Melanoma Cell Death Research Articles

St John's Wort (Hypericum perforatum L.) photomedicine: hypericin-photodynamic therapy induces metastatic melanoma cell death.

Hypericin, an extract from St John's Wort (Hypericum perforatum L.), is a promising photosensitizer in the context of clinical photodynamic therapy due to its excellent photosensitizing properties and tumoritropic characteristics. Hypericin-PDT induced cytotoxicity elicits tumor cell death by various mechanisms including apoptosis, necrosis and autophagy-related cell death. However, limited reports on the efficacy of this photomedicine for the treatment of melanoma have been published. Melanoma is a highly aggressive tumor due to its metastasizing potential and resistance to conventional cancer therapies. The aim of this study was to investigate the response mechanisms of melanoma cells to hypericin-PDT in an in vitro tissue culture model. Hypericin was taken up by all melanoma cells and partially co-localized to the endoplasmic reticulum, mitochondria, lysosomes and melanosomes, but not the nucleus. Light activation of hypericin induced a rapid, extensive modification of the tubular mitochondrial network into a beaded appearance, loss of structural details of the endoplasmic reticulum and concomitant loss of hypericin co-localization. Surprisingly the opposite was found for lysosomal-related organelles, suggesting that the melanoma cells may be using these intracellular organelles for hypericin-PDT resistance. In line with this speculation we found an increase in cellular granularity, suggesting an increase in pigmentation levels in response to hypericin-PDT. Pigmentation in melanoma is related to a melanocyte-specific organelle, the melanosome, which has recently been implicated in drug trapping, chemotherapy and hypericin-PDT resistance. However, hypericin-PDT was effective in killing both unpigmented (A375 and 501mel) and pigmented (UCT Mel-1) melanoma cells by specific mechanisms involving the externalization of phosphatidylserines, cell shrinkage and loss of cell membrane integrity. In addition, this treatment resulted in extrinsic (A375) and intrinsic (UCT Mel-1) caspase-dependent apoptotic modes of cell death, as well as a caspase-independent apoptotic mode that did not involve apoptosis-inducing factor (501 mel). Further research is needed to shed more light on these mechanisms.

Metabotropic glutamate receptor 1 acts as a dependence receptor creating a requirement for glutamate to sustain the viability and growth of human melanomas.

Metabotropic glutamate 1 (mGlu) receptor has been proposed as a target for the treatment of metastatic melanoma. Studies have demonstrated that inhibiting the release of glutamate (the natural ligand of mGlu1 receptors), results in a decrease of melanoma tumor growth in mGlu1 receptor-expressing melanomas. Here, we demonstrate that mGlu1 receptors, which have been previously characterized as oncogenes, also behave like dependence receptors by creating a dependence on glutamate for sustained cell viability. In the mGlu1 receptor-expressing melanoma cell lines, SK-MEL-2 (SK2) and SK-MEL-5 (SK5), we show that glutamate is both necessary and sufficient to maintain cell viability, regardless of underlying genetic mutations. Addition of glutamate increased DNA synthesis while removal of glutamate not only suppressed DNA synthesis, but also promoted cell death in SK2 and SK5 melanoma cells. Using genetic and pharmacological inhibitors we established that this effect of glutamate is mediated by activation of mGlu1 receptors. The stimulatory potential of mGlu1 receptors was further confirmed in vivo in a melanoma cell xenograft model. In this model, subcutaneous injection of SK5 cells with shRNA targeted downregulation of mGlu1 receptors resulted in a decrease in the rate of tumor growth relative to control. We also demonstrate for the first time, that a selective mGlu1 receptor antagonist, JNJ16259685 [(3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone], slows SK2 and SK5 melanoma tumor growth in vivo. Taken together, these data suggest that pharmacological inhibition of mGlu1 receptors may be a novel approach for the treatment of metastatic melanoma.

Quinacrine for extremity melanoma in a mouse model of isolated limb perfusion (ILP).

