The Effects Of Angiopoietin-2 On The Growth Of Oral Squamous Carcinoma And Its Related Mechanism

The concept that solid -tumor depends on angiogenesis is well established. Solid tumors recruit blood vessels from neighboring tissue by angiogenesis with the sprouting capillaries from preexisting vessels that migrate into the tumors and form their own endogenous microcirculation. To stimulates angiogenesis, tumors secretes more than one angiogenic factors that synergistically act on epithelial cells(ECs). Vascular endothelial growth factor and its receptor kinase insert domanic containing receptor (VEGF/KDR) are thought to be the most important regulators in this process. Angiopoietins is a novel family which are recently discovered to play critical roles both on physiological and pathological events, yet its role and mechanism in angiogenesis has not been clarified although many critical roles for VEGF in angiogenesis have been reported.Angiopoietins is a family include both agonist and antagonist, up to date, there are four definitive members having been found: Ang-1, Ang-2, Ang-3 and Ang -4.They were all discovered as ligands for the Tie-2, a family of receptor tyrosine kinases that are selectively expressed within the vascular endothelium, but have different effects. Ang-1 has been suggested to make a positive effect on regulation of microvascular permeability and contractility, and maintain vascular integrity by phosphorylation of Tie-2, while Ang-2 loosens capillary structure and sensitizes endothelial cells to angiogenic stimuli by blocking the action of Ang-1 and consequently disrupting the contact between endothelial cells and pericytes, extracellular matrix, which finally provide a key de-stabilizing signal that reverts the vessels to a more plastic and tenuous state.Now Ang-2 has been detected in many kinds of carcinomas, such as: gastric carcinoma, lung carcinoma, hepatic carcinoma, breast carcinoma, thyroid carcinoma, glioblastoma, prostate carcinoma, ovarial carcinoma ect, and closely associated with tumor microvessel density, clinical stage,lymph node matestasis and its prognosis. With respect to its influence on tumorigenicity, there have been studies showed that in Ang-2 transgenic gastric and breast animal model, over expression of Ang-2 stimulated tumor growth and metastasis, the results were supported by many other experiments. However, there were also other studies, such as in Ang-2 transgenic Lewis lung tumor cells and TAS breast tumor cells, showed that over expression of Ang-2 could induce apoptosis of endothelial cells and tumor cells, which inhibited tumor growth and metastasis. These phenomena indicated that Ang-2 may have different biologic effects on different tumor tissue, depending on its micro-environment. The current studies suggest that Ang-2 has both positive and negative effects on vessel formation, that is destabilization by Ang-2 in the absence of VEGF has been proposed to result in vessel regression, whereas such destabilization in the presence of high VEGF levels may facilitate an angiogenic response. Up to date, many problems still remain unclear and will require additional investigation for clarification, such as: the detail biological effect of Ang-2, its possible mechanism, and the interaction between Ang-2 with other angiogenesis regulators.Malignant tumor in oral-maxlloace is generally a highly vascular tumor apt to metastasis in the early stage. Up to date, there have been few studies that establish a causal role for Angs. In our study, to define a putative role for Ang-2 in oral cancer angiogenesis, we investigate the expression pattern of Ang-2 and its clinical significance. On the basis of the clinical results, we stably transgened Ang-2 into tongue Tca8113 tumor cells and then analyzed its tumorigenicity in nude mice. We focused especially on the VEGF, matrix metalloproteinase (MMPs) and nitric oxide synthase (NOS) with regard to the possible mechanism induced by Ang-2 in tumor angiogenesis.Part I The expression of angiopoietin-2 in oral squamous carcinoma and its clinical significanceObjectiveIn order to investigate the expression pattern of Ang-2 in oral cancinoma and its clinical significance, we detected Ang-2 expression in oral squmaous carcinoma tissue and paired adjacent uninvolved tissue, and analysed its relationship with lymph nodal metastases and clinical stage. Material and method1. MaterialSamples were