Human Melanoma Cell Line G361 Research Articles

Geranylgeranylacetone induces apoptosis via the intrinsic pathway in human melanoma cells.

The aim of this study was to test the anti-cancer effects of geranylgeranylacetone (GGA), an isoprenoid compound, on human melanoma cells. Human melanoma cell lines G361, SK-MEL-2, and SK-MEL-5 were treated with GGA at various doses (1-100μM). Cell viability was measured by crystal violet assay. Western blot analysis was adopted to detect marker proteins of apoptosis. GGA significantly reduced the viability of G361, SK-MEL-2, and SK-MEL-5 human melanoma cells at concentrations above 10μM. Western blot analysis showed the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) after GGA treatment, as well as activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) cleavage. GGA also induced p53 and Bax expression, but did not affect expression of Bcl-2 and MITF. These findings suggest that GGA induces apoptosis through the intrinsic pathway. Accordingly, GGA should be considered for further development as a potential agent for melanoma.

A Novel Series of Highly Potent 2,6,9-Trisubstituted Purine Cyclin-Dependent Kinase Inhibitors

The inhibition of overactive CDKs during cancer remains an important strategy in cancer drug development. We synthesized and screened a novel series of 2-substituted-6-biarylmethylamino-9-cyclopentylpurine derivatives for improved CDK inhibitory activity and antiproliferative effects. One of the most potent compounds, 6b, exhibited strong cytotoxicity in the human melanoma cell line G361 that correlated with robust CDK1 and CDK2 inhibition and caspase activation. In silico modeling of 6b in the active site of CDK2 revealed a high interaction energy, which we believe is due to the 6-heterobiarylmethylamino substitution of the purine moiety.

Growth Inhibition of Human Melanoma Cells by a Recombinant Arginine Deiminase Expressed in Escherichia coli

We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361.

Growth inhibition and apoptosis induction of human melanoma cells by ??-hydroxy fatty acids

We examined the anti-tumor activity and structure-activity requirements of omega-hydroxy fatty acids (omega-HFAs) on the human melanoma cell line G361. The omega-hydroxystearic acid (omega-HSA) had strong growth-inhibiting and cytotoxic activity. Although omega-hydroxypalmitic acid (omega-HPA) also had growth-inhibiting and cytotoxic activity, these effects were relatively low. The effects of both these acids were dose and time dependent. Further, DNA laddering, which is an index of apoptosis, was also observed in G361 cells on treatment with these compounds. On the other hand, the omega-HFAs tested in this study, omega-hydroxymyristic acid and omega-hydroxyeicosanoic acid, had no growth-inhibiting or cytotoxic activity. Treatment for 12 h with 100 microM of omega-HPA and omega-HSA resulted in the expression of caspase-3 activity, and then increased upon 24 h, suggesting that the cell death induced by omega-HPA and omega-HSA was apoptosis. Fatty acids and dicarboxylic acids, which are analogs of omega-HFAs, had no cytotoxicity. However, fatty alcohols and diols, which have a 16- to 18-carbon chain length had weak cytotoxicity. From these results, the most effective carbon chain length is 18. Furthermore, the hydroxyl group at one end of the carbon chain and the carboxyl group at the other end seem to be required for the cytotoxic effect. At least one end of the carbon chain must have a hydroxyl group. The carbon chain length of omega-HFAs appears to be closely related to the cytotoxicity. This study revealed the potent cytotoxic actions of omega-HFAs on the human melanoma cell line G361.

Occurrence of Oligosialic Acids on Integrin α5Subunit and Their Involvement in Cell Adhesion to Fibronectin

Integrin alpha(5)beta(1), a major fibronectin receptor, functions in a wide variety of biological phenomena. We have found that alpha 2-8-linked oligosialic acids with 5 < or = degree of polymerization (DP) < or = 7 occur on integrin alpha(5) subunit of the human melanoma cell line G361. The integrin alpha(5) subunit immunoprecipitated with anti-integrin alpha(5) antibody reacted with the monoclonal antibody 12E3, which recognizes oligo/polysialic acid with DP > or = 5 but not with the polyclonal antibody H.46 recognizing oligo/polysialic acid with DP > or = 8. The occurrence of oligosialic acids was further demonstrated by fluorometric C(7)/C(9) analysis on the immunopurified integrin alpha(5) subunit. Oligosialic acids were also found in the alpha(5) subunit of several other human cells such as foreskin fibroblast and chronic erythroleukemia K562 cells. These results suggest the ubiquitous modification with unique oligosialic acids occurs on the alpha(5) subunit of integrin alpha(5)beta(1). The adhesion of human melanoma G361 cells to fibronectin was mainly mediated by integrin alpha(5)beta(1). Treatment of cells with sialidase from Arthrobacter ureafaciens cleaving alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids inhibited adhesion to fibronectin. On the other hand, N-acetylneuraminidase II, which cleaves alpha 2-3 and alpha 2-6 but not alpha 2-8 linkages, showed no inhibitory activity. After the loss of oligosialic acids, integrin alpha(5)beta(1) failed to bind to fibronectin-conjugated Sepharose, indicating that the oligosialic acid on the alpha(5) subunit of integrin alpha(5)beta(1) plays important roles in cell adhesion to fibronectin.

