Growth inhibition and apoptosis induction of tanshinone II-A on human hepatocellular carcinoma cells

Abstract

Aim: To evaluate the effects of tanshinone II-A on inducing growth inhibition and apoptosis of human hepatocellular carcinoma (HCC) cells.

Methods: The human hepatocellular carcinoma cell line SMMC-7721 was used for the study. The cells were treated with tanshinone II-A at different doses and different times. Cell growth and proliferation were measured by MTT assay, cell count and colony-forming assay. Apoptosis induction was detected by microscopy, DNA ladder electrophoresis and flow cytometry.

Results: In MTT assay, the inhibitory effect became gradually stronger with the passage of time, 24, 48, 72 and 96 h after treatment with tanshinone II-A, and the most significant effect was observed at 72 h. On the other hand, the increase of doses (0.125, 0.25, 0.5, 1.0 mg/L tanshinone II-A) resulted in enhanced inhibitory effect. The growth and proliferation of SMMC-7721 cells were obviously suppressed in a dose- and time-dependent manner. The results of cell count were similar to that of MTT assay. In colony-forming assay, the colony-forming rates were obviously inhibited by tanshinone II-A. In tanshinone II-A group, the morphology of cellular growth inhibition and characteristics of apoptosis such as chromatin condensation, crescent formation, margination and apoptotic body were observed under light and transmission electron microscopes. DNA ladder of cells was presented in electrophoresis. The apoptosis index (AI) was 16.9% (the control group was 4.6%) in flow cytometry. The cells were arrested in G(0)/G(1) phase, and the expressions of apoptosis-related genes bcl-2 and c-myc were down-regulated and fas, bax, p53 up-regulated.

Conclusion: Tanshinone II-A could inhibit the growth and proliferation of HCC cell effectively in vitro by apoptosis induction, which was associated with up-regulation of fas, p53, bax, expression and down-regulation of bcl-2 and c-myc.

Figure 1

Growth-inhibitory effect of Tan II-A on SMMC-7721 cells detected by MTT. A: In a dose-dependent manner detected by MTT; B: In a time-dependent manner detected by MTT.

Figure 2

Morphological characteristics of SMMC-7721 cells before and after Tan II-A treatment. A: Roundish, large and serried SMMC-7721cells (in control group); B: Polygonal, small, detached or sparse, membranous frothed and wizened SMMC-7721cells (II A).

Figure 3

Ultrastructural changes of SMMC-7721 cells with 1.0 mg/L Tan II-A treatment for 96 h under TEM (× 10000). The chromatin condensation, original margination were observed.

Figure 4

DNA agarose gel electrophoresis of SMMC-7721 cells with Tan II-A treatment for 96 h. Lane 1: Marker, Lane 2: Control, Lane 3: 2.0 mg.L^-1, Lane 4: 1.0 mg.L^-1, Lane 5: 0.5 mg.L^-1.

Figure 5

Flow cytometry analysis of apoptosis in SMMC-7721 cells treated with Tan II-A (A) and DMSO (B).