| Mitogen-activated protein kinase (MAPK) is a kind of Serine/Threine kinasewith high preservation. It is one of the important messengers for signal transductionin cell growth, which could be stimulated by various activators. In mammal cells,more researches focus on three parallel MAPK pathways, ERK (extracellular signal-regulated kinase) pathway, JNK (c-jun N-terminal kinase) pathway and p38 pathway.Induced by different stimuli, different cell lines may have corresponding physiologicalresponses.As a growth factor, TPA has close relation with cell growth, differentiation,apoptosis and cell cycle arrest. TPA is the activator of PKC, and PKC-activated couldmodulate cell life. All-trans retinoic acid (ATRA) is the derivative of vitamin A, andits functions cover the following aspects: 1, growth inhibition of cells; 2, induction ofcell differentiation; 3, induction of apoptosis; 4, immunological regulation; 5,repression of blood vessel formation: 6, effect on exprcssions of oncogene or tumorsuppressor gene. In certain cancer cell lines, TPA could work in coordination withATRA, whereas ATRA could effectively inhibit TPA action in other cancer cells.Based on the study of gastric canccr cells BGC-823, we focused on theregulation of some kinases of MAPK signal pathway, as well as cffects of TPA/ATRAon cell cycle regulation. Our research may provide theoretical evidence for furtherunderstanding of gastric cancer occurrence and dcvelopment.First, MTT method was used to detect the effect of TPA/RA on BGC-823 cellgrowth. The results showed that both TPA and ATRA could effectivcly inhibitBGC-823 cell growth with concentration-dependent, indicating that TPA/ATRA havecooperation with each other during the course of cell growth inhibition in BGC-823cells. Further study showed that most of cells was arrcsted in G0/G1 phage by TPA,and this inhibitory effect was strengthened obviously when ceIls were treated withboth TPA and ATRA, simultaneously Thesc data suggested that inhibitory effects ongastric canccr cells by TPA/ATRA might reIate with cell cycle arrest at G0/Gl phage.In addition, Western Blot results revealed that TPA /ATRA might regulate theexpression of some proteins, which is important in G1/S check point regulation. Theseresults were as fOIIowed: cyclinDl and CDK2 were stably expressed, and notregulated by TPA and ATRA; exprcssions of cyclinE and pRB were down-regulatedby TPA and ATRA; especially expression of cyclinE protein was completely inhibitedwhen treatment of TPA combined with ATRA, while this co-effect was not observedon pRb expression inhibition. TPA could up-regulate P2lwafl and P53 proteinexpressions with time-dependent manner. Treated with both TPA and ATRA, thisup-reguIation was strengthened obviousIy Taken togetheC aII these datademonstrated that TPA/ATRA-induced cell arrest at G0/Gl phage was throughrepression of cyclin-CDK kinase activity, and dephosphorylation of Rb protein.Second, in our previous study, treatment of cells with TPA caused thc cIassicalmorphological characteristics of apoptosis. In the present study, detection by Westernblot showed that the expression level of JNK protcin was stimulated rapidly by TPA,reached its highest peak within 3 hC then decreased in a time-dependent mannen butthe level of JNK was always higher than the control. Northern blot anaIysis showedthat cdun mRNA induced patterns were similar to JNK protein. Transienttransfection and CAT assay analysis showed that AP-l transcriptional activity wasinduccd by TPA in a concentration-dependent manner. Pretreatcd with PKC inhibitor,Wortamanin, the level of JNK induced by TPA was lower than TPA treated alone, thenumber of apoptotic cells also decreased markedly These observations indicated thatnot only TPA induced apoptosis through PKC and JNK pathway, but aIso JNKpathway was necessary fOr the apoptosis induced by TPA.Western blot was used to analyze effect of P38 and Rac-l protein ex... |
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