BACKGROUND
adenoid cystic carcinoma  is a common salivary gland cancer subtype accounting for 22% of salivary gland malignancies and 1% of all head and neck cancers  <cit> .
perineural invasion, delayed onset of hematogenous metastasis and poor response to traditional chemotherapies are characteristics of this cancer.
local recurrence and distant metastasis, in which extracellular matrix  and basement membrane degradation are crucial, are responsible for treatment failure.
currently, no systemic therapy is available that effectively inhibits acc progression.
therefore, further understanding of the development and progression of acc and identification of new molecular targets are of great importance.
epidermal growth factor receptor  is a member of the erbb receptor tyrosine kinase family, and upon interacting with its ligand, it undergoes dimerization and autophosphorylation that trigger downstream signaling cascades, such as phosphatidylinositol-3-kinase/akt and mapk activation.
this signaling pathway enhances tumor cell proliferation, invasion, metastasis, and survival.
it is well recognized that matrix metalloproteinase- <dig> , which is closely associated with tumor invasion and metastasis in several human tumors, can be inhibited by egfr signaling pathway blockade  <cit> .
therefore, the egfr signaling pathway is believed to play a vital role in the progression and metastasis of tumors, including acc.
tyrosine kinase inhibitors  were first used to block egfr-mediated signaling pathways.
regretfully, several studies implicated that tki treatment alone cannot achieve impressive clinical outcomes, possibly because of the kinase-independent functions of egfr  <cit> .
however, accumulating evidence indicates that nimotuzumab, a humanized neutralizing g <dig> monoclonal antibody, can suppress cultured epithelial cancer cell proliferation by causing cell cycle arrest.
when this antibody binds to the extracellular domain of egfr, it strongly inhibits egfr-dependent cellular transformation  <cit> .
although anticancer activity in advanced squamous cell carcinoma of nimotuzumab has been previously reported  <cit> , its role in acc progression and metastasis is still unclear.
in this study, we evaluated the effects of nimotuzumab on the growth and invasion of acc- <dig> and acc-m, using a tongue squamous cell carcinoma cell line  as a positive control, and analyzed the possible downstream molecular targets and signaling pathway  <cit> .
RESULTS
nimotuzumab inhibits cell proliferation in vitro
immunohistochemistry was performed to measure the expression of egfr, mapk, and snail in three cell lines.
both egfr and p <dig> immunoreactivity were observed mostly in the cytomembrane and cytoplasm, while snail expression was observed in cell nuclei.
positive staining of three proteins was observed in all three cell lines .
the growth curves showed that the inhibition of proliferation plateaued at doses of  <dig>   <dig>  and 100 μg/ml nimotuzumab in acc- <dig>  acc-m, and tca <dig> cells, respectively.
these concentrations of nimotuzumab were used for all subsequent assays .
cells were immunolabeled with tritc-labeled ki- <dig>  and the cell nuclei were stained with dapi .
ki-67-positive cells were defined as actively proliferating cells.
the percentage of ki-67-positive cells of total dapi-positive cells was  <dig> %,  <dig> % and 86% in tca <dig>  acc-m and acc- <dig> cells, respectively.
however, this percentage decreased to  <dig> %,  <dig> % and  <dig> % in all three nimotuzumab-treated cell lines, confirming that nimotuzumab can suppress cancer cell proliferation in vitro .
nimotuzumab induced g <dig> phase arrest in acc-m and tca <dig> cells
to detect the effect of nimotuzumab on acc cell proliferation, the cell cycle distribution of cells treated with nimotuzumab  and/or hegf  in tca <dig> and acc cell lines were examined.
combined treatment of acc-m and tca <dig> cells with nimotuzumab and hegf resulted in a significant g <dig> phase arrest accompanied by a reduction of the s phase fraction .
after treatment with nimotuzumab and hegf, the percentages of cells in the g <dig> phase increased from  <dig> % to  <dig> % in acc-m and from  <dig> % to  <dig> % in tca8113cells.
in the absence of hegf stimulation, nimotuzumab did not significantly affect the cell cycle distribution .
