cervical cancer is a malignant cervix disease with high incidence and has become the third leading cause of cancer death of females in less developed countries after liver and stomach cancers . in 2012 , cervical cancer led to an estimated 265 700 deaths worldwide . risk factors include weak immune system and unhealthy living habits , but the vast majority of cervical cancer cases are associated with human papillomavirus ( hpv ) infection . persistent infection with hpv in the cervix is considered necessary for the development of cervical cancer ; therefore , certain hpv proteins are effective markers for this disease . molecular tools , such as high - risk viral strains dna hpv 16 and 18 , are being used as an effective screening strategy with obvious advantages over cytology methods . other biomolecular markers , such as high - risk mrnas and dna methylation features , have been investigated [ 5 , 6 ] , offering new methods for the diagnosis of cervical cancer . the treatment of cervical cancer includes surgical treatments , such as hysterectomy , trachelectomy and loop electrical excision procedure , as well as adjuvant radiotherapy and cisplatin - based chemotherapy . however , some cases have local reoccurrence of cervical cancer cells after radiotherapy , due to the low radiosensitivity that may be caused by enhanced dna repair mechanism , hypoxia , cellular glutathione , or other factors , like hpv oncoproteins . researchers have detected lower radiosensitivity in cancer - initiating cells ( cics ) of human cervical cancer cell lines hela , siha , ca ski , and c-4 i , showing that cics are likely to be responsible for the low radiosensitivity of cervical cancer cells . among the frequently - used cell lines in cervical cancer studies , siha cells are less sensitive to radiation , and cell lines me180 and c33a are relatively sensitive to radiation , providing appropriate materials for radiosensitivity research in cervical cancer . still , efficient strategies avoiding the resistance of radiotherapy are of great value for reducing the recurrence rate and improving the outcome of cervical cancer treatment . cd146 , also known as melanoma cell adhesion molecule ( mcam ) , plays key roles in cell adhesion , as well as the pathological progression of inflammation and even cancers . it is defined as a marker of melanoma progression , and its expression allows the interaction between melanoma cells and cellular elements of the vascular system , thus accelerating melanoma growth and metastasis . overexpression of cd146 is also indicated to be a promising marker for cervical cancer , and it is positively correlation with the pathological subtype in this disease . however , no association has been reported between cd146 and radiosensitivity of cervical cancer cells . the purpose of this study was to investigate the potential role of cd146 in modulating radiosensitivity of cervical cancer cells . in the present study , we compared the expression of cd146 protein in cancer tissues sensitive or resistant to radiotherapy and normal tissues in cervical squamous cell carcinoma patients . aa98 , an anti - cd146 monoclonal antibody , was used to inhibit cd146 in siha cells , and the role of cd146 in cell radiosensitivity was reflected in changing degree of cell survival and apoptosis after radiation . these experiments provide new evidence of the effects of inhibiting cd146 on increasing radiosensitivity of cervical cancer cells , and lay the foundation for targeting cd146 in cervical cancer treatment . cervical cancer tissues and adjacent normal tissues were sampled from 50 cervical squamous cell carcinoma patients ages 29 to 73 years ( 49.911.3 ) hospitalized in our institute from december 2013 to november 2014 . no patient had received any treatment for cervical cancer before . according to the staging standard proposed by the international federation of gynecology and obstetrics , the cancer conditions of these patients fell into iib ( 27 cases ) , iiia ( 16 cases ) , and iiib ( 7 cases ) . the tissue samples were quickly frozen in liquid nitrogen and then stored at 80c for protein extraction . the sampling process was approved by the ethics committee of our institute each patient and we obtained consent from all patients . if the cancer remained or relapsed , the corresponding samples were characterized into the lower radiosensitivity group , and the other cancer samples were considered sensitive to the radiotherapy ( higher radiosensitivity group ) . human cervical squamous cell carcinoma siha cells ( atcc , manassas , va ) were cultured in dulbecco s modified eagle s medium / f12 supplemented with 10% fetal bovine serum and 1% penicillin - streptomycin ( gibco , carlsbad , ca ) . the cells were cultured in a humidified atmosphere with 5% co2 at 37c , and divided into 2 groups : the aa98-treated group ( aa98 ) and the control group ( control ) . for the aa98 group , 10 g / ml anti - cd146 monoclonal antibody aa98 ( im1010 , merck millipore , boston , ma ) was added in the medium before the following analysis . for cell survival assay after radiation , siha cells in logarithmic phase were digested into single - cell suspension by trypsin ( gibco ) and 100 cells were seeded in culture plates with culture medium for incubation overnight , after which irradiation was performed using a medical electron linear accelerator ( shinva , zibo , china ) at a dose of 0.3 gy / min for 30 min at room temperature . after irradiation , the plates were incubated in a humidified atmosphere with 5% co2 at 37c for 2 weeks . for each group ( control or aa98 ) , cells without the radiation treatment were designated as the blank control . , the medium was removed and the cells were washed twice in phosphate - buffered saline ( pbs ) , fixed in 4% paraformaldehyde ( vetec , shanghai , china ) for 15 min , incubated in giemsa stain ( harveybio , beijing , china ) for 15 min , washed with water , and air - dried . the cells were then counted under an optical microscope ( leica microsystems , wetzlar , germany ) and the surviving fraction ( sf ) was calculated as ( the colony formation number of control or aa98 group after radiation / the colony formation number of the corresponding blank control ) 100% . cell apoptosis was analyzed at 0 h , 72 h , and 7 days after radiation using flow cytometry and annexin v - fitc apoptosis detection kit i ( univ - bio , shanghai , china ) according to the manufacturer s instructions . briefly , siha cells that underwent radiation treatment as described above were collected by centrifugation . after washing in pbs twice , the cells were resuspended in binding buffer and transferred to 96-well plates ( 110 cells per well ) . annexin v - fitc and propidium iodide ( pi ) were added to each well and the cells were incubated at room temperature in the dark for 15 min , after which the binding buffer was added , and the cells were detected by flow cytometry ( bd biosciences , san jose , ca ) . the fitc - positive and pi - negative cells were considered to be the apoptotic cells . the experiment in each sample was performed in triplicate . the siha cells with or without aa98 treatment received radiation as described above , after which they were collected and lysed in lysis buffer ( beyotime , shanghai , china ) . the same amount of protein samples ( 20 g ) was separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) , and then the protein bands on the gel were transferred to a polyvinylidene fluoride ( pvdf ) membrane . the membrane was first incubated in 5% skim milk at room temperature for 2 h for blockage , and then incubated in aa98 , anti - caspase 3 , anti - bcl - xl , or anti--actin ( ab2302 , ab178844 , ab129348 , abcam , cambridge , uk ) . -actin was used as an internal reference . after washing in tris - buffered saline with tween ( tbst ) , the membrane was incubated in the horseradish peroxidase - conjugated secondary antibody at room temperature for 1 h. signals were developed by ecl plus western blotting substrate ( pierce , carlsbad , ca ) . the grey - scale value of each band was compared to that of the -actin bands , and analyzed by chemidoc xrs system ( bio - rad , hercules , ca ) . data were first analyzed by f test for homogeneity of variance and then the t test for significant difference in spss 20 ( ibm , ny , usa ) . cervical cancer tissues and adjacent normal tissues were sampled from 50 cervical squamous cell carcinoma patients ages 29 to 73 years ( 49.911.3 ) hospitalized in our institute from december 2013 to november 2014 . no patient had received any treatment for cervical cancer before . according to the staging standard proposed by the international federation of gynecology and obstetrics , the cancer conditions of these patients fell into iib ( 27 cases ) , iiia ( 16 cases ) , and iiib ( 7 cases ) . the tissue samples were quickly frozen in liquid nitrogen and then stored at 80c for protein extraction . the sampling process was approved by the ethics committee of our institute each patient and we obtained consent from all patients . if the cancer remained or relapsed , the corresponding samples were characterized into the lower radiosensitivity group , and the other cancer samples were considered sensitive to the radiotherapy ( higher radiosensitivity group ) . human cervical squamous cell carcinoma siha cells ( atcc , manassas , va ) were cultured in dulbecco s modified eagle s medium / f12 supplemented with 10% fetal bovine serum and 1% penicillin - streptomycin ( gibco , carlsbad , ca ) . the cells were cultured in a humidified atmosphere with 5% co2 at 37c , and divided into 2 groups : the aa98-treated group ( aa98 ) and the control group ( control ) . for