Study Of The Antitumor Mechanism Of Monomeric Compound SZ-NCI-C Against Ovarian Cancer

Background:According to 2018 cancer statistics from 185 countries worldwide,the incidence and mortality rate of ovarian cancer ranked eighth among female cancer patients.At the same time,ovarian cancer,one of the reproductive system tumors that threaten women’s health,has the highest mortality among reproductive system tumors.Due to the concealed onset of ovarian cancer,about60%of patients were already in advanced stage of cancer when they were diagnosed firstly.The treatment of ovarian cancer mainly relied on surgical treatment combined with platinum-based and paclitaxel-based chemotherapeutic drugs.However,ovarian cancer patients tended to develop resistance to chemotherapeutic drugs,which affected the effect of chemotherapy and leaded to poor prognosis.Therefore,the urgent need to seek new and effective chemotherapy drugs has become one of the important problems for ovarian cancer treatment.Objective:Our preliminary study showed that SZ-NCI-C,a monomeric compound extracted from mangosteen peel,has a significant inhibitory effect on the proliferation of ovarian cancer cell lines.Therefore,we intend to explore the anti-ovarian cancer effect of SZ-NCI-C through animal and cell models in this paper.Methods:1.Subcutaneous xenograft tumor model and anti-ovarian cancer effect of SZ-NCI-C in vivo:The subcutaneous tumor model of human ovarian cancer cell line Hey A8 was established in nude mice.SZ-NCI-C was intraperitoneal injected every 3 days.The body weight of nude mice and the volume of xenograft tumors was measured continuously.At the end point of the experiment,the tumors were weighed and statistically analyzed to reveal the in vivo anti-ovarian cancer effect of SZ-NCI-C on subcutaneous Hey A8 tumors in nude mice.2.MTS cell proliferation experiment:Gradient concentrations of SZ-NCI-C were used to treat human ovarian cancer cell line（Hey A8,SKOV3）and mouse ovarian cancer cell line(ID8-^luc-sort).After 24,48 and 72 hours of SZ-NCI-C treatment,Cell Titer 96AQ_ueous One Solution Reagent was added to detect absorbance value to verify the inhibitory effect of SZ-NCI-C on ovarian cancer cell viability.3.Plate colony formation assay:Gradient concentrations of SZ-NCI-C were used to treat ovarian cancer cells Hey A8,SKOV3 and ID8-^luc-sort to detect the effect of SZ-NCI-C on the colony formation ability of ovarian cancer cells.Soft Agar colony formation Assay:Gradient concentrations of SZ-NCI-C were used to treat Hey A8 cells grown in Soft Agar to reveal the effect of SZ-NCI-C on the ability of ovarian cancer cells to anchorage-independent growth.4.Flow cytometry cell cycle experiment:Hey A8 and SKOV3 cells were treated with SZ-NCI-C at gradient concentrations for 24,48 and 72 hours,respectively.The effect of SZ-NCI-C on the cell cycle distribution of ovarian cancer cells was detected by PI staining cell cycle detection kit.5.Cell senescence assay:Hey A8 and SKOV3 cells were treated with SZ-NCI-C at gradient concentrations for 10 days,and the effect of SZ-NCI-C on the activity ofβ-galactosidase in ovarian cancer cells was detected using theβ-galactosidase cell senescence kit.Then the effect of SZ-NCI-C on the cell senescence of ovarian cancer could be inferred.6.Detection of protein expression levels by Western blot:In order to explore the anti-ovarian cancer mechanism of SZ-NCI-C,Hey A8 and SKOV3 cells were treated with SZ-NCI-C at gradient concentrations for 24,48 and 72 hours,respectively.Then Western blot was used to detect the expression levels of proteins in various signaling pathways which might affect by SZ-NCI-C treatment.7.Detection of autophagic vacuole numbers by Monodansylcadaverine（MDC）staining:Western blot showed that the expression levels of autophagy-related proteins were regulated by SZ-NCI-C.Ovarian cancer cells Hey A8 and SKOV3 were treated with gradient concentration