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1.  Kaurenic acid: Evaluation of the in vivo and in vitro antitumor activity on murine melanoma 
Indian Journal of Pharmacology  2011;43(6):683-688.
The in vivo and in vitro antitumor activity of kaurenic acid [kaur 16-en-19 oic acid] (KA) in melanoma was evaluated in a murine model in comparison with taxol (Tx).
Materials and Methods:
B16F1 melanoma was developed in C57BL/6 mice and cell cultures. Survival test, tumor growth, dissected-tumor measurements, histology, cytoxicity assay on cultured cells, and changes of apoptotic gene expression at mRNA level were analyzed.
KA showed antitumor effect in vivo and in vitro and compared with Tx, its antimelanoma activity was greater (P < 0.001). These results were confirmed by morphological analysis (P < 0.001). In melanoma cell cultures, KA IC50 was 0.79 μM vs. 18.94 μM for Tx (P < 0.001). RT-PCR analysis demonstrated that Bcl-xL mRNA expression was altered in B16F1 mouse melanoma cells obtained from mice treated with either KA or Tx.
The data suggest that KA is active in animal melanoma models, both in vitro and in vivo, being its cytotoxic effects stronger than those exhibited by Tx. Further trials should be conducted to elucidate its mechanism of action in melanoma with respect to necrosis or apoptotic processes. Our results support other evidences indicating that KA is a potential chemotherapeutic agent against cancer that has to be widely explored.
PMCID: PMC3229785  PMID: 22144774
Antitumor activity; B16F1 melanoma; C57BL/6 mice; kaurenic acid
2.  Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases 
Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.
PMCID: PMC3854348  PMID: 23314342
Kaurenoic acid; Glioblastoma; miR-21; c-FLIP; Apoptosis
3.  The P450-4 Gene of Gibberella fujikuroi Encodes ent-Kaurene Oxidase in the Gibberellin Biosynthesis Pathway 
At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA4. The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3′ consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.
PMCID: PMC93051  PMID: 11472927
4.  Anti-tumour activity of longikaurin A (LK-A), a novel natural diterpenoid, in nasopharyngeal carcinoma 
Longikaurin A is a natural ent-kaurene diterpenoid isolated from Isodon genus. The ent-kaurene diterpenoids isolated from medicinal plants have been shown to have anti-disease effects. The present study was designed to examine the anti-tumour effects of longikaurin A (LK-A) in nasopharyngeal carcinoma in vitro and in vivo.
Apoptosis and cell cycle arrest were determined by flow cytometry analysis of the cells treated with Longikaurin A. The proteins of apoptosis signaling pathway were detected by western blotting analysis. Finally, we examined whether LK-A exhibits anti-tumour activity in xenograft models.
Longikaurin A inhibited the cell growth by inducing apoptosis and cell cycle arrest. At low concentrations, longikaurin A induced S phase arrest and at higher concentrations, longikaurin A induced caspase-dependent apoptosis by regulating apoptotic molecules. Finally, longikaurin A significantly inhibited the tumour growth of CNE2 xenografts in vivo and showed no obvious effect on the body weights of the mice.
Our results suggest that Longikaurin A exhibited anti-tumour activity in nasopharyngeal carcinoma in vitro and in vivo.
PMCID: PMC3847153  PMID: 23985029
Longikaurin A (LK-A); Apoptosis; Caspase; Nasopharyngeal carcinoma
5.  Germination of photoblastic lettuce seeds is regulated via the control of endogenous physiologically active gibberellin content, rather than of gibberellin responsiveness 
Journal of Experimental Botany  2008;59(12):3383-3393.
Phytochrome regulates lettuce (Lactuca sativa L. cv. Grand Rapids) seed germination via the control of the endogenous level of bioactive gibberellin (GA). In addition to the previously identified LsGA20ox1, LsGA20ox2, LsGA3ox1, LsGA3ox2, LsGA2ox1, and LsGA2ox2, five cDNAs were isolated from lettuce seeds: LsCPS, LsKS, LsKO1, LsKO2, and LsKAO. Using an Escherichia coli expression system and functional assays, it is shown that LsCPS and LsKS encode ent-copalyl diphosphate synthase and ent-kaurene synthase, respectively. Using a Pichia pastoris system, it was found that LsKO1 and LsKO2 encode ent-kaurene oxidases and LsKAO encodes ent-kaurenoic acid oxidase. A comprehensive expression analysis of GA metabolism genes using the quantitative reverse transcription polymerase chain reaction suggested that transcripts of LsGA3ox1 and LsGA3ox2, both of which encode GA 3-oxidase for GA activation, were primarily expressed in the hypocotyl end of lettuce seeds, were expressed at much lower levels than the other genes tested, and were potently up-regulated by phytochrome. Furthermore, LsDELLA1 and LsDELLA2 cDNAs that encode DELLA proteins, which act as negative regulators in the GA signalling pathway, were isolated from lettuce seeds. The transcript levels of these two genes were little affected by light. Lettuce seeds in which de novo GA biosynthesis was suppressed responded almost identically to exogenously applied GA, irrespective of the light conditions, suggesting that GA responsiveness is not significantly affected by light in lettuce seeds. It is proposed that lettuce seed germination is regulated mainly via the control of the endogenous content of bioactive GA, rather than the control of GA responsiveness.
PMCID: PMC2529229  PMID: 18653696
Germination; gibberellin metabolism; gibberellin signalling; lettuce
6.  Jaridonin, a Novel Ent-Kaurene Diterpenoid from Isodon rubescens, Inducing Apoptosis via Production of Reactive Oxygen Species in Esophageal Cancer Cells 
Current cancer drug targets  2013;13(6):611-624.
