PP1蛋白在转移及非转移性恶性黑色素瘤中的表达

目的 研究蛋白磷酸酶-1(PP1)对恶性黑色素瘤转移的作用.方法 采用Western blot检测PP1蛋白在非转移性恶性黑色素瘤细胞系-HMCB及转移性恶性黑色素瘤细胞系WM266-4中的表达.用免疫组化的方法检测32例未转移恶性黑色素瘤及20例转移性恶性黑色素瘤标本中PP1的表达.结果 本研究证明非转移性恶性黑色素瘤细胞及组织中PP1蛋白表达明显高于在转移性恶性黑色素瘤细胞及组织中的表达,差异有统计学意义(P<0.05).结论 PP1在转移及未转移恶性黑色素瘤细胞及组织中的差异表达为恶性黑色素瘤转移机制方面的研究开辟新的方向.     

PP1蛋白在转移及非转移性恶性黑色素瘤中的表达

目的：研究蛋白磷酸酶-1（PP1）对恶性黑色素瘤转移的作用。方法：采用Western blot检测PP1蛋白在非转移性恶性黑色素瘤细胞系-HMCB及转移性恶性黑色素瘤细胞系WM266-4中的表达。用免疫组化的方法检测32例未转移恶性黑色素瘤及20例转移性恶性黑色素瘤标本中PP1的表达。结果：本研究证明非转移性恶性黑色素瘤细胞及组织中PP1蛋白表达明显高于在转移性恶性黑色素瘤细胞及组织中的表达,差异有统计学意义（P〈0.05）。结论：PP1在转移及未转移恶性黑色素瘤细胞及组织中的差异表达为恶性黑色素瘤转移机制方面的研究开辟新的方向。     

In vivo evaluation of cell mediated immunity against human melanoma

Evaluation of cell mediated immunity against human melanoma target cells was performed in an in vivo model using human tumor xenografts growing in Balb/c athymic mice. Intraperitoneal inoculation of 1 × 10⁷ human melanoma cells produced peritoneal carcinomatosis which lead to death of the animals at 23.8 ± 2.6 days (N = 12). Peripheral blood lymphocytes (PBL) from normal donors were administered to tumor bearing mice, and survival times of 22.0±2.3 days were observed (N = 8). Peripheral blood lymphocytes from four of five normal donors which had been presensitized on monolayers of melanoma tissue culture cells in vitro failed to prolong host survival times. In contrast, PBL obtained from 15 of 20 melanoma patients were found to prolong survival of the tumor bearing nude mice. Of these 15 patients, 8 were undergoing specific active immunotherapy, while 7 had not been sensitized except by the disease process. The ability of PBL obtained from patients to prolong survival of tumor bearing animals did not appear to correlate with either the stage of the disease or the patient's clinical course. The possible mechanisms for the prolonged survival and usefulness of this model are discussed.     

Expression and function of CD9 in melanoma cells

CD9, a member of the tetraspanin family, functions as an organizer in “tetraspanin webs,” through interacting with other cell adhesion molecules. It plays a role in differentiation, fertilization, and cell migration. We investigated the expression and function of CD9 in melanoma. CD9 protein expression in B16 mouse melanoma and six human melanoma cell lines was decreased compared to normal melanocytes. B16F1 clones stably overexpressing CD9 had reduced ability to form colonies in soft agar; however, paradoxically these overexpressing clones had increased ability to invade Matrigel. Similarly, transient overexpression of CD9 in the human metastatic melanoma cell line WM9 dramatically decreased anchorage‐independent growth, while transient overexpression of CD9 in the radial growth phase cell line SbCl2 resulted in the gain of Matrigel invasion activity. DNA sequencing of CD9 cDNA from all six human melanoma cell lines did not show deletions, insertions, or mutations. Treatment of all six human melanoma cell lines with the histone deacetylase inhibitor trichostatin A increased CD9 levels. The DNA methylation inhibitor 5‐aza‐cytidine also increased CD9 protein levels with greater increases seen in cell lines derived from more malignant melanomas. © 2009 Wiley‐Liss, Inc.     

