Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma

MCAM/MUC18 expression correlates with tumor thickness and metastatic potential of human melanoma cells in nude mice. Moreover, ectopic expression of MUC18 in primary cutaneous melanoma cells leads to increased tumor growth and metastasis in vivo. Here we tested the effect of a fully human anti-MUC18 antibody, ABX-MA1, on angiogenesis, tumor growth, and metastasis. ABX-MA1 had no effect on melanoma cell proliferation rate in vitro. However, when cells of the metastatic melanoma lines A375SM and WM2664 (which express high levels of MUC18) were injected s.c. into nude mice and treated with ABX-MA1 (100 micro g, weekly, i.p. for 5 weeks), tumor growth was significantly inhibited compared with control IgG-treated mice. ABX-MA1 treatment also suppressed experimental lung metastasis of these melanoma cells. ABX-MA1 disrupted spheroid formation by melanoma cells expressing MUC18 (homotypic interaction) and the ability of these cells to attach to human vascular endothelial cells [HUVECs (MUC18 positive)] in vitro. ABX-MA1 treatment of melanoma cells in vitro significantly inhibited the promoter and collagenase activity of matrix metalloproteinase 2, resulting in decreased invasion through Matrigel-coated filters. Decreased expression of matrix metalloproteinase 2 was also observed in the implanted tumors in vivo. Moreover, because HUVECs also express MUC18, ABX-MA1 directly disrupted the tube-like formation by HUVECs in an in vitro vessel formation assay. Collectively, these results point to usefulness of ABX-MA1 as a modality to treat melanoma either alone or in combination with conventional chemotherapy or other antitumor agents.     

Cellular adhesion pathways and metastatic potential of human melanoma

Cellular adhesion molecules of the cadherin, integrin, and immunoglobulin superfamilies are important to both growth and metastasis of many cancers, including malignant melanoma. Malignant melanoma is an excellent model for studying these molecules, due in part to a sequential series of five defineable stages. As the malignant phenotype of melanoma cells changes from the noninvasive radial growth phase to the vertical growth phase, which has high metastatic potential, so does the repertoire of the cellular adhesion molecules expressed on the cells surface. The cellular adhesion molecule MCAM/MUC18 confers metastatic potential and increased tumorigenicity to melanoma cells. MCAM/MUC18 mediates homotypic and heterotypic adhesion between melanoma cells and endothelial cells, respectively. Both types of interaction may promote metastasis at different stages in the metastasis cascade. We developed a fully humanized antibody to MCAM/MUC18 (ABX-MA1) that blocked melanoma metastasis in vivo. Furthermore, ABX-MA1 blocked the homotypic interaction between melanoma cells and endothelial cells as well as the promoter and collagenase activity of MMP-2. During melanoma progression the loss of E-cadherin expression disrupts normal homeostasis in the skin by freeing melanoma cells from structural and functional regulation by keratinocytes. The loss of functional E-cadherin is parallelled by a gain in N-cadherin function that mediates homotypic interaction between melanoma cells, facilitates gap-junctional formation with fibroblasts and endothelial cells and promotes melanoma cell migration and survival. In addition, loss of E-cadherin may affect the beta-catenin/wnt signaling pathways, resulting in deregulation of genes involved in growth and metastasis. The integrin family member alpha(v)beta(3) is widely expressed on melanoma cells in the vertical growth phase. When alpha(v)beta(3) is expressed in melanoma cells in the radial growth phase, this integrin is associated with increased tumor growth in vivo. alpha(v)beta(3) may also promote melanoma invasion, through an interaction with MMP-2, and transendothelial migration, via a heterotypic melanomaendothelial cell interaction. This review summarizes recent knowledge on how changes in these adhesion molecules contribute to the acquisition of the metastatic phenotype in human melanoma.     

高表达MUC1的人食管癌裸鼠移植瘤模型的建立

背景与目的:Mucin-1(MUC1)粘蛋白是一种肿瘤相关抗原,是肿瘤免疫治疗良好的分子靶点之一.本研究建立高表达MUCl的人食管癌裸鼠皮下移植瘤模型,为研究以MUC1为靶点的食管癌的生物治疗提供体内模型.方法:以体外培养的高表达MUC1的人食管癌细胞株EC-109接种于4～5周龄的BALB/c裸小鼠皮下,观察移植瘤生长情况,对移植瘤进行病理组织学检查,采用免疫组化方法检测移植瘤细胞增殖细胞核抗原(proliferating cell nuclear antigens,PCNA),采用流式细胞仪检测移植瘤细胞的细胞周期及MUC1的表达.结果:裸鼠皮下移植瘤成瘤率为86.0%,移植瘤具有与人恶性肿瘤相似的组织学和生物学特点,其平均PCNA标记指数为(63.5 3.6)%、S期细胞百分比(S-phase fraction,SPF)平均值为(37.6±3.7)%、MUC1的平均表达率为97.5%.结论:高表达MUC1的人食管癌裸鼠皮下移植瘤模型具有恶性肿瘤的生物学特性,可用于研究人食管癌的生物学特点,同时为以MUC1为靶点的食管癌的免疫治疗研究提供了良好的体内模型.     

Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on SRC kinase for survival

Sarcomas are rare malignant mesenchymal tumors for which there are limited treatment options. One potential molecular target for sarcoma treatment is the Src tyrosine kinase. Dasatinib (BMS-354825), a small-molecule inhibitor of Src kinase activity, is a promising cancer therapeutic agent with p.o. bioavailability. Dasatinib exhibits antitumor effects in cultured human cell lines derived from epithelial tumors, including prostate and lung carcinomas. However, the action of dasatinib in mesenchymally derived tumors has yet to be shown. Based on our previous findings of Src activation in human sarcomas, we evaluated the effects of dasatinib in 12 cultured human sarcoma cell lines derived from bone and soft tissue sarcomas. Dasatinib inhibited Src kinase activity at nanomolar concentrations in these sarcoma cell lines. Downstream components of Src signaling, including focal adhesion kinase and Crk-associated substrate (p130(CAS)), were also inhibited at similar concentrations. This inhibition of Src signaling was accompanied by blockade of cell migration and invasion. Moreover, apoptosis was induced in the osteosarcoma and Ewing's subset of bone sarcomas at nanomolar concentrations of dasatinib. Inhibition of Src protein expression by small interfering RNA also induced apoptosis, indicating that these bone sarcoma cell lines are dependent on Src activity for survival. These results show that dasatinib inhibits migration and invasion of diverse sarcoma cell types and selectively blocks the survival of bone sarcoma cells. Therefore, dasatinib may provide therapeutic benefit by preventing the growth and metastasis of sarcomas in patients.     

Recombinant nanobody against MUC1 tandem repeats inhibits growth,invasion, metastasis,and vascularization of spontaneous mouse mammary tumors

Alteration in glycosylation pattern of MUC1 mucin tandem repeats during carcinomas has been shown to negatively affect adhesive properties of malignant cells and enhance tumor invasiveness and metastasis. In addition, MUC1 overexpression is closely interrelated with angiogenesis, making it a great target for immunotherapy. Alongside, easier interaction of nanobodies (single‐domain antibodies) with their antigens, compared to conventional antibodies, is usually associated with superior desirable results. Herein, we evaluated the preclinical efficacy of a recombinant nanobody against MUC1 tandem repeats in suppressing tumor growth, angiogenesis, invasion, and metastasis. Expressed nanobody demonstrated specificity only toward MUC1‐overexpressing cancer cells and could internalize in cancer cell lines. The IC50 values (the concentration at which the nanobody exerted half of its maximal inhibitory effect) of the anti‐MUC1 nanobody against MUC1‐positive human cancer cell lines ranged from 1.2 to 14.3 nm. Similar concentrations could also effectively induce apoptosis in MUC1‐positive cancer cells but not in normal cells or MUC1‐negative human cancer cells. Immunohistochemical staining of spontaneously developed mouse breast tumors prior to in vivo studies confirmed cross‐reactivity of nanobody with mouse MUC1 despite large structural dissimilarities between mouse and human MUC1 tandem repeats. In vivo, a dose of 3 µg nanobody per gram of body weight in tumor‐bearing mice could attenuate tumor progression and suppress excessive circulating levels of IL‐1a, IL‐2, IL‐10, IL‐12, and IL‐17A pro‐inflammatory cytokines. Also, a significant decline in expression of Ki‐67, MMP9, and VEGFR2 biomarkers, as well as vasculogenesis, was evident in immunohistochemically stained tumor sections of anti‐MUC1 nanobody‐treated mice. In conclusion, the anti‐MUC1 tandem repeat nanobody of the present study could effectively overcome tumor growth, invasion, and metastasis.     

A unique mucin immunoenhancing peptide with antitumor properties

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.     

