Effects of AZT and RNA-protein complex (FA-2-b-beta) extracted from Liang Jin mushroom on apoptosis of gastric cancer cells

Abstract

Aim: To investigate the synergistic effects of 3'-azido-3'-deoxythymidine (AZT) and FA-2-b-beta extracted from Ling Jin mushroom on apoptosis of gastric cancer cells MKN45 in vitro.

Methods: MTT analysis was made to examine the inhibition rate of MKN45 cells treated with AZT (2.5, 5, 10 and 20 mg/L) and FA-2-b-beta (5, 10, 20 and 40 mg/L) singly and combinatively for 24, 48 and 72 h. Apoptotic effects were evaluated by morphological methods, DNA agarose gel electrophoresis and flow cytometry, respectively. Telomerase activity was estimated by TRAP-ELISA. The mRNA expression of caspase-3 and Bcl-2 were detected by RT-PCR.

Results: AZT and FA-2-b-beta could significantly inhibit MKN45 cell proliferation and induce its apoptosis. MKN45 cells were inhibited in dose- and time- dependent manner. The inhibition effect of AZT combined with FA-2-b-beta was obviously better than that used singly (0.469 +/- 0.022 vs 1.075 +/- 0.055, P < 0.05, 0.325 +/- 0.029 vs 0.469 +/- 0.022 P < 0.01). AZT used singly and combination of FA-2-b-beta could decrease the activity of tumor cell telomerase, and AZT has synergistic function with FA-2-b-beta. A certain concentration of AZT could up-regulate the expression of caspase-3 mRNA (r = 0.9969, P < 0.01), which was positively related to apoptosis rate, and could down-regulate the expression of Bcl-2 mRNA, which was negatively related to apoptosis rate (r = 0.926, P < 0.01). Furthermore, the effect of AZT combined with FA-2-b-beta was significantly higher than that used singly.

Conclusion: Combination of AZT and FA-2-b-beta has an obviously synergetic effect in the gastric cancer cells MKN45, which has provided a new approach to the treatment of gastric cancer clinically.

Figure 1

MKN45 cells, treated or not treated with FA-2-b-β and AZT. A: Untreated cells; B-D: FA-2-b-β (5 mg/L) + AZT 5, 10 and 20 mg/L for 72 h exposure, apoptotic body (B), nuclear fragmentation (C) and chromatin condensation (D) (Acridine orange staining, × 100).

Figure 2

Agarose gel electrophoresis of DNA of MKN45 cells. M: marker, Lane 1: control; Lane 2-5: MKN45 cells treated by FA-2-b-β (5 mg/L), AZT (2.5 mg/L), FA-2-b-β (5 mg/L) + AZT 5, 10 and 20 mg/L for 72 h, respectively.

Figure 3

Effect of FA-2-b-β (5 mg/L) and AZT on apoptosis rate in MKN45 cell for 72 h (^aP < 0.05).

Figure 4

Expression of Caspase-3 mRNA in MKN45 cells treated with AZT and FA-2-b-β. M: marker; Lane 1: control; Lane 2-6: MKN45 cells treated by FA-2-b-β (5 mg/L), AZT (2.5 mg/L), FA-2-b-β (5 mg/L) + AZT 5, 10, 20 mg/L respectively for 72 h.

Figure 5

Expression of Bcl-2 mRNA in MKN45 cells treated with AZT and FA-2-b-β. M: marker; Lane 1-5: MKN45 cells treated by FA-2-b-β (5 mg/L) + AZT 20, 10 and 5 mg/L, AZT (2.5 mg/L), FA-2-b-β (5 mg/L), respectively for 72 h; Lane 6: control.