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. 2018 Feb;15(2):1484-1492.
doi: 10.3892/etm.2017.5591. Epub 2017 Dec 5.

Shikonin Causes Apoptosis by Disrupting Intracellular Calcium Homeostasis and Mitochondrial Function in Human Hepatoma Cells

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Free PMC article

Shikonin Causes Apoptosis by Disrupting Intracellular Calcium Homeostasis and Mitochondrial Function in Human Hepatoma Cells

Hui Wang et al. Exp Ther Med. .
Free PMC article

Abstract

Shikonin is known to suppress proliferation and induce apoptosis in a variety of cancer cell lines. In the present study, SMMC-7721 human hepatocellular carcinoma cells were treated with shikonin (1, 2 or 4 µM) for 12-48 h. Cell morphological alterations and DNA damage were determined. Furthermore, changes in cell cycle, mitochondrial transmembrane potential, calcium homeostasis and levels of reactive oxygen species were measured. Shikonin-treated SMMC-7721 cells exhibited morphological changes and DNA damage. Shikonin inhibited cell proliferation causing cell cycle arrest at the G0/G1 phase and induced apoptosis in a dose- and time-dependent manner. Shikonin-induced apoptosis was associated with activation of caspases-3, -8 and -9, elevated levels of intracellular Ca2+ and reactive oxygen species, reduced mitochondrial membrane potential and enhanced efflux of Ca2+ and K+. Gene expression B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), p53 and caspase-3 was up-regulated, whereas Bcl-2 expression was downregulated. Shikonin caused apoptosis by inhibiting cell cycle progression, disrupting Ca2+ homeostasis, inducing oxidative stress and triggering mitochondrial dysfunction. Activation of caspases-3, -8 and -9, K+ efflux, and regulation of Bax, Bcl-2, p53 and caspase-3 expression are involved in the process. These results provide in-depth insight into the mechanisms of action of shikonin in the treatment of cancer.

Keywords: DNA damage; cell cycle progression; human hepatocellular carcinoma; mitochondrial function; tumorigenesis.

Figures

Figure 1.
Figure 1.
Apoptotic effects of shikonin on SMMC-7721 cells. (A) Chemical structure of shikonin. (B) Cytotoxicity analysis via the MTT assay. Values are expressed as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. corresponding control. (C) DNA fragmentation analysis of cells treated with shikonin for 24 h. (D) Morphological changes of SMMC-7721 cells treated with shikonin for 24 h (magnification, ×100). (E) Transmission electron microscopy images of changes in subcellular structures after 24 h (scale bar, 2 or 5 µm). (F) Morphological observation of nuclear damage in SMMC-7721 cells after 24 h (scale bar, 50 µm). (G) Apoptosis analysis of cells treated with shikonin for 24 h using a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay (magnification, ×40). OD 490, optical density at 490 nm; M, marker.
Figure 2.
Figure 2.
Effect of shikonin on apoptotic rates and cell cycle. (A) Apoptotic rates of SMMC-7721 cells treated with shikonin for 24 h (scatter plot data at 12, 36 and 48 h not shown). (B) Apoptotic rates of SMMC-7721 cells treated with shikonin for 12, 24, 36 and 48 h. (C) Cell cycle progression and sub-G1 content of SMMC-7721 cells after shikonin treatment for 24 h. (D) Distribution of cells in the sub-G1, G0/G1, S and G2/M phases of the cell cycle. Values are expressed as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. corresponding control. PI, propidium iodide; FITC, fluorescein isothiocyanate.
Figure 3.
Figure 3.
Effect of shikonin on cell mitochondrial and intracellular Ca2+. (A and B) Mitochondrial membrane potential of SMMC-7721 cells at 24 h after treatment with shikonin. (C and D) Intracellular Ca2+ homoeostasis of SMMC-7721 cells treated with shikonin for 24 h. A rightward-moving peak indicates an increase in intracellular free Ca2+. (E) Ca2+ flux and (F) K+ flux in SMMC-7721 cells prior to and after treatment with 4 µM shikonin. Values are expressed as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. corresponding control.
Figure 4.
Figure 4.
Analysis of shikonin-induced apoptosis of SMMC-7721 cells. (A) Generation of intracellular reactive oxygen species in SMMC-7721 cells treated with shikonin for 24 h. (B) Caspase-3, −8, and −9 activities were determined in cytosolic extracts of SMMC-7721 cells at 24 h after treatment with shikonin. (C and D) Analysis of Bax, Bcl-2, p53 and caspase-3 mRNA expression levels at 24 h (normalized to GAPDH). Values are expressed as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. corresponding control. OD 405, optical density at 405 nm; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

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References

    1. Gong K, Li W. Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: A potential new treatment for hepatocellular carcinoma. Free Radic Biol Med. 2011;51:2259–2271. doi: 10.1016/j.freeradbiomed.2011.09.018. - DOI - PubMed
    1. Brown JM, Attardi LD. The role of apoptosis in cancer development and treatment response. Nat Rev Cancer. 2005;5:231–237. - PubMed
    1. Chen X, Yang L, Zhang N, Turpin JA, Buckheit RW, Osterling C, Oppenheim JJ, Howard OMZ. Shikonin, a component of Chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1. Antimicrob Agents Chemother. 2003;47:2810–2816. doi: 10.1128/AAC.47.9.2810-2816.2003. - DOI - PMC - PubMed
    1. Hsu PC, Huang YT, Tsai ML, Wang YJ, Lin JK, Pan MH. Induction of apoptosis by shikonin through coordinative modulation of the Bcl-2 family, p27, and p53, release of cytochrome c, and sequential activation of caspases in human colorectal carcinoma cells. J Agric Food Chem. 2004;52:6330–6337. doi: 10.1021/jf0495993. - DOI - PubMed
    1. Min R, Tong J, Wenjun Y, Wenhu D, Xiaojian Z, Jiacai H, Jian Z, Wantao C, Chenping Z. Growth inhibition and induction of apoptosis in human oral squamous cell carcinoma Tca-8113 cell lines by shikonin was partly through the inactivation of NF-kappaB pathway. Phytother Res. 2008;22:407–415. doi: 10.1002/ptr.2340. - DOI - PubMed
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