Nestin 和SOX2 在皮肤恶性黑色素瘤中的表达及临床意义

目的:研究巢蛋白(Nestin)和SOX2在皮肤恶性黑色素瘤中的表达及临床意义。方法:选取2010年1月-2013年1月我院收治的恶性黑色素瘤21例患者(研究组),另选黑色素痣患者21例(对照组),应用免疫组织化学法检测Nestin和SOX2,并比较两组Nestin和SOX2表达,分析其与患者生存期和转移的关系。结果:研究组Nestin和SOX2阳性表达率显著高于对照组,两组比较差异具有统计学意义(P0.05);Nestin、SOX2阳性的恶性黑色素瘤患者生存期显著低于Nestin、SOX2阴性恶性黑色素瘤患者,两组比较差异有统计学意义(P0.05),Nestin、SOX2阳性的恶性黑色素瘤患者与Nestin、SOX2阴性的恶性黑色素瘤患者转移率比较无统计学意义(P0.05)。结论:Nestin和SOX2表达阳性与恶性黑色素有关,且与恶性黑色素瘤的生存期有关。。     

N-Acetylaspartate (NAA) and N-Acetylaspartylglutamate (NAAG) Promote Growth and Inhibit Differentiation of Glioma Stem-like Cells

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.     

Ancient Cytokines,the Role of Astakines as Hematopoietic Growth Factors

Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins (PKs) are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus, astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1, we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named as astakine 2. Both crustacean astakines lack the N-terminal AVIT motif, which is present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. We have found astakine-like sequences in 19 different invertebrate species, and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis, and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation. Astakine 1 can stimulate proliferation of hematopoietic tissue (Hpt) cells (precursor of hemocytes) as well as specifically induce differentiation of Hpt cells along the semigranular cell lineage, whereas astakine 2 plays a role in granular cell differentiation. Moreover, we discuss the impact of the putative structures of different astakines in comparison with the vertebrate prokineticins.     

Prediction of Outcome for Patients with Cutaneous Melanoma

Most patients with primary melanoma are cured by local surgery, but a significant minority develop fatal metastases. The ability to identify patients with progressive disease is central to efficient management: permitting optimal deployment of adjunctive therapy and sparing the non-progressing majority the morbidity of aggressive therapy. Accurate prediction on an individual patient basis is the ideal, but the best current prognosticators permit only assignment to risk categories. Formulaic combinations of well tried correlates of outcome (gender, ulceration, depth, thickness, and mitotic rate increase accuracy of prediction, but not to personalised level. The use of large data bases against which the attributes of individual patients may be compared is useful and amalgamation of data bases will increase the availability of this approach. The development of markers of proliferation fraction (PCNA and MIB-1) and of the metastatic phenotype (PNA-receptor status) will further refine the process. Staging of disease is critical. Ac-curacy of staging is improved by mapping the (sentinel) lymph nodes likely to contain early tumor by lymphoscintigraphy and dye/radiomarker localisation. The application of exquisitely sensitive immunohistochemical and molecular biological techniques to biopsies from tissues likely to be the site of metastases permit assessment of clinical stage with a previously impossible degree of accuracy (ultrastaging).     

Subcutaneous Adipocytes Promote Melanoma Cell Growth by Activating the Akt Signaling Pathway: ROLE OF PALMITIC ACID*

Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt.     

Interactions of Melanocytes and Melanoma Cells With the Microenvironment

Normal or malignant melanocytes interact with the microenvironment through the release of soluble factors from cells and through direct cell-cell contact. Melanoma cells produce a large number of different growth factors and cytokines that affect angiogenesis, stroma formation, motility, and the inflammatory and immune response. Most of the angiogenic growth factors produced by melanoma cells are also mitogenic for fibroblasts. The mechanisms and the receptors involved in direct cell-cell contacts of melanocytes and melanoma cells are largely unknown, but the regulatory role of keratinocytes for melanocytic cells appears at several levels. Keratinocytes induce a dendritic morphology in melanocytes, and control proliferation to maintain a constant keratinocyte/melanocyte ratio during exponential growth. Expression of cell surface adhesion receptors is controlled by keratinocytes on melanocytes and nevus cells but not on advanced melanoma cells. These studies underline the complex interactions between skin cells. The escape of melanocytes from the control by keratinocytes may be a hallmark of nevus cells, and the constitutive production of various growth factors and cytokines appears to represent a major characteristic of melanoma cells.     

