Studies Of The Anti-Tumor Effects And Mechanisms Of Morusin Against Ovarian Cancer

BackgroundOvarian cancer is one of the most common malignant tumors of female genital system.According to the latest statistical data,the incidence of ovarian cancer is in third place in China and in second place in America among all gynecological malignancies,respectively,whereas ovarian cancer is always the leading cause of gynecological cancer-associated death.Due to dormant clinical symptoms at first and lack of highly sensitive and specific biomarkers for early detection,75%of the patients were diagnosed at FIGO stage III or IV.Although cytoreductive surgery and cisplatin-based chemotherapies have been widely used as conventional therapeutic strategies for ovarian cancer,the five-year overall survival rate remains below 45%.Most women present with advanced disease will develop recurrence and even chemoresistance within 18 months.Therefore,identifying novel effective agents and their mechanisms and developing alternative therapeutic strategies are crucial for improving prognosis of patients with advanced disease.Resistance to apoptosis is one of the most common mechanisms of chemoresistance in ovarian cancer.Thus,investigating alternative non-apoptotic cell death pathways and studying their mechanisms may contribute to the development of new therapies against recurrent,chemoresistant advanced ovarian cancer.Paraptosis is a type of programmed cell death that has been discovered in recent years,which is characterized by extensive cytoplasmic vacuolation due to the dilation of the endoplasmic reticulum(ER)and/or mitochondria.Paraptosis lacks the hallmarks of apoptosis such as pyknosis,formation of apoptotic bodies,DNA fragmentation,and caspase activation.Paraptosis is associated with activation of MAPKs and can be inhibited by Alix and a protein synthesis inhibitor called cycloheximide(CHX).In addition,ER stress,reactive oxygen species production and mitochondrial calcium overload have been demonstrated to play a role in paraptosis induced by different stimuli.Despite increasing studies about paraptosis in recent years,the mechanisms underlying paraptosis have not been fully understood yet and need to be further explored.Recent studies have revealed that multifarious natural compounds present anti-cancer effects by inducing paraptosis.Morusin is a natural prenylated flavonoid first isolated from the root bark extracts of Morus australis by Nomura from Japan in 1982,which has been reported to possess various biological activities such as anti-inflammatory,antioxidant,and antibacterial activities.Moreover,previous reports have proved that morusin exhibit anti-tumor effects against different types of solid tumors,including colorectal cancer,cervical cancer,hepatocarcinoma,breast cancer,prostate cancer and so on.Previous studies about the anti-cancer effects of morusin mainly focused on the mechnisms that morusin can cause apoptotic cell death in various human cancer cells through inhibiting NF-?B and STAT3 pathways.So far,whether morusin has potential anti-cancer activity against ovarian cancer and induces non-apoptotic cell death has never been explored before.In the present study,we investigated the anti-cancer effects of morusin on ovarian cancer as well as the ovarian cancer cell death mode and mechanisms induced by morusin for the first time.We further tested our theories on xenografted nude mice.This study will provide novel insights into identifying alternative therapeutic drugs for ovarian cancer.Part ? The anti-cancer activities of morusin against ovarian cancer cells and its effects on apoptosis,necroptosis and autophagyObjective:1.To test whether morusin has anti-cancer effects against A2780,SKOV-3 and HO-8910 ovarian cancer cells.2.To investigate the effects of morusin on apoptosis,necroptosis and autophagy in A2780 and SKOV-3 ovarian cancer cells.Methods:MTT assay was used to test the effects of morusin on cell viability of A2780,SKOV-3 and HO-8910 cells and the effects of z-VAD-fmk,necrostatin-1,3-Methyladenine(3-MA)and chloroquine(CQ)on the cytotoxicity induced by morusin in ovarian cancer cells.Colony formation assay was used to examine the effects on colony formation ability of A2780,SKOV-3 and HO-8910 cells.EdU uptake assay was utilized to explore the effects of morusin on cell proliferation ability of A2780 and SKOV-3 cells.The morphological changes of A2780 and SKOV-3 cells caused by morusin were visualized via optical microscopy.Western blot analysis was used to examine the expression levels of apoptosis-related proteins(PARP,caspase-3/9 and their cleaved forms)and