| ObjectMethionine enkephalin(MENK),an endogenous opioid peptide,which interacts with the opioid receptor,is considered,clinically,as a biological therapy that has potential application for treatment of cancer.The aim of this investigation is to look at whether MENK could inhibit progression and metastasis of human nasopharyngeal carcinoma CNE2 cell,and to explore the mechanisms by which MENK has therapeutic effects on CNE2 cell growth in vivo and in vitro.Methods??Inhibitory effect of MENK on Human Nasopharyngeal carcinoma CNE2 cells1?Inhibitory effect of MENK on Human Nasopharyngeal carcinoma CNE2 cells in VitroIn this study,human nasopharyngeal carcinoma(NPC)CNE2 cells were divided into two groups:negative control group(Ctrl)and experimental group(MENK).The proliferation inhibition of MENK on CNE2 cells was detected by MTT assay,and the growth inhibition rate of MENK on CNE2 cells was calculated.The effect of MENK on the morphology of CNE2 cells was recorded by microscope..The effects of MENK on the cell cycle and apoptosis rate of CNE2 cells were detected by flow cytometry.The the expression of opioid receptors in CNE2 cells was detected by qRT-PCR and the action by MENK was observed.Then the antagonism group was added,and NTX which known as the agonist of opioid receptors was used.Through cell scratches and transwell chambers,the effect of MENK on the migration,invasion and metastasis of CNE2 cells was detected,and compared between the three groups.2?Antitumor effect of MENK on tumor-bearing nude miceThirty BABL/C nude mice were injected subcutaneously with CNE2 cells to establish tumor bearing model,and there were divided into three groups:Ctrl group(normal saline),MENK group and NTX+MENK group.The condition of nude mice and the growth of subcutaneous CNE2 tumor were observed daily.The tumor volume was recorded regularly.The nude mice were killed on the 42nd day,the tumor was removed,weighed,and the tumor size was measured,and compared the difference between the three groups and its significance;HE staining and immunohistochemical staining were used to observe the histopathologic changes of tumor tissues,and the possible mechaniam for the inhibitory effect of MENK on tumor growth in nude mice was analyzed.??Possible Mechanisms of MENK inhibiting invasion and migration of CNE2 cellsWesternblot was used to detect the expressions of invasion and migration related proteins in CNE2 cells,and the possible pathway of MENK was speculated.The CNE2 cells were treated separately with OGFr-siRNA transient interference and TGF-?/smad pathway antagonist SB431542 to observe the effects of MENK on the migration and invasion inhibition of human CNE2 cells of nasopharyngeal carcinoma(NPC).The significance of the decrease of ?-catenin in the inhibitory effect of MENK on the invasion and migration of CNE2 cells was analyzed by immunofluorescence,and the possible mechanisms of MENK on the invasion and migration inhibition of CNE2 cells was discussed.Results??MENK inhibits the growth,invasion and migration of human nasop-haryngeal carcinoma CNE2 cells1?MENK can inhibit the growth,invasion and migration of CNE2 cells in vitro(1).MTT results showed that MENK could inhibit the growth of human nasopharyngeal carcinoma CNE2 cells,and the optimum time was 72 h,IC50 is 4mg/ml.MENK could obviously change the morphology of CNE2 cells.Under inverted microscope,CNE2 cells were found to be flattened fusiform cells,closely aligned with each other.After the treatment of MENK,the cells arranged loosely,the volume became larger,the nuclear membrane appeared more vacuoles,and the floating cells increased.(2).Flow cytometry showed that MENK blocked the division of CNE2 cells and made the majority of CNE2 cells stay in G2/M phase.After 48h of 6mg/ml MENK treatment,the CNE2 cells in G2/M phase were about 29.7%,compared with 20.8%in the control group(p<0.05).The apoptosis rate of CNE2 cells treated with MENK or not was 17.1%and 6.43%,respectively,the difference was significant(p<0.01),which showed that MENK could effectively promote the apoptosis of CNE2 cells.(3).qRT-PCR detected the expression of opioid receptor MOR(?),DOR(8),KOR(?)and ZETA(?)in CNE2 cells,and MENK could up-regulate their expressions.(4).The results of transwell chambers showed that the number of successful penetrating cells in 6mg/mL MENK group was significantly lower than that in control group(p<0.01),and MENK effectively inhibited the invasion of CNE2 cells.The results of scratch test showed that MENK could significantly inhibit the migration of CNE2 cells.The wound healing rate in MENK group was only 17.57%,compared with 62.13%in Ctrl group(p<0.01).The results showed that MENK could inhibit the invasion and migration of CNE2 cells apparently.2?MENK can inhibit the growth of CNE2 tumor in vivo(1)The tumor-bearing model of nude mice was established successfully.(2)Compared with Ctrl group,the growth of CNE2 tumor in MENK group was slower than that in Ctrl group,and the volume and weight of CNE2 tumor in MENK group were significantly lower than those in Ctrl group(p<0.05),The weight of CNE2 tumor in NTX group was higher obviously than that in MENK group,and the difference was significant statistically(p<0.05),which suggesting that MENK inhibited CNE2 tumor growth and NTX could antagonize the inhibition of MENK.(3)The results of HE staining showed,comparing to Ctrl group,that the density of tumor cells in MENK group was lower,the cytoplasm was light stained,the nucleus was balloon degeneration,the nucleolus was obvious,the number of nuclear pyknosis cells was more,the blood vessel between tumor cells was decreased,and the apoptotic cells of bare nucleus could be seen.NTX could antagonize the pathological changes.(4)Immunohistochemical staining showed that the expression of OGFr in cytoplasm and karyotheca in MENK group were increased,and NTX could antagonize the up-regulation.??The possible mechanism of MENK on the invasion and migration inhibition of CNE2 cells(1)Westernblot assay showed that MENK up-regulated the expression of OGFr and E-ca,down-regulated the expression of Slug,Snail,p-Smad3,?-catenin and TGF-?1,but had no significant effect on the expression of Vimentin and N-ca.It could be speculated that MENK may inhibit the invasion and migration of CNE2 cells by regulating the TGF-?/Smad pathway.(2)OGFr-siRNA could successfully interfere with the expression of OGFr in CNE2 cells,and compared to MENK group,the ability of invasion and migration of CNE2 cells interfered by OGFr-siRNA was not significantly decreased by MENK(p>0.05),which indicated that MENK might inhibit the invasion and migration of CNE2 cells by acting on opioid receptor-OGFr.(3)The results of transwell chambers assay showed that the number of successful panetrating CNE2 cells in the SB group was significantly higher than that in MENK group(p<0.01).The results of Westernblot assay showed that,compared to the Ctrl group,the differences in the expression of p-smad3,Slug and Snail in the SB group was significantly smaller than that in MENK group,which indicated that SB431542 could antagonize the inhibitory effect of MENK on the invasion and migration of CNE2 cells.(4)The results of Immunofluorescence assay suggested that the expression of OGFr distributed in the cytoplasm and karyotheca,and the expression of OGFr could be up-regulated by MENK especially in karyotheca,and the expression of ?-catenin could be down-regulated mainly in the karyotheca.Conclusions1.MENK can directly inhibit the proliferation,migration and invasion of human nasopharyngeal carcinoma CNE2 cells and kill CNE2 cells effectively.2.MENK can inhibit the invasion of human nasopharyngeal carcinoma CNE2 cells by OGFr through the TGF-?/SMAD pathway.3.MENK can up-regulate the expression of the opioid receptor OGFr in the CNE2 cell and decrease the expression of ?-catenin in the karyotheca,which effectively inhibit the migration and the invasion of the CNE2 cell. |
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