Astroglial-Conditioned Media and Growth Factors Modulate Proliferation and Differentiation of Astrocytes in Primary Culture

Astroglial conditioned media (ACM) influence the development and maturation of cultured nerve cells and modulate neuron-glia interaction. To clarify mechanisms of astroglial cell proliferation/differentiation in culture, incorporation of [methyl-³H]-thymidine or [5,6-³H]-uridine in cultured astrocytes was assessed. Cultures were pre-treated with epidermal growth factor (EGF), insulin (INS), insulin-like growth factor-I (IGF-I), and basic fibroblast growth factor (bFGF) and subsequently with ACM. DNA labeling revealed a marked stimulatory effect of ACM from 15 days in vitro (DIV) cultures in 30 DIV astrocytes after12 h pre-treatment with growth factors. The main effects were found after INS or EGF pre-treatment in 30 DIV cultures. ACM collected from 15 or 60 or 90 DIV increased RNA labeling of 15 and 30 DIV astrocyte cultures, being the highest value that of 30 DIV cultures added with ACM from 90 DIV. The findings of increased DNA labeling after EGF or INS pre-treatment in 30 DIV cultures, followed by addition of ACM from 15 DIV cultures, suggest that these phenomena may depend by extra cellular signal-regulated kinase 1 (ERK1) activation.     

Meprinα Transactivates the Epidermal Growth Factor Receptor (EGFR) via Ligand Shedding,thereby Enhancing Colorectal Cancer Cell Proliferation and Migration

Meprinα, an astacin-type metalloprotease is overexpressed in colorectal cancer cells and is secreted in a non-polarized fashion, leading to the accumulation of meprinα in the tumor stroma. The transition from normal colonocytes to colorectal cancer correlates with increased meprinα activity at primary tumor sites. A role for meprinα in invasion and metastatic dissemination is supported by its pro-angiogenic and pro-migratory activity. In the present study, we provide evidence for a meprinα-mediated transactivation of the EGFR signaling pathway and suggest that this mechanism is involved in colorectal cancer progression. Using alkaline phosphatase-tagged EGFR ligands and an ELISA assay, we demonstrate that meprinα is capable of shedding epidermal growth factor (EGF) and transforming growth factor-α (TGFα) from the plasma membrane. Shedding was abrogated using actinonin, an inhibitor for meprinα. The physiological effects of meprinα-mediated shedding of EGF and TGFα were investigated with human colorectal adenocarcinoma cells (Caco-2). Proteolytically active meprinα leads to an increase in EGFR and ERK1/2 phosphorylation and subsequently enhances cell proliferation and migration. In conclusion, the implication of meprinα in the EGFR/MAPK signaling pathway indicates a role of meprinα in colorectal cancer progression.     

Effects of a Melanotropic Peptide on Melanoma Cell Growth,Metastasis, and Invasion

Melanocyte stimulating hormone (α-MSH, α-melanotropin),Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Ly-Pro-Val-NH₂, regulates melanogenesis within epidermal melanocytes of many animals. An MSH analogue ([Nle4,D-Phe7]α-MSH) that exhibits superpotency and prolonged biological activity has been synthesized, biologically characterized, and is presently in clinical trials to determine its possible clinical use in tanning of the skin. It also has potential for the diagnosis, localization, and chemotherapy of melanoma. The effects of this analogue on the growth, metastatic behavior, and invasive potential of a melanotic variant of Cloudman S-91 murine melanoma are reported here. In an intracutaneous murine model of melanoma cell tumor growth, the analogue did not increase primary tumor growth (size) after the period of administration of the peptide hormone analogue and did not affect spontaneous lung metastases. Survival times for the control and melanotropin-treated groups were similar, suggesting that overall tumor burden was not affected by treatment with the hormone analogue. Last, melanoma cell invasion through a human amniotic basement membrane in vitro was not enhanced compared to untreated cells.     