this confirmed the hypothesis that combined nimotuzumab and hegf treatment could suppress hegf-induced cell proliferation.
nimotuzumab inhibits egfr and its downstream molecules
serum-starved cells were incubated in medium  or medium containing nimotuzumab for 72 h. qrt-pcr analysis demonstrated that snail mrna levels were  <dig>  ± 2-,  <dig>  ±  <dig> -, and  <dig> ±  <dig> -fold higher in untreated tca <dig>  acc- <dig> and acc-m cell lines than in their treated counterparts, respectively.
conclusively, egfr mrna levels were increased but without statistical significance in all three cell lines.
keratinocyte growth factor  mrna levels were down-regulated by nimotuzumab in tca <dig> and acc- <dig> cells and up-regulated in acc-m cells.
egfr mrna levels were  <dig>  ±  <dig> -,  <dig> ±  <dig> -, and  <dig>  ± 3-fold higher in untreated tca <dig>  acc- <dig>  and acc-m cells than in their treated counterparts, respectively.
kgf mrna levels were  <dig>  ±  <dig> -,  <dig>  ±  <dig> -, and  <dig>  ±  <dig> -fold higher in untreated tca <dig>  acc- <dig>  and acc-m cells than in their treated counterparts, respectively .
p <dig> mrna levels were not affected by treatment in any cell line observed .
the protein levels of egfr and its downstream molecules were assessed by western blotting.
in the first set of studies, we determined whether the in vitro exposure of cells to nimotuzumab decreased pegfr protein expression.
phosphorylated egfr protein levels were significantly decreased in nimotuzumab-treated tca <dig> and acc-m cell lines compared with untreated cells.
for acc- <dig>  the phosphorylated egfr expression level was unchanged .
interestingly, egfr protein levels were down-regulated whole in the three cell lines.
it was reported that egf activates erks mainly through the grb-2-sos-ras-raf-mek-erk pathway  <cit> .
consistent with previous findings, egfr signaling blockade significantly decreased erk and perk production in tca <dig> and acc-m cells   <cit> .
perk and erk protein levels were decreased and increased by nimotuzumab treatment in acc- <dig> cells, respectively.
we also examined p <dig> expression and found that phosphorylated p <dig> did not change significantly in all three cell lines regardless of nimotuzumab treatment.
although nimotuzumab suppressed snail expression in all three cell lines, it did not affect kgf expression in these cells .
to evaluate the proposed mechanism of action of nimotuzumab in vitro, we studied its ability to inhibit egf-induced tyrosine phosphorylation of egfr and mapk by western blotting.
serum-starved cells were treated with medium  or medium containing nimotuzumab for 90 min followed by treatment with hegf  for 15 min.
in agreement with previous findings, besides decreased egfr protein levels, egf-induced egfr activation was abrogated by nimotuzumab treatment in acc-m and acc- <dig> cell lines, resulting in perk down-regulation  <cit> .
unexpectedly, although erk expression was decreased in ligand-stimulated tca <dig> cells, its active form was increased.
no significant differences were found in p <dig> and pp <dig> expression in all three cell lines .
nimotuzumab inhibits cell invasion
to investigate whether nimotuzumab could suppress the invasive phenotypes of tca <dig> and acc cells in vitro, we conducted invasion assays using matrigel-coated transwell chambers.
all three cell lines could migrate across the membrane.
among them tca <dig> and acc- <dig> cells exhibited the highest and lowest capacity to migrate, respectively.
pure cultured or co-cultured cells on the upper chamber were allowed to migrate for 72 h followed by treatment with hegf.
reduced invasive ability of tca <dig> was observed.
the same tendency was found in the acc-m cell line .
the optical density  of the stained cells was measured at 570 nm .
to confirm whether nimotuzumab regulated cell invasion by modulating mmp <dig> expression, which is closely associated with tumor invasion and can be inhibited by egfr signaling blockade, qrt-pcr and western blot analyses were performed.
mmp <dig> mrna and protein levels were decreased significantly by nimotuzumab treatment in all three cell lines .
discussion
acc preferentially metastasizes to the lungs, bone, liver, and brain, unlike squamous cell head and neck cancer   <cit> .
