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Objective To investigate the effect of cetuximab (C225) on the radiosensitivity of colorectal cancer cells CL187 and underlying mechanism.Methods Cell survival was detected by colony forming assay.The levels of apoptosis and cell cycle distribution were determined by flow cytometer.The mitotic ratio was measured by Wright' s-Giemsa mixed coloring method.The protein levels of Bax and Bcl2 were detected by Western blot.Results The sensitizing enhancement ratio of C225 was approximately 1.4.C225 treatment and 125I seed radiation induced G1 cell cycle arrest individually.C225 increased the radiation-induced apoptosis (t =6.6,P ＜ 0.05) and cellular Bax/Bcl-2 ratio (t =9.4,P ＜ 0.05),but did not increase radiation-induced G1 arrest.In addition,there was no difference in mitotic index among different groups.Conclusions C225 sensitizes CL187 to 125I seed irradiation,which might be related with increase of radiation-induced apoptosis.
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Objective To determine the biological effectiveness of 125I radioactive seeds with continuous low dose rate radiation on the human esophageal cancer cell line KYSE150 in vitro and explore the underlying cellular mechanisms.Methods The cells were divided into three cell groups:control group,single dose radiation group (SDR) and 125I radioactive seeds with continuous low dose rate radiation group (125 I-CLDR).The KYSE150 cells were exposed to radiation of X-ray at a high dose rate of 1.052 Gy/min or 125I radioactive seeds at a low dose rate of 2.77 cGy/h.The responses of KYSE150 cells to two modes of irradiation were evaluated by the colony-forming assay,cell apoptosis as well as cell cycle analysis.Furthermore,the expression levels of γ-H2AX and Bax were detected by Western blot.Results KYSE150 cells were more radiosensitive to 125I-CLDR than SDR.The relative biological effectiveness (RBE) for 125I-CLDR related to SDR was 1.56.Compared with SDR,125I-CLDR yielded more proportions of the early and late apoptosis rate (t =4.07,11.08,P ＜0.05) as well as cells at G2/M phase (t =11.25,P ＜0.05).Moreover,γ-H2AX and Bax expression levels in 125I-CLDR significantly increased compared with SDR.Conclusions Compared with the high dose rate X-ray radiation,the continuous low dose rate radiation of 125I radioactive seeds had stronger inhibition effect on KYSE150 esophageal cancer cells by impairing clonogenic capacity,inducing apoptosis and G2/M cell cycle arrest,and increasing radiosensitivity.
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Objective To investigate the inhibition effect of continuous low dose rate radiation by 125Ⅰ radioactive seeds on Hep-2 cells and the corresponding mechanisms.Methods Hep-2 cells were divided into three groups,control group,single dose radiation group with high dose rate form X-rays (SDR) and continuous low dose rate radiation by 125Ⅰ seeds group (125Ⅰ-CLDR).After exposure to SDR and 125Ⅰ-CLDR,colony formation assay was used to determine the radiosensitivity and RBE,trypan blue exclusion assay was used to determine cell proliferation,and flow cytometry was used to detect cell apoptosis and cell cycle arrest.Results The radiosensitivity of Hep-2 cells to 125Ⅰ-CLDR was higher than that to SDR.The RBE of 125Ⅰ-CLDR versus SDR was approximately 1.61.The α/β ratio of 125Ⅰ-CLDR group was higher than that of SDR group.Both SDR and 125Ⅰ-CLDR inhibited cell proliferation (t =30.9,40.7,P＜0.05),in which 125Ⅰ-CLDR was stronger than SDR (t =9.8,P＜0.05).In addition,the incidences of apoptosis and G2/M arrest induced by125Ⅰ-CLDR were also stronger than those induced by SDR (t =5.8,19.8,P ＜ 0.05).Conclusions 125Ⅰ-CLDR generates more serious inhibition effects than SDR on reducing cellular DNA repair capacity,inducing cell apoptosis and G2/M arrest and inhibiting proliferation of Hep-2 cells.