autophagy-related proteins(LC3,p62,CTSB and CTSD)of ovarian cancer cells.DNA ladder assay was utilized to examine the changes of DNA electrophoresis bands of A2780 and SKOV-3 cells after morusin treatment.EBSS was used to activate autophagic flux,while,CQ was used to block autophagic flux.A2780 and SKOV-3 cells were transfected with mRFP-GFP-LC3 adenovirus to monitor the autophagic flux change.Transmission electron microscopy was utilized to observe ultrastructure changes of A2780 and SKOV-3 cells.Results:1.The effects of morusin on cell viability and cell proliferation ability of A2780,SKOV-3 and HO-8910 cells:MTT assay was used to test the effects of morusin on cell viability of ovarian cancer cells,showing that morusin suppressed cell viability of all the ovarian cancer cell lines in a dose-and time-dependent manner.Then EdU uptake assay was utilized to explore the effects of morusin on cell proliferation ability of ovarian cancer cells,revealing that morusin significantly inhibited cell proliferation in ovarian cancer cells.Colony formation assay was used to examine the effects of morusin on colony formation ability of ovarian cancer cells,showing that colony numbers of ovarian cancer cells were significantly decreased when the concentration of morusin was above 20?M.2.The influences of morusin on morphological changes of A2780,SKOV-3 and HO-8910 cells:Morphological changes in A2780,SKOV-3 and HO-8910 cells after incubation with 30 ?M of morusin were visualized via an inverted phase contrast microscope.Extensive cytoplasmic vacuolation was observed in cells after treatment with morusin for 3 h.Moreover,the vacuoles were gradually enlarged and fused within 6 h.3.The effects of morusin on apoptosis and necroptosis in A2780 and SKOV-3 cells:First,western blot analysis was used to examine the expression levels of apoptosis-related proteins(PARP,caspase-3/9 and their cleaved forms)of A2780 and SKOV-3 cells after morusin treatment,showing that morusin treatment was not accompanied by evident expression of cleaved forms of PARP,caspase-3 or caspase-9 compared to apoptosis induced by TNF-a(50ng/mL)/CHX(50?M)for 24 h as a positive control.MTT assay showed that pretreatment with z-VAD-fmk(a pan-caspase inhibitor)or necrostatin-1(a necroptosis inhibitor)did not significantly reverse morusin-induced cell death in ovarian cancer cells.Electrophoresis images of total DNA in A2780 and SKOV-3 cells revealed that no sign of DNA ladder appeared after morusin treatment.Theses results indicate that morusin-induced cell death may be independent of apoptosis and necroptosis.4.The regulation of morusin on autophagy in A2780 and SKOV-3 cells:A2780 and SKOV-3 cells were transfected with mRFP-GFP-LC3 adenovirus to monitor the formation and degradation of autophagosomes.Treatment with morusin caused noticeable formation of LC3 puncta in A2780 and SKOV-3 cells that exhibited yellow overlay of both red and green fluorescence,similar to CQ treatment which blocked autophagy,indicating that autophagic flux is interrupted by morusin treatment.Western blot analysis was used to examine the expression levels of autophagy-related proteins(LC3,p62,CTSB and CTSD)of A2780 and SKOV-3 cells,revealing that morusin increased the ratio of LC3-II/LC3-I in a dose-and time-dependent manner,reduced the expression of p62 at 6 h and then up-regulated it after 12 h in A2780 and SKOV-3 cells.These results indicate that morusin may induce autophagy at an early stage(before 6 h)but then inhibits the activity of autophagic flux at a late stage(after 12 h)in ovarian cancer cells.Furthermore,morusin increased the levels of precursor forms of lysosomal proteases CTSB and CTSD in A2780 and SKOV-3 cells,but decreased the levels of the mature forms,indicating that morusin may block autophagic flux by inhibiting lysosomal proteases activity.MTT assay showed that the cytotoxicity of morusin in A2780 and SKOV-3 cells was not decreased but mildly increased by pretreatment with 3-MA or CQ,indicating that morusin may induce cytoprotective autophagy in ovarian cancer cells.Moreover,3-MA or CQ did not obviously prevent or aggravate cytoplasmic vacuolization caused by morusin,and only a few number of phagophores and autophagosomes were observed while almost no autolysosomes were visualized in TEM images of morusin-treated A2780 and SKOV-3 cells,indicating that morusin-induced cytoplasmic vacuolization is not due to accumulation of autophagosomes or autolysosomes.Conclusions:1.Morusin suppresses cell viability,colony formation ability and cell proliferation ability in ovarian cancer cells.2.Morusin-induced ovarian cancer cell death is not critically