of SZ-NCI-C.MDC staining was used to explore the effect of SZ-NCI-C on the number of autophagic vacuoles in ovarian cancer cells.Results:1.The in vivo assay of subcutaneous Hey A8 tumors showed that the tumor volume of subcutaneous xenograft tumor in SZ-NCI-C treatment group was significantly lower than that in the vehicle group and the chemotherapy drug cisplatin positive control group.The weight of subcutaneous xenograft tumor in the SZ-NCI-C treatment group was also significantly lower than that in the vehicle group.The body weight of nude mice in SZ-NCI-C treatment group was not significantly different from that in vehicle group and chemotherapy drug cisplatin positive control group.These results indicated that SZ-NCI-C significantly inhibited the growth of subcutaneous Hey A8 tumors in nude mice,and had less toxic and side effects on nude mice.2.The MTS cell viability assay showed that SZ-NCI-C significantly inhibited the cell viability of ovarian cancer cells,and this inhibition was in a time-dependent and concentration-dependent manner.The longer treatment time and higher concentration of SZ-NCI-C induced the more obvious inhibitory effect on cell viability.3.plate colony formation assay and soft Agar colony formation assay showed that as the concentration of SZ-NCI-C increased,the colony size of ovarian cancer cells became smaller,suggesting that SZ-NCI-C significantly inhibited the ability of plate colony formation and anchorage-independent growth of ovarian cancer cells.4.Cell cycle assays showed that SZ-NCI-C arrested the cell cycle of Hey A8 at G₂/M phase,and arrested the cell cycle of SKOV3 at S phase in a time-and concentration-dependent manner.The longer time and higher concentration of SZ-NCI-C treatment induced the more obvious effect on cell cycle arrest.5.β-galactosidase cell senescence detection showed that as the concentration of SZ-NCI-C increased,more Hey A8 cells were stained in blue color with increasedβ-galactosidase activity.But SKOV3 cells were barely stained.These data suggested that SZ-NCI-C induced cell senescence of Hey A8.However,whether SZ-NCI-C caused SKOV3 cell senescence remains to be further explored.6.Western blot showed that compared with control group,SZ-NCI-C treatment caused a differential expression of cell cycle-related proteins,including CDK2,CDK4,CDK7,p27,Cyclin D3,GSK-3βand p-GSK-3β,in Hye A8 and SKOV3 cells.These results suggested that SZ-NCI-C might block cell cycle and inhibit cell viability of ovarian cancer cells by regulating the expression levels of these cell cycle-related proteins.Western blot showed that SZ-NCI-C increased the expression levels of Beclin-1,LC3A/B-II and Atg7 in Hey A8 and SKOV3 cells,suggesting that SZ-NCI-C regulated autophagy.In addition,SZ-NCI-C can also affect the expression levels of other proteins in some important signaling pathways,For example,SZ-NCI-C increased the expression levels of TIMP2,Rac1/2/3,Claudin-1 and CD44.8.MDC staining showed that different concentrations of SZ-NCI-C did not significantly induce an increase number of autophagosomes in Hey A8 and SKOV3 cells.Western blot results suggested that SZ-NCI-C might affect autophagy process.However,MDC staining excluded the effect of SZ-NCI-C on increasing the number of autophagic vacuoles in ovarian cancer cells.How SZ-NCI-C affects autophagy remains to be further explored by other experimental methods.Conclusions:We found that SZ-NCI-C significantly slowed the growth of xenograft tumors and inhibited the cell viability and colony formation ability of ovarian cancer cells.SZ-NCI-C induced cell-cycle arrest of ovarian cancer cells and caused cell senescence in Hey A8 cells.Western blot showed that SZ-NCI-C regulated the expression levels of cell-cycle related proteins,autophagy-related proteins and other important proteins.These finding suggest that SZ-NCI-C may be a potential chemotherapy agent against ovarian cancer.