Isodon rubescens, a Chinese herb, has been used as a folk, botanical medicine in China for inflammatory diseases and cancer treatment for many years. Recently, we isolated a new ent-kaurene diterpenoid, named Jaridonin, from Isodon rubescens. The chemical structure of Jaridonin was verified by Infrared (IR), Nuclear magnetic resonance (NMR), and Mass spectrum (MS) data as well as X-ray spectra. Jaridonin potently reduced viabilities of several esophageal cancer cell lines, including EC109, EC9706 and EC1. Jaridonin treatment resulted in typical apoptotic morphological characteristics, increased the number of annexin V-positive staining cells, as well as caused a G2/M arrest in cell cycle progression. Furthermore, Jaridonin resulted in a significant loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, and then activation of Caspase-9 and -3, leading to activation of the mitochondria mediated apoptosis. Furthermore, these effects of Jaridonin were accompanied by marked reactive oxygen species (ROS) production and increased expression of p53, p21waf1/Cip1 and Bax, whereas two ROS scavengers, N-acetyl-L-cysteine (L-NAC) and Vitamin C, significantly attenuated the effects of Jaridonin on the mitochondrial membrane potential, DNA damage, expression of p53 and p21waf1/Cip1 and reduction of cell viabilities. Taken together, our results suggest that a natural ent-kaurenoid diterpenoid, Jaridonin, is a novel apoptosis inducer and deserves further investigation as a new chemotherapeutic strategy for patients with esophageal cancer.
PMCID: PMC3750090  PMID: 23597192
Apoptosis; ent-kaurene diterpenoid; esophageal cancer; mitochondria pathway; rabdosia rubescens; reactive oxygen species
7.  Uveal Melanoma Expression of Indoleamine 2, 3 Deoxygenase: Establishment of an Immune Privileged Environment by Tryptophan Depletion 
Experimental eye research  2007;85(5):617-625.
The enzyme indoleamine 2, 3-dioxygenase (IDO) catalyzes degradation of tryptophan, an essential amino acid required for lymphocyte activation and proliferation. Many tumors express IDO which implied that it acts as a mechanism to evade T cell-mediated immune attack, and also to establish an immunosuppressive tumor microenvironment. The purpose of this study was to determine whether primary and metastatic uveal melanoma expressed the IDO gene and whether uveal melanoma cells could deplete tryptophan. In situ expression of IDO in primary uveal melanoma from tumor bearing eyes and metastatic uveal melanoma liver tissues was determined by immunohistostaining with IDO-specific antibody. Reverse transcription PCR was used to assess IDO gene transcription by primary and metastatic uveal melanoma cell lines. IDO protein expression was determined by Western blot of uveal melanoma cell protein lysate. IDO catalytic activity was assessed by measuring the presence of kynurenine, a product generated by tryptophan degradation, in uveal melanoma culture supernatants.
Primary uveal melanoma from tumor-bearing eyes and metastatic uveal melanoma from the liver did not express IDO in situ. IDO was not constitutively expressed in either primary or metastatic uveal melanoma cell lines. However, stimulation of primary and metastatic uveal melanoma cell cultures with interferon-gamma (IFN-γ) universally upregulated both IDO gene and protein expression. Culture supernatants from IFN-γ treated primary and metastatic uveal melanoma cell cultures contained elevated levels of kynurenine. Addition of the IDO inhibitor 1-methyl DL-tryptophan significantly diminished kynurenine levels in IFN-γ treated uveal melanoma cell cultures. The results from this study suggest that IFN-γ inducible IDO upregulation by primary and metastatic uveal melanoma may generate a local immune privileged microenvironment to promote escape from T cell-mediated immune surveillance.
PMCID: PMC2695208  PMID: 17870068
8.  Down-Regulation of the Interferon Signaling Pathway in T Lymphocytes from Patients with Metastatic Melanoma 
PLoS Medicine  2007;4(5):e176.
Dysfunction of the immune system has been documented in many types of cancers. The precise nature and molecular basis of immune dysfunction in the cancer state are not well defined.
Methods and Findings
To gain insights into the molecular mechanisms of immune dysfunction in cancer, gene expression profiles of pure sorted peripheral blood lymphocytes from 12 patients with melanoma were compared to 12 healthy controls. Of 25 significantly altered genes in T cells and B cells from melanoma patients, 17 are interferon (IFN)-stimulated genes. These microarray findings were further confirmed by quantitative PCR and functional responses to IFNs. The median percentage of lymphocytes that phosphorylate STAT1 in response to interferon-α was significantly reduced (Δ = 16.8%; 95% confidence interval, 0.98% to 33.35%) in melanoma patients (n = 9) compared to healthy controls (n = 9) in Phosflow analysis. The Phosflow results also identified two subgroups of patients with melanoma: IFN-responsive (33%) and low-IFN-response (66%). The defect in IFN signaling in the melanoma patient group as a whole was partially overcome at the level of expression of IFN-stimulated genes by prolonged stimulation with the high concentration of IFN-α that is achievable only in IFN therapy used in melanoma. The lowest responders to IFN-α in the Phosflow assay also showed the lowest gene expression in response to IFN-α. Finally, T cells from low-IFN-response patients exhibited functional abnormalities, including decreased expression of activation markers CD69, CD25, and CD71; TH1 cytokines interleukin-2, IFN-γ, and tumor necrosis factor α, and reduced survival following stimulation with anti-CD3/CD28 antibodies compared to controls.
Defects in interferon signaling represent novel, dominant mechanisms of immune dysfunction in cancer. These findings may be used to design therapies to counteract immune dysfunction in melanoma and to improve cancer immunotherapy.
Prompted by altered expression patterns of interferon-responsive genes in T and B cells, Peter Lee and colleagues find that lymphocytes from melanoma patients have defects in interferon signaling.
Editors' Summary
The immune system, in addition to fighting infections, provides one of the body's main defenses against cancer. During cancer development, normal cells acquire genetic changes that allow them to grow uncontrollably and to move around the body. Some of these changes alter the antigens (proteins recognized by the immune system) expressed on their surface. As a result, the immune system recognizes and eliminates the newly formed cancer cells. Tumors—large masses of cancer cells—occur when this immune surveillance fails. Some tumors, for example, hide from the immune system by altering the antigens they express. Others release factors that shut off the immune response. However, for many tumor types, it is not clear why immune surveillance fails during their development or why global immune suppression develops in most patients with advanced disease.
Why Was This Study Done?
