WWOX Promotes Apoptosis And Inhibits Autophagy In Paclitaxel-treated Ovarian Carcinoma

Background and ObjectiveEpithelial ovarian carcinoma(EOC)is one of the three common malignant tumors in the female reproductive system.It has a high degree of malignancy and a common incidence.It is more common in middle-aged and elderly women.At present,its standard initial treatment plan is a comprehensive treatment such as surgery combined with chemotherapy and radiotherapy.Among them,chemotherapy plays a great role in the treatment of ovarian cancer,and platinum combined with paclitaxel is the current first-line "gold standard" chemotherapy program.Paclitaxel(PTX)was originally derived from the bark and wood of Paclitaxel.It has a good anti-tumor effect on ovarian cancer and endometrial cancer.The anti-tumor mechanism is to inhibit the synthesis and depolymerization of tubulin.However,paclitaxel resistance is common in patients with advanced,recurrent and refractory epithelial ovarian cancer.Paclitaxel resistance can accelerate tumor progression and increase cancer mortality.However,at present,the mechanism of paclitaxel resistance is not very clear.Studies suggest that autophagy may be one of the pathogenesis of paclitaxel resistance.Autophagy is a conservative evolutionary phenomenon that occurs in cells.It is actually a self-protection mechanism of cells;after autophagy is activated,cells can withstand stress and reduce death.At present,a large number of studies have shown that(chemotherapeutic resistance)tumor cells often see a higher level of autophagy,and inhibiting autophagy can increase tumor cell chemotherapy sensitivity and promote cell apoptosis.Therefore,autophagy may be a potential target for exploring tumor treatment.WWOX protein is recognized as a multifunctional tumor suppressor.However,due to the existence of fragile sites,abnormal expression of WWOX is common in tumor cells,including expression loss.Previous studies have proved that phosphorylation and nuclear transfer of WWOX are the prerequisites for WWOX to mediate apoptosis.Recently,studies have found that in tongue cancer,WWOX can participate in the regulation of autophagy and promote tumor cell death.But the confirmation of this new function of WWOX still needs further and stronger evidence.The mammalian target of rapamycin(mTOR)is a serine/threonine kinase that can regulate cell metabolism and promote cell proliferation.The complex mTORC1 composed of mTOR and Raptor and other factors has been widely confirmed to modify the occurrence of autophagy.As a hot molecule WWOX in tumor research,whether it can also promote apoptosis,regulate mTOR pathway and inhibit autophagy in tumor cells in EOC cells under the action of paclitaxel needs to be confirmed by our research.In addition,in addition to the effect of paclitaxel in inducing apoptosis in EOC cells,is it also involved in the regulation of autophagy?What is the direction of this regulation,and whether changes in the level of autophagy are involved in the occurrence of paclitaxel resistance.Therefore,in order to answer the above questions,we studied the relationship between WWOX under the action of paclitaxel and EOC cell apoptosis,autophagy and mTOR pathway.This is useful for clarifying the possible resistance mechanism of paclitaxel in EOC and providing improvements for future medicine.Molecular therapy candidate genes for clinical prognosis of EOC have certain significance.The specific research content of this subject includes the following three parts:1.The expression of WWOX in EOC and its regulatory effect on the apoptosis of epithelial ovarian cancer cells induced by paclitaxel.2.Paclitaxel and WWOX have an effect on epithelial ovarian cancer cells.The influence and research of phagocytosis.3.WWOX resists paclitaxel and regulates the autophagy phagocytosis of epithelial ovarian cancer cells through the mTOR/P70S6K signaling pathway.Part ? The expression of WWOX in EOC and its regulatory effect on the apoptosis of epithelial ovarian cancer cells induced by paclitaxelObjectAs a first-line EOC chemotherapy drug,paclitaxel is believed to destroy cell tubulin by its mechanism.Paclitaxel can inhibit the tubulin dimers that compose microtubules lose their dynamic balance,induce and promote tubulin polymerization,microtubule assembly,and prevent depolymerization,thereby stabilizing microtubules and inhibiting tumor cell mitosis,and ultimately triggering cell apoptosis.Paclitaxel can inhibit tubulin dimers which compose microtubules