The production of the endostatin precursor collagen XVIII in head and neck carcinomas is modulated by CBP2/Hsp47

Tumor progression is dependent in large part on angiogenesis and angiogenesis inhibitors have repeatedly been shown to inhibit tumor growth. The present study sought to determine whether the oral squamous carcinoma cells expressed and produced collagen XVIII, a known precursor of endostatin. Four established cell lines of oral squamous cell carcinoma (SCC) were employed for these studies. Quantitative Real-Time RT-PCR was used to assess the expression of collagen XVIII and CBP2/Hsp47, an ostensible chaperone for fibrillar and basement membrane collagens. Real-Time PCR assessment of collagen XVIII with primers selected to the common region of collagen XVIII revealed variable expression among cell lines of oral SCC. Conversely, the long form of collagen XVIII revealed no products. Comparatively, the lowest level of expression of CBP2/Hsp47 was observed in SCC4 that also had the lowest level of collagen XVIII. However, there was no direct relationship between the levels of CBP2/Hsp47 and collagen XVIII expression across the four cell lines. Treatment of SCC cells with CBP2/Hsp47 antisense phosphorothioate oligonucleotides modulated the production of collagen XVIII but not its expression. These findings imply that CBP2/Hsp47 may play a role in tumor progression by mediating the endogenous processing of collagen XVIII in tumor cells.     

Increased expression of collagen XVIII and its prognostic value in nonsmall cell lung carcinoma

BACKGROUND: Angiogenesis plays a crucial role in tumor growth and metastasis. Recently, some studies have focused on the angiogenesis inhibitor endostatin. However, the biologic role of the precursor of endostatin, collagen XVIII, in human malignancy is unknown. The purpose of the current study was to evaluate whether the expression of collagen XVIII has additional prognostic value for survival in patients with nonsmall cell lung carcinoma (NSCLC). METHODS: The authors investigated the expression of collagen XVIII in 221 patients using immunohistochemical methods. To confirm the specificity of the collagen XVIII polyclonal antibody used in the current study and to test the expression of collagen XVIII in human lung carcinoma, Western blot analysis was performed on a panel of human lung carcinoma cell lines. RESULTS: Collagen XVIII expression was detected in 162 of 221 patients with NSCLC (73%), primarily in the tumor cell cytoplasm. Low collagen XVIII expression levels were found in 75 tumor specimens, while high collagen XVIII expression levels were noted in 87 tumor specimens. The prevalence of positive collagen XVIII expression was greater in T2-4 tumors than in T1 tumors (P = 0.0235). The prognosis for patients with strongly collagen XVIII-positive NSCLC was significantly worse than the prognosis for patients with collagen XVIII-positive or collagen XVIII-negative NSCLC (P = 0.0010). Multivariate analysis indicated that T status, lymph node status, and the overexpression of collagen XVIII were independent prognostic factors. CONCLUSIONS: The results of the current study indicated that the overexpression of collagen XVIII was associated with NSCLC progression and poor outcome. Thus, collagen XVIII expression may serve as a useful prognostic marker in patients with NSCLC.     

促红细胞生成素受体在食管癌组织中的表达及其与微血管密度的相关性

背景与目的:新近多项研究发现,促红细胞生成素受体(erythropoietinreceptor,EPO-R)在多种实体瘤中表达,EPO/EPO-R环路与肿瘤细胞的增殖、新生血管形成、侵袭特性相关。本研究旨在探讨食管癌组织中EPO-R的表达,及其与新生血管形成的关系。方法:采集食管癌标本110例,用免疫组化法检测癌组织中EPO-R蛋白的表达强度;并以Ⅷ因子相关抗原多克隆抗体(FⅧ-R Ag)标记血管内皮细胞,计算微血管密度(microvessel density,MVD),以此量化新生血管形成的程度。结果:110例食管癌组织中均表达EPO-R,EPO-R表达于食管癌细胞的胞浆和(或)胞膜。肿瘤分化级别高组与淋巴结转移组中EPO-R表达强度高;有淋巴结转移组MVD高于无淋巴结转移组(P<0.001),晚期患者(Ⅲ/ⅣA/ⅣB)MVD高于早期患者(Ⅰ/ⅡA/ⅡB)(P=0.022),有吸烟史组MVD高于无吸烟史组(P=0.029)。食管癌中EPO-R表达强度与MVD之间存在显著正相关(r=0.618,P<0.01)。结论:食管癌组织中普遍存在EPO-R的表达,EPO-R水平与食管癌血管生成程度和病情进展呈高度正相关。     

