The sap from Euphorbia peplus is effective against human nonmelanoma skin cancers.

BACKGROUND: The sap from Euphorbia peplus, commonly known as petty spurge in the U.K. or radium weed in Australia, has been used as a traditional treatment for a number of cancers. OBJECTIVE: To determine the effectiveness of E. peplus sap in a phase I/II clinical study for the topical treatment of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and intraepidermal carcinomas (IEC). METHODS: Thirty-six patients, who had refused, failed or were unsuitable for conventional treatment, were enrolled in a phase I/II clinical study. A total of 48 skin cancer lesions were treated topically with 100-300 µL of E. peplus sap once daily for 3 days. RESULTS: The complete clinical response rates at 1 month were 82% (n = 28) for BCC, 94% (n = 16) for IEC and 75% (n = 4) for SCC. After a mean follow-up of 15 months these rates were 57%, 75% and 50%, respectively. For superficial lesions < 16 mm, the response rates after follow-up were 100% for IEC (n = 10) and 78% for BCC (n = 9). CONCLUSIONS: The clinical responses for these relatively unfavourable lesions (43% had failed previous treatments, 35% were situated in the head and neck region and 30% were > 2 cm in diameter), are comparable with existing nonsurgical treatments. An active ingredient of E. peplus sap has been identified as ingenol mebutate (PEP005). This clinical study affirms community experience with E. peplus sap, and supports further clinical development of PEP005 for the treatment of BCC, SCC and IEC.

New cysteine derivatives with antiproliferative activity on melanoma cells.

Here we describe the rational design, computer-aided virtual ligand docking and synthesis of 19 nonpeptidic compounds designed to inhibit histone deacetylases and kill melanoma cells. Compounds were derived from cysteine, fused at the S-terminus to 4-butanoyl hydroxamate, and at the N-terminus to 4-(dimethylamino)benzoic acid. The latter was extended by coupling to amines to form a small library of prospective anti-cancer compounds. Four compounds were cytotoxic at sub-micromolar concentrations against cells of a particularly aggressive human melanoma (MM96L), and nine compounds showed selectivities of >or=5:1 for killing human melanoma instead of normal human fibroblast cells. The most active compounds were shown to cause hyperacetylation of histones due to inhibition of histone deacetylases. Further refinement of these compounds may produce an anti-tumor drug suitable for treating melanoma.

Up-regulation of p21(WAF1/CIP1) by histone deacetylase inhibitors reduces their cytotoxicity.

Histone deacetylase inhibitors show promise as chemotherapeutic agents and have been demonstrated to block proliferation in a wide range of tumor cell lines. Much of this antiproliferative effect has been ascribed to the up-regulated expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). In this article, we report that p21 expression was up-regulated by relatively low doses of the histone deacetylase inhibitor azelaic bishydroxamic acid (ABHA) and correlated with a proliferative arrest. Higher doses of ABHA were cytotoxic. Cells that did not up-regulate p21 expression were hypersensitive to killing by ABHA and died via apoptosis, whereas up-regulation of p21 correlated with reduced sensitivity and a block in the apoptotic mechanism, and these cells seemed to die by necrosis. Using isogenic p21(+/+) and p21(-/-) cell lines and direct inhibition of caspase activity, we demonstrate that the reduced sensitivity to killing by ABHA is a consequence of inhibition of apoptosis by up-regulated p21 expression. These data indicate the enormous potential of therapeutic strategies that bypass the cytoprotective effect of p21 and act on the same molecular targets as the histone deacetylase inhibitors.

Photosensitization of the sunscreen octyl p-dimethylaminobenzoate by UVA in human melanocytes but not in keratinocytes.

Sunscreens penetrate human epidermis and modify the biology of proliferating cells. This study addressed the question whether the UV response of cultured human cells is affected by direct treatment with nontoxic levels of sunscreens. Cell survival following exposure to UVC or unfiltered UBV was not altered by preincubation with 25 micrograms/mL of octyl p-dimethylaminobenzoate (o-PABA), 2-ethylhexyl p-methoxycinnamate (EHMC) or oxybenzone. However, UVA or UVB filtered to reproduce the solar UV spectrum penetrating to the basal layer of the epidermis, highly sensitized cells to killing by o-PABA but not by its hydrolysis product, 4-dimethylaminobenzoic acid. Sensitization was found in all cell types tested, except normal keratinocytes, and could be prevented by certain antioxidants particularly pyruvate and the hydroxyl radical scavenger mannitol. o-PABA and EHMC applied without UV reduced the adherence of cells. The results indicate that sunscreens may increase cell mobility and the combination of o-PABA with solar UV may selectively damage melanocytes in the skin.

