Proteasome inhibitors against amelanotic melanoma.

The incidence of malignant melanoma, the most aggressive skin cancer, is increasing constantly. Despite new targeted therapies, the prognosis for patients with metastatic disease remains poor. Thus, there is a need for new combinational treatments, and antineoplastic agents potentially valuable in this approach are inhibitors of the ubiquitin-proteasome system (UPS). In this work, we analyze the cytotoxicity mechanisms of proteasome inhibitors (MG-132, epoxomicin, and lactacystin) in a specific form of melanoma which does not synthesize melanin-the amelanotic melanoma (Ab cells). We found that the most cytotoxic of the compounds tested was epoxomicin. Caspase-9 activation as well as cytochrome C and AIF release from mitochondria indicated that exposure to epoxomicin induced the mitochondrial pathway of apoptosis. Epoxomicin treatment also resulted in accumulation of Bcl-2 family members-proapoptotic Noxa and antiapoptotic Mcl-1, which were postulated as the targets for bortezomib in melanoma. Inhibition of caspases by BAF revealed that cell death was partially caspase-independent. We observed no cell cycle arrest preceding the apoptosis of Ab cells, even though cdk inhibitors p21Cip1/Waf1 and p27Kip1 were up-regulated. The cell cycle was blocked only after inactivation of caspases by the pan-caspase inhibitor BAF. In summary, this is the first study exploring molecular mechanisms of cell death induced by epoxomicin in melanoma. We found that Ab cells died on the mitochondrial pathway of apoptosis and also partially by the caspase-independent way of death. Apoptosis induction was fast and efficient and was not preceded by cell cycle arrest.

BRAF and KIT somatic mutations are present in amelanotic melanoma.

The genotypic profile of rare amelanotic melanomas (AMs) has been poorly investigated, thus preventing either an accurate identification as a distinctive melanoma subtype or therapy stratification. Here, we investigated the presence of the BRAF(V600E) mutation by real-time quantitative PCR and KIT mutations (exons 11 and 17) by sequencing analysis in 33 AMs. AMs included 'truly' amelanotic lesions (n = 19), with no melanin pigmentation upon dermoscopic inspection and hypomelanotic lesions (n = 14), by definition partially pigmented lesions showing a melanin pigmentation area of less than 25% of the total surface area. The frequency of the BRAF(V600E) mutation was 70.3% in the 33 cases, a percentage that increased to 89% when only the subgroup of thin melanomas (≤ 1 mm in thickness, n = 9) was considered. KIT mutations were found in 12.1% of AMs, all of which developed in nonacral sites. The identification of a relatively high frequency of BRAF(V600E) and KIT mutations in AMs may have important consequences for implementation of the novel targeted therapies now available to treat this life-threatening disease.

Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity.

BACKGROUND: Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype. METHODS: The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored. RESULTS: We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs. CONCLUSION: Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the cell phenotype and can induce a higher sensitivity to tyrosine related anti-blastic drugs.

The activity of caspases in spontaneous and camptothecin-induced death of melanotic and amelanotic melanoma cell.

Loss of pigment in hamster amelanotic melanoma line is accompanied by a faster growth rate, higher tumorigenicity and shorter animal survival time. Thus, the malignancy of melanoma increases during the alteration of melanotic (Ma) into amelanotic (Ab) line. As changes in the ability to undergo a spontaneous or induced apoptosis, and the role of caspases in this process during melanoma progression are not well defined, they were investigated in this work. Our results show that the proportion of spontaneously early apoptotic (caspase+/PI-) cells in the Ab line decreased in comparison to the Ma line. Cytochrome c release into cytosol, and the activation of effector caspases, estimated by PARP degradation clearly showed that during the spontaneous death in the cells from both melanoma lines intrinsic way of apoptosis was activated. Confocal and cytometric flow analyses indicate that camptothecin (CPT) induced apoptosis with caspase activation by the intrinsic way only in the amelanotic melanoma cells, even though cells of the Ma line also underwent CPT-induced apoptosis (the content of TUNEL-positive cells increased). Thus, our results suggest that melanoma progression, associated with a decreased ability to undergo spontaneous apoptosis but an increased susceptibility to CPT-induced apoptosis, relates to different levels of caspase activation; they also show that intrinsic way of apoptotis depends on the phenotype of melanoma cells, being more pronounced in the melanotic melanoma cells. On the other hand, melanotic melanoma cells resistance to camptothecin-induced apoptosis suggests that the melanogenic apparatus or melanin itself may have the protective effect on the ability of the melanoma cells to undergo apoptosis.

