ovarian cancer is the most lethal gynecological malignancy and the second most common gynecologic cancer in the world , with a high incidence of metastasis and a high relapse rate . it caused an estimated 14 270 deaths in 2014 in the usa . because of the lack of effective diagnostics in early stages , the 5-year survival rate for ovarian cancer is only 27% . lysine - specific demethylase 1 ( lsd1 ) is a nuclear enzymatic activity that demethylates mono- and di - methylated histone h3 at lysines 4 and 9 , regulating the transcriptional actions of hormone - liganded nuclear receptors [ 47 ] . lsd1 has been found to be overexpressed in various cancers , including ovarian cancer , breast cancer , colon cancer , and gastric cancer . fda - approved inhibitors of fad amine oxidases such as mitochondrial - associated monoamine oxidase ( mao ) a and b and polyamine oxidase ( pao ) are non - selective inhibitors of lsd1 activity . recently , many selective lsd1 inhibitors have been developed , which can be grouped into 4 different classes based on their chemical structure . s2101 is known to be more selective than other lsd1 inhibitors ( e.g. , pargyline and tcp ) in ovarian cancer cell lines , which can inhibit cell viability and it has potential value in therapeutic use . autophagy is a lysosomal degradative process used to recycle obsolete cellular constituents and eliminate damaged organelles and protein aggregates . when a chemotherapeutic drug induces oxidative stress and dna damage , or when defective vascularization determines hypoxia and starvation , the upregulation of autophagy enables cancer cells to overcome the metabolic stress . in brief when abnormality in the process of autophagy occurs , normal cellular functions become damaged , and the accumulated abnormalities will lead to systematic problems . abundant evidence shows that autophagy is associated with the genesis and development of cancers , and effective therapeutic strategies could induce or inhibit the process of autophagy in various cancers . based on previous studies , this study aimed to investigate the effects of s2101 on viability , apoptosis , and autophagy of skvo3 ovarian cancer cells . the skvo3 is a human ovarian cancer cell line that was donated by the laboratory of pharmacology , harbin medical university , china . cells were cultured in rpmi 1640 medium ( millipore , billerica , ma ) containing 10% fbs , 100 u / ml penicillin , and 100 u / ml streptomycin ( millipore , billerica , ma ) in a humidified chamber at 5% co2 and 37c . lsd1 inhibitor s2101 and autophagy inhibitor 3-methyladenine ( 3-ma ) were purchased from the millipore company . the primary antibodies for bax , bcl-2 , parp , lc3-i / ii , sqstm1/p62 , mtor , phospho - mtor , p70s6k , phospho - p70s6k , akt , phospho - akt , and -actin were purchased from cell signaling technology . the cytotoxicity of s2101 was tested by cell counting kit-8 ( cck-8 ) assay ( beyotime , shanghai , china ) . skov3 cells were prepared and dispersed in 96-well cell culture plates at a cellular density of 1.010 cells / well . the cells in the exponential phase of growth were used . after incubating different concentrations of s2101 ranging from 0 to 200 mol / l for 24 h and 48 h , 10 l of cck-8 solution in pbs was added to each well and incubated at 37c for 2 h. results were calculated as the absorbance of treated cells relative to untreated controls . cellular apoptosis was quantified by flow cytometric analysis using annexin - v / pi staining methods . briefly , 48 h after 100 um s2101 incubation , both floating and attached cells were collected and washed twice with cold pbs . the cells were resuspended in prediluted binding buffer and propidium iodide ( 1 g / ml ) was then added . the samples were immediately detected by a facsort flow cytometer ( bd biosciences , san jose , ca , usa ) . green fluorescent protein ( gfp)-fused - lc3 ( gfp - lc3 ) was used to test autophagy . the skov3 cells were fused to 5 g of gfp - lc3 plasmid using lipofectamine 2000 ( invitrogen ) . the transfected cells were treated with 100 m of s2101 for 48 h. the gfp - lc3 cells were visualized by nikon fluorescence microscopy ( magnification , 100 ; 90i ; nikon corporation , tokyo , japan ) . cell numbers were counted to normalize the measurement , and the percentage of fluorescent cells was calculated . after treatment , skov3 cells were harvested and lysed in radioimmunoprecipitation lysis buffer ( beyotime , shanghai , china ) . the total proteins from each samples were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis , and electro - transferred onto polyvinylidene difluoride membranes ( millipore , billerica , ma ) . after blocking , the membranes were immunoblotted overnight at 4c with primary antibodies , including anti - bax , anti - bcl-2 , anti - parp , anti - lc3-i / ii , anti - sqstm1/p62 , anti - mtor , anti - phospho - mtor , anti - p70s6k , anti - phospho - p70s6k , anti - akt , anti - phospho - akt , and -actin , followed by hrp - conjugated secondary antibodies at 37c for 1 h. signals were detected using an ecl system . the differences were analyzed using the 2-sided t test or anova with spss 17.0 software . the skvo3 is a human ovarian cancer cell line that was donated by the laboratory of pharmacology , harbin medical university , china . cells were cultured in rpmi 1640 medium ( millipore , billerica , ma ) containing 10% fbs , 100 u / ml penicillin , and 100 u / ml streptomycin ( millipore , billerica , ma ) in a humidified chamber at 5% co2 and 37c . lsd1 inhibitor s2101 and autophagy inhibitor 3-methyladenine ( 3-ma ) were purchased from the millipore company . the primary antibodies for bax , bcl-2 , parp , lc3-i / ii , sqstm1/p62 , mtor , phospho - mtor , p70s6k , phospho - p70s6k , akt , phospho - akt , and -actin were purchased from cell signaling technology . the cytotoxicity of s2101 was tested by cell counting kit-8 ( cck-8 ) assay ( beyotime , shanghai , china ) . skov3 cells were prepared and dispersed in 96-well cell culture plates at a cellular density of 1.010 cells / well . the cells in the exponential phase of growth were used . after incubating different concentrations of s2101 ranging from 0 to 200 mol / l for 24 h and 48 h , 10 l of cck-8 solution in pbs was added to each well and incubated at 37c for 2 h. results were calculated as the absorbance of treated cells relative to untreated controls . cellular apoptosis was quantified by flow cytometric analysis using annexin - v / pi staining methods . briefly , 48 h after 100 um s2101 incubation , both floating and attached cells were collected and washed twice with cold pbs . the cells were resuspended in prediluted binding buffer and propidium iodide ( 1 g / ml ) was then added . the samples were immediately detected by a facsort flow cytometer ( bd biosciences , san jose , ca , usa ) . green fluorescent protein ( gfp)-fused - lc3 ( gfp - lc3 ) was used to test autophagy . the skov3 cells were fused to 5 g of gfp - lc3 plasmid using lipofectamine 2000 ( invitrogen ) . the transfected cells were treated with 100 m of s2101 for 48 h. the gfp - lc3 cells were visualized by nikon fluorescence microscopy ( magnification , 100 ; 90i ; nikon corporation , tokyo , japan ) . cell numbers were counted to normalize the measurement , and the percentage of fluorescent cells was calculated . after treatment , skov3 cells were harvested and lysed in radioimmunoprecipitation lysis buffer ( beyotime , shanghai , china ) . the total proteins from each samples were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis , and electro - transferred onto polyvinylidene difluoride membranes ( millipore , billerica , ma ) . after blocking , the membranes were immunoblotted overnight at 4c with primary antibodies , including anti - bax , anti - bcl-2 , anti - parp , anti - lc3-i / ii , anti - sqstm1/p62 , anti - mtor , anti - phospho - mtor , anti - p70s6k , anti - phospho - p70s6k , anti - akt , anti - phospho - akt , and -actin , followed by hrp - conjugated secondary antibodies at 37c for 1 h. signals were detected using an ecl system . the differences were analyzed using the 2-sided t test or anova with spss 17.0 software . when skvo3 cells were treated with s2101 at 0 , 50 , 100 , 150 , and 200 m , the percentages of cellular viability over the control cells ( 100% ) were 98.27% , 88.61% , 79.17% , and 27.17% for 24h treatment , respectively ( figure 1a ) ; the percentages of cellular viability vs. the control cells ( 100% ) were 94.83% , 58.23% , 14.24% , and 12.36% for 48 h treatment , respectively ( figure 1b ) . s2101 inhibits cell growth in dose - dependent and time - dependent manners , indicating an inhibitory effect of s2101 against excessive growth of skov3 cells . annexin - v / pi - stained flow cytometric analysis was used to determine whether the reduced cell viability was due to apoptosis . the proportion of both early apoptotic and late apoptotic cells increased significantly for those treated with s2101 at a concentration of 100 m for 48 h as compared to control cells ( figure 1c ) . moreover , apoptosis - related protein bax and bcl-2 were detected by western blot analysis . treatment of s2101 in skov3 cells resulted in upregulation of bax and downregulation of bcl-2 in a time - dependent manner , indicating that s2101 can induce apoptosis in skov3 ( figure 1d ) . the expression of autophagy - related proteins was assessed by western blot analysis to evaluate the effects of s2101 on autophagy in skov3 cells . autophagosome formation involves the conjugation of cytosolic microtubule - associated protein light chain 3 ( lc3-i ) with phosphatidylethanolamine to form lc3-phosphatidylethanolamine ( lc3-ii ) as an essential process . the conversion of lc3-i to lc3-ii is widely recognized as a marker protein of autophagy . figure 2a shows that there was a downward trend in the expression of p62 when the skov3 cells were treated with 100 m of s2101 for 12 h , 24 h , and 48 h. the conversion of lc3-i to lc3-ii increased significantly at 24 h and 48 h. the percentage of cells with gfp - lc3 puncta was significantly increased along with the number and fluorescence intensity of skov3 cells treated with s2101 compared with the control group ( figure 2b ) . 