sites in the PIK3CA promoter with core sequence (underlined) and the mutagenic primers.

<p>The p53 binding sites in the PIK3CA promoter with core sequence (underlined) and the mutagenic primers.</p

Transcription factor binding sites in PIK3CA promoter*.

<p>Note: *As analysed by the Genomatix software and using references from Astanehe et al, 2008 and Yang et al, 2009.</p

In vitro studies on effect of Cisplatin and paclitaxel on PIK3CA promoter and p53.

<p>2a-d and 2k -2l. <i>PIK3CA</i> promoter activity in drug treated PA1-<i>PIK3CA-fluc2-tdt</i> (PPF), A2780-<i>PIK3CA-fluc2-tdt</i> (APFT) and SKOV3-<i>PIK3CA-fluc2-tdt</i> (SPFT) (2k and l) cells. A dose dependent decrease in luciferase activity (<i>PIK3CA</i> promoter activity) was observed on increasing concentrations of cisplatin treatments in PPF cells (at 3 & 5 µg/ml cisplatin, p<0.03) (2a) and in APFT cells at 20 and 30 µg/ml cisplatin (p<0.003) (2c). Similar dose dependent decrease in luciferase activity was obtained on increasing concentration of paclitaxel treatment in PPF up to 25 µg/ml, p<0.001) (2b) and in APFT cells upto 20 µg/ml paclitaxel, p<0.05 (2d) No such dose dependent change was observed on treatments with increasing concentration of cisplatin and paclitaxel (2k and 2l) in SPFT cells. 2e-2 h. Endogenous expression of p110α and p53 proteins after drug treatments. Western blot analysis of p53 protein from lysates of cisplatin and paclitaxel treated PPF and APFT cells showed induction in p53 level after 24hrs; however no change was observed after 2hrs. The p110α levels did not show any change after drug treatment (2e & 2g). The densitometric analysis representing the same is shown in 2d & 2h. The p53 mutant status in SPFT cells was verified by western blotting (2 m). 2i & 2j. Nuclear localization of p53 after treatment in cisplatin treated cells. p53 protein (red in 3g and green in 3h) showed nuclear localization upon treatment with cisplatin for 2hrs as compared to the vehicle treatment in PPF and APFT cells. DAPI (blue) indicated the nuclear staining.</p

Multimodality imaging of the new generation bi- and tri-fusion vectors in living mice.

<p>1a1 and 1a2. Fluorescence and Bioluminescence imaging. 10×10⁶ of 293T cells transfected with <i>CMV-ttk (A), CMV-fluc2-tdt-ttk (B), CMV-fluc2(C),</i> and <i>CMV-tdt (D)</i> plasmids were implanted subcutaneously in living mice (n = 3) and were imaged first for fluorescence and then for bioluminescence after injection of D- luciferin using IVIS imaging system. Signals were seen only from the cells expressing <i>CMV-tdt</i> and <i>CMV-fluc2-tdt-ttk</i> vector (for fluorescence) and from cells expressing <i>CMV- fluc2</i> and <i>CMV-fluc2-tdt-ttk</i> reporter for bioluminescence. Signals were recorded as max (pixel/sec/cm²/steradian). 1a3. microPET imaging. Mice described in 1a1 were injected with 200 µCi of 18F-FHBG and microPET imaging was performed after 1 hr for 10 minutes. Specific uptake of 18F-FHBG was seen in cells expressing the <i>CMV-ttk</i> and <i>CMV-fluc2-tdt-ttk</i> vectors. High nonspecific accumulation of 18F-FHBG was seen in the gastro-intestinal tract (GI). 1a4. Coronal section of the same microPET image described in 1a3. 1b. Graphical representation of the quantified fluorescence (1b1), bioluminescence (1b2) and microPET (1b3) signals. 1b1 and 1b2. Fluorescence (B and D) and bioluminescence (B and C) signals were calculated for the respective ROIs drawn over the sites of implanted cells. The SEM represents 3 experiments (ns.- statistically non-significant and *indicates p<0.05). 1b3. Percent injected dose (%ID/G) of 18F-FHBG uptakes were calculated for the respective ROIs drawn over the implanted cell (A and B) which showed similar uptake (ns). 1c. Comparative analysis of the fluorescence activity of the new bi and trifusion vectors in cell culture: 293T cells were transiently transfected with <i>CMV-fluc2-tdt-ttk</i>, <i>CMV-mtfl-tdt-ttk, CMV-mtfl-tdt</i> and <i>CMV-fluc2-tdt</i> plasmids and FACS analysis was done from equal number of cells after 24 hrs. All the experiments were performed in triplicate (*indicates p<0.05). 1d & 1e. Fluorescence and bioluminescence imaging of the new bi and triple fusion vector. 10×10⁶ of 293T cells transfected with <i>CMV-fluc2-tdt-ttk</i>, <i>CMV-mtfl-tdt-ttk, CMV-mtfl-tdt</i> and <i>CMV-fluc2-tdt</i> plasmids were implanted on the dorsal side of a nude mouse and imaged for fluorescence (1d) using Maestro system and bioluminescence (1e) as described above. Cells expressing <i>CMV-fluc2-tdt</i> clearly exhibited highest fluorescence and bioluminescence signals among all group of cells.</p