from losing their homeostasis,and induce,promote tubulin polymerization.Paciltaxel also can inhibit microtubule assembly and prevent its depolymerization.Thereby stabilize microtubules and inhibit tumor cells mitosis,and ultimately triggering cell apoptosis.Under the action of stress or apoptosis-inducing factors,the tyrosine 33 of human WWOX gene can be phosphorylated,and the phosphorylated protein of WWOX forms a complex with p53 and JNK1 to participate in the regulation of apoptosis.Phosphorylation and nuclear transfer of WWOX are considered prerequisites for WWOX-mediated apoptosis.In a variety of human tumor cells,WWOX has been proved to be involved in the inhibition of cell proliferation and the promotion of apoptosis.The purpose of this section is to study the expression of WWOX in different EOC cell lines and the effect of paclitaxel on the proliferation and apoptosis of EOC cells,and to explore the regulatory effect of changes in WWOX levels on paclitaxel-induced apoptosis of EOC cells.And verify the conclusions of previous studies:WWOX is a tumor suppressor factor,and its activation is a prerequisite for apoptosis.Contents and methods1.Select human EOC cell lines:A2780 cells,SKOV3 cells and paclitaxel-resistant cells A2780/T cells for culture,and use Western blot analysis to detect the WWOX protein in the next three cell lines Basic expression level.2.Use different concentrations of paclitaxel to treat the above three types of EOC cells for cell proliferation and apoptosis related detection.Including:CCK-8(Cell Counting Kit-8 test)test,cell proliferation and apoptosis assessment under light microscope(further quantitative analysis of cell proliferation by histogram)and flow cytometry(assessment of apoptotic cell rate).3.After treating A2780 cells and A2780/T cells with paclitaxel,Western blot analysis was used to detect WWOX,p-WWOX and apoptosis-related proteins caspase-3(including cleaved-caspase-3),PARP(including cleaved)-PARP)expression.Furthermore,reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was used to analyze the change trend of WWOX expression of EOC cells treated with paclitaxel at the mRNA level.4.Construct a WWOX overexpression vector(plasmid),and after verifying the transfection efficiency,transfect A2780 cells.Treat the A2780 cells before and after transfection with paclitaxel,and analyze the apoptosis rate by flow cytometry again.Results1.Among EOC cells,paclitaxel-resistant A2780/T cells have the highest basal expression level of WWOX,A2780 cells,which are most sensitive to paclitaxel,have a middle expression level of WWOX,while those of SKOV3 cells,which are elongated and similar in shape to mesenchymal cells,have the highest expression level.lowest.2.The CCK-8 experiment indicates that paclitaxel inhibits the proliferation of sensitive cells(A2780 cells and SKOV3 cells)and induces their apoptosis;but it is ineffective against paclitaxel-resistant cells A2780/T,and A2780/T cells show an increase in paclitaxel concentration Proliferation accelerated afterwards.Under the light microscope,it can be seen that A2780 and SKOV3 cells increased with the time of paclitaxel treatment,proliferation was inhibited,and viable cells decreased.The quantitative histogram analysis of viable cells in 5 fields under a light microscope showed that the proportion of viable cells of A2780 and SKOV3 cells decreased significantly with the time of paclitaxel;while the proportion of viable cells of A2780/T cells gradually increased.3.The results of Western blot showed that in A2780 cells,WWOX showed a dose-dependent and time-dependent up-regulation of paclitaxel,and WWOX was activated,showing that the expression of p-WWOX increased gradually.At the same time,it can be seen that the expression of cleaved caspase-3 and cleaved PARP is gradually increasing.In A2780/T cells,the expression of WWOX was down-regulated in a dose and time-dependent manner of paclitaxel treatment;the expression of p-WWOX was constant regardless of the time of paclitaxel treatment,and there was no expression of apoptotic protein.The results of RT-PCR experiments showed that after paclitaxel,the expression of WWOX RNA in A2780 cells was up-regulated,and the expression of WWOX RNA in A2780/T cells was down-regulated.4.The constructed WWOX overexpression vector was verified by western blot experiment,and the results showed that the expression of A2780 cells after transfection increased significantly.Before and after transfection,A2780 cells were treated with 