Multiple structural and epigenetic defects in the human leukocyte antigen class I antigen presentation pathway in a recurrent metastatic melanoma following immunotherapy.

Scant information is available about the molecular basis of multiple HLA class I antigen-processing machinery defects in malignant cells, although this information contributes to our understanding of the molecular immunoescape mechanisms utilized by tumor cells and may suggest strategies to counteract them. In the present study we reveal a combination of IFN-γ-irreversible structural and epigenetic defects in HLA class I antigen-processing machinery in a recurrent melanoma metastasis after immunotherapy. These defects include loss of tapasin and one HLA haplotype as well as selective silencing of HLA-A3 gene responsiveness to IFN-γ. Tapasin loss is caused by a germ-line frameshift mutation in exon 3 (TAPBP(684delA)) along with a somatic loss of the other gene copy. Selective silencing of HLA-A3 gene and its IFN-γ responsiveness is associated with promoter CpG methylation nearby site-α and TATA box, reversible after DNA methyltransferase 1 depletion. This treatment combined with tapasin reconstitution and IFN-γ stimulation restored the highest level of HLA class I expression and its ability to elicit cytotoxic T cell responses. These results represent a novel tumor immune evasion mechanism through impairing multiple components at various levels in the HLA class I antigen presentation pathway. These findings may suggest a rational design of combinatorial cancer immunotherapy harnessing DNA demethylation and IFN-γ response.

ATB-346, a novel hydrogen sulfide-releasing anti-inflammatory drug, induces apoptosis of human melanoma cells and inhibits melanoma development in vivo.

Inflammation plays a key role in tumor promotion and development. Indeed, cyclooxygenase-2 (COX-2) expression is strongly associated with different types of cancer. An emerging class of compounds with significant anti-inflammatory properties is the hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (H2S-NSAIDs). They consist of a traditional NSAID to which an H2S-releasing moiety is covalently attached. We have recently demonstrated that H2S donors inhibit melanoma cell proliferation. In the current study, we evaluated the potential beneficial effects of a new H2S-releasing derivative of naproxen, ATB-346 [2-(6-methoxynapthalen-2-yl)-propionic acid 4-thiocarbamoyl phenyl ester] which inhibits COX activity but also releases H2S. We used cell culture and a mouse melanoma model to evaluate the effect of ATB-346 on: i) in vitro growth of human melanoma cells; ii) in vivo melanoma development in mice. Cell culture studies demonstrated that ATB-346 reduced the in vitro proliferation of human melanoma cells and this effect was associated to induction of apoptosis and inhibition of NF-κB activation. Moreover, ATB-346 had novel Akt signaling inhibitory properties. Daily oral dosing of ATB-346 (43µmol/kg) significantly reduced melanoma development in vivo. This study shows that ATB-346, a novel H2S-NSAID, inhibits human melanoma cell proliferation by inhibiting pro-survival pathways associated with NF-κB and Akt activation. Furthermore, oral treatment with ATB-346 inhibits melanoma growth in mice. In conclusion, the combination of inhibition of cyclooxygenase and delivery of H2S by ATB-346 may offer a promising alternative to existing therapies for melanoma.

A New Medical Device Rigeneracons Allows to Obtain Viable Micro-Grafts From Mechanical Disaggregation of Human Tissues.

Autologous graft is considered the gold standard of graft materials; however, this approach is still limited due to both small amount of tissue that can be collected and to reduced cell viability of cells that can be obtained. The aim of this preliminary study was to demonstrate the efficacy of an innovative medical device called Rigeneracons® (CE certified Class I) to provide autologous micro-grafts immediately available to be used in the clinical practice. Moreover, Rigeneracons® is an instrument able to create micro-grafts enriched of progenitors cells which maintain their regenerative and differentiation potential. We reported preliminary data about viability cell of samples derived from different kind of human tissues, such as periosteum, cardiac atrial appendage biopsy, and lateral rectus muscle of eyeball and disaggregated by Rigeneracons®. In all cases we observed that micro-grafts obtained by Rigeneracons® displayed high cell viability. Furthermore, by cell characterization of periosteum samples, we also evidenced an high positivity to mesenchymal cell markers, suggesting an optimal regenerative potential.

Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: evidence for HDAC2 involvement.

Adult mesenchymal stem cells, such as dental pulp stem cells, are of great interest for cell-based tissue engineering strategies because they can differentiate into a variety of tissue-specific cells, above all, into osteoblasts. In recent years, epigenetic studies on stem cells have indicated that specific histone alterations and modifying enzymes play essential roles in cell differentiation. However, although several studies have reported that valproic acid (VPA)-a selective inhibitor of histone deacetylases (HDAC)-enhances osteoblast differentiation, data on osteocalcin expression-a late-stage marker of differentiation-are limited. We therefore decided to study the effect of VPA on dental pulp stem cell differentiation. A low concentration of VPA did not reduce cell viability, proliferation, or cell cycle profile. However, it was sufficient to significantly enhance matrix mineralization by increasing osteopontin and bone sialoprotein expression. In contrast, osteocalcin levels were decreased, an effect induced at the transcriptional level, and were strongly correlated with inhibition of HDAC2. In fact, HDAC2 silencing with shRNA produced a similar effect to that of VPA treatment on the expression of osteoblast-related markers. We conclude that VPA does not induce terminal differentiation of osteoblasts, but stimulates the generation of less mature cells. Moreover, specific suppression of an individual HDAC by RNA interference could enhance only a single aspect of osteoblast differentiation, and thus produce selective effects.

Isolation and assessment of the in vitro anti-tumor activity of smenothiazole A and B, chlorinated thiazole-containing peptide/polyketides from the Caribbean sponge, Smenospongia aurea.

The study of the secondary metabolites contained in the organic extract of Caribbean sponge Smenospongia aurea led to the isolation of smenothiazole A (3) and B (4), hybrid peptide/polyketide compounds. Assays performed using four solid tumor cell lines showed that smenothiazoles exert a potent cytotoxic activity at nanomolar levels, with selectivity over ovarian cancer cells and a pro-apoptotic mechanism.

AurkA inhibitors enhance the effects of B-RAF and MEK inhibitors in melanoma treatment.

BACKGROUND: Aurora kinase A (AurkA) is over-expressed in melanoma and its inhibition has been observed to limit tumor growth, suggesting a potential role in melanoma treatment. METHODS: A human melanoma cell line with the B-RAF (V600E) mutation (A375mel) was exposed to B-RAF inhibitor (GSK2118436), MEK inhibitor (GSK1120212) and AurkA inhibitor (MLN8054) as single agents or in various combinations (BRAF plus AurkA inhibitor, MEK plus AurkA inhibitor or triple combination BRAF plus MEK plus AurkA inhibitor). Cell proliferation was assessed using xCELLigence technology. Total protein extracts were examined for p53 and c-Myc protein expression by Western blot analysis. Drug anti-tumor effects were further assessed using a 3D-human melanoma skin reconstruction model, in which tissues were incubated with serum-free medium containing control, B-RAF plus MEK inhibitor, MEK plus AurkA inhibitor or the triple combination. RESULTS: AurkA inhibitor plus B-RAF inhibitor, AurkA inhibitor plus MEK inhibitor or triple combination had a markedly greater anti-proliferative effect on A375 (BRAFV600E) melanoma cells than single agents. In the 3D human skin model, the triple combination had a greater anti-tumor effect at the epidermal/dermal junction than control or either double combination. However, S-100 and Ki-67 positively stained spindle-shaped cells were detected in the dermal stratum, suggesting the presence of alive and proliferating melanoma cells. CONCLUSIONS: These findings provide new prospects for melanoma research, including combined B-RAF/AurkA inhibition for B-RAF mutated melanomas and MEK/AurkA inhibitor combination for patients without B-RAF mutations. Moreover, for the first time, we have shown that a B-RAF, MEK and AurkA inhibitor triple drug combination offers increased efficacy against melanoma cell growth and might be considered as a potential treatment strategy for enhancing clinical response in melanoma. However, although this triple drug combination was more effective at the epidermal/dermal junction, the suggested presence of alive and proliferating melanoma cells in the dermal stratum could result in drug resistance and disease recurrence. Molecular characterization of these dermal cells may be critical for the development of novel therapeutic strategies.

