The Effects And Mechanisms Of Nigericin On Human Epithelial Ovarian Cancer Cells

Epithelial ovarian cancer(EOC)is the third most commonly diagnosed cancer among women worldwide,having the highest mortality rate of gynaecological malignancies.Because of lacking effective early screening of EOC,most deaths were a result of multiple metastases caused by tumor migration and invasion.Currently,surgery combined with platinum-based chemotherapy remains the cornerstone of treatment for EOC,while extensive pelvic and peritoneal cavity metastasis increased the difficulty to optimal residual tumor volumes and the incidence of surgical complications.Most of these deaths are caused by metastasis and recurrence due to cisplatin resistance.Despite recent advances in the use of active targeted drugs for treatment,the prognosis of EOC patients remains dismal,and recent data indicated that the 5-year overall survival rate was only 30%.Therefore,it is necessary for us to explore new targeted therapy drugs with potential treatment prospects to improve the prognosis of EOC patients.Epithelial-mesenchymal transition(EMT)is an essential mechanism of metastasis in cancer,including EOC.It is a cellular mechanism characterized by decreased epithelial markers such as E-cadherin,increased mesenchymal markers such as Vimentin,and altered location of transcription factors such as Slug,Snail and Twist.During the EMT process,transformed epithelial tumor cells acquire the ability to propagate,migrate,invade,and resist apoptosis.In recent years,studies found that the most common way for EOC to metastasis is implantation,which is the key step in the process of EOC metastasis.Although the specific molecular mechanism is not very clear in EMT process at present,the mechanism of EMT in tumor invasion and metastasis will be gradually clarified with further research,accordingly,the new strategy of inhibiting EMT tumor metastasis suppressing tumor metastasis will be further developed for the treatment of maliganant tumors.Nigericin is a monocarboxylic polyether antibiotic potassium ionophore that is widely used as a coccidiostatic agent in chickens.It has been recently reported that nigericin could suppress colorectal cancer metastasis and breast cancer stem cells by inhibition of the epithelial-mesenchymal transition,but the specific molecular mechanism remains unclear.In this study,we attempt to ascertain the specific activities of nigericin on human EOC cell lines and to investigate the possible molecular mechanism of nigericin on cell apoptosis,migration,invasion and metastasis.We investigated the anti-tumor mechanisms of nigericin in EOC including four parts.PART ?The role of negericin on proliferation of human epithelial ovarian cancer cellsBackgroundNigericin is a monocarboxylic polyether antibiotic potassium ionophore that is widely used as a coccidiostatic agent in chickens.It has been firstly reported to inhibit breast cancer stem cells at least 100 times more effectively than paclitaxel in mice.Lu et al have reported that nigericin,similar to salinomycin,selectively inhibits the Wnt signalling cascade in HEK293 cells at nanomolar concentrations.Zhou et al have reported that nigericin could suppress colorectal cancer metastasis by inhibition of the epithelial-mesenchymal transition.The role of negericin on proliferation of human epithelial ovarian cancer cells remains unclear.ObjectIn this part of the study we put different doses of nigericin into different types of EOC cell lines,and observed the inhibitory effects using various methods.MethodsThe human EOC cells lines A2780 and SKOV3 were treated with nigericin under specified conditions.The CCK-8 assay was performed to measure cell viability in A2780 and SKOV3 cells.The cell cycle and apoptosis distribution were analyzed by flow cytometry.The expression of proteins related to cell apoptosis was assessed using western blot.Results1.The inhibitory effects of nigericin on EOC cells by CCK-8 assayThe results showed that nigericin exhibited strong toxicity for the A2780 cell line(IC50,16.19±0.26?mol/L,95%CI:1.077 to 1.341).In the SKOV3 cell line,the result is consistent with that of the A2780 cells.SKOV3 cell line was more sensitive to nigericin than A2780 cell line.2.The effects of nigericin on EOC cell cycleCell cycle distribution analysis showed an increase in the number of cells in the G0/G1 phase,indicating that nigericin induces typical cell-cycle arrest at the G0/G1 phase in EOC cells.3.The effects of nigericin on EOC cell apoptosisAnnexin V staining was used to evaluate the effect of nigericin on the apoptosis of EOC cells.With an increasing nigericin concentration,the percentage of Annexin V positive cells increased in a dose-dependent manner.The apoptosis-related proteins expression levels including Bax,Bcl-2,Caspase-3 and PARP were examined by western blot.These results showed that the expression of Bcl-2,Caspase-3 and PARP decreased,while the expression of Bax,Cleaved-Caspase-3 and cleaved-PARP increased in A2780 and SKOV3 cells in a concentration-dependent manner.Conclusions1.Nigericin could inhibit the proliferation of EOC cells.2.Nigericin induced typical cell-cycle arrest at the G0/G1 phase in a dose-dependent manner in EOC cells.3.Nigericin could promote apoptosis in EOC cells.PART IIThe role of nigericin on migration and invasion of EOC cellsBackgroundDue to the lack of effective early screening of epithelial ovarian cancer,most deaths were a result of multiple metastases caused by tumor migration and invasion,having the highest mortality rate of gynaecological malignancies.Recurrence and metastasis of EOC are the main causes of poor prognosis.Invasion and metastasis is a complex pathophysiological process occurring in the late stages of tumor progression,but the exact molecular mechanism is not very clear.Previous studies have reported that nigericin could inhibit the abilities of cell mobility and invasion in vitro in colorectal cancer.It is unknown whether or not nigericin may also play an important role on EOC cell invasion capacity.ObjectIn this part of the study we put different doses of nigericin into different types of EOC cell lines,and observed whether nigericin could inhibit the migration and invasion