Anti-lung cancer activity through enhancement of immunomodulation and induction of cell apoptosis of total triterpenes extracted from Ganoderma luncidum (Leyss. ex Fr.) Karst

Abstract

Ganoderma luncidum (Leyss. ex Fr.) Karst. (GLK) has been used traditionally for the prevention and treatment of cancers or tumors for a long time in Traditional Chinese Medicine. The triterpenes as main effective components of GLK have been found to be beneficial for the efficacy. The purpose of this study was to examine the anti-lung cancer activity of triterpenes of GLK in vitro and in vivo and to explore their anti-lung cancer effects and potential mechanisms. A549 cells and Lewis tumor-bearing mice were used to evaluate the inhibition effects of triterpenes on cell proliferation and tumor growth. The IC50 of triterpenes of GLK on A549 cells was 24.63 μg/mL. Triterpenes of GLK could significantly inhibit tumor growth in mice (30, 60 and 120 mg/kg). The immune organs indexes including spleen and thymus were increased remarkedly by the treatment with triterpenes. Moreover, they were able to stimulate the immune response by increasing the expressions of IL-6 and TNF-α. Flow cytometric analysis revealed that cell arrest caused by triterpenes treatment (7.5, 15 and 30 μg/mL) was in the G2/M phase in A549 cells. Triterpenes induced apoptosis by decreasing the expression of the antiapoptotic protein Bcl-2 and pro-caspase 9 and increasing the levels of cleaved-caspase 9. Our findings suggested that the triterpenes of GLK have anti-lung cancer activity in vitro and in vivo via enhancement of immunomodulation and induction of cell apoptosis. The study provides insights into the mechanism of GLK in the prevention and treatment of lung cancer.

Figure 1

HPLC chromatograms of triterpenes in extract of GLK. (1) ganoderic acid C2; (2) ganoderic acid G; (3) Ganoderic acid C6; (4) ganoderic acid B; (5) ganoderenic acid A; (6) ganoderic acid A; (7) lucideric acid A; (8) ganoderenic acid D and (9) ganoderic acid C1.

Figure 2

The chemical structures of triterpenes of GLK.

Figure 3

The MS² of LC/MS ion fragmentation of triterpenes of GLK.

Figure 4

Proliferation inhibition effect of triterpenes on A549 cells after treatment for 36 h. The IC₅₀ of triterpenes on A549 cells was calculated by the SPSS 11.5 software. All data were expressed as means ± SD (n = 12).

Figure 5

Effect of triterpenes of GLK on tumor growth and immune organ indexes in Lewis tumor-bearing mice. (A) tumor weight; (B) inhibition ratio of tumor growth; (C) spleen index; (D) thymus index. All data were taken from different individual experiments and expressed as means ± SD (n = 8).

Figure 6

Induction of triterpenes of GLK on A549 cells apoptosis. Cells were treated with triterpenes of GLK at a concentration of 7.5 μg/mL, 15 μg/mL and 30 μg/mL for 36 h and then co-stained with PI and FITC-conjugated annexin V.

Figure 7

The effect of triterpenes on cell cycle of A549 cells. Cells were treated with blank control (A); 7.5 μg/mL triterpenes (B); 15 μg/mL triterpenes (C); 30 μg/mL triterpenes (D); positive control CTX (10 μg/mL) (E) for 36 h, and then cell cycle analysis was determined by flow cytometry.

Figure 8

Expressions of apoptosis-related proteins after treating with triterpenes in A549 cells. (A) brands of western blotting; (B) the relative expressions.