Inhibitory Effect Of Shikonin Alone Or In Combination With Cisplatin On Lung Cancer Cells

Objective:To investigate the efficacy of protease body inhibitors L-shikonin combined with cisplatin or L-shikonin alone on Lung cancer A549 cells proliferation and apotosis in vitro; to investigate whether L-shikonin increase the sensitivity to cisplatin, and investigate the relevant mechanism preliminarily.Methods:1. The effect of L-shikonin combined with cisplatin or L-shikonin alone on the activity of purified 20S protease body.Control group: treated with DMSO; L-shikonin group: treated with series doses of L-shikonin (1μmol/L, 10μmol/L, 20μmol/L); L-shikonin combined with cisplatin group: treated with series doses of L-shikonin plus cisplatin (10μmol/L L-shikonin +2μmol/L cisplatin, 10μmol/L L-shikonin + 4μmol/L cisplatin ). Detect the fluorescence of fluorescent substrate by fluorescence dividing apparatus under the condition of 380nm excitation wavelength and 460nm emission wavelength.2. The inhibitory action of L-shikonin combined with cisplatin or L-shikonin alone on cultured Lung cancer A549 cells tested by MTT assay.The first group: Blank control (without any treatment ); the secend group: cisplatin group (treated with series doses of cisplatin); the third group: L-shikonin group ( treated with 2μmol/L or 4μmol/L L-shikonin); the forth group: treated the same with those in the secend group, besides, 2μmol/L L-shikonin was added; the fifth group: treated the same with those in the secend group, besides, 4μmol/L L-shikonin was added. Cells were seeded in a 96 well microplate, after incubation for 24 h or 48 h, the cells were treated with MTT and DMSO solution, the absorbance was measured at 570nm by a microplate reader, the inhibition ratio was calculated with the following formula: IC (inhibition ratio) = (1-the mean value of absorbance in experiment group/ the mean value of absorbance in control group)×100%.3. The effect of L-shikonin combined with cisplatin or administrated alone in inducing apoptosis and regulating cell cycle of Lung cancer A549 cells tested by flow cytometer.Four groups were assigned: control group; 4μmol/L cisplatin group; 4μmol/L L-shikonin group; 4μmol/L L-shikonin+ 4μmol/L L-shikonin group. cells of each group were tested by flow cytometer after treatment.4. RT-PCR were performed to detect transcription level of excision repair genes-1(ERCC-1).Four groups were assigned: control group; 4mg/L cisplatin group; 4mg/L cisplatin+ 2μmol/L L-shikonin group; 4mg/L cisplatin+ 4μmol/L L-shikonin group. After 24h, the total RNA of four groups was extracted, then the levels of ERCC-1 were tested by agarose gel electrophoresis.Results:1. The effect of L-shikonin combined with cisplatin or administrated alone on the activity of purified 20S protease body.L-shikonin inhibited the activitie of 20S protease body Chymotrypsin-1iked from the concentration of 1μmol/L with a dose-response relationship. Inhibition increased with the dose increased( IC50=12.5μmol/L).There was no significant difference of the inhibitory effect on 20S protease body activitie between L-shikonin combined with cisplatin group and L-shikonin group.2. The inhibitory action of L-shikonin combined with cisplatin or L-shikonin alone on cultured Lung cancer A549 cells tested by MTT assay.The inhibition ratioes of cisplatin groups were as follow: 7.31%±0.49%, 12.48%±0.82%, 18.26%±1.22%, 26.62%±1.34%, 35.12%±1.34%. The inhibitory action on A549 cells increased with the increasing doses of cisplatin. There were significant differences of inhibition ratioes on cell proliferation bewteen each two groups (P<0.05).The inhibition ratioes of L-shikonin group: 22.50%±1.01%, 34.12%±1.22%. The inhibition ratioes of L-shikonin(2μmol/L) group: 33.12%±0.86%, 39.21%±0.76%, 43.19%±0.82%, 56.21%±0.54%, 62.28%±0.62%. The inhibition ratioes of L-shikonin(4μmol/L) group: 33.12%±0.86%, 39.21%±0.76%, 43.19%±0.82%, 56.21%±0.54%, 62.28%±0.62%. The inhibition ratioes of L-shikonin(4μmol/L) combined with cisplatin: 44.23%±0.16%,49.18%±0.78%,56.32%±1.77%,62.28%±0.54%,80.28%±1.21%. The inhibition ratioes L-shikonin combined with cisplatin were higher that of cisplatin group and L-shikonin group(P<0.05). With the doses of L-shikonin increasing the the inhibition ratio on A549 cells increased, and there were significant differences between 2μmol/L and 4μmol/L L-shikonin combined with cisplatin group.3. The extent of apoptosis and cell cycle percentage tested by flow cytometer.Control group: 3.21%±0.24%, cisplatin group: 14.46%±1.56%, L-shikonin group: 35.62%±1.37%, L-shikonin combined with cisplatin group: 58.37%±0.97%. L-shikonin combined with cisplatin could induce apoptosis of A549 cells significantly, and the inducing apoptosis rate were higher than control, cisplatin and L-shikonin group(P<0.05). The percentage of S phase increased obviously, and there were significant diffirences of the phase distribution of cell cycles between each two groups(P<0.05).4. The effect of cisplatin and L-shikonin on A549 cells ERCC-1 expression.The results of RT-PCR shows: The relative expression level of Lung cancer A549 cells ERCC-l mRNA in cisplatin group was higher than control, and the ERCC-l transcriptional levels decreased when cisplatin combined with protease body inhibitors L-shikonin.Conclusion:Protease body inhibitors L-shikonin could inhibit the growth of A549 cells, induce apoptosis, combination of L-shikonin and cisplatin had synergistic inhibitory effects on increasing the sensitivity of A549 cells to cisplatin. The potential mechanism may be related with protease body inhibitors L-shikonin down-regulating the expression of ERCC-1 mRNA.