Sestrin2 Protein Positively Regulates AKT Enzyme Signaling and Survival in Human Squamous Cell Carcinoma and Melanoma Cells

Skin cancer is the most common cancer in the United States and is mainly caused by environmental UV radiation. Reducing skin cancer incidence is becoming an urgent issue. The stress-inducible protein Sestrin2 (Sesn2) plays an important role in maintaining redox and metabolic homeostasis and their related pathologies. However, the role of Sesn2 in cancer remains unclear. Here we show that UVB radiation induces Sesn2 expression in normal human keratinocytes, mouse skin, normal human melanocytes, and melanoma cells. In addition, Sesn2 promotes AKT activation through a PTEN-dependent mechanism. Sesn2 deletion or knockdown sensitizes squamous cell carcinoma (SCC) cells to 5-fluorouracil-induced apoptosis and melanoma cells to UVB- and vemurafenib-induced apoptosis. In mice Sesn2 knockdown suppresses tumor growth from injected human SCC and melanoma cells. Last, as compared with normal skin, Sesn2 is up-regulated in both human skin SCC and melanoma. Our findings demonstrate that Sesn2 promotes AKT activation and survival in response to UVB stress and chemotherapeutics and suggest that Sesn2 is oncogenic in skin SCC and melanoma.     

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDH^high CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c. tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFNγ and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.     

Growth and Vascular Structure of Human Melanoma Xenografts

Abstract The growth and the vascular structure of five human melanomas grown in athymic nude mice were studied. Four growth parameters (tumour volume doubling time, fraction of cells in S-phase, growth fraction, cell-loss factor) were analysed against each of four vascular parameters (length of vessels with diameters in the range 5–15 μm, total vessel length, total vessel surface, total vessel volume-all per unit of histologically intact tumour volume). Statistically significant linear correlations between the parameters were found for any of the combinations. However, there was a consistent trend in the data: the tumour volume doubling time and the cell-loss factor tended to decrease while the fraction of cells in S-phase and the growth fraction tended to increase with increasing vascular density, whichever vascular parameter was considered. This finding indicates that the vascular density is among the factors which are decisive for the growth rate of tumours. However, the present work does not exclude the possibility that intrinsic properties of the tumour cells may also be important.     

隆肛蛙皮肤及其腺体的显微结构特征

观察了隆肛蛙(Paa quadranus)皮肤及其腺体的显微结构特点,主要对成体、幼蛙和蝌蚪泄殖腔上方皮肤腺进行了描述和比较。结果表明,隆肛蛙的表皮和真皮内均分布有微血管及黑色素细胞;皮肤腺为泡状腺,腺泡位于真皮浅层的疏松层内,属顶质分泌的粘液腺;雄性成体泄殖腔上方皮肤腺是隆肛蛙的特有结构,属于雄性的第二性征,本文建议称其为肛上腺(supra-anal gland)。文中对肛上腺及其机能、表皮中微血管与皮肤呼吸、黑色素细胞与体温调节等之间的关系进行了讨论。     

星形胶质细胞的体外生长特性

目的研究纯化培养的星形胶质细胞的体外生长特性,作为进一步研究星形胶质细胞功能的依据。方法将纯化的星形胶质细胞分为6h、12h、18h、24h、30h、36h、42h、48h、54h、60h、72h、84h、96h、120h14个时间组,培养不同的时间后,采用细胞计数法绘制细胞生长曲线观察细胞的生长状况,用流式细胞学检测细胞周期各时相的变化并绘制增殖指数曲线,所得数据用SPSS软件进行统计学分析。结果①星形胶质细胞的生长曲线可明显的分为三个阶段:第一阶段为0至24h,在此时间内生长曲线平缓上升,其斜率为0.64。第二阶段为24h至84h,在此时间内生长曲线急剧上升,其斜率为2.69;此阶段中24h至48h时曲线最陡,斜率为3.94,细胞呈指数生长。第三阶段为84h至120h,在此时间内生长曲线走势平直,其斜率为-0.005。②星形胶质细胞的增殖指数在第一个细胞增殖周期内可以反映细胞的增殖状态,第一个细胞周期结束后,细胞的数量继续上升增殖指数却降低了。结论培养的星形胶质细胞的生长过程分为潜伏期、指数生长期和停滞期三个阶段,其潜伏期为0至24h,指数生长期为24h至48h,停滞期为84h以后。单纯用增殖指数等类似的指标作为细胞增殖状态的判断标准是有一定局限性的,一定要将这些指标与细胞数量结合起来分析。     

Distinguishing Tumor and Stromal Sources of MicroRNAs Linked to Metastasis in Cutaneous Melanoma

MicroRNA (miRNA) dysregulation in cancer causes changes in gene expression programs regulating tumor progression and metastasis. Candidate metastasis suppressor miRNA are often identified by differential expression in primary tumors compared to metastases. Here, we performed comprehensive analysis of miRNA expression in The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) tumors (97 primary, 350 metastatic), and identified candidate metastasis-suppressor miRNAs. Differential expression analysis revealed miRNA significantly downregulated in metastatic tumors, including miR-205, miR-203, miR-200a-c, and miR-141. Furthermore, sequential feature selection and classification analysis identified miR-205 and miR-203 as the miRNA best able to discriminate between primary and metastatic tumors. However, cell-type enrichment analysis revealed that gene expression signatures for epithelial cells, including keratinocytes and sebocytes, were present in primary tumors and significantly correlated with expression of the candidate metastasis-suppressor miRNA. Examination of miRNA expression in cell lines revealed that candidate metastasis-suppressor miRNA identified in the SKCM tumors, were largely absent in melanoma cells or melanocytes, and highly restricted to keratinocytes and other epithelial cell types. Indeed, the differences in stromal cell composition between primary and metastatic tumor tissues is the main basis for identification of differential miRNA that were previously classified as metastasis-suppressor miRNAs. We conclude that future studies must consider tumor-intrinsic and stromal sources of miRNA in their workflow to identify bone fide metastasis-suppressor miRNA in cutaneous melanoma and other cancers.     

HDGF的PWWP结构域改变对肿瘤细胞体外及体内增殖的影响

目的:研究肝癌衍生生长因子(Hepatoma-derived growth factor,HDGF)PWWP结构域(PWWP domain)改变对肿瘤细胞体外及体内增殖的影响。方法:构建HDGF的PWWP结构域突变体P24A,利用慢病毒感染细胞筛选稳定细胞系。采用CCK-8法和软琼脂克隆形成实验检测细胞的增殖情况。通过裸鼠皮下成瘤实验检测移植瘤的形成情况。结果:在肝癌Hep G2和结直肠癌DLD1稳定细胞株中,CCK-8法检测结果显示突变体P24A对细胞生长的抑制作用呈时间依赖性,其OD值在24、48、72和96 h均明显低于HDGF稳定细胞株(均P0.001)。克隆形成实验结果显示P24A组克隆数目明显小于HDGF组(P0.01)。异种移植瘤动物模型则证明P24A细胞株的瘤块生长速度(P0.001,P0.01,P0.01),瘤块大小及体积(P0.01)均明显低于HDGF细胞株。结论:PWWP结构域改变可能抑制HDGF发挥促进细胞增殖的作用。     

Features of the Structure and Expression of NPM and NCL Genes in Cutaneous Melanoma

Molecular Biology - Malignant cutaneous melanoma (CM) is an extremely aggressive cancer characterized by a high level of metastatic activity and unfavorable prognosis due to a high incidence of...