A Study Of The Anti-tumor Effects On Ovarian Carcinoma Of A Novel Curcumin Analogue L50H8 In Vitro And In Vivo

Objective: To study the anti-ovarian cancer effect of a novel curcumin analogue L50H8 applying two-dimensional static cell culture mode,three-dimensional dynamic cell culture mode and in-vivo experiments,and to explore the relvevant mechanisms of this analogue,in order to find new pharmic options for ovarian carcinoma and further assess the application of three-dimensional dynamic cell culture mode for the screening and evaluation of anti-tumor drugs.Methods: Under two-dimensional static cell culture mode,the effects of L50H8 on the proliferation of ovarian cancer cells was assessed with MTT assay;the effect of L50H8 on the cell cycle and apoptosis of ovarian cancer cells was evaluated with flow cytometry;the effect of L50H8 on the cell invasiveness was tested with Transwell assay;the effect of L50H8 on the mRNA expression of C yclin A and C yclin B was tested with quantitative real-time PCR(qRT-PCR).Western Blotting was applied to analyse the protein expressions including cell cycle regulatory proteins C yclin A and Cyclin B,apoptotic regulatory proteins Bax,caspase8,cleaved-caspase3 and AIF,endoplasmic reticulum stress(ERS)assosiated proteins GRP78 and CHOP,and cell invasion-assosiated proteins CD147,MMP2,MMP9 and VEGF.Then,under three-dimensional dynamic cell culture mode formed by sodium alginate three-dimensional cell scaffold and Tissue Flex three-dimensional perfusion platform,CASY cell analysis was used to assess the effect of L50H8 on the proliferation of ovarian cancer cells;flow cytometry was used to evaluate the effect of L50H8 on the cell cycle of ovarian cancer cells;laser confocal microscopy was adopted to observe the apoptosis of ovarian cancer cells doubly stained with Hoechst33342 and PI while the apoptotic rate was calculated;the effect of L50H8 on the mRNA expression of Cyclin A and C yclin B in ovarian cancer cells was assessed with qRT-PCR method;Western Blotting was applied to analyse the protein expressions of C yclin A,Cyclin B,Bax,caspase8,cleaved-caspase3,AIF,GRP78,C HOP,CD147,MMP2,MMP9 and VEGF.Finally,in-vivo experiments were conducted in nude mice to observe the effect of L50H8 on the growth of transplanted ovarian cancer in nude mice while the growth curve was drawn and the inhibition rate was calculated.TUN EL method was used to evaluate the effect of L50H8 on the apoptosis of transplanted ovarian cancer cells in nude mice;Western Blotting was used to analyse the protein expressions in the transplanted ovarian cancer tissue including protein C yclin A,C yclin B,Bax,caspase8,cleaved-caspase3,AIF,GRP78,CHOP,CD147,MMP2,MMP9 and VEGF.Results: 1.Under two-dimensional static cell culture mode,L50H8 could significantly inhibit the proliferation of ovarian cancer cells.In SKOV3 cells,the IC50 of L50H8 was 2.71±0.08μM,and in OVCAR3 cells,the IC50 of L50H8 was 3.08±0.08μM.In SKOV3 cells,L50H8 could increase the proportion of S-phase cells;in OVCAR3 cells,L50H8 could increase the proportion of G2/M-phase cells.In SKOV3 cells,L50H8 could down-regulate the mRNA and protein expression of C yclin A;in OVCAR3 cells,L50H8 could down-regulate the mRN A and protein expression of Cyclin B.L50H8 could induce apoptosis in ovarian cancer cells and up-regulate the protein expression of apoptotic factors Bax and AIF.The protein expression of caspase8 was not influenced and cleaved-caspase3 had no expression in ovarian cancer cells.L50H8 could activate ERS and promote the expression of GRP78 and CHOP proteins.L50H8 could dramatically weaken the invasiveness of ovarian cancer cel s,down-regulating the protein expression of CD147,MMP2,MMP9 and VEGF.2.Under three-dimensional dynamic cell culture mode,L50H8 could also significantly inhibit the proliferation of ovarian cancer cells.In SKOV3 cells,the IC50 of L50H8 was 4.73±0.22μM;in OVCAR3 cells,the IC50 was 5.09±0.18μM.In three-dimensional dynamic experiment,the IC50 of L50H8 was higher than that in two-dimensional static experiment(P<0.01).In SKOV3 cells,L50H8 can increase the proportion of S phase cells,and in L50H8-10μM group,it can also increase the proportion of G2/M phase cells;in OVC AR3 cells,L50H8 has no significant impact on the proportion of cells in each cell cycle.In SKOV3 cells,L50H8 could down-regulate the mRNA and protein expression of C yclin A;in OVCAR3 cells,L50H8 could down-regulate the mRNA and protein expression of C yclin B.L50H8 could induce apoptosis in ovarian cancer cells.At the same concentration,the apoptotic rate induced by L50H8 in three-dimensional dynamic experiment was lower than that in two-dimensional static experiment(P<0.05).L50H8 could increase the protein expression of Bax and AIF,but had no impact on the protein expression of caspase8.C leaved-caspase3 had no expression.L50H8 could activate ERS in ovarian cancer cells and promote the expression of GRP78 and CHOP proteins.L50H8 could down-regulate the expression of protein CD147,MMP2,MMP9 and VEGF.3.The in-vivo experiment in nude mice showed L50H8 can dramatically inhibit the growth of transplanted ovarian carcinoma and induce apoptosis in cancer cells.L50H8 could down-regulate the expression of Cyclin B protein,up-regulate the protein expression of apoptotic factors Bax and AIF in transplanted ovarian cancer tissues;it could increase the expression of ERS-assosiated proteins GRP78 and CHOP,and down-regulate the expression of invasion-assosiated proteins CD147,MMP2,MMP9 and VEGF.L50H8 did not show histological damage to the heart,liver and kidney in nude mice,but toxicity in other aspects could not be excluded.Conclusion: 1.L50H8 shows good anti-tumor activity against ovarian ca ncer in vitro and in vivo,and may become a new candidate drug for ovarian ca rcinoma.2.The mechanism of L50H8 may include arresting cell cycle,inducing apoptosis and ERS,and weakening the tumor invasiveness,which deserves further study.3.The apoptotic induction of L50H8 may be accomplished by up-regulating BAX to activate AIF,but not rely on the mitochondrial caspase pathway.4.Three-dimensional dynamic culture can make cells grow in an environment more similar to the state in vivo,which makes it an advantageous mode for in-vitro cell experiment.