| BackgroundEndometrial carcinoma(EC)is a common malignant tumors of the female reproductive system,accounting for 20 to 30%.The incidence of endometrial cancer is closely related to lifestyle.In recent years,there has been a trend of increasing prevalence and younger onset age of EC worldwide.The proportion of childbearing patients under 40 years increased to more than 10%,often accompanied by long-term non-antagonistic estrogen therapy,obesity,hypertension,diabetes,insulin resistance(IR),polycystic ovary syndrome(PCOS).This kind of tumor belong to type I EC,with a high degree of tumor differentiation and a good prognosis,and most of them are stage I according to International Federation of Gynecology and Obstetrics(FIGO).Young patients often lose fertility due to hysterectomy.Therefore,the treatment of preserving the reproductive and physiological functions of young patients is still a clinical issue that urgently needs to be solved.At present,for young EC patients with early low-risk type I who want to preserve fertility,high-dose and long-term application of progesterone is recommended as the first clinical choice.The most common drugs include medroxyprogesterone acetate(MPA,such as Beane)and megestrol acetate(MA,such as Yilizhi).In recent years,Mirena(levonorgestrel intrauterine sustained-release system,LNG-IUD)has increasingly gained patients' acceptance.However,there is a certain recurrence rate after the successful conservative treatment of progesterone.Some patients are insensitive to progesterone or develop progesterone resistance.Besides,the adverse reactions also reduce compliance and limit clinical application of progesterone therapy.Silibinin(SB)is the most active component of silymarin,a polyphenolic flavonoid extracted from milk thistle seeds,and is a natural flavonoid lignans compounds.Silibinin is known in clinical practice as a "natural liver-protecting drug".Recent studies have shown that silibinin plays an anticancer role by inhibiting proliferation,blocking cell cycle and inducing apoptosis of tumor cells through a variety of signaling pathways.Silibinin has progressed into Phase II clinical trial as an anti-tumor drug.Signal transducer and activator of transcription 3(STAT3)is a transcription factor.It is abnormally activated in a variety of tumors and is an intersection point for multiple oncogenic signal transduction pathways.In endometrial cancer,STAT3 regulates cell proliferation,differentiation and apoptosis by altering the expression of target genes.Thus,it is closely related to the occurrence,development and prognosis of EC.It was first reported in 2007 that silibinin can inhibit activation of STAT3 in a prostate cancer model(DU145 cell line)by Agarwal et al.Since then,preclinical data from cancer models and ongoing clinical trial evidence have validated the role of silibinin as a STAT3 inhibitor in cancer.However,the anti-cancer effect of silibinin in endometrial cancer by inhibiting STAT3 has not been reported.The incidence of metabolic syndrome(MS)is rising alarmingly in the modern population.Epidemiology has shown that a variety of malignant tumors are closely related to MS,including sex-hormone-related breast cancer,endometrial cancer,and prostate cancer.The common pathological basis of MS syndrome is obesity,especially central obesity,which leads to insulin resistance and hyperinsulinemia.Abnormal lipid metabolism and insulin resistance are high risk factors for type I EC.Inhibition of abnormal lipid metabolism has become a focus in the treatment of EC.Sterol regulatory element-binding proteins(SREBPs),as a nuclear transcription factor,play an important role in the regulation of cholesterol metabolism and fatty acid metabolism in mammals.Among them,SREBP1 is a main regulator in fat metabolism and metabolic syndrome.The overexpression of SREBP1 causes the disorder of glucose and lipid metabolism,leading to metabolic diseases including obesity,insulin resistance,type 2 diabetes and fatty liver.Moreover,SREBP1 is abnormally expressed in various malignant tumors with abnormal lipid metabolism,especially in hormone-sensitive breast cancer,prostate cancer and endometrial cancer tissues.We previously confirmed that the expression of SREBP1 in EC is significantly higher than normal and atypical hyperplasia endometrium.The