A novel multiplex pyrosequencing assay for genotyping functionally relevant CTLA-4 polymorphisms: potential applications in autoimmunity and cancer.

CTLA-4 expression/function can be affected by single nucleotide polymorphisms (SNPs) of CTLA-4 gene, which have been widely associated with susceptibility or progression to autoimmune diseases and cancer development. In this study, we analyzed six CTLA-4 SNPs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, CT60G>A) in 197 DNA samples from 43 B-lymphoblastoid cell lines (B-LCLs), 40 systemic sclerosis (SSc) patients, 14 pre-analyzed melanoma patients and 100 Italian healthy subjects. Genotyping of -1661A>G, -1577G>A, -658C>T and CT60G>A was performed by newly developed multiplex pyrosequencing (PSQ) assays, whereas -319C>T and +49A>G by T-ARMS PCR and direct sequencing. Genotype/allele frequency were analyzed using χ(2) or Fisher exact test. Our study provides the first multiplex PSQ method that allows simultaneous genotyping of two CTLA-4 SNP pairs (i.e. -1661A>G/-658C>T and -1577G>A/CT60G>A) by two multiplex PSQ reactions. Herein, we show the CTLA-4 genotype distribution in the B-LCLs providing the first and best characterized cell line panel typed for functionally relevant CTLA-4 SNPs. We also report the significant association of the -1661A/G genotype, -1661 & -319 AC-GT diplotype and -319 & CT60 TG haplotype with susceptibility to SSc without Hashimoto's thyroiditis occurrence. Furthermore, we confirmed previous genotyping data referred to melanoma patients and provided new genotyping data for Italian healthy subjects.

NAC, tiron and trolox impair survival of cell cultures containing glioblastoma tumorigenic initiating cells by inhibition of cell cycle progression.

Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.

Chromosomal instability, DNA index, dysplasia, and subsite in oral premalignancy as intermediate endpoints of risk of cancer.

BACKGROUND: Chromosomal instability and aneuploidy may represent biomarkers of oral exposure to damaging agents and early signs of clinical disease according to the theory of "oral field cancerization." METHODS: The hypothesis was tested that the DNA index (DI) values, obtained by high-resolution DNA flow cytometry (DNA-FCM), may potentially contribute to oral cancer risk prediction. For this purpose, the DI of oral fields of normal-appearing mucosa and oral potentially malignant disorders (OPMDs) in 165 consecutive patients was tested for association with dysplasia and/or the oral subsites of tongue and floor of the mouth taken as high-risk intermediate endpoints surrogate of cancer clinical endpoints. The association was evaluated by logistic regression using patient gender, age, tobacco, cigarette smoking habit, and alcohol abuse as confounding variables. RESULTS: Different DI models provided evidence of statistical significant associations. Subdividing the DI values in diploid, near-diploid aneuploid, and high or multiple aneuploid from both OPMDs and oral normal-appearing mucosa, ORs, respectively, of 1, 4.3 (P = 0.001), and 18.4 (P < 0.0005) were obtained. CONCLUSION: Routine DI analysis by high-resolution DNA-FCM seems potentially useful to complement dysplasia and subsite analysis for assessment of oral cancer risk prediction and for a better management of the patients with OPMDs. Work is in progress to validate the present findings in a prospective study with clinical endpoints. IMPACT: Identifying DNA abnormalities in oral premalignancy may lead to biomarkers of oral exposure and cancer risk and potentially to more effective prevention measures.

The chemopreventive retinoid 4HPR impairs prostate cancer cell migration and invasion by interfering with FAK/AKT/GSK3beta pathway and beta-catenin stability.

BACKGROUND: Prostate cancer shows an extremely slow progression, appearing in its metastatic, hormone refractory phenotype mostly in elderly men. The chemopreventive targeting of this tumor could accordingly delay its malignancy over life expectancy. The cancer chemopreventive retinoid N-(4 hydroxyphenyl)retinamide (4HPR) has already been shown to restrain prostate cancer growth in vitro and in vivo, though its mechanisms of action are only partially explained. RESULTS: We found that 4HPR impairs DU145 and PC3 prostate cancer cells migration and invasion by down-regulating FAK and AKT activation and by enhancing beta-catenin degradation, causing the downregulation of target genes like cyclin D1, survivin and VEGF. This non-migratory phenotype was similarly produced in both cell lines by stable silencing of beta-catenin. 4HPR was able to decrease AKT phosphorylation also when powerfully upregulated by IGF-1 and, consequently, to impair IGF-1-stimulated cell motility. Conversely, the expression of constitutively active AKT (myr-AKT) overcame the effects of 4HPR and beta-catenin-silencing on cell migration. In addition, we found that BMP-2, a 4HPR target with antiangiogenic activity, decreased prostate cancer cell proliferation, migration and invasion by down-regulating the pathway described involving AKT phosphorylation, beta-catenin stability and cyclin D1 expression. CONCLUSION: These data point to 4HPR as a negative regulator of AKT phosphorylation, effectively targeting the beta-catenin pathway and inducing a relatively benign phenotype in prostate cancer cells, limiting neoangiogenesis and cell invasion.

Novel antivascular efficacy of metronomic docetaxel therapy in prostate cancer: hnRNP K as a player.