The nonreceptor-type tyrosine phosphatase PTPN13 is a tumor suppressor gene in non-small cell lung cancer.

The aim of the present work was to identify protein tyrosine phosphatases (PTPs) as novel, candidate tumor suppressor genes in lung cancer. Among the 38 PTPs in the human genome that show specificity for phosphotyrosine, we identified six PTPs by quantitative RT-PCR whose mRNA expression levels were significantly down-regulated in lung cancer-derived cell lines (ie, PTPRE, PTPRF, PTPRU, PTPRK, PTPRD, and PTPN13). After validation in primary samples of non-small cell lung cancer (NSCLC), we selected PTPN13 for further studies. The results presented here demonstrate that PTPN13 is a candidate tumor suppressor gene that is frequently inactivated in NSCLC through the loss of either mRNA and protein expression (64/87, 73%) or somatic mutation (approximately 8%). Loss of PTPN13 expression was apparently due to the loss of one or both copies of the PTPN13 locus at 4q (approximately 26% double deletion and approximately 37% single deletion) but not to promoter methylation. Finally, the manipulation of PTPN13 expression in lung cancer cells (ie, NCI-H292, A549) demonstrated that PTPN13 negatively regulates anchorage-dependent and anchorage-independent growth in vitro and restrains tumorigenicity in vivo, possibly through the control of the tyrosine phosphorylation of both EGFR and HER2. In conclusion, the expression screening of PTPs in lung cancer reported here has identified PTPN13 as a novel candidate tumor suppressor in NSCLC whose loss increases signaling from epidermal growth factor receptor and HER2 tyrosine kinase receptors.

Smenamides A and B, chlorinated peptide/polyketide hybrids containing a dolapyrrolidinone unit from the Caribbean sponge Smenospongia aurea. Evaluation of their role as leads in antitumor drug research.

An in-depth study of the secondary metabolites contained in the Caribbean sponge Smenospongia aurea led to the isolation of smenamide A (1) and B (2), hybrid peptide/polyketide compounds containing a dolapyrrolidinone unit. Their structures were elucidated using high-resolution ESI-MS/MS and homo- and heteronuclear 2D NMR experiments. Structures of smenamides suggested that they are products of the cyanobacterial metabolism, and 16S rRNA metagenomic analysis detected Synechococcus spongiarum as the only cyanobacterium present in S. aurea. Smenamides showed potent cytotoxic activity at nanomolar levels on lung cancer Calu-1 cells, which for compound 1 is exerted through a clear pro-apoptotic mechanism. This makes smenamides promising leads for antitumor drug design.

CD133 and CD44 cell surface markers do not identify cancer stem cells in primary human gastric tumors.

Emerging evidence suggests that tumors contain and are driven by a cellular component that displays stem cell properties, the so-called cancer stem cells (CSCs). CSCs have been identified in several solid human cancers; however, there are no data about CSCs in primary human gastric cancer (GC). By using CD133 and CD44 cell surface markers we investigated whether primary human GCs contain a cell subset expressing stem-like properties and whether this subpopulation has tumor-initiating properties in xenograft transplantation experiments. We examined tissues from 44 patients who underwent gastrectomy for primary GC. The tumorigenicity of the cells separated by flow cytometry using CD133 and CD44 surface markers was tested by subcutaneous or intraperitoneum injection in NOD/SCID and nude mice. GCs included in the study were intestinal in 34 cases and diffuse in 10 cases. All samples contained surface marker-positive cells: CD133(+) mean percentage 10.6% and CD133(+)/CD44(+) mean percentage 27.7%, irrespective of cancer phenotype or grade of differentiation. Purified CD133(+) and CD133(+)/CD44(+) cells, obtained in sufficient number only in 12 intestinal type GC cases, failed to reproduce cancer in two mice models. However, the unseparated cells produced glandular-like structures in 70% of the mice inoculated. In conclusion, although CD133(+) and CD133(+)/CD44(+) were detectable in human primary GCs, they neither expressed stem-like properties nor exhibited tumor-initiating properties in xenograft transplantation experiments.

