| BackgroudEsophageal squamous cell carcinoma(ESCC)is the second common cancer in Henan province and is well-known for aggressiveness and dismal prognosis.Adjuvant therapies,chemotherapy,radiotherapy and endoscopic treatment have not improved survival rates in patients with late stage esophageal carcinoma.All-trans retinoic acid(ATRA)is the active ingredient of Vitamin A and affects a wide spectrum of biological processes including development,growth,neural function,immune function,reproduction,and vision.It is one of the most potent therapeutic agents used for treating cancers,especially lung adenocarcinomas.ATRA inhibits metastatic potential and angiogenesis in several tumor models.We investigated the effects of ATRA on the expression of angiopoietin 1(Ang-1),angiopoietin 2(Ang-2)and receptor Tie-2 in EC1 cells in vitro.We also assessed the growth and migration of EC1 cells in vitro.ATRA treatment caused 29.5% and 40.3% reduction of the growth of EC1 cells after 24 hours and 48 hours,relative to untreated cells.After 24 hours ’ treatment,ATRA plus Fluorouracil treatment reduced the viability more strongly by 42.3%,relative to the control.Moreover,ATRA treatment decreased the migration of EC1 cells by 87% in a wound healing assay.Furthermore,ATRAtreatment led to a marked decrease of the transcript levels of Ang-1,Ang-2,and Tie-2,VEGF,and VEGF receptors,as assessed by real-time RT-PCR.Importantly,the protein levels of Ang-1,Ang-2 and Tie-2 were reduced by ATRA treatment.In vivo,we found ATRA treatment suppressed the tumor growth and improved the cachexia of mice.Importantly,ATRA treatment decrease the expression of CD31,Ang-1,Ang-2 and Tie-2 in subcutaneous tumors of EC1 cells.Collectively,our findings demonstrate that ATRA treatment exhibits a dose-and temporal-dependent effect in the metastatic behavior,suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis in EC1 cells.ObjectiveTo investigate the effect of all-trans retinoic acid on cell migration and metastasis,expressions of Ang-Tie-2 pathway in esophageal cancer EC1 cells in vivo and in vitro.MethodsEC1 cells were processed by different concentrations(0、0.1、1、10μmol/L)of ATRA and 100mg/L Fluorouracil.The proliferation of the treated EC1 cells was measured by CCK-8 assay.Wound healing assay was used to observe the effect ATRA showed on cell migration and metastasis.The protein and mRNA expressions of Ang-1,Ang-2,Tie-2 in the cells were detected by western blot and RT-PCR.Results1.CCK-8 resultst: To examine the effects of ATRA on EC1 cell proliferation,cells were cultured for 24 and 48 hours in media including 0.1,1,or 10 mmol/L ATRA,Fluorouracil(100 mg/L)or no treatment.Cells incubated in 0.1 mmol/L ATRA showed 13.7% and 24.2% reduction at 24 and 48 hours,relative to untreated cells,respectively(p < 0.05;Fig 1A).Cells incubated in 1 mmol/L ATRA showed 22.0%and 33.9% reduction at 24 hours and 48 hours,relative to untreated cells,respectively(p < 0.05;Fig 1A).Cells incubated in 10 mmol/L ATRA showed 29.5% and 40.3%reduction at 24 hours and 48 hours,relative to untreated cells.100 mg/L Fluorouracil caused 29.9% and 41.2% reduction at 24 hours and 48 hours,relative to untreated cells,respectively(p < 0.05;Fig 1A).The difference between the reductions caused by 10 mmol/L ATRA and 100 mg/L Fluorouracil were not significant(p > 0.05;Fig1A).We also calculated the dead cell number in each conditions.Cells incubated in0.1 mmol/L ATRA showed 1.24 and 1.18 times increase of dead cells at 24 and 48 hours,relative to untreated cells,respectively(p < 0.05;Fig 1B).Cells incubated in 1mmol/L ATRA showed 2.28 and 2.9 times increase of dead cells at 24 and 48 