| Nitric oxide synthase (NOS) were divided into three types, nNOS(neuronal nitric oxide synthase),iNOS(Inducible nitric oxide synthase) and eNOS(endothelial nitric oxide synthase) respectively. nNOS and eNOS were belonged to Calmodulin dependent type, Mainly exist in tumor cells, neurons, endothelial cells, adrenal cortex cells and platelets, and were adjusted by concentration of calcium ions in Eukaryotic cells. iNOS was non Calmodulin dependent type, Widely exist in mammalian cells especiaaly in tumor cells, its activety could be induced by inflammation but not rely on intracellular calcium ion concentration. All NOS were dimmers, the molecular weight of nNOS was 130-160kDa,of iNOS and eNOS were 130-135kDa. NO was Produced by NOS through oxidation of arginine endogenous, it was an important signal molecules in the cell with close ester and short half-life period of 3-5 seconds, NO could go Through the biological membrane. Different concentration of NO induced by different NOS types, and of course had the different functions. General speaking, high concentration of NO was induced by iNOS, while low concentration was induced by nNOS and eNOS. eNOS was first discovered in vascular endothelium, and displayed a high expression in vascular endothelium. Location expression of eNOS was mainly in cytoplasm, but nuclear expression could be detected at certain condition. Although showing high expression in vascular endothelium, eNOS displayed the lowest expression in other tissues, including tumor tissues when comparing with the other two NOS types. In vascular system, NO induced production by eNOS could activate sGC-cGMP signal pathway, relax The vascular smooth muscle cells and regulate blood pressure. nNOS was first discovered in nervous tissue, and Widely distributed in central nervous system and peripheral nervous system, it could Participate in neuromuscular connection signal transduction and so on. iNOS was activated by inflammatory signals Mainly, when was activated, it could Continue to produce high concentration of NO and acted as an important role in killing tumor cells.Expression of NOS were higher in tumor tissues than in normal tissues, besides, high expression of NOS could promote Tumorigenesis, progression, lymph node metastasis, distant metastasis, and microvascular invasion. for example, nNOS appeared low expression in high differentiation renal clear cell carcinoma, while high expression in Poorly differentiated tumor. the higher expression the nNOS was, the The higher degree the malignant tumor was. Besides, high expression of nNOS also had association with Microvascular invasion and metastasis, and of course could Predictor a poor prognosis. in addition, nNOS was also association with tumor immunology, and a study about 133 melanoma samples showed that high expression of nNOS could Suppress the antitmor immune function, result in Cutting effect of melanoma immunotherapy, it mean that high expression of nNOS could show Inhibition in tumor immune damage. More than this, high expression of nNOS might paticipate in migration and transformation of stem cell, and could Maintain function of hematopoietic stem cell.This was one of the value of our topic. A large amount of studies pointed out that iNOS was mainly promote Inflammation-related tumor proliferation, and nNOS/eNOS played important roles in signal transduction between tumors and blood vessel, regulating tumors progress and invasion. eNOS could Mediated generate VEGF and PGE, induce Tumor angiogenesis and tumor growth, besides,eNOS could also Increase the permeability of blood vessels and made it more easier of promoting tumor growth. Impact on tumor growth by eNOS was not limited to Angiogenesis and permeability changes, it could also induce inhibiting Mediated tumor cells apoptosis by TNF and speed up tumor infiltration. Furthermore,high expression of eNOS was also association with Lymph node metastasis, vessel invasion and of course correlation with low overall survival rate. So targeting intervene eNOS expression could inhibit tumor growth. iNOS was induced by Inflammatory signal, iNOS expression could embellish Tumor related functional protein, Promote cell proliferation and tumor growth, and inhibit expression could down regulate