MDR1-P-glycoprotein behaves as an oncofetal protein that promotes cell survival in gastric cancer cells.

P-glycoprotein (P-gp), traditionally linked to cancer poor prognosis and multidrug resistance, is undetectable in normal gastric mucosa and overexpressed in gastric cancer (GC). We propose that P-gp may be involved in Helicobacter pylori (Hp)-related gastric carcinogenesis by inhibiting apoptosis. Aim of the study was to evaluate the expression of P-gp in fetal stomach and in Hp-related gastric carcinogenesis, the epigenetic control of the multi-drug resistance-1 (MDR1) gene, the localization and interaction between P-gp and Bcl-x(L) and the effect of the selective silencing of P-gp on cell survival. P-gp and Bcl-xl expression was evaluated by immunohistochemistry on 28 spontaneously abortive human fetuses, 66 Hp-negative subjects, 138 Hp-positive chronic gastritis (CG) of whom 28 with intestinal metaplasia (IM) and 45 intestinal type GCs. P-gp/Bcl-x(L) colocalization was investigated by confocal immunofluorescence microscopy and protein-protein interaction by co-immunoprecipitation, in basal conditions and after stress-induced apoptosis, in GC cell lines AGS and MKN-28 and hepatocellular carcinoma cell line Hep-G2. The role of P-gp in controlling apoptosis was evaluated by knocking down its expression with a specific small interfering RNAs in stressed AGS and MKN-28 cell lines. P-gp is expressed in the gastric mucosa of all human fetuses while, it is undetectable in adult normal mucosa and re-expressed in 30/110 Hp-positive non-IM-CG, 28/28 IM-CG and 40/45 GCs. P-gp expression directly correlates with that of Bcl-x(L) and with the promoter hypomethylation of the MDR1 gene. In GC cell lines, P-gp is localized on the plasma membrane and mitochondria where it colocalizes with Bcl-x(L). Co-immunoprecipitation confirms the physical interaction between P-gp and Bcl-x(L) in AGS, MKN-28 and Hep-G2, at both basal level and after stress-induced apoptosis. The selective silencing of P-gp sensitizes GC cells to stress-induced apoptosis. P-gp behaves as an oncofetal protein that, by cross-talking with Bcl-x(L), acts as an anti-apoptotic agent in Hp-related gastric carcinogenesis.

Human primary bone sarcomas contain CD133+ cancer stem cells displaying high tumorigenicity in vivo.

This study aimed to identify, isolate, and characterize cancer stem cells from human primary sarcomas. We performed cytometric analyses for stemness and differentiation antigens, including CD29, CD34, CD44, CD90, CD117, and CD133, on 21 human primary sarcomas on the day of surgery. From sarcoma biopsies, we obtained 2 chondrosarcoma-stabilized cell lines and 2 osteosarcoma stabilized cell lines, on which sphere formation, side population profile, stemness gene expression, and in vivo and in vitro assays were performed. All samples expressed the CD133, CD44, and CD29 markers. Therefore, we selected a CD133(+) subpopulation from stabilized cell lines that displayed the capacity to grow as sarcospheres able to initiate and sustain tumor growth in nonobese diabetic/severe combined (NOD/SCID) mice, to express stemness genes, including OCT3/4, Nanog, Sox2, and Nestin, and to differentiate into mesenchymal lineages, such as osteoblasts and adipocytes. Our findings show the existence of cancer stem cells in human primary bone sarcomas and highlight CD133 as a pivotal marker for identification of these cells. This may be of primary importance in the development of new therapeutic strategies and new prognostic procedures against these highly aggressive and metastatic tumors.

Uterine papillary serous, clear cell, and poorly differentiated endometrioid carcinomas: a comparative study.