hours,relative to untreated cells,respectively(p < 0.05;Fig 1B).Cells incubated in 10mmol/L ATRA showed 3.17 and 2.45 times increase of dead cells at 24 and 48 hours,relative to untreated cells,respectively(p < 0.05;Fig 1B).100 mg/L Fluorouracil caused 3.19 and 2.47 times increase of dead cells at 24 and 48 hours,relative to untreated cells,respectively(p < 0.05;Fig 1B).10 mmol/L ATRA plus 100 mg/L Fluorouracil caused 42.3% reduction of cell viability after 24 hours’ treatment,while ATRA alone caused 21.5% reduction and Fluorouracil alone caused 25.2% reduction,relative to untreated cells,respectively(p < 0.05;Fig 1C).The expression of Cl-PARP in cells treated with ATRA also increased compared with untreated cells(Fig 1D).After 24 hours of ATRA plus Fluorouracil treatment,we checked the expression of Cleaved caspase3(Fig 1E).We compared ATRA treatment,fluorouracil treatment together with ATRA plus fluorouracil treatment.After 24 hours’ treatment,ATRA caused 13.5% increase of the Cl-caspase3 expression,and Fluorouracil caused 33.2%increase,while ATRA plus Fluorouracil caused 66.9% increase,relative to untreated cells,respectively(p < 0.05;Fig 1 F).2.Wound healing assay: To study the effect of ATRA inhibition on EC1 migration,wound healing assays were performed under the same conditions as above,with images captured at 24 hours post wound.We selected 10-15 points at the edge of cells and used the average distance to compare.After 24 hours treatment,the distance of no treatment was 1.457 mM.The distance of 0.1 mmol/L ATRA was 1.227 mM.The distance of 1 mmol/L ATRA was 0.825 mM.The distance of 10 mmol/L ATRA was0.188 mM.The distance of 100 mg/L Fluorouracil was 0.046 mM.Compared with notreatment,the distance between the cells in 0.1 mmol/LATRA has decreased 15.8%,the distance between the cells in 1 mmol/L ATRA has decreased 43.4%,the distance between the cells in 10 mmol/LATRA has decreased 87.1%,the distance between the cells in 0.1 mmol/LATRA has decreased 96.9%.The distances between the wound scratched in the cells were wider with increasing doses of ATRA,and especially 10mmol/L ATRA and 100 mg/L Fluorouracil were significantly different from the no treatment control(Fig 2)(p < 0.05).3.RT-PCR results: To investigate the effects of ATRA on the Angiopioteins-Tie2 pathway,we assessed DNA expression of Ang-1,Ang-2 and Tie-2 in EC1 cells incubated under the same conditions as above.After 24 hours’ treatment,DNA expression level was downregulated compared to controls in each condition(p< 0.05;Fig 3).We also found there are decrease in the expressions of Ang-1.The cells in 0.1mmol/L ATRA had a decrease of 52%.The cells in 1 mmol/L ATRA had a decrease of69%.The cells in 10 mmol/L ATRA had a decrease of 80%.The cells in 100 mg/L Fluorouracil had a decrease of 73%(Fig 3B).When it came to the expression of Ang-2,the cells in 0.1 mmol/LATRA had a decrease of 30%.The cells in 1 mmol/L ATRA had a decrease of 55%.The cells in 10 mmol/L ATRA had a decrease of 69%.The cells in 100 mg/L Fluorouracil had a decrease of 66%(Fig 3C).Furthermore,the expression of Tie-2,the cells in 0.1 mmol/L ATRA had a decrease of 27%.The cells in1 mmol/L ATRA had a decrease of 76%.The cells in 10 mmol/L ATRA had a decrease of 94%.The cells in 100 mg/L Fluorouracil had a decrease of 94%(Fig 3D).We checked the expression of VEGF,the cells in 10 mmol/L AM80 had a decrease of20.8%.The cells in 10 mmol/L ATRA had a decrease of 25.3%.The cells in 100 mg/L Fluorouracil had a decrease of 36.3%.The cells in ATRA and Fluorouracil dual treatment had a decrease of 64.4%,relative to untreated cells,respectively(p < 0.05;Fig 3E).We checked the expression of Flt-1,the