Shikonin induces apoptosis in Caco-2 cells by an increase in caspase-3 and the inhibition of the regulating protein Bcl2

Shikonin is one of the active principles in the root of Lithospermum erythrorhizon Sieb. & Zucc. (Boraginaceae), widely used in traditional Chinese medicine for its anti-inflammatory and wound-healing properties. Recent research highlights shikonin's anticancer properties as well as its preventive ability in acute ulcerative colitis. To evaluate the potential beneficial effects of shikonin on colorectal cancer, a frequent outcome in ulcerative colitis patients, we studied the antiproliferative effect of this naphthoquinone in human colorectal adenocarcinoma cells (Caco-2). Cytotoxicity of shikonin was evaluated using the colorimetric assay described by Mosmann. Flow cytometry was used to study shikonin's proapoptotic activity and to evaluate its effect on cell cycle. Moreover, the study was complemented with the analysis by Western blot of the expression of proteins that play a key role in the apoptotic process.

Our results show that shikonin presents a dose-dependent cytotoxic effect on Caco-2 cells (IC₅₀= 9.84 µM). Cell cycle analysis by flow cytometry demonstrated that this naphthoquinone significantly increased the cell subpopulation in SubG₀ phase, which is compatible with a proapoptotic mechanism. In addition, it decreased populations located in preparative G₀/G₁ and G₂/M phases, an effect that indicates a metabolic activation of quiescent cells. In agreement with these results, Western blot analyses revealed that shikonin has a marked dose-dependent apoptotic mechanism, evidenced by an increase in caspase 3 (a proapoptotic protein) and the inhibition of the regulating protein Bcl2 (antiapoptotic protein). In conclusion, shikonin shows a promising therapeutic potential as an adjuvant in first-line therapy for colorectal cancer.