| BACKGROUNDNasopharyngeal carcinoma(NPC)is an epithelial malignant tumor occurring in the nasopharynx,which is mainly found in southern China and Southeast Asia.Radiotherapy is the main treatment for nasopharyngeal carcinoma.With the improvement of radiotherapy technology,great progress has been made in the treatment outcome of nasopharyngeal carcinoma,and the prognosis of patients has been significantly improved.However,about 25% of patients still have develop into recurrence and/or metastasis clinically after systematic treatment.If these patients achieve re-irradiation,the radiation damage is frequently severe and it is difficult for patients to tolerate.Moreover,platinum-based chemotherapy has severe side effects and the response rate is low.As a result,there is a need to search out more treatment options.Sorafenib,a multi-kinase inhibitor,is an important part of liver and kidney cancer treatment and has been shown to effectively extend median survival of patients with advanced disease.However,many patients with liver cancer often develop into sorafenib resistance after treatment for a period of time.Studies have demonstrated that sorafenib also has anti-tumor effects on recurrent and metastatic nasopharyngeal carcinoma.Scholars have found that compound UC2288 is called trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-[4-(5-trifluoromethyl-pyridin-2-yloxy)-cyclohexyl]-urea and it is an inhibitor of p21,suppressing p21 expression transcriptionally and post-transcriptionally.UC2288 is derived from sorafenib,and structures of both are similar.However,unlike sorafenib,UC2288 has little effects on VEGFR2 and Raf kinase activity.Study has demonstrated that UC2288 inhibits the growth of renal carcinoma cells,but its effect on nasopharyngeal carcinoma and its molecular mechanism remains unclear.Therefore,this study intends to clarify whether UC2288 has anti-tumor effects on nasopharyngeal carcinoma and its specific molecular mechanism,providing a theoretical evidence for the development and application of UC2288.OBJECTIVEThis study investigated the effects of UC2288 on the proliferation,apoptosis,invasion,metastasis and radiosensitivity of nasopharyngeal carcinoma in vitro and in vivo,and further probed into its mechanism.METHODS1.CNE-2R,CNE-2,5-8F and LO2 cells were treated with different concentrations of UC2288,cisplatin and sorafenib.After that,the cell proliferation was detected by CCK8 assay.The effect of UC2288 on the ability of cell colony formation was detected by plate clone formation assay.The changes of cell morphology were observed directly under microscope or by hematoxylineosin staining.The effect of UC2288 on cell apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins were detected by Western blot and immunofluorescence assay.2.The invasion and migration ability of cells after treatment with UC2288 was detected by wound healing assay and transwell assay.The expression levels of EMT markers and MMP2 protein were detected by Western blot assay.3.CCK8 assay and clone formation assay were used to detect the effects of UC2288 on radiosensitivity of CNE-2R cells after X-ray exposure or combination of UC2288 and X-ray exposure.The effects of UC2288 on radiosensitivity in vivo were detected on xenograft tumors which were constructed by CNE-2R cell line.4.To further explore the mechanism of UC2288,the expression levels of MEK/ERK signaling pathway proteins and p21 were detected by Western blot assay.In addition,after treatment with UC2288 combined with or without MEK/ERK pathway inhibitor U0126 and agonist HGF,the proliferation ability of cells was detected by CCK8 assay,and the expression of MEK/ERK signaling pathway proteins and apoptosis-related proteins were detected by Western blot assay.5.The nasopharyngeal carcinoma cell line 5-8F was used to construct the xenograft tumor model in nude mice.The changes of tumor size and body weight of the nude mice were measured periodically after drug treatment to observe the effects of UC2288 on the growth of xenograft tumors.Blood tests were used for the detection of effects of UC2288 on blood routine examination and heart,liver and kidney function in nude mice.The proliferation index of xenograft tumors was detected by immunohistochemical assay.Cell apoptosis index in xenograft tumors was detected by TUNEL assay.The expression levels of MEK/ERK pathway proteins and apoptosis-related proteins in xenograft tumor tissues were detected by Western blot assay.RESULTS1.UC2288 could inhibit the proliferation of nasopharyngeal carcinoma cells in a dose-dependent and time-dependent manner.Specifically,after treatment with DMSO,4μM,6μM,8μM,12μM 16μM UC2288 for 48 h,the survival rate of CNE-2R cells was 100.00±00%,82.35±2.60%,57.62±2.78%,42.50±2.19%,31.64±1.45%,23.65±1.84%,respectively;the survival rate of CNE-2 cells was100.00±00%,86.88±1.64%,55.41±2.20%,49.93±1.80%,37.06±1.05%,30.64±0.35%,respectively;and the survival rate of 5-8F cells was 100.00±00%,77.68±1.05%,38.54±1.35%,21.82±0.35%,7.41±0.88%,4.13±0.28%,respectively.Compared with cisplatin,UC2288 had stronger