Rhein Derivative 4s Inhibits The Malignant Progression And Cisplatin Resistance In Ovarian Cancer

BackgroundOvarian cancer is a malignant tumor occuing in ovarian epithelium,with insidiously onset and high mortality.Surgery and platinum-based chemotherapy are the main treatment methods for ovarian cancer.Cisplatin is widely used in a variety of solid tumors because of its clear anti-tumor effect,and it can also significantly relieve ovarian cancer.However,the toxicity and side effects of cisplatin are frequent,and cisplatin resistance will occur in most ovarian cancer patients after a period of treatment,followed by tumor progression and/or metastasis,which will hinder the subsequent treatment.Therefore,it is necessary to find more efficient and less toxic treatment methods.In order to improve its biological activity,we optimized the anthraquinone structure of Rhein and synthesized a series of novel Rhein derivatives.In vitro antitumor activity experiments showed that the derivative 4s had stronger antitumor effect than Rhein and lower toxicity to normal cells.ObjectiveIn this study,the effects of derivative 4s on the growth,metastasis and cisplatin resistance of ovarian cancer were explored in vitro and vivo,and the mechanism of inducing ferroptosis in ovarian cancer cells was further explored.Methods1.The proliferation activities of ovarian cancer SKOV3,SKOV3/DDP,A2780 cells and normal ovarian IOSE-80 cells treated by Rhein,cisplatin and derivatives were determined by MTT assay.Candidate compounds derivative 4s were identified based on the half maximal inhibitory concentration（IC50）and toxicity to normal ovarian cells.After the liver cancer Hep G2 cells,non-small cell lung cancer A549 cells,breast cancer MDA-MB-231 cells,human umbilical vein endothelial Huvec cells,ovarian cancer SKOV3,and A2780 cells were treated with different concentrations of derivative 4s and Rhein,the effects of the derivative 4s on the proliferation of different tumor cells and vascular endothelial cells were determined.The fluorescence spectrum of the candidate compounds was detected by a full-wavelength microplate reader to obtain its optical properties;the distribution of 4s in cells was observed by a laser confocal microscope;The effect of derivative 4s on cell morphology was observed by hematoxylin-eosin（HE）staining.2.After ovarian cancer SKOV3,SKOV3/DDP,and A2780 cells were treated with different concentrations of derivative 4s,plate cloning assay was used to detect the cell clone formation ability;Annexin V-APC staining and flow cytometry to detect cell apoptosis;DNA dye and flow cytometry was used to detect cell cycle progression;wound healing assay and Transwell assay was used to observe cell migration and invasion;Western blot assay was used to detect the expression of apoptosis-related proteins,epithelial-mesenchymal transition（EMT）markers and matrix metalloproteinases（MMPs）;transmission electron microscope was used to observe cell ultrastructure change.3.After ovarian cancer SKOV3,SKOV3/DDP,and A2780 cells were treated with different concentrations of derivative 4s,plate cloning assay was used to detect the cell clone formation ability;Annexin V-APC staining and flow cytometry to detect cell apoptosis;DNA dye and flow cytometry was used to detect cell cycle progression;wound healing assay and Transwell assay was used to observe cell migration and invasion;Western blot assay was used to detect the expression of apoptosis-related proteins,epithelial-mesenchymal transition（EMT）markers and matrix metalloproteinases（MMPs）;transmission electron microscope was used to observe cell ultrastructure change.4.The resistance index of SKOV3/DDP to cisplatin,paclitaxel,doxorubicin were determined by MTT method,and Western blot assay was used to detect the expression of drug resistance-related proteins in each group of cells to clarify the multidrug resistance characteristics of SKOV3/DDP cells.After SKOV3 and SKOV3/DDP cells were treated with different concentrations of derivative 4s,Seahorse XFe24 analyzer was used to detected the cellular energy metabolism.After ovarian cancer cisplatin-resistant SKOV3/DDP cells were treated with derivative 4s combined with different concentrations of cisplatin,MTT assay was used to detect the proliferation activity compared with treated with cisplatin alone.After SKOV3/DDP cells was treated with derivatives 4s,cisplatin,and 4s combined with cisplatin,plate cloning assay was used to detect colony-forming ability;flow cytometry was used to detect mitochondrial membrane potential and cellular ROS;wound healing assay and Transwell assay was used to observe cell migration and invasion;iron ion detection kit was used to detect the accumulation of intracellular iron ions.Western blot experiments was used to detect the expression of apoptosis related proteins and invasion or metastasis related proteins.5.SKOV3 and SKOV3/DDP cells were used to construct ovarian cancer xenograft model in nude mice.Different concentrations of derivative 4s and/or cisplatin were injected intraperitoneally,xenografts size and mice weight were measured every other day.HE staining was used to observed the effects of 4s on the heart,liver and kidneys of mice;Immunohistochemical assay was used to detect the proliferation index Ki67 and ferroptosis regulatory protein GPX4 in xenografts;TUNEL assay was used to detect the apoptosis index in xenografts;Western blot was used to detect the expression of ferroptosis related proteins in xenografts.Results1.A series of modified derivatives have stronger inhibitory effects on the proliferation of ovarian cancer SKOV3,SKOV3/DDP,and A2780 cells,and the IC₅₀is significantly lower than that of Rhein and cisplatin.Among them,the derivative 4s not only has strong cytotoxicity on ovarian cancer cells,the IC₅₀of the three cell lines were 4.64±1.05μmol/L,4.56±0.59μmol/L,4.44±1.03μmol/L,respectively,its effects on normal ovarian IOSE-80 cells was also relatively weak,with IC₅₀is 34.76±9.21μmol/L,there were significant differences between the groups.Derivative 4s significantly inhibit the proliferation