Role Of MAPK Signaling Pathway In Gastrin-regulated Growth Of Large Intestinal Cancer And Molecular Mechanisms Study

Gastrin (Gastrin, GAS) was found by John as early as1905in the extracts of gastricantral mucosa and its associated with gastric acid secretion, Until1964it was isolated andidentified by Gregory and Tracy and explains its chemical structure, confirmed asgastrointestinal hormones. Gastrin is a kind of important gastrointestinal hormone and akind of gastrointestinal peptide, mainly secreted by gastrointestinal G cell. The G cell is atypical open type cell, up to the antrum. As a kind of nutrition gastrointestinal hormone,not only can stimulate the growth of gastrointestinal tract normal mucosa tissue, but canstimulate the gallbladder carcinoma, pancreatic carcinoma, colorectal carcinoma, gastriccarcinoma and other tumor cell growth. At present, most studies showes that gastrin is anautocrine growth factor through the way of autocrine, paracrine, endocrine to achieve itsfunction. Combined with gastrin and its receptor can regulate tumor cells by intracellularsignal transduction pathway of growth, which is combined with the tumor cells producegastrin and its receptor, exert their biological effects. Recently, studies have demonstratedthat the incidence of colorectal cancer related to the abnormal expression of gastrin, somescholars refer to this type of colorectal cancer as hormone-dependent tumors, whichpromotes colon cancer cell proliferation may be through the mechanism by autonomouslygenerating and secreting gastrin and acts on its own receptor on the cell membrane, whichplay a biological effect, but its effects can be inhibited by gastrin receptor antagonist.However, the regulation of molecular mechanism of proliferation in colon cancer remainsunclear.In recent years, the application of gene chip technology to detect colorectal cancerrelated gene, expressed genes were screened a lot of difference, significant differences ingene expression in many internal and external network, and cell signal transduction pathway is closely related with the inhibition of regulation, directly involved in cellmetabolism, proliferation, differentiation and apoptosis. Protein kinase associated mitogenactivated (mitogen-activated protein kinase, MAPK) signal research attention, MAPK isa intracellular serine/threonine protein kinase, widely present in most mammaliancytoplasm and nucleus, can be the extracellular stimulation signal transduction to the celland its nucleus, and causes cell biological reaction, and regulate of cell proliferation,differentiation, development and apoptosis.MAPK consists of extracellular signal regulatedkinase (ERK), c-Jun N-terminal kinase (JNK)/stress activated protein kinase (SAPK),p38and ERK5/BMK1subgroup MAPK. Multiple signal pathways is made up of thesesubgroups. At present, studies have found that the mammalian cells has three parallelmitogen activated protein kinase pathway. Respectively for the extracellular signalregulated kinase (extracellular-signal regulated protein kinase, ERK) signal pathway, p38MAPK signal pathway, and JNK/SAPK signal pathway. Among them, JNK/SAPK andp38MAPK pathway is related to cellular stress and apoptosis, and ERK pathway is the hubin the MAPK cell signal transduction network is closely related to cell proliferation anddifferentiation. Many studies suggest that MAPK signaling pathway is involved in theregulation of growth of colon cancer cells. Research also found that after combined withgastrin gastrin receptors, through the activation of p38MAPK signaling pathway inducedmRNA and protein expression of urokinase type plasminogen activator (u-PA) expressionenhanced tumor cell invasion, metastasis. Visibly, the MAPK signal transduction pathwayplays an important role in the gastrin-regulated proliferation of colon cancer cells, but themechanism is still unclear.Therefore we assume that firstly using the ELISA method in79cases of colorectalcancer operation resection specimens for detection of expression of gastrin, screening outthe positive and negative specimens of colorectal carcinoma, and than analyze the positiveexpression rate, pathological type, histological differentiation and clinical staging of Duke’s, as to clear the effect of gastrin on colorectal cancer biological characteristics. Secondly,choosing five groups of positive colorectal cancer tissues and adjacent normal mucosa tissue, and another five groups include the negative expression of colorectal carcinoma andthe adjacent normal mucosa, the differential gene expression profiles were analyzed usinggene chip technology, the positive and negative expression groups analysis results werecompared, selected the gene which expression was obvious difference, and selected thesegenes to verify by real-timme PCR. At the same time, all2times more differentiallyexpressed genes input R package, identify differential genes associated with the MAPKsignal transduction pathway. Finally, through the cultivation of human colorectal cancercell line HT-29, usng western blot, RT-PCR technology to detect the differences in geneand protein expression selected in each group respectively which related to signaltransduction pathway of MAPK, and analysed the expression level of gastrin before andafter the impact of its gastrin receptor antagonist proglumide, to explore the molecularmechanisms of gastrin-regulated MAPK signal transduction pathway in large intestinalcancer from the molecular signaling level. To clear the detailed of molecular basis ofgastrin-regulated growth of colorectal cancer from“GAS-MAPK-target genes”signalingpathway, which provide new clues for the effective intervention of the signal transductionpathway links, and for targeted gene therapy provides a new target, and provide atheoretical basis for endocrine therapy of colorectal cancer.Research was divided into three parts:PartⅠ: Detection of gastrin expression and analysis of gastrin andbiological behavior of large intestinal cancerObjective: To explore the rate of positive expression of gastrin in large intestinalcancer, and effects of gastrin on biological characteristics of large intestinal cancer. Suchas pathological type，histological differentiation, clinical staging of Duke ’s and so on.Methods: The protein expression of gastrin was detected on79cases of colorectalcancer operation resection specimens by ELISA and RT-PCR methods. Screening out thepositive and negative of