Melanoma Cells In Tissue Culture Research Articles

Bioactive peptides from several plant species

Bioactive peptides were observed in seeds of several plants. However, the number of peptides characterized in detail is currently small. Nevertheless, it was shown that cationic peptides containing cysteines in their structure exhibit strong protective properties against various types of infectious agents [1, 2]. Such peptides can provide alternatives to antibiotics to which several pathogens have developed resistance. We have shown previously that components of seeds from Apiaceae and Malvaceae plants (peptides and essential oils) exhibit pronounced antimicrobial activity [3–5]. Therefore we isolated peptides from seeds of pepper Capsicum annuum, Nigella sativa, Sesamum indicum, and Cuminum cyminum. Peptides were extracted most completely from ground and defatted seeds using acetic acid (0.05 M). Using mineral acids caused partial destruction of the peptides. Extraction by acetone and buffers was incomplete. The resulting extracts were neutralized with NaOH and centrifuged. The supernatant was lyophilized. Next, extracts were dialyzed twice against water and Tris-HCl buffer (pH 9.0). Then, extracts were separated successively by ion-exchange chromatography over columns of DEAE-servacel 23SN (2 10 cm, Reanal) and CM-TSK (2 5 cm, Tosoh Bioscience) using a stepwise NaCl gradient in ammonium acetate buffer (pH 6.0, 0–0.5 M). Peptide content in the resulting fractions was analyzed by electrophoresis [6] (15% PAAG with 0.1% Na-SDS, pH 8.9). Peptides with molecular weight (MW) 2–10 kDa were found. According to the literature and our results [7, 8], it can be assumed that peptides with MW 2–5 kDa belong to the defensin class; with MW ~10 kDa, to the so-called lipid-transfer proteins. The biological activity of the seed extracts and fractions obtained after separation over CM-TSK gel was checked in tests for fungitoxicity using a culture of pathogenic strains No. 108 Verticillium dahliae and a turbidimetric microplanchette method [9]; for cytotoxicity, KML murine melanoma cells in tissue culture and measurement of 3H-thymidine incorporation into tumor cell DNA [10] (Table 1). According to the results, extract of Nigella sativa containing its total cationic peptides and its separate components obtained by ion-exchange chromatography had the highest biological activity. The pronounced cytotoxic activity of Nigella saliva peptides makes them promising for further in-depth studies of the antitumor activity for various tumor types in both in vitro and in vivo tests.

Comparative expression of NFkappaB proteins in melanocytes of normal skin vs. benign intradermal naevus and human metastatic melanoma biopsies.

Nuclear factor kappa B (NFkappaB) is an essential regulator of gene transcription for hundreds of genes, including many critically involved in apoptosis. NFkappaB complexes containing cRel generally activate pro-apoptotic genes, while those with RelA activate anti-apoptotic genes. We have previously shown that NFkappaB binding by RelA is constitutively elevated in human metastatic melanoma cultures relative to normal melanocytes. Here we extended our investigation to immunohistochemical analysis of human tissue biopsies. We found that RelA expression is significantly elevated in melanocytes of human naevi and melanomas relative to normal skin, but expression of its inhibitor IkappaB-alpha is significantly lower in metastatic melanomas than in intradermal naevi. Antibodies specific for the nuclear localization signal of RelA also showed significantly increased staining in metastatic melanoma biopsies. Notably, in melanomas and in naevi, we also found that RelA is phosphorylated at serine 529, and this activated form accumulates in the nuclei of melanomas. This suggests that increased expression and phosphorylation of RelA occurs at the stage of the benign naevus, but IkappaB-alpha is able to sequester RelA in the cytoplasm and regulate RelA transcriptional transactivation. We also found that antibodies against cRel show a progressive increase in staining from naevi to melanoma. However, staining for IkappaB-epsilon, which primarily inhibits the nuclear localization of cRel was also progressively increased, and cRel expression was predominantly cytoplasmic in melanomas. These results confirm that the altered expression of RelA found in metastatic melanoma cells in tissue culture is relevant to human tumors and offer new insights into the deregulation of NFkappaB signaling.

