Detection of type IV collagenase activity in malignant ascites

AIM: Type IV collagenase participates in invasion and metastasis of cancer cells. Malignant asdtes is a manifestation of advanced malignant disease that is associated with invasion and metastasis of the peritoneal cavity. Thus, it is reasonable to hypothesize that type IV collagenase is linked to malignant ascites. The purpose of our study was to detect type IV collagenase activity in malignant ascites so as to provide the scientific basis for clinic diagnosis and treatment of malignant ascites. METHODS: Cirrhotic ascites (n=36), tuberculous ascites (n=8) and malignant ascites (n=-23) from patients with gastric cancer (n=6), colon cancer (n=5), ovarian cancer (n=8) and other cancers (n=4), including 2 hepatocellular cancers, 1 pancreatic cancer, 1 primary peritoneal carcinoma were collected by paracentesis. The ascites were made cellfree by centrifugation and stored frozen at -70℃ before determination. Type IV collagenase activity was determined by gelatin zymography. RESULTS: The activity of matrix metalloproteinases-2 and -9 could not be detected in ascites of hepatic cirrhosis and tuberculous peritonitis but could be detected in 20 and 18 out of 23 malignant ascites respectively. The positive rate of type IV collagenase (MMP-2, 87.0% and MMP-9, 78.3%) was higher than that by routine ascites tests (P<0.01) in malignant ascites. Furthermore, the activity of MMP-2 was higher than that of MMP-9 (P=0.022<0.05). CONCLUSION: Type IV collagenase is positive in malignant ascites. Detection of type IV collagenase activity is useful in qualitative diagnosis of ascites. Type IV collagenase may play an important role in malignant ascites formation.     

Detection of type Ⅳ collagenase activity in malignant ascites

AIM: Type Ⅳ collagenase participates in invasion and metastasis of cancer cells. Malignant ascites is a manifestation of advanced malignant disease that is associated with invasion and metastasis of the peritoneal cavity. Thus, it is reasonable to hypothesize that type Ⅳcollagenase is linked to malignant ascites. The purpose of our study was to detect type Ⅳ collagenase activity in malignant ascites so as to provide the scientific basis for clinic diagnosis and treatment of malignant ascites.METHODS: Cirrhotic ascites (n=36), tuberculous ascites (n=8) and malignant ascites (n=23) from patients with gastric cancer (n=6), colon cancer (n=5), ovarian cancer (n=8) and other cancers (n=4), including 2 hepatocellular cancers, 1 pancreatic cancer, 1 primary peritoneal carcinoma were collected by paracentesis. The ascites were made cellfree by centrifugation and stored frozen at -70℃ before determination. Type Ⅳ collagenase activity was determined by gelatin zymography.RESULTS: The activity of matrix metalloproteinases-2 and -9 could not be detected in ascites of hepatic cirrhosis and tuberculous peritonitis but could be detected in 20 and 18 out of 23 malignant ascites respectively. The positive rate of type Ⅳ collagenase (MMP-2, 87.0 % and MMP-9, 78.3 %) was higher than that by routine ascites tests (P<0.01) in malignant ascites. Furthermore, the activity of MMP-2 was higher than that of MMP-9 (P=0.022<0.05).CONCLUSION: Type Ⅳ collagenase is positive in malignant ascites. Detection of type Ⅳ collagenase activity is useful in qualitative diagnosis of ascites. Type Ⅳ collagenase may play an important role in malignant ascites formation.     

siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo

BACKGROUND/AIMS: We have investigated whether siRNA targeted against VEGF inhibits functional properties of endothelial cells in vitro and HCC tumor growth and blood vessel formation in vivo. METHODS: The influence of siRNA-VEGF on endothelial cell proliferation, apoptosis and tube formation were analyzed in vitro. Antitumoral effects were examined in an orthotopic tumor model after ex vivo transfer or intraperitoneal treatment of siRNA, respectively. Intratumoral microvessel density was assessed by CD31 staining. RESULTS: VEGF expression was inhibited in Hepa129 by 70% and in SVEC4-10 by 48% within two days after transfection. In vitro, endothelial cell proliferation and tube formation was reduced by 23% and 38%, respectively. Interference with VEGF signaling was demonstrated by reduced pAKT in hepatoma cells. Tumor growth was inhibited by ex vivo transfer or intraperitoneal application of siRNA-VEGF by 83% or 63% in orthotopic tumors within 14 days. VEGF protein was reduced in both models by 29% and 44%. Microvessel density dropped to 34% for tumors from ex vivo transfected cells and 39% for systemic treated tumors. CONCLUSIONS: The results show that VEGF knockdown can be associated with reduced endothelial cell proliferation and tube formation in vitro and decreased tumor growth and microvessel density in vivo.     