MUC1 and MUC2 expression in human gallbladder carcinoma: a clinicopathological study and relationship with prognosis

Mucins are a group of high molecular weight glycoproteins consisting of a mucin core protein and O-linked carbohydrates. To date, nine apomucins (MUC1-8, and MUC5B) have been identified. Recent studies have demonstrated that MUC1 is expressed in tumors of various human organs, and may function as an anti-adhesion molecule that inhibits cell-to-cell adhesion, inducing tumor metastasis. MUC2 is a major secreted mucin of colon and is known to be expressed in cells showing intestinal metaplasia in the stomach and other organs. MUC2 expression in the mucosal epithelia is an apparently abnormal phenomenon related to the neoplastic process. In this study, we examined MUC1 and MUC2 expression in human gallbladder adenocarcinoma and its clinicopathological significance and relationship with the prognosis of the patients. MUC1 immunoreactivity was detected not only in the cancer cells but also in the cancer stroma. Cytoplasmic MUC1 expression was significantly relation to lymphatic invasion and lymph node metastasis (p < 0.001), and was associated with a poor outcome. In contrast, MUC2 was rarely expressed in gallbladder carcinomas, and its immunoreactivity was detected only in the cancer goblet cells. Overexpression of MUC2 was not significantly related to lymphatic invasion or lymph node metastasis, or prognosis of patients. These observations suggested that MUC1 expression plays a more important role than MUC2 expression in cancer cell growth and metastasis of human gallbladder adenocarcinomas.     

RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells.

MUC1 is a highly glycosylated, type I transmembrane protein expressed by normal ductal epithelial cells of the pancreas, breast, lung, and gastrointestinal tract, and overexpressed in many cases of adenocarcinoma. We down-regulated MUC1 expression by RNA interference and investigated the effects on malignant and metastatic potential of a human pancreatic cancer cell line, S2-013. MUC1-suppressed clones, S2-013.MTII.C1 and S2-013.MTII.C2, were established by targeting a sequence 3,151 bp from the initiation codon and characterized in vitro for proliferation, invasion, and adhesion. We evaluated the effects of MUC1 suppression in vivo on tumor growth and metastatic properties following implantation into the cecum or pancreas of athymic mice. MUC1-suppressed clones showed significantly decreased proliferation in vitro and in vivo. Global gene expression was evaluated by oligonucleotide microarray analysis. Surprisingly, genes predicted to increase doubling times (cyclin B1 and cyclin D3) were overexpressed in MUC1-suppressed clones. There were alterations in expression of several genes that may affect the malignant properties of pancreatic cancer. Adhesion of MUC1-suppressed cells in vitro to type IV collagen and fibronectin was slightly increased, and adhesion was slightly decreased to type I collagen and laminin. Results of implantation to cecum and pancreas showed significant reduction of metastasis to lymph nodes, lung, or peritoneal sites compared with S2-013.gfp-neo control cells. These results support the hypothesis that MUC1 contributes significantly to growth and metastasis, and that down-regulation of MUC1 protein expression decreases the metastatic potential of pancreatic adenocarcinoma.     

Cotargeting tumor and stroma in a novel chimeric tumor model involving the growth of both human prostate cancer and bone stromal cells

Stromal-epithelial interaction contributes to local prostate tumor growth, androgen-independent progression and distant metastasis. We have established in vitro coculture and in vivo chimeric tumor models to evaluate the roles of stromal cells isolated from either osteosarcoma or normal bone, a site where prostate cancer cells frequently metastasize, in contributing to the growth and survival of human prostate cancer cells. We have evaluated extensively the effects of toxic gene therapy using luciferase-tagged chimeric human prostate cancer models both in vitro and in vivo. In the in vitro cocultured cell model, we assessed cancer cell growth and residual cellular proteins after targeting either prostate cancer epithelial cells alone or both prostate cancer and bone stromal cells. In the in vivo animal model, we measured tumor volume and serum prostate-specific antigen (PSA) in mice bearing chimeric prostate tumors comprised of human prostate tumor cells and normal bone stromal cells. Our results demonstrated that: (1) The rate of human prostate cancer cell growth in vitro is accelerated by coculturing with human and rat osteosarcoma or normal mouse bone marrow stromal cell lines. No growth stimulation was noted when cocultured with a human prostate epithelial cell line. (2) Disabling the growth of normal bone stromal cells using transgenic targeting with a bystander gene, herpes simplex virus thymidine kinase (hsv-TK), plus the pro-drug ganciclovir (GCV) or acyclovir markedly depressed the growth of cocultured human prostate cancer cells in vitro and human prostate cancer-mouse normal bone stroma chimeric tumors in vivo. (3) By cotargeting both human prostate cancer and normal mouse bone stromal cells in vitro with an adenoviral construct, Ad-hOC-TK (a replication-defective Ad5 vector with the bystander transgene hsv-TK under the control of a human osteocalcin (hOC) promoter) plus GCV4, we observed greater inhibition of tumor cell growth than by targeting a single cell compartment with Ad-PSA-TK (a vector construct similar to Ad-hOC-TK except that the transgene expression is under regulation by a full-length human PSA promoter). These results, taken together, established a basic principle that cotargeting both tumor and its supporting stroma is more efficacious than targeting a single cell compartment in the treatment of human prostate cancer bone metastasis. This principle can be applied to other clinical conditions of cancer growth where stroma contribute to the overall growth and survival potential of the cancer.     