Scientists want to understand the molecular basis of immune dysfunction in patients with cancer because if they knew what had gone wrong with the immune system, they might be able to repair it. Also, there is considerable interest in immunotherapy for cancer—for example, treatment with interferons (proteins made by certain immune system cells that activate other immune cells and also kill tumor cells) and the development of vaccines to stimulate antitumor immune responses. So far, immunotherapy has not been very successful, probably because of the underlying dysfunction of the immune system in patients with cancer. Understanding this dysfunction might lead to improvements in immunotherapy, so in this study the researchers have investigated the molecular mechanism responsible for immune dysfunction in patients with metastatic melanoma, a deadly form of skin cancer.
What Did the Researchers Do and Find?
The researchers purified lymphocytes (immune cells that are involved in antitumor responses) from the blood of patients with metastatic melanoma and healthy people and examined their patterns of gene expression using a technique called microarray expression profiling. CD8 T cells (which kill cells expressing foreign or altered antigens), CD4 T cells (which help other T and B lymphocytes do their jobs), and B cells (which make antibodies, proteins that recognize antigens and label cancer cells for destruction by the immune system) from patients with melanoma all expressed lower levels of 24 genes, and higher levels of one gene, than those from healthy individuals. 17 of these genes were interferon-stimulated genes, which encode proteins responsible for the effects of interferons. Therefore, the researchers checked the functional responses of patient and control lymphocytes to interferon. When interferon binds to lymphocytes, it triggers the addition of a phosphate group to the protein STAT1, which then induces changes in gene expression. STAT1 phosphorylation occurred in a lower percentage of patient lymphocytes than control lymphocytes in response to interferon-α (which is sometimes used to treat melanoma). The lymphocytes from one-third of the patients responded well to interferon-α, but those from the other patients showed little response. Furthermore, prolonged treatment with high concentrations of interferon-α partly overcame the defect in interferon signaling in patient lymphocytes. Finally, T cells from the patients failed to make the normal markers of immune cell activation or cytokines (proteins that mediate the killing of tumor cells) after exposure to activating stimuli and had reduced survival compared to control lymphocytes.
What Do These Findings Mean?
These results indicate that for patients with metastatic melanoma defects in interferon signaling are an important contributor to immune dysfunction. They also show that T cells from patients with melanoma (particularly those who respond poorly to interferon-α) have functional abnormalities that make them less likely to recognize and deal with melanoma cells. These results need confirming in many more patients, but they nevertheless represent an important step toward understanding the immune dysfunction associated with advanced melanoma and possibly other tumors. In addition, the identification of two subgroups of patients—interferon responders and poor interferon responders—may explain why only some patients with melanoma benefit from treatment with interferon-α. It might, therefore, be possible to pre-select those who would benefit from this treatment (which has some serious side effects) by examining patient lymphocytes for interferon responsiveness.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute information (in English and Spanish) for patients on the immune system and its involvement in cancer, and for patients and professionals on melanoma
American Cancer Society information for patients on immunotherapy
Cancer Research Institute (New York) web-based book on cancer and the immune system
MedlinePlus encyclopedia pages on melanoma (in English and Spanish)
Cancer Research UK patient information on melanoma, including information on immunotherapy
PMCID: PMC1865558  PMID: 17488182
9.  Melanoma-Associated Cancer-Testis Antigen 16 (CT16) Regulates the Expression of Apoptotic and Antiapoptotic Genes and Promotes Cell Survival 
PLoS ONE  2012;7(9):e45382.
Cancer-testis (CT) antigens are predominantly expressed in testis or placenta, but absent in most adult tissues. During malignant transformation CT genes are often activated. CT antigen 16 (CT16, PAGE5) is frequently expressed in advanced melanoma but its biological function has been unknown. To examine the role of CT16 in cell survival we knocked it down in A2058 melanoma cells using specific siRNAs and exposed the cells to cancer drug cisplatin known to induce apoptosis. As a result, cell survival was markedly decreased. To study the effects of CT16 on cell survival in more detail, the cellular gene expression profiles were investigated after CT16 silencing in CT16 positive A2058 melanoma cells, as well as after CT16 overexpression in CT16 negative WM-266-4 melanoma cells. Among the 11 genes both upregulated by CT16 silencing and downregulated by CT16 overexpression or vice versa, 4 genes were potentially apoptotic or antiapoptotic genes. CT16 was recognized as a positive regulator of antiapoptotic metallothionein 2A and interleukin 8 genes, whereas it inhibited the expression of apoptosis inducing dickkopf 1 (DKK1) gene. In addition CT16 enhanced the expression of fatty acid binding protein 7, a known promoter of melanoma progression. The effect of CT16 on DKK1 expression was p53 independent. Furthermore, CT16 did not regulate apoptotic genes via DNA methylation. In twenty melanoma metastasis tissue samples average DKK1 mRNA level was shown to be significantly (p<0.05) lower in high CT16 expressing tumors (n = 3) when compared to the tumors with low CT16 expression (n = 17). Thus, our results indicate that CT16 promotes the survival of melanoma cells and is therefore a potential target for future drug development.
PMCID: PMC3448647  PMID: 23028975
10.  Functional Conservation of the Capacity for ent-Kaurene Biosynthesis and an Associated Operon in Certain Rhizobia 
Journal of Bacteriology  2014;196(1):100-106.
Bacterial interactions with plants are accompanied by complex signal exchange processes. Previously, the nitrogen-fixing symbiotic (rhizo)bacterium Bradyrhizobium japonicum was found to carry adjacent genes encoding two sequentially acting diterpene cyclases that together transform geranylgeranyl diphosphate to ent-kaurene, the olefin precursor to the gibberellin plant hormones. Species from the three other major genera of rhizobia were found to have homologous terpene synthase genes. Cloning and functional characterization of a representative set of these enzymes confirmed the capacity of each genus to produce ent-kaurene. Moreover, comparison of their genomic context revealed that these diterpene synthases are found in a conserved operon which includes an adjacent isoprenyl diphosphate synthase, shown here to produce the geranylgeranyl diphosphate precursor, providing a critical link to central metabolism. In addition, the rest of the operon consists of enzymatic genes that presumably lead to a more elaborated diterpenoid, although the production of gibberellins was not observed. Nevertheless, it has previously been shown that the operon is selectively expressed during nodulation, and the scattered distribution of the operon via independent horizontal gene transfer within the symbiotic plasmid or genomic island shown here suggests that such diterpenoid production may modulate the interaction of these particular symbionts with their host plants.
