Tyrosinase suppresses vasculogenic mimicry in human melanoma cells

Melanoma is a type of skin cancer that derives from melanocytes; this tumor is highly metastatic and causes poor clinical outcomes in patients. Vasculogenic mimicry (VM), a vascular-like network that is formed by tumor cells instead of endothelial cells, promotes the growth and metastasis of tumors by providing tumors with oxygen- and nutrient-containing blood. VM correlates with a poor prognosis in patients with melanoma, but the melanoma-specific mechanisms of VM are unknown. The present study revealed that treatment with the melanogenesis stimulators 3-isobutyl 1-methylxanthine (IBMX) and α-melanocyte-stimulating hormone (α-MSH) significantly inhibited VM in MNT-1 human pigmented melanoma cells. Tyrosinase (TYR), an essential enzyme in melanin production, was upregulated on treatment with α-MSH and IBMX, prompting an examination of the association between TYR and VM. A TYR inhibitor, arbutin, promoted VM in melanoma cells. Furthermore, CRISPR/Cas9-mediated knockout (KO) of TYR increased VM by melanoma cells. Notably, even in non-pigmented melanoma cells, TYR attenuated VM. Although re-expression of wild-type TYR suppressed VM in TYR-KO cells, T373K TYR, a frequently detected mutation in individuals with albinism, failed to inhibit VM. Overall, these results demonstrated that TYR negatively regulates VM, providing novel insights into the antioncogenic function of TYR in melanomas.     

Inhibitors of melanogenesis increase toxicity of cyclophosphamide and lymphocytes against melanoma cells

High mortality rate for metastatic melanoma is related to its resistant to the current methods of therapy. Melanogenesis is a metabolic pathway characteristic for normal and malignant melanocytes that can affect the behavior of melanoma cells or its surrounding environment. Human melanoma cells in which production of melanin pigment is dependent on tyrosine levels in medium were used for experiments. Peripheral blood mononuclear cells were derived from the buffy coats purchased from Lifeblood Biological Services. Cell pigmentation was evaluated macroscopically, and tyrosinase activity was measured spectrophotometrically. Cell proliferation and viability were measured using lactate dehydrogenase release MTT, [³H]‐thymidine incorporation and DNA content analyses, and gene expression was measured by real time RT‐PCR. Pigmented melanoma cells were significantly less sensitive to cyclophosphamide and to killing action of IL‐2‐activated peripheral blood lymphocytes. The inhibition of melanogenesis by either blocking tyrosinase catalytic site or chelating copper ions sensitized melanoma cells towards cytotoxic action of cyclophosphamide, and amplified immunotoxic activities of IL‐2 activated lymphocytes. Exogenous L‐DOPA inhibited lymphocyte proliferation producing the cell cycle arrest in G1/0 and dramatically inhibited the production of IL‐1beta, TNF‐alpha, IL‐6 and IL‐10. Thus, the active melanogenesis could not only impair the cytotoxic action of cyclophosphamid but also has potent immunosuppressive properties. This resistance to a chemotherapeutic agent or immunotoxic activity of lymphocytes could be reverted by the action of tyrosinase inhibitors. Thus, the inhibition of melanogenesis might represent a valid therapeutic target for the management of advanced melanotic melanomas. © 2008 Wiley‐Liss, Inc.     

Molecular biology of human melanoma development and progression

In the United States, Australia, Northern Europe, and Canada, malignant melanoma is increasing at a faster rate than any other cancer, with the exception of lung cancer in women. Major advances have been made in the molecular biology and immunology of melanoma. These advances in basic science have led to more rational approaches to specifically targeting melanoma cells, with promising results in the clinic. An increased understanding of how melanoma spreads has led to more selective, less invasive surgical procedures that do not compromise patient health. Combinations of chemotherapy and immunotherapy are now available for patients with advanced melanoma that affect both the length and quality of the patients' lives. This review of the molecular biology of melanoma development and progression discusses the disease's etiology, molecular genetics, cell-surface antigens, experimental models, biological markers, and new forms of treatment. As we continue to learn more about malignant melanoma, we will be able to devise more specific and effective treatments that will give patients with this potentially deadly disease longer and more productive lives. Mol. Carcinog. 23:132–143, 1998. © 1998 Wiley-Liss, Inc.     

