| Objective: Invasion and metastasis of the tumor is closely related with the survival and prognosis of patients.Anoikis refers to cells divorced from original growth site,loss of cell-cell,cell-extracellular matrix adhesion and then occurrence of a procedural death.However,malignant tumor cells often obtain resistance to anoikis through a variety of programs,which will facilitate tumor invasion and metastasis correspondingly.Previous data report that cells will loss of sensitivity to anoikis when undergo Epithelial-Mesenchymal Transition(EMT).Because resistance to anoikis and EMT are tightly linked events involved in metastasis,exploring the relationship between these two processes is thus a promising way for better understanding metastasis.In the current study,cells underwent EMT were obtained by continuously treated with TGF-β1,and the ability of cell migration and anoikis resistance was detected further.In addition,the expressions of EMT-related molecules were examined when cells cultured in detached condition.Our aim was to explore the relationship between anoikis resistance and EMT in NSCLC cells.Method:1 Cell culture:Human Non-small cell lung cancer(NSCLC)cell lines H358 and A549,were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum,100U/m L penicillin and 100U/m L streptomycin.Both of the cells were incubated in a humidified atmosphere of 5% CO2 in air at 37?C.2 Treated cells with TGF-β1:H358 and A549 cells were continuously treated with TGF-β1 for 21 days,and the expression of EMT-related molecules were detected to confirm that cells had undergone EMT,which were referred as H358-TGF-β or A549-TGF-β cells.3 Cell culture under forced detachment condition:Poly-HEMA,a non-adhesive substratum,was prepared using a solution of 10 mg/m L and was added to 6-well culture plate at a volume of 1.5 m L per well,allowed to evaporate to dryness at room temperature overnight.H358,H358-TGF-β,A549 or A549-TGF-β cells at certain concentration were plated and incubated at 37℃ with 5% CO2,respectively.4 Real-time PCR assay:Total RNA was extracted from the above cells when cultured under adherent(Attached)and Suspension(Detached),respectively.The expressions of EMT-related molecules,including CDH1,CDH2,Vimentin,Snail,ZEB1 and ZEB2 in each group were detected by Real-time PCR.The relative expression level of the target gene was calculated using the comparative Ct(ΔΔCt)method provided by Applied Biosystems.5 Western Blot assay:Total protein was extracted from cells in each group under attached and detached condition,and the expressions of EMT-related molecules,including E-Cadherin,N-Cadherin,Vimentin and Snail protein were detected by Western blot assay.6 Wound healing assay:The growth of cells on both sides of the scribing line was observed within0-72 h after scratches,and the cell migration was observed under microscope.Each experiment was repeated three times.7 Transwell assay:2~6×104 cells were plated in medium without serum in the top chamber of a transwell.After incubation for some times,the cells that had migrated to the lower surface of the membrane were fixed with formalin,stained with crystal violet,and photographed under microscope.Cell numbers were counted under a light microscope.Experiments were carried out at least three times.8 Apoptosis detection by flow cytometry:The apoptosis rates of the cells under adherent or suspension status weredetermined by flow cytometry using Annexin-V/PE kit.FlowJo 7.6 software was used for data analysis.9 Trypan blue staining to indentify dead cells:Cells in each group were stained with trypan blue to distinguish the dead cell from live cells.10 Statistical analysis:SPSS Statistics 17.0 software was used to analyze the statistical significance.Data are presented as mean ± SD.Statistical comparisons between experimental groups were analyzed by t-test.P<0.05 was taken to indicate statistical significance.Results:1 Establishment EMT cell model of H358 and A549 cells by continuously treated with TGF-β11.1 Cell morphological changes:The appearance of cell shape was changed obviously after continuously treated with TGF-β1 for 21 days both in H358 and A549.Compared with the original cells,H358-TGF-β and A549-TGF-β cells became elongated,like spindle shaped appearance,indicated that cells underwent EMT.1.2 Expression of EMT related molecules:1.2.1 H358 cells continuously treated with TGF-β1:Compared with H358 cells,the expression of the epithelial marker CDH1 m RNA was significantly decreased,while the expression of the mesenchymal markers CDH2,Vimentin,Snail,ZEB1 and ZEB2 were significantly increased by using Real-time PCR assay.Western blot results further confirmed that E-cadherin protein was significantly decreased,N-cadherin and vimentin protein were significantly increased after H358 cells continuously treated with TGF-β1 for 21 days.The above results suggested that H358 cells underwent EMT after continuously treated with TGF-β1 for 21 days.The cells were labeled as H358-TGF-β.1.2.2 A549 cells continuously treated with TGF-β1:Upon TGF-β1 treatment for 21 days,the expression of the epithelialmarker E-cadherin in A549 cells was inhibited significantly both in m RNA and protein levels,while the levels of CDH2,vimentin Snail,ZEB1 and ZEB2were significantly increased at m RNA level,At 14 days of stimulation,the EMT markers were lower than those stimulated for 7 days,and the change was most significant at 21 days after TGF-β1 treatment.At the level of protein,the levels of N-cadherin,Vimentin and Snail protein were significantly increased in TGF-β1-treated cells.Considering the m RNA level and protein expression results,TGF-β1 was continuously administered for 21 days as EMT cell model,and the cells were labeled A549-TGF-β.1.3 Cell migration assays:Monolayer