150ng/ml paclitaxel for 48h and the apoptosis rate was detected by flow cytometry.The results showed that high expression of WWOX can significantly increase the sensitivity of paclitaxel induction:A2780 group:46.4%,WWOX-A2780 group:63.6%.Research conclusions1.The highest WWOX expression is observed in A2780/T cells,and the lowest in SKOV3.It is suggested that there may be abnormal expression of WWOX protein inactivation in drug-resistant cells A2780/T,or the existence of related antagonists.WWOX may have an inhibitory effect on epithelial-mesenchymal cell transformation,so SKOV3,which has mesenchymal cell morphology,has the lowest expression level of WWOX.2.Paclitaxel induce apoptosis in sensitive EOC cells,but it is not effective on drug-resistant cells.3.Increased expression and activation of WWOX(p-WWOX)is a prerequisite for paclitaxel to induce apoptosis in EOC cells.4.Up-regulation of WWOX can sensitize the apoptosis-inducing effect of paclitaxel on EOC cells.Part ? The effects of paclitaxel and WWOX on autophagy in EOC cellsObjectThe mechanisms of paclitaxel resistance in EOC have not yet fully understood.Previous studies have found that tumor cells often show increased levels of autophagy compared with normal cells,and inhibition of autophagy can effectively weaken the proliferation activity of tumor cells and promote apoptosis induced by chemotherapy.Current studies have found that autophagy is involved in the development of drug resistance of a variety of chemotherapeutic drugs,and inhibition of autophagy is expected to become a new target for overcoming tumor drug resistance.A 2015 study on tongue cancer suggested that WWOX protein can inhibit the occurrence of autophagy.Therefore,this part of the study aims to explore whether WWOX has an autophagy inhibitory effect on EOC cells based on previous experiments(clarifying the pro-apoptotic effect of WWOX);at the same time,to study the effect of paclitaxel on the autophagy level of EOC cells,and then to further explore The possible mechanism of paclitaxel resistance in EOC cells,as well as the role and molecular significance of WWOX in the field of anti-tumor.Contents and methods1.To investigated the change of autophage in A2780 cells and A2780/T cells,western blot was used to detect the expression of autophagy key protein Beclin-1,Atg12-5,LC3(?,?)and autophagy substrate P62 after 0,6,12,24,48 and 72 hours 150ng/ml or 1600ng/ml paclitaxel.2.To inhibit autophagy(lysosomal)degradation autophagy inhibitor chloroquine(CQ)was used,the we detected the expression of LC3? in A2780 cells under the action of NC,CQ,CQ+paclitaxel and paclitaxel by western blot.3.We used adenovirus containing red-green fluorescently labeled LC3 protein(GFP mRFP LC3)to infect A2780 and A2780/T cells after pre-experimentation to find the best infection conditions.Afterwards,confocal microscopy was used to observe the changes in red and green fluorescence of the transfected cells after treatment with 150ng/ml or 1600ng/ml paclitaxel.4.To observe the number of autophagic vesicles in A2780 and A2780/T cells before and after the treatment of150ng/ml and 1600ng/ml paclitaxel,transmission electron microscopy(TEM)was used.5.A WWOX knockout transfection vector(plasmid)was instructed,and after verifying its efficacy,we used it to transfect A2780 cells.Then western blot was used to detect the expression of LC3(?,?)and Beclin-1.Resluts1.Paclitaxel up-regulated the expression of autophagy-related proteins in EOC cells.After paclitaxel treatment,A2780 and A2780/T cells all showed autophagy-related key proteins:Atg12-5,Beclin-1 and LC3 II expression levels increased in a time-dependent manner,and autophagy substrate P62 was time-dependent Sexual expression is down-regulated.2.The use of chloroquine verifies the authenticity of paclitaxel-induced autophagy.We used chloroquine to verify the authenticity of paclitaxel-induced autophagy:the expression of LC3 ? increased after the use of CQ,and the expression of LC3 ?was further increased after the superimposed treatment with paclitaxel.3.Confocal electron microscope observations showed that after paclitaxel treatment,the number and volume of red and green fluorescent spots in both A2780 and A2780/T cells showed an increasing and increasing trend(the change in A2780 was especially obvious).In addition,it was showed that paclitaxel-resistant A2780/T cells showed a significantly high level of LC3-related fluorescent spots before paclitaxel treatment(significantly higher than which in A2780 