PMCID: PMC3911121  PMID: 24142247
11.  Molecular subtyping of metastatic melanoma based on cell ganglioside metabolism profiles 
BMC Cancer  2014;14(1):560.
In addition to alterations concerning the expression of oncogenes and onco-suppressors, melanoma is characterized by the presence of distinctive gangliosides (sialic acid carrying glycosphingolipids). Gangliosides strongly control cell surface dynamics and signaling; therefore, it could be assumed that these alterations are linked to modifications of cell behavior acquired by the tumor. On these bases, this work investigated the correlations between melanoma cell ganglioside metabolism profiles and the biological features of the tumor and the survival of patients.
Melanoma cell lines were established from surgical specimens of AJCC stage III and IV melanoma patients. Sphingolipid analysis was carried out on melanoma cell lines and melanocytes through cell metabolic labeling employing [3-3H]sphingosine and by FACS. N-glycolyl GM3 was identified employing the 14 F7 antibody. Gene expression was assayed by Real Time PCR. Cell invasiveness was assayed through a Matrigel invasion assay; cell proliferation was determined through the soft agar assay, MTT, and [3H] thymidine incorporation. Statistical analysis was performed using XLSTAT software for melanoma hierarchical clustering based on ganglioside profile, the Kaplan-Meier method, the log-rank (Mantel-Cox) test, and the Mantel-Haenszel test for survival analysis.
Based on the ganglioside profiles, through a hierarchical clustering, we classified melanoma cells isolated from patients into three clusters: 1) cluster 1, characterized by high content of GM3, mainly in the form of N-glycolyl GM3, and GD3; 2) cluster 2, characterized by the appearance of complex gangliosides and by a low content of GM3; 3) cluster 3, which showed an intermediate phenotype between cluster 1 and cluster 3. Moreover, our data demonstrated that: a) a correlation could be traced between patients’ survival and clusters based on ganglioside profiles, with cluster 1 showing the worst survival; b) the expression of several enzymes (sialidase NEU3, GM2 and GM1 synthases) involved in ganglioside metabolism was associated with patients’ survival; c) melanoma clusters showed different malignant features such as growth in soft agar, invasiveness, expression of anti-apoptotic proteins.
Ganglioside profile and metabolism is strictly interconnected with melanoma aggressiveness. Therefore, the profiling of melanoma gangliosides and enzymes involved in their metabolism could represent a useful prognostic and diagnostic tool.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-560) contains supplementary material, which is available to authorized users.
PMCID: PMC4132924  PMID: 25085576
Ganglioside; Melanoma; N glycolyl GM3; Sialidase; Survival
12.  Transcriptome Analysis of Medicinal Plant Salvia miltiorrhiza and Identification of Genes Related to Tanshinone Biosynthesis 
PLoS ONE  2013;8(11):e80464.
Salvia miltiorrhiza Bunge, a perennial plant of Lamiaceae, accumulates abietane-type diterpenoids of tanshinones in root, which have been used as traditional Chinese medicine to treat neuroasthenic insomnia and cardiovascular diseases. However, to date the biosynthetic pathway of tanshinones is only partially elucidated and the mechanism for their root-specific accumulation remains unknown. To identify enzymes and transcriptional regulators involved in the biosynthesis of tanshinones, we conducted transcriptome profiling of S. miltiorrhiza root and leaf tissues using the 454 GS-FLX pyrosequencing platform, which generated 550,546 and 525,292 reads, respectively. RNA sequencing reads were assembled and clustered into 64,139 unigenes (29,883 isotigs and 34,256 singletons). NCBI non-redundant protein databases (NR) and Swiss-Prot database searches anchored 32,096 unigenes (50%) with functional annotations based on sequence similarities. Further assignments with Gene Ontology (GO) terms and KEGG biochemical pathways identified 168 unigenes referring to the terpenoid backbone biosynthesis (including 144 MEP and MVA pathway genes and 24 terpene synthases). Comparative analysis of the transcriptomes identified 2,863 unigenes that were highly expressed in roots, including those encoding enzymes of early steps of tanshinone biosynthetic pathway, such as copalyl diphosphate synthase (SmCPS), kaurene synthase-like (SmKSL) and CYP76AH1. Other differentially expressed unigenes predicted to be related to tanshinone biosynthesis fall into cytochrome P450 monooxygenases, dehydrogenases and reductases, as well as regulatory factors. In addition, 21 P450 genes were selectively confirmed by real-time PCR. Thus we have generated a large unigene dataset which provides a valuable resource for further investigation of the radix development and biosynthesis of tanshinones.
PMCID: PMC3834075  PMID: 24260395
13.  Genomic and in vivo evidence of synergy of a herbal extract compared to its most active ingredient: Rabdosia rubescens vs. oridonin 
Rabdosia rubescens is a herbal root extract of traditional Chinese medicine (TCM) used to treat inflammatory diseases and oral cancers. A key principle of TCM is that multiple ingredients in a plant extract are more effective and less toxic than a single purified active ingredient or a purified drug derived from a plant product. Rabdosia rubescens extract (RRE) contains terpenoids and flavonoids, but the most active ingredient within the extract attributed to the inhibition of cancer is the kaurene diterpene, oridonin. In order to research synergy with a complete plant extract, the effects of RRE on the inhibition of prostate cancer cell proliferation were compared to the effects of pure oridonin alone in vitro. Three groups of 8 SCID mice bearing human prostate cancer xenografts (LAPC-4) were administered either RRE containing 0.02 mg/g oridonin, pure oridonin at a dose of 0.02 mg/g, or pure oridonin at a dose of 0.1 mg/g, by gavage for 5 days/week for 4 weeks. RRE and pure oridonin at 0.1 mg/g inhibited tumor growth to a similar extent, while oridonin at a dose of 0.02 mg/g did not. Therefore, in comparison to RRE, five times more pure oridonin was required to obtain equivalent prostate xenograft growth inhibition. Since the nuclear factor-κB signaling pathway and inflammation are implicated in prostate carcinogenesis, gene microarray analysis was conducted and demonstrated activation of genes by RRE that was not evident with oridonin treatment alone. This study demonstrated that genomic methods and xenograft studies are capable of demonstrating the benefits of the synergy of whole plant extracts rather than active ingredients isolated and purified as drugs.