of EOC cells.MethodsThe cell migration assay was performed using Boyden chambers with 8-?m-pore filters.Cells were quantified by counting the number of stained nuclei in five random fields by fluorescence microscopy.Results1.Nigericin could inhibit the migration abilities of EOC cells.After incubation for 48 h,different concentrations of nigericin induced a remarkable reduction in the number of cells migrating through the membrane without Matrigel relative to the vehicle-treated controls in A2780 and SKOV3 cells.2.Nigericin could inhibit the invasion abilities of EOC cells.Compared to the control group,the number of A2780 and SKOV3 cells invading through the Matrigel-coated membrane also decreased in the nigericin-treated groups.ConclusionsNigericin could inhibit the migration and invasion of EOC cells.PART ?The role of nigericin on tumor angiogenesis of EOC cellsBackgroundThe reason why EOC with high heterogeneity may due to peritoneal metastasis,a growing number of studies have shown that angiogenesis is an important factor for the process of epithelial ovarian cancer development.In cancer cells,the EMT programme is aberrantly initiated by extracellular stimuli such as hypoxia,inflammatory cytokines and vascular endothelial growth factor(VEGF)derived from the tumor microenvironment.The influence of the tumor microenvironment is also potentially associated with vasculogenic mimicry(VM)formation,and hypoxia inducible factor-la(HIF-1?)is the key regulator of hypoxia in tumor cells.Studies from many groups have shown that the presence of VM is closely associated with an increased risk of metastasis,which results in poor prognosis in EOC and other malignant tumors.Though anti-angiogenesis drugs are used in clinical treatments,their curative effects remain unsatisfactory,potentially due to the formation of VM.EMT progress and VM formation may explain why VM-positive EOC patients have an elevated risk of metastasis and tumor recurrence and shorter survival periods.It is still a challenge to explore the novel therapies both for tumor metastasis and targeting VM.ObjectIn this part of the study we put different doses of nigericin into different types of EOC cell lines,aiming to investigate whether nigericin plays a role in the EOC tumour microenvironment and VM formation.MethodsTo investigate the effect of nigericin on tumor microenvironment and VM formation,A2780 and SKOV3 cells were treated with different concentrations of nigericin.Western blot was performed to investigate the expression of VEGF,HIF-la and VE-cadherin.Results1.Nigericin decreased the expression ofVEGF and HIF-1? of EOC cells.The protein levels of VEGF and HIF-1? of A2780 and SKOV3 cells were determined by western blot.The results showed that the expression of VEGF and HIF-la of A2780 and SKOV3 cells were decreased in a concentration-dependent manner.2.Nigericin significantly decreased the expression of VM related transcription factor VE-cadherin.The protein levels of VE-cadherin of A2780 and SKOV3 cells were determined by western blot.The results showed that the expression of VE-cadherin was also decreased in a concentration-dependent manner.ConclusionsNigericin could inhibit angiogenesis and vasculogenic mimicry of EOC cells.PART ?Suppression of EMT by nigericin via the Wnt/?-catenin signalling pathway in EOC cellsBackgroundEMT is a biological process characterized by decreased epithelial markers and increased mesenchymal markers.During the EMT process,transformed epithelial tumor cells acquire the ability to propagate,migrate,invade,and resist apoptosis.Studies have shown that EMT related gene expression of primary tumor metastatic probability is higher,but the patient's progression free survival time is shorter.Therefore,inhibition of EMT process will be a very promising treatment options.It has been reported that nigericin could suppress colorectal cancer metastasis by inhibition of the epithelial-mesenchymal transition.The first two parts of the experimental results have confirmed that nigericin could significantly reduce EOC cell proliferation,migration and invasion ability,but the specific molecular mechanism is not very clear.ObjectIn this part of the study we put different doses of nigericin into different types of EOC cell lines,aiming to investigate the possible molecular mechanism of nigericin on EOC cell apoptosis,migration,invasion and metastasis.MethodsTo explore the molecular mechanism under which nigericin inhibits migration and invasion of EOC cells,we evaluated the effects of nigericin treatment on the characteristics of the EMT phenotype by using Western blot and Real-time PCR.Then,we detected the expression of EMT-related transcription factors Slug,Snail,and Twist to further illustrate the suppression of EOC cell metastasis by inhibition of EMT.The expression of proteins related to Wnt/?-catenin signalling pathway was assessed by western blot.ResultsI.Nigericin inhibits EMT phenotype of EOC cells.After drug treatments,the expression of epithelial marker(E-cadherin)and mesenchymal marker(Vimentin)of EMT were evaluated by Western blot analysis.These results confirmed that increased expression of E-cadherin and decreased expression of Vimentin in A2780 and SKOV3 cells treated with nigericin showed a concentration dependence.2.Nigericin significantly decreased the expression of EMT-related transcription factorsWe then detected the protein levels of EMT-related transcription factors by western blot.The expression levels of Slug,Snail and Twist were reduced in A2780 and SKOV3 cells in a dose-dependent manner.3.Nigericin could inhibit the Wnt/?-catenin signalling pathway in EOC cells.We firstly examined Gsk3? phosphorylation at Ser9,a state of inactivation,by western blot.The results showed that the expression of phosphorylated-Gsk3? was significantly decreased,dependent on concentration,while the total Gsk3? protein levels were not altered.We next detected the expression of ?-catenin in A2780 and SKOV3 cells treated with nigericin by western blot.Our results revealed prominently decreased expression of ?-catenin with the treatment of nigericin.ConclusionsNigericin could inhibit EMTduring cell invasion and metastasis through the canonical Wnt/?-catenin signalling pathway.