obtained from patients with oral squmaous carcinoma in the first affiliated hospital of zhejiang university from May 2003 to May 2006, all samples included carcinoma tissue and paired adjacent uninvolved tissue.2. Method(1) Real-time RT-PCR using for detection the expression of Ang-2 mRNA, VEGF mRNA(2) Imunohistochemistry using for detection the expression of Ang-2, VEGF protein. Results1.The expression of Ang-2 mRNA, VEGF mRNA in oral carcinoma tissues To assess a critical role of Ang-2, VEGF as modulators of tumor angiogenesis in oral carcinoma, the mRNA expression of Ang-2, VEGF were examined by qRT-PCR assay. The result showed that Ang-2 mRNA, VEGF mRNA level in the oral carcinoma tissue were 6.86 ± 1. 37 and 9. 36 ± 2. 75, which were significantly higher than those in paired adjacent uninvolved tissue(7.95 ±2.08 and 11.03±2.67)(P<0.05)2.The expression of Ang-2, VEGF protein in oral carcinoma tissues The distribution and cellular location of Ang-2, VEGF protein were examined by immunohistochemistry staining. The results showed these 2 proteins expressed not only in endothelia cells but also in rumor cells. semiquantitative analysis revealed higher positive expression of Ang-2, VEGF protein in neoplasm specimen (53.57% and 60.71%) than those in paired adjacent uninvolved tissue(24.00% and 28.00%) (P<0.05) , the results were completely paralleled the data from qRT-PCR.3. The biological effect of Ang-2 on clinical pathological features of oral carcinoma. In lymph nodal positive metastases and negative metastases patients, the expression of Ang-2mRNA were 6.60±1. 21 and 7.07 ± 1.50 (P>0.05) . With respect to tumor clinical stage, Ang-2 mRNA also showed no variation between I , II stage (7. 17±1. 58 ) and III, IV stage (6. 59±1. 14) (P>0.05) . Conclusion1. The expression of Ang-2 mRNA and VEGF mRNA in tumor tissue were significantly higher compared with rumor uninvolved tissue, which suggested that Ang-2,VEGF take part in the process of angiogenesis in the oral carcinoma.2. Ang-2, VEGF protein was produced not only by ECs, but also by tumor cells, the expression level was higher in neolpasm tissue than in paired adjacent uninvolved tissue, which further demonstrated its role in tumor angiogenesis.3. our study of the relationship between the expression of Ang-2 mRNA and the clinicopathological features disclosed that high expression of Ang-2 mRNA had no correlation with lymph nodal metastases and clinical stage. These findings need further to be confirmed in a large series of oral cancer patients.Part II The effects of angiopoietin-2 on the growthof tongue transplanted carcinomaObjectiveIn order to investigate the impact of Ang-2 on oral tumor formation and vascular angiogenesis, we developed Ang-2-transgenic Tca8113 cell line and Ang-2-transgenic animal model. Material and method1. Material(1) nude mice: 27 female healthy BALB/C nude mice divided into 3 group (2) .cells: human tongue squamous carcinoma Tca8113 cell lines. (3) Ang-2 cloning plasmid: obtained from Invivogen Company.2. Method(1) Constructing pcDNA3.1(-)B/Ang-2 recombined expression plasmid by subcloning Ang-2 into a pcDNA3.1(-)B vector.(2) Trancfecting pcDNA3.1(-)B/Ang-2 into Tca8113 cell line by lipofection method, and detected Ang-2 mRNA expression by RT-PCR and qRT-PCR.(3) Developing Ang-2-transgenic nude mice.3 × 10~6 cells of Tca8113/Ang-2 cell, Tca8113/pcDNA3.1 (-)B or Tca8113 cells were injected s.c. in back of nude mice. The time of tumor forming and tumor growth were carefully measured.(4) Detecting the expression of Ang-2 protein by Immunohistochemical staining in tumor tissue.Result1. successfully constructing of pcDNA3.1(-)B/Ang-2purified Ang-2cDNA were subcloned into pcDNA3.1(-)B vector, which were demonstrated by RT-PCR and sequence detection.2. successfully constructing of Ang-2 stably trancgenic Tca8113 cell line pcDNA3.1(-)B/Ang-2 and pcDNA3.1(-)B were transfected into Tca8113 cells by lipofection method. After transfected, over-expression of Ang-2 mRNA were confirmed by RT-PCR and qRT-PCR, Tca8113/Ang-2 cells had 7690 times greater exogenous Ang-2 production than endogenous Ang-2 production..3. Tca8113/Ang-2 transplanted tumor were successfully established in BALB/C nude miceimmunohistochemistry staining demonstrated amount of Ang-2 protein expressed in the Tca8113/Ang-2 transplanted tumor tissue, while only slightly in Tca8113 and Tca8113/pcDNA3.1(-)B transplanted ones.4. The effect of Ang-2 on the time of Tca8113 transplanted tumor formingThe time of tumor forming in Tca8113/Ang-2 group was longer than those in Tca8113/pcDNA3.1(-)B and Tca8113 groups (P<0.05)5. The effect of Ang-2 on Tca8113 transplanted tumor growthTumors in the mice treated with the Tca8113/Ang-2 cells grew slower than those in the Tca8113/pcDNA3.1(-)B group and Tca8113 group (P<0.05). The weight of transplantationtumor on the treatment day 35 from Tca8113/Ang-2. Tca8113/pcDNA3.1(-)B ,Tca8113 were 0.531 ± 0. 