tumor growth, including Glioma, breast cancer, colon cancer, lung cancer and bile duct cancer and so on. because of Proinflammatory characteristics, iNOS was considered to have bidirectional in regulating tumor growth, in other word, iNOS might appear function of tumor suppression at certain conditon, for example, iNOS gene transfer could inhibit Melanoma and renal cancer cells growth, and over expression of iNOS could down regulate growth of renal cancer and Mouse fibrosarcoma. In fact, the role of iNOS in tumor growth process was decided by various factors, such as cell microenvironment, redox, state in the cell, NO concentration and time of duration. NOS played such an important role in the development of tumor, so to illustrate expression of three NOS types and their regulation on tumor growth was significant.NO function in Tumor biological was mainly depends on source, concentration, the types of nitric oxide synthetase, organization reactivity and so on. low concentration of NO was combined with sGC, and lead to Increased levels of cGMP in intracellular, then VEGF was induced to expression, result in neovascularization and tumor growth. high concentration of NO induced by iNOS could displayed a function of inhibiting tumor growth by preventing tumor cell division cycle and Induction apoptosis. NO producted by tumor cells could promote tumor growth, but appeared inhibition function when producted by stroma cell. In a word, NO promote tumor growth at most time, but suppress tumor growth at some conditions.during the world wide, Colon cancer showed a rising trend morbidity, and took the third place of all malignant tumors. Surgical resection was a mainly treatment for colon cancer, when combining with chemotherapy, radiation therapy, molecular targeted drugs and China medicine, colon cancer’s 5-year survival rate could become more extend. Although colon cancer’s 5-year survival rate was step up year by year, Mortality rate remains high, taking the third place of all malignant tumors, so to Clarify the relation of NOS and development of colon cancer mechanism is great significance. Colon cancer model showed that Inflammation was closely related to the occurrence of colon cancer. cancer suppressor gene APC and oncogenes Ras mutations could be easy induced by tumor Inflammation, leading to Colon epithelial hyperplasia and P53 gene mutation, which resulted in tumor formation. A research by IHC technology showed that NOS displayed a more hgher expression in colon cancer tissue than in normal tissue. high expression of iNOS acted as an important role in early stage colon cancer, but As the disease progress, its expression decline, whose reason might be as follows:iNOS expression could induce P53 gene mutation and facilitate colon cancer formation, but absent from development, after tumor formation, iNOS could establish relation with T-lymphocytes and inhibit tumor growth by inducing Inflammation. A large number of studies have shown that Physiological doses of NO concentration was induced product by eNOS and nNOS, among that, eNOS showed expression mainly in blood vessel endothelium,with the function of inducing neovascularization,and expression of nNOS was enhance in colon cancer cells, although the Mechanism was still unclear. Cytology in vitro experiments showed that low concentration NO induced by eNOS and nNOS could Promote tumor cells proliferation, migration, and displayed Induction of cell apoptosis resistance function. in recent years,some researchs showed that NO could induce stem cell differentiation and proliferation, so study the role of eNOS and nNOS in colon cancer is of great significance. in order to ananlysis the relation of eNOS/nNOS with staging and prognosis of colon cancer,we Used GEO public resources database and did analysis with 657 colon cancer transcriptome chips, results reminded that mRNA expression quantity of iNOS was drop as Stage increased, and had positive correlation with survival time. whereas, mRNA expression quantity of eNOS/nNOS was increase as Stage increased, and had negative correlation with survival time, which reminded us that eNOS/nNOS might play important roles in colon cancer development. combining with the Preliminary experiments, which indicated that