| Relationship between RACK1 expression and chemotherapy resistance in esophageal squamous cell carcinoma and its underlying mechanismsBackground:Esophageal cancer is one of the most common gastrointestinal malignant tumors worldwide,with dismal prognosis,even when diagnosed in early stages.In Asia,esophageal squamous cell carcinoma(ESCC)occupies for about 90%of esophageal carcinomas.Although surgical techniques and perioperative treatments have been advanced,the prognosis of ESCC still remains poor on account of the local recurrence and distant metastasis.Most patients with ESCC are diagnosed at advanced or late stages,who can only receive chemotherapy,radiotherapy and adjuvant treatments.However,only responders benefit from chemotherapeutic drugs,while resisters remain a major defect in the clinical applications.Therefore,it is very imperative to discover the key markers and elementary mechanism affecting the chemotherapy response.In 1989,RACK1 was firstly cloned from a chicken genomic DNA clusters and human B-lymphoblastoid cell line cDNA library,which was sharing high homology with the beta subunit of G-proteins(Gβ).RACK 1 is a member of the tryptophan-aspartate(WD40)repeat protein family which adopts a highly conserved 7-bladed beta-propeller structure.RACK1 plays an important role in anchoring proteins at particular locations,shuttling proteins from one cellular compartment to another,participating in transcriptional and translational events and modulating binding protein activity.It serves as a scaffold protein that is able to interact simultaneously with many protein kinases and membrane-bound receptors.And it also plays a crucial role in a large quantity of biological processes,including cell growth,development,adhesion,migration,differentiation and immune responses.Increased RACK1 expression has been observed in various kinds of carcinomas and regarded as an independent biomarker of breast cancer,non-small-cell lung cancer(NSCLC),hepatocellular carcinoma(HCC),oral squamous carcinoma,etc.We have already conf-irmed that RACK1 predicted poor prognosis in ESCC with promoting tumor progression and EMT.It is confirmed that RACK1 can regulate several apoptosis-related genes and drug-related genes and then lead to the occurrence of chemotherapy resistance.Furthermore,recent study showed that the expression level of RACK1 in tumor tissue was correlated with the chemoresistance of hepatocellular cancer.Purpose:This study aimed to investigate the role of RACK 1 gene in the proliferation,apoptosis and chemotherapeutic effect of esophageal squamous carcinoma cell lines such as Eca109 and EC9706,and to explore the potential mechanism of RACK1 andchemosensitivity to esophageal squamous cell carcinoma.Methods:1.Cell culture and transfection:The human esophageal carcinoma cell lines Eca109 and EC9706 cells were cultured in RPMI 1640 Medium.Plasmid with shRNA or shNC or Open Reading Frame targeting RACK1 gene was transfected into Eca109-and EC9706 cell lines with lipofectamine 2000 reagent.Cells were transfected when the plating cells were 70-90%confluent,and harvested for subsequent assays within 48h-72 h after transfection.2.Selected stable transfectants:Stably transfected cells were selected with 400 ug/mL of G418 antibiotic for 2 weeks and maintained in 300 ug/ml of G418 culture medium.3.Detection of transfection efficiency:The efficiency of RACK1 overexpression and knockdown were evaluated by real-PCR and Western Blot Assay.4.Colony formation assay:Cells were trypsinized into single-cell suspensions with the treatment of 0.25%Trypsin.About 500 cells were planted into six-well plates per well with the density of 250/ml cells.After incubating cells in a humidified environment at 37 ℃ containing 5%C02 for 14 days,the cells were fixed in anhydrous ethanol for 10 minutes and then stained with 0.1%crystal violet for half an hour.Colonies consisting of more than 50 cells were counted.And relative colony numbers were obtained.The ability of colony-forming was evaluated by the ratio comparing the number of colonies formed in transfected cells with that in control group,multiplied by a hundred.5.Chemotherapy resistance assay:To assess the response to chemotherapy