Increased level of p27 subunit of proteasomes and its co-localization with tyrosinase in amelanotic melanoma cells indicate its direct role in the regulation of melanin biosynthesis.

Proteasomes have been shown to be involved in the regulation of melanin biosynthesis in melanoma cells. Here we report on the correlation between proteasome subunits and Tyrosinase (Tyr) activity in different cell phenotypes, and thereby regulation of melanin biosynthesis in B16F10 mouse melanoma cells. Our results indicated that the quantity of proteasome subunit p27 is higher and that of the enzyme Tyr and its activity are lower in amelanotic melanoma cells, while the reverse is true in melanotic melanoma cells. Proteasome subunit p27, compared to another subunit p31, shows increased co-localization with Tyr and Tyrosinase related protein 1 (Trp1) in amelanotic cells to a greater extent than that in melanotic cells. On exposure to cycloheximide, increased Tyr degradation was seen in amelanotic cells, as indicated by increased co-localization of p27 and Tyr. Further, exposure of amelanotic melanoma cells with proteasome-specific inhibitor MG132 resulted in an increased Tyr activity, increased levels of Tyr and Trp1, leading to increased melanin synthesis. These results therefore suggest that proteasomes, particularly p27 subunit, are directly involved in the regulation of melanin biosynthesis in mouse melanoma cells.

Improvement of the tumor-suppressive effect of boron neutron capture therapy for amelanotic melanoma by intratumoral injection of the tyrosinase gene.

Boron neutron capture therapy (BNCT) is successful when there is a sufficient (10)B concentration in tumor cells. In melanoma, (10)B-para-boronophenylalanine (BPA) accumulation is proportional to melanin-producing activity. This study was done to confirm enhancement of the tumor-suppressive effect of BNCT on amelanotic melanoma by intratumoral injection of the tyrosinase gene. D178 or FF amelanotic melanomas were implanted s.c. in Syrian hamsters. One group of D178- or FF-bearing hamsters (TD178 or TFF group) received intratumoral injections of pcDNA-Tyrs constructed as a tyrosinase expression plasmid. The other hamsters (pD178 and pFF groups) were injected with pUC119, and control hamsters (D178 and FF groups) only with transfection reagents. All the groups underwent immunofluorescence analysis of tyrosinase expression and BPA biodistribution studies. BNCT experiments were done at the Kyoto University Research Reactor. Tyrosinase expression increased in the tumors of the TD178 and TFF groups but remained the same in the pD178 and pFF groups. Tumor boron concentrations in the TD178 and TFF groups increased significantly (TD178: 49.7 +/- 12.6 versus D178: 27.2 +/- 4.9 microg/g, P < 0.0001; TFF: 30.7 +/- 6.6 versus FF: 13.0 +/- 4.7 microg/g, P < 0.0001). The BNCT tumor-suppressive effect was marked in the TD178 and TFF groups. In vivo transfection with the tyrosinase gene increased BPA accumulation in the tumors, the BNCT tumor-suppressive effect on amelanotic melanoma being significantly enhanced. These findings suggest a potential new clinical strategy for the treatment of amelanotic melanoma with BNCT.

Microtubule-dependent matrix metalloproteinase-2/matrix metalloproteinase-9 exocytosis: prerequisite in human melanoma cell invasion.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave and degrade a wide spectrum of extracellular matrix components. By enhancing turnover of extracellular matrix, MMP activity is also known to play a key role in tumor cell invasion. Because extracellular protease activity requires efficient release of these proteases to the cellular surface, we investigated storage, transport, and exocytosis of MMP-2 and MMP-9 in human melanoma cells using immunofluorescence, electrical, and biochemical techniques. Immunolabeling of melanoma cells with antibodies specific for MMP-2 and MMP-9 led to the identification of two distinct populations of small cytoplasmatic vesicles containing MMP-2 or MMP-9, respectively. In combination with alpha-tubulin-specific antibodies, both vesicle populations were found to be aligned along the microtubular network. Moreover, the molecular motor protein kinesin is shown to be localized on most of these vesicles, providing evidence that the identified vesicles are actively propelled along microtubules toward the plasma membrane. The functional relevance of these findings is demonstrated using low dosage (5.9 nmol/L) of paclitaxel to affect the microtubular function of melanoma cells. Although cell proliferation is not altered, paclitaxel treatment impairs secretion of MMP-2/MMP-9 and significantly reduces invasive activity in our new cell invasion assay. In conclusion, we demonstrate in melanoma cells that microtubule-dependent traffic of MMP-containing vesicles and exocytosis are critical steps for invasive behavior and therefore are potential targets for specific antitumor drugs.

Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity.

Pigmentation of the hair, skin, and eyes of mammals results from a number of melanocyte-specific proteins that are required for the biosynthesis of melanin. Those proteins comprise the structural and enzymatic components of melanosomes, the membrane-bound organelles in which melanin is synthesized and deposited. Tyrosinase (TYR) is absolutely required for melanogenesis, but other melanosomal proteins, such as TYRP1, DCT, and gp100, also play important roles in regulating mammalian pigmentation. However, pigmentation does not always correlate with the expression of TYR mRNA/protein, and thus its function is also regulated at the post-translational level. Thus, TYR does not necessarily exist in a catalytically active state, and its post-translational activation could be an important control point for regulating melanin synthesis. In this study, we used a multidisciplinary approach to examine the processing and sorting of TYR through the endoplasmic reticulum (ER), Golgi apparatus, coated vesicles, endosomes and early melanosomes because those organelles hold the key to understanding the trafficking of TYR to melanosomes and thus the regulation of melanogenesis. In pigmented cells, TYR is trafficked through those organelles rapidly, but in amelanotic cells, TYR is retained within the ER and is eventually degraded by proteasomes. We now show that TYR can be released from the ER in the presence of protonophore or proton pump inhibitors which increase the pH of intracellular organelles, after which TYR is transported correctly to the Golgi, and then to melanosomes via the endosomal sorting system. The expression of TYRP1, which facilitates TYR processing in the ER, is down-regulated in the amelanotic cells; this is analogous to a hypopigmentary disease known as oculocutaneous albinism type 3 and further impairs melanin production. The sum of these results shows that organellar pH, proteasome activity, and down-regulation of TYRP1 expression all contribute to the lack of pigmentation in TYR-positive amelanotic melanoma cells.

Activity of antioxidant enzymes and concentrations of thiobarbituric acid reactive substances (TBARS) in melanotic and amelanotic Bomirski melanoma tissues in the golden hamster (Mesocricetus auratus, Waterhouse).

The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSHPx), as well as the concentration of thiobarbituric acid reactive substances (TBARS) in tissues of transplantable melanoma in the golden hamster were measured and compared. Ten inbred male hamsters were used for the experiment. They were divided into two groups and were given Bomirski melanoma cells subcutaneously. The first group was given melanotic (Ma) melanoma cells. The second group was given amelanotic (Ab) melanoma cells. Thirty days after the transplantation the hamsters were dissected and the tumor tissues were taken and homogenized. A statistically significantly higher activity of the measured antioxidant enzymes was found in homogenates of Ma tumor than in homogenates of the Ab tumor. Activity of SOD is 8% higher in melanotic melanoma, 24% higher in CAT, and 45% higher in GSHPx. Statistically significant differences between TBARS concentrations were not confirmed. The higher activity of antioxidant enzymes in the melanotic tumor is a result of increased generation of oxygen-derived free radicals. It is presumed that it is strictly connected with intensified production of quinone and semiquinone radicals in the process of melanogenesis.

Development of cutaneous amelanotic melanoma in the absence of a functional tyrosinase.

Lack of characteristic pigmentation and a wide range of clinical presentations account for the diagnostic challenge associated with amelanotic malignant melanoma. Experimental studies of this important human cancer have been hampered by the lack of an appropriate animal model. We previously described a transgenic mouse line (TG-3) that spontaneously develops pigmented cutaneous melanoma. F1 crosses were generated with TG-3 and several albino strains, and backcrosses were then made with the albinos. In the present report, we describe the restricted development and characterization of cutaneous amelanotic melanoma in these albino transgenic backcrosses. The incidence and behavior of melanoma in these mice were monitored. A high incidence (80-100%) of spontaneous amelanotic melanoma was observed in albino transgenic mice derived from backcrosses with A, AKR, FVB, and SJL strains. The lowest incidence (30%) was obtained in BALB/c-derived crosses. No tumors were observed in non-transgenic mice. Immunohistochemical and western blot analyses using antibodies against three melanocyte-specific markers of the tyrosinase family of proteins confirmed that the tumors were composed of amelanotic melanocytes. Furthermore, the presence of numerous premelanosomes observed by electron microscopy further supported the melanocytic origin of these tumors. Previous in vitro studies on human melanoma have suggested that cutaneous amelanotic melanoma was evolving from preexisting pigmented cutaneous melanoma. However, our results indicate that it can occur directly, as evidenced by the appearance of cutaneous amelanotic melanoma in the tyrosinase-deficient albino mice. These mice represent a potentially valuable model for studying the mechanistic, diagnostic, and therapeutic features of this highly malignant neoplasm.