associated with apoptosis,necroptosis or autophagic cell death.Part ? The study of morusin-induced paraptosis and mechanisms in ovarian cancer cellsObjective:1.To investigate the cell death type caused by morusin in ovarian cancer cells.2.To investigate the mechanisms of paraptosis induced by morusin in ovarian cancer cells.Methods:ER-Tracker Red and Mito-Tracker Green staining were used to monitor the morphological changes and distribution of the ER and mitochondria in morusin-treated A2780 and SKOV-3 cells.Transmission electron microscopy was utilized to observe ultrastructure changes of A2780 and SKOV-3 cells.Western blot analysis was used to examine the expression levels of paraptosis inhibitor Alix and ER stress markers(BiP,CHOP,IRE1a and p-eIF2a)in A2780 and SKOV-3 cells.Flow cytometry analysis was utilized to examine the cytosolic and mitochondrial calcium levels,reactive oxygen species(ROS)production and mitochondrial membrane potential.The changes of mitochondrial membrane potential in ovarian cancer cells after morusin treatment was examined by flow cytometry analysis and observed via fluorescence microscope after JC-1 staining.N-acetyl-L-cysteine(NAC)was used to inhibit ROS accumulation in A2780 and SKOV-3 cells.DIDS was used to inhibit mitochondrial calcium influx through voltage-dependent anion channel(VDAC).BAPTA was used to inhibit cytosolic calcium influx.MTT assay was utilized to examine the effects of DIDS or BAPTA on the cytotoxicity induced by morusin in A2780 and SKOV-3 cells.Results:1.The influences of morusin on morphology of the ER and mitochondria in A2780 and SKOV-3 cells:We utilized ER-Tracker Red staining and Mito-Tracker Green staining to explore the origination of cytoplasmic vacuoles in morusin-treated A2780 and SKOV-3 cells,showing that the vacuolar membranes appeared positive for not only ER-specific marker but also mitochondria-specific marker,suggesting that morusin-induced cytoplasmic vacuoles originate from both the ER and mitochondria.Transmission electron microscopy was utilized to observe ultrastructure change of A2780 and SKOV-3 cells,revealing extensive swelling of the ER and mitochondria in ovarian cancer cells after morusin treatment.2.The effects of expression levels of paraptosis inhibitor Alix and ER stress markers in A2780 and SKOV-3 cells:Western blot analysis was used to examine the expression levels of paraptosis inhibitor Alix and ER stress markers(BiP,CHOP,IRE1? and p-eIF2?)in A2780 and SKOV-3 cells,showing that exposure to morusin significantly reduced Alix protein expression,and dramatically increased the expression levels of BiP,CHOP,IREla and p-eIF2a,all in a time-and dose-dependent manner.These results indicate that morusin induces paraptosis and ER stress in ovarian cancer cells.3.The effects of morusin on cytosolic and mitochondrial calcium levels,ROS production levels and mitochondrial membrane potential in A2780 and SKOV-3 cells:Flow cytometry analysis was utilized to examine the cytosolic calcium levels,mitochondrial calcium levels and ROS production levels in A2780 and SKOV-3 cells,showing that morusin slightly increased the cytosolic calcium concentration,dramatically increased the mitochondrial calcium concentration and ROS production levels in ovarian cancer cells.Flow cytometry analysis and fluorescence microscopy using JC-1 staining both showed that morusin caused loss of mitochondrial membrane potential in ovarian cancer cells.4.The study of mechanisms of paraptosis induced by morusin in A2780 and SKOV-3 cells:Flow cytometry analysis showed that pretreatment with DIDS,a chemical blocker of VDAC,effectively prevented morusin-induced mitochondrial calcium overload in A2780 and SKOV-3 cells.MTT assay showed that morusin-induced ovarian cancer cell death was markedly blocked by DIDS pretreatment,but not calcium chelator BAPTA pretreatment,revealing that morusin-incuced ovarian cancer cell death is due to elevated levels of mitochondrial calcium instead of cytosolic calcium,EdU uptake assay showed that DIDS could not reverse morusin-induced inhibition of cell proliferation in ovarian cancer cells,indicating that morusin-induced mitochondrial calcium overload mainly causes ovarian cancer cell death instead of cell proliferation inhibition.Moreover,pretreatment with DIDS prevented the cytoplasmic vacuolization induced by morusin treatment in ovarian cancer cells.Western blot analysis showed that DIDS pretreatment also inhibited morusin-stimulated accumulation of ER stress markers,LC3II and p62 in ovarian cancer cells and reversed the decrease of Alix expression.These results indicate that morusin-induced cell death