Oridonin increases anticancer effects of lentinan in HepG2 human hepatoblastoma cells

Abstract

The aim of the present study was to investigate whether oridonin is able to increase the effects of lentinan (LNT) in HepG2 human hepatoblastoma cells by MTT, flow cytometry, reverse transcription-quantitative polymerase chain reaction and western blot analysis. The in vitro results demonstrated that 20 µg/ml of oridonin was a nontoxic concentration for L02 normal liver cells and HepG2 liver cancer cells. Furthermore, treatment with 0-200 µg/ml LNT was only able to decrease the viability of HepG2 liver cancer cells. The growth inhibitory rate of the LNT-L (100 µg/ml) treatment group was 20.7% and the rate of the LNT-H (200 µg/ml) treatment group was 54.8%. Notably, the growth inhibitory rate of the oridonin + LNT-H group was 84.3%. The highest percentage of apoptotic cells was observed in the oridonin + LNT-H group (20 µg/ml oridonin and 200 µg/ml LNT). The percentage of apoptotic cells in the oridonin + LNT-H group was significantly different from the percentage of apoptotic cells in the LNT-H (26.1%) and the LNT-L (16.8%) groups. Treatment with LNT produced an increase in caspase-3, caspase-9, Bcl-2-like protein 4, p53, p21, nuclear factor κB inhibitor-α mRNA and protein expression and a decrease in B-cell lymphoma 2 and nuclear factor-κB expression in HepG2 cells compared with untreated control cells. Treatment with a combination of oridonin and LNT-H induced a further increase in expression with the biggest differences in expression observed between the oridonin + LNT-H group and control. It was observed that treatment with oridonin was able to increase the anticancer effects of LNT in HepG2 cells. Therefore, oridonin may be used to sensitize cells to LNT.

Keywords: HepG2 human hepatoblastoma cells; expression; growth inhibition; lentinan; oridonin.

Figure 1.

Effect of oridonin treatment on the viability of human normal liver cells (A) L02 and (B) HepG2 human hepatoblastoma cells as determined by MTT assay.

Figure 2.

Effect of lentinan treatment on the viability of human normal liver cells (A) L02 and (B) HepG2 human hepatoblastoma cells as determined by MTT assay.

Figure 3.

Flow cytometric analysis of the percentage of apoptotic HepG2 human hepatoblastoma cells. The DNA content of the sub-G1 phase was evaluated. ^a-d*Mean values with different letters over the bars are significantly different (P<0.05) according to Duncan's multiple range test. Oridonin + LNT-H, 20 µg/ml oridonin + 200 µg/ml lentinan; LNT-L, 100 µg/ml lentinan; LNT-H, 200 µg/ml; LNT, lentinan.

Figure 4.

Expression of caspase-3 and caspase-9 in HepG2 human hepatoblastoma cells by reverse transcription-quantitative polymerase chain reaction and western blot analysis. (A) mRNA and (B) protein expression. *P<0.05 vs. control. Oridonin + LNT-H, 20 µg/ml oridonin + 200 µg/ml lentinan; LNT-L, 100 µg/ml lentinan; LNT-H, 200 µg/ml lentinan; LNT, lentinan.

Figure 5.

Expression of Bax and Bcl-2 in HepG2 human hepatoblastoma cells by (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis. *P<0.05 vs. control. Oridonin + LNT-H, 20 µg/ml oridonin + 200 µg/ml lentinan; LNT-L, 100 µg/ml lentinan; LNT-H, 200 µg/ml lentinan; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-like protein 4; LNT, lentinan.

Figure 6.

Expression of p53 and p21 in HepG2 human hepatoblastoma cells by (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis. *P<0.05 vs. control. Oridonin + LNT-H, 20 µg/ml oridonin + 200 µg/ml lentinan; LNT-L, 100 µg/ml lentinan; LNT-H, 200 µg/ml lentinan; LNT, lentinan.

Figure 7.

Expression of NF-κB and IκB-α in HepG2 human hepatoblastoma cells by (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis. *P<0.05 vs. control. Oridonin + LNT-H, 20 µg/ml oridonin + 200 µg/ml lentinan; LNT-L, 100 µg/ml lentinan; LNT-H, 200 µg/ml lentinan; LNT, lentinan; NF-κB, nuclear factor κB; IκB-α, nuclear factor κB inhibitor α.