gastrin expression in colorectal cancer tissue specimens. Toanalysis the positive expression rate of gastrin, and effects of gastrin on the extent ofpathological type, tissue differentiation and Duke’s staging. Results: The positive expression rate of GAS mRNA was46.8%in79cases ofcolorectal cancer tissues, and the positive expression rate of GAS protein was40.5%. ThemRNA expression of gastrin is positive correlated with the protein (P<0.01). The mRNAand protein expression of gastrin in poorly differentiated cancers of large intestinal wassignificantly higher than the high and moderate differentiation (P<0.05). The mRNA andprotein expression of gastrin in papillary adenocarcinoma and tubular adenocarcinomawere significantly lower than that in mucinous signet ring cell carcinoma andundifferentiated carcinoma (P<0.05). The mRNA and protein expression of gastrin inDukes A,B were lower than that in Dukes C,D (P<0.05).Conclusion: There is the expression of gastrin in some of large intestinal carcinoma.Abnormal expression of gastrin is closely related to the extent of tissue differentiation,pathological type and clinical staging of Duke ’s. Detection of gastrin can be rection somebiological characteristics of colorectal cancer. Gastrin may be to evaluate target one of thebiological behavior of clinical large intestinal cancer.PartⅡ:Differential geneScreening of MAPK signal transductionpathway between the positive and negative expression of gastrin in largeintestinal cancerObjective: To differential gene expression analysis of MAPK signal transductionpathway between the positive and negative expression of gastrin in large intestinal cancer.Methods: The gene chip technology was used for analyzing the differential geneexpression profiling in the positive expression of gastrin of cancer tissues and normaltissues adjacent to cancer and the gastrin negative expression of cancer tissues and normaltissues adjacent to cancer. The positive and negative sets of results were compared,screening of differentially expressed genes,and the selection of differentially expressedgenes were verified by real-time PCR. At the same time, all2times more differentiallyexpressed genes input R package, identify differential genes associated with the MAPKsignal transduction pathway. Results: Genechip research result showed differential geneincreased more than2times of3529,2times of the down regulated genes were3328 between positive expression of gastrin of cancer tissues and adjacent normal mucosa.Expression of gastrin-negative cancer tissue and adjacent normal mucosa tissue microarrayanalysis showed differential gene increased more than2times with2867,2times of thedown regulated genes were2549. The difference between the two groups of genes withfluorescence cancer marker, and carries on the analysis, analysis of differential gene ofgastrin related results showed differential gene increased more than2times with567,2times of the down regulated genes were342. And screening out related genes of MAPKsignal transduction pathway were78. Respectively, to select up-regulated and downregulated genes were3through gene verification. The analysis results of PCR and the chipwas consistent.Conclusion: The cDNA microarray method can be used for screening related genesassociated with gastrin in colorectal cancer, the difference of gene differential gene has astrong representation. There are differential genes expression between the positive andnegative expression of gastrin in colorectal carcinoma. MAPK signal transduction pathwayis involved in the gastrin-regulated growth of large intestinal cancer cells.Part Ⅲ: Role of MAPK signaling pathway in gastrin-regulated cellproliferation and apoptosis of large intestinal cancer and mechanismsstudyObjective: To explore the relationship between the cells proliferation and apoptosis oflarge intestinal cancer induced by gastrin and signal transduction pathway of MAPK.Methods: HT-29cells were incubated in the different medium according on differentrequirement.MTT assay investigated the growth change of HT-29cells and determined theoptimal concertration of pentagastrin(PG) and proglumide(PGL) to intervene the cellgrowth. Cells proliferation and apoptosis of HT-29were analyzed with Annexin V-FITCflow cytometry at the optimal concertration of drugs. RT-PCR detected the expression ofgastrin receptor CCK-BR mRNA in HT-29cells and the mRNA expression of ERK1/2,K-ras, p38, bax and bcl-2in each group cells. The protein expression of ERK1/2, K-ras,p38, bax and bcl-2, and ERK1/2, K-ras and p38phosphorylation levels were detected by Western blot.Results: Gastrin could promote the proliferation of HT-29cells and inhibited cellsapoptosis in a dose dependent manner（6.25-100.00mg/L）,and the optimal concentrationwas25mg/L (P<0.05). Proglumide had no obvious effect on the HT-29cells, but it couldremarkablely inhibit the cells proliferation of gastrin stimulating large intestinal cancer in adose dependent manner(8.00～128.00mg/L), and the optimal dose is32mg/L (P<0.05).The proliferation index(PI) of gastrin group was significantly higher than that of controlgroup and combined treatment group(P<0.01). The apoptotic ratio(AR) of PG group wassignificantly lower than that of control group and combined treatment group(P<0.05).RT-PCR test results showed that there is a clear expression of mRNA about CCK-BR inthe HT-29cells. The mRNA and protein expression of bax and the levels of phosphorylatedp38protein among gastrin group was significantly lower than the control group and thegastrin+proglumid group(P<0.05). On the contrary, the mRNA and protein expression ofbcl-2and the levels of phosphorylated ERK1/2and K-ras protein among gastrin group wassignificantly higher than the control group and the gastrin+proglumidgroup(P<0.05).Compared with gastrin group,the mRNA and protein expression ofERK1/2、K-ras and p38in the other groups have not obviously changed.Conclusion: Gastrin could promote the human large intestinal cancer cell line HT-29proliferation and inhibit their apoptosis,but which can be restrained by gastrin receptorantagonist proglumide. Gastrin perhaps regulates the cells proliferation and apoptosis oflarge intestinal cancer by signal transduction pathway of ERK-MAPK inRas→Raf→MEK1/2→ERK1/2ways up-regulation ERK1/2and K-ras proteinphosphorylation levels and signal transduction pathway of p38-MAPK inp38MAPK→bcl-2/bax ways down-regulation p38protein phosphorylation levels.