Antisense oligonucleotide targeting Livin induces apoptosis of human bladder cancer cell via a mechanism involving caspase 3

Abstract

Background and aim: in recent years, Livin, a new member of IAPs family, is found to be a key molecule in cancers. Researchers consider Livin may become a new target for tumor therapy; however, the role of it in bladder cancer is still unclear. The purpose of this article is to investigate Antisense Oligonucleotide (ASODN) of Livin on treating bladder cancer cell and underlying mechanisms.

Methods: Phosphorathioate modifying was used to synthesize antisense oligonucleotides targeting Livin, followed by transfection into human bladder cancer cell 5637. After transfection, Livin mRNA and protein level, cell proliferation and apoptosis changes, caspase3 level and its effect on human bladder cancer transplantable tumor in nude mice were measured.

Result: results showed Livin ASODN effectively inhibited Livin expression and tumor cell proliferation, and these effects probably through enhanced caspase3 activity and apoptosis of tumor cells. In nude mice transplantable tumor model, Livin expressions were inhibited meanwhile caspase3 expression was increased. Tumor growth slowed down and apoptosis was enhanced.

Conclusion: Our data suggest that Livin plays an important role in inhibiting apoptosis of bladder cancer cells. Livin ASODN may promote cell apoptosis, inhibit bladder cancer growth, and become one of the methods of gene therapy for bladder cancer.

Figure 1

Inhibitory rate of 5637 cells transfected with Livin ASODN. After the transfection of bladder cancer cell lines with different concentrations of Livin antisense oligonucleotides, the inhibition effect on cell growth was determined by MTT and a clear dose-dependence was found.

Figure 2

Livin mRNA and protein expression level in each group of cells. After transfected with Livin antisense oligonucleotides, (a) the Livin mRNA was decrease significantly (Lane 1: antisense group; 2: missense group; 3: Lipo group; 4: PBS group) and (b) the Livin protein was decrease significantly(Lane 1: PBS group; 2: missense group; 3: Lipo group; 4: antisense group;), while the other three groups did not have significant difference.

Figure 3

Using confocal laser scanning microscope detects Livin Expression and location. After the transfection of Livin ASODN, the green fluorescence for marking Livin significantly decreased with uneven distribution. It was observed that the volume of some cells significantly reduced with no green fluorescence at all, while the other three groups did not have significant difference. (a: PBS group; b: Lipo group; c: missense group; d: antisense group).

Figure 4

The morphologic changes of each group cells observed by electromicroscope. Antisense group showed more cell degeneration and necrosis, with cell volume enlargement, chromatin margination and dissolving and lipid droplets within the cytoplasm increase, endoplasmic reticulum dilation, and swelling of mitochondria like vacuoles. Occasional plasma membrane rupture and cell collapse were also seen. (a: control group(original magnification × 10000 b: antisense group(original magnification × 4000).

Figure 5

Cell apoptosis rate measurement. Antisense group showed increase of cell apoptosis rate (46.39 ± 9.23) %, while the other three groups did not have significant difference. *, p < 0.05.

Figure 6

Caspase3 activities in the cells of each group. The results of kinase method to detect Caspase3 activity showed that after Livin ASODN transfection with 5637 cells, the Caspase3 activity was significantly increased with the relative activity of 0.062 ± 0.018. Compared with missense group (0.025 ± 0.011), liposome group (0.029 ± 0.016) and PBS group (0.032 ± 0.016), the difference was significant with P < 0.05. The latter three groups had no significant difference *, p < 0.05.

Figure 7

Comparison of tumor volume in nude mice injected with MSODN and ASODN. After injection of Livin ASODN, tumor volume was significantly smaller in ASODN group than in MSODN group from 18 to 30 days. Tumor growth was inhibited by injection of ASODN compared with injection of MSODN. *, p < 0.05.

Figure 8

Apoptosis in tumor tissue of nude mice observed by TUNEL staining. The tumor cell nucleus of Livin ASODN injection group was stained brown-red with nuclear enrichment. And the cytoplasm was dispersedly and slightly stained. Randomly select 10 high power fields for each case to calculate the apoptotic index (AI). The antisense group apoptotic index was 19.60 ± 5.91, which was significantly higher than the control group (3.48 ± 2.35), and the difference was significant with P < 0.05(a Control group, b Livin ASODN group) (original magnification ×400).

Figure 9

Livin and caspase 3 expression level in tumor tissue of nude mice. After injection of Livin ASODN, the Livin expression level in tumor tissue was significantly inhibited (a control group compare b ASOND group) and Caspase 3 expression was significantly increased (c control group compare with d ASOND group).