Human malignant ascites and histamine-induced protein leakage from the normal microcirculation

The accumulation of malignant ascites is a significant cause of morbidity and mortality in patients with intraabdominal malignancies. However, the cause of malignant ascites is unknown. In this study, we used the rat cremaster muscle preparation to determine if and how malignant ascites could produce protein leakage from normal blood vessels which would lead to fluid accumulation in the peritoneal cavity. The rat cremaster muscle, with nerves and blood vessels to the animal intact, was prepared for microscopic observations of the microcirculation. Serum albumin was tagged to fluorescein isothiocyanate and injected into the rat. Fluorescent microscopy was used to quantitate leakage of the tagged albumin into the interstitial tissue. Malignant ascites was collected from a patient with metastatic breast cancer. The ascites fluid was placed on the cremaster muscle and it induced protein leakage from the normal blood vessels of this tissue. Protein leakage was partially blocked by diphenhydramine (10(-4) M) and by mast cell depletion with compound 48/80. There was a high level of C3a in the malignant ascites solution but C3a did not increase during the exposure period. These data suggest that activated complement in malignant ascites may release histamine from mast cells to cause protein leakage of the normal vasculature. The movement of protein into the peritoneal cavity would be followed by water, thus increasing the volume of the ascites and exacerbating the clinical condition.     

VEGF、CEA联合检测在腹水鉴别诊断中的价值

目的 探讨测定腹水中血管内皮生长因子(VEGF)、癌胚抗原(CEA)在良、恶性腹水鉴别诊断中的价值,以及二者联合检测的临床意义.方法 收集腹水标本61例,分为良性腹水组和恶性腹水组,采用ELISA的方法测定VEGF,放免法测定CEA.结果 恶性腹水组VEGF水平明显高于良性腹水组(P＜0.01);恶性腹水组CEA水平明显高于良性腹水组(P＜0.01).卵巢癌引起的腹水中VEGF的水平显著高于肝癌、胃癌、结肠癌组(P＜0.05).不同肿瘤引起的腹水中CEA的含量无明显差异(P＞0.05).VEGF诊断恶性腹水的敏感性为82.1%,特异性为87.9%,准确率为85.2%.CEA诊断恶性腹水的敏感性为64.3%,特异性为90.9%,诊断准确率为78.7%.同时检测患者腹水VEGF和CEA,其诊断恶性腹水的敏感性为92.9%,特异性为84.8%,诊断准确率为88.5%.结论 腹水中VEGF、CEA测定有助于良、恶性腹水的鉴别诊断;恶性腹水中VEGF的含量对恶性腹水的组织来源可能有一定的鉴别诊断作用;联合检测恶性腹水中的VEGF、CEA水平,可明显提高诊断恶性腹水的敏感性.     

The stem cell marker prominin-1/CD133 interacts with vascular endothelial growth factor and potentiates its action

Prominin-1, a pentaspan transmembrane protein, is a unique cell surface marker commonly used to identify stem cells, including endothelial progenitor cells and cancer stem cells. However, recent studies have shown that prominin-1 expression is not restricted to stem cells but also occurs in modified forms in many mature adult human cells. Although prominin-1 has been studied extensively as a stem cell marker, its physiological function of the protein has not been elucidated. We investigated prominin-1 function in two cell lines, primary human endothelial cells and B16-F10 melanoma cells, both of which express high levels of prominin-1. We found that prominin-1 directly interacts with the angiogenic and tumor survival factor vascular endothelial growth factor (VEGF) in both the primary endothelial cells and the melanoma cells. Knocking down prominin-1 in the endothelial cells disrupted capillary formation in vitro and decreased angiogenesis in vivo. Similarly, tumors derived from prominin-1 knockdown melanoma cells had a reduced growth rate in vivo. Further, melanoma cells with knocked down prominin-1 had diminished ability to interact with VEGF, which was associated with decreased bcl-2 protein levels and increased apoptosis. In vitro studies with soluble prominin-1 showed that it stabilized dimer formation of VEGF₁₆₄, but not VEGF₁₂₁. Taken together, our findings support the notion that prominin-1 plays an active role in cell growth through its ability to interact and potentiate the anti-apoptotic and pro-angiogenic activities of VEGF. Additionally, prominin-1 promotes tumor growth by supporting angiogenesis and inhibiting tumor cell apoptosis.     