cells),showing the avtive autophagy.4.Transmission electron microscopy observational experiments indicated that the number of basal autophagic vesicles in drug-resistant cells A2780/T cells were significantly more than that in A2780 cells.After treatment with paclitaxel,the number of autophagic vesicles in A2780 and A2780/T cells increased,indicating that the level of autophagy increased under the pacitaxel treatment and the active autophagy in paclitaxel-resistant EOC cells.5.After up-regulating the expression of WWOX in A2780 cells,it was found that the expression of autophagy-related proteins LC3 and Beclin-1 was significantly down-regulated,suggesting the inhibitory effect of WWOX on autophagy.Results1.The basic autophagy level of paclitaxel-resistant EOC cells were significantly increased,suggesting that autophagy is involved in the mechanism of paclitaxel resistance.2.Paclitaxel up-regulate the autophagy tide of EOC cells,suggesting the increase of autophagy,which creates certain conditions for the occurrence of EOC resistance after paclitaxel treatment.3.WWOX inhibit the occurrence of autophagy in EOC cells and provide a molecular basis for the treatment of drug resistance.Part ? WWOX resist the regulation effect on autophagy from paclitaxel in EOC cells via mTOR/P70S6K signalingObjectThe mammalian target of rapamycin(mTOR)is a threonine/serine kinase that can regulate cell metabolism and promote cell proliferation.The complex mTORC1 composed of factors such as mTOR and Raptor and its substrates have been shown to regulate the occurrence of autophagy.The purpose of this part of our experiment is to study the relationship between paclitaxel and the mTOR/P70S6K pathway,and to explore the possible mechanism of paclitaxel regulating autophagy in EOC cells;at the same time,to analyze the relationship between WWOX and mTOR to try to speculate on the regulation of paclitaxel The role of WWOX provides a new theoretical basis for in vitro experiments for reversing the chemotherapy resistance of tumors at the molecular level.Contents and methods1.In A2780 cells and A2780/T cells,Western blot was used to detect the mTOR pathway(mTOR-p70S6K pathway)under the action of 150ng/ml and 1600ng/ml paclitaxel at 0,6,12,24,48,72h time points.The protein expression levels of mTOR,p-mTOR,p-P70S6K and p-4E-BP-1.By analyzing the relationship between the various factors in this pathway and paclitaxel,the regulatory effect of paclitaxel on the mTOR pathway in these two cells was analyzed,and the possible mechanism of paclitaxel regulating autophagy was further analyzed.2.After constructing the WWOX knock-out transfection vector(plasmid),and verifying its efficacy,it was transfected with the WWOX overexpression vector constructed in the previous part of the experiment,respectively,to transfect the two EOC cells selected in the experiment.Western blot was used to analyze the change trend of mTOR and p-mTOR after the level of WWOX was changed.Therefore,it is speculated that WWOX may have a possible regulatory effect on the mTOR pathway,and then combined with the second part of the experimental conclusions to analyze the possible mechanism of WWOX resisting paclitaxel to enhance EOC autophagy.Results1.Paclitaxel inhibit the mTOR/p70S6K pathway of EOC sensitive cells and drug-resistant cells.The results of Western blot experiments showed that after paclitaxel treatment for different periods of time,A2780 cells(paclitaxel sensitive cells)and A2780/T cells(paclitaxel resistant cells)both exhibited inhibition of mTOR/p70S6K pathway,specifically:p-mTOR Paclitaxel treatment time-dependent expression down-regulation,p-P70S6K always no expression state and p-4E-BP-1 expression up-regulated with paclitaxel treatment time.2.WWOX resist the inhibitory effect of paclitaxel on the mTOR pathway.After the transfection experiment,the results of Western blot experiment showed that:after up-regulating WWOX,the expression of p-mTOR can be increased.This regulatory effect on p-mTOR could resist the down-regulation effect of paclitaxel on p-mTOR.In contrast,after knocking out WWOX,p-mTOR was down-regulated.Conclusion1.Paclitaxel can inhibit the mTOR/P70S6K pathway in both Paclitaxel-sensitive and drug-resistant EOC cells.We speculate that paclitaxel regulates autophagy in EOC cells by regulating mTOR/P70S6K.2.WWOX may inhibit paclitaxel and promote autophagy through the mTOR/P70S6K pathway,thereby reducing the level of autophagy in EOC,showing a multifunctional tumor-inhibiting function.