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.
Antitumor activity; B16F1 melanoma; C57BL/6 mice; kaurenic acid
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.
Kaurenoic acid; Glioblastoma; miR-21; c-FLIP; Apoptosis
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.
Longikaurin A (LK-A); Apoptosis; Caspase; Nasopharyngeal carcinoma
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.
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.
Apoptosis; ent-kaurene diterpenoid; esophageal cancer; mitochondria pathway; rabdosia rubescens; reactive oxygen species
Harmine is a beta-carboline alkaloid from the plant Peganum harmala. Previous studies found that harmine inhibited metastasis of B16F-10 melanoma cells. This study aims to elucidate the role of harmine in apoptosis of B16F-10 cells.
B16F-10 melanoma cells were treated in the presence and absence of harmine in vitro. Morphological changes, cell cycle and expression of various pro and anti- apoptotic genes were analyzed for the study of apoptosis.
Morphological observation and DNA laddering assay showed that harmine treated cells displayed marked apoptotic characteristics, such as nuclear fragmentation, appearance of apoptotic bodies and DNA laddering fragment. TUNEL assay and flow cytometric analysis also confirmed apoptosis. Furthermore, RT-PCR analysis showed that harmine induced apoptosis in B16F-10 melanoma cells by up-regulating Bax and activating Caspase-3, 9 and p53 and down-regulating Bcl-2. Harmine also up-regulated Caspase-8 and Bid, indicating that harmine affected both extrinsic and intrinsic pathways of apoptosis. This study also showed inhibitory effects of harmine on some transcription factors and pro- inflammatory cytokines that protect cell from apoptosis.
Harmine activates both intrinsic and extrinsic pathways of apoptosis and regulates some transcription factors and pro-inflammatory cytokines.
The fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces metabolites of biotechnological interest, such as gibberellins, bikaverins, and carotenoids. Gibberellin and bikaverin productions are induced upon nitrogen exhaustion, while carotenoid accumulation is stimulated by light. We evaluated the effect of nitrogen availability on carotenogenesis in comparison with bikaverin and gibberellin production in the wild type and in carotenoid-overproducing mutants (carS). Nitrogen starvation increased carotenoid accumulation in all strains tested. In carS strains, gibberellin and bikaverin biosynthesis patterns differed from those of the wild type and paralleled the expression of key genes for both pathways, coding for geranylgeranyl pyrophosphate (GGPP) and kaurene synthases for the former and a polyketide synthase for the latter. These results suggest regulatory connections between carotenoid biosynthesis and nitrogen-controlled biosynthetic pathways in this fungus. Expression of gene ggs1, which encodes a second GGPP synthase, was also derepressed in the carS mutants, suggesting the participation of Ggs1 in carotenoid biosynthesis. The carS mutations did not affect genes for earlier steps of the terpenoid pathway, such as fppS or hmgR. Light induced carotenoid biosynthesis in the wild type and carRA and carB levels in the wild-type and carS strains irrespective of nitrogen availability.
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.
prostate cancer; inflammation; nuclear factor-κB
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.
REPRESSION OF SHOOT GROWTH; GA 20-oxidase; gibberellin biosynthesis; feedback regulation; signaling; transcription factor; histone modification
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.
inhibitor of apoptosis protein; ML-IAP; melanoma; naevus; expression
Wheat (Triticum aestivum) and rice (Oryza sativa) are two of the most agriculturally important cereal crop plants. Rice is known to produce numerous diterpenoid natural products that serve as phytoalexins and/or allelochemicals. Specifically, these are labdane-related diterpenoids, derived from a characteristic labdadienyl/copalyl diphosphate (CPP), whose biosynthetic relationship to gibberellin biosynthesis is evident from the relevant expanded and functionally diverse family of ent-kaurene synthase-like (KSL) genes found in rice (OsKSL). Here we report biochemical characterization of a similarly expansive family of KSL from wheat (the TaKSLs). In particular, beyond ent-kaurene synthases (KS), wheat also contains several biochemically diversified KSLs. These react either with the ent-CPP intermediate common to gibberellin biosynthesis or with the normal stereoisomer of CPP that also is found in wheat (as demonstrated by the accompanying description of wheat CPP synthases). Comparison with a barley (Hordeum vulgare) KS indicates conservation of monocot KS, with early and continued expansion and functional diversification of KSLs in at least the small grain cereals. In addition, some of the TaKSLs that utilize normal CPP also will react with syn-CPP, echoing previous findings with the OsKSL family, with such enzymatic promiscuity/plasticity providing insight into the continuing evolution of diterpenoid metabolism in the cereal crop plant family, as well as more generally, which is discussed here.
