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    Awarded Grants
    Use of Antisense Oligodeoxynucleotides and Molecular Imaging in the Detection, Suppression and Evaluation of Multidrug Resistance in Breast Cancer

    Scientific Abstract:
    USE OF ANTISENSE OLIGODEOXYNUCLEOTIDES AND MOLECULAR IMAGING IN THE DETECTION, SUPPRESSION AND EVALUATION OF MULTIDRUG RESISTANCE IN BREAST CANCER BACKGROUND Chemotherapy is currently one of the most widely-used treatment for both locally-advanced and metastatic breast tumors. However, repeated chemotherapy cycles may also induce multidrug resistance (MDR), which results in treatment failure. P-glycoprotein (PGP)-mediated MDR is the most common form of MDR in breast cancer, and has been extensively studied as a potential target for pharmaceutical development. A combined treatment of chemotherapeutic agents and a non-toxic PGP inhibitor for cancer patients is currently the ideal method for overcoming MDR during chemotherapy. Antisense oligodeoxynucleotide (asODN) therapy targets specific gene mRNA instead of protein as with traditional drugs. Once they have been radiolabeled, asODNs are thus also good tools for in vivo imaging of transcripts coding for a particular protein. Hence, utilizing asODN against mdr1/PGP is a promising tool with diagnostic and therapeutic applications in MDR of breast cancer. In this project, we will use an asODN directed against the translation start site of mdr1 mRNA to inhibit PGP translation in breast cancer cells, which in turn will reverse PGP-mediated MDR in breast tumors. In addition, this will be developed for use as a noninvasive imaging technique by using radio-labeled mdr1 asODN and sestamibi to monitor the gene expression and functional activity of PGP/mdr1 in breast tumors during chemotherapy. OBJECTIVE/HYPOTHESIS The main objective of this project is to employ an asODN specifically against mdr1 mRNA, which will allow us to image drug-resistant tumors in a non-invasive manner, as well as suppress the translation of the gene product PGP in breast cancer cells and tumors. We will also examine if PGP suppression can lead to sensitivity to chemotherapeutic drugs, which in turn would reverse multidrug resistance. SPECIFIC AIMS 1) To construct an antisense ODN directed against the mRNA of target genes, and to use the antisense ODN as a radiopharmaceuticals by labeling with 99mTc. 2) To investigate the uptake and retention of the radiopharmaceutical by human breast cancer cells in vitro as a function of treatment-modulated gene expression. 3) To evaluate the impact of treatment on the tumor and normal tissue uptake and imaging properties of the antisense ODN in vivo in mice implanted with s.c. human breast cancer xenografts. STUDY DESIGN This project will include an in vitro cell culture study and an in vivo study using a tumor xenograft mouse model to assess the diagnostic and therapeutic profiles of mdr1 asODN. The human breast cancer cell line MCF-7, which expresses a low basal level of PGP/mdr1 and is sensitive to chemotherapeutic drugs, will be used in this study. Another MCF-7 derived cell line, MCF-7/ADR, which overexpresses PGP/mdr1 and the MDR phenotype will be used for comparison. MCF-7 and MCF-7/ADR cells will also be implanted in athymic nude mice for the growth of drug sensitive and resistant tumors respectively. The specificity and toxicity of mdr1 asODN will be examined by using MTT assay. The efficacy of targeting mdr1/PGP as well as reversal of MDR in vitro and in vivo will be evaluated using various approaches, including Western blots, RT-PCR, cell uptake study, tumor growth study and real-time molecular imaging. The use of radio-labeled mdr1 asODN as a chemoresistance detection probe will also be evaluated in the same cell culture and tumor models. Comparisons between drug sensitive and resistant cells or tumors, and between antisense and control sense or mismatch ODNs will be made using Student’s t-test (p<0.05). POTENTIAL OUTCOMES AND BENEFITS OF THE RESEARCH Expected results from this project will reveal the suitability and efficacy of using mdr1 asODN as a novel PGP modulator to enhance the tumor responsiveness to chemotherapeutic drugs, and as a sensitive marker to detect the tumor chemoresistance in vivo. The beneficial outcome from this project may provide critical information revealing mdr1 asODN as a PGP modulator that should be further developed, which in turn may improve MDR reversal during chemotherapeutic treatment in breast cancer patients.

    Lay Abstract:
    USE OF ANTISENSE OLIGODEOXYNUCLEOTIDES AND MOLECULAR IMAGING IN THE DETECTION, SUPPRESSION AND EVALUATION OF MULTIDRUG RESISTANCE IN BREAST CANCER Today, in clinical breast cancer treatment, chemotherapy remains the major defense line for breast cancer patients. While, some patients can be treated successfully by chemotherapeutic means, others respond transiently or incompletely. These phenomena may be due to some complicated factors. Among them, multidrug resistance (MDR) is believed to be the major obstacle leading to the treatment failure. MDR is defined as the ability of cancer cells to become simultaneously resistant to different types of chemotherapeutic drugs. It is believed that repeated cycles of drug treatment results in increased levels of one significant drug transporter called P-glycoprotein (PGP). Currently, many studies have focused on suppression of PGP expression in tumors to overcome MDR during cancer chemotherapy. In our project, we will employ a novel strategy that allows both treatment and detection of breast tumors. These studies will utilize so-called antisense oligodeoxynucleotide (asODN), which can hybridize to a transcript of the human genome encoding PGP, in order to suppress its expression. By labeling the same asODN with a low energy radioactive element, we can use it as a probe to detect the location and expression of genes that encode PGP in tumors prior to treatment. The effectiveness of asODN as a novel anti-MDR treatment will be tested first using human breast cancer cells grown in culture dishes. The extent of the inhibitory effect will be tested by evaluating the level of PGP expression, functional activity and the ability to reverse the cells’ sensitivity against chemotherapeutic drugs. Similar end points will be measured in mice which bear an implanted human breast cancer tumor. The effect of using radio-labeled asODN to detect tumor drug sensitivity will also be tested in these mice by using a gamma camera to acquire nuclear images. About 50% of total cancer patients need to be treated with chemotherapy after failure of local excision or radiation therapy. This non-invasive molecular imaging detection method can help to screen the drug sensitivity of tumors prior to processing for chemotherapy, and can provide the information to develop a treatment strategy for each individual patient. In addition, asODN itself can be used as a novel anti-MDR drug with more advanced properties than traditional transporter-targeting drugs. With higher efficiency and specificity, but lower toxicity and side effects, asODN has the potential for replacing traditional drug in the trials for improving the effectiveness of chemotherapy. This project may require two years to complete the in vitro cell study and in vivo animal study. Clinical trials will be ready to start after the successful completion of this project.