PMCID: PMC3445985  PMID: 22993634
prostate cancer; inflammation; nuclear factor-κB
14.  bZIP transcription factor RSG controls the feedback regulation of NtGA20ox1 via intracellular localization and epigenetic mechanism 
Plant Signaling & Behavior  2011;6(1):26-28.
Gibberellins (GAs) are phytohormones that regulate growth and development throughout the life cycle of plants. Negative feedback contributes to homeostasis of GA levels. DELLA proteins are involved in this process. Since DELLA proteins do not have apparent DNA binding motifs, other DNA binding proteins might act as a mediator downstream of DELLA proteins in the GA feedback regulation. In this review, we highlight the mechanisms of GA feedback regulation, specifically the differential regulation of GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox) by transcription factors. RSG (REPRESSION OF SHOOT GROWTH) is a tobacco (Nicotiana tabacum) transcriptional activator with a basic leucine zipper domain that controls the levels of endogenous GAs through the regulation of GA biosynthesis genes. Recently we reported that RSG not only regulates the expression of ent-kaurene oxidase gene but is also involved in the negative feedback of NtGA20ox1 by GAs. RSG plays a role in the homeostasis of GAs through direct binding to the NtGA20ox1 promoter triggered by a decrease in GA levels in the cell. Furthermore, decreases in GA levels promote modifications of active histone marks on the NtGA20ox1 promoter. We have developed a hypothetical model to explain how RSG regulates dual target genes via epigenetic regulation.
PMCID: PMC3122000  PMID: 21248488
REPRESSION OF SHOOT GROWTH; GA 20-oxidase; gibberellin biosynthesis; feedback regulation; signaling; transcription factor; histone modification
15.  Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53 
British Journal of Cancer  2009;100(2):322-333.
Malignant melanomas are highly resistant to chemotherapy. First-line chemotherapeutics used in melanoma therapy are the methylating agents dacarbazine (DTIC) and temozolomide (TMZ) and the chloroethylating agents BCNU and fotemustine. Here, we determined the mode of cell death in 11 melanoma cell lines upon exposure to TMZ and fotemustine. We show for the first time that TMZ induces apoptosis in melanoma cells, using therapeutic doses. For both TMZ and fotemustine apoptosis is the dominant mode of cell death. The contribution of necrosis to total cell death varied between 10 and 40%. The O6-methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg−1 protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and fotemustine. MGMT inactivation by O6-benzylguanine sensitized all melanoma cell lines expressing MGMT to TMZ and fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O6-alkylguanines are critical lesions involved in the initiation of programmed melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to fotemustine. Although these data indicate that methylating drug resistance of melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in melanoma cells induced by TMZ and fotemustine was accompanied by double-strand break (DSB) formation (as determined by H2AX phosphorylation) and caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for fotemustine a correlation was not found. Melanoma lines expressing p53 wild-type were more resistant to TMZ and fotemustine than p53 mutant melanoma lines, which is in marked contrast to previous data reported for glioma cells treated with TMZ. Overall, the findings are in line with the model that in melanoma cells TMZ-induced O6-methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner.
PMCID: PMC2634706  PMID: 19127257
temozolomide; fotemustine; melanoma therapy; apoptosis; MGMT; mismatch repair
16.  Biochemical mechanism of Caffeic Acid Phenylethyl Ester (CAPE) selective toxicity towards melanoma cell lines 
In the current work, we investigated the in-vitro biochemical mechanism of caffeic acid phenylethyl ester (CAPE) toxicity and eight hydroxycinnamic/caffeic acid derivatives in-vitro, using tyrosinase enzyme as a molecular target in human SK-MEL-28 melanoma cells. Enzymatic reaction models using tyrosinase/O2 and HRP/H2O2 were used to delineate the role of one- and two-electron oxidation. Ascorbic acid (AA), NADH and GSH depletion were used as markers of quinone formation and oxidative stress in CAPE induced toxicity in melanoma cells. Ethylenediamine, an o-quinone trap, prevented the formation of o-quinone and oxidations of AA and NADH mediated by tyrosinase bioactivation of CAPE. The IC50 of CAPE towards SK-MEL-28 melanoma cells was 15μM. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased CAPE’s toxicity towards SK-MEL-28 cells indicating quinone formation played an important role in CAPE induced cell toxicity. Cyclosporin-A and trifluoperazine, inhibitors of the mitochondrial membrane permeability transition pore (PTP), prevented CAPE toxicity towards melanoma cells. We further investigated the role of tyrosinase in CAPE toxicity in the presence of a shRNA plasmid, targeting tyrosinase mRNA. Results from tyrosinase shRNA experiments showed that CAPE led to negligible anti-proliferative effect, apoptotic cell death and ROS formation in shRNA plasmid treated cells. Furthermore, it was also found that CAPE selectively caused escalation in the ROS formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 cells which express functional tyrosinase. In contrast, CAPE did not lead to ROS formation and ICG depletion in amelanotic C32 melanoma cells, which do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in CAPE’s selective toxicity towards melanocytic melanoma cell lines. Our findings suggest that the mechanisms of CAPE toxicity in SK-MEL-28 melanoma cells mediated by tyrosinase bioactivation of CAPE included quinone formation, ROS formation, intracellular GSH depletion and induced mitochondrial toxicity.
PMCID: PMC2932756  PMID: 20685355
Caffeic acid; melanoma; SK-MEL-28; cancer; quinone; GSH; CAPE
17.  Modulation of extracellular matrix/adhesion molecule expression by BRG1 is associated with increased melanoma invasiveness 
Molecular Cancer  2010;9:280.
Metastatic melanoma is an aggressive malignancy that is resistant to therapy and has a poor prognosis. The progression of primary melanoma to metastatic disease is a multi-step process that requires dynamic regulation of gene expression through currently uncharacterized epigenetic mechanisms. Epigenetic regulation of gene expression often involves changes in chromatin structure that are catalyzed by chromatin remodeling enzymes. Understanding the mechanisms involved in the regulation of gene expression during metastasis is important for developing an effective strategy to treat metastatic melanoma. SWI/SNF enzymes are multisubunit complexes that contain either BRG1 or BRM as the catalytic subunit. We previously demonstrated that heterogeneous SWI/SNF complexes containing either BRG1 or BRM are epigenetic modulators that regulate important aspects of the melanoma phenotype and are required for melanoma tumorigenicity in vitro.