The neuroprotective and neuronal rescue effects of (-)-deprenyl.

The pharmacological effects of (-)-deprenyl is multi-fold in its nature (dopamine sparing activity, neuroprotective and neuronal rescue effects), which cannot be explained solely by the irreversible MAO-B inhibitory action of the substance. Deprenyl slightly inhibits the re-uptake of noradrenaline and dopamine, but methylamphetamine, the metabolite of the inhibitor, by one order of magnitude is more potent in this respect, than the parent compound. Neither the metabolite nor (-)-deprenyl acts on the uptake of serotonin. The inhibitor has an intensive first pass metabolism after oral treatment. The in vivo pharmacokinetic studies with (-)-deprenyl, using the double labelled radioisotope technique (1.5 mg/kg; orally) in rats revealed that the molar concentration of methylamphetamine can reach the level suitable to induce a significant inhibition of amine uptake. Deprenyl, but especially methylamphetamine pre-treatment can prevent the noradrenaline release induced by the noradrenergic neurotoxin DSP-4. The uptake inhibitory effect of (-)-deprenyl and the metabolites is reversible. After repeated administration of (-)-deprenyl (1.5 mg/kg daily, for 8 days) sustained concentration of its metabolites was detected, compared to that of the acute studies. This can at least partly explain why (-)deprenyl should be administered daily to evoke therapeutic effects in Parkinson's disease. Administration of (-)-deprenyl in a low dose, following the toxic insult, can rescue the damaged neurones. The neuronal rescue effect of the drug was studied on M-1 human melanoma cells in tissue culture. The inhibitor reduced the apoptosis of serum-deprived M-1 cells, but the (+)-isomer failed to exert this effect. The (+/-)-desmethyl-deprenyl almost lacks the property to inhibit apoptosis. For neuroprotection and neuronal rescue an optimal dose of (-)-deprenyl should be administered, because to reach a well balanced concentration of the metabolites in tissues is critical.

HLA antigen expression on uveal melanoma cells in vivo and in vitro

Immunotherapy with cytokines may be an additional option in the treatment of primary uveal melanoma or melanoma metastases. A study of the effect of cytokines on cultured uveal melanoma cells may predict the effect that cytokines may have in vivo. Knowledge about the influence of cytokines on HLA expression may be especially beneficial, as HLA expression is essential for immune recognition. However, little is known about the normal expression of the HLA antigens on uveal melanoma cells in tissue culture. We therefore determined the HLA expression on short-term cultures of uveal melanoma cells and compared the results to the expression on tissue sections of the original tumors. In vivo and in vitro expression of the monomorphic HLA class I determinants and of HLA-A ( R = 0.77) correlated well. A slightly lower agreement was observed for expression of HLA-B ( R = 0.68). In vitro growth was associated with a decrease in expression of the class II determinant HLA-DR. We conclude that expression of HLA class I on cultured melanoma cells corresponds to the expression on the original tumor, allowing the applicability of cultured cells as predictors of responsiveness to cytokines in vivo.

Photodynamic therapy: a 5-year study of its effectiveness in the treatment of posterior uveal melanoma, and evaluation of haematoporphyrin uptake and photocytotoxicity of melanoma cells in tissue culture