398g. 1.427±0.903g and 1.552 ±0. 663g, which indicated Ang-2 inhibited tumor growth.. Conclusion1. we successfully constructed pcDNA3.1(-)B/Ang-2, Ang-2 transgenicTca8113 cell line, and further Ang-2 transgenic Tca8113 transplanted tumor in BALB/C nude mice, which established a causal role for later Ang-2 study in oral cancer.2. Transgenic Ang-2 delayed the time of Tca8113 tumor forming in BALB/C nude mice.3. Transgenic Ang-2 inhibited Tca8113 tumor growth in BALB/C nude mice.Part III Mechanism of angiopoietin-2 inhibiting thegrowth of tongue transplanted carcinomaObjectiveTo further study the mechanism about overexpression of Ang-2 inhibiting the growth of Tca8113 carcinoma, we investigate the effect of Ang-2 on the proliferation and apoptosis of the tumor cells; the effect of Ang-2 on tumor angiogenesis; and its impact on the expression of VEGF, matrix metalloproteinase (MMP-2. MMP-9) and nitric oxide synthase (iNOS. eNOS) during tumor angiogenesis. Material and method1. MaterialThe transplanted tumors arising from Tca8113/Ang-2 cells. Tca8113/pcDNA3.1(-)B, Tca8113, (see Part II)2. Method(1) Real-time RT-PCR: detecting the expression of VEGF, MMP-2, MMP-9, iNOS, eNOS mRNA.(2) Immunohistochemistry: staining for PCNA. α -SMA and FⅧ.(3) TUNEL: detecting tumor apoptosisResult 1.Effect of Ang-2 transgene on tumor vessel's stability and it's microvessel countsThe microvessel density in tumors derived from Tca8113/Ang-2 , Tca8113/pcDNA3.1(-)B and Tca8113 were 5.88 ± 1.46, 5.33±2.00 and 5.86± 2.41 respectively, which showed no significant difference among three groups (P>0.05 ) .But the result of immunohistochemistry for α -SMA showed less expression of α-SMA in Ang-2-transgenic group,. which indicated Ang-2 reverts the vessels into a more tenuous and.de-stabilizing state.2. Effect of Ang-2 transgene on PCNA expression and tumor cell apoptosisImmunohistochemistry staining for PCNA revealed that LI of PCNA-positive cells was less in the tumors from Tca8113/Ang-2 group(34.63 ± 9.40%) than in Tca8113/pcDNA3.1(-)B and Tca8113 groups(57.61 ±12. 26% and 54.71± 12. 82%) (P<0.05). In consistence with this, more TUNEL-positive tumor cells were present in Tca8113/Ang-2 group(19.00±5.48%) than in the tumors from the other two groups(10.22±3. 42% and 10.71±3.30%)(P<0.05).,3. Effect of Ang-2 transgene on VEGF mRNA expressionThe results from the qRT-PCR assay showed VEGF mRNA level in tumors arising from Tca8113/Ang-2, Tca8113/pcDNA3.1(-)B and Tca8113 were4.37±2. 73. 4. 63+2. 96 and 5. 09±3. 94, which indicated no significant difference among three groups (P>0.05 ) .4. Effect of Ang-2 transgene on MMP-2, MMP-9 mRNA expressionQRT-PCR assay showed the expression of MMP-2 mRNA in Tca8113/Ang-2, Tca8113/pcDNA3.1 (-)B , Tca8113 were 6.84±3. 01, 6. 76±4. 17 and 8. 27±5. 24: the expression of MMP-9 mRNA in Tca8113/Ang-2, Tca8113/pcDNA3.1(-)B , Tca8113 were 8. 57±5.13. 7. 76±4. 20 and 9. 23±6. 10, neither of them showed significantly difference among three group (P>0.05)5. Effect of Ang-2 transgene on iNOS, eNOS mRNA expressionThe expression of iNOS mRNA in Tca8113/Ang-2, Tca8113/pcDNA3.1(-)B and Tca8113 tumors were 8.68±5. 89, 10.13,4. 85 and 9. 78 ± 5. 76; and eNOS mRNA expression in Tca8113/Ang-2,Tca8113/pcDNA3.1(-)B and Tca8113 tumors were 10.55 ± 1.77, 12. 18 ± 2.43 and 10. 78 ± 3. 51, respectively. Compared to Ang-2 non-transgenic tumor,Ang-2-transgenic tumor present no significant variation in iNOS and eNOS mRNA expression(P>0.05) Conclusion:1. Transgenic Ang-2 resulted in aberrant tumor angiogenesis in Tca8113 transplanted tumor, but not their MVD.2. Transgenic Ang-2 increased apoptosis of tumor cells and decreased their proleferation in Tca8113 transplanted tumor.3. Transgenic Ang-2 had no significant effect on the expression of VEGF mRNA in Tca8113 transplanted tumor.4. Transgenic Ang-2 had no significant effect on the expression of MMP-2, MMP-9, iNOS and eNOS mRNA in Tca8113 transplanted tumor.