Vascular endothelial growth factor: the key mediator in pleural effusion formation

Pleural effusion is common in clinical practice. Increased vascular permeability and leakage play a principal role in the development of exudative pleural effusions. In vitro and in vivo evidence have solidly established vascular endothelial growth factor (VEGF), a potent inducer of vascular permeability, as a crucial mediator in pleural fluid formation. VEGF is present in high quantities in human effusions. In the pleural space, mesothelial cells, infiltrating inflammatory cells, and (in malignant pleuritis) cancer cells contribute to the VEGF accumulation in the pleural fluids. Pleural fluid VEGF is biologically active and may promote tumor growth and chemotaxis. Strategies to antagonize the VEGF activity at various target points of its signaling pathway have shown success in vitro and in animal models of malignant pleural or peritoneal effusions. Novel agents targeting VEGF activities are undergoing clinical trials. Regulation of VEGF activity and vascular permeability represent a rapidly expanding field of research, which is likely to provide further insight in the pathophysiology of pleural fluid formation.     

Detection of free malignant cells in the peritoneal cavity before and after resection of colorectal cancer

PURPOSE: This study was designed to select the best monoclonal antibody to stain malignant cells in peritoneal wash fluid, and to investigate the incidence of free malignant cells in preresection and postresection colorectal cancer peritoneal washings using a combination of conventional cytology and immunocytochemistry. METHODS: Peritoneal washings were taken from 35 consecutive patients undergoing colorectal cancer resection. RESULTS: Malignant cells were isolated on a density gradient and identified by conventional cytology and an indirect immunoperoxidase stain. Malignant cells were identified in peritoneal washings from 15 patients (preresection only n=3, postresection only n=4, both n= 8). The origin of free malignant peritoneal cells in 11 preresection-positive washings must be the serosa. The origin of these cells in the four postresection-positive patients is uncertain: serosal and luminal spillage were considered unlikely and no circulating cells were found in the mesenteric vessels near the tumor. CONCLUSION: Tumor cells may have leaked out from lymphatics cut during the dissection.     

FP therapy for controlling malignant ascites in advanced pancreatic cancer patients

BACKGROUND/AIMS: Malignant ascites is one of the poor prognostic factors for pancreatic cancer, and causes serious symptoms and treatment-related toxicity. We conducted a retrospective analysis to evaluate the efficacy of 5-fluorouracil (5-FU) plus cisplatin (FP therapy) for controlling malignant ascites in patients with advanced pancreatic cancer. METHODOLOGY: This analysis was based on 28 consecutive chemotherapy-naive advanced pancreatic cancer patients with cytologically proven malignant ascites who were treated with FP therapy from November 1991 to April 2003. RESULTS: No patients achieved measurable tumor responses. The objective improvement of ascites was seen in 35.7% of the patients (N = 10/28, 95% confidence interval, 18.0 to 53.4%), but there was no patient with complete disappearance of ascites. The median time to disease progression and the median survival time were 1.7 months and 2.7 months, respectively. In all pretreatment variables, the presence of distant metastasis other than peritoneal dissemination was an unfavorable predictive factor for the objective improvement of ascites (Fisher's exact test: P = 0.002). CONCLUSIONS: FP therapy was modestly effective for controlling malignant ascites but insufficient in shrinking for measurable metastatic lesions. Systemic chemotherapy for controlling malignant ascites might be worth while for palliative management in advanced pancreatic cancer patients, especially in patients without distant metastasis.     

Effects of MDM2 inhibitors on vascular endothelial growth factor-mediated tumor angiogenesis in human breast cancer

Background

Mouse double minute 2 (MDM2) is overexpressed in many malignant tumors, and MDM2 levels are associated with poor prognosis of several human cancers, including breast cancer. In the present study, we investigated the function of MDM2 in vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis of breast cancer and the potential value of MDM2 as an anti-angiogenic therapy target for cancer therapy by inhibiting MDM2 with antisense oligonucleotides (ASO) or other antagonist nutlin-3.