Expression of vascular endothelial growth factor in patients with testicular germ cell tumors as an indicator of metastatic disease

BACKGROUND: Angiogenesis is essential for tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP)/platelet-derived endothelial cell growth factor (PD-ECGF) are involved in increased angiogenic activity and disease progression in solid tumors. However, there is no information regarding the association of these angiogenic factors with clinicopathologic findings in testicular germ cell tumors (GCTs). METHODS: The authors examined the expression of VEGF and TP as well as microvessel density in GCTs and their association with clinicopathologic findings. Expression of VEGF and TP and microvessel density were examined immunohistochemically in 80 GCTs, including 33 seminomas (25 tumors with organ-confined disease and 8 with metastasis) and 47 nonseminomatous testicular GCTs (NSGCTs) (20 tumors with organ-confined disease and 27 with metastasis). Expression of VEGF also was examined in four GCTs and one nonneoplastic testis by immunoblotting. RESULTS: VEGF protein was expressed more highly in GCTs compared with nonneoplastic testes. VEGF expression in GCTs was correlated significantly with microvessel count (P < 0.001). Both VEGF expression and microvessel count were correlated with metastasis in seminoma (P = 0.008 and P < 0.001, respectively), but only VEGF expression was identified as statistically significant by multiple regression analysis (P = 0.006). Conversely, four variables (VEGF expression, microvessel count, the presence of venous invasion, and the presence of embryonal carcinoma elements in the primary tumor) were correlated with metastasis in NSGCT (P < 0.001, P < 0.001, P = 0.004, and P = 0.029, respectively). However, multiple regression analysis revealed that only VEGF expression and microvessel count were significant factors for metastasis (P < 0.007 and P < 0.001, respectively). In contrast, high levels of TP were observed in infiltrating cells, but not in the majority of cancer cells. CONCLUSIONS: The findings of the current study suggest that VEGF expression is involved in tumor development, angiogenesis, and metastasis in GCT.     

Expression pattern and circulating levels of endostatin in patients with pancreas cancer

Endostatin is a potent inhibitor of angiogenesis that is cleaved from the basement membrane protein type XVIII collagen. Expression of endostatin has recently been shown by Western blot analysis of tissue lysates in normal pancreas and pancreas cancer tissue. We show here that the expression pattern of type XVIII collagen/endostatin is shifted from a general basement membrane staining and is mainly located in the vasculature during tumor progression. This shift in type XVIII collagen/endostatin expression pattern coincides with an up-regulation of MMPs involved in endostatin processing in the tumor microenvironment, such as MMP-3, MMP-9 and MMP-13. The circulating levels of endostatin was analyzed in patients with pancreas cancer and compared to that of healthy controls, as well as after surgical treatment or in a group of nonoperable patients after intraperitoneal fluorouracil (5-FU) chemotherapy. The results show that patients with pancreas cancer have increased circulating levels of endostatin and that these levels are normalized after surgery or intraperitoneal chemotherapy. These findings indicate that endostatin could be used as a biomarker for pancreas cancer progression.     

Levels of soluble vascular endothelial growth factor (VEGF) receptor 1 in astrocytic tumors and its relation to malignancy, vascularity, and VEGF-A.

