Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

Table 1 Specific inhibition of SMMC-7721 human hepatoma cell growth by alpha-fetoprotein (AFP) antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) in vitro

Concentration (μmol/L)	Hepatoma cell growth (absorbance at 570 nm)
	Sense	Antisense	Oligo dT15
0	0.83 ± 0.10	0.83 ± 0.10	0.83 ± 0.10
6.3	0.84 ± 0.08	0.50 ± 0.08 (39.8)a	0.82 ± 0.11
12.5	0.76 ± 0.06	0.44 ± 0.04 (46.9)a	0.87 ± 0.07
25.0	0.72 ± 0.13	0.42 ± 0.03 (49.4)a	0.88 ± 0.18

SMMC-7721 cells were incubated with various concentrations of S-ODNs for 48 h in vitro. A specific growth inhibition was observed by the treatment with antisense S-ODNs. Values in parentheses: percent inhibition. Each value represents mean ± SD of 3 separate experiments.

^aP < 0.05 (Student's t test) compared with 0 μmol/L.

Table 2 Inhibition of AFP protein expression and apoptosis induction in SMMC-7721 human hepatoma cells by AFP antisense S-ODNs in vitro

Concentration (μmol/L)	AFP protein expression (absorbance at 460 nm)			Apoptosis (%)
	Antisense	Sense	Oligo-dT15
0	0.32 ± 0.04	0.32 ± 0.04	0.32 ± 0.04	7.3 ± 5.2
6.3	0.22 ± 0.03 (31.5)a	ND	ND	ND
12.5	0.19 ± 0.05 (40.6)a	ND	ND	26.7 ± 7.8a
25.0	0.15 ± 0.02 (53.1)a	0.31 ± 0.06	0.31 ± 0.02	47.9 ± 7.5a

Cells were incubated with different S-ODNs for 48 h in vitro. AFP gene expression was determined by using the avidin-biotin-peroxidase complex immunocytochemical assay. Antisense S-ODNs specifically blocked AFP gene expression in the cells. The cells were also analyzed for apoptotic cell population with a fluorescence-activated cell sorter. AFP antisense S-ODNs induced apoptosis of the hepatoma cells. Values in parentheses: percent inhibition. Each value represents mean ± SD of 2 separate experiments.

^aP < 0.05 (One-way analysis of variance for protein expression and Student's t test for apoptosis) compared with 0 μmol/L. ND: Not done.

Table 3 Therapeutic efficacy of AFP S-ODNs in SMMC-7721 hepatoma bearing nude mice

Group (mg/day × day)	Mean tumor weight (mg)								Toxic deaths	AFP content (% inhibition)
	0*	2	4	6	8	10	12	14
Control (0)	96.7 ± 51.1	191.4 ± 48.3	336.9 ± 67.8	504.8 ± 80.2	636.3 ± 68.8	823.9 ± 187.6	1056.7 ± 182.0	1201.6 ± 208.0	0/6	-
Antisense (0.25 ± 8)	83.1 ± 33.7	145.2 ± 43.8 (24.2)	215.8 ± 49.6 (35.9)a	286.5 ± 49.2 (43.2)a	356.7 ± 52.5 (43.9)a	520.0 ± 56.1 (36.9)a	653.1 ± 128.8 (38.2)a	776.6 ± 61.3 (35.4)a	0/6	68.9a
Antisense (0.50 ± 8)	84.8 ± 34.6	130.3 ± 36.9 (31.9)a	202.3 ± 47.9 (39.9)a	254.2 ± 43.2 (49.6)a	310.8 ± 56.9 (51.2)a	449.9 ± 108.3 (45.4)a	593.9 ± 72.3 (43.8)a	737.3 ± 146.7 (38.6)a	0/6	73.8a
Sense (0.25 ± 8)	100.5 ± 33.5	201.4 ± 44.2	339.1 ± 71.1	510.4 ± 114.1	629.6 ± 109.4	829.5 ± 164.5	1034.9 ± 198.1	1191.9 ± 181.3	0/6	3.4
Sense (0.50 ± 8)	86.9 ± 25.6	187.7 ± 42.3	359.7 ± 59.6	509.5 ± 111.8	659.7 ± 101.7	837.7 ± 147.3	1011.0 ± 142.9	1232.3 ± 149.0	0/6	0

Mice were treated the eighth day after tumor cell implantation with S-ODNs following different schedules. Untreated mice received normal saline only. *Time after S-ODN administration (day). Values in parentheses: percent inhibition. Each value represents mean ± SD of 6 samples.

^aP < 0.05 (Mann-Whitney U test) compared with untreated mice and mice treated with sense S-ODNs.

Table 4 Reversal of SMMC-7721 human hepatoma cell growth inhibition of AFP antisense S-ODNs by hybridization competition in vitro

Antisense (μmol/L)	Sense (μmol/L)	Hepatoma cell growth (absorbance at 570 nm)	Suppression (%)
0	0	1.00 ± 0.08
0	10	0.95 ± 0.07	5
10	0	0.46 ± 0.02	54
10	10	0.51 ± 0.05	49
10	100	0.95 ± 0.05	5

SMMC-7721 cells were incubated with combined AFP sense and antisense S-ODNs for 48 h in vitro. The cell growth suppression induced by antisense S-ODNs was reversed by hybridization competition of sense S-ODNs. Each value represents mean ± SD of 3 separate experiments.