the aa98 group , 10 g / ml anti - cd146 monoclonal antibody aa98 ( im1010 , merck millipore , boston , ma ) was added in the medium before the following analysis . for cell survival assay after radiation , siha cells in logarithmic phase were digested into single - cell suspension by trypsin ( gibco ) and 100 cells were seeded in culture plates with culture medium for incubation overnight , after which irradiation was performed using a medical electron linear accelerator ( shinva , zibo , china ) at a dose of 0.3 gy / min for 30 min at room temperature . after irradiation , the plates were incubated in a humidified atmosphere with 5% co2 at 37c for 2 weeks . for each group ( control or aa98 ) , cells without the radiation treatment were designated as the blank control . , the medium was removed and the cells were washed twice in phosphate - buffered saline ( pbs ) , fixed in 4% paraformaldehyde ( vetec , shanghai , china ) for 15 min , incubated in giemsa stain ( harveybio , beijing , china ) for 15 min , washed with water , and air - dried . the cells were then counted under an optical microscope ( leica microsystems , wetzlar , germany ) and the surviving fraction ( sf ) was calculated as ( the colony formation number of control or aa98 group after radiation / the colony formation number of the corresponding blank control ) 100% . cell apoptosis was analyzed at 0 h , 72 h , and 7 days after radiation using flow cytometry and annexin v - fitc apoptosis detection kit i ( univ - bio , shanghai , china ) according to the manufacturer s instructions . briefly , siha cells that underwent radiation treatment as described above were collected by centrifugation . after washing in pbs twice , the cells were resuspended in binding buffer and transferred to 96-well plates ( 110 cells per well ) . annexin v - fitc and propidium iodide ( pi ) were added to each well and the cells were incubated at room temperature in the dark for 15 min , after which the binding buffer was added , and the cells were detected by flow cytometry ( bd biosciences , san jose , ca ) . the fitc - positive and pi - negative cells were considered to be the apoptotic cells . the siha cells with or without aa98 treatment received radiation as described above , after which they were collected and lysed in lysis buffer ( beyotime , shanghai , china ) . the same amount of protein samples ( 20 g ) was separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) , and then the protein bands on the gel were transferred to a polyvinylidene fluoride ( pvdf ) membrane . the membrane was first incubated in 5% skim milk at room temperature for 2 h for blockage , and then incubated in aa98 , anti - caspase 3 , anti - bcl - xl , or anti--actin ( ab2302 , ab178844 , ab129348 , abcam , cambridge , uk ) . -actin was used as an internal reference . after washing in tris - buffered saline with tween ( tbst ) , the membrane was incubated in the horseradish peroxidase - conjugated secondary antibody at room temperature for 1 h. signals were developed by ecl plus western blotting substrate ( pierce , carlsbad , ca ) . the grey - scale value of each band was compared to that of the -actin bands , and analyzed by chemidoc xrs system ( bio - rad , hercules , ca ) . data were first analyzed by f test for homogeneity of variance and then the t test for significant difference in spss 20 ( ibm , ny , usa ) . the protein expression level of cd146 was compared in cervical cancer cells and the adjacent normal cells from patients . results showed that cd146 protein was significantly increased in cervical cancer cells with higher radiosensitivity compared to normal cells ( p<0.001 , figure 1a , 1b ) , and the cancer cells with lower radiosensitivity possessed even higher cd146 protein level than the cancer cells sensitive to radiotherapy ( p<0.01 ) . these results indicate that cd146 was overexpressed in cervical cancer cells , especially in cells with lower radiosensitivity , suggesting its association with radiosensitivity of cervical cancer cells . the expression pattern of cd146 led us to speculate about the relationship between cd146 expression and the radiosensitivity of cervical cancer cells . the anti - cd146 monoclonal antibody aa98 was applied to cultured siha cells , a human cervical squamous carcinoma cell line with relatively high resistance to radiotherapy , to investigate the effects of cd146 on cell radiosensitivity . colony formation assay showed that sf of cells treated with aa98 was much lower than in cells without aa98 treatment ( p<0.05 , figure 2 ) , indicating that inhibiting cd146 could improve the effects of radiation on suppressing cell proliferation and survival . further , cell apoptosis assay was performed using flow cytometry and results showed that at 72 h and 7 d after radiation , the cell apoptosis of siha cells treated by aa98 was promoted