eNOS showed very low expression in tumor cells but high expression in Tumor vascular endothelial, we focused our research on nNOS, to clarified the relationship of nNOS with malignant degree, prognosis and so on, then verified its regulation effect on cell proliferation and migration, Explore its development meaning in colon cancerExperiment Objectives1. Comparing analysis different expression and functions of promoting proliferation and migration of the three subtypes NOSs in colon cancer cell lines, clear and definite the leading position of nNOS in colon cancer growth process.2. Analysis the correlation of nNOS expression in colon cancer samples with clinical indexs, then discuss function of nNOS in promoting colon cancer development.Statistical approachSPSS 16.0 statistical software was used to analysis all the datas, test level at P< 0.05 or P< 0.001 was considered significant difference. first all datas were done F test, and then completely random single factor analysis of One-Way ANOVA, Chi-squared test or t test of homogeneity of variance were used to compare the different expression between these groupsExperiment resultsPart I expression and function analysis of NOSs in colon cancer cell lines1. locational and Quantitative analysis of NOSs expression in four colon cancer cell lines and related function research1) Quantitative analysis of NOSs expression in four colon cancer cell lines: Western blot was used to analysis different expression of nNOS,iNOS and eNOS in four colon cancer cell lines. Results showed that the expression of NOS in RKO/LOVO/SW480/SW620 were as follows:nNOS:0.59/0.69/0.62/0.32; iNOS: 0.20/0.09/0.29/0.23; eNOS0.08/0.03/0.12/0.07;.one-way ANOVA, P<0.01)FCM:fluorescent antibodies were co-culture with colon cancer cell lines and FACS was used to analysis NOS fluorescent mean value,among these cell lines, nNOS showed the highest expression, iNOS showed the second highest expression, and eNOS showed the lowest expression, (nNOS:RKO/LOVO/SW480/SW620: 489.7/239.9/350.7/366.4. iNOS:250.6/44.75/42.6/76.9.eNOS:68.3/13.25/37.9/57.2. one-way ANOVA, P<0.01)Real time PCR:Expression of mRNA leavel by real time PCR showed a discord results with Western blot and FCM in six colon cancer cell lines, in which eNOS appeared a high expression in RKO, SW620, HT29, while iNOS appeared a high expression in LoVo, SW480, M5, nNOS showed a low expression in almost all the six cell lines,. There may exst a difference between transcription level and protein translation, which leads to different expression between NOS protein and mRNA leveal.these datas gave us an impression that nNOS showed the highest expression in the four colon cancer cell lines,and might play an important role in colon cancer growth.2) Locational analysis of NOSs expression in four colon cancer cell lines:then IF technology were used to analysis different location expression of the three kinds NOS subtypes in four colon cancer cell lines,the results were as follows:NOS was mainly expression in Cytoplasm,nucleus expression could be seen in a part cell lines (nNOS:15%, iNOS:12.6%, eNOS:0%).2.function analysis of promoting colon cancer growth by NOSs1) Function analysis of promoting proliferation:the effect of NOS expression on cell proliferation was verified by EDU,CCK-8 and Tablet cloning assays.Similar as above, cells were incubated with NOS inhibitors(L-NAME,N-PLA,1400W) and then experiments were done.Results were showed as follows:EDU assay:L-NAME,N-PLA and 1400W groups showed inhibition effect in colon cancer proliferation when comparing with control group (control:0.67005, 1400W:0.57905, N-PLA:0.1761, L-NAME:0.10925), L-NAME and N-PLA groups had Statistically difference(t-test, p<0.05), among them, L-NAME showed the strongest inhibition and N-PLA showed the second strongest inhibition.CCK-8 assay:L-NAME,N-PLA and 1400W groups showed inhibition effect in colon cancer proliferation when comparing with control group (control:0.77, 1400W:0.73, N-PLA:0.53, L-NAME:0.48), L-NAME and N-PLA groups had Statistically difference((t-test, P=0.0044,0.0092)), among them, L-NAME showed the strongest inhibition and N-PLA showed the second strongest inhibition, and 1400W showed the weskest inhibition(t-test, P=0.1561).Tablet cloning