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts.

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma.

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell carcinoma and squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio = 3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information wained through observation of pigmentation phenotype.

Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors.

The class III POU gene brn-2, encoding the Brn-2/N-Oct-3 transcription factor, is widely expressed in the developing mammalian central nervous system. Brn-2 has also been found to regulate the melanocytic phenotype with N-Oct-3 DNA binding activity elevated in malignant melanoma, however, its mode of action is yet to be defined. The functional role of the Brn-2 transcription factor has been investigated through the analysis of protein-protein interactions it forms with a number of basal and melanocytic transcriptional regulatory proteins. In vivo interactions were tested by gene-cotransfection using the mammalian GAL4-Herpes Simplex viral protein 16 (VP16) two hybrid formation and direct protein binding by in vitro glutathione S-transferase (GST)-pull down assay. The Brn-2 protein was found to homodimerize in vivo with high affinity, using Brn-2 deletion constructs dimer complex formation was found to be dependent on the presence of both the homeodomain and linker regions of the POU-domain. However, the POU-homoedomain was dispensable for the formation of the dimerization interface in one of the partner molecules but not both, when the POU-linker region was removed the ability to interact was lost irrespective of the presence of the homeodomain. Dimerization of Brn-2/N-Oct-3 was also found to occur in DNA binding assays using melanoma cell line nuclear extracts and a recently reported dimer target sequence probe, which may have significant consequences for gene regulation in melanocytic tumours. Low affinity Brn-2 protein contacts have also been found with the basal transcription complex, including TATA binding protein (TBP) and the transcriptional coactivator p300, and with the Sox-10 and Pax-3 transcription factors that are known to play an important role in melanocyte cell formation.

Anti-malarial effect of histone deacetylation inhibitors and mammalian tumour cytodifferentiating agents.

The histones of Plasmodium falciparum represent a potential new target for anti-malarial compounds. A naturally occurring compound, apicidin, has recently been shown to inhibit the in vitro growth of P. falciparum. Apicidin was shown to hyperacetylate histones, suggesting that its mode of action is through histone deacetylase inhibition. We have tested the ability of known histone deacetylase inhibitors, mammalian tumour suppressor compounds, and cytodifferentiating agents to inhibit the in vitro growth of a drug sensitive and resistant strain of P. falciparum. Seven of the tested compounds had microM IC50 values, and trichostatin A, a histone deacetylation inhibitor and cytodifferentiating agent, was active at low nM concentrations. One compound, suberic acid bisdimethylamide, which selectively arrests tumour cells as opposed to normal mammalian cells, had an in vivo cytostatic effect against the acute murine malaria Plasmodium berghei, and one round of treatment with the compound failed to select for resistant mutations. These results suggest a promising role for histone deacetylase inhibitors and cytodifferentiating agents as antimalarial drug candidates.

Histone deacetylase inhibitors trigger a G2 checkpoint in normal cells that is defective in tumor cells.

Important aspects of cell cycle regulation are the checkpoints, which respond to a variety of cellular stresses to inhibit cell cycle progression and act as protective mechanisms to ensure genomic integrity. An increasing number of tumor suppressors are being demonstrated to have roles in checkpoint mechanisms, implying that checkpoint dysfunction is likely to be a common feature of cancers. Here we report that histone deacetylase inhibitors, in particular azelaic bishydroxamic acid, triggers a G2 phase cell cycle checkpoint response in normal human cells, and this checkpoint is defective in a range of tumor cell lines. Loss of this G2 checkpoint results in the tumor cells undergoing an aberrant mitosis resulting in fractured multinuclei and micronuclei and eventually cell death. This histone deacetylase inhibitor-sensitive checkpoint appears to be distinct from G2/M checkpoints activated by genotoxins and microtubule poisons and may be the human homologue of a yeast G2 checkpoint, which responds to aberrant histone acetylation states. Azelaic bishydroxamic acid may represent a new class of anticancer drugs with selective toxicity based on its ability to target a dysfunctional checkpoint mechanism in tumor cells.

Inhibition of melanin synthesis by cystamine in human melanoma cells.