compared to the untreated cells , and significant differences were found at these 2 time points ( p<0.01 , figure 3 ) , indicating that inhibiting cd146 improved the effects of radiation on promoting cell apoptosis . to confirm the cell apoptosis change detected by flow cytometry , 2 apoptosis indicators , caspase 3 and bcl - xl , were analyzed by western blot . results showed that after radiation , caspase 3 protein was up - regulated and bcl - xl protein was down - regulated in aa98-treated siha cells ( figure 4a ) , with significant difference compared to the untreated cells ( p<0.001 , figure 4b ) , indicating that inhibiting cd146 by aa98 could further promote caspase 3 and inhibit bcl - xl . because caspase 3 is a pro - apoptotic factor and bcl - xl is anti - apoptotic in various cancers , including cervical cancer , the changing patterns of them was consistent with the promoted cell apoptosis in aa98-treated cells , further confirming that inhibiting cd146 can improve the effects of radiation on siha cell apoptosis . the protein expression level of cd146 was compared in cervical cancer cells and the adjacent normal cells from patients . results showed that cd146 protein was significantly increased in cervical cancer cells with higher radiosensitivity compared to normal cells ( p<0.001 , figure 1a , 1b ) , and the cancer cells with lower radiosensitivity possessed even higher cd146 protein level than the cancer cells sensitive to radiotherapy ( p<0.01 ) . these results indicate that cd146 was overexpressed in cervical cancer cells , especially in cells with lower radiosensitivity , suggesting its association with radiosensitivity of cervical cancer cells . the expression pattern of cd146 led us to speculate about the relationship between cd146 expression and the radiosensitivity of cervical cancer cells . the anti - cd146 monoclonal antibody aa98 was applied to cultured siha cells , a human cervical squamous carcinoma cell line with relatively high resistance to radiotherapy , to investigate the effects of cd146 on cell radiosensitivity . colony formation assay showed that sf of cells treated with aa98 was much lower than in cells without aa98 treatment ( p<0.05 , figure 2 ) , indicating that inhibiting cd146 could improve the effects of radiation on suppressing cell proliferation and survival . further , cell apoptosis assay was performed using flow cytometry and results showed that at 72 h and 7 d after radiation , the cell apoptosis of siha cells treated by aa98 was promoted compared to the untreated cells , and significant differences were found at these 2 time points ( p<0.01 , figure 3 ) , indicating that inhibiting cd146 improved the effects of radiation on promoting cell apoptosis . to confirm the cell apoptosis change detected by flow cytometry , 2 apoptosis indicators , caspase 3 and bcl - xl , were analyzed by western blot . results showed that after radiation , caspase 3 protein was up - regulated and bcl - xl protein was down - regulated in aa98-treated siha cells ( figure 4a ) , with significant difference compared to the untreated cells ( p<0.001 , figure 4b ) , indicating that inhibiting cd146 by aa98 could further promote caspase 3 and inhibit bcl - xl . because caspase 3 is a pro - apoptotic factor and bcl - xl is anti - apoptotic in various cancers , including cervical cancer , the changing patterns of them was consistent with the promoted cell apoptosis in aa98-treated cells , further confirming that inhibiting cd146 can improve the effects of radiation on siha cell apoptosis . this study found up - regulated expression of cd146 protein in cervical cancer cells of cervical squamous cell carcinoma patients , especially in the cancer cells with lower sensitivity to radiotherapy . aa98 , an anti - cd146 monoclonal antibody , was used to investigate the role of cd146 in the radiosensitivity of siha cells . we found that inhibiting cd146 in siha cells aggravates the cell survival inhibition and cell apoptosis promotion by radiation , indicating improved radiosensitivity of siha cells . cd146 is a potential therapeutic target for a variety of diseases , including gastric cancer and breast cancer , where it functions as an epithelial - mesenchymal transition inducer [ 2022 ] . a previous study has shown that cd146 mrna expression can be induced by cisplatin in cisplatin - sensitive cancer cell lines , suggesting its involvement in resistance to cytotoxic chemotherapeutic agents . cd146 protein was up - regulated in cervical cancer cells compared to normal cells , which is consistent with its elevation in cervical squamous cell carcinoma , as reported previously , and was further increased in the cervical cancer cells with lower radiosensitivity , indicating the association of cd146 with radiosensitivity of cervical cancer cells , as verified in this study in aa98-treated siha