The Apoptosis Of Lung Cancer And Liver Cancer Cells And Their Cancer Stem Cells Induced By Realgar Nanoparticle

Objective:Realgar nanoparticle (nano-realgar) and raw realgar can kill the tumor cells by inducing to apoptosis or suppressing the synthesis of nucleic acid in tumor cells. The goal of this study is to investigate the proliferation-inhibiting and apoptosis-inducing effects of Nano-realgar and raw realgar on lung cancer A549 cells and liver cancer HepG2 cells, and compare the difference in apoptosis-inducing effects of nano-realgar between lung and liver cancer cell population and their CSCs, and explore the molecular mechanism of apoptosis induced by nano-reagar in A549 lung cancer cells and HepG2 liver cancer cells.Methods:Preparation of nano-realgar was mechanically milled using a high-energy planetary ball mill. Particle size is observed by scanning electron microscope(SEM) and transmission electron microscope(TEM). The A549 cells and HepG2 cells were used as the model cells. MTT colormetric assay was used to detect the proliferating activity of A549 cells and HepG2 cells. Lung cancer cells'morphological change was observed by inverted phase contrast microscope. The apoptosis of the A549 cells, HepG2 cells and the CSCs were investigated with Annexin V and propidium iodide (PI) double staining. Flow cytometry (FCM) was employed to assess the expressions of intracellular Bax, Bcl-2, P53 P-glycoprotein(P-gp), breast cancer resistance protein(BCRP) and the activity of Caspase-3.Results: 1.10-100μg/ml nano-realgar significantly inhibited the proliferation of A549 cells and the inhibition ratios showed positive correlative to both concentration and time (P＜0.01). Realgar also suppressed the proliferation of A549 cells, However, the killing effect was obviously lower than the nano-realgar. After exposure to 50μg/ml and 100μg/ml nano-realgar for 48 hours, the apoptotic rate of A549 cells detected by Annexin V/PI staining was increased to 12.53% and 69.19%, respectively. After the same concentration of realgar treated A549 cells, the apoptotic rate of A549 cells was 11.18% and 45.6%.0μg/ml,20μg/ml and 50μg/ml of nano-realgar dealed with the cells for 24h, the activity of Caspase-3 was (2.25±0.17)%, (3.84±0.63)% and (7.35±0.325)%, respectively. After 20μg/ml and 50μg/ml nano-realgar treated the cells for 48 hours, the expression of Bax protein in A549 cells increased from 80.0% to 92.1% and 96.9%. Both Bax and Bcl-2protein expressions in A549 cells also increased with the raised concentration of nano-realgar. Administrated the cells for 48h with 20μg/ml,50μg/ml and 100μg/ml nano-realgar, the relative content of CSCs in the overall A549 cell population increased from(4.35±0.18)% to (10.46±2.15)% and (21.25±1.05)%, Meanwhile the apoptotic rate of cancer stem cells were also obviously up to (4.28±0.42)%, (9.17±1.11)% and (30.71±2.82)%, respectively. The sensitivity of the CSCs to nano-realgar was remarkablely lower than that of the overall A549 cell population; Nano-realgar had little effect on the expressions of BCRP and P-gp protein in lung A549 cancer cells.2. Nano-realgar significantly inhibited the proliferation of HepG2 cells at both concentration-and time-dependent manner (P＜0.01). Raw realgar also restrained the proliferation of HepG2 cells, and its IC50 are 621.31μg/ml,997.02μg/ml and 455.11μg/ml, respectively. After exposure to 100μg/ml and 200μg/ml nano-realgar for 48 hoμrs, the apoptosis of HepG2 cells detected by Annexin V/PI staining was increased, the apoptotic rate of HepG2 cells was 0.14%,7.60% and 14.47%, respectively. But after the same concentration of realgar treated HepG2 cells for the same time, the apoptotic rate of the cells hardly changed. After exposured to 100μg/ml and 200μg/ml nano-realgar for 48 hours, the expression of Bax protein increased from 73.1% to 85.7% and 83.3%, and that of Bcl-2 protein also increased from 75.5% to 83.8% and 97.1% , respectively. Treated with 50μg/ml,100μg/ml and 200μg/ml nano-realgar for 24h, the activity of Caspase-3 in HepG2 cells was clearly up from (6.4±2.05)% to (12.8±0.70)%, (14.9±0.10)% and (25.75±2.45)%, and at 48h, the expression of P53 protein changed from (5.73±0.20)% to (8.08±0.50)%, (8.46±0.80)% and (13.55±1.25)% , and the relative content of CSCs in overall HepG2 cell population dramaticaly increased from (5.48±0.3)% to (16.75±2.05)%, (26.85±1.55)% and (32.3±5.5)%, Meanwhile the apoptotic rate of CSCs increased from (0.3±0.01)% to (8.69±0.66)%, (7.57±0.05)% and (17.34±0.79), respectively. HepG2 cells overexpressed BCRP and P-gp protein. The basic expressions of BCRP and P-gp in HepG2 cells were(3.74±0.56)% and (4.21±0.46)%. After treated with 50μg/ml, 100μg/ml and 200μg/ml nano-realgar, the expressions of them were (7.91±1.71)%, (11.85±1.05)% and (14.95±1.05)%, and (7.88±1.25)%, (14.55±1.75)% and (16.05±0.55)%, respectively. In addition, there also existed co-expressed P-gp and BCRP (P-gp+ BCRP+) in HepG2 cells about (0.61±0.02)%, (2.18±0.54)%, (4.33±0.70)% and (6.04±0.36)%, respectively.Conclusion:Realgar nanoparticle can significantly inhibit the proliferation of A549 cells and HepG2 cells, and induce the cancer cells to apoptosis with a caspases-dependent manner via mitochondrial pathway as well as the cancer stem cells. The growth-inhibiting and apoptosis-inducing effects of realgar nanoparticle are more powerful than that of raw realgar in both A549 cells and HepG2 cells, and lung cancer A549 cells are more sensitive to nano-realgar than liver cancer HepG2 cells.