INTRODUCTION: Uterine papillary serous and clear cell carcinomas (UPSCs/CCs) show a different spreading from that of poorly differentiated endometrioid carcinomas (PDECs) and are usually thought to be prognostically more aggressive than PDECs. On the contrary, it has been recently claimed that UPSC/CC and PDEC have a similar prognosis. In this retrospective study on 2 institutional databases, the surgical-pathological data and survival have been compared in patients with UPSC/CC and PDEC. METHODS: A total of 139 surgically staged consecutive patients, 63 with UPSC/CC (37 UPSC; 26 CC) and 76 with PDEC clinically limited to the uterine corpus, have been compared for nuclear ploidy, myometrial invasion, (occult) cervical extension, peritoneal, and lymph node metastasis. Prognostic factors have been correlated through multivariate analysis with survival (disease-specific [DSS] and disease-free [DFS]). RESULTS: Peritoneal metastases and aneuploidy were found to be the only parameters significantly different in the 2 groups: peritoneal metastases 28.6% in UPSC/CC (extrapelvic 19%) and 7.9% in PDEC (extrapelvic 2.6%) (P = 0.001), aneuploidy 48.6% in UPSC/CC and 30.6% in PDEC (P = 0.05). Five-year DSS was 57.9% versus 75.2% (P = 0.02), and DFS was 52.3% versus 71.4% (P = 0.04) for UPSC/CC and PDEC, respectively. All but cervical and lymph node involvement were significant predictors of survival. After multivariate analysis, histotype (DSS: hazard ratio [HR], 1.98; 95% confidence interval [CI], 1.02-3.86; P = 0.04; DFS: HR, 1.94; 95% CI, 1.04-3.63; P = 0.04), stage (DSS: HR, 2.26; 95% CI, 1.10-4.65; P = 0.03; DFS: HR, 2.21; 95% CI, 1.12-4.38; P = 0.02), and myometrial invasion (DSS: HR, 2.86; 95% CI, 1.22-6.69; P = 0.01; DFS: HR, 3.96; 95% CI, 1.63-9.62; P = 0.002) were independent risk factors for survival. CONCLUSIONS: Uterine papillary serous and clear cell carcinomas spread to abdominal peritoneum more frequently than PDEC; multivariate analysis confirms UPSC/CC as an independent, unfavorable predictor of outcome.

Involvement of the soluble urokinase receptor in chondrosarcoma cell mobilization.

High levels of urokinase receptor (uPAR) in tissue and serum of patients with chondrosarcoma correlate with poor prognosis. First, we analyzed the uPAR levels in tissues and plasma of five patients affected by chondrosarcoma. Interestingly, very high levels of uPAR and its soluble forms (SuPAR) were found on tumor cell surfaces and plasma, respectively, of two patients with lung metastases. Therefore, to investigate the role of SuPAR in chondrosaromas, we generated a primary cell culture from a chondrosarcoma tissue overexpressing uPAR on cell surfaces. We found that chondrosarcoma-like primary culture cells release a large amount of SuPAR in the medium. In vitro, SuPAR elicits chondrosarcoma cell migration likely through its uPAR(88-92) sequence, since the DII(88-183) or DIIDIIR(88-284) uPAR domains retain motogen effect whereas DI(1-87) or DIII(184-284) domains, both lacking the uPAR(88-92) sequence, are ineffective. Chondrosarcoma cells cross matrigel in response to SuPAR, and their invasion capability is abrogated by RERF peptide which inhibits uPAR(88-92) signalling. These findings assign a role to uPAR in mobilizing chondrosarcoma cells and suggest that RERF peptide may be regarded as a prototype to generate new therapeutics for the chondrosarcoma treatment.

Endoscopic ultrasound-guided drainage of a fungal liver abscess using a lumen-apposing metal stent: case report and literature review.

Liver abscesses are rare entities for which percutaneous drainage is traditionally employed. The technique is simple, but associated with a significant rate of side effects and patient discomfort. We herein report a case of fungal left liver lobe abscess that was successfully treated by using EUS-guided drainage, with insertion of a large caliber lumen-apposing metal stent. The literature review we performed on the topic seems to favor, at least for abscess in the left and/or caudate liver lobes, EUS as compared to percutaneous drainage.

Identification and expansion of human osteosarcoma-cancer-stem cells by long-term 3-aminobenzamide treatment.