Quinacrine is a relatively non-toxic drug, once given almost exclusively for malaria. However, recent studies show that quinacrine can suppress nuclear factor-κB (NF-κB), and activate p53 signaling. We investigated the anti-cancer effect of quinacrine, using a novel mouse model of isolated limb perfusion (ILP) for extremity melanoma. Female C57BL/6 mice (22-25 g) were injected with B16 melanoma cells (1 × 10(5)) subcutaneously in the distal thigh. After 7 days of tumor establishment, mice were perfused with either PBS, melphalan (90 µg), or quinacrine (3.5 and 4.5 mg) through the superficial femoral artery for 30 min at either 37 or 42 °C in a non-oxygenated circuit. We analyzed morbidity, toxicity, tumor apoptosis, and responses. Melanoma cell death following in vitro exposure to quinacrine was dose and time dependent. A significant decrease in mean tumor volume was observed after perfusion with low-dose and high-dose quinacrine (both P = 0.002) at 37 °C as well as after perfusion with low-dose quinacrine (P = 0.0008) at 42 °C. Quinacrine has demonstrable efficacy against melanoma cells in vitro and in a clinically relevant model of ILP. Further studies to evaluate the optimal conditions for quinacrine usage are warranted.

Raman spectroscopy as an analytical tool for melanoma research.

Raman spectroscopy is an optical noninvasive screening technology that generates individual fingerprints of living cells by reflecting their molecular constitution. To discriminate melanoma cells from melanocytes, to identify drug-induced melanoma cell death stages (apoptosis, necrosis, autophagy) and to assess the susceptibility of melanoma cells to anticancer therapy. We used Raman spectroscopy on normal and melanoma cells, and on wild-type (WT) and mutant melanoma cells, to investigate whether the technique could distinguish between different types of cells, identify mutations and evaluate response to anticancer therapy. Using the multivariate principal component analysis of the Raman spectra, melanocytes could be distinguished from melanoma cells, and WT melanoma cells could be distinguished from melanoma cells with BRAF or NRAS mutations. When we used the apoptosis inducer staurosporine, the necrosis inducer 3-bromopyruvate and the autophagy inducer resveratrol to induce cell death in SKMEL28 melanoma cells, Raman spectroscopy clearly distinguished between these three types of cell death, as confirmed by immunoblotting. Finally, the technique could discriminate between different melanoma cell lines according to their susceptibility to high-dose ascorbate. Raman spectroscopy is a powerful noninvasive tool to distinguish between melanocytes and melanoma cells, to analyze the specific type of cell death in melanoma cells, and to predict the susceptibility of melanoma cells to anticancer drugs.

HPV based photodynamic therapy: a new approach for anti-cancer therapy (VAC12P.1019)

Abstract A new method of targeted photodynamic therapy (PDT) is proposed for potentiating anti-tumor immunity. Human papillomavirus (HPV) virus-like particles (VLPs) bind a broad range of tumor types both, in vitro and in vivo, but do not bind healthy, intact tissues. We have linked a photosensitizer dye, IR700, to the HPV VLPs and applied the dye-coupled particles (IR700-VLP) to tumor cells. When exposed to 690nm light, the bound conjugates induced immediate necrotic-like cell death in ovarian, lung, melanoma and cervical cancer cell lines in vitro, and in melanoma and lung tumors in vivo. We are currently expanding these studies to examine long-term tumor growth/survival and the generation of anti-tumor immunity in C57Bl/6 mice using the TC-1 tumor model, which expresses the HPV oncogenes, E6 and E7. This model allows us to track the T-cell responses generated against the HPV oncogenes, in both primary and metastatic tumor settings. Treatment with IR700-VLP and light exposure induced an influx of CD8+ and CD4+ T-cells into the treated tumors. Ex vivo cultures of whole tumor cell suspensions showed IFN-γ production in PMA/ionomycin-stimulated CD8+ and CD4+ T cells and to a lower extent without stimulation, suggesting that these T-cells may have been primed against the tumor antigens. Our data suggest that local anti-tumor immunity can be induced through the rapid tumor cell death caused by IR700, in conjunction with the ability of HPV VLP to induce an innate immune response.