drugs,Eca109 and EC9706 cell lines were cultured in 96-well plates with the density of 1 x 104 cells per well,and then cells were treated with a series of different concentrations of cisplatin or 5-FU for 48 or 72 hours.The CCK8 assay was used to detect the cell viability.About 1 × 105 cells were planted into six-well plates per well with the density of 5×104/ml cells for 24 hours,and then they were treated with cisplatin(1～320umol/L)or 5-FU(1～10umol/L).After 24,48,72 hours,cells were harvested for detection the expression of mRNA and protein changes.6.Cell Proliferation and Cytotoxicity Assay(CCK8 assay):Cell proliferation was assessed with a CCK8 assay kit.In brief,cells transfected with the shRACK1 plasmid,control plasmid and overexpressed plasmid were seeded into 96-well culture plates.Preincubate the plate for 24 hours in a humidified incubator.And then cells were treated with different concentrations of cisplatin or 5-FU for 48 or 72 hours.Spectrometric absorbance at 450nm wavelength was measured on a microplate reader(Bio-Rad)after 1 hour of lOul CCK8 reagents incubation.The cell viability was figured with the quantitative value multiplied by 100%.The proliferation rate was recorded based on the OD value.7.Flow cytometry analysis:Esophageal carcinoma cells were harvested and washed by PBS twice,then incubated in phycoerythrin and 7-Amino-Actinomycin staining buffer at 25 ℃ for 15 min.The apoptotic percentage of samples was determined on a BD FACSAriaⅡ instrument and data were analyzed by FlowJo software.Results:1.Upregulation and downregulation of RACK1 mRNA and protein levels by transfection in ESCC cell lines.To investigate the importance of RACK1 in ESCC,we initially constructed two transfected cell lines,transfected Eca109 and EC9706 cells with a plasmid expressing shRACK1 or nonsense shNC or RACK1--overexpression and established stable cell lines as described by G418 selection.Quantitative real-time PCR confirmed the mRNA levels of RACK1 in the overexpression,control,shNC and shRACK1 ESCC cell lines,respectively.It was upregulated in overexpression Eca109 cells(6.76±1.46,P=0.0209)and EC9706 cells(7.76±0.31,P=0.0007)compared to control cells.Besides,in contrast with control cells,mRNA expression levels of RACK1 in the shRACK1 cells decreased to 0.44±0.06(P=0.0037)and 0.33±0.07(P=0.0041).Moreover,RACK1 protein expression levels in Eca109 and EC9706 cells were detected by western blot.The RACK1 expression level increased in both clone E109-Overexpression and E9706-Overexpression groups compared to the control cells.Additionally,after transfection by shRACK1,RACK1 protein level reduced to 65%and 44%compared to the control group in both Eca 109 and EC9706 cells.2.Downregulation of RACK1 inhibited cell colony formation ability of ESCC cells in vitro.To investigate the roles of RACK1 in the colony formation properties of esophageal carcinoma cells,we then conducted colony formation assay in vitro.The results showed that downregulation of RACK1 by shRACK1 transfection significantly suppressed cell proliferation in human esophageal carcinoma cells(Eca109:254±11 vs 343± 15,P=0.0013;EC9706:187±23 vs 353±7,P=0.0003,Figure 2a and b).Moreover,the number of formation colony increased in clone E 109-Overexpression(393±14 vs 343±15,P=0.0148)and E9706-Overexpression(369±12 vs 353±7,P=0.0280)groups compared with the control one.3.RACK1 increased proliferation ability of esophageal carcinoma cells with the treatment of chemotherapy drugs.CCK-8 assay was conducted to validate the role of RACK1 on the proliferation ability of ESCC cells.Eca109 and EC9706 were exposed to different concentrations of cisplatin(0-320μM)or 5-fluorouracil(Eca109,0-10μM,EC9706,0-320μM)for 48h and 72h,respectively.The results showed that the proliferation ability of ESCC cells improved in the overexpression RACK1 groups(P<0.001)and decreased in the cells transfected shRACK1(P<0.001)groups compared with the control ones.4.RACK1 regulated the chemosensitivity of ESCC in vitro.Since RACK1 was found to be capable of regulating the apoptotic response to chemotherapy in breast cancer and hepatocellular carcinoma,we then detected the impacts of RACK1 in the chemosensitivity of ESCC in