人骨肉瘤原位移植模型的建立及生物学特征

目的 用人骨肉瘤细胞系HOS-98建立人骨肉瘤裸鼠胫骨原位移植模型,以探讨宿主器官微环境对人骨肉瘤细胞侵袭及转移等生物学行为的影响。方法 将人骨肉瘤细胞系HOS-98接种于裸鼠皮下,形成移植瘤,用传代移植瘤组织作为移植材料,进行胫骨原位移植及皮下移植。分别于移植后4周和8周处死小鼠,进行病理形态学检查,并对两种方法在成瘤率、生长方式及侵袭、转移等生物学行为比较。结果 两种移植方式在成瘤率及形态学上无明显不同,胫骨原位移植的潜伏期较短,并且生长快于皮下移植方式。皮下移植瘤呈局限性膨胀生长,有不完整的纤维包膜,瘤内类骨基质较少见,未见肺转移,观察8周时无明显消瘦;而胫骨原位移植瘤侵袭周围组织,可见发生肺转移,8周明显消瘦。原位移植的裸鼠血清ALP水平高于皮下移植者。原位移植的X线检查有明显的类似于人的骨性反应。结论 用人骨肿瘤细胞系HOS-98皮下接种的移植瘤作为移植材料是建立肿瘤异位移植的可行途径,裸鼠胫骨微环境较皮下组织更适合于人骨肉瘤的侵袭及转移表达,裸鼠胫骨原位移植模型的恶性生物学行为更接近临床骨肉瘤患者的体内侵袭及转移实际,该原位移植模型为今后的实验研究提供了更加接近患者实际的实验模型。     

CD44s、MMP-9、Ki-67的表达与骨肉瘤侵袭、转移、复发的关系

背景及目的：近期研究表明,细胞表面粘附分子CD44和基质金属蛋白酶类（MMPs)在多种肿瘤组织中有高水平表达,且与肿瘤的侵袭转移关系密切;Ki-67是一种细胞增殖核抗原,表示肿瘤细胞的增殖速率;但二者与骨肉瘤的局部侵袭、远处转移及术后是否复发之间的关系尚不清楚,本研究拟探讨细胞表面粘附分子标准型CD44蛋白（CD44s)以及基质金属蛋白酶－9（MMP－9）和细胞增殖核抗原（Ki-67)在骨肉瘤中的表达情况及其与骨肉瘤侵袭、转移、复发的关系。方法：采用免疫组织化学SP法检测了62例骨肉瘤（初发性骨肉瘤51例、复发性骨肉瘤11例,确诊时即伴有肺转移者9例、不伴有肺转移者53例）以及骨纤维结构不良和正常对照组织中CD44s、MMP－9、Ki-67的表达,并根据阳性瘤细胞占肿瘤细胞总数的比率进行半定量量化分析和非参数秩和检验。结果：CD44s、MMP－9、Ki-67在骨肉瘤中的阳性表达率分别为71．0％、75．8％和35．5％,与三种分子在骨良性病变和正常对照组织中的阳性表达率相比,其表达水平显著性升高（P值均小于0．05）。CD44s和MMP－9在初发性和复发性骨肉瘤之间的表达差异无显著性意义,但Ki-67在术后复发的骨肉瘤中的阳性率为81．8％,比初发性骨肉瘤中的阳性率显著性升高。CD44s和Ki-67在伴有肺转移的骨肉瘤中的阳性率分别为88．9％和66．7％,均显著高于两者在不伴有肺转移的骨肉瘤中的表达水平;但MMP－9在有无肺转移骨肉瘤之间的表达差异无显著性意义。CD44s和MMP－9在低分化性骨肉瘤中的阳性率分别为76．3％和79．7％,均显著高于两者在高分化性骨肉瘤中的表达水平;但Ki-67在不同分化骨肉瘤之间的表达差异无显著性意义。Spearman相关分析证明CD44s、MMP－9、Ki-67三者之间的表达均有显著性相关关系。结论：骨肉瘤组织中CD44s、MMP－9和Ki-67的表达水平均升高,三者共同促进肿瘤细胞的生长和局部的侵袭破坏。骨肉瘤术后是否复发与肿瘤的增殖状况有关,而与CD44s和MMP－9的表达无关;骨肉瘤是否出现早期远处转移受粘附分子CD44s的表达量和细胞增殖状况的影响,而与MMP－9的关系不大;CD44s和MMP－9的表达水平随骨肉瘤细胞分化程度的降低而升高,但不同分化程度的骨肉瘤组织之间的增殖速率无显著性差异。     