PTEN基因蛋白在恶性黑素瘤中的表达及意义

目的探讨PTEN基因蛋白在恶性黑素瘤中的表达及其意义.方法采用免疫组化EnVision检测PTEN基因蛋白在51例恶性黑素瘤以及30例皮内痣组织中的表达情况.结果恶性黑素瘤组织中PTEN表达强度低,总体阳性率52.9%(27/51),其中Ⅰ、Ⅱ、Ⅲ各期阳性率分别为59.4%(17/32)、38.5%(5/13)、33.3%(2/6);而皮内痣中PTEN均呈强阳性,表达阳性率为100%(30/30),二者差异具有显著性(P＜0.01).结论恶性黑素瘤的发生可能与PTEN基因蛋白的低表达有关,PTEN基因蛋白表达缺失或低下者更易发生远处转移,标记PTEN可作为判断黑素瘤预后的一项指标.     

人黑色素瘤细胞系基因突变的研究

目的　揭示金属蛋白酶类 (MMPs) ,层粘连蛋白受体 (LN R)与人黑色素瘤细胞侵袭转移的关系 ,并探讨MMPs ,LN R用以判断肿瘤细胞侵袭转移的可能性。方法　通过流式细胞术 (FCM )定量研究和蛋白酶活性分析 (zy mography) ,对具有不同潜在转移能力的人黑色素瘤细胞进行研究。 结果　早期WM 3 5不产生MMPs ;WM 13 4 1B仅产生MMP 2不产生MMP 9;进展期WM 983A和远处转移瘤株WM 45 1既产生MMP 2又产生MMP 9。瘤细胞表面67KDLN R的荧光阳性率和全部细胞的平均荧光强度大小顺序为WM 45 1>WM 983A >WM 13 4 1B >WM 3 5。结论　MMPs和LN R与人黑色素瘤细胞系的侵袭转移能力获得之间存在着密切关系 ,并可作为一种较特异的肿瘤侵袭转移标记物被应用于肿瘤研究与治疗中。     

Malignant melanoma in a blind eye

A 62-year-old man developed a malignant melanoma in his left eye that had been blind due to trauma for 35 years. This case illustrates the difficulties in detection and early diagnosis of occult malignant melanoma of the uveal tract in phthistic globes. The necessity for long-term follow-up of posttraumatic eyes is stressed.     

Cytotoxic effect of a new 1,3,4-thiadiazolium mesoionic compound (MI-D) on cell lines of human melanoma

The structural characteristics of mesoionic compounds, which contain distinct regions of positive and negative charges associated with a poly-heteroatomic system, enable them to cross cellular membranes and interact strongly with biomolecules. Potential biological applications have been described for mesoionic compounds. 1,3,4-Thiadiazolium mesoionic compound (MI-D), a new mesoionic compound, has been demonstrated to be extremely cytotoxic to B16-F10 murine melanoma cells when compared to fotemustine and dacarbazine, drugs of reference in melanoma treatment protocols, describing inhibition of tumours grown in vitro and in vivo. We now evaluate the effects of mesoionic compound MI-D on different human melanoma cell lines. The drug decreased the viability and proliferation of MEL-85, SK-MEL, A2058 and MEWO cell lines in vitro, showing a considerable cytotoxic activity on these human cells. Adhesion of MEL-85 cells was evaluated in the presence of the drug using different extracellular matrix (ECM) constituents. MI-D decreased MEL-85 adhesion to laminin, fibronectin and matrigel. The morphology and actin cytoskeleton organisation of MEL-85 cells were also modified on MI-D treatment. These results on human melanoma cell lines indicate that MI-D is a very encouraging drug against melanoma, a tumour that is extremely resistant to chemotherapy.     

Functional genomics of calcium channels in human melanoma cells

Ca(2+)-signaling of human melanoma is in the focus of intensive research since the identification of the role of WNT-signaling in melanomagenesis. Genomic and functional studies pointed to the important role of various Ca(2+) channels in melanoma, but these data were contradictory. In the present study we clearly demonstrate, in a number of different ways including microarray analysis, DNA sequencing and immunocytochemistry, that various human melanoma cell lines and melanoma tissues overexpress ryanodine receptor type 2 (RyR2) and express P2X(7) channel proteins as compared to melanocytes. These channels, although retain some of their usual characteristics and pharmacological properties, display unique features in melanoma cells, including a functional interaction between the two molecules. Unlike P2X(7), RyR2 does not function as a calcium channel. On the other hand, the P2X(7) receptor has an antiapoptotic function in melanoma cells, since ATP-activation suppresses induced apoptosis, while knock down of the gene expression significantly enhances that.     