local and distant recurrence can occur concurrently irrespective of initial local control, suggesting that distant and local recurrence is a separate disease  <cit> .
acc is typically controlled through surgery and postoperative adjuvant radiotherapy.
chemotherapy has also been attempted with extremely variable and inconsistent outcomes  <cit> .
to date, the most effective chemotherapy treatment is the combination of cyclophosphamide plus doxorubicin and cisplatin  <cit> .
vascular egfr -targeted therapies have been suggested to increase tumor invasiveness.
vegfr and egfr signaling pathways appear to be closely related, casting doubt on the effectiveness of anti-egfr therapy  <cit> .
furthermore, the efficacy of small-molecule tyrosine kinase inhibitors including gefitinib, imatinib, and lapatinib has also been investigated with disappointing results  <cit> .
therefore, new therapeutic regimens should be developed to treat advanced acc.
many preclinical studies suggest that nimotuzumab can be used as part of a multimodality treatment scheme.
combined radiation therapy and egfr inhibition is considered a standard concept for scchn  <cit> .
targeted therapies such as nimotuzumab have also attracted attention for the treatment of acc.
in the present study, nimotuzumab inhibited tumor cell proliferation in vitro via an egfr-dependent signaling transduction mechanism.
to facilitate cell motility, the invading cells must alter their cell-cell adhesion properties and the ecm environment.
in this study, we observed that tca <dig>  acc- <dig> and acc-m cell lines expressed mmp <dig>  snail, p <dig> and egfr in accordance with previous findings  <cit> .
unlike vandetanib, which suppresses cell growth via a direct pro-apoptotic mechanism, nimotuzumab inhibited cellular proliferation as confirmed by cck- <dig> assay and ki- <dig> immunofluorescence  <cit> .
nimotuzumab inhibited the phosphorylation of egfr and erk, two downstream effectors of the egfr signaling pathway that promote cellular proliferation and invasive potential.
furthermore, nimotuzumab down-regulated the mrna and protein expression of snail, which can mediate emt.
we hypothesized that binding of nimotuzumab to egfr impeded the interaction between egf and egfr, thus inhibiting the activation of downstream cell signals and preventing increased cellular proliferation and invasive potential.
p <dig> protein levels were not altered by nimotuzumab in the nimotuzumab group or combination group, suggesting that the inhibitory function of this antibody did not involve p <dig> mapk signaling.
interestingly, egfr mrna in the experimental group increased but was not statistically significant, compared with the control group.
however, egfr protein levels were significantly down-regulated in both the nimotuzumab group and combination group.
we hypothesized that the down-regulation was caused by nimotuzumab blocking the binding site for the antibody used for western blotting.
CONCLUSIONS
two unique features of accs are a predilection for perineural infiltration and a low incidence of lymph node metastasis.
kgf expression was not affected by nimotuzumab treatment.
thus, the predilection of acc for perineural invasion may be explained by the activity of other adhesion-related molecules.
mmps facilitate cancer cell invasion and metastasis by degrading ecm proteins.
under normal conditions, mmp <dig> expression is low or absent in acc cells.
however, the egfr signaling pathway has been implicated in mmp <dig> up-regulation in scchn  <cit> .
in this study, mmp <dig> was expressed in both carcinoma cell lines.
its expression was inhibited by nimotuzumab treatment.
as tumor cell invasion is mmp-dependent, mmps likely participate in the antitumor effects of nimotuzumab.
in summary, we demonstrated that nimotuzumab could inhibit the growth and invasion of acc cell lines induced by egf, probably through the inactivation of erk phosphorylation, suggesting that the in vivo efficacy of nimotuzumab against metastatic acc should be examined.
nimotuzumab should be considered as a promising novel agent for the treatment of acc.