expression of SREBP1 is negatively correlated with the differentiation stage of EC.It suggested that overexpression and activation of SREBP1 may be involved in the progression of EC.Studies have shown that silibinin can benefit MS patients.It enhances insulin sensitivity through anti-oxidative stress and lipid toxicity reduction,and inhibits SREBP-lc and genes related to fat synthesis enzymes,so that it interferes with lipid metabolism in the liver and alleviates insulin resistance.Nambiar et al.suggested that SREBP1 may become a novel target of silibinin and play a critical role in silibinin inhibitory effects on prostate cancer.Therefore,as a drug with dual characteristics of anti-tumor and anti-metabolic diseases,the inhibitory effect of silibinin on SREBP1 is expected to provide a new way for the conservative treatment of early low-risk type I ECThis study aims to reveal that silibinin regulates the corresponding signaling pathways by targeting STAT3 and SREBP1,and plays an anticancer role in inhibiting EC cell proliferation,promoting apoptosis and reducing abnormal lipid metabolism.Silibinin provides new options and feasibility for the medicine conservative treatment of young female attacked by early-stage low-risk type I EC,thereby achieving the purpose of inhibiting tumor growth,slowing the disease progression,and creating opportunities for retaining fertility.Part One The mechanism of silibinin affecting the proliferation and apoptosis of type I endometrial carcinoma cells via STAT3 pathway Objective:This part aims to investigate the effects of silibinin on the proliferation,clone formation ability,apoptosis and cell cycle on type I EC cell lines(Ishikawa and RL-952),to detect the expression of STAT3,p-STAT3,and downstream target genes involved in cell cycle and apoptosis at protein and mRNA levels in both EC cells regulated by silibinin,so as to explore the molecular mechanism by which silibinin represses STAT3 signaling pathway and its downstream related genes.Methods:1.The survival rates of Ishikawa and RL-952 cells were respectively examined by MTT assay after silibinin treatment with different concentration for 24,48 and 72 hours.2.The effects of silibinin treatment on cell colony formation ability of Ishikawa and RL-952 cells were tested by colony formation assay.3.The effects of silibinin treatment on apoptosis and cell cycle of Ishikawa and RL-952 cells were detected by flow cytometry.4.After silibinin treatment on Ishikawa and RL-952 cells for 48 h,Western blot was performed to examine the protein expression of STAT3,p-STAT3 and downstream related genes including Caspase-3,Cleaved Caspase-3,Bcl-2,Bax,Survivin,CyclinDl and CyclinBl in both cells.5.After silibinin treatment on Ishikawa and RL-952 cells for 48 h,qRT-PCR was performed to determine the mRNA expression of STAT3 and downstream related genes including Caspase-3,Bcl-2,Bax,Survivin,Ki67,CyclinD1 and CyclinB1 in both cells.Results:1.Results of MTT assay showed that silibinin significantly inhibited the proliferation of type I EC cells(Ishikawa and RL-952)in a time-and dose-dependent manner,P<0.05;The IC50 values of silibinin for Ishikawa and RL-952 cells were 162 ?M and 136.7 ?M after treatment for 48h,respectively.2.Results of colony formation assays showed that,the number of colony formation of Ishikawa and RL-952 cells decreased significantly after silibinin treatment with different concentration compared with the control group,P<0.05.3.Results of flow cytometry showed that the increased concentrations of silibinin signifificantly increased the proportion of cells in G0/G1 phase in Ishikawa,while the percentage of cells in G2/M phase was obviously added up in RL-952,P<0.001.4.Results of flow cytometry showed that as the silibinin concentration increased,the percentage of early apoptosis and late apoptosis cells increased significantly in both cells,P<0.05 and P<0.01.5.Western-blot was performed to detect the effect of silibinin on the protein expression levels of STAT3,p-STAT3 and downstream genes including apoptosis-related proteins and cell cycle regulatory proteins in Ishikawa and RL-952 cells.The results showed that after two cells were treated with silibinin for 48h,the protein expression levels of total STAT3 remained unchanged compared with the control groups,P>0.05,while