The Research On Toyocamycin Induced A549 Apoptpsis And Its Endoplasmic Reticulum Stress-related Mechanisms

Objective:To explore the effects of toyocamycin on cell proliferation and apoptpsis,then evaluate the antitumor effects and its endoplasmic reticulum stress-related mechanisms.Methods:1. Study on the antitumor effects of toyocamycin and its concentration-dependent manner.A549 cells were maintained in RPMI-1640 Medium supplemented with 10% fetal bovine serum.The cells were divided into 6 groups,and made the Final concentration of toyocamycin was 0μM,0.05μM,0.1 μM,0.5uM,1 μM.Cells weremaintained in an anaerobic chamber with 1%O2 and 5%CO2, then used the cck8 cell-counting kit for detecting the proliferation of A549 cells after 24 hours.2.To explore the antitumor effect of toyocamycin and its time-dependent manner.Divided the cells into control groups and toyocamycin groups, added the DMSO and toyocamycin to the corresponding groups,and put the A549 cells into the incubator with 1%O2 and 5%CO2.Used the cck8 cell-counting kit for detecting the proliferation of A549 cells afer 0h,3h,6h,12h,24h,36h.3.To explore the pro-apoptotic effects of toyocamycin.Divide the cells into Normoxic groups, Normoxic+toyocamycin groups, Hypoxia groups, Hypoxia+toyocamycin groups.For all hypoxia experiments, cells maintained in an anaerobic chamber with 1%O2.then use the Annexin V-APC/7-AAD Apoptosis Detection Kit for detecting the apoptosis of cells.4.Reserch on the pro-apoptotic mechanisms of toyocamycin. Divided the cells into Normoxic groups, Normoxic+toyocamycin groups, Hypoxia groups, Hypoxia+toyocamycin groups. Western-Blot technology detected the expression of XBPls and some apoptotic factors(like CHOP,JNK, CASPASE-4), and RT-qPCR technology detected the mRNA of XBPls and the apoptotic factors after 24h.Result:1.The OD value of each group was 1.384±0.014,0.884±0.042, 0.802±0.053,0.763±0.021,0.774±0.040.It indicated that as the concentration of toyocamycin increasing,the number of viable cells were reduced. Toyocamycin can inhibit the proliferation of A549 cells in a concentration-dependent manner.2.In control groups the OD value of each group was 0.220±0.025, 0.255±0.012,0.299±0.019,0.373±0.017,0.666±0.057,1.050±0.016, the A549 cells were approximate a exponential-growth, while the OD value of toyocamycin groups was 0.218±0.016,0.268±0.016,0.285± 0.009,0.316±0.010,0.316±0.010,0.114±0.015, It indicated that toyocamyc-in can inhibition the proliferation of A549 cells obviously,and it works in a time-dependent manner.3.The rate of early apoptosis was 1.58%±0.07% and the late apoptosis was 1.67%±0.07% in control groups.And in hypoxia groups,the early apoptosis rate was 1.62%±0.12%, the late apoptosis rate of was 1.62%±0.12%.However,the apoptosis rates including the early apoptosis and the late apoptosis were both increased significantly in Normoxic+toyocamycin groups, the early apoptosis rate was 1.83%±0.10% and the late apoptosis rate was 3.86%±1.15%. While the highest rate of apoptosis was 2.82%±0.27%,4.77%±0.20% for the early and late apoptosis respectively in Hypoxia+toyocamycin groups.4.In Normoxic and Normoxic+toyocamycin groups,the relative amount of IRE1 a RNA was 1.00±0.01,0.97±0.13 respectly.While the RNA amount was increased in hypoxic conditions, the relative amount was 1.89±0.06,1.93±0.04 in Hypoxia groups and Hypoxia+ toyocamycin groups. The relative amount of XBPls RNA was 1.00± 0.05 in Normoxic, and 1.59±0.06 in Hypoxia groups,however, the XBPls was decreased when deal with toyocamycin,the relative amout of XBP1s RNA was 0.40±0.02 in Normoxic+toyocamycin groups,and 0.45 ±0.04 in Hypoxia+toyocamycin groups.Then we detected the Endoplasmic reticulum stress-related apoptosis factors,the relative amount of CHOP protein was 0.31±0.01,0.29±0.02 in Normoxic and Hypoxia groups,and the relative amount of CHOP RNA was 1.00±0.08,1.06±0.08.However, in Normoxic+toyocamycin groups and Hypoxia+toyocamycin groups the relative amount of CHOP protein was 0.51±0.03,0.48±0.03, and the relative amounts of CHOP RNA was 1.44±0.09,2.08±0.05,both of them were increased comparing to the Normoxic and Hypoxia groups.The same trend could be found in other Endoplasmic reticulum stress-related apoptosis factors. These increased Endoplasmic reticulum stress-related apoptosis factors was shown that the pro-apoptosis effect of toyocamycin was closely related to Endoplasmic reticulum stress.Conclusion:1.Toyocamycin can significantly inhibit the proliferation of A549 cells and induce A549 cells apoptosis.2.Toyocamycin can active Endoplasmic reticulum stress-related apoptosis pathway via inhibit the expression of XBPls,then lead to A549 cell apoptosis. Toyocamycin has a stronger cytotoxicity for the cells which were cultured in hypoxia conditions.