I. Woelfel1, K. P. Terracina3, S. Lima5, C. Oyeniran5, J. Newton5, H. Aoki3, D. Avni5, P. Mukhopadhyay3, N. Hait5, A. Raza3, X. Wu4, H. Yamamoto4, S. Spiegel5, K. Takabe2,3,5 1Virginia Commonwealth University,School Of Medicine,Richmond, VA, USA 2VCU Massey Cancer Center,Richmond, VA, USA 3Virginia Commonwealth University,Department Of Surgery,Richmond, VA, USA 4Osaka University,Suita, Osaka, Japan 5Virginia Commonwealth University,Department Of Biochemistry And Molecular Biology,Richmond, VA, USA
Introduction:
Gene therapy as an effective treatment modality for cancer has been sought for decades. Its full therapeutic potential has not been realized due to many barriers, including efficiency and cost. Small interfering RNA (siRNA) has emerged as a successful technology for specifically silencing gene expression in vitro. However, due to the small size and delicate nature of these molecules, an effective delivery system that allows these molecules to reach cancer and thus be applicable in patient treatment is necessary. Recent innovations in nanoparticle technology have enabled the development of a new delivery system: super carbonate apatite (sCA). sCA system is extremely inexpensive, and has been reported to deliver genes specifically to cancer due to the characteristic size and leakiness of peritumoral vessels. Sphingosine-1-phosphate, generated by Sphingosine kinase 1 (SphK1), has been established as a key lipid signaling molecule in cancer progression and is integral to cell survival, proliferation, migration, angiogenesis, and lymphangiogenesis. We investigated the ability of this new nanoparticle delivery system (sCA) with SphK1 siRNA to effectively knockdown SphK1 and suppress its physiological functions in vitro.
Methods:
A carbonate apatite inorganic nanoparticle delivery solution was created using CaCl2 and NaCO3 ions. 4T1 murine breast cancer cells were treated with 2 ug/mL of SphK1 siRNA, Non-targeting siRNA or a control containing no siRNA using the inorganic solution with a 4-hour serum free incubation time. At 4 hours 1 mL of 10% FBS media was added. 24 hours after the initial application of inorganic solution the old media was removed and 2 mL of DMEM with 10% FBS was added. mRNA was harvested at the 48 hour time for qPCR. Cell survival was measured using WST-8 assay. To study the effects of silencing on cell migration, a point scratch assay was conducted using established technique with images collected at 0, 15 and 24 hours. Analysis of the images was conducted using Image J.
Results:
We successfully introduced SphK1 siRNA into the 4T1 cells using sCA system, demonstrated through qPCR with a statistically significant reduction of SphK1 message production (67%, p value 0.008). We found that sCA transfection of SphK1 siRNA significantly decreased 4T1 cell survival compared to vector treated cells. We also observed faster closure of scratch area in cells treated with a non-targeting vector, as opposed to cells treated with SphK1 siRNA.
Conclusion:
We have shown that our new inorganic nanoparticle delivery system, sCA, was successful in gene silencing of SphK1 in vitro and suppressed 4T1 cell proliferation and migration. Considering the advantage of sCA in gene delivery to cancer, we have high expectations regarding the application of this approach in vivo, which has the potential to transform the treatment frontier and to positively impact patient outcomes in the future.
