Molecular Mechanism Of Anthraquinone Derivative P Induced Paraptosis-Like Cell Death Through Endoplasmic Reticulum Stress In The Hepatocellular Carcinoma Cells

Objectives:1.The morphological changes of human hepatocellular carcinoma Hep G2and Hep3B cells which treated with anthraquinone derivative P were studied systematically,and the mode of cells death induced by anthraquinone derivative P was speculated.2.The molecular characteristics of paraptosis were identified by comparing and analyzing the differences between typical apoptosis and autophagic death.3.Combined with paraptosis inhibitor,the signal transduction pathway of paraptosis induced by anthraquinone derivative P in hepatocellular carcinoma cells was preliminarily explored.Methods:In this research,human hepatocellular carcinoma Hep G2 and Hep3B cells were used as research objects and anthraquinone derivative P was used as the test compound.1.MTT assay was applied to detect the proliferation of Hep G2 and Hep3B cells.2.The morphological changes of the cells were observed through light microscope and HE staining.3.The ER-Tracker Red staining to verify if the vacuoles were derived from the endoplasmic reticulum under the laser confocal microscopy.Mitochondrial probe（Mito-Tracker Green）was used to detect the damage of mitochondrial.4.Changes in cell organelles were observed by transmission electron microscopy（TEM）5.The distribution of cell cycle and the apoptosis rate were detected by flow cytometry.6.The protein levels of apoptosis-related proteins PARP,Bcl-2 and Bax;Autophagy-related proteins LC3 and p62,paraptosis protein Alix and endoplasmic reticulum stress-related proteins GRP78,PERK,ATF6,IRE1,ATF4,e IF2αand p-e IF2αwere measured by Western Blotting assay.7.Dual fluorescent lentivirus stub RFP-sens GFP-LC3 was used to monitor the formation of autophagy flux.Results:1.MTT results showed that the anthraquinone derivative P exhibited good inhibition activities on the proliferation of Hep G2 and Hep3B cells,and the IC₅₀was 6.2μM and 12.8μM,respectively,which was better than Rhein.Apoptosis inhibitor Z-VAD could not reverse the cell death induced by anthraquinone derivative P.However,paraptosis inhibitor CHX could significantly increase the survival rate of Hep G2 cells（P<0.05）.2.Under light microscope,it was found that anthraquinone derivative P could induce a large number of vacuoles and cells damage were irreversible.According to the results of HE staining,it was found that the vacuoles appeared around the cells nucleus,and with the extension of time,the vacuoles increased and the cytoplasm lost.3.Under laser confocal microscope,the vacuoles caused by anthraquinone derivative P appeared at the edge of the nucleus and were surrounded by positive ER-specific markers,indicating that the vacuoles originated from the endoplasmic reticulum.Mito-Tracker Green staining showed that the mitochondria changed from rod to dot after treated with anthraquinonederivative P which indicating the cells mitochondria were damaged.4.After treatment with anthraquinone derivative P,the results of transmission electron microscope showed that the endoplasmic reticulum swelled,expanded moderately to severely and formed a large number of cytoplasmic vacuoles with a small amount of flocculent in the vacuoles.The ribosomes on the severely dilated rough endoplasmic reticulum were partially lost,and the swelling of mitochondria and disappearance of cristae were observed,but the structure of cell membrane remained intact and the nucleus did not shrink obviously.No apoptotic bodies and autophagolysosomes were observed.5.The results of flow cytometry showed that the proportion of G2/M phase of control group was 20.67%in Hep G2 cell.After treated with 4,8,16μM anthraquinone derivative P for 24 h,the proportion of G2/M phase was increased to 21.68%,29.79%and 37.61%,respectively.In Hep3B cells,the proportion of G2/M phase of control group was 20.39%.Under the same conditions,the proportion of G2/M phase was increased to 39.65%,43.99%and 45.37%,respectively.The results of apoptosis detection showed that after treated with 4,8,16μM anthraquinone derivative P in Hep G2 cells for 48 h,the early apoptosis rates were 3.88%,7.30%and 8.61%,respectively.When Caspase inhibitor Z-VAD combined with 8μM anthraquinone derivative P,the rate of early apoptosis was still 8.22%.After treated with 10μM cisplatin,the early apoptosis rate of Hep G2 cells was 64.94%,which was significantly higher than that of the control group（6.00%）.When cisplatin combined with Caspase inhibitor Z-VAD,the apoptosis rate was significantly down-regulated to 32.00%（P<0.05）.Similar results were observed in Hep3B cells.The rate of early apoptosis was 5.95%,6.25%and 8.88%,respectively.And the cells treated with 4,8,16μM anthraquinone derivative P for 48 h.The combination of Z-VAD and 8μManthraquinone derivative P showed that the rate of early apoptosis was 7.81%,and there were no significant changes in the rate of apoptosis.The early apoptosis rate of Hep3B cells treated with cisplatin 10μM was 49.36%,which was significantly higher than that of the control group（5.00%）.Combined with Z-VAD,the rate of apoptosis was significantly down-regulated to 24.72%（P<0.05）.6.After treated with anthraquinone derivative P on Hep G2 and Hep3B cells,we did not detect cleaved apoptosis-related protein PARP in the cells.The expression levels of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax did not change significantly.The autophagic-related protein LC3-II was obviously activated,the ratio of LC3-II/LC3-I increased,and the expression level of P62 gradually increased or had no significant change with time.Anthraquinone derivative P can significantly inhibited the expression of Alix protein after treated with 6h and 12 h（p<0.05）,while the level of GRP78protein was continuously up-regulated.Endoplasmic reticulum stress-related proteins PERK,p-e IF2αand ATF4 were first activated,and the protein expression level was maintained at a high level for 48 h,among which the activation of ATF4 was the most obviously.However,there was no significant change in ATF6 and IRE1 protein after treatment with anthraquinone derivative P at different time points.When CHX,an inhibitor of paraptosis,was combined with anthraquinone derivative P,the expression of Alix protein was increased,while the expression of GRP78 and ATF4 protein was inhibited.7.The results of autophagy flux showed that the number of yellow spots in Hep G2 and Hep3B cells treated with anthraquinone derivative P,autophagy inhibitor chloroquine and autophagy activator rapamycin was more than that of the control group.In the cells treated with rapamycin,in addition to the increase of yellow spots,the number of red fluorescent spots also increased significantly（P<0.05）.When anthraquinone derivative P combined with rapamycin,the red fluorescent spots decreased.It is suggested that there is no autophagic death in the cells after treated with anthraquinone derivative P.Conclusions:1.Anthraquinone derivative P showed good anti-liver cancer activity.2.The morphological changes of anthraquinone derivative P induced the cancer cell death are consistent with the characteristics of paraptosis.3.Paraptosis induced by anthraquinone derivative P is an independent Caspase death and without the occurrence of apoptosis and autophagic death.4.Paraptosis induced anthraquinone derivative P is related to endoplasmic reticulum stress.The PERK-e IF2α-ATF4 pathway may be the signaling pathway of paraptosis induced by anthraquinone derivative P.