cells in AM80 had a decrease of22.9%.The cells in ATRA had a decrease of 31.1%.The cells in Fluorouracil had a decrease of 34.0%.The cells in ATRA and Fluorouracil dual treatment had a decrease of 62.0%,relative to untreated cells,respectively(p < 0.05;Fig 3F).We checked the expression of KDR,the cells in AM80 had a decrease of 18.0%.The cells in ATRA had a decrease of 30.0%.The cells in Fluorouracil had a decrease of 40.2%.The cellsin ATRA and Fluorouracil dual treatment had a decrease of 55.0%,relative to untreated cells,respectively(p < 0.05;Fig 3G).4.Western blot results: To assess the effects of ATRA and fluorouracil on the angiopoietin-Tie2 pathway in EC1 cells,Ang-1,Ang-2 and Tie-2 protein expression was assessed by western blot under the conditions above(Fig 4A).There was a trend of decline of Ang-1,Ang-2 and Tie-2 protein expression with increasing dose of ATRA,and also with 100 mg/L fluorouracil treatment(Fig 4B).Compared the expression of Ang-1 with no treatment,the cells in 0.1 mmol/L ATRA had a decrease of 36%.The cells in 1 mmol/L ATRA had a decrease of 40%.The cells in10mmol/LATRA had a decrease of 35%.The cells in 100 mg/L Fluorouracil had a decrease of 87%(Fig 4C).Compared the expression of Ang-2 with no treatment,the cells in 0.1 mmol/LATRA had a decrease of 36%.The cells in 1 mmol/LATRA had a decrease of 40%.The cells in 10 mmol/LATRA had a decrease of 35%.The cells in100 mg/L Fluorouracil had a decrease of 87%(Fig 4D).Compared the expression of Tie-2 with no treatment,the cells in 0.1 mmol/L ATRA had a decrease of 15%.The cells in 1 mmol/L ATRA had a decrease of 0.6%.The cells in 10 mmol/LATRA had a decrease of 22%.The cells in 100 mg/L Fluorouracil had a decrease of 68%(Fig 4E).5.Pre-clinical studies were performed to assess the effects of ATRA on EC1 cells in vivo.To evaluate the efficacy,Mice were injected subcutaneous with EC1 cells,and we divided 25 mice into five treatment groups to replicate the in vitro experiments above.When tumors became visible,mice were gavaged with different concentrations(0.1,1,10 mg/Kg)of ATRA,(50 mg/Kg/day)fluorouracil,or PBS and DMSO as placebo.We weighted the body weight of mice every week.After 10 days’ treatment,the body weight of 0.1 mg/Kg ATRA treatment group have 4% increase compared with no treatment.The body weight of 10 mg/Kg ATRA treatment group have 9% increase compared with no treatment(Fig 5A).Mice in the placebo group exhibited cachexia comparted to the treatment groups(Fig 5B).Mice in the treatment groups produced smaller tumors compared to control.ATRA can significantly decrease the area size of xenograft tumors.The average tumor size of no treatment are 2.2 times larger than 10 mg/Kg ATRA group.Compared with the tumor size of 10mg/Kg ATRA group,0.1 mg/Kg ATRA group has 30% increase and 0.1 mg/Kg ATRAgroup has 34% increase.When compared with 10 mg/Kg ATRA with 50 mg/Kg/day fluorouracil,there is a 50% increase(Fig 5C,Fig 5D).We checked the expression of CD31,Ang-1,Ang-2,and Tie-2 in subcutaneous tumor tissue(Fig 5E).Conclusion1.ATRA can induce cell apoptosis,inhibit the proliferation of EC1 cells.The inhibition of ATRA showed on EC1 cells has a dose and time dependent manner.2.ATRA can inhibit EC1 cell migration,also inhibit the protein and mRNA expressions of Ang-1,Ang-2,Tie-2,VEGF and VEGFR via RAR.The effect also has a dose and time dependent manner.3.ATRA may inhibit EC1 tumor angiogenesis in nude mice by down-regulation expression levels of Ang-1,Ang-2,Tie-2 and CD31 gene.4.ATRA treatment suppresses the growth of xenograft tumors of EC1 cells and improves the cachexia of mice. |
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