Shikonin, a natural naphthoquinone pigment isolated from Lithospermum erythrorhizon, has been reported to suppress growth of various cancer cells. This study was aimed to investigate whether this chemical could also inhibit cell growth of lung cancer cells and, if so, works via what molecular mechanism. To fulfill this, A549 lung cancer cells were treated with shikonin and then subjected to microscopic, biochemical, flow cytometric, and molecular analyses. Compared with the controls, shikonin significantly induced cell apoptosis and reduced proliferation in a dose-dependent manner. Specially, lower concentrations of shikonin (1–2.5 μg/mL) cause viability reduction; apoptosis and cellular senescence induction is associated with upregulated expressions of cell cycle- and apoptotic signaling-regulatory proteins, while higher concentrations (5–10 μg/mL) precipitate both apoptosis and necrosis. Treatment of cells with pifithrin-α, a specific inhibitor of p53, suppressed shikonin-induced apoptosis and premature senescence, suggesting the role of p53 in mediating the actions of shikonin on regulation of lung cancer cell proliferation. These results indicate the potential and dose-related cytotoxic actions of shikonin on A549 lung cancer cells via p53-mediated cell fate pathways and raise shikonin a promising adjuvant chemotherapeutic agent for treatment of lung cancer in clinical practice.

Background: Endothelial dysfunction is one of the underlying causes for vascular diseases. tert-Butyl hydroperoxide (t-BHP), a short-chain lipid hydroperoxide analog, has been reported to cause adverse effects in different systems. However, the adverse actions of t-BHP on inducing endothelial dysfunction are unclear and remain under investigation. Aim of the present study was to identify the pathobiological mechanisms of t-BHP in rat aortic endothelial cells and thoracic aorta. Methods: Primary cultured cells were treated with vehicle or t-BHP (50, 100, 250, 500, and 1,000 µM). Cells were harvested and specific analyses regarding cellular apoptosis, necrosis, and senescence were conducted. Additionally, t-BHP (0.1, 0.2, and 0.4 mmol/kg body weight) or vehicle were administered to male rats (the young group at 6 weeks of age and the mature adult group at 24 weeks of age) daily through intraperitoneal injections. At 10 days after the first drug treatment apoptotic endothelial toxicity was evaluated by biochemical, histological, and immunofluorescent staining analyses. Results: Dose-dependent effects of t-BHP were observed for the reduction of cell viability, deterioration of cell toxicity, initiation of cell cycle arrest, and triggering of apoptosis and necrosis. Moreover, increase of cells stained positive for senescence-associated beta-galactosidase (SA-β-Gal), amelioration of telomerase activity, and precipitations of necrotic, cell cycle, and apoptotic signaling regulatory proteins were also found in the in vitro model. In the in vivo study, results indicated that t-BHP at higher doses enlarged the intima-medial thickness of descending aorta in the mature adult group, but led to aortic narrowing in the young group. Increased injuries were observed by upregulating endothelial apoptosis-and senescence-positive staining, along with caspase-3 activity and down-regulating telomerase activity. Conclusion: These results confirmed that t-BHP impaired aortic endothelial cell survival at least partially by the activation of p53-mediated signaling pathways, inhibition of cell cycle regulatory proteins, and initiation of cellular senescence-related signaling pathways. In conclusion, t-BHP was found to be a major trigger for impairing aortic endothelial cell survival and deteriorating vascular dysfunction in experimental practice.