A human melanoma cell line RVH-421 which selectively demonstrates melanin synthesis when cultured in L15 Leibowitz medium but not in RPMI medium was used as a model to examine haematoporphyrin derivative (HPD) uptake and the photocytotoxicity of photodynamic therapy (PDT). Confocal scanning microscopy and extraction fluorometry showed HPD uptake in both non-pigmented and pigmented melanoma cells. Phototoxicity was determined by incubating pigmented and non-pigmented monolayer cells with HPD, exposing them to variable periods of white fluorescent light and calculating the number of viable cells in the samples relative to the controls. Both the non-pigmented and pigmented melanoma cells were affected by light-dependent cytotoxicity which was greater in the non-pigmented cells. Melanin or other substances may reduce the photo-oxidative effects of PDT. Posterior uveal melanomas in 36 patients were treated with PDT with the longest duration of tumour control being 6.5 years. Kaplan-Meier survival analysis showed that 76% of melanomas were not growing at the end of the first year, declining to 62% at the end of the second year, with 38% showing no signs of growth at the end of the fifth year. No eyes were lost as a result of PDT. Cox's hazards analysis showed that the degree of tumour pigmentation and patient age at therapy significantly influence the tumour response to PDT.

Occupational Sunlight Exposure and Melanoma in the U.S. Navy

Melanoma is the second most common cancer, after testicular cancer, in males in the U.S. Navy. A wide range of occupations with varying exposures to sunlight and other possible etiologic agents are present in the Navy. Person-years at risk and cases of malignant melanoma were ascertained using computerized service history and inpatient hospitalization files maintained at the Naval Health Research Center. A total of 176 confirmed cases of melanoma were identified in active-duty white male enlisted Navy personnel during 1974-1984. Risk of melanoma was determined for individual occupations and for occupations grouped by review of job descriptions into three categories of sunlight exposure: (1) indoor, (2) outdoor, or (3) indoor and outdoor. Compared with the U.S. civilian population, personnel in indoor occupations had a higher age-adjusted incidence rate of melanoma, i.e., 10.6 per 100,000 (p = .06). Persons who worked in occupations that required spending time both indoors and outdoors had the lowest rate, i.e., 7.0 per 100,000 (p = .06). Incidence rates of melanoma were higher on the trunk than on the more commonly sunlight-exposed head and arms. Two single occupations were found to have elevated rates of melanoma: (1) aircrew survival equipmentman, SIR = 6.8 (p less than .05); and (2) engineman, SIR = 2.8 (p less than .05). However, there were no cases of melanoma or no excess risk in occupations with similar job descriptions. Findings on the anatomical site of melanoma from this study suggest a protective role for brief, regular exposure to sunlight and fit with recent laboratory studies that have shown vitamin D to suppress growth of malignant melanoma cells in tissue culture. A mechanism is proposed in which vitamin D inhibits previously initiated melanomas from becoming clinically apparent.

Tumour necrosis factor in serum and synovial fluid of patients with active and severe rheumatoid arthritis.

Fifteen serum samples and 29 synovial fluids of patients with rheumatoid arthritis (RA) were examined for the presence of tumour necrosis factor (TNF). The assay for TNF was based on the cytotoxic activity of this cytokine for human melanoma cells in tissue culture. High concentrations of TNF were found in serum samples of patients with severe RA, who had increased erythrocyte sedimentation rate and serum alpha 2 macroglobulin, but decreased haemoglobin and serum iron concentrations. Tumour necrosis factor was also found in the synovial fluid of 16 out of 29 patients. High TNF concentrations were found in fluids with greater than 10(10) leucocytes/l. Tumour necrosis factor was not detected in the serum of normal subjects or in synovial fluid of patients with osteoarthritis. A mediator of inflammation, such as TNF, may contribute to the severity of RA.

Transmission and scanning electron microscopy and freeze-fracture replication of normal human melanocytes and human melanoma cells in tissue culture.

Basic LM, TEM, SEM, and FFR appearances of a pure line of normal human melanocytes derived from foreskin, and a human melanoma line, in cell culture are described. Normal melanocyte cultures exhibit side by side, cells of widely different melanogenic activities--possible clones--and melanosomes of bizarre shape and internal structure are frequent. Aggregates of melanosomes, with or without associated amorphous material, and with no discernible limiting membrane are present within many cells, and occasional simple specialised contacts occur between apposed cells. On replicas of plasma membrane of normal melanocytes, particle densities and diameters on P and E fracture faces were within the ranges for cells in general, and equivalent data for the melanoma cells were not significantly different. Similarly, there was no difference in density of distribution or diameter of nuclear pores between the normal and the tumoural cells.