 adenoid cystic carcinoma is a common salivary gland cancer subtype accounting for 22% of salivary gland malignancies and 1% of all head and neck cancers. perineural invasion, delayed onset of hematogenous metastasis and poor response to traditional chemotherapies are characteristics of this cancer. local recurrence and distant metastasis, in which extracellular matrix and basement membrane degradation are crucial, are responsible for treatment failure. currently, no systemic therapy is available that effectively inhibits acc progression. therefore, further understanding of the development and progression of acc and identification of new molecular targets are of great importance. epidermal growth factor receptor is a member of the erbb receptor tyrosine kinase family, and upon interacting with its ligand, it undergoes dimerization and autophosphorylation that trigger downstream signaling cascades, such as phosphatidylinositol-3-kinase/akt and mapk activation. this signaling pathway enhances tumor cell proliferation, invasion, metastasis, and survival. it is well recognized that matrix metalloproteinase-, which is closely associated with tumor invasion and metastasis in several human tumors, can be inhibited by egfr signaling pathway blockade. therefore, the egfr signaling pathway is believed to play a vital role in the progression and metastasis of tumors, including acc. tyrosine kinase inhibitors were first used to block egfr-mediated signaling pathways. regretfully, several studies implicated that tki treatment alone cannot achieve impressive clinical outcomes, possibly because of the kinase-independent functions of egfr. however, accumulating evidence indicates that nimotuzumab, a humanized neutralizing g monoclonal antibody, can suppress cultured epithelial cancer cell proliferation by causing cell cycle arrest. when this antibody binds to the extracellular domain of egfr, it strongly inhibits egfr-dependent cellular transformation. although anticancer activity in advanced squamous cell carcinoma of nimotuzumab has been previously reported, its role in acc progression and metastasis is still unclear. in this study, we evaluated the effects of nimotuzumab on the growth and invasion of acc- and acc-m, using a tongue squamous cell carcinoma cell line as a positive control, and analyzed the possible downstream molecular targets and signaling pathway.  nimotuzumab inhibits cell proliferation in vitro immunohistochemistry was performed to measure the expression of egfr, mapk, and snail in three cell lines. both egfr and p immunoreactivity were observed mostly in the cytomembrane and cytoplasm, while snail expression was observed in cell nuclei. positive staining of three proteins was observed in all three cell lines. the growth curves showed that the inhibition of proliferation plateaued at doses of and 100 μg/ml nimotuzumab in acc- acc-m, and tca cells, respectively. these concentrations of nimotuzumab were used for all subsequent assays. cells were immunolabeled with tritc-labeled ki- and the cell nuclei were stained with dapi. ki-67-positive cells were defined as actively proliferating cells. the percentage of ki-67-positive cells of total dapi-positive cells was %, % and 86% in tca acc-m and acc- cells, respectively. however, this percentage decreased to %, % and % in all three nimotuzumab-treated cell lines, confirming that nimotuzumab can suppress cancer cell proliferation in vitro. nimotuzumab induced g phase arrest in acc-m and tca cells to detect the effect of nimotuzumab on acc cell proliferation, the cell cycle distribution of cells treated with nimotuzumab and/or hegf in tca and acc cell lines were examined. combined treatment of acc-m and tca cells with nimotuzumab and hegf resulted in a significant g phase arrest accompanied by a reduction of the s phase fraction. after treatment with nimotuzumab and hegf, the percentages of cells in the g phase increased from % to % in acc-m and from % to % in tca8113cells. in the absence of hegf stimulation, nimotuzumab did not significantly affect the cell cycle distribution. this confirmed the hypothesis that combined nimotuzumab and hegf treatment could suppress hegf-induced cell proliferation. nimotuzumab inhibits egfr and its downstream molecules serum-starved cells were incubated in medium or medium containing nimotuzumab for 72 h. qrt-pcr analysis demonstrated that snail mrna levels were ± 2-, ± -, and ± -fold higher in untreated tca acc- and acc-m cell lines than in their treated counterparts, respectively. conclusively, egfr mrna levels were increased but without statistical significance in all three cell lines. keratinocyte growth factor mrna levels were down-regulated by nimotuzumab in tca and acc- cells and up-regulated in acc-m cells. egfr mrna levels were ± -, ± -, and ± 