Critical role of notch signaling in osteosarcoma invasion and metastasis.

PURPOSE: Notch signaling is an important mediator of growth and survival in several cancer types, with Notch pathway genes functioning as oncogenes or tumor suppressors in different cancers. However, the role of Notch in osteosarcoma is unknown. EXPERIMENTAL DESIGN: We assessed the expression of Notch pathway genes in human osteosarcoma cell lines and patient samples. We then used pharmacologic and retroviral manipulation of the Notch pathway and studied the effect on osteosarcoma cell proliferation, survival, anchorage-independent growth, invasion, and metastasis in vitro and in vivo. RESULTS: Notch pathway genes, including Notch ligand DLL1, Notch1 and Notch2, and the Notch target gene HES1, were expressed in osteosarcoma cells, and expression of HES1 was associated with invasive and metastatic potential. Blockade of Notch pathway signaling with a small molecule inhibitor of gamma secretase eliminated invasion in Matrigel without affecting cell proliferation, survival, or anchorage-independent growth. Manipulation of Notch and HES1 signaling showed a crucial role for HES1 in osteosarcoma invasiveness and metastasis in vivo. CONCLUSION: These studies identify a new invasion and metastasis-regulating pathway in osteosarcoma and define a novel function for the Notch pathway: regulation of metastasis. Because the Notch pathway can be inhibited pharmacologically, these findings point toward possible new treatments to reduce invasion and metastasis in osteosarcoma.     

MTA1基因表达与人骨肉瘤细胞浸润和转移的关系

Objective： To compare the expression level of metastasis associated-1 （MTA1） in the higher and lower metastasis sublines of human osteosarcoma cells （MG63）, and to investigate the relationship between the expression level of MTA1-EGFP and in vitro invasion and metastasis of human osteosarcoma cells. Methods： The expression level of MTA1 in two sublines of MG63 cells was detected by semi-quantitative RT-PCR, and cell invasion assay and cell proliferation assay were used to evaluate the invasive capacity in vitro in two sublines. The lower metastasis line of MG-63 cells were transfected with MTA1-EGFP full-length cDNA expression plasmid by lipofectamine. The changes of the MTA1-EGFP expression and in vitro invasion potential were measured after transfection. Results： M8 subline expressed significantly higher level of MTA1 than that of M6 subline by RT-PCR. The invasive potentials of low metastasis MG63 cell line were increased after MTA1 gene transfection. Conclusion： There may be a relationship between MTA1 and invasive potentials of human osteosarcoma cells, and MTA1 may play a role in the molecular mechanism of tumor metastases and be a potential target for gene therapy of osteosarcoma. Further studies of MTA1 in human ostersarcoma cell metastasis are needed.     

Pancreatic ductal adenocarcinoma mice lacking mucin 1 have a profound defect in tumor growth and metastasis

MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G(2)-M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro.     

Functional aspects of three molecules associated with metastasis development in human malignant melanoma

To identify molecules which may be functionally associated with the development of metastasis in human melanoma, monoclonal antibodies which discriminate between benign and malignant melanocytic lesions in situ were selected. Biochemical studies and cDNA cloning identified the antigens HLA-DR, ICAM-1 and MUC18 which showed an expression pattern on primary tumors correlating with vertical tumor thickness, the most predictive parameter for the development of metastasis in melanoma. ICAM-1 and MUC18 show sequence similarity to a family of cell adhesion molecules which include the neural cell adhesion molecule NCAM. Both HLA-DR and ICAM-1 can be induced on melanoma cells by lymphokines, suggesting a role of the mononuclear cell infiltrate in the control of tumor cell phenotype. Knowledge of the normal function of these molecules allows one to hypothesize how they may contribute to the successful development of metastases.     