inhibitory effect on5-8F cell proliferation,and UC2288 had less toxicity on normal hepatic epithelial cell line LO2.In addition,UC2288 was more toxic to nasopharyngeal carcinoma cells compared with sorafenib.Moreover,UC2288 significantly suppressed the colony formation ability of the nasopharyngeal carcinoma cells.Morphological assay results showed that,after incubation with UC2288,the cells shrank;the volume became smaller;the nucleus was smaller and condensed.The results of flow cytometry assay revealed that the cell apoptosis rate increased significantly with the increase of drug concentration.Specifically,after treated with DMSO,4μM,6μM,8μM,12μM UC2288 for 48 h,the apoptosis rate of CNE-2R cells was1.94±0.67%,8.81±0.99%,38.29±2.46%,59.23±3.10%,71.44±2.99%,respectively;the apoptosis rate of CNE-2 cells was 5.37±0.93%,6.83±0.79%,50.03±4.60%,63.21±5.29%,75.67±4.59%,respectively;the apoptosis rate of 5-8F cells was 10.95±2.81%,64.17±4.06%,78.00±4.06%,82.76±0.50%,84.95±1.54%,respectively.Western blot analysis showed that the expression levels of anti-apoptotic proteins Bcl-2 and survivin were decreased,and the levels of pro-apoptotic proteins Bax and cleaved caspase3 were increased in UC2288 treated cells.The results of immunofluorescence assay and western blot analysis showed that the expression level of γ-H2 AX,a marker of DNA double-strand break,was increased,suggesting that UC2288 induced DNA damage.2.The results of wound healing assay and transwell assay revealed that the invasion and metastasis ability of nasopharyngeal carcinoma cells decreased after UC2288 treatment.Furthermore,western blot analysis showed that the expression of epithelial marker E-cadherin increased,while the expression of mesenchyme markers N-cadherin and vimentin and MMP2 protein decreased,further indicating that UC2288 could suppress the invasion and metastasis ability of nasopharyngeal carcinoma cells.3.The results of clone formation assay showed that the survival fraction of combination group was lower than that of the single irradiation group at different irradiation doses,and the radiosensitization ratio of combination group to single irradiation group was 1.60 >1,indicating that UC2288 could increase the radiosensitivity of CNE 2R cells.Moreover,CCK8 assay results demonstrated that the proliferation ability of cells in the combination group was significantly reduced compared with that in the single irradiation group,which also suggested that the radiosensitivity of cells was increased after UC2288 treatment.In addition,combination of UC2288 and irradiation also significantly inhibited the growth of xenograft tumors in nude mouse model.4.In terms of molecular mechanism,western blot analysis showed that UC2288 inhibited the phosphorylation of MEK and ERK.CCK8 assay results revealed that U0126 could further increase the proliferation inhibition induced by UC2288.Western blot analysis showed that U0126 further decreased the expression of anti-apoptotic protein Bcl-2 and increased pro-apoptotic protein cleaved-caspase3 induced by UC2288.The MEK/ERK pathway agonist HGF partially reversed UC2288-induced dephosphorylation of MEK and ERK.CCK8 assay results indicated that HGF could decrease the proliferation inhibition induced by UC2288.Western blot assay revealed that HGF partially reversed the decrease of the expression of anti-apoptotic protein Bcl-2 and the increase of the expression of pro-apoptotic protein cleaved-caspase3 induced by UC2288.5.UC2288 significantly inhibited the growth of xenograft tumor in nude mice compared with the control group.There was no significant difference in body weight between the two groups.The results of blood test showed that UC2288 had no obvious toxicity to blood routine and heart,liver and kidney function.Immunohistochemical assay results indicated that the expression of Ki-67 decreased in the UC2288 group.The TUNEL assay showed that the apoptosis index of xenograft tumors was increased in the UC2288-treated group.Western blot analysis results revealed that the expression levels of the phosphorylation proteins of MEK/ERK pathway and anti-apoptotic protein Bcl-2 and survivin were decreased,and the expression level of pro-apoptotic protein Bax was increased in UC2288 group.CONCLUSIONS This study confirmed that UC2288 could inhibit the proliferation,induce apoptosis,suppress the invasion and metastasis,and increase the radiosensitivity of nasopharyngeal carcinoma cells,and effectively retarded the growth of xenograft tumors of nasopharyngeal carcinoma in nude mice.UC2288 possessed strong anti-nasopharyngeal carcinoma effects in vitro and in vivo.Mechanism studies showed that UC2288 could inhibit the activation of MEK/ERK pathway,thereby decreasing cell proliferation and inducing cell apoptosis.This study verified the anti-nasopharyngeal cancer effect of UC2288 in vitro and in vivo,and explored the molecular mechanism of UC2288,which provided a theoretical evidence for the development of UC2288 as an effective anti-nasopharyngeal carcinoma drug. |
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