Anti-insulin-like growth factor-IIP3 DNAzymes inhibit cell proliferation and induce caspase-dependent apoptosis in human hepatocarcinoma cell lines

Abstract

Background

Insulin-like growth factor II (IGF-II) is a fetal growth protein and an important proangiogenic factor controlled by four promoters (P), of which P2–P4 are inactive in the adult liver. Reactivation and dysregulation of IGF-IIP3 in particular is associated with the attenuation of apoptosis and increased proliferation in a number of liver cancer cell types. Its involvement in experimental liver carcinogenesis makes it a potential target for cancer gene therapy. We designed two IGF-IIP3 specific DNAzymes (DRz1 and DRz2) that target IGF-IIP3 messenger RNA (mRNA) with the aim of reducing IGF-II expression through promoter 3.

Methods

IGF-IIP3 mRNA and protein expression levels were assessed using real-time polymerase chain reaction and gel electrophoresis/western blotting after transfection with Lipofectamine® in SMMC-7721, Huh7, and HepG2 cell lines. Cell proliferation was determined via MTT assay; apoptosis was evaluated by fluorescence microscopy and with flow cytometry; procaspase-3 and -9 expression were detected via western blotting; and caspase activity was assayed colorimetrically. Standard procedures were used to calculate means and standard deviations, and P-values below 0.05 were considered to indicate significant differences.

Results

DRzs were transfected into hepatocellular carcinoma cells and the results showed that DRz1, in particular, could decrease the expression of IGF-IIP3 by nearly 50%. Furthermore, DRz1 significantly inhibited cell proliferation and induced apoptosis. In addition, the down-regulation of IGF-IIP3 expression was associated with increased caspase-3 and -9 activity in SMMC-7721 cells after 24 hours of transfection. In all experiments, the efficacy of DRz2 to influence IGF-IIP3 levels and associated effects remained second to DRz1.

Conclusion

Overall, these results suggest that DRz1-based targeting of IGF-IIP3 mRNA might have antitumorigenic activity and may potentially provide the basis for a novel therapeutic intervention in liver cancer treatment, although further development is required.

Keywords:

• DNAzymes
• proliferation
• apoptosis
• IGF-II
• caspase
• hepatocellular carcinoma
• cancer

Acknowledgments

This work was partially supported by the governmental technology department of Jilin province under grant #200505249, the governmental education department of Jilin province under grant #2013188 (MZ and SL) and a Grant for External Collaborative Research AGE-2009 (GD).

Disclosure

GPCD is supported and partially exempted by Bio & Nano-Solutions, Düsseldorf, Germany to pursue external collaborative fundamental scientific research. The authors report no other conflicts of interest in this work.