Tumor growth requires a competent vascular supply and angiogenesis is now considered a potential target for cancer treatment. Chemotherapeutic drugs, and docetaxel in particular, chronically administered using a frequent schedule at low dose (metronomic dosing), can cause potent antiangiogenic effects by targeting the endothelial cells of newly growing blood vessels. Because the exposure to cytotoxic drugs could target both endothelial and tumor cells, we investigated the effects of "metronomic docetaxel" on hormone refractory prostate carcinoma cells. In vitro, metronomic therapy lowered tumor cell viability, inducing apoptosis and reducing the invasive potential at 10- to100-fold lower concentrations as compared with the maximum tolerated dose. Metronomic regimens resulted in a significant reduction of vascular endothelial cell growth factor expression and up-regulation of endogenous angiogenesis inhibitors. Our studies suggest that heterogeneous nuclear ribonucleoprotein K is a mediator of the effects we observed. Targeting heterogeneous nuclear ribonucleoprotein K may serve as a specific antimetastasis and antiangiogenic therapy and could be a potential predictive marker to determine the optimal dose and schedule for metronomic chemotherapy regimens. These findings highlight the multiple effects that may characterize antiangiogenic metronomic chemotherapy and suggest that docetaxel might act as antitumor compound by affecting both cancer and endothelial cells at the same drug concentration. Careful optimization of drug scheduling and dosages will be required to maximize antitumor responses with metronomic approaches.

Antileukemia effects of xanthohumol in Bcr/Abl-transformed cells involve nuclear factor-kappaB and p53 modulation.

The oncogenic Bcr-Abl tyrosine kinase activates various signaling pathways including phosphoinositide 3-kinase/Akt and nuclear factor-kappaB that mediate proliferation, transformation, and apoptosis resistance in Bcr-Abl+ myeloid leukemia cells. The hop flavonoid xanthohumol inhibits tumor growth by targeting the nuclear factor-kappaB and Akt pathways and angiogenesis. Here, we show that xanthohumol has in vitro activity against Bcr-Abl+ cells and clinical samples and retained its cytotoxicity when imatinib mesylate-resistant K562 cells were examined. Xanthohumol inhibition of K562 cell viability was associated with induction of apoptosis, increased p21 and p53 expression, and decreased survivin levels. We show that xanthohumol strongly inhibited Bcr-Abl expression at both mRNA and protein levels and show that xanthohumol caused elevation of intracellular reactive oxygen species and that the antioxidant N-acetylcysteine blunted xanthohumol-induced events. Further, we observed that xanthohumol inhibits leukemia cell invasion, metalloprotease production, and adhesion to endothelial cells, potentially preventing in vivo life-threatening complications of leukostasis and tissue infiltration by leukemic cells. As structural mutations and/or gene amplification in Bcr-Abl can circumvent an otherwise potent anticancer drug such as imatinib, targeting Bcr-Abl expression as well as its kinase activity could be a novel additional therapeutic approach for the treatment of Bcr-Abl+ myeloid leukemia.

Targeted delivery of tumor necrosis factor-alpha to tumor vessels induces a therapeutic T cell-mediated immune response that protects the host against syngeneic tumors of different histologic origin.

PURPOSE: We sought to demonstrate that a single systemic administration of L19mTNFalpha (a fusion protein constituted by the scFv L19 specific for the oncofetal ED-B domain of fibronectin and tumor necrosis factor alpha, TNFalpha) in combination with melphalan induced complete and long-lasting tumor eradication in tumor-bearing mice and triggered the generation of a specific T cell-based immune response that protects the animals from a second tumor challenge, as well as from challenges with syngeneic tumor cells of different histologic origin. EXPERIMENTAL DESIGN AND RESULTS: Treatment with L19mTNFalpha, in combination with melphalan, induced complete tumor regression in 83% of BALB/c mice with WEHI-164 fibrosarcoma and 33% of animals with C51 colon carcinoma. All cured mice rejected challenges with the same tumor cells and, in a very high percentage of animals, also rejected challenges with syngeneic tumor cells of different histologic origin. In adoptive immunity transfer experiments, the splenocytes from tumor-cured mice protected naive mice both from C51 colon carcinoma and from WEHI-164 fibrosarcoma. Similar results were also obtained in adoptive immunity transfer experiments using severely immunodepressed mice. Experiments using depleted splenocytes showed that T cells play a major role in tumor rejection. CONCLUSIONS: The results show that the selective targeting of mTNFalpha to the tumor enhances its immunostimulatory properties to the point of generating a therapeutic immune response against different histologically unrelated syngeneic tumors. These findings predicate treatment approaches for cancer patients based on the targeted delivery of TNFalpha to the tumor vasculature.

Cryosurgery for advanced malignant melanoma of the facial skin. A case report.

BACKGROUND: Cryosurgery is safely employed for the treatment of skin precancerous and malignant lesions of the head and neck in selected patients. The case of a 101-year-old female patient with advanced malignant melanoma of the facial skin, undergoing cryosurgery, is reported in order to assess the feasibility and tolerability of the technique, as well as the biological implications of cryosurgical treatment in this specific neoplasm. CASE REPORT: A 101-year-old woman, with a large (pT4b N0 M0) cutaneous melanoma of the facial skin on the right cheek, was treated at the Division of Surgical Oncology of the National Cancer Research Institute, Italy, from June to August 2003. The treatment was accomplished by means of serial cryosurgical applications which were performed within three months; the bulk of the lesion was cryotreated with a liquid nitrogen cryoprobe, while the residual disease was treated with a nitrous protoxide cryoprobe, by means of the insertion technique. The treatment was well tolerated, with a good aesthetic result, and the patient is recurrence- and distant-disease-free two years after the initial cryosurgical application. CONCLUSION: Cryosurgery is feasible in the treatment of head and neck melanoma, mostly for mucosal melanomas and cutaneous lesions in anatomically critical sites, as well as in high-risk surgical patients. Here, a good aesthetic result was obtained in a very elderly patient with a large cutaneous melanoma of the facial skin, avoiding skin flap transposition for tissue repair and postoperative complications (e.g., serious bleeding or postoperative pain), with a satisfactory functional and oncological outcome at two years.