3-methyladenine ( 3-ma ) was used to investigate the role of autophagy in s2101-induced growth suppression . as figure 3a shows , pre - treatment with 3-ma resulted in reduced conversion of lc3-i to lc3-ii as compared with the s2101 alone group . autophagy may play a double - acting role in regulation of cell death , which can either promote or inhibit cell death . skov3 cells were pre - treated by 3-ma , followed by different concentrations of s2101 for 48 h. cck-8 assay results showed that the group treated with s2101 alone significantly inhibited cell proliferation compared with s2101 and 3-ma ( figure 3b ) . our results indicate that s2101 suppressed the phosphorylation of mtor and p70s6k , but exerted little effect on total protein levels ( figure 4 ) . akt participated in the regulation of various signaling pathways , including cell proliferation , growth , survival , angiogenesis , and chemoresistance . our results show that the phosphorylation of akt and the ratio of p - akt / akt were decreased after treatment with various doses of s2101 for 48 h. when skvo3 cells were treated with s2101 at 0 , 50 , 100 , 150 , and 200 m , the percentages of cellular viability over the control cells ( 100% ) were 98.27% , 88.61% , 79.17% , and 27.17% for 24h treatment , respectively ( figure 1a ) ; the percentages of cellular viability vs. the control cells ( 100% ) were 94.83% , 58.23% , 14.24% , and 12.36% for 48 h treatment , respectively ( figure 1b ) . s2101 inhibits cell growth in dose - dependent and time - dependent manners , indicating an inhibitory effect of s2101 against excessive growth of skov3 cells . annexin - v / pi - stained flow cytometric analysis was used to determine whether the reduced cell viability was due to apoptosis . the proportion of both early apoptotic and late apoptotic cells increased significantly for those treated with s2101 at a concentration of 100 m for 48 h as compared to control cells ( figure 1c ) . moreover , apoptosis - related protein bax and bcl-2 were detected by western blot analysis . treatment of s2101 in skov3 cells resulted in upregulation of bax and downregulation of bcl-2 in a time - dependent manner , indicating that s2101 can induce apoptosis in skov3 ( figure 1d ) . the expression of autophagy - related proteins was assessed by western blot analysis to evaluate the effects of s2101 on autophagy in skov3 cells . autophagosome formation involves the conjugation of cytosolic microtubule - associated protein light chain 3 ( lc3-i ) with phosphatidylethanolamine to form lc3-phosphatidylethanolamine ( lc3-ii ) as an essential process . the conversion of lc3-i to lc3-ii is widely recognized as a marker protein of autophagy . figure 2a shows that there was a downward trend in the expression of p62 when the skov3 cells were treated with 100 m of s2101 for 12 h , 24 h , and 48 h. the conversion of lc3-i to lc3-ii increased significantly at 24 h and 48 h. the percentage of cells with gfp - lc3 puncta was significantly increased along with the number and fluorescence intensity of skov3 cells treated with s2101 compared with the control group ( figure 2b ) . 3-methyladenine ( 3-ma ) was used to investigate the role of autophagy in s2101-induced growth suppression . as figure 3a shows , pre - treatment with 3-ma resulted in reduced conversion of lc3-i to lc3-ii as compared with the s2101 alone group . autophagy may play a double - acting role in regulation of cell death , which can either promote or inhibit cell death . skov3 cells were pre - treated by 3-ma , followed by different concentrations of s2101 for 48 h. cck-8 assay results showed that the group treated with s2101 alone significantly inhibited cell proliferation compared with s2101 and 3-ma ( figure 3b ) . our results indicate that s2101 suppressed the phosphorylation of mtor and p70s6k , but exerted little effect on total protein levels ( figure 4 ) . akt participated in the regulation of various signaling pathways , including cell proliferation , growth , survival , angiogenesis , and chemoresistance . our results show that the phosphorylation of akt and the ratio of p - akt / akt were decreased after treatment with various doses of s2101 for 48 h. although various treatments for