In studies to determine whether pigmentation can be regulated physiologically by thiols, human melanoma cells (MM418c5) and melanocytes were found to become depigmented when cultured continuously in 50 microM cystamine. Cystamine was depleted from the culture medium and the treatment was nontoxic and reversible. Cysteamine, dithiothreitol, and phenylthiourea were less effective, and glutathione, cysteine, and cystine were inactive. Tyrosinase (dopa oxidase) activity was not greatly affected except for induction of a lag period. In contrast, tyrosinase activity in an amelanotic melanoma cell line (MM96L) was rapidly inhibited without consumption of cystamine/cysteamine, in association with the generation of free thiol in the culture medium, and could be enhanced by the cystine transport inhibitor, glutamate. Tyrosinase expressed by a recombinant vaccinia virus was inhibited by cystamine treatment of MM96L and HeLa cells. Cystamine treatment lowered the degree of cross-linking of the pigmentation antigen gp75/TRP-1 in MM418c5 cells. Tyrosinase protein and mRNA levels in MM418c5 cells were not affected by cystamine. The results show that cystamine at a concentration close to physiologic levels has multiple effects on the melanogenic pathway. In amelanotic cells, tyrosinase has a short half-life and is readily inhibited by cystamine/cysteamine whereas tyrosinase in the more mature melanosomes of the pigmented cell appears to be less accessible to proteolytic and thiol attack. Inhibition of melanin synthesis in the latter cell type may arise to a significant degree from reduction of cystamine to cysteamine, which sequesters quinones.

Determinants of melanocyte density in adult human skin.

The distribution of melanocytes in human skin has been observed to vary within and among individuals, yet little is known of the factors that determine the density of these pigment cells. These factors were explored in a molecular epidemiological study conducted among a population-based sample of 97 male subjects aged over 50 years in Queensland, Australia. Information relating to environmental and phenotypic factors was collected through face-to-face interviews and physical examination of all participants. In addition, 2-mm biopsies of representative skin were taken from the dorsum of the hand and another anatomical site. Melanocytes were identified by cytoplasmic staining with the B8G3 (anti-TRP1) monoclonal antibody using standard immunohistochemical techniques. Melanocyte counts were performed blind by two observers. On crude analysis, melanocyte density decreased with advancing age (P = 0.0002), and increased with increasing number of naevi (P = 0.01). Other pigmentary characteristics (such as hair and eye colour and depth of tan) were not associated with epidermal melanocyte density. Melanocyte density varied significantly by anatomical site (P = 0.02), with highest densities observed on the back/shoulders (n = 50, 17.1 +/- 8.8 cells/mm, mean +/- SD) followed by the upper limbs (n = 11, 12.6 +/- 8.8 cells/mm) and lower limbs (n = 14, 14.4 +/- 5.9 cells/mm). Lowest melanocyte densities were recorded on the anterior trunk (n = 3, 3.2 +/- 2.4 cells/mm). These findings confirm the results of earlier studies in which site-specific differences in melanocyte density have been found. We speculate that the unequal distribution of melanocytes may partially explain the site-specific incidence of melanoma, offering fresh perspectives on the aetiology of this cancer.

Critical targets of protein kinase C in differentiation of tumour cells.

The ultimate target of pharmacological research is to find new drugs for treating human diseases such as cancer. Agents causing differentiation and thus growth arrest should be particularly useful in this regard. A potential target for such anticancer therapy is the enzyme family protein kinase C (PKC), which is involved in the transduction of signals for cell proliferation, differentiation, and apoptosis. Our recent work showing the induction of differentiation in melanoma cells by an activator of one PKC isoform, PKCdelta, touches on several important areas of investigation, which will form the basis of this review: the role of individual isoforms of PKC, their downstream targets and their specific substrates, the mechanism of activation of specific genes involved in the differentiation process, and the molecular basis for the morphological changes associated with differentiation. The central role that PKC plays in these processes points to the need for a greater understanding of the signalling pathways utilized by individual isoforms of this family of enzymes.

Anti-tumour activity in vitro and in vivo of selective differentiating agents containing hydroxamate.

A series of hydroxamates, which are not metalloprotease inhibitors, have been found to be selectively toxic to a range of transformed and human tumour cells without killing normal cells (fibroblasts, melanocytes) at the same concentrations. Within 24 h of treatment, drug action is characterized by morphological reversion of tumour cells to a more normal phenotype (dendritic morphology), and rapid and reversible acetylation of histone H4 in both tumour and normal cells. Two hydroxamates inhibited growth of xenografts of human melanoma cells in nude mice; resistance did not develop in vivo or in vitro. A third hydroxamate, trichostatin A, was active in vitro but became inactivated and had no anti-tumour activity in vivo. Development of dendritic morphology was found to be dependent upon phosphatase activity, RNA and protein synthesis. Proliferating hybrid clones of sensitive and resistant cells remained sensitive to ABHA, indicating a dominant-negative mechanism of sensitivity. Histone H4 hyperacetylation suggests that these agents act at the chromatin level. This work may lead to new drugs that are potent, and selective anti-tumour agents with low toxicity to normal cells.