To characterize BRG1 expression during melanoma progression, we assayed expression of BRG1 in patient derived normal skin and in melanoma specimen. BRG1 mRNA levels were significantly higher in stage IV melanomas compared to stage III tumors and to normal skin. To determine the role of BRG1 in regulating the expression of genes involved in melanoma metastasis, we expressed BRG1 in a melanoma cell line that lacks BRG1 expression and examined changes in extracellular matrix and adhesion molecule expression. We found that BRG1 modulated the expression of a subset of extracellular matrix remodeling enzymes and adhesion proteins. Furthermore, BRG1 altered melanoma adhesion to different extracellular matrix components. Expression of BRG1 in melanoma cells that lack BRG1 increased invasive ability while down-regulation of BRG1 inhibited invasive ability in vitro. Activation of metalloproteinase (MMP) 2 expression greatly contributed to the BRG1 induced increase in melanoma invasiveness. We found that BRG1 is recruited to the MMP2 promoter and directly activates expression of this metastasis associated gene.
We provide evidence that BRG1 expression increases during melanoma progression. Our study has identified BRG1 target genes that play an important role in melanoma metastasis and we show that BRG1 promotes melanoma invasive ability in vitro. These results suggest that increased BRG1 levels promote the epigenetic changes in gene expression required for melanoma metastasis to proceed.
PMCID: PMC3098014  PMID: 20969766
18.  Functional modulation of insulin-like growth factor binding protein-3 expression in melanoma 
Insulin-like growth factor binding protein-3 (IGFBP3) is a member of the IGFBP family, which regulates mitogenic and anti-apoptotic effects of insulin-like growth factors. In this report we evaluated the role of IGFBP3 in melanoma. Quantitative real-time PCR (qRT-PCR), western and ELISA analysis indicated a significant downregulation of IGFBP3 expression in melanoma cell lines as compared to a normal melanocyte cell line. Melanoma cell lines treated with the demethylating agent 5-AZA-2′ deoxycytidine re-expressed IGFBP3 at the mRNA and protein level. Chromatin immunoprecipitation assays revealed enrichment of acetylated histone H3, H4, H3 di- and tri-methylated lysine 4 on the unmethylated IGFBP3 promoter. The IGFBP3 promoter region was highly methylated in human melanoma samples as compared to normal nevi. Overexpression of IGFBP3 in melanoma cells in vitro suppressed tumor cell survival, induced apoptosis, reduced colony formation and invasion, and induced expression of the pro-apoptotic genes p21, PUMA, and BAX. IGFBP3 overexpression also resulted in cleavage of caspase 3 and reduced expression of phosphorylated-AKT. Stable overexpression of IGFBP3 suppressed tumor cell growth in vivo. Our results indicate that silencing of IGFBP3 in melanoma is due to the methylation of its promoter, and that overexpression of IGFBP3 induces apoptosis and suppresses cell survival and growth.
PMCID: PMC3023991  PMID: 20357812
IGFBP3; methylation; melanoma; cell growth; tumor suppressor
19.  Improving Melanoma Classification by Integrating Genetic and Morphologic Features 
PLoS Medicine  2008;5(6):e120.
In melanoma, morphology-based classification systems have not been able to provide relevant information for selecting treatments for patients whose tumors have metastasized. The recent identification of causative genetic alterations has revealed mutations in signaling pathways that offer targets for therapy. Identifying morphologic surrogates that can identify patients whose tumors express such alterations (or functionally equivalent alterations) would be clinically useful for therapy stratification and for retrospective analysis of clinical trial data.
Methodology/Principal Findings
We defined and assessed a panel of histomorphologic measures and correlated them with the mutation status of the oncogenes BRAF and NRAS in a cohort of 302 archival tissues of primary cutaneous melanomas from an academic comprehensive cancer center. Melanomas with BRAF mutations showed distinct morphological features such as increased upward migration and nest formation of intraepidermal melanocytes, thickening of the involved epidermis, and sharper demarcation to the surrounding skin; and they had larger, rounder, and more pigmented tumor cells (all p-values below 0.0001). By contrast, melanomas with NRAS mutations could not be distinguished based on these morphological features. Using simple combinations of features, BRAF mutation status could be predicted with up to 90.8% accuracy in the entire cohort as well as within the categories of the current World Health Organization (WHO) classification. Among the variables routinely recorded in cancer registries, we identified age < 55 y as the single most predictive factor of BRAF mutation in our cohort. Using age < 55 y as a surrogate for BRAF mutation in an independent cohort of 4,785 patients of the Southern German Tumor Registry, we found a significant survival benefit (p < 0.0001) for patients who, based on their age, were predicted to have BRAF mutant melanomas in 69% of the cases. This group also showed a different pattern of metastasis, more frequently involving regional lymph nodes, compared to the patients predicted to have no BRAF mutation and who more frequently displayed satellite, in-transit metastasis, and visceral metastasis (p < 0.0001).
Refined morphological classification of primary melanomas can be used to improve existing melanoma classifications by forming subgroups that are genetically more homogeneous and likely to differ in important clinical variables such as outcome and pattern of metastasis. We expect this information to improve classification and facilitate stratification for therapy as well as retrospective analysis of existing trial data.
Boris Bastian and colleagues present a refined morphological classification of primary melanomas that can be used to improve existing melanoma classifications by defining genetically homogeneous subgroups.
Editors' Summary
Skin cancers—the most commonly diagnosed cancers worldwide—are usually caused by exposure to ultraviolet (UV) radiation in sunlight. UV radiation damages the DNA in skin cells and can introduce permanent genetic changes (mutations) into the skin cells that allow them to divide uncontrollably to form a tumor, a disorganized mass of cells. Because there are many different cell types in the skin, there are many types of skin cancer. The most dangerous type—melanoma—develops when genetic changes occur in melanocytes, the cells that produce the skin pigment melanin. Although only 4% of skin cancers are melanomas, 80% of skin cancer deaths are caused by melanomas. The first signs of a melanoma are often a change in the appearance or size of a mole (a pigmented skin blemish that is also called a nevus) or a newly arising pigmented lesion that looks different from the other moles (an “ugly duckling”). If this early sign is noticed and the melanoma is diagnosed before it has spread from the skin into other parts of the body, surgery can sometimes provide a cure. But, for more advanced melanomas, the outlook is generally poor. Although radiation therapy, chemotherapy, or immunotherapy (drugs that stimulate the immune system to kill the cancer cells) can prolong the life expectancy of some patients, these treatments often fail to remove all of the cancer cells.
