| BackgroundNasopharyngeal carcinoma(NPC)is a kind of malignant tumor derived from nasopharyngeal epithelial cells.Its malignant degree is often very high,and easily diagnosed at lymph node metastasis.Although after radiation treatment,patients with early 5-year survival rate is 80%-90%,more and more evidences have shown that full radiation therapy can achieve a good local control,but there is still as high as 10-20% of the patients occur recurrence and distant metastasis.Thus,facing the fact that current treatment effect is significantly lower,some new therapy strategies and novel drugs are urgently needed.Chinese herbal medicine in clinical application has a long history in China.They display multiple drug effects on tumor cell proliferation,cancer cell apoptosis,cancer metastasis and other medicinal effects.In addition,they have the advantages of safety,economy,development of safe and effective resistance to nasopharyngeal carcinoma current,which is an urgent need to be solved in the research field of Chinese medicine,especially for the recurrent nasopharyngeal carcinoma invasion clinical problem.Due to these characters including its simple structure,small side effects,low cost factors,traditional Chinese medicine monomers display significantly more potential applications.Matrix metalloproteins(MMPs)are a general family,and several members of this family have been reported to be highly expressed in a variety of tumors,which are related to tumor invasion,migration and prognosis.However,there is no relevant report on which MMPs are mainly expressed in nasopharyngeal carcinoma.This study aims to identify the highly expressed MMPs in nasopharyngeal carcinoma and reveal its molecular mechanism of action.In addition,drug screening of traditional Chinese medicine monomers was performed to discover some hits that can inhibit both the proliferation of nasopharyngeal carcinoma and the high expression of MMPs in nasopharyngeal carcinoma.Our research will provide necessary empirical research basis and effective reference for the development of anti-nasopharyngeal carcinoma traditional Chinese medicine.Purpose and significance:(1)To explore the high expression of MMPs in nasopharyngeal carcinoma tissue cells,and conduct follow-up studies through experimental verification.Screening the monomer of Chinese medicine against nasopharyngeal carcinoma;(2)To explore the mechanism of action of the lead drug cucurbitin B against nasopharyngeal carcinoma,and to provide necessary basic research for the clinical application of cucurbitin B against nasopharyngeal carcinoma.The research content mainly includes the following four parts:(1)Bioinformatics screening and experimental verification of high expression of MMPs in nasopharyngeal carcinoma;(2)High-throughput screening of traditional Chinese medicine monomers that can inhibit the proliferation of nasopharyngeal carcinoma and inhibit the expression of MMP3;(3)To investigate the anti-nasopharyngeal carcinoma effect of cucurinin B in vivo and in vitro;(4)Based on network pharmacology and experiments,the mechanism of cucurinin B on proliferation,apoptosis,invasion and migration of nasopharyngeal carcinoma cells was verified.Research Methods:(1)The MMPs with high expression in nasopharyngeal carcinoma were identified by bioanalysis,and then the MMPs with high expression were verified by experiments.(2)We collected 60 kinds of Chinese medicine monomers with abundant yield and anti-tumor effect in Guangxi through literature search,and then screened out the Chinese medicine monomers with inhibitory effect on nasopharyngeal carcinoma by Cell Titter Glo luminescence method;Fluorescence quantitative PCR was used to further screen drugs that down-regulated MMP3.(3)After screening out the drugs with inhibitory effect on differentially expressed MMPs,CCK8,plate cloning test,flow cytometry test and subcutaneous tumor-forming test in nude mice were used to test the inhibitory effect of the drugs on the proliferation of NPC cells;The apoptosis induced effect of the drugs on nasopharyngeal carcinoma cells was examined by apoptosis fluorescence assay and flow cytometry assay.The effects of drugs on invasion and migration of nasopharyngeal carcinoma cells were tested by scratch test and Transwel test.