of liver cancer,lung cancer,breast cancer and vascular endothelial cells with IC₅₀between 2.49-5.86μmol/L,which are significantly lower than28.64-163.96μmol/L of Rhein.The full-wavelength microplate reader detected that derivative 4s had the maximum absorption at 386 nm,the maximum excitation wavelength was 427 nm,and the maximum emission wavelength was508 nm.According to the fluorescence wavelength range,the derivative 4s was observed by a laser confocal microscopy that it can easily enter the cells,mainly distributed in the cytoplasm,and less in the nucleus.HE staining showed that the cells shrank,became smaller and slender,the nuclei were deformed irregularly,and the cytoplasm was shrunken after treated with derivative 4s.2.The colony formation experiment showed that after the treatment of derivative 4s,the number of ovarian cancer cell clones decreased in a dose-dependent manner,the size of the colonies formed was reduced,and the cells were looser.Flow cytometry showed that the apoptotic rate was increased in a dose-dependent manner after the treatment of derivative 4s.Western blot experiments showed that the expression of the anti-apoptotic protein Survivin was decreased,and the expression of cleaved PARP appeared,which were further proved the apoptosis-inducing effect.In addition,derivative 4s altered cell cycle progression and significantly increased the ratio of G2/M phase in SKOV3 cells and G0/G1 phase in SKOV3/DDP cells and A2780 cells,which could lead to cell cycle arrest.The results of wound healing assay and Transwell assay showed that the invasion and migration ability of cells were significantly reduced after the treatment of derivative 4s.Western blot results showed that the EMT was inhibited and the expression of MMPs family was reduced,which further confirmed that derivative 4s inhibited the invasion and metastasis of ovarian cancer cells.The results of transmission electron microscopy showed that the derivative 4s made mitochondria pyknosis and increased electron density,suggesting that the ovarian cancer cells death pattern caused by derivative 4s may be related to the destruction of mitochondria.3.Using Erastin as a positive control,it was found that the damage to the cell ultrastructure by derivative 4s was similar to that of Erastin,which may be related to ferroptosis.Flow cytometry showed that derivative 4s significantly decreased mitochondrial membrane potential and increased intracellular ROS,which is similar to Erastin.The results of intracellular iron ions showed that the derivative 4s induced the accumulation of intracellular iron ions,which is similarly to Erastin.The results of Western blot experiments showed that the derivative 4s activated the endoplasmic reticulum stress protein GRP78,but inhibited the expressions of downstream PERK,p-e IF2α,CHOP and IRE1,suggesting that 4s may not induce endoplasmic reticulum stress.Derivative 4s up-regulated the ratio of LC3II/LC3I and decreased the expression of P62,suggesting that derivative 4s may cause autophagy.In addition,derivative 4s inhibited the expression of ferroptosis related proteins p-m TOR,GPX4,HIF-1α,activates the expression of p-ERK1/2,which is consistent with the positive control Erastin,suggesting that the mechanism of derivative 4s may be the induction of ferroptosis.4.Compared with parental SKOV3 cells,SKOV3/DDP cells were significantly more resistant to cisplatin,paclitaxel,and doxorubicin,and had higher expression levels of MRP1 and LRP,which confirmed the drug resistance characteristics of SKOV3/DDP cells.After the intervention of derivative 4s,the expressions of MRP1 and LRP in SKOV3/DDP cells were down-regulated,and cellular energy metabolism was inhibited,suggesting that4s could reduce cell drug resistance.This was confirmed by the combined effect of 4s and cisplatin.Under the combined efffect,the reversal fold of cisplatin resistance was up to 16.76 times,and the combination index（CI）<1,indicating that 4s and cisplatin had a synergistic effect on ovarian cancer.Compared with cisplatin alone,the combined effect of 4s and cisplatin further promoted apoptosis,inhibited invasion and metastasis,reduced mitochondrial membrane potential,and induced the accumulation of intracellular ROS and iron ions.It is suggested that 4s may enhance cisplatin sensitivity through the ferroptosis pathway.5.In the nude mouse xenograft model,compared with the control group,the derivative 4s significantly inhibit the growth of xenografted tumors,and the tumor inhibition rate is similar to that of cisplatin.There was no significant difference in the mice weight between derivative 4s and the control group.The mice weight of the 4s combined with cisplatin group was decreased,but still higher than that of the cisplatin group.HE staining showed that the derivative 4s had no obvious toxicity to the heart,liver and kidney of mice,and reduce the toxicity of cisplatin.Immunohistochemistry showed that derivative 4s reduced the the expression of proliferation index Ki67 and ferroptosis regulatory protein GPX4 in xenografts.TUNEL assay showed that the treatment of derivative 4s induced the apoptosis in the xenografts.Western blot showed that the derivative4s inhibited the expression of p-m TOR,GPX4,HIF-1α,and activated the expression of p-ERK1/2,which was consistent with the in vitro experiments.ConclusionsThis study demonstated that the derivative 4s,which is screened from a series of Rhein derivatives,inhibited the proliferation,induced apoptosis,inhibited cell invasion and metastasis,and increased the cisplatin sensitivity of ovarian cancer cells by triggering the ferroptosis pathway.It effectively weaken the growth of ovarian cancer xenografts in nude mice,has strong anti-ovarian cancer effect in vitro and in vivo,and has low toxicity to normal cells and vital organs of nude mice.This experiment clarified the anti-ovarian cancer effect of derivative 4s in vitro and in vivo,and preliminarily investigated its molecular mechanism,which provided a theoretical basis for the development of derivative4s as an effective anti-ovarian cancer drug.