cells . significant disparities were detected between the aa98-treated and untreated siha cells in cell survival and apoptosis after radiation . cells treated with aa98 possessed higher sensitivity to radiation based on the more obvious changes in the reduced cell sf and increased cell apoptosis , which show that inhibiting cd146 by aa98 improved siha cell radiosensitivity , and that cd146 is an inducer of radioresistance in cervical cancer cells . the monoclonal antibody aa98 is identified as anti - cd146 and capable of inhibiting proliferation and migration of human umbilical vein endothelial cells , thereby suppressing angiogenesis and tumor growth , possibly via regulating nuclear factor-b ( nf-b ) . numerous studies used aa98 for the inhibition of cd146 to reveal roles of cd146 in tumor angiogenesis . for example , cd146 dimerization , which is required for tumor angiogenesis , is dramatically decreased by aa98 , thus helping to reduce the risk of malignancy in living cells . knockdown of cd146 allows the suppression in migration and proliferation of vascular endothelial cells , suggesting the possibility of using cd146 as a target in anti - angiogenesis therapy . in addition to these findings , this study established the connections between cd146 and radiosensitivity in cervical cancer cells using aa98 to inhibit cd146 . when the cd146 molecules on siha cell surface were blocked , the cells became more sensitive to radiation , reflecting the anti - radiation functions of cd146 and the possibility of targeting cd146 to increase radiosensitivity of cervical cancer cells and to improve the outcome of radiotherapy . it has been reported that the mechanism involving ubiquitin protein ligase e3a and p53 is close related to the radiosensitivity of cervical cancer . the mechanism of cd146 in affecting cell radiosensitivity has not been defined in previous studies , although its roles in facilitating tumor angiogenesis are likely to include the regulation of nf-b , the direct interaction with vascular endothelial growth factor receptor 2 , or the up - regulation of p53 in breast cancer . in this study , inhibiting cd146 was shown to affect the changing degrees of 2 apoptotic factors , caspase 3 and bcl - xl , after irradiation , showing that cd146 might regulate cell apoptosis pathways . it is also possible that cd146 influenced cell apoptosis via modulating other cellular processes , since suppressed dna repair is closely related to enhanced radiosensitivity of cancer cells . further studies will concentrate on the association between cd146 and radiation resistance mechanisms , such as dna repair , for a better understanding of the mechanisms . this study shows the relationship between cd146 and cervical cancer radiosensitivity . inhibiting cd146 with its antibody aa98 obviously improves the radiosensitivity of siha cells . these results suggest that cd146 is an anti - radiation factor and a potential target for cervical cancer treatment . detailed mechanism studies are still necessary for a comprehensive understanding of the role of cd146 in radiosensitivity .
backgroundcervical cancer is one of the major causes of cancer death of females worldwide . 
 radiotherapy is considered effective for cervical cancer treatment , but the low radiosensitivity found in some cases severely affects therapeutic outcomes . 
 this study aimed to reveal the role of cd146 , an important adhesion molecule facilitating tumor angiogenesis , in regulating radiosensitivity of cervical cancer cells.material/methodscd146 protein expression was compared in normal cells , cervical cancer cells with lower radiosensitivity , and cervical cancer cells with higher sensitivity from cervical squamous cell carcinoma patients . 
 anti - cd146 monoclonal antibody aa98 was used to inhibit cd146 in human cervical cancer siha cells with relatively low radiosensitivity , and then the cell survival and apoptosis changes after radiation were detected by colony formation assay and flow cytometry.resultscd146 protein was significantly up - regulated in cervical cancer cells ( p<0.001 ) , especially in cancer cells with lower radiosensitivity . 
 the siha cells treated with aa98 showed more obvious inhibition in cell survival ( p<0.05 ) and promotion in cell apoptosis ( p<0.01 ) after radiation , compared to the untreated cells . 
 more dramatic changes in apoptotic factors caspase 3 and bcl - xl were also detected in aa98-treated cells.conclusionsthese results indicate that inhibiting cd146 improves the effect of radiation in suppressing siha cells . 
 this study shows the potential of cd146 as a target for increasing radiosensitivity of cervical cancer cells , which might allow improvement in treatment outcome in cervical cancer . 
 further studies are necessary for understanding the detailed mechanism of cd146 in regulating radiosensitivity .