Cell cycle delay, mitochondrial stress and uptake of hydrophobic cations induced by sunscreens in cultured human cells.

Reports of systemic absorption of sunscreens prompted a study of the effects of emulsions of 3 commonly used sunscreens on cultured human cells; vegetable oil and paraffin oil were used as controls. Ethylhexyl p-methoxycinnamate (EHMC), octyl p-dimethylaminobenzoate (PABA) and oxybenzone (OB) inhibited cell growth and DNA synthesis and retarded cycle progression from G1 in the dose range 25-100 micrograms/mL. An extended period of exposure (up to 24 h) was required for maximum uptake of sunscreens and for inhibition of cell growth. Melano-cytes and fibroblasts tended to be more resistant than tumor cell lines (melanoma, cervical carcinoma). Sunscreens had no major effects on the transcription of certain genes, as judged by the activity of reporter constructs driven by the p53, c-fos and metal response (sheep metallothionein Ia promoter) elements and transfected into a human melanoma cell line (MM96L). The activity of the cytomegalovirus promoter was also not affected. A cell line (CI80-13S) with mitochondrial dysfunction was significantly more sensitive to growth inhibition by EHMC and PABA than the other cell lines tested. Treatment of MM96L with the mitochondrial inhibitor ethidium bromide sensitized the cells to killing by cotreatment with sunscreens, in association with increased cellular uptake of ethidium bromide. These results established conditions for studying the action of sunscreens on cultured human cells. Further studies are required to determine whether the mitochondrial stress and changes in drug uptake associated with sunscreens in the above cell lines are relevant to their action in vivo.

Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers.

Epidemiology shows a relationship between solar exposure and all types of skin cancer. Understanding the mechanisms of skin cancer requires knowledge of the photomolecular events that occur within the relevant epidermal cell types in vivo. Studies to date have focused on UVR-induced DNA lesions in keratinocytes, the majority epidermal cell population which gives rise to most skin cancers. Malignant melanoma, arising from melanocytes (5%-10% of epidermal cells), accounts for most skin cancer deaths. We report on new techniques to detect DNA photolesions in human epidermal melanocytes in situ. Previously nonexposed buttock skin of volunteers of skin types I/II was exposed to clinically relevant doses of narrow bandwidth UVB (300 nm) and UVA (320 nm, 340 nm, 360 nm) radiation. Biopsies were taken immediately afterwards and processed for routine histology. Microscope sections were prepared and double-stained with fluorescent-tagged monoclonal antibodies for thymine dimers and melanocytes. UVR dose-response curves for dimer levels within melanocyte nuclei were determined by image analysis and compared with dimer levels in adjacent basal cell keratinocytes. Our data show that UVB and UVA readily induce thymine dimers in melanocytes at levels that are comparable with those found in adjacent keratinocytes. This new technique will enable melanocyte specific studies, such as DNA repair kinetics, to be done in vivo.

p53 expression and risk factors for cutaneous melanoma: a case-control study.

Epidemiological data strongly implicate sunlight as the principal environmental cause of melanoma; however, critical molecular targets for ultraviolet (UV)-induced melanoma remain to be identified. The p53 tumor suppressor gene is one possible target, being abnormally expressed in 20-40% of primary melanomas. We undertook a population-based molecular epidemiological study with the aim of determining the environmental and phenotypic factors associated with p53-positive and p53-negative melanomas. One hundred fifty cases of melanoma were randomly ascertained from the Queensland Cancer Registry and matched to 150 electoral roll controls. Data on environmental and phenotypic exposures were collected through interviews and physical examination of all participants. Sections of tumor tissue were obtained from 134 (89%) cases and stained with the anti-p53 DO-7 monoclonal antibody (MAb) following microwave antigen retrieval. Of 121 useable sections, 22 tumors (18%) had more than 1% cells with positive staining consistent with abnormalities in p53 expression. Strongest predictors of p53-positive melanoma were inability to tan [odds ratio (OR) 6.8], history of non-melanoma skin cancer (OR 3.2) and site of melanoma: head/neck (OR 2.2) and lower limbs (OR 2.3). In contrast, factors such as nevus density and freckling propensity were strongly associated only with p53-immunonegative melanoma (OR 8.6 for >25 moles; OR 3.0 for heavy facial freckling). Overall, the determinants of p53-positive and p53-negative melanomas were independent and complementary, the former being associated with features of sun-sensitivity and chronic sun exposure, the latter with phenotypic markers of melanocytic proliferation. Our findings are consistent with at least 2 independent pathways in the pathogenesis of melanoma, characterized by environmental induction and p53 overexpression on the one hand and pigment cell instability on the other.