Effect of shikonin on multidrug resistance in HepG2: The role of SIRT1

Abstract

Context: Overexpression of SIRT1 is considered to enhance the resistance of HepG2 cells to irradiation. Shikonin, a naturally occurring naphthoquinone compound, displays anticancer effects and circumvents cancer drug resistance.

Objectives: This study investigated the MDR reversal effect of shikonin induced by the overexpression of SIRT1.

Materials and methods: The overexpression of SIRT1 in HepG2 cells was established by lentivirus infection. Five days after transduction, real-time quantitative polymerase chain reaction and western blotting were used to detect the expression of SIRT1 and MDR1/P-gp. Drug resistance was also evaluated by flow cytometry after rhodamine-123 staining. On day 5, the multidrug resistance cells were treated by shikonin (10(-7), 10(-6), and 10(-5) µmol/L) one time. The cell viability was detected by the MTT assay, and apoptosis was evaluated by Hoechst 33342 staining and caspase-3 activity 24 h after shikonin treatment.

Results: Overexpression of SIRT1 decreased rhodamine-123 staining and successfully produced the R-HepG2 cell line. Compared with HepG2, the expression of MDR1/P-gp mRNA (3.45 ± 0.35) and protein (1.40 ± 0.05) were both upregulated in R-HepG2. Shikonin inhibited cell viability (from 93.9 ± 2.1 to 66.7 ± 1.5%), induced apoptosis of R-HepG2 (apoptotic ratio from 3.5 ± 0.8 to 47.5 ± 2.7%, caspase-3 activity from 103.5 ± 1.9 to 329.2 ± 14.9%, respectively), downregulated the mRNA and protein expression of SIRT1 and MDR1/P-gp, and decreased rhodamin 123 efflux.

Discussion and conclusion: In the present study, we demonstrated that shikonin is able to overcome drug resistance in hepatocellular carcinoma cells, and the mechanism is related to the SIRT1-MDR1/P-gp signaling pathway.

Keywords: Apoptosis; HepG2; NAD+-dependent deacetylase; caspase-3; hepatocellular cancer; multidrug resistance 1/P-gp; paclitaxel; rhodamin-123; sirtuin 1.