ovarian cancer including surgery and chemotherapy have been developed , its long - term outcome remains poor . our results show that s2101 inhibited skov3 cells viability in a concentration- and time - dependent manner , and increased the apoptosis and autophagy in skov3 , indicating that s2101 , one of the most active inhibitors of lsd1 , can kill ovarian cancer cells in vitro . autophagy is a necessary response to extracellular stimulus , allowing the elimination of toxic metabolites , intracellular pathogens , and damaged proteins and organelles , and providing energy and amino acids necessary for vital functions during metabolic stress [ 2125 ] . emerging evidence shows that the induction of autophagy is a promising therapeutic strategy in cancer treatment . gfp - tagged lc3-expressing cells have been used as a specific marker to monitor autophagy . our results show that the punctate pattern of gfp - lc3 was increased in skov3 cells treated with s2101 . western blot analysis also showed a marked change in the expression of lc3-i / lc3-ii and p62 in response to s2101 treatment . although autophagy and apoptosis are 2 different physiological processes , they are often interconnected through various crosstalk mechanisms . the common methods to assess the relationship between autophagy and apoptosis rely on : i ) the presence of autophagic features in apoptotic cells , and ii ) rescue of cell apoptosis via changed level of autophagy . to explore whether autophagy was involved in s2101-induced cell apoptosis , the pharmacological inhibition of autophagy ( 3-ma ) the results of cck-8 assay showed that s2101 suppressed the growth of skov3 cells , which was partially reversed by pre - treatment with 3-ma , indicating that autophagy may co - occur with apoptosis in skov3 cells treated with s2101 . several studies have shown that the suppression of the mtor pathway can induce autophagy in several human cancer cell lines , including lung cancer , liver cancer , ovarian cancer , and gastric cancer . our results showed the negative effect of s2101 on the mtor / p70s6k pathway , indicating that inhibition of the mtor pathway may be a mechanism of s2101-triggered autophagy in skov3 cells . as an upstream positive regulator of mtor , akt inactivates tuberous sclerosis complex 2 by phosphorylating it on 4 residues ; the resulting compound is a negative regulator of mtor , thereby activating mtor . our results suggest that the inhibition of akt / mtor / p70s6k signaling pathway , at least in part , participated in the induction of autophagy by treatment of s2101 . further , the akt - mtor axis may be involved in s2101-induced autophagic cell death as apoptosis was promoted , following by the induction of autophagy . our study provides evidence that s2101 inhibited the viability of human ovarian cells by inducing autophagy and apoptosis . in addition , the anticancer effect of 2101 was associated with suppression of the akt / mtor pathway . these results suggest that s2101 is an attractive therapeutic agent for developing alternative treatment protocols , and possibly for combining with other anticancer agents to overcome drug resistance and achieve better outcomes . in vivo studies using cancer animal models
backgrounds2101 is one of the most potent lsd1 inhibitors , which can inhibit ovarian cancer cells viability . 
 this study aimed to detect the mechanism behind the anticancer properties of s2101 in skov3 ovarian cells.material/methodscell viability was tested by cell counting kit-8 ( cck-8 ) assay . 
 cellular apoptosis and autophagy were evaluated by flow cytometric analysis using annexin - v / pi staining methods and green fluorescent protein ( gfp)-fused - lc3 ( gfp - lc3 ) , respectively . 
 western blotting was performed for analyzing the bax , bcl-2 , mtor , p - mtor , p62 , lc3-i , lc3-ii , akt , and p - akt protein expression.resultsour results show that the proportion of early apoptotic and late apoptotic cells increased significantly for cells treated with s2101 at a concentration of 100 m for 48 h. treatment of s2101 in skov3 cells resulted in upregulation of bax and downregulation of bcl-2 in a time - dependent manner , indicating that s2101 can induce apoptosis in skov3 . there was a downward trend in the expression of p62 when the skov3cells were treated with 100 m s2101 for 12 h , 24 h and 48 h. the conversion of lc3-i to lc3-ii was increased significantly at 24 h and 48 h. autophagy was induced by s2101 in skov3 cells , evidenced by an increase in punctuate localization of gfp - lc3 and a change in expression of autophagy - related proteins.conclusionss2101 treatment decreased the levels of phosphorylated akt and mtor . 
 s2101 inhibits skov3 cells viability and induces apoptosis and autophagy . the akt / mtor signaling pathway was found to be affected by s2101 .