| Objective:In the previous study,using Rhein as the lead compound,a new Rhein derivative 4a was designed and synthesized by Schlenk technology.The most important feature of this compound is that it causes the endoplasmic reticulum and mitochondria of ovarian cancer cells to swell and produce a large number of vacuoles,but the mechanism is unclear.The main purpose of this study is to investigate the mechanism of non-apoptotic programmed death induced by the new Rhein derivative 4a and its inhibition of invasion and metastasis in ovarian cancer cells.Methods:1.The fluorescence excitation spectrum and emission spectrum of derivarite4a was measured using ultraviolet spectrophotometer.HE staining was applied to observe the morphological changes.After different concentrations of rhein derivative 4a treatment SKOV3 and SKOV3-PM cells,the proliferation of cells was tested by the CCK8 essay.The distribution of cell cycle was detected by flow cytometry.2.Changes in cell organelles were observed by transmission eletron miscroscope（TEM）.The source of intracellular vacuoles was detected by the ER–tracker red probe.The cell apoptosis rate were analyzed by flow cytometer.The protein levels of apoptosis-related proteins（caspase3,caspase9,PARP）,endoplasmic reticulum stress-related proteins（Bip,PERK,IRE1,ATF6,P-eif2α,ATF4）and MAPK related proteins（JNK,P38,ERK and p-JNK,p-P38,p-ERK）were detected by Western blot.3.Cellular invasion and migration abilities were assayed by Transwell and scratch test.The changes of cytoskeleton filament F-actin treated with Rhein derivative 4a were detected by Immunofluorescence.Epithelial mesenchymal transition（EMT）protein such as Vimentin,β-cantenin,E-caderin,Metalloproteinase（MMP2,MMP9）and Rac1protein were detected by Western blot.Results:1.The maximum wavelength of rhein derivative 4a is 520nm,maximum absorption wavelength is 380nm and the maximum wavelength of excitation light is 400-450nm.Rhein derivative 4a can significantly inhibit the proliferation of ovarian cancer SKOV3 and SKOV3-PM4 cells,the half inhibition rate(IC50)is（5.79±1.81）μmol/L and（4.56±3.99）μmol/L respectively.Under light microscope,it was found that Rhein derivative 4a could induce a large number of vacuoles in SKOV3 and SKOV3-PM cells.The cell cycle is blocked at the G2/M phase.2.After treatment with Rhein derivative 4a,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 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 autophagolysosome were observed.Under laser confocal microscope,the vacuoles caused by Rhein derivative 4a appeared at the edge of the nucleus and were surrounded by positive ER-specific markers,indicating that the vacuoles originated from the ER.There was no significant change in the rate of apoptosis with the increase of the concentration of Rhein derivative 4a.The results of Western blotting showed that the derivative 4a-treated group did not involved in cleaved-caspase protein（including caspase3 and caspase9）and poly-ADP-ribose polymerase（PARP）protein activation,whereas cisplatin significantly activated them.The expression of p62 and LC3II in SKOV3 and SKOV3-PM4 cells increased,but LC3I decreased.The results of autophagy flow showed that ovarian cancer cells transfected with（m RFP–GFP-LC3）were more green fluorescent spots and less red fluorescent spots in the cells,compared with the autophagy inducer（RAPA）.It is suggested that there is no autophagic death in the cells treated with Rhein derivative 4a.Western Blot results showed that Rhein derivative 4a up-regulated the endoplasmic reticulum stress marker protein BIP78 in ovarian cancer cells and activated the PERK-p-EIF2α-ATF4signaling pathway.Meanwhile,Rhein derivative 4a could downregulate Alix and activated the phosphorylation of ERK,JNK and P38 of MAPK kinase,leads to apoptosis-like death of ovarian cancer cells.3.Cell scratch test showed that the treatment with rhein derivative 4a significantly inhibited the rate of cell scratch healing and it was in dose-and time-dependent.Transwell assay show that rhein derivative 4a has stronger inhibitory effect on the invasion of ovarian cancer cells than the lead compound Rhein.F-actin staining showed that the ovarian cancer cells treated with Rhein derivative 4a had microfilament breakage,cytoskeleton rearrangement and cytoplasmic vacuolization.After SKOV3 and SKOV3-PM4 cells were treated with derivative 4a for 24 h,the expression of Rac1 protein decreased,the expression of E-caderin protein,the marker of EMT,and the expression ofβ-cantenin protein decreased significantly（P<0.05）.It is suggested that the process of Epithelial mesenchymal transition in ovarian cancer cells was inhibited.The expression levels of MMP2 and MMP9 of matrix metalloproteinases were also significantly lower than those of the blank group（P<0.05）.Conclusions:1.Rhein derivative 4a significantly inhibited the proliferation of ovarian cancer cells.2.Rhein derivative 4a can cause mitochondrial damage and endoplasmic reticulum swelling in ovarian cancer cells,resulting in the accumulation of unfolded or wrong proteins and a large number of vacuoles in ovarian cancer cells.By activating the PERK-p-EIF2α-ATF4 endoplasmic reticulum stress signal pathway,resulting in the phosphorylation of ERK,JNK and P38 of MAPK kinase,and finally inducing the apoptosis-independent programmed death mode-paraptosis.3.Rhein derivative 4a can inhibit the migration and invasion of ovarian cancer cells.The mechanism may be that it down-regulates the expression of Rac1 protein,inhibits the secretion of matrix metalloproteinases,induces cytoskeletal rearrangement,up-regulates E-caderin,a key molecule in EMT process,and down-regulates the expression of Vimentin andβ-cantenin. |
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