PURPOSE: Vascular endothelial growth factor (VEGF)-A isa key mediator of angiogenesis in malignant gliomas. Soluble VEGF receptor 1 (sVEGFR-1) can complex VEGF-A and reduce its bioavailability. In several animal models sVEGFR-1 inhibited angiogenesis and tumor growth. We analyzed the levels of endogenous sVEGFR-1 in gliomas of different malignancy grades in relation to tumor vascularity and VEGF-A. EXPERIMENTAL DESIGN: The concentration of sVEGFR-1 was determined by ELISA in 104 gliomas and normal brain. Levels of sVEGFR-1 were compared with malignancy grade, microvessel density, and VEGF-A concentration. Effects of sVEGFR-1 on glioma extract-induced endothelial cell chemotaxis were analyzed in vitro. RESULTS: The concentration of sVEGFR-1 correlated with the malignancy grade and was 12-fold higher in glioblastomas than in diffuse astrocytomas (P < 0.001), with intermediate levels for anaplastic astrocytomas. VEGF-A levels were 30-fold higher (P < 0.001) in glioblastomas than in diffuse astrocytomas. The sVEGFR-1:VEGF-A ratio was 0.27 in glioblastomas and 0.70 in diffuse astrocytomas. Both sVEGFR-1 and VEGF-A correlated with microvessel density (P < 0.001) and with each other (P < 0.001); sVEGFR-1 and VEGF-A also correlated with each other when only glioblastomas were analyzed (P = 0.001). In vitro, recombinant sVEGFR-1 inhibited endothelial cell chemotaxis induced by tumor extracts. CONCLUSIONS: Although absolute levels of sVEGFR-1 are increased in the more malignant gliomas, the sVEGFR-1:VEGF-A ratio is decreased 2.6-fold in glioblastomas compared with diffuse astrocytomas, suggesting that the ensuing increased bioavailability of VEGF-A favors angiogenesis. The inhibition of tumor extract-induced endothelial chemotaxis by sVEGFR-1 suggests that sVEGFR-1 could be useful as an angiogenesis inhibitor in the specific context of human gliomas.     

Antiangiogenic treatment with endostatin inhibits progression of AML in vivo.

Increased vessel density in the bone marrow of patients with acute myeloid leukemia as well as elevated expression of proangiogenic factors by leukemic cells implies a central role of angiogenesis in hematological malignancies. Endostatin (ES), a fragment of collagen XVIII, is an endogenous inhibitor of angiogenesis that has shown therapeutic activity in solid tumors in various preclinical models. Using microencapsulation technology, we studied the therapeutic effect of ES in AML. While ES had no effect on proliferation of M1 murine leukemic cells in vitro, ES producing microbeads significantly inhibited growth of subcutaneous chloromas in SCID mice as compared to controls. In a leukemia model using M1 cells the concomitant treatment of mice with ES microbeads prolonged median survival significantly. Histological analysis revealed a decreased microvessel density and a reduced number of CD31-positive single cells, putatively endothelial progenitor cells, in the bone marrow of treated animals. Taken together, ES has inhibitory effects on neo-angiogenesis in the bone marrow and on progression of leukemia in vivo. These experiments suggest a possible therapeutic role of antiangiogenic gene therapy with ES in AML.     

Prognostic relevance of collagen XVIII expression in metastatic gastric carcinoma

Collagen XVIII is a component of vascular and epithelial basement membranes. The C-terminal fragment of the protein is termed endostatin, and is a potent inhibitor of angiogenesis. No reports on the clinical implications of collagen XVIII expression in human gastric cancer are currently available. Here, we investigate the clinical significance of collagen XVIII expression in gastric cancer. Seven gastric cancer cell lines were subjected to Western blotting. Collagen XVIII expression was examined in 118 gastric carcinoma tissues via immunohistochemistry. Western blotting revealed the presence of the 22-kDa collagen XVIII protein in four of seven gastric cancer cell lines. Immunohistochemistry detected collagen XVIII expression in the tumor cytoplasm in 115 of 118 gastric carcinoma patients (97%). No correlation was evident between collagen XVIII expression score and clinicopathologic findings when all patients were considered together. However, on subgroup analysis, 42 of 70 patients with distant metastasis were classified into low or moderate collagen XVIII expression groups, whereas the remaining 28 patients were grouped as showing high collagen XVIII expression. The prognosis for patients with high collagen XVIII-expressing gastric carcinoma was significantly worse than that for patients displaying low or moderate collagen XVIII expression (median survival time, 7.8 months vs. 18.3 months [log-rank, p = 0.01]; median time to progression, 3 months vs. 8 months [log-rank, p = 0.01]). High expression of collagen XVIII is associated with poor prognosis in patients with metastatic gastric carcinoma. Further studies on larger patient populations are warranted to validate the utility of collagen XVIII as a prognostic biomarker in gastric carcinoma.     

