Modulation of cell growth and cisplatin sensitivity by membrane gamma-glutamyltransferase in melanoma cells

The plasma membrane enzyme gamma-glutamyltransferase (GGT) is regarded as critical for the maintenance of intracellular levels of glutathione (GSH). GGT expression has been implicated in drug resistance through elevation of intracellular GSH. The dependence of intracellular GSH on GGT expression was not conclusively ascertained. The present study was designed to investigate the role of GGT and of intracellular GSH levels in modulating proliferation and sensitivity to cisplatin of melanoma cells. GGT transfection resulted in increased growth, both in vitro and in tumour xenografts. In addition, GGT-transfected cells exhibited reduced sensitivity to cisplatin associated with lower DNA platination. A decrease in intracellular GSH levels, rather than an increase, was observed in GGT-transfected cells; moreover, in cysteine-deficient conditions, the expression of GGT did not provide transfected cells with the ability of utilising extracellular GSH. In conclusion, these results indicate that GGT activity confers a growth advantage unrelated with intracellular glutathione supply, and are consistent with the interpretation that cisplatin resistance is the consequence of modifications of cellular pharmacokinetics as a result of extracellular drug inactivation by thiol metabolites originated by GGT-mediated GSH cleavage.     

Erythropoietin receptor contributes to melanoma cell survival in vivo

Erythropoietin (Epo) is widely used clinically to treat anemia associated with various clinical conditions including cancer. Data from several clinical trials suggest significant adverse effect of Epo treatment on cancer patient survival. However, controversy exists whether Epo receptor (EpoR) is functional in cancer cells. In this study, we demonstrated that EpoR mRNA expression was detectable in 90.1% of 65 melanoma cell lines, and increased copy number of the Epo and EpoR loci occurred in 30 and 24.6% of 130 primary melanomas, respectively. EpoR knockdown in melanoma cells resulted in diminished ERK phosphorylation in response to Epo stimulation, decreased cell proliferation and increased response to the inhibitory effect of hypoxia and cisplatin in vitro. EpoR knockdown significantly decreased melanoma xenograft size and tumor invasion in vivo. On the contrary, constitutive activation of EpoR activated cell proliferation pathways in melanoma cells and resulted in increased cell proliferation and resistance to hypoxia and cisplatin treatment in vitro. EpoR activation resulted in significantly larger xenografts with increased tumor invasion of surrounding tissue in vivo. Daily administration of recombinant Epo fails to stimulate melanoma growth in vivo, but the treatment increased vascular size in the xenografts. Increased local recurrence after excision of the primary tumors was observed after Epo treatment. Epo induced angiogenesis in Matrigel plug assays, and neutralization of Epo secreted by melanoma cells results in decreased angiogenesis. These data support that EpoR is functional in melanoma and EpoR activation may promote melanoma progression, and suggest that Epo may stimulate angiogenesis and increase survival of melanoma cells under hypoxic condition in vivo.     

Upregulated ankyrin repeat-rich membrane spanning protein contributes to tumour progression in cutaneous melanoma

Background:

We have previously demonstrated that overexpression of ankyrin repeat-rich membrane spanning (ARMS) protein facilitates melanoma formation via conferring apoptotic resistance. This study aims to investigate whether ARMS contributes to melanoma progression.

Method:

Using immunohistochemistry, we graded the expression level of ARMS in 54 cases of primary melanoma and 46 cases of metastatic melanoma. The immunointensity of ARMS was statistically correlated with individual clinicopathological characteristics. By RNA interference, stable melanoma cell clones with ARMS-knockdown were constructed, and were used for in vitro scratch wound, transwell invasion assays, and in vivo lung metastasis experiment.

