Effects of CXCR4 Silencing by Sirna Engrafted Cotionized Dextran in Mouse Models of Colorectal Cancer and Liver Metastasis

Liver metastasis is the main cause of colorectal cancer related mortality. CXCR4 is necessary for the outgrowth of colon cancer micrometastasis. CXCR4 gene expression and serum total lactate dehydrogenase, creatine kinase and alkaline phosphatase levels are often increased in patients with colorectal cancer. RNA interference is a well recognized pathway involved in cellular defense against viral invasion and post transcriptional regulation. This technology has emerged as a promising new strategy for the study of functional genomics and drug target validation. It is currently being evaluated in clinical trials as a potential therapy for cancers. This study aims to evaluate the transfection efficiency of three biodegradable polymers as carriers for CXCR4 siRNAI, II to treat liver metastasis from colorectal cancer in vitro on mouse colon cancer cells (CT26.WT) and human colon cancer cells (HT29) and in vivo balb/c mice. In this study, dextran spermine, pullulan spermine and dextran hexamine were used as non-viral vectors for in vitro and in vivo CXCR4 siRNAs. Characterization of the morphology, size and stability of CXCR4 siRNAs cationized dextran were performed using transmission electron microscopy, particle sizer and zeta potential. CXCR4 expression in human colorectal cell line HT29 was measured by real-time reverse transcription PCR and immunocytochemistry. Cell proliferation assay, cell cycle analysis, acridine orange/propidium iodide staining and ultrastructural changes of cells using transmission electron microscopy were also studied as biological evaluations. Among three carriers studied, dextran spermine showed smallest size 99.25±4.3 nm with suitable zeta potential 34.15±1.55 mV. These findings were further supported by RT PCR that showed more silencing has been achieved by CXCR4 pool siRNA/dextran spermine in comparison to pullulan spermine and dextran hexamine. The percentage of viability for dextran spermine was higher than pullulan spermine and dextran-hexamine. Animal study demonstrated that inhibition of CXCR4 gene with CXCR4 siRNAI, II/dextran spermine was more efficient than naked CXCR4 siRNAI, II and also post treatment follow transfection of tumor cells more efficient than just transfection cells treatment with naked CXCR4 siRNAI, II or CXCR4 siRNAI, II/dextran spermine.CXCR4 expression was correlated with serum total lactate dehydrogenase, alkaline phosphatase and creatine kinase. These data show that CXCR4 expression lactate dehydrogenase, alkaline phosphatase and creatine kinase may be useful markers to predict liver metastasis in colorectal cancer. Dextran spermine demonstrated improved transfection efficiency in siRNA therapy.

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