vitro.Upregulating RACK1 significantly suppressed cisplatin-induced apoptosis in Eca109 and EC9706 cells,while downregulation of RACK1 promoted the sensitivity compared to the control group(Eca109:P<0.001 for shRACK1,P<0.01 for shNC and P<0.001 for over group;EC9706:P<0.001 for shRACK1,P<0.001 for shNC and P<0.05 for over,respectively).We observed the similar results in 5-fluorouracil-treated Eca109 and EC9706 cells(Eca109:P<0.001 for shRACKl,P<0.01 for shNC,P<0.05 for over;EC9706:P<0.001 for shRACK1,P<0.001 for shNC,P<0.05 for over).These data indicate that RACK1 plays an important regulatory role in the chemosensitivity of ESCC in vitro.5.Elementary mechanism involved in the regulation of cell chemosensitivity by RACK1 in vitro.To explore the preliminary mechanism of RACK1 regulation on cell chemosensitivity in ESCC cells,in the present study,we focused on the AKT,Erk1/2,BcI-2 and Bim molecules by western blot.Our data found notable changes in the phosphory AKT levels and expression levels of cell anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim.Afterwards,we examined the effects of cisplatin on the expression of AKT,pAKT,ERK1/2,Bcl-2 and Bim.Downregulation of RACK1 in ESCC cells significantly inhibited the activation of AKT and ERK1/2,reduced Bcl-2 expression and increased Bim expression compared to the control groups.In contrast,upregulation of RACK1 in Eca109 and EC9706 cells promoted the activation of AKT and ERK1/2,facilitated Bcl-2 expression and suppressed Bim expression.After 24h,48h and 72h treatment with cisplatin,proteins were extracted and analyzed by western blot,respectively.Compared to control cells,we observed that esophageal carcinoma cells transfected by shRACKl led to a significant suppression of expression levels of p-AKT and Bcl-2,while the expression of Bim was observably increased.In addition,the expression level of Bim became increasingly higher with the extension of effect time.These results provided the clues of RACK1 targeting Bcl-2 and Bim to promote chemoresistance of the esophageal carcinoma cell through PI3K pathway activation.Conclusion:Our findings showed that RACK1 could active PI3K/AKT pathway and upregulate expression of Bcl-2,which subsequently lead to the chemotherapy resistance.Inhibition of RACK1 largely abolished the chemoresistance.In brief,our findings showed that RACK1 might be available as a marker of the chemotherapy resistance and poor survival in ESCC ultimately.Therefore,the treatment of RACK1 provides a new way to improve the efficacy of chemotherapy in ESCC.Relationship between RACK1 and radiosensitivity of esophageal squamous cell carcinomaBackground:Esophageal cancer is one of the most common malignant tumors in the world.The latest epidemiological data of malignant tumors shows that the number of annual esophageal cancer incidence was 477,900 people and the number of deaths was375,000 people in China.In Asia,90% pathological types of esophageal cancer patients are esophageal squamous cell carcinoma(ESCC).Radiation therapy is one of the major treatments widely used in ESCC.Nevertheless,Radiation resistance is an important factor leading to radiation therapy failure caused by local recurrence and distant metastasis in ESCC.Therefore,it is an urgent problem to find new radiation resistance indicators and effective radiation sensitizers to improve strategies of radiation therapy.The receptor for activated C-kinase 1(RACK1)is served as a gene encoding a36 kDa activated C kinase(PKC)-binding protein.As a member of seven Trp-Asp 40(WD40)repeat protein family,RACK1 interacts with a wide range of proteins and participates in many signalling pathways,such as PKC,JNK,SRC,NF-kB and so on.Moreover,RACK1 plays a dramatic role in the occurrence,development and metastasis of various cancers.Our previous study found that RACK1 predicted poor prognosis and promoted progression of ESCC through its epithelial-mesenchymal transition.At present,there is little research on RACK1 and radiation resistance.Bergami et al.found that upregulation the expression level of RACK 1 in melanoma cells enhanced its ability in inhibition of UV-induced apoptosis,leading to radiation resistance.At the same time,selective expression of