Antitumor activity of 3-ingenyl angelate: Plasma membrane and mitochondrial disruption and necrotic cell death

Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodes-sication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 mug) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; it = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; it = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Fox1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD90 for PEP005 for a panel of tumor cell lines was 180-220 muM. Electron microscopy showed that treatment with PEP005 both ill vitro (230 tot) and ill vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. Cr-51 release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 muM) treatment of tumor cells ill vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.

Gene-expression profiling reveals distinct expression patterns for classic versus variant Merkel cell phenotypes and new classifier genes to distinguish Merkel cell from small-cell lung carcinoma

Merkel cell carcinoma (MCC) is a rare aggressive skin tumor which shares histopathological and genetic features with small-cell lung carcinoma (SCLC), both are of neuroendocrine origin. Comparable to SCLC, MCC cell lines are classified into two different biochemical subgroups designated as 'Classic' and 'Variant'. With the aim to identify typical gene-expression signatures associated with these phenotypically different MCC cell lines subgroups and to search for differentially expressed genes between MCC and SCLC, we used cDNA arrays to pro. le 10 MCC cell lines and four SCLC cell lines. Using significance analysis of microarrays, we defined a set of 76 differentially expressed genes that allowed unequivocal identification of Classic and Variant MCC subgroups. We assume that the differential expression levels of some of these genes reflect, analogous to SCLC, the different biological and clinical properties of Classic and Variant MCC phenotypes. Therefore, they may serve as useful prognostic markers and potential targets for the development of new therapeutic interventions specific for each subgroup. Moreover, our analysis identified 17 powerful classifier genes capable of discriminating MCC from SCLC. Real-time quantitative RT-PCR analysis of these genes on 26 additional MCC and SCLC samples confirmed their diagnostic classification potential, opening opportunities for new investigations into these aggressive cancers.

The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

Brca1 inactivation induces p27Kip1-dependent cell cycle arrest and delayed development in the mouse mammary gland

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.

Interferon-α-2b and oral cytarabine ocfosfate for newly diagnosed chronic myeloid leukaemia

Background: Treatment with interferon and subcutaneous cytarabine produces superior cytogenetic responses in chronic myeloid leukaemia (CML) than treatment with interferon alone, but at the expense of greater toxicity. Cytarabine ocfosfate (YNK01) is an oral precursor of cytarabine that may overcome some of the inconvenience and toxicities associated with subcutaneous cytarabine administration. Patients and methods: We studied the efficacy and tolerability of combination therapy with interferon-alpha-2b and YNK01 in patients with newly diagnosed, untreated CML. Forty patients were treated with interferon-alpha-2b (5 MU/m(2)/day) plus monthly courses of YNK01 (600 mg/day for 10 days) for I year. Results: The 6-month complete haematological response rate was 63% and the 1-year major cytogenetic response rate was 30%, with 10% of cytogenetic responses being complete. With a median follow-up of 57 months, the estimated 5-year overall survival was 86% (95% confidence interval 70% to 94%). Treatment tolerability was poor, with toxicity leading to discontinuation of one or both drugs in 60% of cases. The median daily dose of interferon alpha-2b was 7.75 MU and the median dose of YNK01 was 600 mg/day for each 10-day treatment cycle. Conclusions: Interferon-alpha-2b and YNK01 produce cytogenetic responses comparable to those achieved with interferon-alpha-2b and parenteral cytarabine, although toxicity was excessive. Alternate dosing strategies may enhance the tolerability of YNK01.

Long-term outcome after intensive therapy with etoposide, melphalan, total body irradiation and autotransplant for acute myeloid leukemia

Intensive therapy and autologous blood and marrow transplantation (ABMT) is an established post-remission treatment for acute myeloid leukemia (AML), although its exact role remains controversial and few data are available regarding longer-term outcomes. We examined the long-term outcome of patients with AML transplanted at a single center using uniform intensive therapy consisting of etoposide, melphalan and TBI. In all, 145 patients with AML underwent ABMT: 117 in first remission, 21 in second remission and seven beyond second remission. EFS and OS were significantly predicted by remission status (P

Concurrent binding of anti-EphA3 antibody and ephrin-A5 amplifies EphA3 signaling and downstream responses: Potential as EphA3-specific tumor-targeting reagents

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands form a unique cell-cell contact-mediated system for controlling cell localization and organization. Their high expression in a wide variety of human tumors indicates a role in tumor progression, and relatively low Eph and ephrin levels in normal tissues make these proteins potential targets for anticancer therapies. The monoclonal antibody IIIA4, previously used to isolate EphA3, binds with subnanomolar affinity to a conformation-specific epitope within the ephrin-binding domain that is closely adjacent to the low-affinity ephrin-A5 heterotetramerization site. We show that similar to ephrin-A5, preclustered IIIA4 effectively triggers EphA3 activation, contraction of the cytoskeleton, and cell rounding. BIAcore analysis, immunoblot, and confocal microscopy of wild-type and mutant EphA3 with compromised ephrin-A5 or IIIA4-binding capacities indicate that IIIA4 binding triggers an EphA3 conformation which is permissive for the assembly of EphA3/ephrin-A5-type signaling clusters. Furthermore, unclustered IIIA4 and ephrin-A5 Fc applied in combination initiate greatly enhanced EphA3 signaling. Radiometal conjugates of ephrin-A5 and IIIA4 retain their affinity, and in mouse xenografts localize to, and are internalized rapidly into EphA3-positive, human tumors. These findings show the biological importance of EphA3/ ephrin-A5 interactions and that ephrin-A5 and IIIA4 have great potential as tumor targeting reagents.