Unique proteins defined by monoclonal antibodies specific for human melanoma: Some potential clinical applications

A panel of monoclonal antibodies, designated HMB 18, 45, and 50, have been isolated that are highly specific for malignant melanoma. When tested on fixed, paraffin-embedded tissue sections, they reacted with 97 percent of melanomas tested (58 of 60), including pigmented, unpigmented, primary, and metastatic melanoma. The specificity in differentiating melanomas from other malignant tumors, including 112 carcinomas, 35 lymphomas, and 39 sarcomas, was 100 percent. Normal melanocytes were unreactive, although some benign melanocytic lesions were recognized. Using immunoprecipitation and SDS-PAGE analysis of 35S-methionine-labeled melanoma cells in tissue culture, a previously undescribed protein of approximately 10 kd was recognized by all three antibodies. HMB 50 also precipitated two high molecular weight proteins of 97 kd and 110 kd from the conditioned medium of melanoma cells. These monoclonal antibodies are the most sensitive and specific antibodies generated against human melanoma to date. Their clinical application in diagnostic surgical pathology and potential use in immunotherapy are discussed.

Ultrastructural and cell surface studies of cancer epidermal cells following vitamin E administration

It has been postulated that vitamin E (α-tocopherol) plays an important role in the prevention and treatment of cancer. Thus, an inhibition of growth of melanoma cells in tissue culture, of NB cells in culture and in nude mice, was previously reported following vitamin E succinate administration. However, the exact mechanism of the antitumor activity of vitamin E is still unknown. The present study deals with the effects of natural vitamin E (α-tocopherol acetate) on the ultrastructure and cell surface of cancer cells from squamous cell carcinomas induced by 3-methylcholanthrene (MCA) in mice.

Effect of Dicarboxylic Acids on Harding-Passey and Cloudman S91 Melanoma Cells in Tissue Culture

Clinically, dicarboxylic acids have a cytotoxic effect on the abnormally hyperactive and malignant epidermal melanocyte, and diacids from C8 to C13 have been shown to inhibit mitochondrial oxidoreductases. Here, their effect on the growth kinetics and ultrastructure of murine melanoma cells in culture is examined. Cultures of Harding-Passey and Cloudman S91 melanoma cells were exposed to single doses of the disodium salts of C12, C9, and C6 (which does not significantly inhibit mitochondrial enzymes) dicarboxylic acids at concentrations of 10(-3) M to 10(-1) M. With C12 and C9, viability and cell proliferation over 3 days were significantly affected by concentrations greater than 10(-2) M. With exposure to C6 at 10(-1) M and to medium to which NaCl was added to produce equal osmolarity, the effect was much less. Electron microscopy of cells exposed to C9 at 10(-1) M for 1 h and 6 h revealed massive swelling of mitochondria with destruction of cristae, but plasma and nuclear membranes and membranes of endoplasmic reticulum were intact. Similar damage was not seen with C6 at 10(-1) M nor with equiosomolar NaCl. The results confirm (1) the cytotoxicity of dicarboxylic acids for malignant melanocytes, and (2) that the mitochondrion is a prime target for their action.