experiment:L-NAME,N-PLA and 1400W groups showed inhibition effect in Monoclonal formation when comparing with control group (control:29.3,1400W:23.9, N-PLA:12, L-NAME:5.7),L-NAME and N-PLA groups had Statistically difference (t-test, P=0.007'0.018), among them, L-NAME showed the strongest inhibition and N-PLA showed the second strongest inhibition, and 1400W showed the weskest inhibition(t-test, P=0.125).Datas above pointed out, colon cancer proliferation could be promoted by NOS, inhibit expression of NOS especially nNOS inhibition could delay colon cancer growth.2) Function analysis of promoting migration:the effect of NOS expression on cell migration was verified by Transwell and Cell scratch healing assay, similar as above, cells were incubated with NOS inhibitors and then experiments were done, results were showed as follows:Transwell assay:L-NAME,N-PLA and 1400W groups showed inhibition effect in colon cancer migration when comparing with control group (Cloning numbers:Control:27.6,1400W:22, N-PLA:7.6,L-NAME:5.3), L-NAME and N-PLA groups had Statistically difference((t-test, P=0.02,0.03)), among them, L-NAME showed the strongest inhibition, N-PLA showed the second strongest inhibition, and 1400W showed the weskest inhibition(t-test, P=0.109).Cell scratch healing assay:L-NAME,N-PLA and 1400W groups showed inhibition effect on colon cancer migration when comparing with control group (Control:0.07,1400W:0.16, N-PLA:0.65,L-NAME:0.73), L-NAME and N-PLA groups had Statistically difference((t-test, P=0.0006,0.002)), among them, L-NAME showed the strongest inhibition, N-PLA showed the second strongest inhibition, and 1400W showed the weskest inhibition.Datas showed that nNOS might also play the most important role in promoting colon cancer migration.3. Research on mechanisms of promoting colon cancer growth by NOSsin order to clarify different functions of the three kinds NOSs, select NOS inhibitors(N-PLA,1400W) and non-select NOS inhibitor (L-NAME)were utilized to intervene NOS expression, then cells were Cracked and protein was extracted, some proliferation-related factors(C-myc, beta-catenin,Cyclin D1, EGFR, P-AKT,C-JUN) were analysis by western blot, results showed that L-NAME and N-PLA could down regulate the leveal of EGFR、p-AKT、wnt Downstream transcription factors beta-catenin, C-MYC and Cyclin D1, but showed limit effect on C-JUN.1400W showed a tiny inhibition on these factors. It gave us an impression that nNOS might be the most important subtype in regulating colon cancer growth. N-PLA and L-NAME could inhibit these proliferation-related factors expression obviously, and 1400W only showed a weak suppression effect.Part Ⅱ Analysis of regulating colon cancer stem cell function by NOSs1.Comparable expression of NOSs in stem cells and non stem cells1) Comparable expression of NOSs in CD133+/CD133-cell subgroups:FACS analysis:RKO, LoVo, SW480, SW620 cell lines were labeled with fluorescent antibody of CD133/nNOS/iNOS/eNOS, then Co-expression cells flu orescent mean value of NOS and CD133 were detected by FACS. Results sh owed that NOS displayed a more higher expression in CD133+ stem cells tha n in CD 133-non stem cells, among them, nNOS/eNOS showed a statistical d ifference while iNOS show a non statistical difference.Western blot analysis:similar analysis were detected by Western blot, CD1 33+ stem cells and CD133-non stem cells were sorted by FACS especially, protein were extracted and Western blot was used to detected the different ex pression of NOS in stem cells and non stem cells. Results showed that nNO S displayed a more higher expression in CD133+ stem cells than in CD133-non stem cells, while iNOS/eNOS displayed a weak and not so obvious expr ession in them.2) Comparable expression of NOSs in ALDH1+/ALDH1- cell subgroups:FACS analysis:RKO, LoVo, SW480, SW620 cell lines were labeled with fluorescent antibody of ALDHl/nNOS/iNOS/eNOS, then Co-expression cells fl uorescent mean value of NOS and ALDH1 were detected by FACS. Results showed that NOS displayed a more higher expression in ALDH1+stem cells than in ALDH1- non stem cells, among them, nNOS showed a statistical dif ference while iNOS/eNOS show a non statistical difference.2. Study of colon cancer stem cells regulation by NOSs1) Regulation