Cloning and expression of the large zebrafish protocadherin gene, Fat

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat 1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals. (c) 2005 Elsevier B.V. All rights reserved.

Randomized trial of 8 Gy in 1 versus 20 Gy in 5 fractions of radiotherapy for neuropathic pain due to bone metastases (Trans-Tasman Radiation Oncology Group, TROG 96.05)

Background and purpose: Despite numerous randomized trials investigating radiotherapy (RT) fractionation schedules for painful bone metastases, there are very few data on RT for bone metastases causing pain with a neuropathic component. The Trans-Tasman Radiation Oncology Group undertook a randomized trial comparing the efficacy of a single 8 Gy (8/1) with 20 Gy in 5 fractions (20/5) for this type of pain. Materials and methods: Eligible patients had radiological evidence of bone metastases from a known malignancy with no change in systemic therapy within 6 weeks before or anticipated within 4 weeks after RT, no other metastases along the distribution of the neuropathic pain and no clinical or radiological evidence of cord/cauda equina compression. All patients gave written informed consent. Primary endpoints were pain response within 2 months of commencement of RT and time to treatment failure (TTF). The hypothesis was that 8/1 is at least as effective as 20/5 and the planned sample size was 270 patients. Results: Between February 1996 and December 2002, 272 patients were randomized (8/1:20/5 = 137:135) from 15 centres (Australia 11, New Zealand 3, UK 1). The commonest primary cancers were lung (31%), prostate (29%) and breast (8%); index sites were spine (89%), rib (9%), other (2%); 72% of patients were males and the median age was 67 (range 2989). The median overall survival (95% CI) for all randomized patients was 4.8 mo (4.2-5.7 mo). The intention-to-treat overall response rates (95% Cl) for 8/1 vs 20/5 were 53% (45-62%) vs 61% (53-70%), P = 0.18. Corresponding figures for complete response were 26% (18-34%) vs 27% (19-35%), P = 0.89. The estimated median TTFs (95% CI) were 2.4 mo (2.0-3.3 mo) vs 3.7 mo (3.1-5.9 mo) respectively. The hazard ratio (95% Cl) for the comparison of TTF curves was 1.35 (0.99-1.85), log-rank P = 0.056. There were no statistically significant differences in the rates of re-treatment, cord compression or pathological fracture by arm. Conclusions: 8/1 was not shown to be as effective as 20/5, nor was it statistically significantly worse. Outcomes were generally poorer for 8/1, although the quantitative differences were relatively small. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Characterization of the human cytochrome P450 forms involved in metabolism of tamoxifen to its alpha-hydroxy and alpha,4-dihydroxy derivatives

Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial. cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include alpha-hydroxytamoxifen, alpha,4-dihydroxytamoxifen, and alpha-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing a-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250,mu M) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as alpha-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No alpha,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100,mu M) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of a,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.

Repeated occurrence of basal cell carcinoma of the skin and multifailure survival analysis: Follow-up data from the nambour skin cancer prevention trial

The aim of this study was to apply multifailure survival methods to analyze time to multiple occurrences of basal cell carcinoma (BCC). Data from 4.5 years of follow-up in a randomized controlled trial, the Nambour Skin Cancer Prevention Trial (1992-1996), to evaluate skin cancer prevention were used to assess the influence of sunscreen application on the time to first BCC and the time to subsequent BCCs. Three different approaches of time to ordered multiple events were applied and compared: the Andersen-Gill, Wei-Lin-Weissfeld, and Prentice-Williams-Peterson models. Robust variance estimation approaches were used for all multifailure survival models. Sunscreen treatment was not associated with time to first occurrence of a BCC (hazard ratio = 1.04, 95% confidence interval: 0.79, 1.45). Time to subsequent BCC tumors using the Andersen-Gill model resulted in a lower estimated hazard among the daily sunscreen application group, although statistical significance was not reached (hazard ratio = 0.82, 95% confidence interval: 0.59, 1.15). Similarly, both the Wei-Lin-Weissfeld marginal-hazards and the Prentice-Williams-Peterson gap-time models revealed trends toward a lower risk of subsequent BCC tumors among the sunscreen intervention group. These results demonstrate the importance of conducting multiple-event analysis for recurring events, as risk factors for a single event may differ from those where repeated events are considered.

Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia

Purpose: Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer. Experimental Design: RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry. Results: We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARa, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and Pan IN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early Pan IN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors. Conclusions: We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.