RACK 1 could increase sensitivity to UV melanoma cells.There is a potential relationship between RACK1 expression levels and radiation resistance.However,whether the expression level of RACK 1 affects the radiosensitivity of tumor cells is still vacant in this field.Purpose:In the present study,we aimed to investigate the role of RACK1 on the radiotherapy response of ESCC and the underlying mechanism of RACK1 effected radiation resistance in ESCC.Methods:1.Cell culture: The human esophageal carcinoma cell lines Ecal09 and EC9706 were cultured in RPMI 1640 Medium supplemented with 10% fetal bovine serum,100 μg/ml streptomycin and 100 U/ml penicillin at 37°C in a humidified 5% CO2atmosphere.2.Cell transfection: Ecal09 and EC9706 cell lines were transfected by plasmids with shRNA or shNC or ORF targeting RACK1 gene with Iipofectamine 2000 reagent,then harvested cells within 48h-72 h.Stably transfected cells were selected with 400ug/mL of G418 antibiotic for 2 weeks and maintained in 300 ug/ml of G418 culture medium.The efficiency of RACK1 overexpression and knockdown were evaluated by real-PCR and Western Blot Assay.3.Colony formation assay: Cells were trypsinized to generate single-cell suspensions and seeded in six-well plates about 500 cells per well.A single radiation dose of 0,2,4,6,8 Gy was delivered to cells using a medical linear accelerator(varian 23 EX).After incubation for 14 days,cells were fixed in anhydrous ethanol,stained with crystal violet for half an hour and counted the colonies containing 50 cells or more.The adherence rate was refered to the ratio comparing the number of colonies formed to the number of cells planted.Survival fraction(SF)is refered to the adherence rate at each dose divided by that at OGy.The Colony formation ability meaned the ratio of the SF of the transfected cells to the control cells under the same dose of radiation.4.Cell Proliferation and Cytotoxicity Assay(CCK8 assay): ESCC cell lines transfected with the shRACK1 plasmid,control plasmid and overexpressed plasmid were cultured in 96-well plates and incubated for 24 hours.Cells were treated with a single radiation dose of 0,2,4,6,8 Gy and cultured for 48 hours.lOul CCK8 reagents and lOOul medium were added into each well followed by 1 hour incubation.The spectrometric absorbance of cells at 450 nm wavelength was measured with automatic microplate reader(BioRad).The proliferation rate was recorded based on the OD value.Percentage of viable cells =(ODtransfected-ODblank)/(ODcontrol-ODbIank)*100.5.Flow cytometry analysis: Ecal09 and EC9706 cells were seeded into 6-well plates and irradiated with single dose of 4Gy rays.After cultured for 24 h,the cells were harvested and then incubated with phycoerythrin(PE)Annexin V and7-Amino-Actinomycin(7-AAD)staining buffer at room temperature for 15 min.The cell apoptosis was employed by a BD FACSCalibur.Data analysis was performed by FlowJo software.PE Annexin V and 7-AAD negative cells are considered to be viable;PE Annexin V positive and 7-AAD negative cells are served as early apoptosis cells;and both PE Annexin V and 7-AAD positive cells are corresponded to late apoptosis or already dead cells.6.Statistical analysis: Statistical analyses were performed using the SPSS 19.0software.The data were expressed as the mean 土 standard error and statistical comparisons were made using the two-tailed Student’s t-test and Oneway ANOVA analysis.Multitarget-single hitting model was fit to dose survival curve with GraphPad Prism 5.01 version software.All the data were repeated at least three independent experiments to be effective.P values < 0.05(*)or < 0,01 were considered statistically significant difference.Results:1.Knockdown or Overexpress RACK1 gene in ESCC cell lines: We respectively used RT-PCR and western blot techniques to detect the expression of protein and mRNA levels of RACK1 in transfected cells.Compared with shNC control cells,mRNA levels of RACK1 were upregulated to 3,76±0.31 times(P=0.0002)in over-RACK1 Ecal09 cells and 5.43 ±0.30 times(P< 0.0001)in over-RACK1 EC9706 cells;mRNA levels of RACK1 were downregulated to 0.44 + 0.06(P=0.0032)in shRACK1 Ecal09 cells and 0.33±0.07(P=0.0056)in shRACK1 EC9706 cells.Additionally,the