A novel cancer stem-like cell line (3AB-OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG-63 cells by long-term treatment (100 days) with 3-aminobenzamide (3AB). 3AB-OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle-shaped, polygonal-shaped, and rounded-shaped. With respect to MG-63 cells, 3AB-OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self-renewal ability and much higher levels of cell cycle markers which account for G1-S/G2-M phases progression. Differently from MG-63 cells, 3AB-OS cells can be reseeded unlimitedly without losing their proliferative potential. They show an ATP-binding cassette transporter ABCG2-dependent phenotype with high drug efflux capacity, and a strong positivity for CD133, marker for pluripotent stem cells, which are almost unmeasurable in MG-63 cells. 3AB-OS cells are much less committed to osteogenic and adipogenic differentiation than MG-63 cells and highly express genes required for maintaining stem cell state (Oct3/4, hTERT, nucleostemin, Nanog) and for inhibiting apoptosis (HIF-1alpha, FLIP-L, Bcl-2, XIAP, IAPs, and survivin). 3AB-OS may be a novel tumor cell line useful for investigating the mechanisms by which stem cells enrichment may be induced in a tumor cell line. The identification of a subpopulation of cancer stem cells that drives tumorigenesis and chemoresistance in osteosarcoma may lead to prognosis and optimal therapy determination. Expression patterns of stem cell markers, especially CD133 and ABCG2, may indicate the undifferentiated state of osteosarcoma tumors, and may correlate with unfavorable prognosis in the clinical setting.

Gefitinib induction of in vivo detectable signals by Bcl-2/Bcl-xL modulation of inositol trisphosphate receptor type 3.

PURPOSE: To test whether epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) induce detectable signals in tumor cells and whether such signals may reveal alterations of the apoptotic program. EXPERIMENTAL DESIGN: Tumor cells were treated with gefitinib or erlotinib and tested for their ability to accumulate 99mTc-Sestamibi, a radiolabeled lipophilic cation that localizes in mitochondria. Then we tested whether Bcl-2 and Bcl-xL alter the pattern of drug-dependent tracer accumulation while reducing tumor cell sensitivity to EGFR TKIs. The mechanism underlying the pattern of tracer accumulation was elucidated. Finally, imaging studies were done in animal models and lung cancer patients before and after treatment with EGFR TKIs using single-photon emission computed tomography and 99mTc-Sestamibi. RESULTS: Gefitinib increases accumulation of 99mTc-Sestamibi in Bcl-2-overexpressing cells and enhances the physical interaction of phosphorylated Bcl-2 with inositol trisphosphate receptor type 3 (IP3R3). Consequently, a relative increase of cytosolic and mitochondrial calcium levels occurs. Similarly, lung cancer cells showed an increase of tracer uptake and an enhanced interaction of Bcl-xL with IP3R3 on exposure to erlotinib concentrations achievable in plasma. The occurrence of these interactions was associated with an enhanced EGFR TKI-induced apoptosis resistance. Posttreatment imaging studies in nude mice bearing control and Bcl-2-overexpressing breast carcinomas showed a high tumor uptake of the tracer whereas baseline studies failed to visualize tumors. Similarly, an enhancement of tracer uptake could be detected in patients with lung cancer treated with erlotinib. CONCLUSION: EGFR TKIs generate detectable signals by Bcl-2/Bcl-xL modulation of IP3R3 in tumor cells.

Antioxidant and anti-proliferative properties of extracts from heterotrophic cultures of Galdieria sulphuraria.

This study explores the possibility to use the extremophilic microalga Galdieria sulphuraria (strain 064) as a source of natural biomolecules with beneficial and protective effects on human health. Galdieria was cultivated in heterotrophy conditions and cells extracts for their antioxidant and anti-proliferative properties were tested. Galdieria extracts showed high antioxidant power tested through ABTS assay and revealed high glutathione and phycocyanin contents. Based on Annexin-V FITC/propidium iodide and MTT analysis, algae extracts inhibited the proliferation of human adenocarcinoma A549 cells (51.2% inhibition) through the induction of apoptosis without cell cycle arrest. Besides, cytotoxicity and cytometry assays showed a positive pro-apoptotic mechanism. On these bases, we suggest that G. sulphuraria from heterotrophic culture, for its therapeutic potential, could be considered a good candidate for further studies with the aim to isolate bioactive anti-cancer molecules.