phosphorylated STAT3 were decreased,P<0.05;the protein expression levels of Survivin,Bcl-2 and Bcl-2/Bax were decreased,P<0.05;Caspase-3 were under-expressed,whereas Cleaved caspase-3 protein levels were over-expressed;in addition,the protein expression levels of CyclinDl were decreased in silibinin-treated Ishikawa cells,while the CyclinB 1 were decreased in silibinin-treated RL-952 cells,P<0.05.6.qRT-PCR was performed to detect the mRNA levels of STAT3 and downstream genes including apoptosis-related genes and cell cycle regulatory genes in Ishikawa and RL-952 cells treated by silibinin.The results showed that after 48 hours of treatment with the increased concentration of silibinin,the mRNA levels of STAT3,Bcl-2/Bax,Survivin and Ki67 were significantly decreased compared with the control cells in both cells,P<0.01;pro-Caspase-3 were significantly decreased,P<0.05;the mRNA levels of CyclinD1 were reduced in silibinin-treated Ishikawa cells,while CyclinB 1 were reduced in silibinin-treated RL-952 cells,P<0.001Conclusion:1.Silibinin significantly inhibited the viability and proliferation of type I EC cells.The IC50 values of silibinin at 48 hour for Ishikawa and RL-952 cells were 162?M and 136.7 ?M,respectively.2.Silibinin can inhibit the colony formation ability of Ishikawa and RL-952 cells3.Silibinin blocked cell cycle of Ishikawa cell in G0/G1 phase,while blocked RL-952 cell in G2/M phase.4.Silibinin significantly promoted apoptosis in Ishikawa and RL-952 cells.5.Silibinin obviously inhibited STAT3 phosphorylation in type I EC cells,regulated apoptosis-associated and cell cycle-related genes in downstream signaling pathways at protein and mRNA levels.6.The current study confirmed that silibinin can inhibit the activation of STAT3,cause cell proliferation inhibition,apoptosis promotion and cell cycle arrest in type ?EC cells.It is expected to become a new way for the medicine conservative treatment of type ? EC,Part Two The mechanism of silibinin regulating SREBP1-mediated abnormal lipid metabolism of type ? endometrial carcinoma cellsObjective:This portion targets to investigate the effects of silibinin on the expression of protein and mRNA levels of SREBP1 and its downstream target genes involved in lipid metabolism in type ? EC cell lines(Ishikawa and RL-952),to detect the changes of SREBP1 expression in the nucleus after treatment with silibinin,to examine the changes of intracellular lipid synthesis and accumulation after treatment with silibinin,and to explore the molecular mechanism of silibinin on the anti-tumor effects of type ?EC by inhibiting abnormal lipid metabolism pathway.Methods:1.Western blot was used to detect the protein expression of SREBP1 in Ishikawa and RL-952 cells.After treatment with 150?M silibinin for 48 hours,the protein expressions of SREBP1 in both cells were detected by Western blot.2.After silibinin treatment on Ishikawa and RL-952 cells for 48 h,Western blot was used to detect the protein expressions of SREBP1 and downstream related genes including ACLY,p-ACLY,SCD-1 in both cells.3.After silibinin treatment on Ishikawa and RL-952 cells for 48 h,qRT-PCR was used to detect the mRNA levels of SREBP1,SCAP and downstream related genes including FASN,ACLY,SCD-1 and HMGCR in both cells.4.Cell immunofluorescence assay was used to detect the expression of nuclear expression levels of SREBP1 in RL-952 cells with high SREBP1 expression levels after treatment with 150 ?M silibinin for 48 hours.5.After treatment with 150 ?M silibinin for 48 hours,the distribution of intracellular lipid droplets in Ishikawa and RL-952 cells were detected by Oil red O staining.Results:1.Western-blot was used to detect the protein levels of SREBP1 and its downstream genes in Ishikawa and RL-952 cells.The results showed that the protein levels of SREBP1 in RL-952 cells were higher than that in Ishikawa cells;After treatment with the increased concentration of silibinin for 48 h,the protein levels of SREBP1,SCD-land p-ACLY were decreased compared with the control groups,P<0.05,while total ACLY remained unchanged,P>0.05.2.qRT-PCR was used to detect the mRNA levels of SREBP1,SCAP,and downstream genes in Ishikawa and RL-952 cells treated by silibinin.The results showed that after treatment with the increased concentration of silibinin for 48 h,the mRNA levels