Shikonin induces apoptosis in the human gastric cancer cells HGC-27 through mitochondria-mediated pathway

Abstract

Background: Gastric cancer (GC) is one of the most frequently occurring digestive tract cancers and fewer chemotherapeutic drugs for GC have shown promising results. In this study, we investigated the anti-tumor activity of shikonin, a natural compound isolated from the Chinese plant Lithospermum erythrorhizon, against the human GC cell line HGC-27.

Materials and methods: HGC-27 cells treated with shikonin at a concentration of 30μM or above showed significant growth inhibition compared to control cells. Shikonin-treated cells also underwent apoptosis as detected by flow cytometric analysis and microscopic examination of cellular morphology. Further investigation into the underlying mechanism of apoptosis by western blot showed that the shikonin promoted the activation of poly-(ADP-ribose)-polymerase, caspase-3 and caspase-9 following 24 h or 48 h of treatment time, as well as the activation of caspase-8, but only after 48 h of treatment time. Furthermore, the levels of mitochondrial membrane potential, B-cell lymphoma 2 (Bcl-2) and Bcl-extra large were reduced following shikonin treatment while the level of Bax was increased. In addition, shikonin also caused a significant reduction of the protein Survivin, while having little effect on the expression on X-linked inhibitor of apoptosis protein.

Conclusion: Taken together, these results showed that the shikonin exhibited its anti-tumor activity against HGC-27 cells through inhibiting cell growth and promoting apoptosis by targeting mitochondrial-related signaling pathway. Our finding may represent a positive step in finding a natural and effective compound that could be important implication for future development of chemotherapeutic and/or chemopreventive agent against GC.

Keywords: Apoptosis; HGC-27 cells; caspases; mitochondrial pathway; shikonin.

Figure 1

Structure of shikonin

Figure 2

Growth inhibition of HGC-27 cells by shikonin. HGC-27 cells were treated with different concentrations (1, 3, 10, 30 or 100 μM) of shikonin for 24 h and 48 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell viability was expressed as a percentage of surviving cells relative to control cells (no treatment). Data are the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control cells

Figure 3

Shikonin-induced apoptosis in HGC-27 cells. (a) Sub-G1 DNA content in HGC-27 cells as analyzed by flow cytometry after treatment without or with shikonin. (b) Morphology of HGC-27 cells following treatment without or with shikonin. Cells were first stained with Hochest-33258 and then observed under a fluorescence microscope

Figure 4

Effects of shikonin on the levels of poly-(ADP-ribose)-polymerase (PARP), caspases and mitochondrial membrane potential (MMP) in HGC-27 cells. (a) Changes in the levels of activated PARP, caspase-3, caspase-8 and caspase-9. HGC-27 cells were treated with shikonin (3, 10 or 30 μM) for 24 h or 48 h, and the total proteins were extracted and analyzed by Western blot analysis. (b) Changes in the level of MMP. HGC-27 cells were treated with shikonin (30 μM) for 6, 12, 24 and 48 h. Disruption of MMP was determined according to changes in fluorescence density using rhodamine-123

Figure 5

Effect of shikonin on apoptosis related proteins. HGC-27 cells were treated with shikonin (3, 10 and 30 μM) for 24 h and 48 h. Total cell lysates or cytosolic fractions were prepared and the levels of B-cell lymphoma 2 family proteins as well as of Surivin and XIAP were determined by western blot