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Objective To investigate the effect of C225 on DNA repair and molecular pathways in CL187 colorectal cancer cells after irradiated by 125Ⅰ radioactive seeds.Methods In the experiment involved were four groups:control group,100 nmol/L C225 treatment group,125Ⅰ radioactive seeds continuous low-dose rate irradiation group and C225 combined with 125Ⅰ radioactive seeds continuous lowdose rate irradiation group.Cells were collected at 48 h after 4 Gy irradiation,and γH2AX foci/cell and γH2AX foci positive cells were counted with immunofluorescence.At the same time,DNA repair proteins were detected by Western blot.Cells were lyzed immediately after 4 Gy irradiation,and changs in EGFR downstream signaling molecules were detected by Western blot.Results Compared with 125Ⅰ seeds irradiated cells,cells treated with C225 and 125Ⅰ seeds irradiation showed more γH2AX foci per cell (t =8.0,P =0.05),and more γH2AX foci positive cells (t =6.8,P ＜ 0.05) and less expression of Ku70 (t =6.6,P ＜ 0.05) and DNA-PKcs (t =5.6,P ＜ 0.05).Combined with 125Ⅰ-CLDR irradiation,C225 reduced cellular EGFR level(t =4.9,P ＜0.05) and inhibited the activation of Akt(t =5.5,P ＜0.05).Conclusions In the condition of 125Ⅰ seeds irradiation,C225 reduced the expression of Ku70 and DNA-PKcs,inhibited the activation of Akt and attenuated the DNA damage repair capacity in CL187 colorectal cancer cells.
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Objective To investigate the effect and underlying mechanism of single,fractioned and continuous low dose rate radiation on CL187 colorectal cancer cell line.Methods CL187 cells were exposed to 6 MV X-rays at a high dose rate of 4 Gy/min and 125Ⅰ seed at a low dose rate of 2.77 cGy/h with three groups:single dose radiation group (SDR),fractioned dose radiation group (FDR) by 2 Gy/f,and continuous low dose rate radiation group (CLDR).The radiation doses were 0,2,4 and 8 Gy.Total cell number and cell viability were determined by trypan blue.Clone forming assay was used to evaluate the cell proliferation ability.The percentage of apoptosis cells was analyzed by flow cytometry.Western blot was used to detect the protein expression levels of PHLPP2,PTEN and Bax.Results Compared with SDR and FDR groups,the total cell number and survival fraction of CLDR group decreased.The relative biological effect (RBE) for 125Ⅰ seeds compared with 6 MV X-rays was 1.41.The percentage of apoptosis cells of CLDR group was significantly increased (t =-15.08,-11.99,P ＜ 0.05).The expression level of Bax increased in CLDR group,while no obvious changes were observed on PHLPP2 and PTEN among three groups.Conclusions The expression level of PHLPP2 increaseS in SDR,FDR and CLDR group,while it seems that it was not influenced by dose rate.The expression level of Bax increased in three groups,while more colorectal CL187 cells in CLDR group may be killed due to the increase of Bax expression.
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Objective To investigate the combination effect of cetuximab and irradiation on colorectal carcinoma CL187 cell line and underlying molecular mechanism.Methods CL187 cells with or without cetuximab treatment were irradiated by 0,4 and 8 Gy X-rays,then cell death percentage was determined by MTT 24 and 48 h post-irradiation.Clone forming assay was used to evaluate the cell reproliferation ability.Cell cycle distribution,apoptosis,and necrosis were analyzed by flow cytometry.Western blot was used to detect the protein expressions of DNA-PKcs,Ku70 and Ku80.Results The cetuximab enhanced the percentage of radiation-induced cell death,while descreased the cloning formation capacity and increased radiosenvtivity (t =-6.14、-6.53,P ＜0.05).The SER of cetuximab on CL187 cell line approached to 1.38.In addition,cetuximab also increased radiation-induced G0/G1 phase arrest (t=-4.64,P＜0.05) and the percentage of apoptosis and necrosis (t=-9.16,P ＜0.05),but it descreased the expression levels of DNA-PKcs,Ku70 and Ku80 proteins.Conclusions The cetuximab treatment might enhance the inhibitory effect of irradiation on colorectal carcinoma CL187 cell line by influencing cell cycle distribution,cell apoptosis,and the expression of DNA repair proteins.