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in angiogenesis and clinical outcome of human gastric cancer

BACKGROUND AND OBJECTIVES: It has been recognized that inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) produce important endogenous factors of human tumors such as nitric oxide (NO) and prostaglandins, which is involved in the process of carcinogenesis and tumor progression. This study aimed to evaluate the association of clinicopathologic factors, microvessel density, and patient survival with the expression of iNOS and COX-2 in patients with gastric adenocarcinoma. MATERIALS AND METHODS: Seventy-nine specimens, resected from patients with gastric adenocarcinoma, were investigated by immunohistochemical stain against iNOS and COX-2. Microvessels were stained using anti-CD34 antibody and counted as microvessel density. RESULTS: Positive iNOS and COX-2 expressions were significantly correlated with microvessel density by multivariate analysis, respectively (P = 0.0127 vs. P = 0.0214). There was significant difference among the four groups (both iNOS and COX-2 positive, iNOS positive only, COX-2 positive only, and both negative) in serosal invasion (P = 0.038), lymph node metastasis (P = 0.038), Helicobacter pylori infection (P = 0.025), vascular invasion (P = 0.035), and microvessel density (P = 0.019). In patients with gastric cancer that co-expressed iNOS and COX-2, prognosis was significantly poorer than in those that expressed either iNOS or COX-2, or did not express both of them (P = 0.01738). The Cox proportional hazard regression analysis indicated that iNOS expression, vascular invasion, serosal invasion, and microvessel density are independent prognostic factors for patients with gastric cancer. CONCLUSIONS: iNOS and COX-2 expression of gastric cancer are related to tumor angiogenesis, tumor progression, and patient survival in human gastric cancer.     

Overexpression of vascular endothelial growth factor (VEGF) and its cellular receptor KDR (VEGFR-2) in the bone marrow of patients with acute myeloid leukemia.

Vascular endothelial growth factor (VEGF) and its cellular receptor VEGFR-2 have been implicated as the main endothelial pathway required for tumor neovascularization. However, the importance of the VEGF/VEGFR-2 system for angiogenesis in hematologic malignancies such as AML remains to be elucidated. In 32 patients with newly diagnosed untreated AML, we observed by immunohistochemical analysis of bone marrow biopsies significantly higher levels of VEGF and VEGFR-2 expression than in 10 control patients (P <0.001). In contrast, VEGFR-1 staining levels in AML patients were in the same range as in the controls. Expression of VEGF and VEGFR-2 was significantly higher in patients with a high degree of microvessel density compared to those with a low degree (VEGF: P =0.024; VEGFR-2: P =0.040) and correlated well with bone marrow microvessel density (r(s)=0.566 and 0.609, respectively; P <0.001). Furthermore, in patients who achieved a complete remission following induction chemotherapy VEGFR-2 staining levels decreased into the normal range. In conclusion, our results provide evidence for increased expression of VEGF/VEGFR-2 of leukemic blasts and correlation with angiogenesis in the bone marrow of AML patients. Thus, VEGF/VEGFR-2 might constitute promising targets for antiangiogenic and antileukemic treatment strategies in AML.     

Increased levels of tissue endostatin in human malignant gliomas.

