Shikonin Induces Apoptosis, Necrosis, and Premature Senescence of Human A549 Lung Cancer Cells through Upregulation of p53 Expression

Figure 5

Mediating effect of p53 in shikonin-induced cell growth inhibition, apoptosis, necrosis, senescence, and apoptotic-regulatory protein expressions. (a) MTT study. Pretreatment of A549 cells with pifithrin-α (45 and 60 μM) decreases shikonin (SHK) (2.5 μg/mL-) induced inhibition of cell proliferation. (b) Flowcytometric study. Pretreatment of A549 cells with pifithrin-α (45 and 60 μM) blocks SHK-induced apoptosis and provokes necrosis after 24 hours of treatment. (c) Quantitative analysis of cellular senescence from SA-β-Gal staining. The ratio of positive labeling cells was significantly inhibited by pifithrin in shikonin-treated cells by quantitative analysis of cellular senescence in cells. Values were obtained from three independent experiments and represented as mean ± S.E.M. Significant difference is indicated by and versus Cont. (d) Immunoblotting analysis. Expression of p53 and p16 significantly increased by shikonin was ameliorated by the p53 inhibitor pifithrin-α. Representative sets of data are shown from three independent immunoblotting analyses. (e) Immunofluorescent microscopy. Cellular distribution of p53 and cytochrome proteins in A549 lung cancer cell pretreated with pifithrin prior to administration of shikonin at 2.5 μg/mL. Red signal and green fluorescence indicate the locations of p53 and cytochrome , and blue color represents the cells counterstained with DAPI. Scale bar indicated as 20 μm. These results indicated that the effect of SHK on A549 cells was mediated at least partially by cellular p53.

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