| Background and objectives:Cervical cancers(CC)are one of the leading causes of cancer-related death among women in developing countries,which are classified into squamous cell carcinomas and adenocarcinomas according to their cellular origins.Surgery is still the first choice of CC treatment,but frequent relapse and metastasis lead to poor prognosis of CC patients,especially those with advanced tumors.Chemotherapy has been widely used to prevent recurrence in postoperative management of CCs.However,drug resistance is very common and the toxicities severe damage patients' life quality.It is therefore of clinical values to explore more reliable and less toxic therapeutic approach in the treatment of cervical carcinomas.In recent years,searching and extract from Chinese herbal medicine and natural plant active ingredients of the prevention and treatment of tumor has become a new way for the treatment of tumor.Resveratrol(Res)is widely exist in peanuts and grape skins,wine and a variety of medicinal plants in more than a kind of phenolic compounds,its biological effect mainly has anti-tumor,anti-inflammatory,anti-oxidation,anti-aging,cardiovascular protection,reducing blood fat,regulating immune function,etc.Previous studies have found that resveratrol can obviously induce apoptosis of leukemia,liver cancer,breast cancer,colon cancer,ovarian cancer,prostate cancer,gastric cancer,laryngeal cancer and other tumor cells.Our previous work showed that human medulloblastoma cells were sensitive to resveratrol in terms of growth arrest,neuron-oriented differentiation and distinct apoptosis.And the growth of transitional cell carcinomas in mouse urinary bladder can be efficiently suppressed by regular resveratrol installation.More importantly,resveratrol has little harmful effect on glial cells and neurons in central nervous system and the tumor surrounding uro-epithelium,suggesting its potential values in the treatments of those cancers.In the case of cervical cancers,resveratrol exerts radiosensitizing and anti-proliferative effects on them,but its underlying molecular mechanism remains to be investigated.Studies of the role of resveratrol in cervical cancer at home and abroad are rare?So far,cisplatin is the first-line chemotherapeutic drug for cervical cancer but its efficacy is not promising because of the drug resistance and the serious adverse reactions.It is,therefore,in urgent need to explore a safer and more effective agent against cervical cancers.The current study thus aims to check 1)cervical squamous carcinoma,adenocarcinoma and normal cervical tissues from the uterine fibroid uterine all cutting operations in Wnt,Notch and STAT3 signaling pathway and the expression of related genes 2)the statuses of STAT3-,Notch-and Wnt-mediated signaling in a squamous carcinoma cell line,SiHa,and an adenocarcinoma cell line,HeLa,of the cervix,4)the influence of resveratrol in the biological activities of the three signaling pathways and 4)the biological consequence(s)of selective inhibition of individual signaling to the two cell lines.Materials and methods:The archived paraffin blocks of 106 cervical cancers and,where possible,the tumor surrounding tissues were provided by the Division of Clinical Pathology,Shen-Jing Hospital,China Medical University at Shenyang,China.Human cervical adenocarcinoma cell line HeLa and human cervical squamous carcinoma cell line SiHa were provided by Cell Bank of Academia Sinica,Shanghai.This study based on paraffin tissue microarray technology,using immunohistochemical staining to detect Notch,Wnt and STAT3 signaling pathways and the related genes in cervical cancer.By flow cytometry,trypan blue,immunocytochemistry and immunofluorescence,RT-PCR and western-blotting methods to detect Notch,Wnt and STAT3 signaling pathway in cervical cancer cell lines both before and after resveratrol treatmen.Applicated three corresponding inhibitors and using the above methods to detect the changes of above signaling pathways.Experimental data using SPSS 12.0 software package used Kruskal Wallis and Mamm Whitney test methods for statistical analysis.Results:1.Frequent expression of Notch,Wnt and STAT3 signaling components in cervical cancers1)The results of immunohistochemical staining performed on 25 cervical adenocarcinomas,43 squamous cell carcinomas and 38 normal cervical tissues showed that Notchl was expressed in 7 of 38(18.4%)CC surrounding premalignant tissues,in 18 of 25(72.0%,p<0.01)adenocarcinomas and in 31 of 43(72.1%,p<0.01)squamous cell carcinomas.Similarly,the frequencies of Notch2,Hesl,Wnt2,Wnt5a,?