Indomethacin Sensitizes TRAIL-Resistant Melanoma Cells to TRAIL-Induced Apoptosis through ROS-Mediated Upregulation of Death Receptor 5 and Downregulation of Survivin

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention owing to its selective killing of tumor cells but not normal cells. Melanoma shows weak response to TRAIL because of its low level of TRAIL death receptors. Here, we investigated whether indomethacin, a nonsteroidal anti-inflammatory drug, can potentiate TRAIL-induced apoptosis in melanoma cells. We showed that indomethacin was capable of promoting TRAIL-induced cell death and apoptosis in A375 melanoma cells. Mechanistically, indomethacin induced cell surface expression of death receptor 5 (DR5) in melanoma cells and also in various types of cancer cells. DR5 knockdown abolished the enhancing effect of indomethacin on TRAIL responses. Induction of the DR5 by indomethacin was found to be p53 independent but dependent on the induction of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP abolished indomethacin-induced DR5 expression and the associated potentiation of TRAIL-mediated cell death. In addition, indomethacin-induced reactive oxygen species (ROS) production preceded upregulation of CHOP and DR5, and consequent sensitization of cells to TRAIL. We also found that indomethacin treatment downregulated survivin via ROS and the NF-κB-mediated signaling pathways. Interestingly, indomethacin also converted TRAIL-resistant melanoma MeWo and SK-MEL-5 cells into TRAIL-sensitive cells. Taken together, our results indicate that indomethacin can potentiate TRAIL-induced apoptosis through upregulation of death receptors and downregulation of survivin.

Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (mPTP) opening

Here we studied the role of mitochondrial permeability transition pore (mPTP) opening in curcumin’s cytotoxicity in melanoma cells. In cultured WM-115 melanoma cells, curcumin induced mitochondrial membrane potential (MPP) decrease, cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) (two mPTP components) mitochondrial association and cytochrome C release, indicating mPTP opening. The mPTP blocker sanglifehrin A (SfA) and ANT-1 siRNA-depletion dramatically inhibited curcumin-induced cytochrome C release and WM-115 cell death. CyPD is required for curcumin-induced melanoma cell death. The CyPD inhibitor cyclosporin A (CsA) or CyPD siRNA-depletion inhibited curcumin-induced WM-115 cell death and apoptosis, while WM-115 cells with CyPD over-expression were hyper-sensitive to curcumin. Finally, we found that C6 ceramide enhanced curcumin-induced cytotoxicity probably through facilitating mPTP opening, while CsA and SfA as well as CyPD and ANT-1 siRNAs alleviated C6 ceramide’s effect on curcumin in WM-115 cells. Together, these results suggest that curcumin-induced melanoma cell death is associated with mPTP opening.

Discovery of a Novel Series of Tetrahydro‐β‐carbolines Inducing Autophagic Cell Death in Human Metastatic Melanoma

Herein, we report the synthesis of novel tetrahydro-β-carbolines that induce cell death via the autophagic pathway. Five of the new compounds induced cell death in a panel of patient-derived human metastatic melanoma cells. The autophagic pathway was confirmed using LC3 autophagosome markers; the involvement of ATG7 and Beclin 1 autophagy regulating genes was confirmed using infection with short hairpin RNA (shRNA) to silence Beclin 1 and ATG7. Compound VIII (IC50 = 2.34-5.15 μM) displayed activities greater than cisplatin against a panel of patient-derived human metastatic melanoma cell lines. The structure-activity relationship (SAR) of this class and the role of the absolute stereochemistry and geometrical isomerism are evaluated.