Paroxysmal nocturnal hemoglobinuria: significant association with specific HLA-A, -B, -C, and -DR alleles in an Italian population.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of a PIG-A mutated hematopoietic stem cell. An immune-mediated origin has been suggested for this disease. Because HLA genes represent a susceptibility factor for autoimmunity, we investigated HLA genotype in 42 Italian PNH patients compared with 301 control subjects of the same ethnic origin. A significantly increased frequency of the HLA class I alleles A*0201 (p < 0.05), B*1402 (p < 0.001), and Cw*0802 (p < 0.005), and of the HLA class II DRB1*1501 (p < 0.01) with the linked DQB1*0602 (p

Effect of ABT-888 on the apoptosis, motility and invasiveness of BRAFi-resistant melanoma cells.

Melanoma is a molecularly heterogeneous disease with many genetic mutations and altered signaling pathways. Activating mutations in the BRAF oncogene are observed in approximately 50% of cutaneous melanomas and the use of BRAF inhibitor (BRAFi) compounds has been reported to improve the outcome of patients with BRAF-mutated metastatic melanoma. However, the majority of these patients develop resistance within 6-8 months following the initiation of BRAFi treatment. In this study, we examined the possible use of the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, ABT-888 (veliparib), as a novel molecule that may be successfully employed in the treatment of BRAFi-resistant melanoma cells. Sensitive and resistant to BRAFi dabrafenib A375 cells were exposed to increasing concentrations of ABT-888. Cell viability and apoptosis were assessed by MTT assay and Annexin V-FITC analysis, respectively. The cell migratory and invasive ability was investigated using the xCELLigence technology and Boyden chamber assays, respectively. ABT-888 was found to reduce cell viability and exhibited pro-apoptotic activity in melanoma cell lines, independently from the BRAF/NRAS mutation status, in a dose-dependent manner, with the maximal effect being reached in the 25-50 µM concentration range. Moreover, ABT-888 promoted apoptosis in both the sensitive and resistant A375 cells, suggesting that ABT-888 may be useful in the treatment of BRAFi-resistant subsets of melanoma cells. Finally, in accordance with the involvement of PARP1 in actin cytoskeletal machinery, we found that the cytoskeletal organization, motility and invasive capability of both the A375 and A375R cells decreased upon exposure to 5 µM ABT-888 for 24 h. On the whole, the findings of this study highlight the pivotal role of PARP1 in the migration and invasion of melanoma cells, suggesting that ABT-888 may indeed be effective, not only as a pro-apoptotic drug for use in the treatment of BRAFi-resistant melanoma cells, but also in suppressing their migratory and invasive activities.

The Hydrogen Sulfide Releasing Molecule Acetyl Deacylasadisulfide Inhibits Metastatic Melanoma.

Melanoma is the most common form of skin cancer. Given its high mortality, the interest in the search of preventive measures, such as dietary factors, is growing significantly. In this study we tested, in vitro and in vivo, the potential anti-cancer effect of the acetyl deacylasadisulfide (ADA), a vinyl disulfide compound, isolated and purified from asafoetida a foul-smelling oleo gum-resin of dietary and medicinal relevance. ADA markedly suppressed proliferation of human melanoma cell lines by inducing apoptosis. Moreover, treatment of melanoma cells with ADA reduced nuclear translocation and activation of NF-κB, decreased the expression of the anti-apoptotic proteins c-FLIP, XIAP, and Bcl-2 and inhibited the phosphorylation and activation of both AKT and ERK proteins, two of the most frequently deregulated pathways in melanoma. Finally, the results obtained in vitro were substantiated by the findings that ADA significantly and dose-dependently reduced lung metastatic foci formation in C57BL/6 mice. In conclusion, our findings suggest that ADA significantly inhibits melanoma progression in vivo and could represent an important lead compound for the development of new anti-metastatic agents.