Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>Advanced non-small cell lung cancer (NSCLC) has a dismal prognosis and remains the most common cause of cancer death worldwide. One of the primary reasons for its poor survival rate is its high level of resistance to chemotherapy. Expression of the microtubule protein αIII-tubulin is associated with an aggressive and drug resistant disease in NSCLC. Recently, we demonstrated for the first time that targeting αIII-tubulin using siRNA sensitizes NSCLC cells to chemotherapy and reduces tumour growth in mice. However, therapeutic delivery of siRNA to cancer cells in vivo remains problematic. Previously our group has designed novel non-viral nanoparticles (iNOP-7) which can complex and deliver siRNA in vivo. However, whether these nanoparticles can deliver siRNA targeting αIII-tubulin to NSCLC cells is unknown. Therefore, the aims of this study were to determine whether iNOP-7 could deliver siRNA targeting αIII-tubulin to lung cancer cells in vitro and in vivo. Methods: Two NSCLC cell lines (H460 and Calu-6) were treated with increasing amounts of iNOP-7 complexed to siRNA. Cell viability was assessed using a colorimetric cell counting assay. Cellular uptake of iNOP-7-siRNA complexes was determined by confocal microscopy and flow cytometry. Gene silencing efficiency of iNOP-7-αIII-tubulin siRNA complexes was measured by real time PCR and Western blotting. To determine whether iNOP-7 could deliver siRNA to lung tumors, 1x106 stably expressing luciferase H460 cells were orthotopically implanted into the lungs of Balb/c nude mice. Tumor growth was monitored weekly using the Xenogen IVIS System. Twelve days post-tumor implantation mice were treated systemically with iNOP-7 complexed to fluorescently labelled siRNA or αIII-tubulin siRNA (1.5mg/kg once a day for three days). Twenty four hours after the final injection tumor tissue was collected and uptake and gene silencing efficiency was evaluated using confocal microscopy and real-time PCR. Results: iNOP-7 did not affect cell viability and was able to effectively deliver high levels of siRNA to both cell lines. Importantly, iNOP-7-αIII-tubulin siRNA complexes potently silenced αIII-tubulin mRNA and protein levels by &gt;85% (n=3, p&lt;0.001) when compared to cells treated with non-silencing siRNA or iNOP-7 alone. Moreover, iNOP-7 delivered siRNA to the lung in mice harbouring lung tumors. Finally, preliminary data demonstrated that iNOP-7-αIII-tubulin siRNA silenced αIII-tubulin mRNA levels in lung tumor tissue by &gt;65% (n=2 animals per group) when compared to controls. Conclusions: This is the first study to show that RNA interfering nanoparticles can silence αIII-tubulin expression in NSCLC cells both in vitro and in vivo. Future studies will address the therapeutic potential of iNOP-7-αIII-tubulin siRNA complexes alone and in combination with chemotherapeutic agents in an orthotopic lung cancer mouse model.</jats:p>
<jats:p>Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1969. doi:1538-7445.AM2012-1969</jats:p>