Melatonin Combined with Endoplasmic Reticulum Stress Induces Cell Death via the PI3K/Akt/mTOR Pathway in B16F10 Melanoma Cells

This study investigated B16F10 melanoma cell death induced by melatonin combined with endoplasmic reticulum (ER) stress through the PI3K/Akt/mTOR pathway. Cell viability was significantly decreased after treatment with melatonin combined with ER stress from thapsigargin or tunicamycin compared to no treatment or treatment with melatonin only. Combined melatonin and ER stress also significantly reduced expression of p85β, p-Akt (Ser473, Thr308), and p-mTOR (Ser2448, Ser2481) compared to treatment with melatonin only. The ER stress protein p-PERK and p-eIF2α were significantly increased under combined melatonin and ER stress treatment compared to no treatment or treatment with melatonin only. Combined melatonin and ER stress significantly reduced Bcl-2 protein and augmented Bax protein compared to melatonin-only treatment. Also, the combined treatment significantly lowered expression of catalase, Cu/Zn-SOD, and Mn-SOD proteins compared to melatonin only. Expression of p85β was significantly more decreased under treatment with melatonin and thapsigargin or tunicamycin plus the PI3K inhibitors LY294002 or wortmannin than under treatment with only melatonin or a PI3K inhibitor. The PI3K downstream target p-Akt (Ser473, Thr308) showed significantly decreased expression under treatment with melatonin and thapsigargin or tunicamycin plus PI3K inhibitors than under treatment with melatonin or PI3K inhibitors only. These results indicate that survival of B16F10 melanoma cells after combined treatment with melatonin and ER stress inducers is suppressed through regulation of the PI3K/Akt/mTOR pathway. Melatonin combined with thapsigargin or tunicamycin appears to be a promising strategy for effective melanoma treatment.

BH3-mimetic gossypol-induced autophagic cell death in mutant BRAF melanoma cells with high expression of p21Cip1

AimsThe aim of the present study was to identify the potential therapeutic effects of BH3-mimetic gossypol on melanoma cells with acquired resistance to BRAF inhibitors. Main methodsThe IC50 values of gossypol were determined using MTT assays in three melanoma cell lines with different resistances to BRAF inhibitor. The effects of gossypol on three melanoma cell lines were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. The functional role of autophagy in gossypol-induced growth inhibition was investigated using siRNA-mediated knockdown of Beclin-1. Key findingsGossypol retained its efficacy in BRAF-V600E melanoma clones with acquired resistance to BRAF inhibitors through a mechanism independent of MEK–ERK inhibition. Gossypol caused G2/M arrest in both BRAF mutant A375P and A375P/Mdr cells with high expression of p21Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells. In particular, alleviation of autophagy by knockdown of Beclin-1 partially caused a resistance to gossypol-induced cell cycle arrest at G2/M in BRAF-V600E cells with a concomitant decreased induction of apoptosis. SignificanceTaken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21Cip1 is a prerequisite for a positive response to its clinical application.

A lectin from Bothrops leucurus snake venom raises cytosolic calcium levels and promotes B16-F10 melanoma necrotic cell death via mitochondrial permeability transition

BlL, a galactose-binding C-type lectin purified from Bothrops leucurus snake venom, exhibits anticancer activity. The current study was designed to elucidate the cellular mechanisms by which BlL induces melanoma cell death. The viabilities of B16-F10 melanoma cells and HaCaT keratinocytes treated with BlL were evaluated. Necrotic and apoptotic cell death, cytosolic Ca2+ levels, mitochondrial Ca2+ transport and superoxide levels were assessed in B16-F10 melanoma cells exposed to BlL. We found that treatment with BlL caused dose-dependent necrotic cell death in B16-F10 melanoma cells. Conversely, the viability of non-tumorigenic HaCaT cells was not affected by similar doses of BlL. BlL-induced B16-F10 necrosis was preceded by a significant (2-fold) increase in cytosolic calcium concentrations and a significant (3-fold) increase in mitochondrial superoxide generation. It is likely that BlL treatment triggers B16-F10 cell death via mitochondrial permeability transition (MPT) pore opening because the pharmacological MPT inhibitors bongkrekic acid and Debio 025 greatly attenuated BlL-induced cell death. Experiments evaluating mitochondrial Ca2+ transport in permeabilized B16-F10 cells strongly supported the hypothesis that BlL rapidly stimulates cyclosporine A-sensitive Ca2+-induced MPT pore opening. We therefore conclude that BlL causes selective B16-F10 melanoma cell death via dysregulation of cellular Ca2+ homeostasis and Ca2+-induced opening of MPT pore.

