Abstract 2562: Role of Pyruvate Kinase isozyme type M2 in response and resistance to oxaliplatin in HT29 human colon adenocarcinoma cell lines
Authors: Alba Ginés, Eva Martinez-Balibrea, Anna Martinez-Cardús, Albert Abad
Journal: Cancer Research (2010)

Abstract BACKGROUND: Resistance to platinum-based chemotherapy is a multifactorial and complex process resulting in a major obstacle for effective treatment. In a previous work we identified low levels of PKM2 (mRNA and protein) as a putative oxaliplatin-resistance marker by using an in vitro model of resistance and a comparative proteomic approach. Our results were also validated in advanced colorectal cancer patients treated with oxaliplatin (Martinez-Balibrea, Molecular cancer therapeutics, 2009). PKM2 has been reported to translocate to the nucleus and activate apoptosis after H2O2, UV or a somatostatin analog treatment. The aim of this work was to study the role of PKM2 in the molecular mechanisms involved in response and resistance to oxaliplatin and identify proteins related to PKM2 expression levels. EXPERIMENTAL DESIGN: We silenced PKM2 expression by using siRNA techniques in the human adenocarcinoma cell line HT29. Validation of knockdown was done by Western Blotting (WB) and RTQPCR. To study the effect of PKM2 silencing in the resistance phenotype an MTT assay was performed to compare the viability between PKM2-silenced cells and those transfected with an unspecific siRNA (NTC). Cells were treated 24 hours with oxaliplatin at 15 µM. To verify the PKM2 nuclear translocation and its possible role in activating apoptosis after H2O2 and oxaliplatin treatment, HT29 and HCT116 cells were treated with H2O2 and oxaliplatin at equivalent doses and harvested at 0, 2 and 4h after treatment. Sub-cellular fractionation and WB as well as immunoflorescenece techniques were performed to visualize nuclear translocation of PKM2. To study possible changes in the proteome of HT29 cells as a consequence of PKM2 silencing, 2D- DIGE and Mass Spectrometry techniques were performed. RESULTS: Forty-eight hours after transfection we obtained a 95% of PKM2 knockdown by RTQPCR (mRNA level) and also by WB (protein level). A slightly increase of viability was observed in PKM2-silenced cells in comparison to NTC cells after oxaliplatin exposition (50% vs. 35%). Preliminary results showed that both oxaliplatin and H2O2 induced PKM2 nuclear translocation in HT29 and HCT116 cells 4 hours after the exposition to these agents. By 2D-DIGE we determined changes in the proteome of HT29 cells as a consequence of PKM2 silencing. The identification and validation of those proteins showing up- or down-regulation are in process and will be presented during the annual meeting. CONCLUSIONS: PKM2 knockdown alters sensitivity to oxaliplatin and protein expression patterns in HT29 cells. Oxaliplatin induces nuclear translocation of PKM2. Further studies are guaranteed to evaluate the role of PKM2 in activating apoptosis after oxaliplatin exposure. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2562.