Correlation of molecular human leukocyte antigen typing and outcome in high‐risk melanoma patients receiving adjuvant interferon

BACKGROUND:

Interferon is approved for adjuvant treatment of patients with stage IIB/III melanoma. The identification of predictive markers that would permit selection of patients would be beneficial. Specific human leukocyte antigen (HLA) class I and II antigens have previously shown an association with response to therapy or overall survival of patients with metastatic melanoma.

METHODS:

A total of 284 high‐risk melanoma patients participating in a randomized trial and 246 healthy controls were molecularly typed for HLA class I and II. Specific allele frequencies were compared between the healthy and patient populations, as well as presence or absence of these in relation to recurrence. Alleles related to autoimmune disease were also investigated.

RESULTS:

No significant differences were found between the distribution of HLA genotype in the melanoma population compared with healthy controls. Correlations between nonrecurrence and the presence of HLA‐Cw*06 allele were noted present in 19.3% of melanoma patients. The median relapse‐free survival of the Cw*06‐positive cohort was 100.2 months versus 37.3 months in the Cw*06‐negative cohort (P = .013). The median overall survival for the Cw*06‐positive cohort has not yet been reached, versus 78.9 months in the Cw*06‐negative cohort (P = .025). HLA‐Cw*06 was present in 29.79% of patients in the autoimmunity group and 15.38% of patients in the nonautoimmunity group (P = .049).

CONCLUSIONS:

No allele was associated with absence of recurrence in patients receiving adjuvant interferon with the exception of HLA‐Cw*06, an allele correlated with psoriasis. HLA‐Cw*06‐positive patients have better relapse‐free and overall survival. Cancer 2010. © 2010 American Cancer Society.     

Drug-resistance in human melanoma

Advanced malignant melanoma has a poor prognosis since chemotherapy is mostly ineffective due in part to the intrinsic and/or extrinsic resistance of melanoma cells to systemic treatment with anti-neoplastic agents. The reasons for the chemoresistant phenotype are unknown. The relevance of well-analyzed drug-resistance mechanisms, e.g., intracellular/extracellular transport and induction of certain enzyme systems, is reviewed. Most anti-cancer drugs kill susceptible cells through induction of apoptosis. Therefore, it appears that differences in the apoptotic pathways which lead to apoptotic deficiency may account for the ability of some tumor cells to resist drug therapy. Human melanomas, which are characteristically drug-resistant, are more likely to have altered apoptotic pathways and fewer pro-apoptotic molecules. Tumor cells with these characteristics are seldom sensitive to drugs. The complexity of the molecular variants involved in signal transduction along apoptotic pathways suggests that the cell may have a variety of possibilities for regulating apoptosis and generating apoptotic deficiency. Thus, apoptosis and apoptotic deficiency should be analyzed to better clarify the mechanisms of melanoma resistance.     

Expression and prognostic significance of iNOS in uveal melanoma

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Disease metastasis occurs in half of the patients and is uniformly fatal despite systemic therapy. Inducible nitric oxide synthase (iNOS) is associated with disease progression in various malignancies including cutaneous melanoma. In this retrospective cohort, we examined the prognostic value of iNOS in UM by performing immunohistochemistry on paraffin‐embedded sections of primary tumors (90 patients) and matched primary and metastatic hepatic tumors (19 patients) with complete histopathological and clinical data. We show that iNOS is expressed in UM (57% of the patients) and high iNOS levels significantly (p = 0.04; hazard ratio (HR) = 2.3) predict disease‐specific survival (DSS) as assessed by Kaplan‐Meier analysis and univariate Cox's proportional hazards regression model. Furthermore, high iNOS expression in the UM primary tissue was significantly associated with metastatic disease and vice versa. Expression of iNOS in hepatic metastases significantly (p = 0.02) predicted a shortened survival as assessed by Kaplan‐Meier analysis. However, iNOS did not appear to be a significant (p = 0.16; HR = 1.9) factor in the multivariate Cox's regression analysis performed together with the clinical parameters tumor diameter, tumor cell type, and tumor location in which only tumor diameter predicted DSS. In conclusion, iNOS predicts DSS in UM and may play a role in disease progression but it is not an independent prognostic factor.     