PURPOSE: Malignant gliomas are typically angiogenic and express greater amounts of angiogenic factors. We examined glioma tissues for their expression of an endogenous inhibitor of angiogenesis, endostatin, a COOH-terminal fragment of collagen XVIII. EXPERIMENTAL DESIGN: We examined frozen tissues from 51 patients with astrocytic tumors (grade 2, 13; grade 3, 9; and grade 4, 29). Frozen tissues were subjected to immunoblot analysis and immunohistochemistry for endostatin. Tumor vascular density was determined by calculating the percentage of tumor capillary vessel areas/tissue section area. Tissue concentrations of vascular endothelial growth factor and basic fibroblast growth factor were examined by enzyme immunoassay. RESULTS: The levels of endostatin protein estimated by immunoblotting were significantly higher in grade 4 than lower-grade glioma tissues. The immunoreactive bands for endostatin were identified as the fragment derived from noncollagenous domain 1 of collagen XVIII, a peptide 15 residues longer than endostatin toward the NH(2)-terminal end, by NH(2)-terminal amino acid sequencing. In addition to an intense immunoreactivity for endostatin in tumor blood vessels, sections from malignant gliomas showed widely distributed immunoreactivity around tumor cells near the hyperplastic microvessels. The tumor vascular density and the levels of vascular endothelial growth factor in grade 4 glioma tissues were significantly higher than grade 2 and grade 3 gliomas, whereas the levels of basic fibroblast growth factor were the same. CONCLUSIONS: The results indicate a positive correlation between the levels of tissue endostatin and malignancy grades in gliomas. The endostatin may be released near the tumor blood vessels with hyperplasia to counteract angiogenic stimuli in malignant gliomas.     

Overexpression of collagen XVIII is associated with poor outcome and elevated levels of circulating serum endostatin in non-small cell lung cancer.

PURPOSE: The aim of this study was to determine whether collagen XVIII expression is correlated with circulating serum endostatin and whether this has any prognostic value in patients with non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Serum endostatin levels were measured quantitatively by a competitive enzyme immunoassay, and collagen XVIII expression in tumor tissue was investigated with an immunohistochemical method in a series of 94 patients who underwent surgery for NSCLC. RESULTS: Sixty cases (63.8%) had positive immunohistochemical staining with anticollagen XVIII polyclonal antibodies, including strongly positive staining in 11 (11.7%) cases. The mean (+/- SD) serum endostatin level was 41.6 +/- 34.4 ng/ml in the patient group and 16.3 +/- 10.3 ng/ml in the control group (P < 0.0001). The 11 cases who were strongly collagen XVIII-positive had significantly higher serum endostatin levels than the cases who were negative or weakly positive (P = 0.0297). The 5-year survival rates of negative, weakly positive, and strongly positive patients were 77.8%, 56.9%, and 43.8%, respectively. The cases with strongly positive collagen XVIII expression had a significantly poorer outcome than cases with negative expression (P = 0.0027). A multivariate analysis with Cox proportional hazards model for disease-specific survival revealed that expression of collagen XVIII (strongly positive versus negative; weakly positive versus negative), tumor classification, and regional lymph node classification were independent prognostic factors. CONCLUSIONS: Our results suggest that expression of collagen XVIII in tumor tissue is strongly associated with a poorer outcome in NSCLC and correlates with elevated levels of circulating serum endostatin.     

Coexpression of inducible nitric oxide synthase and COX-2 in hepatocellular carcinoma and surrounding liver: possible involvement of COX-2 in the angiogenesis of hepatitis C virus-positive cases.

Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) has been reported to be responsible for enhanced tumor growth and angiogenesis in various tumors. However, the relationships between tumor vascularity and COX-2 and iNOS expression have not been evaluated in hepatocellular carcinoma (HCC). In this study, we examined the expression of iNOS and COX-2 and microvessel density (MVD) by immunohistochemical staining in 100 tissue sections collected from HCC patients. iNOS expression was significantly higher in hepatitis C virus (HCV)-positive HCCs (P = 0.011). COX-2 expression was significantly correlated with iNOS expression (P = 0.046) and tumor MVD (P = 0.011) in HCV-positive HCCS: iNOS expression was neither correlated with MVD nor had any influence on patient survival; however, combined negative expression of iNOS and COX-2 had a significant impact on patient survival (P = 0.041 and 0.018, log-rank test for overall and recurrence-free survival rate, respectively). The present findings suggest that combined expression of iNOS and COX-2 may play an important role in prognosis of HCV-positive HCC patients and that this could be partially attributable to modulation of angiogenesis by COX-2.     