Methods

Anti-MDM2 ASO and nutlin-3 were evaluated for their in vitro and in vivo anti-angiogenesis activities in different human breast cancer models with a different p53 status: MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53. MCF-7 and MDA-MB-468 cells were incubated with different concentrations of ASO or nutlin-3 for various periods of time. VEGF gene and protein expression in tumor cells was measured by qPCR and Western blot. The level of VEGF protein secreted in the culture supernatant of treated cells was quantified by enzyme-linked immunosorbent assay (ELISA). Nude mouse xenograft models were further established to determine their effects on tumor growth and angiogenesis. Serum levels of VEGF were measured by ELISA. VEGF expression and microvessel density in tumor tissues were studied by immunohistochemistry. Both angiogenesis and tumor growth were digitally quantified.

Results

In both MCF-7 and MDA-MB-468 cells, VEGF expression and secretion were reduced, resulting from specific inhibition of MDM2 expression by ASO. In vivo assay, after administration of ASO, VEGF production reduced and anti-angiogenesis activity occurred in nude mice bearing MCF-7 or MDA-MB-468 xenograft. However, in both models treated with nutlin-3, VEGF production was not changed and anti-angiogenesis activity was not observed.

Conclusion

In summary, the ASO construct targeting MDM2 specifically suppresses VEGF expression in vitro and VEGF-mediated tumor angiogenesis in vivo in breast cancer. Furthermore, the suppression of VEGF expression subsequent to inhibition of MDM2 in p53 mutant cells suggests that MDM2 has a regulatory role on VEGF expression through a p53-independent mechanism.     

Co-expression of vascular endothelial growth factor and its receptor flt-1 in malignant pleural mesothelioma

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent angiogenic protein with a selective mitogenic effect on endothelial cells known to be involved in many normal and pathological processes. Coexpression of VEGF and its receptor flt-1 has been reported in different types of malignant tumors. OBJECTIVE: In the present study we investigated the expression of VEGF and flt-1 in 90 cases of diffuse malignant pleural mesotheliomas. METHODS: VEGF and flt-1 expression was analyzed by immunohistochemistry and non-radioactive in situ hybridization. Results: VEGF expression was visualized immunohistochemically in tumor cells. flt-1 expression correlated with histological differentiation (p < 0.013). Furthermore, expression of flt-1 was detected in tumor cells, macrophages and microvessels adjacent to tumor cells. VEGF and flt-1 expression were confirmed by in situ hybridization. CONCLUSION: There was a statistically significant correlation between VEGF and flt-1 expression (p < 0.001). The observed coexpression of VEGF and flt-1 possibly suggests a potential autocrine loop for malignant pleural mesothelioma cells.     

三氧化二砷对裸鼠结肠癌腹腔转移的抑制作用

目的　研究Ａｓ２Ｏ３ 对裸鼠结肠癌腹腔转移的影响及其作用机制。方法　ＢＡＬＢ／Ｃｎｕ／ｎｕ裸鼠腹腔内接种结肠癌Ｌｏｖｏ 细胞后,随机分为５ 组,分别腹腔内注射生理盐水、表阿霉素及不同剂量的Ａｓ２Ｏ３ ,观察各组的致瘤率、腹水生成及生存时间。结果　表阿霉素、低剂量Ａｓ２Ｏ３ 与对照组相比可明显抑制裸鼠腹水的生长,延长生存期（ Ｐ＜ ０．０１） ;中高剂量Ａｓ２Ｏ３ 除上述作用外还可通过诱导肿瘤细胞凋亡消除腹腔内肿瘤细胞,抑制腹水生成,并使裸鼠寿命明显延长。结论　Ａｓ２Ｏ３ 可诱导结肠癌细胞凋亡,抑制或消除裸鼠肿瘤性腹水的生成,不同程度地延长生存期。     

In vitro and in vivo angiogenesis in PC12 pheochromocytoma cells is mediated by vascular endothelial growth factor.

Angiogenesis, the formation of new blood vessels from existing vascular endothelium, is essential for tumor growth. Vascular endothelial growth factor (VEGF) is an endotheliumspecific mitogen and regulator of angiogenesis. Angiogenesis has been associated to the malignant phenotype of pheochromocytomas and is readily observed in experimental pheochromocytomas. Although VEGF gene expression has already been demonstrated in the rat PC12 cell line, the detailed mechanisms of action are not known. We have, therefore, studied angiogenesis in the rat PC12 pheochromocytoma cell line in vitro and in vivo. VEGF gene expression and accumulation of VEGF protein in cytoplasm and conditioned medium of PC12 cells was found. Conditioned medium from PC12 cells significantly increased proliferation of VEGF-dependent endothelial cells from human umbilical veins, and this effect reversed upon addition of a neutralizing anti-VEGF antibody. Dexamethasone and nerve growth factor (NGF) increased VEGF mRNA expression and accumulation of VEGF protein of PC12 subclones with established metastatic activity in vivo. PC12 cells xenotransplanted to nude mice had marked VEGF expression and induced host angiogenesis, confirmed by the presence of CD34-positive endothelial cells in the experimental PC12 tumors. When NGF-primed PC12 cells were immobilized in Matrigel supplemented with rising concentrations of the growth factor and xenotransplanted, increasing NGF resulted in tumors with smaller areas of necrosis and increased vital tumor volume. These results suggest that VEGF is a mediator of angiogenesis in the PC12 pheochromocytoma cell line, and that dexamethasone and NGF affect VEGF expression. Our data further suggest that NGF may contribute to angiogenesis in experimental pheochromocytoma.     