3-fold higher in untreated tca acc- and acc-m cells than in their treated counterparts, respectively. kgf mrna levels were ± -, ± -, and ± -fold higher in untreated tca acc- and acc-m cells than in their treated counterparts, respectively. p mrna levels were not affected by treatment in any cell line observed. the protein levels of egfr and its downstream molecules were assessed by western blotting. in the first set of studies, we determined whether the in vitro exposure of cells to nimotuzumab decreased pegfr protein expression. phosphorylated egfr protein levels were significantly decreased in nimotuzumab-treated tca and acc-m cell lines compared with untreated cells. for acc- the phosphorylated egfr expression level was unchanged. interestingly, egfr protein levels were down-regulated whole in the three cell lines. it was reported that egf activates erks mainly through the grb-2-sos-ras-raf-mek-erk pathway. consistent with previous findings, egfr signaling blockade significantly decreased erk and perk production in tca and acc-m cells. perk and erk protein levels were decreased and increased by nimotuzumab treatment in acc- cells, respectively. we also examined p expression and found that phosphorylated p did not change significantly in all three cell lines regardless of nimotuzumab treatment. although nimotuzumab suppressed snail expression in all three cell lines, it did not affect kgf expression in these cells. to evaluate the proposed mechanism of action of nimotuzumab in vitro, we studied its ability to inhibit egf-induced tyrosine phosphorylation of egfr and mapk by western blotting. serum-starved cells were treated with medium or medium containing nimotuzumab for 90 min followed by treatment with hegf for 15 min. in agreement with previous findings, besides decreased egfr protein levels, egf-induced egfr activation was abrogated by nimotuzumab treatment in acc-m and acc- cell lines, resulting in perk down-regulation. unexpectedly, although erk expression was decreased in ligand-stimulated tca cells, its active form was increased. no significant differences were found in p and pp expression in all three cell lines. nimotuzumab inhibits cell invasion to investigate whether nimotuzumab could suppress the invasive phenotypes of tca and acc cells in vitro, we conducted invasion assays using matrigel-coated transwell chambers. all three cell lines could migrate across the membrane. among them tca and acc- cells exhibited the highest and lowest capacity to migrate, respectively. pure cultured or co-cultured cells on the upper chamber were allowed to migrate for 72 h followed by treatment with hegf. reduced invasive ability of tca was observed. the same tendency was found in the acc-m cell line. the optical density of the stained cells was measured at 570 nm. to confirm whether nimotuzumab regulated cell invasion by modulating mmp expression, which is closely associated with tumor invasion and can be inhibited by egfr signaling blockade, qrt-pcr and western blot analyses were performed. mmp mrna and protein levels were decreased significantly by nimotuzumab treatment in all three cell lines. discussion acc preferentially metastasizes to the lungs, bone, liver, and brain, unlike squamous cell head and neck cancer. local and distant recurrence can occur concurrently irrespective of initial local control, suggesting that distant and local recurrence is a separate disease. acc is typically controlled through surgery and postoperative adjuvant radiotherapy. chemotherapy has also been attempted with extremely variable and inconsistent outcomes. to date, the most effective chemotherapy treatment is the combination of cyclophosphamide plus doxorubicin and cisplatin. vascular egfr -targeted therapies have been suggested to increase tumor invasiveness. vegfr and egfr signaling pathways appear to be closely related, casting doubt on the effectiveness of anti-egfr therapy. furthermore, the efficacy of small-molecule tyrosine kinase inhibitors including gefitinib, imatinib, and lapatinib has also been investigated with disappointing results. therefore, new therapeutic regimens should be developed to treat advanced acc. many preclinical studies suggest that nimotuzumab can be used as part of a multimodality treatment scheme. combined radiation therapy and egfr inhibition is considered a standard concept for scchn. targeted therapies such as nimotuzumab have also attracted attention for the treatment of acc. in the present study, nimotuzumab inhibited tumor cell proliferation in vitro via an egfr-dependent signaling transduction mechanism. to facilitate cell motility, the invading cells must alter their cell-cell adhesion properties and the ecm environment. in this study, we observed that tca acc- and acc-m cell lines expressed mmp snail, p and egfr