colon cancer stem cells propotion by NOSs:RKO colon cancer line was treated with NOS inhibitors 1400W, N-PLA, L-NAME especially and was made into single-cell suspension, labeled with P E-CD133 antibody and APC/CY7 antibody, then was divided into two subgro ups (CD133+ stem cell and CD133-non stem cell subgroup, ALDH1+ stem cell and ALDH1- non stem cell subgroup) by FACS, then proportion were de tected by FACS and comparable analysis were as follows, CD133+ stem cells proportion:Control (3.73%±0.9%),1400W (1.73%±0.2%), N-PLA (0.83 %±0.2%), L-NAME (0.4%±0.1%). ALDH1±stem cells proportion::Co ntrol (5.87%±1.5%),1400W (3.03%±1%), N-PLA (0.97%±0.1%), L-NA ME (0.1%±0.01%). It gave us an impression that when comparing with Co ntrol group, N-PLA and L-NAME could down regulate stem cells proportion with statistical difference while 1400W only show a tiny inhibition effect.2) NOSs regulate stem cell proliferation:sphere formation techonology was used to analysis stem cell proliferation re gulation by NOS.1000 cells were cultivated in 6 well plate and medium wer e added into to form sphere, NOS inhibitors were added into at the same ti me, sphere formation were harvested and calculated with 1/2*(long+wide) 14 days later, results showed that N-PLA and L-NAME could obvious inhibit sp here formation while 1400W show a weak inhibition.3) NOSs regulate stem cell factors expression:stem cell factors Nanog、Sox2、Oct4 paly an important role in stem cell s proliferation and differentiation.in this experiment, Nanog、Sox2、Oct4 wer e detected by Western blot after treating with NOS inhibitors in RKO cell li ne, results showed that Nanog and Sox2 appeared strong expression while Oc t4 appeared weak expression, in addition, N-PLA and L-NAME could suppres s the expression of Sox2 violently while suppress Nanog and Oct4 expression weakly, and 1400W only appeared a tiny suppression on Sox2 expression.3.meaning of NOSs co-expression with CD133+ stem cellpre-experimentshad showed that NOS appeared much more high expressi on in stem cells than in non stem cells. CD133 and NOS were marked by fluorescent antibody at the same time and detected by FACS, results showe d that when conbining with CD133, all the three NOS types could divided cell line into two characteristic subgroups, which were high expression of N OS/CD 133 subgroup and low expression of NOS/CD133 subgroup, we called it CD133+/NOS+ and CD133-/NOS-subgroups, the proportion was about 3:5. Whether the CD133+/NOS+ subgroup have stem cell characteristic remind s a further study.Part Ⅲ Analysis of nNOS expression in colon cancer clinical samples1. NOSs expression in colon cancer tissue samples and analysis of the relationship with clinical elementsthen 27 clinical colon cancer samples were gathered, classified and Immune stained by Immunohistochemistry technology, Immunohistochemical results were evaluated by independent double-blind method by experienced pathologist, Evaluation criteria with refer to the following documents (Li J,Int J Cancer. 2012;131:1863-73), Positive cells rates were evaluated as follows:negative Cells was defined as 0 points,1%～9% positive cells was defined as 1 points,10%～50% positive cells was defined as 2 points,51%～100% positive cells was defined as 3 points. Besides, Non-staining is 0 points, pale yellow is 0 points, yellow is 2 points, claybank is 3 points. Finally, two scored multiplication, then 0-4 points were divided into lower expression,6-9 points were divided into high expression.Results showed as follows:1) NOSs expression in colon cancer samples:27 clinical colon cancer samples were gathered and 10 samples both have carcinoma and para-carcinoma were selective to do IHC, staining with nNOS/iNOS/eNOS antibodys and the expression condition were ananlysis, results appeared a similar expression with cell lines, in which NOS show mainly expression in cytoplasm an partly in nuclear, besides, nNOS showed the highest expression while eNOS show the lowest expression.2) Discriminating expression of nNOS in carcinoma and para-cacinoma:10 clinical colon cancer samples were gathered, classified and Immune stained by Immunohistochemistry technology, Results showed that the expression of nNOS in carcinoma was more higher than in para-carcinoma,and