Acetyl Shikonin Inhibits Proliferation And Induces Apoptosis By Triggering Endoplasmic Reticulum Stress In Esophageal Squamous Cell Carcinoma

Objective:In this study,KYSE180 and KYSE450 cells of human esophageal cancer(HEC)cells were used as research objects to discover the molecular mechanism of acetyl shikonin(AS)induced-endoplasmic reticulum stress(ER stress),and to elucidate the relationship between ER stress and apoptosis with acetyl shikonin treatment in esophageal cancer cells.Our data identified the anticancer action of AS,which strengthened it as promising candidate in HEC chemotherapy.Methods:1.In this study,esophageal cancer cells were treated with different concentrations of acetyl shikonin(0,0.625,1.25,2.5,5,10 and 20 μM)respectively.The proliferation of KYSE180,KYSE450,KYSE510,EC109,EC9706 and TE10 cells was observed after AS treatment for 24 hours.The sensitive cell lines of HEC and the optimal concentration of acetyl shikonin were determined by CCK-8 assay.2.KYSE 180 cells were treated with indicated acetyl shikonin(0,0.15,0.3,0.6,1.2and 2.4 μM)for 24 hours,and then cultured in complete medium for 2 weeks.The quantity of clones were observed.3.KYSE 180 and KYSE 450 cells were treated with different concentrations of acetyl shikonin for 24 hours.Cells were then washed with PBS and fixed in 70% ethanol at 4°C overnight.The cells were incubated in PBS containing RNase A and PI for 30 min at 37°C.Cells were collected on a nylon mesh filter and cell cycle phases were assayed with a flow cytometer.4.KYSE 180 and KYSE 450 cells were treated with different concentrations of acetyl shikonin for 24 hours.Cells were then washed with PBS twice.The cells were incubated with Annexin V-FITC/PI double staining and incubated for 15 min at 25 °C in the dark.Flow cytometry was applied to detect apoptosis.5.KYSE180 and KYSE450 cells were treated with different concentrations of acetyl shikonin for 24 hours or one concentration of acetyl shikonin for different hours.The expression of apoptosis-related proteins and endoplasmic reticulum stress response-related proteins were detected by Western Blotting.6.The nude mice bearing tumors were established by intraperitoneal injection of different concentrations of acetyl shikonin.The anti-tumor activity of acetyl shikonin in vivo was measured by observing the changes of tumor volume,weight and weight of mice.7.Tumor tissues of mice were pathologically prepared by routine methods.The pathological characteristics of the tumors were observed under microscopy by hematoxylin-eosin(HE)staining and immunohistochemistry.8.KYSE 180 and KYSE 450 cells were treated with different concentrations of acetyl shikonin for 24 hours.Total RNA was isolated from KYSE180 and KYSE 450 cells,and the expression of endoplasmic reticulum stress-related genes were calculated by the comparative Ct method.And the activation of IRE1/XBP1 was detected by enzyme digestion reaction.Results:1.After 24 hours of acetyl shikonin treatment,the viability of esophageal cancer cells KYSE180,KYSE510,EC109,EC9706,TE10 and KYSE450 was decreased in a dose-and time-dependent manner.The IC50 of esophageal cancer cells was between 1.69 and 13.25μM.Respectively the IC50 values of KYSE180 and KYSE450 cells were less than other cell lines,which were 3.72 and 1.69 μM.These results suggested that KYSE180 and KYSE450 cells were more sensitive to acetyl shikonin.2.Cell clones assay showed that the number of clones formed by acetyl shikonintreated KYSE180 cells was significantly reduced in a dose-dependent manner.The number of clones in the control group was 301.When the concentration of acetyl shikonin reached1.2 μM,KYSE180 cells could hardly form clones.3.Cell cycle arrest occurred in KYSE180 cells and KYSE450 cells 24 hours after treatment with acetyl shikonin.KYSE180 cells significantly increased the percentage of cells in G1 phase at low concentration and increased the percentage of cells in S phase at medium and high concentration.But KYSE450 cells increased the percentage of cells in S phase at low concentration and increased the percentage of cells in G1 phase at medium and high concentration.Acetyl shikonin can effectively block cell cycle suggesting that acetyl shikonin can inhibit the proliferation of KYSE180 cells