Clinical significance of the RT-PCR positive sentinel node in melanoma

BACKGROUND: The clinical relevance of RT-PCR positivity for melanoma markers in the sentinel node remains controversial. Our purpose was to determine whether patients with a histologically negative but RT-PCR positive node were at an increased risk for recurrence than their RT-PCR negative counterparts. METHODS: Thirty-nine adult patients underwent sentinel node biopsies for melanoma between 1998 and 2000. Each sentinel node was bivalved. Half was serially sectioned and examined by routine hematoxylin and eosin (H&E) and immunohistochemistry (IHC; S100, HMB-45, melanA, and tyrosinase). The other half was analyzed by a nested RT-PCR assay for tyrosinase. RESULTS: Patients were followed for recurrence with a mean follow-up of 71.1 months. The odds ratio of recurrence for RT-PCR positive versus RT-PCR negative patients was 1.39 (0.34, 5.62; p = 0.73). Within the histology negative subgroups, the risk of recurrence in the RT-PCR positive group (26.7%) was not significantly different from the risk of recurrence in the RT-PCR negative group (22.2%) (p = 0.33 chi-squared). RT-PCR of the sentinel node was not a predictor for recurrence on multivariate analysis (p = 0.65). CONCLUSION: Sentinel node RT-PCR positivity did not risk stratify histologically negative melanoma patients beyond routine pathologic examination in this series.     

Surgical approach to malignant melanoma in the gastrointestinal tract

The gastrointestinal (GI) tract is a common site for malignant melanoma. Diagnosis of lesions in the GI tract is usually delayed until complications occur, such as obstruction, bleeding, or perforation of the GI tract. Of 348 patients with malignant melanoma treated during a 10-year period, 11 had GI involvement either in a metastatic form or as a primary melanoma. Three of these patients were treated surgically for metastatic lesions in the small bowel causing intussusception, two for peritonitis secondary to perforation of the small bowel, and one for massive bleeding from metastatic melanoma in the stomach. Another patient had a primary melanoma in the esophagus and underwent esophagectomy. Three patients had primary melanomas of the anal canal and one of the rectum. Three of them underwent abdominoperineal resections, and two had bilateral groin dissection in addition. Six of the patients are alive 6 months to 4 years following diagnosis. The remaining five died of metastatic melanoma from 6 months to 4 years post-surgery.     

A novel AKT3 mutation in melanoma tumours and cell lines

Recently, a rare activating mutation of AKT1 (E17K) has been reported in breast, ovarian, and colorectal cancers. However, analogous activating mutations in AKT2 or AKT3 have not been identified in any cancer lineage. To determine the prevalence of AKT E17K mutations in melanoma, the most aggressive form of skin cancer, we analysed 137 human melanoma specimens and 65 human melanoma cell lines for the previously described activating mutation of AKT1, and for analogous mutations in AKT2 and AKT3. We identified a single AKT1 E17K mutation. Remarkably, a previously unidentified AKT3 E17K mutation was detected in two melanomas (from one patient) as well as two cell lines. The AKT3 E17K mutation results in activation of AKT when expressed in human melanoma cells. This represents the first report of AKT mutations in melanoma, and the initial identification of an AKT3 mutation in any human cancer lineage. We have also identified the first known human cell lines with naturally occurring AKT E17K mutations.     

Nitrogen-containing bisphosphonates inhibit cell cycle progression in human melanoma cells

Cutaneous melanoma is one of the highly malignant human tumours, due to its tendency to generate early metastases and its resistance to classical chemotherapy. We recently demonstrated that pamidronate, a nitrogen-containing bisphosphonate, has an antiproliferative and proapoptotic effect on different melanoma cell lines. In the present study, we compared the in vitro effects of three different bisphosphonates on human melanoma cell lines and we demonstrated that the two nitrogen-containing bisphosphonates pamidronate and zoledronate inhibited the proliferation of melanoma cells and induced apoptosis in a dose- and time-dependent manner. Moreover, cell cycle progression was altered, the two compounds causing accumulation of the cells in the S phase of the cycle. In contrast, the nonaminobisphosphonate clodronate had no effect on melanoma cells. These findings suggest a direct antitumoural effect of bisphosphonates on melanoma cells in vitro and further support the hypothesis of different intracellular mechanisms of action for nitrogen-containing and nonaminobisphosphonates. Our data indicate that nitrogen-containing bisphosphonates may be a useful novel therapeutic class for treatment and/or prevention of melanoma metastases.     