ent-kaurene synthase; phytoalexin; phytoanticipin; allelochemicals; natural products biosynthesis; plant defense
Brain metastases are a common pre-terminal event in patients with metastatic melanoma and require radiation therapy. Our group has previously demonstrated that human GRM1 (hGRM1) expressing melanoma cells release excess extracellular glutamate and are growth inhibited by riluzole, an inhibitor of glutamate release. Riluzole treated cells accumulate in G2/M phase of the cell cycle at 24 hours, and then undergo apoptotic cell death. We evaluated whether riluzole enhanced radiosensitivity in melanoma cells.
Clonogenic assays were performed to evaluate clonogenic survival after treatment in hGRM1 expressing and non-expressing melanoma cells. Western immunoblots were performed to confirm apoptotic cell death. A xenograft mouse model was used to validate the in vitro experiments. Tumors harvested from the xenografts were fixed and stained for apoptosis and DNA damage markers.
In the hGRM1-positive cell lines C8161 and UACC903, riluzole enhanced the lethal effects of ionizing radiation; no difference was seen in the hGRM1-negative UACC930 cell line. C8161 cells treated with riluzole plus irradiation also showed the highest levels of the cleaved forms of PARP and caspase-3; excised C8161 xenografts demonstrated the greatest number of apoptotic cells by immunohistochemistry (p<0.001). On cell cycle analysis, a sequence-dependent enrichment in the G2/M phase was demonstrated with the combination of riluzole and irradiation. Xenografts treated with riluzole and weekly radiation fractions demonstrated significant growth inhibition and revealed markedly increased DNA damage.
We have demonstrated, in vitro and in vivo, that the combination of riluzole and ionizing radiation leads to greater cytotoxicity. These results have clinical implications for patients with brain metastases receiving whole brain radiation therapy.
Glutamate; Melanoma; riluzole; Cell Surface Receptors; Ionizing Radiation
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.
Apaf-1; tissue microarray; melanoma; gene expression
Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a pro-apoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 together with the adoptive transfer (AT) of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: 1) increase in MHC and tumor-associated antigen (TAA) expression by tumor cells; 2) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and 3) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein-2 (TRP2), which also demonstrated a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes.
Rodent; T cells; tumor immunity; dendritic cells; vaccination
Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.
Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRAS or BRAF have been identified in ∼85% of melanomas and components of this pathway have been developed as molecular targets for therapeutic intervention. We and others have demonstrated that inhibition of this pathway with specific small molecule MEK inhibitors induces a wide range of apoptotic responsiveness in human melanoma cells both in vitro and in vivo. To define the molecular mechanism underlying variable apoptotic sensitivity of melanoma cells to MEK inhibition, we examined the expression and subcellular localization of Bcl-2 family members in a comprehensive set of human melanoma cell lines. While the proapoptotic protein Bim was activated and localized to the mitochondrial membrane in all cell lines regardless of apoptotic sensitivity, Bmf activation and cytosolic translocation was exclusive to sensitive cells. In resistant cells, Bmf remained sequestered to the cytoskeleton through dynein light chain 2 (DLC2) binding. Overexpression of Bmf in resistant cells did not enhance apoptosis, whereas expression of mutant BmfA69P, which has decreased binding to DLC2, promoted cell death. Expression of BmfA69P mutants possessing the BH3 domain mutation L138A, which impairs BH3 interactions, did not enhance apoptosis in resistant cells. RNA interference targeting Bim and Bmf provided protection from apoptosis induced by MEK inhibition. These results demonstrate a novel role for Bmf in promoting apoptosis and provide insight into the mechanism of apoptotic resistance to MEK inhibition in melanoma.
