In the United States, breast cancer is the most common cancer occurring in women (excluding cancers of the skin) and the second most common cause of death from cancer in women after lung cancer. Men can also develop breast cancer, but male breast cancer is rare, accounting for less than 1% of all breast cancer cases . Normal breast cells have GLUT1 only. But in breast cancer cells overexpression of GLUT1 as well as expression of GLUT2, GLUT3, GLUT4 and GLUT5 [1, 3, 5, 6] has been observed. This overexpression causes increased uptake of glucose/fructose and may allow for uncontrolled proliferation of cancer cells.
Figure 1-RT-PCR product obtained from different breast cancer cell lines (MCF7, HTB26, HTB122 and HTB126) and normal breast cell line (MCF-10A) fractionated on 1.2% agarose gel. The expected size of the RT-PCR product is 357bp. The primers were created from GLUT5 sequence obtained from NCBI (Accession number: NM_003039). Primers were used at a concentration of 10pm/Ál and the RT-PCR annealing temperature was 58˚C with 40 cycles of amplification (18 cycles for beta-Actin). To calculate the size of RNAs, a 100bp ladder was ran parallel to the samples. Beta-Actin (NCBI, Accession number: AK223055) was used for normalization.
Figure 2-Sequence alignment of RT-PCR product (AnjuSeq). ClustalW alignment of the 357bp band (AnjuSeq) obtained from RT-PCR revealed homology to GLUT5 (NCBI: Accession number: NM_003039).
Figure 3-Western blot analysis of different breast cancer cell lines (MCF7, HTB26, HTB122 and HTB126) and normal breast cell line (MCF10A) using anti-GLUT5 antibody (at a concentration of 3.5mg/ml). The anti-GLUT5 antibody recognized the 55kDa GLUT5 protein in all breast cancer cell lines (MCF7, HTB26, HTB122 and HTB126) but not in normal breast cell line (MCF10A). The antibody does not cross react with other GLUT isoforms. GAPDH (50kDa) is used for normalization and the size of the recognized protein was detailed using a protein molecular weight marker.
RT-PCR and sequencing experiments showed the 357bp band obtained in all the breast carcinoma cell lines and the normal breast cells as GLUT5 (NCBI database: Accession number: NM_003039) (Fig. 1 and Fig. 2). The expression of GLUT5 in MCF-10A cells is intriguing. The experiment was performed six times and in all cases the result was consistent, GLUT5 was always expressed in the normal cell line. Previous reports have indicated absence of GLUT5 in normal cell lines . The most probable explanation for the presence of GLUT5 mRNA and protein is the cell line might have transformed during its storage and is now showing positive result.
The second ~500 bp band obtained in MCF-7 cells is interesting. According to several reports, MCF7 cells overexpress GLUT1, GLUT2 and GLUT3 [1, 9, 16]. It is possible that the primers have amplified another GLUT isoform because there is a 50-70% homology between the GLUT isoforms  or 500bp product is an isoform of GLUT5 that was spliced differently. Further investigation is required to explain the expression of this ~500bp product in MCF7.
In case of HTB 122 cell line both the normalization samples, actin in RT-PCR and GAPDH in western blotting is expressed more than in other cells (Fig. 1b and Fig3). This might be because in both cases the sample for HTB122 was loaded more than in other cell lines. Hence a comparative analysis of HTB-122 cells with other cell lines cannot be made. The RT-PCR product expression in HTB126 is highest, followed by HTB26 and MCF7 has the least (Fig. 1a). It is known that MCF-7 is highly invasive and HTB 26 is non-invasive. Disregarding the result of HTB122 in RT-PCR, it can be concluded from the remaining cell lines that expression of GLUT5 is inversely related to invasiveness. Grover-McKay and colleagues have also reported that in MCF-7 (tumorogenic non invasive breast cancer cell), HTB-26 (tumorogenic highly invasive breast cancer cells) and MDA-MB-435 (tumorogenic invasive breast cancer cells), the invasiveness of human breast cancer cells increase the expression of GLUT1 but decreases the expression of GLUT2 and GLUT5 .
Zamora-Leon and colleagues have reported from immunolocalization study that normal breast cells lack expression of GLUT5 . However the western blotting experiment does not agree with this report (Fig. 3). Faint staining was observed in the normal cell line, MCF-10A. The remaining cell lines have comparatively higher amount of GLUT5 protein than the normal cell line. In this case as well, the result for HTB-122 cannot be interpreted because the amount of GAPDH appears more than that of other cell lines. The remaining three cell lines MCF-7 and HTB-26 have almost equal amount of staining while HTB126 has comparatively less amount of staining. This result cannot be correlated with the RT-PCR result that shows highest amount of PCR product in HTB126 and less in HTB-26 and MCF-7. More investigation is required to confirm the western blotting result and the correlation between expression of GLUT5 mRNA and GLUT5 protein in relation to invasivenes.
Additional investigation is required to find what the second band observed in MCF7 cell line is, to understand the regulation mechanism involved that controls the increase and decrease of GLUT5 expression in different stages of cancer. In addition the relationship between GLUT5 protein expression and invasiveness of the cancer cell has to be confirmed in the normal breast cell line, MCF-10A cell lines, and other breast cancer cell lines
In conclusion, there appears to be a potential for using GLUT5 as a diagnostic marker to identify the different stages of cancer and to see if the tumor has reached an invasiveness stage or not. GLUT5 can also be targeted for decreasing the invasiveness of the cancer cells for producing therapeutic benefit.
I would like to thank Jinsong Qiu and Brian P. Fox for their help, patience and support throughout the project. I would also like to thank Dr. Berish Rubin for his guidance, providing an opportunity to do this project and allowing us to use the lab. Thanks also goes out to Dr. Raj Kandpal for providing the cells.
|This document was last modified 01/31/2006.|
This site is powered by the versatile Zope platform.
|This is a project of the Biology Department of Fordham University