Serum endostatin levels correlate with enhanced extracellular matrix degradation and poor patients' prognosis in bladder cancer

Endostatin, the proteolytic fragment of collagen XVIII, is an inhibitor of angiogenesis and tumor growth. Interestingly, elevated circulating endostatin levels have been found to correlate with poor patients' prognosis in several cancers. The aim of this study was to assess the prognostic value of endostatin in bladder cancer (BC) and to gain insight into the mechanisms involved in its production. This retrospective study included a total of 337 patients with BC and 103 controls. Collagen XVIII gene expression was analyzed using real-time PCR (n = 82). Endostatin tissue localization was assessed by immunohistochemistry (n = 27). Endostatin serum (n = 87) and urine (n = 153) levels were determined by ELISA. In 12 cases, both serum and paraffinized tissue samples from the same patients were available. We found decreased collagen XVIII tissue expression and increased endostatin urine and serum concentration in samples of patients with BC compared to controls. High serum endostatin levels correlated with the presence of lymph node metastases and MMP-7 concentrations and were independently associated with poor metastasis-free and disease-specific survival. Immunohistochemical analysis revealed a strong endostatin staining in the wall of tumor associated blood vessels in superficial but not in muscle-invasive BCs. Based on these, we concluded that elevated endostatin levels in patients with BC are the consequence of enhanced extracellular matrix degradation and are independent from collagen XVIII expression. Furthermore, serum endostatin levels may provide prognostic information independent from histopathological parameters and may therefore help to optimize therapy decisions. Loss of endostatin expression in tumor associated blood vessels might represent an important step supporting tumor-induced angiogenesis.     

Angiogenesis in gastric cancer: importance of the thymidine phosphorylase expression of cancer cells as an angiogenic factor

Thymidine phosphorylase (TP) has been reported to stimulate angiogenesis in a variety of human malignancies. We investigated TP expression and its association with angiogenesis in 73 cases of resected gastric cancer. In addition, we compared the expression of the other angiogenesis related factors (VEGF, eNOS and p53) with that of TP with respect to angiogenesis. TP expression was not detected in most of the non-tumoral glandular epithelial cells except for 5 cases. TP expression of the cancer cells and the stroma was assessed separately. The stromal TP expression was not associated with the TP expression of the cancer cells. The mean percent of TP reactive cancer cells was 18.36+/-2.61 (median, 10.00; range, 0-90) and cases showing a percentage higher than the mean were considered as bearing high reactivity. The mean microvessel score assessed was 90.44+/-3.69 (median, 86; range, 31-174). The TP expression of cancer cells was strongly associated with microvessel density (p=0.030), but the stromal TP expression was not. The microvessel density of the tumor showed strong correlation with VEGF expression (p<0.001), but a marginally significant association with eNOS (p=0.055). On the contrary, there was no association with p53 expression and microvessel density of the tumor. No significant correlation was detected between lymph node metastasis and tumoral or stromal TP expression or VEGF/TP coexpression. In gastric cancer, TP expression of the cancer cells, not stromal cells may play an important role in tumor growth by microvessel formation.     

Immunohistochemical expression of vascular endothelial growth factor correlates with positive surgical margins and recurrence in T1 and T2 squamous cell carcinoma (SCC) of the lower lip

Tumor angiogenesis is associated with tumor progression and aggressiveness in a number of malignancies, and vascular endothelial growth factor (VEGF) is considered as a leading candidate in this process. The aim of the present study was to investigate the role of VEGF immunohistochemical expression and tumor angiogenesis in predicting local recurrence in surgically treated lip SCC. We performed a retrospective analysis of 50 patients with lip SCC in order to investigate whether VEGF immunohistochemical expression and tumor angiogenesis correlate with clinicopathologic parameters and outcome. Tumor angiogenesis was estimated by determining microvessel density (MVD) with the use of CD34 antibody. Our results showed that VEGF was strongly correlated with tumor invasion towards the surgical margin. There was also a significant association of high VEGF expression with a higher incidence of local recurrence (p < 0.001). We suggest that VEGF expression may be used as an index to distinguish patients with higher risk of relapse.     

Expression of thrombospondin-derived 4N1K peptide-containing proteins in renal cell carcinoma tissues is associated with a decrease in tumor growth and angiogenesis.