Why Was This Study Done?
Now, however, scientists have identified some of the genetic alterations that cause melanoma. For example, they know that many melanomas carry mutations in either the BRAF gene or the NRAS gene, and that the proteins made from these mutated genes (“oncogenes”) help cancer cells to grow uncontrollably. The hope is that targeted drugs designed to block the activity of oncogenic BRAF or NRAS might stop the growth of those melanomas that make these altered proteins. But how can the patients with these specific tumors be identified in the clinic? The expression of altered proteins is likely to affect the microscopic growth patterns (“histomorphology”) of melanomas. However, the current histomorphology-based classification system for melanomas, which distinguishes four main types of melanoma, does not help clinicians choose the best treatment for their patients. In this study, the researchers have tried to improve melanoma classification by looking for correlations between histomorphological features and genetic alterations in a large collection of melanomas.
What Did the Researchers Do and Find?
The researchers examined several histomorphological features in more than 300 melanoma samples and used statistical methods to correlate these features with the mutation status of BRAF and NRAS in the tumors. They found that some individual histomorphological features were strongly associated with the BRAF (but not the NRAS) mutation status of the tumors. For example, melanomas with BRAF mutations had more melanocytes in the upper layers of the epidermis (the outermost layer of the skin) than did those without BRAF mutations (melanocytes usually live at the bottom of the epidermis). Then, by combining several individual histomorphological features, the researchers built a model that correctly predicted the BRAF mutation status of more than 90% of the melanomas. They also found that, among the variables routinely recorded in cancer registries, being younger than 55 years old was the single most predictive factor for BRAF mutations. Finally, in another large group of patients with melanoma, the researchers found that those patients predicted to have a BRAF mutation on the basis of their age survived longer than those patients predicted not to have a BRAF mutation using the same criterion.
What Do These Findings Mean?
These findings suggest that an improved classification of melanomas that combines an analysis of known genetic factors with histomorphological features might divide melanomas into subgroups that are likely to differ in terms of their clinical outcome and responses to targeted therapies when they become available. Additional studies are needed to investigate whether the histomorphological features identified here can be readily assessed in clinical settings and whether different observers will agree on the scoring of these features. The classification model defined by the researchers also needs to be validated and refined in independent groups of patients. Nevertheless, these findings represent an important first step toward helping clinicians improve outcomes for patients with melanoma.
Additional Information.
Please access these Web sites via the online version of this summary at
A related PLoS Medicine Research in Translation article is available
The MedlinePlus encyclopedia provides information for patients about melanoma
The US National Cancer Institute provides information for patients and health professionals about melanoma (in English and Spanish)
Cancer Research UK also provides detailed information about the causes, diagnosis, and treatment of melanoma
PMCID: PMC2408611  PMID: 18532874
20.  BRG1 promotes melanoma survival in UV-irradiated melanoma cells by cooperating with MITF to activate the melanoma inhibitor of apoptosis gene 
Pigment cell & melanoma research  2013;26(3):377-391.
Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of anti-apoptotic genes and promotes lineage specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death. BRG1 prevents apoptosis in UV- irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP). Down-regulation of ML-IAP compromises BRG1 mediated survival of melanoma cells in response to UV-radiation. BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter. The alternative catalytic subunit, BRM and the BRG1 associated factor, BAF180 were found to be dispensable for elevated expression of ML-IAP in melanoma cells. Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an anti-apoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the pro-survival function of MITF.
SWI/SNF enzymes interact with the Microphthalmia-associated transcription factor (MITF) a lineage addiction oncogene, to promote MITF target gene expression in melanoma cells. In this study we determined that the SWI/SNF component, BRG1, promotes melanoma survival in response to UV-radiation, by activating expression of the melanoma inhibitor of apoptosis, ML-IAP gene. Our data show that BRG1 and MITF co-operate to establish permissive chromatin structure on the ML-IAP promoter and alter the association of other epigenetic regulators. Thus, we have elucidated a mechanism by which a component of the SWI/SNF complex promotes the pro-survival function of MITF. We further demonstrate that the BRG1 associated factor, BAF180, is not required for the activation of ML-IAP, suggesting that a specific configuration of the SWI/SNF complex mediates distinct activities. These results provide insight into how SWI/SNF function is deregulated in melanoma.
PMCID: PMC3633630  PMID: 23480510
melanoma; MITF; SWI/SNF enzymes; chromatin remodeling; ultraviolet-radiation; apoptosis; ML-IAP
21.  Macroscopic spectral imaging and gene expression analysis of the early stages of melanoma. 
Molecular Medicine  1999;5(12):785-794.
BACKGROUND: The stages of melanocytic progression are defined as atypical (dysplastic) nevus, melanoma in situ, melanoma in the radial growth phase (RGP), melanoma in the vertical growth phase (VGP), and melanoma in the metastatic growth phase (MGP). Melanoma in situ and RGP melanoma often develop in contiguous association with atypical nevi. This frequently poses a problem with respect to their early detection. Furthermore, unlike cells obtained from VGP and MGP melanomas, cells derived from melanoma in situ and RGP melanoma do not proliferate in vitro. Thus, compared to the late stages of the disease, less information is available regarding genes expressed in the early stages. MATERIALS AND METHODS: To determine whether spectral imaging, a recently developed optical imaging technique, can detect melanoma in situ and RGP melanoma arising in melanoma precursor lesions, atypical nevi in patients with a clinical history of melanoma were subjected to noninvasive macroscopic spectral imaging. To determine at what stage in the progression pathway of melanoma genes having important biological functions in VGP and MGP melanomas are activated and expressed, lesions of melanoma in situ were analyzed by immunohistochemistry and in situ hybridization for expression of some of these known molecular and immunologic markers. RESULTS: The present study demonstrates the capability of noninvasive spectral imaging to detect melanoma in situ and RGP melanoma that arise in contiguous association with atypical nevi. Furthermore, the study provides evidence that genes and antigens expressed in VGP and MGP melanoma are also expressed in melanoma in situ. CONCLUSIONS: Because of the dark and variegated pigmentation of atypical nevi, melanoma in situ and RGP melanoma that arise in these melanoma precursor lesions are often difficult to recognize and thus frequently go unnoticed. The application of new optical screening techniques for early detection of melanoma and the identification of genes expressed in the early stages of melanoma development are two important avenues in the pursuit of melanoma prevention. The investigations presented here document that macroscopic spectral imaging has the potential to detect melanoma in its early stage of development and that genes essential for the proliferation and cell adhesion of VGP and MGP melanoma are already expressed in melanoma in situ.