and KYSE450 cells by blocking cell cycle.4.The morphology under microscope of KYSE180 cells and KYSE450 cells changed dramatically after 24 h of treatment with acetyl shikonin,and the apoptotic rate increased in a dose-dependent manner.The effect of acetyl shikonin on apoptosis of esophageal cancer cells was confirmed by flow cytometry.After 24 hours of treatment with acetyl shikonin,the apoptotic rate of KYSE180 cells and KYSE450 cells increased in a dose-dependent manner.These results suggest that acetyl shikonin can significantly induce apoptosis of esophageal cancer cells.5.Western blotting showed that the expression of c PARP in KYSE180 cells and KYSE450 cells were increased in a dose-dependent manner.And cleaved caspase-3 began to activate significantly after treatment with acetyl shikonin above 3 μM.However,the change of BAX is not obvious.These results suggest that acetyl shikonin can induce apoptosis of esophageal cancer cells in a time-and dose-dependent manner.6.Acetyl shikonin treatment significantly suppressed the growth of KYSE180 xenografts.The results showed that the difference of tumor volume between the two groups was more significant.The weight of tumors showed that acetyl shikonin could significantly reduce the weight of tumors in a dose-dependent manner.However,the weight of mice in the experimental group began to decrease slightly after 10 days’ treatment,and the specific mechanism needs further study.7.Tumor tissues were detected by HE staining and immunohistochemistry.After treatment with acetyl shikonin,the expression of Ki67 in tumor tissue decreased significantly.These results suggest that the proliferation of esophageal cancer cells decreases with the increase of acetyl shikonin concentration.Compared with the control group,the expression of endoplasmic reticulum stress-related proteins BIP and CHOP increased with the increase of acetyl shikonin concentration,suggesting that acetyl shikonin may induce apoptosis of tumor cells through endoplasmic reticulum stress.8.Western blotting detected the expression of a series of endoplasmic reticulum stress-related proteins.The results showed that BIP was significantly up-regulated with the treatment of acetyl shikonin in a time-and dose-dependent manner.p-e IF2α(S51)increased significantly after 1 hour of acetyl shikonin treatment and lasted for nearly 24 hours.This suggests that acetyl shikonin activates the PERK/e IF2α signaling pathway in endoplasmic reticulum stress.In addition,the protein levels of IRE1α,Ero1-Lα,CHOP and PDI in KYSE180 and KYSE450 cells were significantly increased,while the expression level of Calnexin was significantly decreased after acetyl shikonin treatment.9.With the increase of acetyl shikonin concentration,XBP1,which could not be digested by Pst I,increased gradually in KYSE180 and KYSE450 cells.These results suggest that acetyl shikonin can activate IRE1/XBP1 signaling pathway.10.The expression of endoplasmic reticulum stress-related genes after acetyl shikonin treatment was detected by Real-time PCR.Except BIP,the expression of CHOP,ATF3 and ATF4 were up-regulated in a dose-dependent manner.The expression of ATF3 and CHOP were increased most significantly,but ATF6 did not change significantly.Acetyl shikonin may up-regulate the expression of ATF3 and CHOP through endoplasmic reticulum stress and promote cell apoptosis.The specific mechanism needs further study.Conclusion:1.Acetyl shikonin significantly inhibited the proliferation of esophageal cancer cell lines,and its half inhibitory concentration IC50 ranged from 1.69 to 13.25 μM.It also induced cell arrest and inhibited the cloning ability of cancer cells.2.Acetyl shikonin can activate caspase 3,promote PARP shear and induce apoptosis,but the change of BAX is not obvious,suggesting that BAX may not be involved in apoptosis induced by acetyl shikonin.3.Acetyl shikonin significantly inhibited the proliferation of tumors in nude mice,reduced the volume and weight of tumors.4.Acetyl shikonin induces endoplasmic reticulum stress in esophageal cancer cells,up-regulates the expression of BIP and PDI,down-regulates the expression of Calnexin,and activates PERK/e IF2α and IRE1/XBP1 pathways,and then induces apoptosis by increased the expression of ATF3 and CHOP.