Stimulation of S91 melanoma tyrosinase activity by superpotent α-melanotropins

α-Melanocyte-stimulating hormone (α-MSH, α-melanotropin), [Nle 4,D-Phe 7]-α-MSH and related fragment analogues, Ac-[Nle 4,D-Phe 7]-α-MSH 4–11-NH 2 and Ac-[Nle 4,D-Phe 7]-α-MSH 4–10-NH 2, were studied for their ability to stimulate tyrosinase activity in Cloudman S91 mouse melanoma cells in tissue culture. All of the melanotropins stimulated tyrosinase activity in a dose-dependent manner. [Nle 4,D-Phe 7]-α-MSH was about 100 times more active than a-MSH as determined from the minimal effective dose (MED) required to activate the enzyme above control (basal) levels. The MED of this analogue to significantly stimulate tyrosinase activity at 24, 48 and 72 h of incubation was 10 −11 M whereas the MED of α-MSH was 10 −9 M at each of these times. The maximum tyrosinase activity achieved from the time of initial incubation in the presence of [Nle 4,D-Phe 7]-α-MSH was approximately 3-, 5- and 6-fold greater than control levels at 24, 48 and 72 h, respectively. The 2 [Nle 4,D-Phe 7]-substituted fragment analogues were at least as active as the tridecapeptide analogue and therefore at least 100-fold more active than a-MSH in stimulating enzyme activity. These [Nle 4,D-Phe 7]-substituted analogues were more active in the melanoma tyrosinase assay than in the melanoma adenylate cyclase assay or other normal melanocyte (frog and lizard skin) bioassays.

A Comparison of the Cyclic Anhydride and Mixed Anhydride Methods for 111In-DTPA Chelation to Monoclonal Antibodies

The cyclic anhydride (CA) and the mixed anhydride (MA) of DTPA were synthesized and used to chelate 111In to an antimelanoma monoclonal antibody. The CA and MA methods showed mean labeling efficiencies of 25.7 and 20.5%, respectively (p = NS). The binding efficiency of labeled antibody to human melanoma cells in tissue culture also was similar (means = 52 and 50%, respectively, p = NS), as was tumor uptake in nude mice at 96 hrs post-injection (16%-CA vs 12%-MA). The method required less complicated chemical syntheses, much less preparation time, and the product was stable over a much longer period. The results suggest that the CA method is preferable for bifunctional chelate labeling of monoclonal antibodies with 111In-DTPA.

Chromosome abnormality in a human TCGF-dependent T-lymphocyte culture generated against autologous melanoma cells in vitro

Six T-cell growth promoting factor (TCGF)-dependent T-cell cultures and phytohemagglutinin-stimulated peripheral blood lymphocytes from a patient with metatastic malignant melanoma were karyotyped. Three of the TCGF-dependent cultures were noncloned, started after in vitro sensitization against autologous malignant melanoma cells in tissue culture. A fourth was a subculture of one of these. The two remaining T-cell cultures were generated by in vitro sensitization of the same individual's lymphocytes against allogeneic Epstein-Barr virus-transformed B-cell lines. One of the cultures (AV7) and its subculture (AV/LDP39) showed a 17q+ anomaly in 100% of metaphases studied. All other TCGF-dependent T-cell cultures and fresh blood lymphocytes had normal karyotypes. The possible mode of origin of the abnormality and its significance are discussed.

Adjuvant Immunotherapy or Chemotherapy for Malignant Melanoma: Preliminary Report of the National Cancer Institute Randomized Clinical Trial

A preliminary analysis is presented of a clinical trial to determine whether adjuvant immunotherapy or chemotherapy can prolong the disease-free interval and survival of patients with poor prognosis Stage I malignant melanoma or Stage II melanoma. Postsurgical patients were randomized to receive either no further treatment, bacille Calmette Guerin (BCG); BCG plus neuraminidase-treated, allogeneic, tissue-cultured melanoma cells; or methyl-CCNU. No statistically significant improvement in disease-free interval or overall survival has been noted.

Tumor-Associated Antibodies in the Serum of Patients with Ocular Melanoma: IV. Correction for Smooth Muscle Antibodies

Serum samples from patients with choroidal malignant melanoma, carcinoma metastatic to the choroid and “normal” controls containing tumor-associated antibodies (TAA) to tissue cultured melanoma cells were examined for the presence of smooth muscle antibody (SMA). Sera found to contain SMA were absorbed with actin or thrombosthenin and reassayed for TAX With this modification, 72% (78% before absorption) of patients with uveal melanoma, 26% (78% before absorption) of patients with carcinoma metastatic to the choroid and 13% (24% before absorption) of “normal” controls were found to have tumor-associated antibodies in their serum.