Graveoline Isolated from Ethanolic Extract of Ruta graveolens Triggers Apoptosis and Autophagy in Skin Melanoma Cells: A Novel Apoptosis-Independent Autophagic Signaling Pathway

Anti-cancer drugs generally kill cancer cells by apoptosis but fail to do so when they become resistant and escape apoptosis signals. But these resistant cells can still be killed by autophagy. Therefore, drugs having both apoptotic and autophagic abilities are solicited in effective cancer management. In search of such a drug, we examined the efficacy of graveoline, a bioactive compound isolated from Ruta graveolens on skin melanoma A375 cells through the use of specific signaling cascades and their inhibitors. Cytotoxicity of graveoline was tested by conducting MTT assay. Induction of autophagy and apoptosis was checked. Expression of related proteins and their localization were studied by conducting immunoblot assay and through confocal microscopy, respectively. We found graveoline-induced Beclin-1 associated autophagy in A375 cells and 3-methyladenine, an inhibitor of autophagy did not affect apoptosis. Conversely, caspase inhibitor that blocked apoptosis did not affect autophagic cell death, suggesting thereby that these two were independent events. Use of reactive oxygen species (ROS) scavengers inhibited cell death, but blocking autophagy did not affect graveoline-induced ROS generation, suggesting that ROS generation ensued autophagy. Thus, graveoline-induced both apoptotic and autophagic cell death in skin melanoma cells, a desirable quality in effective anti-cancer drug design.

Abstract A264: Response of CNS melanoma to 4-demethyl-4-cholestrylcarbonylpenclomedine (DM-CHOC-PEN) and cell death.