Gp100 mRNA is more sensitive than tyrosinase mRNA for RT-PCR amplification to detect circulating melanoma cells in peripheral blood of melanoma patients

Two different melanocyte-specific mRNAs are studied as markers for circulating melanoma cells in vitro using the human melanoma cell line G361 and in vivo using blood samples from Japanese melanoma patients at different clinical stages. These mRNAs encode tyrosinase, the most essential enzyme for melanin synthesis, and gp100, a melanosomal matrix glycoprotein recognized by monoclonal antibody HMB-45. We used reverse-transcription polymerase chain reaction (RT-PCR) to detect tyrosinase mRNA and gp100 mRNA in peripheral blood. Since melanocytes would not normally be present in peripheral blood, the detection of those transcripts should indicate the presence of circulating melanoma cells. RT-PCR detection of these two mRNAs was highly sensitive and specific. Our in vitro study showed that as few as 10 melanoma cells in 0.125 ml normal blood could be detected. In in vivo study, 130 blood samples from 55 melanoma patients gave positive and variably sensitive results, whereas no samples from healthy controls or patients with other cancers gave positive results. Tyrosinase mRNA was not detected in any of the melanoma patients. gp100 mRNA was detected in 12 of 55 melanoma patients, in none of five stage I patients (0%), in four of 26 stage II patients (15.4%), in one of six stage III patients (16.7%) and in seven of 18 stage IV patients (38.9%). Thus gp100 mRNA is a more sensitive marker for detecting circulating melanoma cells compared with tyrosinase mRNA.

Superantigen-induced lysis of melanoma cells.

Superantigens like the Staphylococcus enterotoxin A (SEA) can direct cytotoxic T lymphocytes expressing certain T cell receptor V beta regions to lyse MHC class II-positive target cells. This superantigen-dependent cellular cytotoxicity (SDCC) has been extended to MHC class II-negative tumour cells by targeting T cells via conjugates of a tumour-specific monoclonal antibody (moAb) and a superantigen. In the present study the MHC class II-negative human melanoma cell lines G361 and MaRI were tested for susceptibility to SDCC in vitro. Antibodies recognizing the disialoganglioside GD3 and the CD10 antigen were linked to SEA either by a recombinant protein A-SEA fusion protein or an anti-kappa moAb-SEA chemical conjugate. Specific lysis of melanoma cells was dose- and effector to target (E:T) cell ratio-dependent. Introduction of a point mutation into the SEA gene (producing SEAm9) in order to reduce MHC II affinity of the superantigen, which has already been shown to severely diminish superantigen-dependent binding and lysis of MHC class II-positive cells, did not influence antibody-targeted SDCC. Cytotoxicity was equal with both antibodies (anti-GD3 and anti-CD10) and independent of whether protein A-SEA, protein A-SEAm9 or anti-kappa-SEA were used.

Synergistic Activation of Interleukin-8 Gene Transcription by All-trans-retinoic Acid and Tumor Necrosis Factor-α Involves the Transcription Factor NF-κB

Induction of interleukin-8 (IL-8) by IL-1 or tumor necrosis factor (TNF), and repression by interferons or glucocorticoids have been shown to involve sequences between nucleotides -94 and -71 of the 5'-flanking region, and the transcription factors NF-IL-6 and NF-kappaB. The A3 cell line was derived from the human melanoma cell line G-361 by stable transfection with part of the IL-8 promoter (nucleotides -101 to +40 from transcription start) fused to the luciferase coding region. These regulatory sequences were sufficient for transcriptional activation by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid, IL-1beta, or TNF-alpha. Simultaneous treatment of A3 cells with ATRA and TNF-alpha resulted in a dose- and time-dependent synergistic increase in luciferase expression and IL-8 mRNA levels. Transient transfections of the parental cell line demonstrated that the NF-kappaB binding site is essential for this synergistic transactivation. Electrophoretic mobility shift assays with nuclear extracts of A3 cells showed that stimulation with ATRA and TNF-alpha for more than 16 h resulted in enhanced NF-kappaB binding compared to that induced by TNF-alpha alone. The simultaneous treatment with ATRA and TNF-alpha also resulted in changes in the composition of NF-kappaB complexes bound to the IL-8 NF-kappaB site, preventing the formation of two TNF-alpha-inducible binding activities. We suggest that these complexes consist of repressive factors which, when removed, allow enhanced binding of NF-kappaB to its cognate site.