Abstract 905: Molecular dissection of platinum resistance through functional analysis
Authors: Pan Amy W. , Wang Sisi , Zhang Hongyong , Vinall Ruth , Lin Tzu-yin , Malfatti Michael , Zimmermann Maike , Scharadin Tiffany , Turteltaub Kenneth , White Ralph de Vere , Pan Chong-xian , Henderson Paul 
Published: May 2015

Abstract
BACKGROUND: Platinum (Pt) agents (cisplatin, carboplatin and oxaliplatin) are active in many cancers including bladder cancer. Chemoresistance is the most common cause of treatment failure. This study is to determine the feasibility of using ultrasensitive accelerator mass spectrometry (AMS) to identify chemoresistance after cancer cells or patients are treated with one non-toxic microdose (1/100th of therapeutic dose) of Pt agents. The long-term goal is to identify chemoresistance before cancer patients receive toxic chemotherapy, and to determine the underlying resistance mechanisms to design personalized chemotherapy.
METHODS: Cellular sensitivity to chemotherapeutic agents was determined by the MTT assay. Platinum-induced DNA adduct formation and repair of adducts was measured with AMS after cells were exposed 14C-labeled carboplatin and oxaliplatin. AMS quantifies the 14C label that is attached to genomic DNA when the 14C-labled drug forms adducts with DNA. Cell uptake and efflux was measured by liquid scintillation counting. Intracellular glutathione levels were measured by colorimetric analysis.
RESULTS: Compared to the parental bladder cancer 5637 cells, chemoresistant 5637R cells are resistant to oxaliplatin (IC50: 2.45 µM versus 27.27 µM, p&lt;0.0001), and cisplatin (0.59 µM versus 2.99 µM, p=0.049), carboplatin (24.34 µM versus 72.18 µM, p&lt;0.0001), and gemcitabine (0.12 µM versus 1.44 µM, p=0.0015). Both 5637 and 5637R cells are still sensitive to other chemotherapeutic agents commonly used in treating bladder cancer, such as doxorubicin, methotrexate and vinblastine. Consistent with our hypothesis, chemoresistant 5637R cells have low oxaliplatin-induced DNA adduct levels than the parental 5637 cells (AUC of 943 versus 2,772 adducts per 109 nucleotide-hour for 5637, p=0.001). This low level of oxaliplatin-DNA adduct formation might be secondary to the pre-DNA damage mechanisms, such as decreased uptake (AUC of 4.42 versus 5.12 X 109 oxaliplatin molecules per cell for 5637, p=0.037) and increased intracellular inactivation of oxaliplatin by glutathione (53.91 versus 46.93 nmol/mg protein for 5637, p=0.003), plus increased repair of oxaliplatin-DNA adducts (3.48 versus 1.34 adducts per 108 nucleotides per hour for 5637, p=0.0004). We found the same correlation of low Pt-DNA adduct levels and chemoresistance in non-small cell lung (NSCLC) and breast cancer cell lines, and determined the same resistant mechanisms, such as cell uptake/efflux, intracellular inactivation and DNA repair. Carboplatin had partially different resistant mechanisms.
CONCLUSION: Functional analysis of major resistant steps can identify some chemoresistance mechanisms that can potentially help design personalized chemotherapy to overcome resistance. This approach can be applied to several different cancer types. A Phase 0 microdosing clinical trial is currently going on in patients with NSCLC and bladder cancer.
Citation Format: Amy W. Pan, Sisi Wang, Hongyong Zhang, Ruth Vinall, Tzu-yin Lin, Michael Malfatti, Maike Zimmermann, Tiffany Scharadin, Kenneth Turteltaub, Ralph de Vere White, Chong-xian Pan, Paul Henderson. Molecular dissection of platinum resistance through functional analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2014-905