伊马替尼治疗78例KIT变异的晚期黑色素瘤的疗效和安全性

目的:旨在探讨酪氨酸激酶抑制剂伊马替尼在KIT突变/扩增的晚期黑色素瘤患者中的疗效及安全性.方法:回顾性分析2009年11月至2015年9月北京大学肿瘤医院肾病黑色素瘤科78例KIT突变/扩增的晚期黑色素瘤患者的临床资料,患者接受伊马替尼口服(400 mg/日)治疗,直至疾病进展或发生不能耐受的不良反应.结果:78例患者可评价疗效.全组患者客观缓解率22.4％,疾病控制率60.6％.17例部分缓解患者中,有11例为11或13号外显子突变.全组患者中位无疾病进展时间3.9个月(95％ CI:2.1 ～5.8个月),中位总生存时间13.2个月(95％ CI:10.1～16.3个月);1年生存率57％,2年生存率36％,3年生存率19％.最常见的不良反应包括水肿、乏力、食欲减退、皮疹、粒细胞下降(发生率均≥10％),未发现致命性药物不良反应.结论:伊马替尼治疗KIT突变/扩增的晚期黑色素瘤具有一定的疗效,且安全性良好.     

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。     

Temozolomide induces senescence but not apoptosis in human melanoma cells

Temozolomide (TMZ), a DNA alkylating agent used in the treatment of melanoma, is believed to mediate its effect by addition of a methyl group to the O(6) position of guanine in DNA. Resistance to the agent may be in part due to the activity of O(6)-methylguanine-DNA methyl transferase (MGMT). In the present study, we show that sensitivity of melanoma cells to TMZ was dependent on their p53 status and levels of MGMT. Analysis of the mechanisms underlying reduced viability showed no evidence for induction of apoptosis even though marked levels of apoptosis was seen in TK6 lymphoma cells. Sensitivity of melanoma cells was associated with p53-dependent G2/M cell cycle arrest and induction of senescence. To verify the role of p53, the assays were repeated in presence of pifithrin-alpha, an inhibitor of p53. This resulted in increased viability of melanoma cells with wild-type p53 and reversed G2/M cell cycle arrest. Paradoxically, apoptosis was increased in melanoma but decreased as expected in TK6 lymphoma cells. These results are consistent with the view that TMZ is relatively ineffective against melanoma due to defective apoptotic signalling resulting from activation of p53. The nature of the defects in apoptotic signalling remains to be explored.     

The bisphosphonate pamidronate induces apoptosis in human melanoma cells in vitro

Pamidronate belongs to the class of nitrogen-containing bisphosphonates that are potent inhibitors of bone resorption frequently used for the treatment of osteoporosis and cancer-induced osteolysis. The inhibition of osteoclasts' growth has been suggested as the main mechanism of the inhibitory effect of pamidronate on bone metastases. Recent findings indicated that bisphosphonates also have a direct apoptotic effect on other types of tumour cells. Nitrogen-containing bisphosphonates were shown to inhibit farnesyl diphosphate synthase, thus blocking the synthesis of higher isoprenoids. By this mechanism they inactivate monomeric G-proteins of the Ras and Rho families for which prenylation is a functional requirement. On the background of the known key role of G-proteins in tumorigenesis, we investigated a possible beneficial use of pamidronate in the treatment of malignant melanoma. Our results indicate that pamidronate inhibits the cell growth and induces apoptosis in human melanoma cells in vitro. Susceptibility to pamidronate did not correlate to CD95 ligand sensitivity or p53 mutational status. Furthermore it is interesting to note that overexpression of bcl-2 did not abolish pamidronate-induced apoptosis. These data suggests that pamidronate has a direct anti-tumour effect on malignant melanoma cells, independently of the Bax/Bcl-2 level.     

BRAF,a target in melanoma

The successful translation of therapies targeting signal‐transduction pathways that are activated by oncogenes has provided a model for molecularly targeted therapy, and the identification of mutations in v‐raf murine sarcoma viral oncogene homolog B1 (BRAF), a serine/threonine kinase, has turned the attention of the melanoma field toward this concept. The current review indicated that BRAF represents an important target in cancer, in part because it is present in 7% of all cancers and also because it represents the first intracellular signaling molecule that is activated by point mutations for which single‐agent therapy appears to have efficacy. Therapy for advanced melanoma has progressed slowly over the past 3 decades, although significant advances have been made in other cancers with the application of cytotoxic chemotherapy and targeted therapies. However, in melanoma, cytotoxic chemotherapies have severe limits, chemotherapy does not convincingly improve on the natural history of metastatic disease and has no role in the adjuvant setting, and cytokine therapy may have a niche in both the adjuvant and metastatic settings but confers only a modest benefit to a small proportion of patients at the cost of severe toxicity. Thus, there are few other cancers in which completely novel therapies are so highly prioritized in clinical research. Understanding network of signal‐transduction pathways and how that network may adapt to BRAF inhibition or mitogen‐activated protein kinase kinase inhibition will point to the next generation of clinical trials investigating rational combination regimens. The current investigations in melanoma will create a set of hypotheses to be tested in each cancer that harbors BRAF mutations. Cancer 2010. © 2010 American Cancer Society.     