The contribution of innate immunity responsible for aggressive β-cell destruction in human fulminant type 1 diabetes is unclear.
RESEARCH DESIGN AND METHODS
Islet cell expression of Toll-like receptors (TLRs), cytoplasmic retinoic acid–inducible gene I (RIG-I)-like receptors, downstream innate immune markers, adaptive immune mediators, and apoptotic markers was studied in three autopsied pancreata obtained 2 to 5 days after onset of fulminant type 1 diabetes.
RIG-I was strongly expressed in β-cells in all three pancreata infected with enterovirus. Melanoma differentiation–associated gene-5 was hyperexpressed in islet cells, including β- and α-cells. TLR3 and TLR4 were expressed in mononuclear cells that infiltrated islets. Interferon (IFN)-α and IFN-β were strongly expressed in islet cells. Major histocompatibility complex (MHC)-class I, IFN-γ, interleukin-18, and CXC motif ligand 10 were expressed and colocalized in affected islets. CD11c+ MHC-class II+ dendritic cells and macrophage subsets infiltrated most islets and showed remarkable features of phagocytosis of islet cell debris. CD4+ forkhead box P3+ regulatory T cells were not observed in and around the affected islets. Mononuclear cells expressed the Fas ligand and infiltrated most Fas-expressing islets. Retinoic acid–receptor responder 3 and activated caspases 8, 9, and 3 were preferentially expressed in β-cells. Serum levels of IFN-γ were markedly increased in patients with fulminant type 1 diabetes.
These findings demonstrate the presence of specific innate immune responses to enterovirus infection connected with enhanced adoptive immune pathways responsible for aggressive β-cell toxicity in fulminant type 1 diabetes.
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.
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.
IGFBP3; methylation; melanoma; cell growth; tumor suppressor
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.
The tumor suppressor candidate gene RASSF1A encodes a microtubule-associated protein that is implicated in the regulation of cell proliferation, migration, and apoptosis. Several studies indicate that down-regulation of RASSF1A resulting from promoter hypermethylation is a frequent epigenetic abnormality in malignant melanoma. In this study, we report that compared with melanocytes in normal skins or benign skin lesions, RASSF1A is down-regulated in melanoma tissues as well as cell lines, and its expression negatively correlates with lymph node metastasis. Following ectopic expression in RASSF1A–deficient melanoma A375 cell line, RASSF1A reduces cell viability, suppresses cell cycle progression but enhances apoptotic cell death. In vivo, RASSF1A expression inhibits the tumorigenic potential of A375 cells in nude mice, which also correlates with decreased cell proliferation and increased apoptosis. On the molecular level, ectopic RASSF1A expression leads to differential expression of 209 genes, including 26 down-regulated and 183 up-regulated ones. Among different signaling pathways, activation of the apoptosis signal-regulating kinase 1 (ASK1)/p38 MAP kinase signaling is essential for RASSF1A-induced mitochondrial apoptosis, and the inhibition of the Akt/p70S6 kinase/eIF4E signaling is also important for RASSF1A-mediated apoptosis and cell cycle arrest. This is the first study exploring the biological functions and the underlying mechanisms of RASSF1A during melanoma development. It also identifies potential targets for further diagnosis and clinical therapy.