Chi-squared test showed a statistically significant difference (P=0.021),wich displayed a key role of nNOS in regulating colon cancer growth.2. The related elements with nNOS expression in in 27 colon cancer clinical samples1) nNOS was associated with clinical staging and differentiation degree:high expression of nNOS accounted for73.3% and 8% in low and high Differentiation respectively (chi-square test P=0.0014). high expression of nNOS accounted for 75% and 20% in T3-4 stages and Tl-2 stage respectively (chi-square test P= 0.0071). high expression of nNOS accounted for 81.8% and 18.75% in Dukes’stage Ⅲ-Ⅳ and stage Ⅰ-Ⅱ respectively (chi-square test P=0.002). it gave us an impression that high expression of nNOS have positive association with poor differentiation and advanced stage in colon cancer.2) nNOS might involve in Promoting colorectal cancer metastasis: high expression of nNOS accounted for75% and 31.6% in Lymph node metastasis stages N2-3 and stages NO-1 respectively (chi-square test P=0.087). high expression of nNOS accounted for 35% and 71.4% in Distant metastases M0 and M1 stage respectively (chi-square test P=0.185).3) nNOS has not association with sex and age:high expression of nNOS accounted for 35.3% and 60% in Male and female respectively (chi-square test P=0.2566). high expression of nNOS accounted for 41.2% and 40% in< 45 years age and≥45 years age respectively (chi-square test P ＝1);3. The analysis of nNOS expression and prognosis of 567 cases of colon cancer (GSE39582)1) Relation of nNOS expression with colon cancer stage:567 colon cancer samples were divided into four stages and NOS expression was analysis in it. Results showed that the worse the stage was, the high expression of nNOS appeared, on the contrary, iNOS appeared a more low expression as the stage becomes worse. eNOS appeared not association with it.2) Relation of nNOS expression with survival time:567 colon cancer samples were divided into NOS high expression group and NOS low expression group, and being analysis the relation of NOS expression condition with survival time. Results showed that the expression of nNOS/eNOS had negative association with survival time, while iNOS had positive association with survival time.3) Relation of nNOS expression with colon cancer key control factors:gene expression association analysis discovered that nNOS/eNOS could appear suppression on PTEN/APC gene expression as well as promoting Sox2/C-myc factors expression, and therefore promoting colon cancer growth\, while iNOS appeared not association with these gene expression. It gave us a hint that nNOS might promot colon cancer growth by inhibiting the expression of PTEN and APC gene.Conclusion:1. the three kinds of NOSs appeared the different expression in colon cancer cell lines, and could promote colon cancer proliferation and migration, among them, nNOS appeared the highest expression and.displayed the strongest effect2. nNOS appeared high expression in colon cancer stem cell subgroup, and it could increase colon cancer stem cell proportion and enhanced stem cell factors expression; nNOS could co-expression with stem cell markers CD133/ALDHl,and therefore could distinguish the stem cell subgroup, leading to a new therapeutic strategies on colon cancer by targeting this subgroup cells3.analysis by IHC showed that nNOS appeared a higher expression in carcinoma than para-carcinoma. Besides, high expression nNOS had positive correlation with The degree of tumor malignant, and of course, high expression f nNOS could Predict a poor prognosis. In addition, high expression of nNOS might be association with metastasis,but had not association with sex and age.567 chips data showed that high expression of nNOS had negative association with survival. It revealed that nNOS might participate in regulating colon cancer progression.4.:nNOS showed inhibition on PTEN,APC gene expression, regulating the expression of EGFR,P-AKT, CyclinD1 and C-myc, then might participate in regulating Multiple signaling pathways and promote colon cancer proliferation, migration and stem cell function. To verity the Control targets of nNOS might be a new therapeutic strategy of treat Colon cancer in the future. |
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