Results:

Stronger immunointensity of ARMS was observed mostly in melanomas with Breslow tumour thickness >1.0 mm (Fisher''s exact test, P=0.002) or with nodal metastasis (Fisher''s exact test, P=0.026), and was correlated with a worse overall survival in melanoma patients (log-rank test, P=0.04). Depletion of ARMS inhibited migration, invasion, and metastatic potential of melanoma cells in vitro and in vivo. Moreover, ARMS mediated melanoma cell migration and invasion through activation of the extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signalling pathway.

Conclusion:

Ankyrin repeat-rich membrane spanning expression, conjunctly with tumour thickness or ulceration, may serve as a prognostic factor in patients with cutaneous melanoma.     

Chromatin and cell surface receptors mediate melanoma cell growth response to nerve growth factor.

Growth response to nerve growth factor (NGF) was tested in the primary melanoma cell line WM 164, which expressed a low level of NGF cell-surface receptor, and in WM 164 cells transfected with cDNA for the cell-surface receptor (TrWM 164 cells), which expressed a higher level of the cell-surface receptor. Neither cell line expressed the chromatin receptor for NGF or internalized NGF. Both cell lines were stimulated to growth by NGF. After 10 d of exposure to NGF, a 230,000 Mr chromatin protein (receptor) was induced in both cell lines; as a result, NGF bound to the chromatin, and ribosomal RNA synthesis and cell proliferation were inhibited. We suggest that the cell-surface and chromatin receptors each mediate a different function of NGF.     

Anhydroretinol induces oxidative stress and cell death.

The retro-retinoid anhydroretinol (AR), a physiological metabolite of retinol (vitamin A), induces cell death in multiple in vitro systems. AR-induced cell death is blocked by retinol and its metabolite 14-hydroxy-4,14-retro-retinol. AR has been shown also to prevent mammary cancer induced by N-methyl-N-nitrosourea in rats. We report that AR kills cells by generating reactive oxygen species. Direct measurements show that the addition of AR to lymphoblastoid cells increases the intracellular oxidative stress in a time- and dose-dependent manner. Furthermore, the amount of induced oxidative stress directly correlates with the number of dying cells. The addition of retinol, 14-hydroxy-4,14-retro-retinol, or the antioxidant, alpha-tocopherol (vitamin E), decreases AR-induced oxidative stress and proportionally reduces AR-induced cell death. In contrast, pretreatment with caspase inhibitors, known to inhibit apoptosis, has no effect on AR-induced cell death. This is the first demonstration of cellular reactive oxygen species production by a natural retinoid.     

The apurinic/apyrimidinic endonuclease activity of Ape1/Ref-1 contributes to human glioma cell resistance to alkylating agents and is elevated by oxidative stress.

Alkylating agents are standard components of adjuvant chemotherapy for gliomas.We provide evidence here that Ape1/Ref-1, the major mammalian apurinic/apyrimidinic endonuclease (Ap endo), contributes to alkylating agent resistance in human glioma cells by incising DNA at abasic sites. We show that antisense oligonucleotides directed against Ape1/Ref-1 in SNB19, a human glioma cell line lacking O(6)-methylguanine-DNA-methyltransferase, mediate both reduction in Ape1/Ref-1 protein and Ap endo activity and concurrent reduction in resistance to methyl methanesulfonate and the clinical alkylators temozolomide and 1,3-(2-chloroethyl)-1-nitrosourea. An accompanying increase in the level of abasic sites indicates that the DNA repair activity of Ape1/Ref-1 contributes to resistance. Conversely, we also show that exposure of SNB19 cells to HOCl, a generator of reactive oxygen species (ROS), results in elevated Ape1/Ref-1 protein and Ap endo activity, enhanced alkylator resistance, and reduced levels of abasic sites. Given current evidence that heightened oxidative stress prevails within brain tumors, the finding that ROS increase resistance to clinical alkylators in glioma cells may have significance for the response of gliomas to alkylating agent-based chemotherapy. Our results may also be relevant to the design of therapeutic regimens using concurrent ionizing radiation (a generator of ROS) and alkylating agent-based chemotherapy.     