The adhesion molecule L1 (CD171) promotes melanoma progression

The adhesion molecule L1 is expressed in primary melanomas and cutaneous metastases in contrast to melanocytic nevi and melanocytes, and is significantly associated with metastatic spread. Recent studies have demonstrated that in carcinomas L1 expression is associated with sustained activation of the extracellular signal-regulated kinase (ERK) pathway and upregulation of ERK-dependent, motility- and invasion-associated gene products including alphavbeta3 integrin. The objective of this study was to further investigate the role of the adhesion molecule L1 in melanoma progression, and to evaluate whether targeting the L1 adhesion molecule would have therapeutic effects against invasive melanoma growth. Using human melanoma cells from different stages of progression in monolayer and organotypic human skin culture mimicking the pathophysiological environment of cutaneous melanoma, we found that (1) L1 expression mostly correlates with melanoma progression and alphavbeta3 integrin expression, (2) overexpression of L1 in early radial growth phase melanoma cells promotes conversion from radial to vertical growth phase melanoma without upregulation of alphavbeta3 integrin expression, and (3) suppression of L1 function significantly reduces migration and invasion of melanoma cells, but does not completely block invasive melanoma growth. Altogether, L1 plays a critical role in melanoma invasion and progression and offers therapeutic potential in combination with conventional anticancer agents.     

Oncogenic NRAS has multiple effects on the malignant phenotype of human melanoma cells cultured in vitro

Activating mutations in the NRAS gene, which occur predominantly in codon 61 (Q61R, Q61K) are among the most common genetic events in malignant melanoma. NRAS protein with oncogenic codon 61 mutations may therefore be good therapeutic targets. In the present study, we used gene expression profiling as a method for global characterization of gene expression alterations that resulted from treatment of melanoma cells with siRNA specifically targeting NRAS(Q61R). Sixteen probe sets representing 15 unique genes were identified whose expression was significantly altered by siRNA against NRAS(Q61R) in 2 melanoma cell lines. The genes with altered expression are involved in several functions, including modulation of cell growth, invasion and migration. The results suggest that downregulation of cyclin E2 and cyclin D1 and also upregulation of the negative cell-cycle regulator HBP1 in NRAS(Q61R) knockdown cells contribute to the inhibition of cell proliferation. Furthermore, suppression of oncogenic NRAS results in reduced migration and invasion, which is accompanied by downregulation of EphA2 (a receptor tyrosine kinase), uPAR (urokinase receptor) and cytoskeleton proteins such as leupaxin, paxillin and vinculin. These studies support the concept that suppression of oncogenic NRAS by siRNA can induce growth arrest and inhibit invasion of human melanoma cells by modulating the levels of these gene products.     

Systemic therapy of malignant melanoma

The present status of medical treatment of malignant melanoma is briefly reviewed, both with regard to adjuvant therapy for individuals with high-risk melanoma and a high probability of harbouring subclinical rnicrometastases, as well as to therapy for establised disseminated (macrometastatic) disease. At present, disseminated, macrometastatic melanoma is incurable in the majority of cases. Single agent chemotherapy has modest effects and results in disease remission in a minority of patients, usually of short duration, Combination chemotherapy, or the combination of chemotherapeutic drugs and cytokines, results in increased response rates and occasionally remissions of prolonged duration. So far, no regimen has demonstrated improved survival compared to single agent therapy in disseminated melanoma. New insights into the mechanisms of resistance to chemotherapeutic drugs may lead to development of predictive tests that can identify individuals with tumors sensitive to a specific agent, as well as to the development of strategies to circumvent drug resistance. It has recently been shown that adjuvant therapy of high-risk melanoma with large doses of interferon-α2b significantly prolongs relapse-free and overall survival, at the price of considerable toxicity. Ongoing studies aim to define the optimum dose and duration of adjuvant interferon therapy. Recent advances in molecular biology and immunology may lead to the development of new treatment modalities, such as improved vaccines and other biologic therapies, which may benefit patients with malignant melanoma.