Interferon as an inhibitor of cell growth: studies with mouse melanoma cells.

SummaryInterferon treatment was found to inhibit the growth of Cloudman mouse melanoma cells in tissue culture. This was not associated with a block in the G2 or M phases of the cell cycle. A highly purified interferon preparation, approximately 30% pure, was as effective as a less purified interferon preparation, less than 1% pure, in inhibiting melanoma cell growth. This suggests that interferon rather than contaminants in the preparation is responsible for the effects seen. The growth of the melanoma cells in DBA/2 mice was also retarded upon interferon treatment.

The synthesis and cytotoxic activity of some haloacetamidoalkyl β- D-xylopyranosides

2-Hydroxyethyl 2,3,4-tri- O-acetyl-β- D-xylopyranoside was prepared from 2,3,4-tri- O-acetyl-α- D-xylopyranosyl chloride by the action of 1,2-ethanediol and mercuric acetate. Subsequent mesylation and azide displacement gave 2-azidoethyl 2,3,4-tri- O-acetyl-β- D-xylopyranoside, which was hydrogenated over palladiumon-charcoal and the amine acylated with various haloacetyl halides, to afford 2-(haloacetamido)ethyl 2,3,4-tri- O-acetyl-β- D-xylopyranosides. Deprotection to obtain the free sugars was carried out with 5m M ethanolic sodium ethoxide. 2-(Chloroacetamido)ethyl 2,3,4-tri- O-acetyl-β- D-xylopyranoside was further modified by sequential azide displacement, hydrogenation, and subsequent acylation with various haloacetyl halides to afford 2-[(haloacetamido)acetylamino]ethyl 2,3,4-tri- O-acetyl-β- D-xylopyranosides, which were also deprotected to give the corresponding free sugars. The effects of these haloacetamido analogs on the growth of the melanoma cells in tissue culture was evaluated.

Detection of tumor-associated antigen in human melanoma cell line supernatants.

Spent tissue culture medium (CDM-S) removed from a single cell line of human malignant melanoma grown in serum-free CDM, contained tumor-associated antigenic activity. Antibodies to CDM-S measured by complement fixation were detected in 44% (31/70) melanoma, 55% (15/27) sarcoma, 63% (24/38) carcinoma and 15% (11/72) normal sera. Delayed cutaneous hypersensitivity reactions (DCHR) were demonstrated in 4/5 melanoma patients at a 500 mug dose, 3/5 at a 100 mug dose and in 1/7 carcinoma patients at the 500 mug dose. One ml of CDM-S was shown to contain antigen equivalent to that obtained from the membranes of 2.9 X 10(7) tissue-cultured melanoma cells. After purification, 84% (16/19) sera from melanoma patients, 66% (12/18) from sarcoma and carcinoma patients and 8% (2/26) from normal controls were positive to the antigen by complement fixation.

Melanogenesis in melanoma cells in tissue culture.

The relationship between cell proliferation and differentiation was studied using C341 cells' isolated from a culture cell line of mouse melanoma B-16. The C341 cells' rate of melanin formation is intimately related to active cell proliferation. The cells were examined with light and electron microscopes on the 4 th day of cultivation when the cells were proliferating exponentially and on the 7 th day when the culture was in the stationary phase. The 4-day-old culture consisted of dark, light and melanosome-containing cells. Melanosomes, mostly in the early stages of development, were found to be present in the light cells as well as in the melanosome-containing cells. In cells of the 7 th day culture, there were numerous melanosomes in various developmental stages and also melanosome complexes. The morphological evidence obtained is compatible with the biochemical evidence previously recorded.