(4)Study the mechanism of action of drugs on nasopharyngeal carcinoma through network pharmacology.Western Blot was used to verify the pathway involved in the drug and the expression of related proteins affecting the proliferation,apoptosis and invasion of nasopharyngeal carcinoma.Immunohistochemistry was used to verify the expression of proteins related to cell proliferation after drug treatment.Results:(1)Through bioinformatics analysis,MMP1,MMP3,MMP12 in nasopharyngeal carcinoma tissues from normal nasopharyngeal tissue expression significantly increases,fluorescence quantitative PCR results showed that relatively normal nasopharyngeal epithelial cells,MMP3 expression significantly increased in four nasopharyngeal carcinoma(NPC)cell line,while MMP1 and MMP12 expression in four lines are no significant difference;WB results showed that,compared with normal nasopharyngeal epithelial cells,the expression of MMP3 was increased in CNE1,CNE2 and 5-8F of nasopharyngeal carcinoma,while there was no significant difference in 6-10 B cells.(2)Five monomers with proliferation inhibitory effect on nasopharyngeal carcinoma were screened out,including gossyol acetate,cucurbitin B,dihydrotanshinone,saikuratinin A and saikuratinin D.The inhibitory effect of cucurbitin B on the expression of MMP3 in two types of nasopharyngeal carcinoma cells with high malignancy was detected by qt-PCR,and then cucurbitin B was selected for further study.(3)CCK8 results showed that the IC50 of CNE2 and 5-8F cells of nasopharyngeal carcinoma after 24 h,48h and 72 h treatment was much lower than that of HNEPC cells.The plate cloning experiment showed that cucurbitin B could inhibit the cloning ability of two kinds of nasopharyngeal carcinoma cell lines.Flow cytometry showed that cucurbitin B could induce G2 phase arrest in both nasopharyngeal carcinoma cell lines.In vivo experiment results showed that intratumoral injection group,intravenous injection group and intraperitoneal injection group could inhibit the proliferation of nasopharyngeal carcinoma CNE2 cell tumors in nude mice.The effect of intratumoral injection group was the most obvious.There was no significant difference in body weight among the groups.Apoptosis fluorescence assay and flow cytometry showed that cucurbitin B could induce apoptosis of two kinds of nasopharyngeal carcinoma cell lines.The scratch test,Transwel migration and invasion test showed that cucurbitin B could inhibit the invasion and migration of two kinds of nasopharyngeal carcinoma cell lines.(4)Through network pharmacology and bioinformatics analysis,MAPK1 is the key gene of cucurbitin B,MAPKK pathway can not only affect cell proliferation and apoptosis,but also inhibit tumor invasion and migration by inhibiting the expression of MMPs.According to WB verification,compared with the control group,P-ERK /ERK,Bcl2,CDK1 and c-myc showed a decreasing trend with the increasing concentration of cucurinin B after 48 h treatment,while BAX and Cleaved Caspase-3 showed an increasing trend with the increasing concentration of cucurinin B.However,P-ERK/ERK,c-JUN,MMP3 and Vimentin showed a decreasing trend with the increase of concentration after 24 h treatment with calucinin B at different concentrations(0.05 u M,0.1u M,0.2u M).Immunohistochemistry showed that the expressions of c-myc and CDK1 in the intraperitoneal injection group and the caudal venous injection group were lower than those in the control group.Conclusion:(1)MMP3 was highly expressed in nasopharyngeal carcinoma tissues and each cell line of nasopharyngeal carcinoma;(2)Gossypol acetate,dihydrotanyshinone,saparosaponin A,saparosaponin D and cucurbitin B all had good inhibitory effects on the proliferation of the four NPC cell lines,but cucurbitin B had A stronger inhibitory effect on the expression of MMP3 in NPC cells.(3)Cucurbitin B can play an anti-nasopharyngeal carcinoma role by inhibiting cell proliferation,inducing cell apoptosis,and inhibiting invasion and migration;(4)Cucurbitin B can inhibit the proliferation of NPC cells and induce the apoptosis of NPC cells through MAPK-ERK1/2 pathway;The expression of MMP3 in NPC can be inhibited by MAPK-ERK1/2 pathway,and the invasion and migration of NPC cells may be inhibited by inhibiting EMT of NPC cells.(5)Reverse screening combined with routine screening may be helpful for the clinical development and application of anti-nasopharyngeal carcinoma drugs. |
PDF Full Text Request