Non-invasive imaging of PIK3CA promoter modulation in tumor xenografts of living mice after cisplatin treatment.

<p>3a. Bioluminescence signal of PIK3CA promoter in PPF tumors. 3a1. Graphical representation of the kinetics of <i>PIK3CA</i> promoter modulation. The luciferase activity in the control group of mice (n = 3) increased with time while that of the treated group (n = 3) attenuated from the fourteenth day of treatment till the 22^nd day (end point) after one treatment (8 mg/kg) (shaded area represented the days of treatment). At 22^nd day, measurable attenuation in the bioluminescence signal between the control and treated mice was evident however it did not reach statistical significance. (Day 1 represents the day prior to treatment). 3a2. Graphical representation of fold-changes in the bioluminescence signals. The temporal fold change in bioluminescence signal (post treatment signal/pre treatment signal) demonstrated augmented bioluminescence in the control group and attenuation in the treated group. 3a3. Representative bioluminescent images of the mice bearing PPF tumors. Mouse from the control and treated group exhibited specific and similar intensity signals which decreased after treatment (arrowhead). 3b. Bioluminescence signal of PIK3CA promoter in APFT tumors after cisplatin treatment. 3b1. Graphical representation of the kinetics of <i>PIK3CA</i> promoter modulation. The bioluminescence signal in the control mice (n = 3) increased with time while that of the treated group (n = 4) showed a slight decrease at 11th day after first treatment and significant attenuation at 15^th day after second treatment (p<0.025). 3b2. Graphical representation of fold-changes in the bioluminescence signals. The temporal fold change in bioluminescence signals (post treatment signal/pre treatment signal) demonstrated augmented bioluminescence in the control group and but attenuation in the treated (Day 10 represented signal prior to treatment). 3b3. Representative bioluminescent images of the mice bearing APFT tumors. Mouse from control and treated group exhibited specific signals which decreased only in treated mouse as shown by an arrow.</p

Staining intensities from 40 breast cancer samples stained with GLUT 5-antibody.

<p>Staining intensities from 40 breast cancer samples stained with GLUT 5-antibody.</p

Cytochalasin B treatment affects fructose uptake in MDA MB 468 cells.

<p>MDA MB 468 cells were exposed to 100 µM cytochalasin B for 30 mins. Then 1 µCi of C14 fructose or glucose was added to the cells, and the uptake was continued for 10 mins in HBSS. The activity associated with the cells was then counted and normalized to activity in the medium and to total protein mass. * indicates p values <0.05. The error bars represent S.E.M of triplicates.</p

GLUT 5 mRNA levels by quantitative real time PCR.

<p>RNA from MCF7, MDA MB 468 and MCF10A were extracted, converted to cDNA and quantified by real time PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026902#pone-0026902-g001" target="_blank">fig 1A</a>) and the products also visualized on a 1% agarose gel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026902#pone-0026902-g001" target="_blank">fig 1B</a>).</p

Knocking down GLUT 5 mRNA in MCF7 cells has no effect on fructose uptake.

<p>GLUT 5 specific siRNA and negative control siRNA were transfected into MCF7 cells. 48 hrs after transfection cells were simultaneously interrogated for both mRNA levels (using quantitative real time PCR) (right Y axis) and for fructose uptake (left Y axis) by incubating with C14 labeled fructose. The error bars represent S.E.M of triplicates.</p