in accordance with previous findings. unlike vandetanib, which suppresses cell growth via a direct pro-apoptotic mechanism, nimotuzumab inhibited cellular proliferation as confirmed by cck- assay and ki- immunofluorescence. nimotuzumab inhibited the phosphorylation of egfr and erk, two downstream effectors of the egfr signaling pathway that promote cellular proliferation and invasive potential. furthermore, nimotuzumab down-regulated the mrna and protein expression of snail, which can mediate emt. we hypothesized that binding of nimotuzumab to egfr impeded the interaction between egf and egfr, thus inhibiting the activation of downstream cell signals and preventing increased cellular proliferation and invasive potential. p protein levels were not altered by nimotuzumab in the nimotuzumab group or combination group, suggesting that the inhibitory function of this antibody did not involve p mapk signaling. interestingly, egfr mrna in the experimental group increased but was not statistically significant, compared with the control group. however, egfr protein levels were significantly down-regulated in both the nimotuzumab group and combination group. we hypothesized that the down-regulation was caused by nimotuzumab blocking the binding site for the antibody used for western blotting.  two unique features of accs are a predilection for perineural infiltration and a low incidence of lymph node metastasis. kgf expression was not affected by nimotuzumab treatment. thus, the predilection of acc for perineural invasion may be explained by the activity of other adhesion-related molecules. mmps facilitate cancer cell invasion and metastasis by degrading ecm proteins. under normal conditions, mmp expression is low or absent in acc cells. however, the egfr signaling pathway has been implicated in mmp up-regulation in scchn. in this study, mmp was expressed in both carcinoma cell lines. its expression was inhibited by nimotuzumab treatment. as tumor cell invasion is mmp-dependent, mmps likely participate in the antitumor effects of nimotuzumab. in summary, we demonstrated that nimotuzumab could inhibit the growth and invasion of acc cell lines induced by egf, probably through the inactivation of erk phosphorylation, suggesting that the in vivo efficacy of nimotuzumab against metastatic acc should be examined. nimotuzumab should be considered as a promising novel agent for the treatment of acc. 
antagonists of egfr can suppress the growth of several malignancies; however, their therapeutic effect in adenoid cystic carcinoma is controversial. epidermal growth factor receptor adenoid cystic carcinoma nimotuzumabmonoclonal antibodymatrix metalloproteinase epithelial- mesenchymal transition invasioncancer therapy the increased proliferation of two acc cell lines induced by egf-treatment was reversed by nimotuzumab. the effects of egf were down-regulated by nimotuzumab treatment. these results suggest that nimotuzumab can inhibit the growth and invasion of acc cells induced by egf, probably through inactivation of erk phosphorylation. thus, nimotuzumab should be considered as a promising novel agent for the treatment of acc. regardless of egf stimulation, nimotuzumab-treated acc cells were arrested in g phase and showed decreased expression of ki in addition, egf activated the mapk-dependent pathway and up-regulated the expression of matrix metalloproteinase- and snail, enhancing the invasive potential of an acc cell line. epidermal growth factor receptor is involved in the development of many human malignant tumors and plays an important role in tumor growth and metastasis.
 epidermal growth factor receptor is involved in the development of many human malignant tumors and plays an important role in tumor growth and metastasis. antagonists of egfr can suppress the growth of several malignancies; however, their therapeutic effect in adenoid cystic carcinoma is controversial.  the increased proliferation of two acc cell lines induced by egf-treatment was reversed by nimotuzumab. regardless of egf stimulation, nimotuzumab-treated acc cells were arrested in g phase and showed decreased expression of ki in addition, egf activated the mapk-dependent pathway and up-regulated the expression of matrix metalloproteinase- and snail, enhancing the invasive potential of an acc cell line. the effects of egf were down-regulated by nimotuzumab treatment.  these results suggest that nimotuzumab can inhibit the growth and invasion of acc cells induced by egf, probably through inactivation of erk phosphorylation. thus, nimotuzumab should be considered as a promising novel agent for the treatment of acc. epidermal growth factor receptor adenoid cystic carcinoma nimotuzumabmonoclonal antibodymatrix metalloproteinase epithelial- mesenchymal transition invasioncancer therapy 