腺病毒介导VEGF-siRNA对荷人骨肉瘤裸鼠移植瘤生长的影响

目的:观察腺病毒介导的VEGF-siRNA对荷人骨肉瘤细胞株MG63裸鼠移植瘤生长的影响。方法:将Ad-VEGF-siRNA感染MG63,用噻唑蓝(thiazolyl blue,MTT)法检测细胞体外增殖活力,反转录-聚合酶链反应(reverse transeription-poly-merase chain reaction,RT-PCR)法检测VEGF抑制效果。建立裸鼠移植瘤模型,定期瘤内注射Ad-VEGF-siRNA,观察裸鼠致瘤率、肿瘤体积、抑瘤率及免疫组化法检测肿瘤组织中bcl-2的表达。结果:感染Ad-VEGF-siRNA能明显抑制MG63细胞的生长,瘤灶内注射Ad-VEGF-siRNA后裸鼠移植瘤的质量和体积均显著低于对照组(P<0.01)。TUNEL染色显示肿瘤细胞凋亡增加,免疫组化结果提示肿瘤组织中bcl-2表达明显减少(P<0.01)。结论:Ad-VEGF-siRNA可有效而特异地阻断VEGF基因表达,抑制裸鼠移植瘤生长,促进肿瘤细胞的凋亡。     

MUC1的表达与乳腺癌细胞粘附的关系

背景与目的:近年研究认为,肿瘤的转移与肿瘤细胞膜上粘液型糖蛋白的大量形成有关。本实验研究糖基化抑制剂苯甲基-α-N-乙酰半乳糖胺对粘蛋白-1(mucin1,MUC1)形成的抑制作用及其对乳腺癌细胞株MDA-MB-231细胞的粘附、侵袭转移能力的影响。方法:免疫细胞化学法检测MUC1表达,噻唑蓝比色法(MTT)检测细胞对人工基底膜(Matrigel)的粘附能力,明胶酶谱法(zymography)检测MDA-MB-231细胞MMP-2、MMP-9的分泌变化。结果:经苯甲基-α-N-乙酰半乳糖胺去糖基化处理后的肿瘤细胞组与相应对照组比较,MUC1表达降低,对基底膜的粘附力明显降低(P<0.01),同时细胞中MMP-2和MMP-9的分泌量显著下降。结论:MUC1表达水平与肿瘤细胞的粘附能力相关,MUC1的抑制使乳腺癌MDA-MB-231细胞粘附能力,分泌Ⅳ型胶原酶能力明显减弱。     

Clinical significance of MUC1 and MUC2 mucin and p53 protein expression in colorectal carcinoma

BACKGROUND: Up-regulation of MUC1, down-regulation of MUC2 and p53 overexpression are seen in colorectal carcinomas. However, there have been few reports about the associations between MUC1, MUC2 and p53 expression and metastatic potential. The aim of this study was to investigate MUC1, MUC2 and p53 expression in colorectal carcinoma with special reference to regional and distant metastasis. METHODS: Eighty-six colorectal carcinomas were collected from patients undergoing tumor resection. Sections were used for MUC1, MUC2 and p53 immunostaining. Cancers were regarded as MUC1 or MUC2 positive when the positive cells were beyond 30% of cancer cells. Cancers with diffuse or nested patterns were regarded as having p53 overexpression. RESULTS: Of 86 cancers, 37 (43%) were MUC1 positive, 28 (33%) were MUC2 positive and 59 (69%) showed p53 overexpression. A difference was observed only in the frequency of MUC1 positivity with respect to depth of tumor invasion. Neither depth of tumor invasion nor histological differentiation had a positive correlation with MUC1, MUC2 and p53 overexpression. The frequency of MUC1 positive cells in Dukes' C and D tumors was significantly higher than that in Dukes' A and B tumors. The frequency of MUC1 positivity in tumors with hepatic involvement was significantly higher than that in tumors without hepatic involvement (100 vs 39%; p < 0.01). There was no difference in the frequency of MUC2 or p53 positivity in Dukes' stage or hepatic metastasis. MUC1 immunoreactivity of the surface was identical with that of the whole tumor in 81% (70/86) of carcinomas, MUC 2 in 87% and p53 in 100%. CONCLUSIONS: The results suggest that up-regulation of MUC1 is involved in the progression from the non-metastatic to the metastatic stage and that p53 abnormality is not directly involved in it. The data also imply that immunostaining of preoperative biopsy samples is useful for evaluating the immunoreactivity of the whole tumor.