Abstract Introduction: DM-CHOC-PEN is a polychlorinated pyridine cholesteryl carbonate, which has completed a Phase I clinical trial in patients with advanced cancer - IND 68,876 (AACR 1185, 2013). DM-CHOC-PEN produced responses with improved cerebral progression free intervals (CPFI) in patients with CNS melanoma, breast, and lung cancers. CNS melanoma is of especial interest since there is no acceptable therapy for this stage of disease. DM-CHOC-PEN's principle MOA is via alkylation of DNA with N7-guanine, however, in melanoma, there is also generation of reactive oxygen species (ROS) via the DOPA/melanin pathway resulting in melanin encapsulation of melanoma cells and cell death. In mice the drug accumulated in intracerebrally/cerebellar implanted tumors, and not in normal CNS tissue, with no Purkinje cell degeneration; in contrast to drugs such as penclomedine, phencyclidine, ibogaine, etc. that also accumulate in the cerebellum but with cellular destruction. Patients with metastatic CNS cancer treated with DM-CHOC-PEN demonstrated some euphoria, but no CNS toxicity. Methods: B-16 melanoma cells were cultured in RPMI media with 5% FBS and pen/strep at 37oC in a CO2 incubator. Drugs were added to the cells during growth phase and removed after 16 hrs; responses were monitored. Adult female C57BL mice in groups of 5-6 with growing B-16 melanoma were dosed IP daily (75-200 mg/kg) for 5 days with DM-CHOC-PEN or saline and monitored daily until death or moribund and sacrificed. Animals were sacrificed, brain tumor and normal tissue removed and prepared for histology exams. Patients in the Phase I trial (DTI-021) with CNS melanoma were treated with 86.5 mg/m2 IV q 21 days and CNS lesions were monitored with MRI during the Phase I clinical trial (DTI-021) [AACR 1185, 2013]. Results: In vitro, DM-CHOC-PEN had an IC50 0.5 µg/mL against B-16 melanoma cells. Mice bearing B-16 melanoma treated with DM-CHOC-PEN (200 mg/kg/d x 5d, IP) alone vs. saline controls demonstrated %ILS of 142% - supporting the in vitro observations. Patients with CNS melanoma treated with DM-CHOC-PEN demonstrated objective responses and improved CPFI with associated melanin deposits similar to those seen with mice. DM-CHOC-PEN induced ROS via DOPA/DOPA oxidase-melanin deposits and cell death in mice with melanoma. Discussions - DM-CHOC-PEN bound to erythrocytes crosses the BBB and accumulates in CNS melanoma (not normal tissue) with intracellular DOPA-DOPA quinone and melanin formation that interrupts cancer cell metabolism and cellular death via ROS. The drug is not recycled into the systemic circulation. Electronic modeling studies support DM-CHOC-PEN's ability to act as a pyridinium co-factor in the transfer of electrons from DOPA to cytochrome c and the intermediary metabolism pool with generation of ROS. Oxidative stress and ROS generation from a DM-CHOC-PEN/DOPA-melanin pathway has now been confirmed. Cerebellar lesions appear to be especially sensitive. Conclusion: Responses and CPFI for patients with CNS cancers (and no CNS toxicity) seen in the Phase I study have been reported [AACR 1185, 2013]. The principle human toxicity was hepatic. The role for the DOPA/DOPA quinone-melanin pathway with ROS formation will be presented as a key MOA for the drug, with emphasis on cerebellar melanoma lesions. Supported in part by: NCI/ SBIR grants R43/44CA85021 and R43/44CA132257. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A264. Citation Format: Lee Roy Morgan, Edmund Benes, Andrew H. Rodgers, Roy S. Weiner, Marcus L. Ware, Philip Friedlander, Yvonne Saenger, Craig Gordon, Tallat Mahmood, Branko Jursic, Gerard Bastian. Response of CNS melanoma to 4-demethyl-4-cholestrylcarbonylpenclomedine (DM-CHOC-PEN) and cell death. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A264.