Effects of incubation with liposomes at different temperatures on cultured melanoma cells (M14)

A melanoma cell line (M14) was used in order to investigate the effect of hyperthermia on the mechanisms of interaction between liposomes and cultured cells. The treatment was performed by adding different concentrations of multilamellar liposomes (L-alpha-dipalmitoylphosphatidylcholine, stearylamine and cholesterol in the ratio 7:2:1) to cell cultures which were then incubated at 37·0 or 41·5°C for 2 h. The damage induced by liposome treatment in normothermia or hyperthermia was evaluated by determining cell survival and by electron microscopy. When different concentrations of liposomes were used, a dose-dependent impairment of cell survival was observed. An enhancement of the cytotoxic effect was observed when the treatment was performed at 41·5°C. This effect went on even after 24 h from the end of the treatment, but the difference between cells treated in normothermia and hyperthermia was remarkably reduced. The mechanism of the liposome-plasma membrane interaction has been investigated by electron microscopy. Our observations demonstrated that the outer bilayer of the multilamellar liposomes was capable of fusing with the plasma membrane, inducing changes in its fluidity and molecular organization. Following this process the inner liposomal bilayers entered the cell. These effects seemed to be favoured when the treatment was performed under mild hyperthermic conditions, accounting for the synergic cytotoxic action displayed by the liposome-hyperthermia association.     

皮肤黑色素瘤中IDH1基因突变分析

目的:应用癌症基因组图谱(TCGA)数据库分析皮肤黑色素瘤中IDH1基因突变情况.方法:从癌症基因组图谱(TCGA)数据库收集皮肤黑色素瘤数据集,统计患者临床信息、应用cBioPortal网站对数据集进行突变、离散基因、基因共表达、生存期等分析.结果:287个病例中,IDH1基因突变15例,其中,R132C突变10例,R132L突变2例,P33S/E361K突变各1例,X174缺失1例.结论:IDH1基因突变在皮肤黑色素瘤中有一定发生率,占比5.2%,且以R132突变为主,此位点有可能成为靶向药物作用的靶点.     

Intercellular contact and the radiation response of cells of human melanoma xenografts

Cells from two human melanoma xenografts (E.E. and V.N.) were irradiated at various times up to 24 hours after dispersion of the tumors. The surviving fraction for cells exposed to 5.0 (E.E.) and 4.0 Gy (V.N.) did not change with time, and was not significantly different for cells kept at 4 degrees C and 37 degrees C in the time interval between tumor dispersion and irradiation. The complete survival curves for cells irradiated 1.5, 6, 12, and 24 hours (E.E.) and 1.5, 6, and 18 hours (V.N.) after tumor dispersion were not significantly different. It is concluded that an intercellular contact effect similar to that reported for V79 Chinese hamster spheroids and murine KHT sarcomas is not present for E.E. and V.N. human melanoma xenografts.     

SIRT1 promotes proliferation and inhibits the senescence-like phenotype in human melanoma cells

SIRT1 operates as both a tumor suppressor and oncogenic factor depending on the cell context. Whether SIRT1 plays a role in melanoma biology remained poorly elucidated. Here, we demonstrate that SIRT1 is a critical regulator of melanoma cell proliferation. SIRT1 suppression by genetic or pharmacological approaches induces cell cycle arrest and a senescence-like phenotype. Gain and loss of function experiments show that M-MITF regulates SIRT1 expression, thereby revealing a melanocyte-specific control of SIRT1. SIRT1 over-expression relieves the senescence-like phenotype and the proliferation arrest caused by MITF suppression, demonstrating that SIRT1 is an effector of MITF-induced proliferation in melanoma cells. Interestingly, SIRT1 level and activity are enhanced in the PLX4032-resistant BRAF^V600E-mutated melanoma cells compared with their sensitive counterpart. SIRT1 inhibition decreases melanoma cell growth and rescues the sensibility to PLX4032 of PLX4032-resistant BRAF^V600E-mutated melanoma cells. In conclusion, we provide the first evidence that inhibition of SIRT1 warrants consideration as an anti-melanoma therapeutic option.