in ovarian cancer cells is closely associated with the mitochondrial calcium influx through VDAC and mitochondrial calcium overload,but not the cytosolic calcium influx.5.The study of mechanisms of increased ROS levels and decreased mitochondrial membrane potential induced by morusin in A2780 and SKOV-3 cells:Flow cytometry analysis was utilized to examine mitochondrial calcium levels,ROS production levels and mitochondrial membrane potential,revealing that pretreatment with DIDS partially reversed the morusin-induced ROS production and loss of mitochondrial membrane potential in ovarian cancer cells.However,pretreatment with antioxidant NAC did not prevent mitochondrial calcium overload induced by morusin in ovarian cancer cells,indicating that mitochondrial calcium overload may act upstream of ROS generation and loss of mitochondrial membrane potential in morusin-treated ovarian cancer cells.Conclusions:1.Morusin induced paraptosis in ovarian cancer cells.2.Morusin-induced mitochondrial calcium influx through VDAC and subsequent mitochondrial calcium overload cause mitochondrial dysfunction,leading to paraptosis in ovarian cancer cells.Part ? Therapeutic effects of morusin on subcutaneous ovarian cancer xenografts in nude mice and the study of mechanismsObjective:1.To establish a subcutaneous xenograft model of ovarian cancer in nude mice and evaluate the therapeutic effects of morusin on xenografted nude mice in vivo.2.To explore mechanisms of the anti-tumor effects in vivo of morusin.Methods:We established a xenograft nude mouse model of ovarian cancer through subcutaneous inoculation of SKOV-3 cells into nude mice under arms.The xenografted mice were injected with vehicle control,DIDS,morusin,or morusin in combination with DIDS each day respectively.The tumor volume of each mouse was measured by vernier caliper every three days.And the body weight of each mouse was measured via electronic scale every three days.All tumor-bearing mice were sacrificed after 21 days and the tumor masses were photographed and weighed.Paraffin sections of tumor tissues were made and immunohistochemistry(IHC)staining was used to detect the expression levels of ER stress markers(BiP and CHOP)in tumor tissues.Results:1.Establish of the model of subcutaneous ovarian cancer cell SKOV-3 xenografts in nude mice:Nodules about the size of a grain of rice with certain tenacity could be touched at the injection site of nude mice after subcutaneous inoculation of SKOV-3 cells for 18 days,and the tumor formation rate was 100%.Xenografted nude mice were in good condition and the subcutaneous xenografts growed fast.The nude mice were trandomly assigned for intraperitoneal injection of different drugs when the average tumor volume came up to 100 mm3 after 28 days of inoculation.During 21 days of injection,nude mice of every group tolerated the drugs well without obvious side effects or adverse reactions.2.The effects of morusin on growth of subcutaneous ovarian cancer xenografts in nude mice:Tumor growth curve showed that tumor growth in vivo was significantly inhibited after 12 days of morusin treatment,and the mean weight of tumor masses in morusin-treated mice was also markedly reduced compared to the control group.However,no obvious change of body weight was observed in the morusin-treated group,indicating that morusin causes no obvious toxicity in nude mice.3.The study of the mechanisms of the anti-tumor effects in vivo of morusin:Tumor growth curve showed that the mean tumor volume of DIDS-treated mice had no significant difference compared to the control group,but the mean tumor volume of DIDS and morusin co-treatment group was larger compared to the morusin-treated mice after 15 days of injection.The mean weight of tumor masses in DIDS and morusin co-treatment group was also heavier compared to the morusin-treated group,indicating that morusin inhibits ovarian cancer growth in vivo through mitochondria calcium overload.No obvious change of body weight was observed in the DIDS-treated group and DIDS and morusin co-treatment group compared to the control group.IHC staining analysis demonstrated that expression levels of BiP and CHOP were increased in morusin-treated group compared to the control group and this effect was reversed by co-treatment with morusin and DIDS,indicating that morusin can trigger ER stress in vivo through interruption of intracellular calcium homeostasis of ovarian cancer cells.Conclusions:1.Morusin can inhibit the growth of subcutaneous ovarian cancer xenografts in vivo.2.Morusin possesses anti-tumor effects and triggers ER stress in vivo through mitochondria calcium overload.