RASSF1A; tumor suppressor gene; melanoma; apoptosis; cell cycle
Increased expression of genes, silenced by methylation of their promoters, could have relevance for increasing effects of not only interferons (IFNs) but also APO2L/TRAIL, cytotoxics and immunotherapeutics for melanoma and other malignancies. A resistant melanoma cell line, A375, lacked APO2L/TRAIL or apoptosis induction by either IFN-α2 or IFN-β. However, apoptosis was induced by IFNs in A375 cells by 5-aza, 2′deoxycytidine, evaluated based upon the postulate that promoter methylation might be silencing pro-apopoptotic IFN-stimulated genes (ISGs). RASSF1A, commonly methylated at high frequency in many tumors including melanoma, which we discovered to be also an IFN-regulated gene, was increased by 5-Aza-dC. RASSF1A was important in enhancing apoptotic effects of not only IFNs and APO2L/TRAIL but also cisplatin. Unraveling epigenetic regulatory mechanisms, as yet only partially identified, will result in new biological insights and improved strategies for therapeutic use of IFNs or ISGs such as APO2L/TRAIL.
APO2L/TRAIL; azacytidine; apoptosis
Treatments for primary and metastatic melanomas are rarely effective. Even therapeutics such as retinoic acid (RA) that are successfully used to treat several other forms of cancer are ineffective. Recent evidence indicates that the antiproliferative effects of RA are mediated by the transcription factor SOX9 in human cancer cell lines. As we have previously shown that SOX9 is expressed in normal melanocytes, here we investigated SOX9 expression and function in human melanomas. Although SOX9 was expressed in normal human skin, it was increasingly downregulated as melanocytes progressed to the premalignant and then the malignant and metastatic states. Overexpression of SOX9 in both human and mouse melanoma cell lines induced cell cycle arrest by increasing p21 transcription and restored sensitivity to RA by downregulating expression of PRAME, a melanoma antigen. Furthermore, SOX9 overexpression in melanoma cell lines inhibited tumorigenicity both in mice and in a human ex vivo model of melanoma. Treatment of melanoma cell lines with PGD2 increased SOX9 expression and restored sensitivity to RA. Thus, combined treatment with PGD2 and RA substantially decreased tumor growth in human ex vivo and mouse in vivo models of melanoma. The results of our experiments targeting SOX9 provide insight into the pathophysiology of melanoma. Further, the effects of SOX9 on melanoma cell proliferation and RA sensitivity suggest the encouraging possibility of a noncytotoxic approach to the treatment of melanoma.
Our understanding of the molecular pathways that underlie melanoma remains incomplete. Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies. Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways.
Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules. Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients. The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis. An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study. Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active. Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro.
This analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis. Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma biological processes (e.g., proliferation) but not others (e.g., immune response). The gene expression modules identified here, and the RNAs predicted by Bayesian network inference to be upstream of these modules, are potential prognostic biomarkers and drug targets.
Numerous immune genes are epigenetically silenced in tumor cells and agents such as histone deacetylase inhibitors (HDACi), which reverse these effects, could potentially be used to develop therapeutic vaccines. The conversion of cancer cells to antigen presenting cells (APCs) by HDACi treatment could potentially provide an additional pathway, together with cross-presentation of tumor antigens by host APCs, to establish tumor immunity.
HDACi-treated B16 melanoma cells were used in a murine vaccine model, lymphocyte subset depletion, ELISpot and Cytotoxicity assays were employed to evaluate immunity. Antigen presentation assays, vaccination with isolated apoptotic preparations and tumorigenesis in MHC-deficient mice and radiation chimeras were performed to elucidate the mechanisms of vaccine-induced immunity.
HDACi treatment enhanced the expression of MHC class II, CD40 and B7-1/2 on B16 cells and vaccination with HDACi-treated melanoma cells elicited tumor specific immunity in both prevention and treatment models. Cytotoxic and IFN-γ-producing cells were identified in splenocytes and CD4+, CD8+ T cells and NK cells were all involved in the induction of immunity. Apoptotic cells derived from HDACi treatments, but not H2O2, significantly enhanced the effectiveness of the vaccine. HDACi-treated B16 cells become APCs in vitro and studies in chimeras defective in cross presentation demonstrate direct presentation in vivo and short-term but not memory responses and long-term immunity.
The efficacy of this vaccine derives mainly from cross-presentation which is enhanced by HDACi-induced apoptosis. Additionally, epigenetic activation of immune genes may contribute to direct antigen presentation by tumor cells. Epigenetically altered cancer cells should be further explored as a vaccine strategy.