Synergistic effect of oxymatrine and angiogenesis inhibitor NM-3 on modulating apoptosis in human gastric cancer cells

Abstract

Aim: To investigate the synergistic effect of oxymatrine (OM) and angiogenesis inhibitor NM-3 on modulating apoptosis in human gastric cancer cell lines SGC-7901, MKN-45, MKN-74.

Methods: Human gastric cancer lines SGC-7901, MKN-45, MKN-74 were treated with OM in the absence and presence of NM-3. The inhibitory rates were detected by MTT assay. Synergistic effect of OM and NM-3 on the growth of survivin, bcl-2, bax and p53 in SGC-7901 cells were examined by semiquantitative RT-PCR and Western blotting, and their growth inhibitory effects were also observed on SGC-7901 tumor xenograft in nude mice.

Results: OM combined with NM-3 exhibited a synergistic inhibitory effect on the growth of SGC-7901, MKN-45 and MKN-74 cells in a time-dependent manner. Twenty-four hours after treatment with OM, NM-3 alone and their combination, mRNA expression of survivin and bcl-2 in SGC-7901 cells decreased, p53 mRNA expression increased. OM (4 g/L) combined with NM-3 significantly increased the expression of p53 mRNA and decreased the expression of survivin and bcl-2 compared with either agent alone (193% +/- 34% vs 129% +/- 12%; 44% +/- 18% vs 92% +/- 18%; 36 +/- 17% vs 93% +/- 23%, P < 0.05). Western blotting showed that the synergistic effect of OM and NM-3 on protein translation of survivin, bcl-2 and p53 was in accordance with their mRNAs. Furthermore, OM/NM-3 combination obviously exhibited antitumor growth effect in xenografted human gastric cancer cells SGC-7901 compared with either agent alone.

Conclusion: OM combined with NM-3 has synergistic inhibitory effects on human gastric cancer cells in vitro and can suppress the growth of xenografted human gastric cancer cells SGC-7901 in vivo.

Figure 1

Growth inhibitory effects of OM alone and in combination with NM-3 on 3 gastric cancer cell lines, SGC-7901, MKN-45 and MNK-74. A: MTT assays were performed to examine the growth inhibitory effect of OM alone. Clear growth inhibition was observed in a time- and dose- dependent manner in each of the gastric cancer cell lines; B: Growth inhibitory effects of OM combined with NM-3. The concentrations of OM used are indicated for each symbol.

Figure 2

After 24 h treatment identification of differentially expressed survivin, bcl-2, bax, p53, β-actin genes in human gastric cancer line SGC-7901 by means of semiquantitative RT-PCR. NM-3 signifies NM-3 alone; OM 1 mg/mL signifies OM 1 mg/mL combined with NM-3; OM 2 mg/mL signifies OM 2 mg/mL combined with NM-3; OM 4 mg/mL signifies OM 4 mg/mL combined with NM-3.

Figure 3

Differentially expressed mRNA histograms of survivin, bcl-2, bax, p53, β-actin genes in human gastric cancer line SGC-7901 after 24 h treatment. There are significant differences in expression of survivin bcl-2 and p53 mRNA between the NM-3 and NM-3 combined OM 4 mg/mL groups (P < 0.05).

Figure 4

Western blotting analysis showing expression of survivin bcl-2 bax and p53 in human gastric cancer line SGC-7901 after 24 h treatment. NM-3 signifies NM-3 alone; OM 1 mg/mL signifies OM 1 mg/mL combined with NM-3; OM 2 mg/mL signifies OM 2 mg/mL combined with NM-3; OM 4 mg/mL signifies OM 4 mg/mL combined with NM-3.

Figure 5

Curve lines of mean tumor volume (mm³) of nude mice with xenografted human gastric cancer in different groups, effects of NM-3 alone and 1 mg/mL, 2 mg/mL, 4 mg/mL OM combined with NM-3 on gastric tumor volume in 36 d.