Antiangiogenic effects of melatonin in endothelial cell cultures

Endothelial cells represent one of the critical cellular elements in tumor microenvironment playing a crucial role in the growth and progression of cancer through controlling angiogenesis. Vascular endothelial growth factor (VEGF) produced from tumor cells is essential for the expansion of breast cancer and may function in both paracrine and autocrine manners to promote proliferation, growth, survival and migration of endothelial cells. Since melatonin regulates tumor microenvironment by decreasing the secretion of VEGF by malignant epithelial cells and also regulates VEGF expression in human breast cancer cells, the aim of the present study was to investigate the anti-angiogenic activity of melatonin against the pro-angiogenic effects of breast cancer cells.In this work, we demonstrate that melatonin strongly inhibited the proliferation as well as invasion/migration of human umbilical vein endothelial cells (HUVECs). Melatonin disrupted tube formation and counteracted the VEGF-stimulated tubular network formation by HUVEC. In addition, conditioned media collected from human breast cancer cells were angiogenically active and stimulated tubule length formation and this effect was significantly counteracted by the addition of anti-VEGF or melatonin. Melatonin also disintegrated preformed capillary network.All these findings demonstrate that melatonin may play a role in the paracrine interactions that take place between malignant epithelial cells and proximal endothelial cells. Melatonin could be important in reducing endothelial cell proliferation, invasion, migration and tube formation, through a downregulatory action on VEGF. Taken together, our findings suggest that melatonin could potentially be beneficial as an antiangiogenic agent in breast cancer with possible future clinical applications.     

磁激活细胞分选术联合混合抗体提高模拟恶性腹水中游离癌细胞检出效率的分析

背景磁激活细胞分选术(magnetic activated cell sorting,MACS)是一种新的免疫磁性分离技术.其原理是基于抗体对抗原的特异性识别,将50 nm磁性微珠直接或者间接耦联在抗体上,与有相应抗原表达的细胞相连,在高强度、高梯度磁场中达到细胞磁性分离的目的.该法具有分离纯度和回收率均较高的优势,也能分离出体液中存在的少量肿瘤细胞.目的评价MACS联合一组肿瘤细胞标志物的方法对提高模拟恶性腹水中游离癌细胞检出效率的作用.方法 选择5种与恶性腹水病因有关的肿瘤细胞株作为研究对象,采用免疫荧光反应和流式细胞仪(FCM)检测上皮相关抗原(epithlial-related antigen,EpCAM)、CA125、癌胚抗原和TAG-72共4种单克隆抗体及其混合抗体在各肿瘤细胞的表达.将肿瘤细胞以不同比例掺入单个核细胞中模拟恶性腹水细胞成分,与联合单个抗体进行分选对比,观察MACS术联合混合抗体分选肿瘤细胞的效率.结果 FCM结果表明,混合抗体在5种肿瘤细胞的阳性表达率均高于4种抗体单独反应的阳性率.MACS术联合混合抗体检出模拟恶性腹水中肿瘤细胞的得率比联合单个抗体的得率要高,其中以2种胃癌细胞和结肠癌细胞的平均得率提高较多(分别为69.18%±20.84%比45.23%±11.54%、78.75%±15.42%比59.73%±16.64%和85.63%±12.30%比76.88%±8.65%),卵巢癌细胞次之(32.49%±3.58%比31.79%±4.82%),肝癌细胞最少(11.78%±0.43%比7.16%±0.46%).结论 与联合单个抗体进行分选相比,应用MACS术联合一组混合抗体的方法可有效提高恶性腹水中游离癌细胞的检出效率,尤其对胃肠道肿瘤所致的恶性腹水有潜在的临床应用价值.