knockdown or overexpress efficiency of RACK1 was also confirmed by western blot.2.Downregulation of RACK1 reduced the colony formation ability of cells after radiation;whereas upregulation of RACK1 promoted the colony formation of cells after radiation: the number of formed cell clones decreased,with a single dose of radiation increasd gradually.Moreover,under the same dose of radiation,the number of clones formed in the shRACK group was decreased compared with the shNC group,which was less than that in over-RACK1 group.Similar results were obtained in both Ecal09 cells and EC9706 cells.The results showed that downregulated RACK1 could reduce the colony formation ability of cells after irradiation;whereas upregulated RACK1 could promote the colony formation after irradiation.3.To detect the survival ability of cells after radiation,we used multi-target single-hitting model to fit the dose survival curve.SF(survival fraction)= 1-(1-e-kD)n.SER(sensitive enhancing ratio)=(Dq in shNC group)/(Dq in shRACK 1 or over-RACK1 group).We figured out the parameter k,n,DO,Dq,D37,SF and SER.We found that knockdown of RACK1 could increase the SER in both Ecal09 cells and EC9706 cells.On the contrary,Overexpression of RACK1 could decrease the SER in ESCC cells.4.Knockdown RACK1 reduced the proliferation ability of ESCC cells in the eifection of irradiation;while overexpressed RACK1 promoted cells proliferation under the influence of irradiation: The cells were exposed to single radiation dose of 0,2,4,6,8 Gy and followed by culturing for 48 hours.The survival curves from CCK-8assay showed that overexpressed RACK1 could enhance the percentage of vible cells compared with control cells(P< 0.001),suggesting that RACK1 could promote tumor cells proliferation under the influence of radiation.On the contrary,when RACK1 was knockdown,the percentage of vible cells compared with shNC cells was significantly decreased(P<0.001)in both Ecal09 and EC9706 cells.These results indicated that RACK1 could promoted the radiation resistance of ESCC cells,and treatment of RACK 1 overexpression could increase the sensitivity of tumor cells to radiation therapy.5.High expression of RACK1 promoted resistance of ESCC cells to radiation by reducing the quantity of apoptotic cells: We performed the PE Annexin-V 7-AAD staining for flow cytometry analysis of ESCC cells,which were pretreated with 4Gy radiation and 48 hours incubation.Under single dose of 4Gy irradiation;,the apoptotic cells percentage of overexpression-RACK1 was obviously decreased than in control group(Ecal09: over: 23.49%±1.56% vs control: 31.08%±1.23%s P=0.0187;EC9706:over:17.21%±1.15% vs control:26.14%± 1.59%,P=0.0105).In contrast,downregulation of RACK1 apparently enhanced the radiation-related apoptosis in transfected-shRACK 1 ESCC cells compared with the control cells(Ecal 09:shRACKl: 42.35%±1.04% vs control: 31.08%±1.23% P=0.0022;EC9706:shRACK1: 42.64%±2.91% vs control: 26.14%±1.59%,P=0.0076).These data indicated that RACK1 played an important regulatory role in the radiotherapy sensitivity of ESCC in vitro.6.Knockdown RACK1 inhibited the expression of pAKT and Bcl-2,and enhanced the Bim expression: We detected the changes of apoptosis association proteins after4 Gy dose radiation within western blot assay.We found that compared with the control group,downregulation expression of RACK 1 could observably decrease the expression of pAKT and Bcl-2,accompanied by an increase in Bim expression levels in both Ecal09 and EC9706 cells.Oppositely,our data demonstrated that overexpression of RACK 1 could markedly facilitate the activity of pAKT,upregulate Bcl-2 expression levels and downregulate Bim expression in ESCC cell lines.Conclusion:Inhibition of RACK1 largely enhanced the sensitivity of radiation therapy and abolished the radioresistance in esophageal cancer.We discovered that the underlying mechanism of resulting in radioresistance was that RACK1 activated the pAKT and upregulated the Bcl-2 expression along with downregulation of Bim.Taken together,these findings emphasize a potentially key role of RACK 1 in the responses of radiation therapy in esophageal carcinoma. |
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