PURPOSE: We have reported that the 4N1K peptide (KRFYVVMWKK) from thrombospondin (TSP) 1 has antiangiogenic activities. The goal of this study was to examine whether the expression of 4N1K-containing proteins correlates with reduced growth of human renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: We examined the expression of 4N1K-containing proteins and TSP1, microvessel density, proliferation index, and apoptotic index in 119 surgically excised RCC tissues by immunohistochemical techniques. The correlation between the above variables and clinicopathological features was analyzed statistically. RESULTS: The antibody raised against the 4N1K peptide recognized protein fragments of matrix metalloproteinase 3-digested TSP1 and positively stained the sections of renal cancer tissues. These reactions disappeared when the antibody was preincubated with immobilized 4N1K peptide, suggesting that the reactions were 4N1K peptide specific. Although TSP1 expression did not correlate with various clinicopathological features and tumor size, all 4N1K-positive tumors were locally confined and of significantly smaller size (median, 3.3 cm; range, 2.0-4.4 cm) than 4N1K-negative tumors (median, 5.2 cm; range, 2.8-8.8 cm; P < 0.001). 4N1K-positive tumors exhibited significantly lower microvessel density and higher apoptotic index of tumor cells than 4N1K-negative tumors (P < 0.001 and P = 0.042, respectively). CONCLUSIONS: Our data suggest that expression of 4N1K-containing proteins in tumor tissues is associated with reduced angiogenesis and tumor growth; thus, it would be a potentially predictive marker for progression of RCC.     

Thymidine phosphorylase expression in tumor stroma of uterine cervical carcinomas: histological features and microvessel density

Immunohistochemical staining was performed on 91 cases of uterine neoplasm in order to determine if expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase (TP) correlates with tumor microvessel density (MVD) and histological parameters of uterine carcinomas in tumor cells and in tumor stroma. The sample group consisted of 72 primary invasive squamous cell carcinomas of the cervix (ISC) and 19 cervical intraepithelial neoplasms (CIN) of the uterus. In ISC of the cervix, TP expression in tumor stroma showed a significant correlation with a non-keratinizing histological subtype (P < 0.001) and with an infiltrating invasive pattern (P < 0.001). However, in tumor cells the TP expression showed a higher correlation with a keratinizing histological subtypes (P = 0.009). MVD was significantly higher (P = 0.002) in tumors showing high TP expression in stroma than in tumors with low expression. These findings suggest that the TP expression in stromal cells, rather than in tumor cells, may play a role in promoting microvessel growth in cervical squamous cell carcinoma, and angiogenesis may also have an association with tumor cell invasion.     

Thrombospondin-1 associated with tumor microenvironment contributes to low-dose cyclophosphamide-mediated endothelial cell apoptosis and tumor growth suppression

Low-dose cyclophosphamide (LDC) induces selective apoptosis of endothelial cells within the vascular bed of tumors. Here, we investigated a hypothesis that the effect of LDC is mediated by the pro-apoptotic action of endogenous inhibitors of angiogenesis. Tumors treated with LDC demonstrate similar expression of matrix metalloproteinases and also basement membrane-derived angiogenesis inhibitors when compared with wild-type tumors, whereas the expression of thrombospondin-1 (TSP-1) is significantly elevated in LDC-treated tumors. We used mice with an absence of type XVIII collagen (endostatin) or type IV collagen alpha3 chain (tumstatin) or TSP-1 to assess the contribution of these endogenous inhibitors of angiogenesis on LDC-mediated tumor suppression. Lack of TSP-1 in the host in addition to tumor cells leads to diminished capacity of LDC to suppress tumor growth, whereas the absence of endostatin and tumstatin did not alter the effect of LDC. LDC treatment predominantly induces selective expression of TSP-1 in tumor cells and peri-vascular cells and facilitates apoptosis of proliferating endothelial cells, with minimal direct effect on tumor cells and peri-vascular cells. These studies indicate that TSP-1 contributes to tumor growth suppression induced by LDC and suggest that tumors that express high basal level of TSP-1 may be more susceptible to tumor suppression by such a regimen. This study also makes a strong case for TSP-1 expression levels as a potential predictive marker for the successful use of LDC in cancer patients.