MIF produced by bone marrow-derived macrophages contributes to teratoma progression after embryonic stem cell transplantation

Although stem cell therapy holds promise as a potential treatment in a number of diseases, the tumorigenicity of embryonic stem cells (ESC) and induced pluripotent stem cells remains a major obstacle. In vitro predifferentiation of ESCs can help prevent the risk of teratoma formation, yet proliferating neural progenitors can generate tumors, especially in the presence of immunosuppressive therapy. In this study, we investigated the effects of the microenvironment on stem cell growth and teratoma development using undifferentiated ESCs. Syngeneic ESC transplantation triggered an inflammatory response that involved the recruitment of bone marrow (BM)-derived macrophages. These macrophages differentiated into an M2 or angiogenic phenotype that expressed multiple angiogenic growth factors and proteinases, such as macrophage migration inhibitory factor (MIF), VEGF, and matrix metalloproteinase 9, creating a microenvironment that supported the initiation of teratoma development. Genetic deletion of MIF from the host but not from ESCs specifically reduced angiogenesis and teratoma growth, and MIF inhibition effectively reduced teratoma development after ESC transplantation. Together, our findings show that syngeneic ESC transplantation provokes an inflammatory response that involves the rapid recruitment and activation of BM-derived macrophages, which may be a crucial driving force in the initiation and progression of teratomas.     

Susceptibility of human melanoma cells to oxidative stress including UVA radiation

Ultraviolet radiation, and in particular UVA (320–400 nm), induces significant oxidative stress to human skin. Ferritin and glutathione have been shown to be among the more important molecules within human skin cells providing protection against this damage, the presence of lower levels of these anti-oxidants giving rise to increased cellular sensitivity to stress. We compared endogenous levels of ferritin and glutathione in human melanoma cells with normal human skin fibroblasts and keratinocytes, also the response of melanoma cells to oxidative stress with fibroblasts and keratinocytes. Ferritin levels were heterogenous in the untreated melanoma cell lines tested and remained the same following oxidative stress (UVA radiation) or hemin treatment. Epidermal keratinocytes were unaffected, as were the melanoma cell lines, but skin fibroblasts showed dose-dependent ferritin depletion. Similar results were seen for glutathione alterations resulting from UVA radiation: melanoma cell lines and epidermal skin keratinocytes remained unchanged following UVA radiation, while skin fibroblasts showed dose-dependent depletion. Our results show that human melanoma cells have low ferritin and glutathione levels, yet are resistant to oxidative stress. © 1996 Wiley-Liss, Inc.     

Stromal fibroblasts synergize with hypoxic oxidative stress to enhance melanoma aggressiveness

On the basis of recent advances indicating a key role of microenvironment for tumor progression, we investigated the role of fibroblasts, macrophages and hypoxia, for primary melanoma aggressiveness. Our data indicate a key role of hypoxia in stromal reactivity, acting on both myofibroblasts and machrophages differentiation. Hypoxic myofibroblasts are more active than macrophages in inducing melanoma invasiveness and exploit their oxidative stress due to hypoxia to secrete soluble factors favouring melanoma invasion and chemotaxis. We underscore the key role of microenviroment on melanoma malignancy, highlighting reactive fibroblasts, intratumoral hypoxia and oxidative stress as promising targets for melanoma antimetastatic strategies.     

高三尖杉酯碱增强慢性粒细胞白血病细胞对于伊马替尼敏感性的研究*

目的: 研究高三尖杉酯碱（HHT）增强慢性粒细胞白血病（CML）细胞对伊马替尼（IM）敏感性的机制。 方法: 采集1例CML患者骨髓血标本,经体外筛选、克隆建立人白血病细胞株NPHA1（初治）和NPHA2（复发）。RNA干扰NPHA2细胞的EphB4蛋白表达,建立NPHA2-EphB4-sh细胞株。 结果: 与NPHA1细胞相比,耐IM的NPHA2细胞中EphB4过表达;在NPHA2-EphB4-sh细胞中,EphB4表达显著低于NPHA1细胞和NPHA2细胞（P<0.001）。NPHA2细胞对IM有明显耐药性（IC₅₀=5.45 mg/L）,但NPHA2-EphB4-sh对IM敏感性增加（IC₅₀=0.93 mg/L,P<0.001）。HHT与IM共处理后,NPHA2细胞对IM的IC50值降至1.17 mg/L（P<0.001）。蛋白磷酸化测定表明HHT抑制了EphB4/RhoA通路表达。 结论: EphB4/RhoA是一个新的IM耐药途径。HHT通过抑制EphB4/RhoA途径,使IM治疗获得优势。      

Cellular response to oxidative stress and ascorbic acid in melanoma cells overexpressing gamma-glutamyltransferase

The extracellular gamma-glutamyltransferase-mediated metabolism of glutathione has been implicated in prooxidant events which may have impact on cellular functions including drug resistance. This study was performed in two GGT-transfected melanoma clones to explore the hypothesis that GGT expression in tumour cells is implicated in modulation of cell behaviour under stress conditions. Our results show that GGT-overexpression in melanoma cells was associated with resistance to oxidative stress produced by prooxidant agents such as hydrogen peroxide and ascorbic acid. In GGT-overexpressing cells, ability to tolerate oxidative stress was evidenced by the presence of a moderate level of ROS and lack of DNA damage response following treatment with H(2)O(2). Cellular response to oxidative stress induced by ascorbic acid was detectable only in the clone with low GGT activity which also exhibited an increased susceptibility to apoptosis. The increased resistance of the GGT-overexpressing clone was not related to intracellular GSH content but rather to the increased expression of catalase and to a reduced efficiency of iron-mediated formation of toxic free radicals. Taken together, these findings are consistent with a contribution of GGT in the mechanisms of drug resistance, because induction of oxidative stress is a relevant event in the apoptotic response to cytotoxic agents.     

Targeting glutamine transport to suppress melanoma cell growth

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF^WT (C8161 and WM852) and BRAF^V600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2‐mediated glutamine transport is a potential therapeutic target for both BRAF^WT and BRAF^V600E melanoma.     

ASNA1, an ATPase targeting tail-anchored proteins, regulates melanoma cell growth and sensitivity to cisplatin and arsenite

Purpose  ASNA1 is homologous to E. coli ArsA, a well characterized ATPase involved in efflux of arsenite and antimonite. Cells resistant to arsenite and antimonite are cross-resistant to the chemotherapeutic drug cisplatin. ASNA1 is also an essential ATPase for the insertion of tail-anchored proteins into ER membranes and a novel regulator of insulin secretion. The aim of this study was to determine if altered ASNA1 levels influenced growth and sensitivity to arsenite and cisplatin in human melanoma cells. Methods  Cultured melanoma T289 cells were transfected with plasmids containing sense or antisense ASNA1. Cells were exposed to cisplatin, arsenite and zinc. Cell growth and chemosensitivity were evaluated by the MTT assay and apoptosis by a TUNEL assay. Results  ASNA1 expression was necessary for growth. T289 clones with decreased ASNA1 expression exhibited 51 ± 5% longer doubling times than wildtype T289 (P = 0.0091). After exposure to cisplatin, ASNA1 downregulated cells displayed a significant increase in apoptosis. The cisplatin IC₅₀ in ASNA1 underexpressing cells was 41.7 ± 1.8% compared to wildtype (P = 0.00097) and the arsenite IC₅₀ was 59.9 ± 3.2% of wildtype IC₅₀ (P = 0.0067). Conclusions  Reduced ASNA1 expression is associated with significant inhibition of cell growth, increased apoptosis and increased sensitivity to cisplatin and arsenite.