Inhibition of L-tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells.

It was previously found that L-tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM L-tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. L-Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of L-tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high L-tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of L-tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM L-tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from L-tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of L-tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.

Nitric oxide synthase activity is up-regulated in melanoma cell lines: a potential mechanism for metastases formation.

Nitric oxide (NO) may be an important mediator of tumour angiogenesis and metastasis formation. Tumour cell derived NO may be important in the regulation of angiogenesis and vasodilatation of the blood vessels surrounding a tumour. The aims of the present study were, firstly, to determine whether malignant melanoma cells and normal melanocytes had nitric oxide synthase (NOS) activity (measured by the conversion of L-arginine to L-citrulline) and, secondly, to determine whether there was a difference in NOS activity between malignant and normal cell types. This paper assays NOS activity directly in lysates from normal human melanocyte and malignant melanoma cell lines. The enzyme activity was not inducible with bacterial lipopolysaccharide and could be heat denatured. The activity of NOS was demonstrated to be both NADPH- and calcium-dependent and it was inhibitable in a dose-dependent manner by the NOS inhibitor Nw-nitro-L-arginine methyl ester. We conclude that melanoma and melanocyte cells express a constitutive form of NOS. Finally, nitric oxide synthase activity in melanoma cell lines was found to be significantly greater than in normal melanocytes. These findings suggest that NO synthesis is elevated in malignant melanoma. An elevated NO concentration in melanoma is expected to promote metastases by maintaining a vasodilator tone in the blood vessels in and around the melanoma.

Approaches to prove the melanoma origin in amelanotic human melanoma cell lines.

The human melanoma B-HM8 cell line was derived from highly pigmented malignant skin melanoma. After 5 weeks of cultivation it entirely lost the pigmentation and has remained amelanotic since. Electron microscopy revealed neither premelanosomes nor melanosomes and the cells did not release detectable amount of dopa-oxidase activity into culture medium. Immunocytochemical studies using the polyclonal anti-S-100 antibody and detection of alpha-mannosidase activity in culture medium proved the melanoma origin of B-HM8 cells. Chromosomal changes in the karyotype of these cells were typical for human melanoma with chromosomes No. 1, 5, 7, 9, and 11 involved most frequently.

Alteration of tyrosinase activity in human melanocytes and melanoma cells by histamine H2 and H3 ligands.

Nontoxic doses of the histamine H2 antagonists ranitidine, cimetidine, lamtidine and mifentidine rapidly and reversibly increased tyrosinase activity in an amelanotic human melanoma cell line (MM96L) with low constitutive activity. The H2 antagonists, famotidine and MGTI, and the imidazol(in)e receptor ligand clonidine had no effect either alone or in competition with ranitidine, whilst metiamide decreased tyrosinase activity. Lysosomotropic amines had a similar effect to ranitidine, except that induction reached a plateau at 6 h and was insensitive to amiloride. Human melanocytes and pigmented human melanoma cell lines exhibited minimal levels of tyrosinase induction, which was dependent on protein synthesis but not on RNA or DNA synthesis. Constitutive tyrosinase activity in MM96L cells was much less stable than in melanocytes and pigmented melanoma cells. No change was observed in expression of gp75, neural specific octamer binding proteins, or in mRNA levels of tyrosinase, Pmel-17 and gp75 (TRP-1). Tyrosinase was inhibited by the H3 agonist imetit but not by alpha-methylhistamine or the H3 antagonist thioperamide. Overall, this work showed that certain H2 antagonists activate an unstable form of tyrosinase in amelanotic melanoma cells by a post-transcriptional mechanism dependent on protein synthesis. An imidazoline/guanidinium receptor site rather than the H2 receptor appeared to be involved.