wound healing assay and Transwell assay were performed to observe cell ability of migration.It showed that the speed which cells migrated towards the scratch was higher in H358-TGF-β cells when compared with the parallel cells(62.6±2.5%v.s.9.3±0.83%,P<0.05).Compared with A549 cells,A549-TGF-β cells also showed increased migration ability by wound healing assay(47.7±2.08% v.s.98.6±0.65%,P<0.05).Transwell assay further indicated that the number of H358-TGF-β or A549-TGF-β cells migrated into the lower chamber was significantly higher than that in the parallel cells,respectively.Taken together,the observations suggested that treatment of TGF-β1induced EMT transition in H358 and A549 cells,accompanied with enhanced ability of cell migration.2 The effects of EMT on Anoikis resistance in NSCLC cells2.1 Changes in cell morphology under detached conditions:Cells morphology was observed under microscope after cultured in detached condition for 48 hours.All the cells spread in microspheres with different sizes.H358 cells are closely bound,bead-like aggregation,H358-TGF-β cells are not closely bound,scattered in the distribution.Compared with the original cells,A549-TGF-β cells arranged more loosen.2.2 Detection of apoptosis under attached or detached condition:The cell suspension was divided into two groups to culture underattached or detached conditions,respectively.Cell apoptosis was detected by Annexin V-PE staining after 48 hours.2.2.1 Apoptosis of cells in the adherent state:Compared with H358 cells,the apoptotic rate of H358-TGF-β cells was significantly decreased(11.67±1.36% V.S.5.52±0.22%,P<0.05).However,the apoptotic rate of A549-TGF-β cells did not change significantly compared with A549 cells(P<0.05).2.2.2 Apoptosis of cells in detached state:Compared with cells under adherent condition,the apoptosis rates of cells in each group were all increased obviously when under detached state(All P<0.05).[H358: 11.67%±1.36%(A),51.35%±1.34%(D).H358-TGF-β:5.52%±0.22%(A),22.0%±0.42%(D).A549: 5.2±0.28%(A),24.22±3.75%(D).A549-TGF-β: 4.24±1.56%(A),10.65±0.21%(D)].2.2.3 Resistance to anoikis when cells underwent EMT The apoptotic rate of H358-TGF-β cells(22%±0.42%)was significantly lower than that of H358 cells(51.35%±1.34%,P<0.05).In addition,the apoptotic rate of A549-TGF-β cells was also significantly lower than that of A549 cells(10.65±0.21% V.S.24.22±3.75%,P<0.05).The above results suggested that EMT cells could avoid apoptosis caused by detached condition,which manifested as ankiosis resistance.2.3 Dead cell counting by Trypan blue staining:Cells in each group were stained with trypan blue to distinguish the dead cell from live cells.The results showed that there were no significant differences between cells cultured in attached and detached condition,indicating that cells were viable even in detached condition for 48 hours.3 Expression of EMT-related molecules of cells under detached situation Total RNA and protein were extracted from cells both in attached and detached conditions,and the expressions of EMT-related molecules both at m RNA and protein levels were detected by Real-time PCR and Western blot assay,respectively.3.1 H358/ H358-TGF-β cells :3.1.1 H358 cells:Compared with the adherent state(Referred as 1),the expression of Snail m RNA(FC=2.57)and ZEB2(FC=1.56)m RNA were increased,while CDH2 m RNA was decreased(FC=0.10)in H358 cells under detached conditions.At protein level,the expression of Snail and Vimentin protein was increased significantly in the Detach group.The expression of the epithelial marker E-Ca protein did not show significantly change.3.1.2 H358-TGF-β cells Compared with the adherent cells,the expression of the epithelial marker CDH1 m RNA was significantly decreased(FC=0.21),while the expressions of the mesenchymal markers Snail,ZEB1 and ZEB2 m RNA were significantly increased in the detached cells.Western bolt results showed the same effects as in Real-time PCR assay.These above results suggested that the expressions of the mesenchymal markers in H358 and H358-TGF-β cells were increased upon detachment,especially in H358-TGF-β cells.3.2 A549/A549-TGF-β cells3.2.1 A549 cells:The levels of CDH1 m RNA and E-Ca protein were significantly lower in detached cells than that in attached ones.On the contrary,the level of Vimentin,Snail,ZEB1 and ZEB2 m RNA were all increased in detached cells.Additional,expression of Vimentin and Snail protein were also increased in detached cells than that in attached ones.3.2.2 A549-TGF-β cells:Under detached condition,CDH1 m RNA level in A549-TGF-β cells was inhibited to certain extent,but E-Ca protein did not show difference in attached or detached cells.As for the mesenchymal markers,the expression of Vimentin,Snail,ZEB1 and ZEB2 m RNA were all increased in detached cells.In addition,the expression of Snail protein was also increased in detached cells than that in attached ones.Combining the PCR and Western Blot results,the expressions of the mesenchymal markers in A549 and A549-TGF-β cells were increased upon detachment mainly.Conclusion:1 Cells were induced EMT transition after continuously treated with TGF-β1 for 21 days both in H358 and A549,accompanied by enhanced ability of cell migaration.2 The apoptosis rates of H358,H358-TGF-β,A549 and A549-TGF-βcells in detached state were significantly higher than that in adherent condition.One of the characteristics of EMT cells is resistance to anoikis.3 The expressions of some mesenchymal markers in H358,H358-TGF-β,A549 and A549-TGF-β cells were increased upon detachment,with or without the decreased expression of the epithelial marker E-cadherin,which indicated that cells might keep in(partial)EMT status under detached condition. |
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