PMCID: PMC2230496  PMID: 10666478
22.  Melanoma inhibitor of apoptosis protein is expressed differentially in melanoma and melanocytic naevus, but similarly in primary and metastatic melanomas 
Journal of Clinical Pathology  2005;58(10):1081-1085.
Background: Malignant melanoma is highly resistant to current treatments. The inhibitor of apoptosis protein (IAP) family member, melanoma IAP (ML-IAP), is overexpressed in some melanoma cell lines, rendering them resistant to apoptotic signals. Targeting ML-IAP is a promising approach to treating melanoma. However, the status of ML-IAP expression in human melanoma tissues and the difference in expression between melanoma and melanocytic naevus are not known.
Aims: To investigate these issues.
Methods: ML-IAP expression in 48 archived patient samples (34 melanomas and 14 dermal naevi) was assessed by immunohistochemistry and by in situ hybridisation and reverse transcription polymerase chain reaction (RT-PCR) assays developed for the study.
Results: Expression of ML-IAP was detected in 47.6–70.6% (10 of 21 to 24 of 34) of the melanomas, varying with detection methods. The expression rate in melanoma was much higher than that in melanocytic naevus (10.0–21.4%; one of 10 to three of 14). No significant difference was seen between primary and secondary melanomas. ML-IAP expression rates assessed by the three methods were in agreement.
Conclusions: The ML-IAP expression rate in archived melanoma tissues is around 50–70%, with no difference between primary and secondary melanomas. A small number of dermal naevi (∼ 20%) also expressed ML-IAP.
PMCID: PMC1770742  PMID: 16189155
inhibitor of apoptosis protein; ML-IAP; melanoma; naevus; expression
23.  Selenocysteine derivative overcomes TRAIL resistance in melanoma cells: evidence for ROS-dependent synergism and signaling crosstalk 
Oncotarget  2014;5(17):7431-7445.
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), as one of the most promising targeted drug for new cancer therapeutics, is limited in clinical application by the evolution of resistance in many cancer cell lines, especially in malignant melanoma. Thus, it is urgently needed to identify chemosensitizers to enhance the apoptotic inducing efficacy of TRAIL and overcome resistance of malignant melanoma cells. Herein, we reported that 3,3′-diselenodipropionic acid (DSeA), a Selenocysteine derivative, could synergistically enhance the growth inhibitory effect of TRAIL on A375 melanoma cells though induction of ROS-dependent apoptosis with involvement of PTEN-mediated Akt inactivation and DNA damage-mediated p53 phosphorylation, which subsequently activated mitochondrial and death receptor apoptotic pathways. Moreover, silencing of p53 down-regulated the expression levels of p53-inducible genes, and effectively blocked the cell apoptosis. Suppression of PI3K significantly increased the apoptotic cell death. In contrast, antioxidants effectively reversed the cell apoptosis through regulation of Akt and p53 signaling pathways. Taken together, the combination of DSeA and TRAIL could be a novel strategy to overcome TRAIL resistance in malignant melanoma, and DSeA may be candidates for further evaluation as a chemosensitizer in clinical trails.
PMCID: PMC4202134  PMID: 25277183
Chemosensitizer; TRAIL; Selenium; apoptosis; ROS; p53
24.  Reduced Apaf-1 expression in human cutaneous melanomas 
British Journal of Cancer  2004;91(6):1089-1095.
Malignant melanoma is a life-threatening skin cancer due to its highly metastatic character and resistance to radio- and chemotherapy. It is believed that the ability to evade apoptosis is the key mechanism for the rapid growth of cancer cells. However, the exact mechanism for failure in the apoptotic pathway in melanoma cells is unclear. p53, the most frequently mutated tumour suppressor gene in human cancers, is a key apoptosis inducer. However, p53 mutation is only found in 15–20% of melanoma biopsies. Recently, it was found that Apaf-1, a downstream target of p53, is inactivated in metastatic melanoma. Specifically, loss of heterozygosity (LOH) of the Apaf-1 gene was found in 40% of metastatic melanoma. To determine if loss of Apaf-1 expression is indeed involved in melanoma progression, we employed the tissue microarray technology and examined Apaf-1 expression in 70 human primary malignant melanoma biopsies by immunohistochemistry. Our data showed that Apaf-1 expression is significantly reduced in melanoma cells compared with normal nevi (χ2=6.02, P=0.014). Our results also revealed that loss of Apaf-1 was not associated with the tumour thickness, ulceration or subtype, patient's gender, age and 5-year survival. In addition, our in vitro apoptosis assay revealed that overexpression of Apaf-1 can sensitise melanoma cells to anticancer drug treatment. Taken together, our data indicate that Apaf-1 expression is significantly reduced in human melanoma and that Apaf-1 may serve as a therapeutic target in melanoma.
PMCID: PMC2747705  PMID: 15305193
Apaf-1; tissue microarray; melanoma; gene expression
25.  Vemurafenib potently induces endoplasmic reticulum stress-mediated apoptosis in BRAFV600E melanoma cells 
Science signaling  2013;6(260):ra7.
The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase (MAPK) signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of anti-apoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress-induced response, vemurafenib decreased the abundance of the ER chaperone protein GRP78, increased the abundance of the spliced isoform of the transcription factor X-box protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress-related genes. Knockdown of the ER stress response protein ATF4 significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance.
PMCID: PMC3698985  PMID: 23362240

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