of SREBP1,SCAP,FASN,ACLY,SCD-1 and HMGCR were downregulated in both cells compared with the control cells,P<0.053.The results of cell immunofluorescence staining on RL-952 cells with high SREBP1 expression after treatment with 150 ?M silibinin for 48 hours showed that compared with the control cells,mean density of SREBP1 in the nucleus was calculated and the expression of intranuclear SREBP1 of silibinin-treated RL-952 cells was significantly decreased,P<0.014.The results of the effect of silibinin on lipid accumulation in Ishikawa and RL-952 cells detected by Oil red O staining showed that after treatment with 150 ?M silibinin for 48 hours,the intracellular distribution area of lipid droplets in both cells were significantly decreased compared with the control cells,P<0.01.Conclusion:1.Silibinin inhibited the expression of SREBP1 and its downstream lipid metabolism-related genes in type I EC cells lines(Ishikawa and RL-952),thereby blocking abnormal lipid metabolism pathway mediated by SREBP1.2.Silibinin inhibited the expression of nuclear SREBP1 of RL-952 cells and reduced lipid synthesis and accumulation both in Ishikawa and RL-952 cells indicated that it hindered the synthesis and metabolism of lipid mediated by SREBP1.3.The current study verified that silibinin can inhibit abnormal lipid metabolism and lipid synthesis in type I EC cells,and synergistically exerts anticancer propertiesPart three The effects of silibinin on tumor proliferation and apoptosis of endometrial carcinoma in vivoObjective:This part aims to determine the effects of silibinin on the proliferation and apoptosis in subcutaneous xenograft tumor in nude mice subcutaneously injected by RL-952 cells,and to detect the expression of p-STAT3 and SREBP1 in the tumor tissues by immunohistochemical staining.Investigation on in-vivo effect of silibinin can further verified the anti-tumor mechanism of silibinin on type I EC.Methods:1.To established tumor models of human endometrial carcinoma,RL-952 cells were subcutaneously implanted into BALB/C female nude mice.The effects of different doses of silibinin on tumor growth including volume and weight in nude mice were observed and measured.2.Apoptosis of RL-952 cells in subcutaneously transplanted tumor tissues of nude mice with different doses of silibinin were detected by TUNEL assay.3.After the intragastric administration of silibinin at different doses,transplanted tumors were taken from nude mice in each group for immunohistochemical detection of p-stat3 and SREBP1 expression in tumor tissues.4.Serum GOT,GPT,urea nitrogen(Bun)and creatinine(Cr)of each group of nude mice were detected by biochemical kit.Results:1.Four groups given different doses(50,100,150,200 mg/kg)of silibinin by gavage,the growth volume of the tumors started to decrease from 15 days after administration compared with the control group,P<0.05.When tumors were removed after 21 days of administration,the low-dose groups(50,100mg/kg)showed no significant difference in tumor weight reduction compared with the control group,while the high-dose groups(150,200mg/kg)showed significant reduction in tumor weight,P<0.05.2.The effect of silibinin on apoptosis in subcutaneous transplanted tumor tissues of nude mice detected by TUNEL assay showed that the apoptosis rate of tumor cells in the low dose groups(50,100 mg/kg)did not increase significantly,while the apoptosis rate of tumor cells in the high dose(150,200 mg/kg)groups increased significantly,P<0.01.3.The results from immunohistochemistry assays showed the expression of p-STAT3 and SREBP1 in the silibinin-treated groups significantly decreased with the increased doses compared with the control group P<0.01.4.There was no significant difference in serum GOT,GPT,Bun and Cr in nude mice of each group,P>0.05Conclusion:1.Silibinin can inhibit the growth of EC xenograft tumor and promote the apoptosis of tumor tissue.2.Silibinin inhibited the expression of p-STAT3 and SREBP1 in subcutaneous transplanted tumor tissues of nude mice.3.In vivo assays demonstrated that feasibility study of silibinin can be carried out for clinical application of the conservative treatment of type I endometrial carcinoma by inhibiting STAT3 activation and SREBP1 to restrain the development of EC. |
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