Metabolism under the spotlight in senescence

Mammals and poikilotherms share twomelanized pigment cell types, the mela-nocytes derived from the neural crestand the retinal pigmented epithelium(RPE) derived from the brain (Bhartiet al., 2006). Zebrafish, chicken andhuman studies have identified some ofthe main genes involved in melanin pro-duction in the RPE; however, the extentto which the transcriptome of thesetwo melanized cell types is sharedremains unclear. In addition to the mel-anized cells, fish and other poikilo-therms have other pigment cellsderived from the neural crest, includingiridophores. The characteristic shinysilver appearance of the iridophores isdue to their cytoplasmic reflecting plate-lets, membrane-bound organelles con-taining guanine crystals that areorganized at tightly regulated fixed dis-tances from each other to generate theiridescence by a thin-layer interferencemechanism (Bagnara and Matsumoto,2006). We still do not know how theseplatelets are organized, and how thesecells synthesize abundant guanine forthe platelets while maintaining enoughpurines for nucleic acid production. Thecharacterization of the transcriptomesof iridophores and melanocytes (bothtypes) by Higdon et al. sheds light onthe genes regulating the interestingproperties of the iridophore, as well asthe similarities between the melano-cytes.Higdon et al. developed a method toisolate populations of each of threepigment cells – RPE, and neural crest-derived melanocytes and iridophores– from zebrafish embryos. Cells wereisolated from 3 days post-fertilizationembryos by density gradient centrifuga-tion, using the differences in density ofmelanin and guanine to separate pig-ment cells from the cells of the rest ofthe embryo. To isolate the RPE, embry-onic eyes were dissected, disaggregat-ed and then separated by densitygradient centrifugation. NC-derived mel-anocytes and iridophores were isolatedby density gradient centrifugation ofwhole disaggregated embryos afterremoval of eyes, followed by takingadvantage of the reflective properties ofiridophores to use FACS to separateiridophores from melanocytes. Indepen-dent pooled samples of each pigmentcell were isolated, cDNA extracted andthe transcriptomes sequenced by RNA-seq. Unfortunately, for technical rea-sons, their method was unsuitable forisolating the fourth zebrafish pigmentcell type, the xanthophore.Higdon et al. first aimed to identifygenes shared between the three pig-ment cells, which might indicate sharedfunctions of all pigment cell types. Theyidentified 28 genes, including someencoding ribosomal proteins, and geneswhose role in any pigment cell remainsunclear and will require further study(crfb5 or ghitm). One surprise in this listwas the zebrafish albino gene (slc45a2),which is interesting because albino fishhave reported no iridophore defect; theauthors suggest that it may have a rolein pH regulation in reflecting plateletsas well as in melanosomes.A comparison of genes sharedbetween pairs of pigment cell typesreveals shared functions, but also islikely to reflect shared developmentalorigin. Unsurprisingly, they found 214genes enriched in both melanocytesand RPE, but not iridophores. As bothcells produce the same pigment, mela-nocytes and RPE co-express manygenes involved in melanin synthesisand melanosome regulation, such asdct, tyrp1, pah and tyrp1b. They alsoshare transcription factors from theforkhead box and homeobox-containinggroups (foxo1b, foxp4, hmx1, otx1a,etc.). More unexpected, however, wasthe finding that the 38 most highlyexpressed genes in both melanocytesand RPE are also expressed (albeit atmuch lower levels) in iridophores. Theyalso identified 62 genes enriched inmelanocytes and iridophores, butnot RPE. The authors note that thesefindings likely reflect the shared originsof melanocytes and iridophores fromthe neural crest, because the lists ofshared genes include several well-known NC and pigment cell regulatorsand markers (sox10, crestin, mc1r andthe cell adhesion molecule pcdh10a).Moreover, some transcription factorsthat have not been studied previously,such as foxo1a or cdk15, were alsoenriched in both pigment cells and willwarrant further study using targetedgene mutagenesis. Only one gene wasenriched in RPE and iridophores, whichwas expected because they do notshare a close developmental origin orpigment type.Finally, Higdon et al. have identifiedgenes specific to each cell type. Theiranalysis identified 108 genes specificallyenriched in melanocytes, and 24 in theRPE. However, the most interestingresults concern the characterization ofthe iridophore transcriptome, which pro-vides insights into the genetics underly-ing these highly specialized cells.The authors identify 346 genes whoseexpression is significantly enriched iniridophores. However, by filtering the listto include only highly enriched genes (atleast 30-fold greater than melanocytesand RPE, and 100-fold greater thanwhole embryos), they highlight 30 geneslikely to play major roles in iridophorespecialization. This list includes genes,such as slc23 l, that may act as a guan-ine transporter, although whether thisacts in the plasma membrane or in thereflecting platelet membrane remains tobe determined. More surprisingly, thelist also includes gpnmb, encoding atransmembrane glycoprotein previouslydescribed in melanocytes as both aplasma membrane protein and a mela-nosome component that regulates theexpression of melanosomal proteins andmelanosome formation; the authorsspeculate that it may have a similar func-tion in the generation of the iridophorereflecting platelet organelle.One striking finding of their study isthe enrichment of transcripts encodingenzymes involved in the purine synthe-sis pathway, from extracellular glucoseimport and glycolysis, through de novosynthesis and purine retrieval. Upregula-tion of certain genes in the purineCoverage on: Higdon, C.W., Mitra, R.D.,and Johnson, S.L. (2013). Gene expres-sion analysis of zebrafish melanocytes,iridophores, and retinal pigmented epi-thelium reveals indicators of biologicalfunction and developmental origin.PLoS ONE 8(7), e67801. doi:10.1371/journal.pone.0067801