-catenin and p-STAT3 of CC samples were significantly higher than the noncancerous group(p<0.01).2)The expression rates of the protein inhibitor of activated STAT3/PIAS3 were 52.6%(20/38)among noncancerous epithelial tissues but reduced to 16.0%(4/25,p<0.01)and 11.6%(5/43,p<0.01)among cervical adenocarcinomas and squamous carcinomas,respectively.2.The functions of resveratrol on three signaling pathways of the two cervical cancer cell lines1)Resveratrol could inhibit the growth of cervical cancer cells and induce apoptosisHE morphologic staining showed that HeLa and SiHa cells showed reduction of cell numbers and distinct apoptotic phenotypes after 100?M resveratrol treatment for 48 hours.Trypan blue cell discrimination assay revealed increased fraction of cell death in time-related pattern(p<0.01)and the total cell number were significantly decreased after treated by 100 ?M resveratrol for 48 hours(p<0.05).Flow cytometry demonstrated that resveratrol-treated HeLa and SiHa cells exhibited S phase arrest accompanied with increased apoptotic fraction.2)The influence of resveratrol on Notch signal pathway in cervical cancer celllinesImmunocytochemical/ICC staining showed that Notch1 and Notch2 were expressed in HeLa and SiHa cells and mainly distributed in cytoplasm,while immunocytochemical/ICC and immunofluorescent/IF staining showed Hesl,was mainly located in the nuclei.When the two cell lines were treated with 100 ?M resveratrol,the expression level of all above proteins were significantly reduced.RT-PCR and western-blotting showed that Notch1,Notch2 and Hesl were significantly down-regulated after treated with 100 ?M resveratrol for 48 hours.3)The influence of resveratrol on Wnt signal pathway in cervical cancer cell linesImmunocytochemical/ICC staining showed that Wnt2 and Wnt5a were expressed in HeLa and SiHa cells and mainly distributed in cytoplasm,while immunocytochemical/ICC and immunofluorescent/IF staining shwoed ?-catenin was mainly located in the nuclei.When the two cell lines were treated with 100 ?M resveratrol,the expression level of all above proteins were significantly reduced.RT-PCR and western-blotting showed that Wnt2,Wnt5a and ?-catenin were significantly down-regulated after resveratrol treatment.4)The influence of resveratrol on STAT3 signal pathway in cervical cancer cell linesThe levels of STAT3 expression remained largely unchanged between the HeLa and SiHa cells without and with 100 ?M resveratrol treatment by RT-PCR,But the amounts of p-STAT3 proteins were significantly reduced after resveratrol treatment.In normally cultured HeLa and SiHa cells,the protein inhibitor of activated STAT3/PIAS3 was expressed in low level,which was up-regulated after resveratrol treatment.3.Variable sensitivities of HeLa and SiHa cells to three pathway inhibitors1)The results of IF revealed that 8 ?M L-685,458 blocked Notch activation in the two cell lines in terms of reduced cytoplasmic distribution and almost diminished nuclear translocation of Hesl proteins.However,this treatment neither brought about growth arrest and cell death nor the cell cycle alteration.2)The results of IF revealed that 10?M XAV-939 blocked Wnt activation in the two cell lines in terms of reduced cytoplasmic distribution and almost diminished nuclear translocation of ?-catenin proteins.However,this treatment neither brought about growth arrest and cell death nor the cell cycle alteration.3)Upon 80 ?M AG490 treatment,STAT3 phosphorylation was inhibited in HeLa and SiHa cells.Furthermore,61.95%of HeLa cells were arrested in S-phase and 40.22%underwent apoptosis,which were significantly higher than that(34.14%and 0%)of normally cultured HeLa cells(p<0.05).In AG490 treated SiHa cells,the S-phase and apoptotic cells were 43.54%and 27.25%respectively in comparison with 17.60%and 0%of their normally cultured counterparts(p<0.05).Conclusions:1.Notch,Wnt and STAT3 signaling pathway were all in the activated state in cervical cancers,so the development and incidence of cervical cancer were proboblely has certain correlations with Notch,Wnt and STAT3 signaling pathways.2.Resveratrol could induce apoptosis of cervical cancer cells by way of time dependence.Resveratrol made the cervical adenocarcinoma cancer HeLa and cervical squamous cancer SiHa cell cycle blocked in S phase.These findings further confirm the multiple targeting features of resveratrol and,meanwhile,suggest potential therapeutic effectiveness of resveratrol on both squamous cell carcinomas and adenocarcinomas of human cervical uterus.3.Resveratrol could down-regulate Notch 1,Notch2,Hesl,Wnt2,Wnt5a,?-catenin and p-STAT3 expression in SiHa and HeLa cells,at the same time up-regulate the expression of PIAS3.The obove results prompted that resveratrol inhibit the actication of Notch,Wnt and STAT3 signaling pathways of cervical cancer cells.4.L-685,458,the inhibitor of Notch signaling pathway,blocked Notch activation in HeLa and SiHa.However,this treatment neither brought about growth arrest and cell death nor the cell cycle alteration.5.XAV-939 was the inhibitor of Wnt signaling pathway,it could block Wnt activation in the two cell lines.However,this treatment also neither brought about growth arrest and cell death nor the cell cycle alteration.6.The inhibitor of STAT3 signaling pathway was AG490,it could block STAT3 activation in HeLa and SiHa.At the same time,it could induce apoptosis and S phase arrest of the two cervical cell lines.These results strongly suggest that among the three resveratrol inhibited signaling pathways so far checked,inactivation of STAT3 signaling is the lethal molecular event in Hela and SiHa cells.7.Alternatively,the activated STAT3 signaling is crucial for the proliferation and survival of human cervical cancers although Notch signaling may also play certain active roles in those processes.In this context,STAT3 targeting strategy would be applicable in the management of this sort of woman malignancies and the less toxio resveratrol with multifaceted actions is the ideal candidate. |
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