The Herbal Compound Cryptotanshinone Restores Sensitivity in Cancer Cells That Are Resistant to the Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis and kills cancer cells but not normal cells. However, TRAIL resistance due to low level of TRAIL receptor expression is widely found in cancer cells and hampers its development for cancer treatment. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are urgently needed. We investigated whether tanshinones, the major bioactive compounds of Salvia miltiorrhiza (danshen), can up-regulate TRAIL receptor expression. Among the major tanshinones being tested, cryptotanshinone (CT) showed the best ability to induce TRAIL receptor 2 (DR5) expression. We further showed that CT was capable of promoting TRAIL-induced cell death and apoptosis in A375 melanoma cells. CT-induced DR5 induction was not cell type-specific, as DR5 induction was observed in other cancer cell types. DR5 knockdown abolished the enhancing effect of CT on TRAIL responses. Mechanistically, induction of the DR5 by CT was found to be p53-independent but dependent on the induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). Knockdown of CHOP abolished CT-induced DR5 expression and the associated potentiation of TRAIL-mediated cell death. In addition, CT-induced ROS production preceded up-regulation of CHOP and DR5 and consequent sensitization of cells to TRAIL. Interestingly, CT also converted TRAIL-resistant lung A549 cancer cells into TRAIL-sensitive cells. Taken together, our results indicate that CT can potentiate TRAIL-induced apoptosis through up-regulation of DR5.

Flipping the phenotypic switch on a novel antimelanoma differentiation strategy

Flipping the phenotypic switch on a novel antimelanoma differentiation strategy

PUMA: A Puzzle Piece in Chloroquine’s Antimelanoma Activity

Chloroquine can induce cell death in a subset of cancer cell lines, and some melanoma cell lines are quite susceptible. While it is well known that chloroquine impairs lysosomal function and can serve as an autophagy inhibitor, the molecular target of chloroquine and the subsequent cascade of events that leads to cell death are not fully understood. Recent evidence indicates that in melanoma cell lines, chloroquine induces apoptosis by preventing degradation of the pro-apoptotic BH3-only protein PUMA. This finding adds to the unfolding story of chloroquine’s mechanism of action as a cancer therapeutic agent.

Nanocarriers with ultrahigh chromophore loading for fluorescence bio-imaging and photodynamic therapy

We describe the design of original nanocarriers that allows for ultrahigh chromophore loading while maintaining the photo-activity of each individual molecule. They consist in shells of charged biocompatible polymers grafted on gold nanospheres. The self-organization of extended polymer chains results from repulsive charges and steric interactions that are optimized by tuning the surface curvature of nanoparticles. This type of nano-scaffolds can be used as light-activated theranostic agents for fluorescence imaging and photodynamic therapy. We demonstrate that, labeled with a fluorescent photosensitizer, it can localize therapeutic molecules before triggering the cell death of B16-F10 melanoma with an efficiency that is similar to the efficiency of the polymer conjugate alone, and with the advantage of extremely high local loading of photosensitizers (object concentration in the picomolar range).

Impaired PIASy–Tip60 signaling weakens activation of p53 in melanoma

The tumor suppressor p53 plays a central role in preventing tumor development by promoting transcription of genes that stall cell cycle and induce cell death. Although the majority of melanomas express wild-type p53, the molecular mechanisms that impede its activation remain unclear. We previously reported that the SUMO E3 ligase PIASy and the histone acetyltransferase Tip60 signaling cascade promote p53-dependent autophagy and apoptosis. We hypothesized that impairment in this signaling attenuates p53, thus disabling its apoptotic function in melanoma. Here, we show that human melanoma patient samples and cell lines maintain p53 expression but PIASy and/or Tip60 are frequently lost. We observed dysregulation of Tip60-mediated p53 transcription program in melanoma cell lines. Reconstitution of PIASy and Tip60 in melanoma cells increased genotoxic stress-induced apoptosis. Our study provides a clinical link of how sumoylation signaling may activate p53-mediated cell death in melanoma.

Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation.