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Supplementary Methods from MicroRNA-21 in Pancreatic Cancer: Correlation with Clinical Outcome and Pharmacologic Aspects Underlying Its Role in the Modulation of Gemcitabine Activity

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MicroRNA-21 (miR-21) was reported to be overexpressed and contributes to invasion and gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to evaluate whether miR-21 expression was associated with the overall survival (OS) of PDAC patients treated with gemcitabine and to provide mechanistic insights for new therapeutic targets. miR-21 expression was evaluated in cells (including 7 PDAC cell lines, 7 primary cultures, fibroblasts, and a normal pancreatic ductal cell line) and tissues (neoplastic specimens from 81 PDAC patients and normal ductal samples) isolated by laser microdissection. The role of miR-21 on the pharmacologic effects of gemcitabine was studied with a specific miR-21 precursor (pre-miR-21). Patients with high miR-21 expression had a significantly shorter OS both in the metastatic and in the adjuvant setting. Multivariate analysis confirmed the prognostic significance of miR-21. miR-21 expression in primary cultures correlated with expression in their respective tissues and with gemcitabine resistance. Pre-miR-21 transfection significantly decreased antiproliferative effects and apoptosis induction by gemcitabine, whereas matrix metalloproteinase (MMP)-2/MMP-9 and vascular endothelial growth factor expression were upregulated. Addition of inhibitors of phosphoinositide 3-kinase and mammalian target of rapamycin resulted in decrease of phospho-Akt and prevented pre-miR-21–induced resistance to the proapoptotic effects of gemcitabine. miR-21 expression correlated with outcome in PDAC patients treated with gemcitabine. Modulation of apoptosis, Akt phosphorylation, and expression of genes involved in invasive behavior may contribute to the role of miR-21 in gemcitabine chemoresistance and to the rational development of new targeted combinations. Cancer Res; 70(11); 4528–38. ©2010 AACR.

Introduction: In colorectal cancer (CRC) surveillance, adenoma characteristics such as size, number of adenomas, dysplasia, and villous components, are used as indicators for risk of developing metachronous cancer and guide the surveillance interval. The current risk groups (e.g., advanced adenomas) cannot optimally distinguish high risk from low risk, which may result in suboptimal surveillance strategies. Specific DNA copy number aberrations are associated with risk of colorectal adenoma-to-carcinoma progression, but these aberrations are present in only a subset of advanced adenomas. Therefore, we hypothesize that specific DNA copy number aberrations may better predict the risk of CRC than advanced adenoma. Aim: To evaluate whether a molecularly-defined high-risk adenoma is a better risk factor for CRC than the presently used advanced adenoma. Materials and methods: DNA copy number profiles were determined, by means of low-coverage whole genome sequencing, on a series of 529 adenomas, selected from a Norwegian adenoma cohort. We retrieved detailed information on adenoma characteristics and whether adenoma patients were subsequently diagnosed with CRC or not. By univariate and multivariate regression analysis we estimated the odds ratio for association between the development of CRC and baseline presence of advanced adenoma versus presence of a molecularly-defined high-risk adenoma, respectively. Results: Molecular high-risk features were observed in 85/267 (32%) of advanced adenomas and in 27/262 (10%) of non-advanced adenomas. The odds ratio for developing metachronous CRC was 3.58 (p=2.84E-8) and 1.90 (p=0.012), when an advanced adenoma or molecular high-risk adenoma was detected at the baseline colonoscopy, respectively. In the multivariate regression analysis only advanced adenoma was as a significant risk factor for CRC. Conclusion: Molecularly-defined high-risk adenomas are associated with an increased risk of CRC. However, the advanced adenoma is a stronger predictor for risk of CRC. Citation Format: Henriette C. Jodal, Eddymurphy U. Akwiwu, Leticia G. Leon, Margriet Lemmens, Pien Delis-van Diemen, Dagmar Klotz, Meike de Wit, Remond Fijneman, Monique van Leerdam, Evelien Dekker, Manon C. W. Spaander, Gerrit A. Meijer, Michael Bretthauer, Magnus Løberg, Veerle M. H. Coupé, Mette Kalager, Beatriz Carvalho. Molecular adenoma features to predict metachronous colorectal cancer risk: A nested-case control study [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A004.

Chronic lymphocytic leukemia (CLL) is a disease with heterogeneous clinical and biological characteristics. Differences in Ca2+ levels among cases, both basal and upon B-cell receptor (BCR) stimulation, may reflect heterogeneity in the pathogenesis due to cell-intrinsic factors. Our aim was to elucidate cell-intrinsic differences between BCR-responsive and -unresponsive cases. We therefore determined BCR responsiveness ex vivo based on Ca2+ influx upon alpha-IgM stimulation of purified CLL cell fractions from 52 patients. Phosphorylation levels of various BCR signaling molecules, and expression of activation markers were assessed by flow cytometry. Transcription profiling of responsive (n=6) and unresponsive cases (n=6) was performed by RNA sequencing. Real-time quantitative polymerase chain reaction analysis was used to validate transcript level differences in a larger cohort. In 24 cases an alpha-IgM response was visible by Ca2+ influx which was accompanied by higher phosphorylation of PLC gamma 2 and Akt after alpha-IgM stimulation in combination with higher surface expression of IgM, IgD, CD19, CD38 and CD43 compared to the unresponsive cases (n=28). Based on RNA sequencing analysis several components of the canonical nuclear factor (NF)-kappa B pathway, especially those related to NF-kappa B inhibition, were expressed more highly in unresponsive cases. Moreover, upon alpha-IgM stimulation, the expression of these NF-kappa B pathway genes (especially genes coding for NF-kappa B pathway inhibitors but also NF-kappa B subunit REL) was upregulated in BCR-responsive cases while the level did not change, compared to basal level, in the unresponsive cases. These findings suggest that cells from CLL cases with enhanced NF-kappa B signaling have a lesser capacity to respond to BCR stimulation.

T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

Several oncogenic drivers have been identified in non-small cell lung cancer. Targeted therapies for these aberrations have already been successfully developed and implemented in clinical practice. Owing to improved sensitivity in genetic testing, more and more tumors with multiple driver mutations are identified, resulting in dilemmas for treating physicians whether and which targeted therapy to use. In this case series, we provide an overview of patients with intrinsic double mutations in oncogenic drivers and their reported response to targeted therapies, with a focus on epidermal growth factor receptor, anaplastic lymphoma kinase, cMET, and Kirsten rat sarcoma viral oncogene. We also include an unpublished case report on a patient with an epidermal growth factor receptor L858R and cMET exon 14 skipping.

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL). In vivo models of CTL exhaustion yield very few exhausted CTL, limiting the analysis can be done on these cells. Furthermore, the milieu in these models can obscure the phenotype of exhausted CTL. An in vitro system could therefore greatly facilitate the study of this cell state. Purified OT-I CTL were cultured and repeatedly stimulated with OVA(257–264) SIINFEKL peptide to induce exhaustion. On day 5, inhibitory receptors (IRs) and cytokine production were assayed. Sorted live CTL were transferred into WSN-OVA influenza infected mice and on day10 post infection the number of the donor CTL were measured. Gene expression changes were determined by RNAseq and DNA methylation sequence. In vitro exhausted CTL exhibited upregulation of multiple IRs as well as impaired polyfunctionality. They expanded less than controls in vivo. In vitro exhausted CTL exhibited all the transcriptomic characteristics of in vivo exhausted CTL. Repeated stimulation of CTL with their specific peptide, induced all the functional, phenotypic and transcriptomic characteristics of exhausted CTL. This in vitro system can be used to identify genes and signalling pathways involved in exhaustion and facilitate the screening of reagents that prevent/reverse CTL exhaustion.

Cytotoxic CD8+ T lymphocytes (CTLs) play a major role in protection against chronic viral infections and tumors. Chronic antigen stimulation in both conditions leads to CTL exhaustion and failure of CTLs to efficiently eradicate tumors and virally infected cells. miRNAs are small, non-coding RNA molecules that participate in post-transcriptional gene regulation. This study aimed to determine the core miRNA signature of CTL exhaustion in mouse models of both tumors and chronic infection. Exhausted anti-tumor CTLs were isolated from tumors of mice that received 10^6 mouse mesothelioma cells (AE17sOVA) followed by an adoptive transfer of 10^4 CD45.1+ OT-I cells. Effector anti-viral CTLs were isolated from mice adoptively transferred with 10^4 CD45.1+ OT-I cells or P14 cells and infected with WSN-OVA or LCMV Armstrong respectively. For exhausted anti-viral CTLs, mice were adoptively transferred with 10^4 CD45.1+ P14 cells and infected with LCMV-Cl13. Single cell suspensions were prepared from the different tissues, live CTLs were FACS sorted, RNA isolated and small RNA-seq was performed. Principle component analysis (PCA) of miRNA expression revealed that exhausted anti-tumor CTLs and anti-LCMV Cl13 CTLs cluster apart from effector anti-viral CTLs (WSN-OVA and LCMV Armstrong). Differential expression analysis identified 73 miRNAs commonly shared between the exhausted day anti-tumor CTL and anti-viral CTL. Using RNA sequencing of two different models of exhaustion, distinct RNA profiles common in exhausted CTLs were found. This core miRNA signature can now be used to manipulate the expression of specific miRNA to potentially prevent or revert CTL exhaustion and improve the CTL response against tumors and/or chronic infections.

Respiratory viral infections contribute substantially to global infant losses and disproportionately affect preterm neonates. Using our previously established neonatal murine model of influenza infection, we demonstrate that three-day old mice are exceptionally sensitive to influenza virus infection and exhibit high mortality and viral load. Intranasal pre- and post-treatment of neonatal mice with Lactobacillus rhamnosus GG (LGG), an immune modulator in respiratory viral infection of adult mice and human preterm neonates, considerably improves neonatal mice survival after influenza virus infection. We determine that both live and heat-killed intranasal LGG are equally efficacious in protection of neonates. Early in influenza infection, neonatal transcriptional responses in the lung are delayed compared to adults. These responses increase by 24 hours post-infection, demonstrating a delay in the kinetics of the neonatal anti-viral response. LGG pretreatment improves immune gene transcriptional responses during early infection and specifically upregulates type I IFN pathways. This is critical for protection, as neonatal mice intranasally pre-treated with IFNβ before influenza virus infection are also protected. Using transgenic mice, we demonstrate that the protective effect of LGG is mediated through a MyD88-dependent mechanism, specifically via TLR4. LGG can improve both early control of virus and transcriptional responsiveness and could serve as a simple and safe intervention to protect neonates., Author summary Viral lung infections are the number one reason for hospitalization of children under the age of 5 and are a major public health concern. Premature babies, or those born before 37 weeks gestation, are particularly susceptible to viral infections, but exact mechanisms for this susceptibility have not been determined. Here, using a pre-clinical infant mouse model of influenza virus infection, we have found increased neonatal susceptibility to respiratory viral infection compared to adults, and we demonstrate that a probiotic administered intranasally prior to infection provides dramatic protection to infant mice by inducing production of a key anti-viral cytokine, type I interferons.

Background: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease, where patients often suffer from fatigue. Biological pathways underlying fatigue are unknown. In this study aptamer-based SOMAscan technology is used to identify potential biomarkers and treatment targets for fatigue in pSS. Methods: SOMAscan® Assay 1.3k was performed on serum samples of healthy controls (HCs) and pSS patients characterized for interferon upregulation and fatigue. Differentially expressed proteins (DEPs) between pSS patients and HC or fatigued and non-fatigued pSS patients were validated and discriminatory capacity of markers was tested using independent technology. Results: Serum concentrations of over 1,300 proteins were compared between 63 pSS patients and 20 HCs resulting in 58 upregulated and 46 downregulated proteins. Additionally, serum concentrations of 30 interferon positive (IFNpos) and 30 interferon negative (IFNneg) pSS patients were compared resulting in 25 upregulated and 13 downregulated proteins. ELISAs were performed for several DEPs between pSS patients and HCs or IFNpos and IFNneg all showing a good correlation between protein levels measured by ELISA and relative fluorescence units (RFU) measured by the SOMAscan. Comparing 22 fatigued and 23 non-fatigued pSS patients, 16 serum proteins were differentially expressed, of which 14 were upregulated and 2 were downregulated. Top upregulated DEPs included neuroactive synaptosomal-associated protein 25 (SNAP-25), alpha-enolase (ENO1) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1). Furthermore, the proinflammatory mediator IL36a and several complement factors were upregulated in fatigued compared to non-fatigued pSS patients. ROC analysis indicated that DEPs showed good capacity to discriminate fatigued and non-fatigued pSS patients. Conclusion: In this study we validated the use of aptamer-based proteomics and identified a novel set of proteins which were able to distinguish fatigued from non-fatigued pSS patients and identified a so-called “fatigue signature.”

Immunophenotyping is a widely used method for a precise diagnosis and classification of haematopoietic neoplasia in human beings and also in dogs. The gold standard for cell preparation is density gradient centrifugation of mononuclear cells. Alternatively, another way to separate human leukocytes is carrying out whole blood lysis. The aim of this study was to validate whole blood lysis as an alternative method in clinical veterinary procedures using an immunophenotype panel of leukocytes designed by our group. Flow cytometry study of adult canine leukocytes subset groups, using whole blood lysis or mononuclear cells tested against an array of canine leukocyte antibodies were done. Besides differential white blood cell counts were done. Also immunophenotyping studies in whole blood samples stored at 4 °C for 48 h were performed. The Coefficient Variation values were less than 20%, for most of the comparison. Consistent results were observed in phenotyping canine peripheral blood leukocytes. Stability results indicated that whole blood samples might be stored for 48 h without a significant difference in the data compared to samples processed immediately after blood collection. This study shows that whole blood lysis represents an efficient and quick alternative for canine leukocyte preparation. In addition, samples can be analysed immediately or stored for 48 h without a significant difference between them. This is relevant for veterinary medicine considering the lack of facilities in many laboratories to process samples. Flow cytometry, peripheral blood, leukocyte subsets, long-term stability

Adult Mesenchymal Stem Cells (MSC) are cells that can be defined as multipotent cells able to differentiate into diverse lineages, under appropriate conditions. These cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Initially discovered in bone marrow, MSC can now be isolated from a wide spectrum of adult and foetal tissues. Studies to evaluate the therapeutic potential of these cells are based on their ability to arrive to damaged tissues. In this paper we have done a comparative study analyzing proliferation, surface markers and OCT4, SOX9, RUNX2, PPARG genes expression in MSC cells from Bone marrow (BMMSC) and Adipose tissue (ASC). We also analyzed the role of Stem Cell Factor (SCF) on MSC proliferation and on ASCs metalloproteinases MMP-2, MMP-9 secretion. Healthy dogs were used as BMMSC donors, and ASC were collected from omentum during elective ovariohysterectomy surgery. Both cell types were cultured in IMDM medium with or without SCF, 10% Dog Serum (DS), and incubated at 38 °C with 5% CO2. Growth of BMMSCs and ASCs was exponential until 25–30 days. Flow citometry of MSCs revealed positive results for CD90 and negative for CD34, CD45 and MCH-II. Genes were evaluated by RT-PCR and metalloproteinases by zymografy. Our findings indicate morphological and immunological similarities as well as expression of genes from both origins on analyzed cells. Furthermore, SCF did not affect proliferation of MSCs, however it up-regulated MMP-2 and MMP-9 secretion in ASCs. These results suggest that metalloproteinases are possibly essential molecules pivoting migration.

Malignant pleural mesothelioma (MPM) is a lethal disease with scarce therapeutic options, and preclinical studies on new targeted-agents are warranted. Because previous studies reported high c-Met expression and alterations in the microtubules network in most MPM samples, we evaluated the activity of tivantinib, which has been recently suggested to affect microtubule polymerization in addition to inhibiting c-Met. In four MPM cell lines tivantinib inhibited both c-Met activity and microtubule polymerization, resulting in inhibition of cell-growth with IC 50 s ranging between 0.3 µM (MSTO-211H) and 2.4 µM (H2052). Furthermore tivantinib synergistically enhanced the antiproliferative and proapoptotic activity of pemetrexed, as detected by sulforhodamine-B-assay and flow cytometry. The synergistic interaction was associated with reduction of thymidylate synthase expression and inhibition of migratory activity. In aggregate, these data show the ability of tivantinib to specifically target key pathways in MPM cells and synergistically interact with pemetrexed, supporting further studies on this therapeutic approach.

Using several reactions that include homologations and asymmetric epoxidations as well as Ugi and Huisgen couplings, we generated a small focused library of new derivatives from the labdane-type diterpene grindelic acid. These compounds were evaluated as cytotoxic agents against a panel of five human solid tumor cell lines (HBL-100, HeLa, SW1573, T-47D, and WiDr). The presence of the diamide functionalizations enhanced the cytotoxic effect. N-Benzyl-N-(1-(benzylamino)-2-methyl-1-oxopropan- 2-yl)grindelicamide, proved to be the most active product in all cell lines tested, with values of 0.95 (0.38) mM against HBL-100 cells. Fil: Reta, Guillermo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Investigaciones en Tecnología Química; Argentina Fil: Chiaramello, Alejandra Ilda. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Departamento de Química. Area de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Investigaciones en Tecnología Química; Argentina Fil: García, Celina. Universidad de la Laguna. Departamento de Química Orgánica; España. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "Antonio Gonzalez"; España Fil: León, Leticia G.. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "Antonio Gonzalez"; España Fil: Martín, Víctor S.. Universidad de la Laguna. Departamento de Química Orgánica; España. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "Antonio Gonzalez"; España Fil: Padrón, José M.. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "Antonio Gonzalez"; España Fil: Tonn, Carlos Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Investigaciones en Tecnología Química; Argentina Fil: Donadel, Osvaldo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Investigaciones en Tecnología Química; Argentina

Malignant pleural mesothelioma (MPM) is a very hypoxic malignancy, and hypoxia has been associated with resistance towards gemcitabine. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis. Therefore we investigated the combination of a new LDH-A inhibitor (NHI-1) with gemcitabine in MPM cell lines. Under hypoxia (O2 tension of 1%) the cell growth inhibitory effects of gemcitabine, were reduced, as demonstrated by a 5- to 10-fold increase in IC50s. However, the simultaneous addition of NHI-1 was synergistic (combination index < 1). Flow cytometry demonstrated that hypoxia caused a G1 arrest, whereas the combination of NHI-1 significantly increased gemcitabine-induced cell death. Finally, the mRNA expression levels of the human equilibrative transporter-1 (hENT1) were significantly down-regulated under hypoxia, but treatment with NHI-1 was associated with a recovery of hENT1 expression. In conclusion, our data show that hypoxia increased MPM resistance to gemcitabine. However, cell death induction and modulation of the key transporter in gemcitabine uptake may contribute to the synergistic interaction of gemcitabine with the LDH-A inhibitor NHI-1 and support further studies for the rational development of this combination.

A new family of heterometallic compounds 3-6 containing ferrocenyl and platinum(II) centers has been synthesized by reaction of 1-β-aminoethylferrocene (1) and 1,1'-bis(β-aminoethyl)ferrocene (2) with Pt(II) precursors. Using K(2)[PtCl(4)] as the Pt(II) source, the cis-square-planar neutral compounds [Fe{η(5)-C(5)H(4)(CH(2))(2)NH(2)}(2)PtCl(2)] (3) and [{Fe(η(5)-C(5)H(4)(CH(2))(2)NH(2))(η(5)-C(5)H(5))}(2)PtCl(2)] (5) were obtained. Reaction of cis-[PtCl(2)(dmso)(2)] with 1 and 2 resulted in the displacement of dmso and chloride ligands from the platinum coordination sphere, affording the cationic and neutral compounds [Fe{η(5)-C(5)H(4)(CH(2))(2)NH(2)}(2)Pt(dmso)Cl]Cl (4) and [Fe(η(5)-C(5)H(4)(CH(2))(2)NH(2))(η(5)-C(5)H(5))Pt(dmso)Cl(2)] (6). Compounds 3-6 were thoroughly characterized using multinuclear ((1)H, (13)C, (195)Pt) NMR, IR spectroscopy, ESI mass spectrometry and elemental analysis. Single-crystal X-ray analysis of heterometallic 6 confirmed the cis geometry of the molecule and revealed that the platinum atom is held in a perfect square-planar geometry. The electrochemical behaviour of the heterometallic compounds 3-6, which has been examined by cyclic (CV) and square wave (SWV) voltammetries in dichloromethane and dmso solution, is characterized by the reversible one-electron oxidation of the ferrocene moieties. The results of the biological activity studies revealed that the organometallic complex 5 is active against all cell lines with GI(50) values in the range 1.7-2.3 μM. When compared to the standard anticancer drug cisplatin, heterotrimetallic 5, possessing two aminoethylferrocenyl units coordinated to the Pt(II) center, showed a greater activity profile in the colon cancer cell line. Cell cycle studies revealed that the new mixed compound exhibits a mechanism of action different to cisplatin.

A Phenotypic Drug Discovery strategy was applied to study a set of pyrimidine analogs prepared by means of intramolecular oxidation-reduction reactions of N-substituted-N-(2,6-disubstituted-5-nitro-4-pyrimidinyl) aminoacetic acid methyl esters in basic media. The combined and rational use of specific assays allowed in short time reducing from all possible cellular targets to those involved in metaphase to anaphase transition. © 2011 Elsevier Ltd. All rights reserved., Co-financed by the European Social Fund (FEDER): the Spanish MICINN (CTQ2008-06806-C02-01/BQU), the Spanish MSC (RTICC RD06/0020/1046 and RD06/0020/0041), the Canary Islands ACIISI (PI 2007/021), the Canary Islands FUNCIS (PI 43/09), and by the Lithuanian Research Council: Global Grant Programme (Grant No. VP1-3.1-SMM-07-K-01-002). L.G.L. thanks the Spanish MSC-FIS for a Sara Borrell contract.

The biological evaluation of new Ru(II) complexes carrying dmoPTA (dmoPTA=3,7-dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) ligands is reported. The results on the biological activity revealed that the organometallic complexes are active against all cell lines with GI(50) values in the range 1.1-2.6 μM. When compared to the standard anticancer drug cisplatin, the bimetallic Ru(II) complexes showed a greater activity profile. The cell cycle analysis revealed that the new compounds induced arrest in G(1) phase. Contrary to cisplatin, these Ru(II) complexes do not interact with DNA. This result suggests that DNA might not be the key pharmacological target.

Highly invasive tumor cells are characterized by a metabolic switch, known as the Warburg effect, from "normal" oxidative phosphorylation to increased glycolysis even under sufficiently oxygenated conditions. This dependence on glycolysis also confers a growth advantage to cells present in hypoxic regions of the tumor. One of the key enzymes involved in glycolysis, the muscle isoform of lactate dehydrogenase (LDH-A), is overexpressed by metastatic cancer cells and is linked to the vitality of tumors in hypoxia. This enzyme may be considered as a potential target for new anticancer agents, since its inhibition cuts cancer energetic and anabolic supply, thus reducing the metastatic and invasive potential of cancer cells. We have discovered new and efficient N-hydroxyindole-based inhibitors of LDH-A, which are isoform-selective (over LDH-B) and competitive with both the substrate (pyruvate) and the cofactor (NADH). The antiproliferative activity of these compounds was confirmed on a series of cancer cell lines, and they proved to be particularly effective under hypoxic conditions. Moreover, NMR experiments showed that these compounds are able to reduce the glucose-to-lactate conversion inside the cell.

T-cell prolymphocytic leukemia (T-PLL) is a rare type of neoplasm with an adverse prognosis due to poor responsiveness to conventional chemotherapies. Accurate and rapid diagnosis of T-PLL is problematic due to the heterogeneous nature of the disease. Therefore, further molecular insights of T-PLL are needed. Most of the T-PLL cases (80%) harbor chromosome 14 inversions and translocations or activating mutations in the JAK-STAT pathway. Small non-coding RNAs (sncRNA) are defined as single stranded RNAs that are smaller than 200nt in length and include e.g. microRNAs (miR), small nucleolar RNA (snoRNA) and PIWI-interacting RNAs (piRNA). To increase the molecular understanding of T-PLL, we performed sncRNA expression profiling with the Illumina platform on a well-characterized panel of 21 T-PLL samples. Notably, detailed molecular, cytogenetic, morphologic and clinical data are available of all T-PLL patients included in this study. Of the selected samples, we performed gene expression profiling by microarrays, cytogenetics, VDJ-recombination and multi-color flow cytometric analysis. In short, we sorted CD4+ and CD8+ naïve (CD45RA+, CD27+), effector (CD45RA+, CD27-) and memory (CD45RO+, CD27+) T-cells, and TCRγδ+ T-cells from Ficoll separated PBMCs of buffy coat blood. Total RNA of leukemic cells isolated from the blood was extracted and cDNA libraries for sequencing sncRNAs were generated. For the annotation of the sequence reads we used the DASHR database of sncRNAs and miRbase database of mature miRs. SnoRNA (16%) and ribosomal (r) RNAs (25%) were the most abundant, whereas miRs were only 12% of the total sncRNA fraction. Next, we performed a correlation study using the expression profiles of the different types of sncRNAs and found a clear separation of T-PLL samples from the normal T-cell fractions when miRNA, piRNA and snoRNA expression profiles were used in the analysis. In addition, based on these data we could already identify potential subgroups of T-PLL, indicating that T-PLL is not a homogeneous disease. Finally, we noted aberrant expression of different types of sncRNAs compared to normal T-cell fractions, which may be indicative for essential roles of sncRNA species in the pathogenesis of T-PLL. Currently, we are in the process of characterizing sncRNA species that are aberrantly regulated specifically in T-PLL. Finally, we are selecting candidate sncRNAs for further functional characterization and for testing their diagnostic and/or prognostic value. Citation Format: Leticia G. Leon, Steven C. Koetzier, Fabiënne van Opstal, Joyce Schilperoord-Vermeulen, Anton W. Langerak, Stefan J. Erkeland. A comprehensive genome-wide analysis of small noncoding RNAs in T-cell prolymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2466.

This study investigated the interaction between the novel ceramide analog AL6 and gemcitabine in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, harboring different polymorphic variants of the gemcitabine catabolism enzyme cytidine deaminase (CDA). AL6 dose-dependently inhibited cell growth, induced apoptosis and synergistically enhanced the cytotoxic activity of gemcitabine. Moreover, it triggered apoptosis, which was significantly enhanced by the combination, and increased the ratio between gene expression of the activating enzyme deoxycytidine kinase (dCK) and CDA, potentially favoring gemcitabine activity. In conclusion, AL6 displays synergistic cytotoxic activity, enhances apoptosis, and favorably modulates enzymes involved in gemcitabine metabolism, supporting future investigation of this combination in pancreatic cancer.

A series of analogs were synthesized in a straightforward manner from naturally available sesquiterpenes ilicic acid and tessaric acid. The in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780, HBL-100, HeLa, SW1573, T-47D and WiDr. The most potent analog induced considerably growth inhibition in the range 1.9-4.5 microM. Cell cycle studies for tessaric acid derivatives indicated a prominent arrest of the cell cycle at the G(2)/M phase. Damage to the cells was permanent as determine by the so called 24+24 drug schedule.

A series of 2,4-disubstituted 6-aryl-7 H -pyrrolo[3,2- d ]pyrimidin-7-one 5-oxides were synthesized and in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780, HBL-100, HeLa, SW1573, T-47D, and WiDr. The most potent analog induced considerably growth inhibition in the range 0.35–2.0 μM. Cell cycle studies in the breast and lung cancer cells revealed arrest in the G 2 /M compartment. The results showed that the title compounds bearing alkylamino or dialkylamino moieties in position 2 of the pyrimidine ring are more active than those bearing hydrogen or methylthio groups.

Novel antiproliferative β′‐acyloxy‐α,β ‐unsaturated ketones were obtained by means of an iron(III)‐catalyzed multicomponent domino process (ABB′ 3CR). The most active derivatives displayed GI50 values in the range of 0.5–3.9 μ M against a panel of representative human solid tumor cell lines: A2780, SW1573, HBL‐100, T‐47D and WiDr. Analysis of cells following 24 h exposure to these drugs showed cell cycle arrest in the S and G2/M phase, in a dose‐dependent manner. Our data indicate that the β ′‐acyloxy‐α,β ‐unsaturated ketones cause permanent damage to the cells and induce apoptosis.

A series of trans-2-alkyl-4-halopiperidines and 2-alkyl-4-halo-1,2,5,6-tetrahydropyridines were prepared by means of an iron(III) catalyzed process. The in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780 (ovarian cancer), SW1573 (non-small cell lung cancer), and WiDr (colon cancer). The results on the biological activity revealed that, in general, the 2-alkyl-4-halo-1,2,5,6-tetrahydropyridine analogs are more potent than the trans-2-alkyl-4-halopiperidine derivatives. A remarkable selectivity of the aza compound 5f for the resistant cell line WiDr was observed. Cell cycle studies revealed a G2/M phase arrest for 5f.

In this study, we synthesized a series of enantiomerically pure (2R,3R)-disubstituted tetrahydropyrans with diverse functional groups. The in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780 (ovarian cancer), SW1573 (non-small cell lung cancer), and WiDr (colon cancer). Overall, the results show the relevance for antiproliferative activity of the α,β-unsaturated ester side chain at position 2 of the THP ring.

A series of cis-2,6-dialkyl-4-chloro-tetrahydropyrans were prepared by means of an iron(III)-catalyzed process. The in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780, SW1573, and WiDr. The results show that the presence of bulky substituents favors the Prins cyclization leading to new products with better activity profile against all cell lines tested.

Malignant pleural mesothelioma(MPM) is an aggressive cancer, which incidence has constantly increased over the past two decades and is expected to peak in 2020. The overall prognosis is poor, and predictive biomarkers of drug activity are missing. Given the heterogeneous and complex nature of MPM, it is likely that genomic aberrations changing the expression of several genes, might affect therapeutic response. Therefore, the aim of the present study was to identify genes whose copy number alterations might predict the MPM prognosis. Recurrent copy number alterations of genes were analyzed by high-resolution whole-genome sequencing in DNA obtained from a “discovery cohort” of 26 resected MPM patients treated with pemetrexed-based chemotherapy (8 with progressive disease, vs. 10 with stable disease and 8 with partial response). Prognostic markers identified by Copy Number Variation analysis with Nexus, Control-FREEC and ReadDepth software were validated by PCR gene copy number and gene expression analyses both in the “discovery” and in two “validation” cohorts of pemetrexed-treated and untreated patients (N=45 and 40). The role of emerging genes was evaluated through siRNA and pharmacological studies using proliferation, migration and apoptosis assays in MPM cells. As reported previously we observed copy number loss of CDKN2A (15.4%) and BAP1 (7.7%). Interestingly, copy number gain of NOTCH2 was observed in 50% of samples of the patients who underwent progression, whereas losses of PTP4A3 were associated with clinical benefit (SD+PR). The prognostic relevance of NOTCH2 was confirmed by PCR analysis. NOTCH-2 silencing reduced MPM cell migration and enhanced apoptosis induction by pemetrexed, while a PTP4A3 inhibitor overcame pemetrexed resistance in MPM cells characterized by high NOTCH2 and PTP4A3 expression. These results support the role of NOTCH2 as a novel prognostic/predictive biomarker for MPM, prompting prospective randomized trials for its validation. Moreover, preclinical data suggest that NOTCH2 and PTP4A3 are oncogenes suitable for effective therapeutic targeting in pemetrexed-resistant MPM cells. Citation Format: Leticia G. Leon, Maria Gemelli, Paolo A. Zucali, Christos Zoumadakis, Elisa Giovannetti. Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2460. doi:10.1158/1538-7445.AM2017-2460

Oncogenic KRAS signaling is the main driving force behind pancreatic ductal adenocarcinoma (PDAC); however, targeting this pathway has proven to be difficult. Conversely, the PI3K/Akt pathway represents an exciting new target, because it has been associated with poor prognosis and chemoresistance, and several inhibitors are under development. In particular, perifosine prevents Akt translocation to the cell membrane, while MK-2206 is an Akt allosteric inhibitor and BEZ-235 is a dual PI3K/mTOR inhibitor. Therefore, we investigated the prognostic role of phospho (p)-Akt in PDAC tissues, as well as the molecular mechanisms underlying the interaction of Akt inhibitors with gemcitabine, using PDAC cells, primary cultures and spheroids. Immunohistochemistry of tissue microarrays with specimens from radically-resected patients (n=100) revealed a correlation between high p-Akt1 expression and worse outcome. Patients with low p-Akt expression (as detected by digital scoring) had a median overall survival (OS) of 16.2 months (95% CI, 14.8-20.1), while patients with high expression had a median OS of 12.0 months (95% CI, 9.0-14.9, P=0.03). Parallel immunocytochemistry studies revealed high expression levels in LPC028 primary cells, while LPC006 were characterized by low p-Akt1. Akt inhibitors reduced cancer cell growth in monolayers and spheroids, and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028 (e.g., combination index CI of 0.2, in the gemcitabine-perifosine combination for 72h, at fixed IC50 ratio), while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a 5-fold reduction in the expression of the main gemcitabine target ribonucleotide reductase. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. However, the combination of Akt inhibitors with gemcitabine increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. The Akt signaling is involved in the expression/localization of the key glucose transporter Glut1, and increased glucose metabolism was associated to resistance to axitinib (Hudson et al, Cell Death Dis 2014). Remarkably, the resistant LPC006 cells were characterized by overexpression of Glut1, which was not reduced after exposure to Akt inhibitors. However, the novel Glut1 inhibitor PGL13 enhanced perifosine and perifosine/gemcitabine-induced cell death. In conclusion, our findings support the analysis of phospho-Akt1 expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies. Citation Format: Daniela Massihnia, Amir Avan, Niccola Funel, Carlotta Granchi, Filippo Minutolo, Leticia Leon, Godefridus Peters, Elisa Giovannetti. Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2017-4731

A set of γ-lactones α,β-fused and β,γ-fused to carbocycles have been synthesized and evaluated for their in vitro antiproliferative activities using the human cancer cell lines SW1573 (lung), T-47D (breast) and WiDr (colon). The compounds are obtained by intramolecular ring closing metathesis of the corresponding dienes. Active compounds exhibited GI 50 values in the range 8–18 μM. A structure–activity relationship is also discussed.

A phytochemical study has been carried out on the aerial parts of Salvia pachyphylla and S. clevelandii. From S. pachyphylla, the known diterpenes carnosol (2), rosmanol, 20-deoxocarnosol (3), carnosic acid, isorosmanol (4), 7-methoxyrosmanol, 5,6-didehydro-O-methylsugiol (5), 8beta-hydroxy-9(11),13-abietadien-12-one (6), 11,12-dioxoabieta-8,13-diene, and 11,12-dihydroxy-20-norabieta-5(10),8,11,13-tetraen-1-one were isolated, together with the new diterpene pachyphyllone (1). From S. clevelandii, the known diterpenes rosmadial (7), 16-hydroxycarnosol (8), abieta-8,11,13-triene, and taxodone were obtained, together with carnosol (2), rosmanol, and carnosic acid. The structure of the new compound (1) was identified on the basis of spectroscopic data analysis. Several of these compounds (1-8) were evaluated against a small panel of human cancer cell lines.

FireDB (http://firedb.bioinfo.cnio.es) is a curated inventory of catalytic and biologically relevant small ligand-binding residues culled from the protein structures in the Protein Data Bank. Here we present the important new additions since the publication of FireDB in 2007. The database now contains an extensive list of manually curated biologically relevant compounds. Biologically relevant compounds are informative because of their role in protein function, but they are only a small fraction of the entire ligand set. For the remaining ligands, the FireDB provides cross-references to the annotations from publicly available biological, chemical and pharmacological compound databases. FireDB now has external references for 95% of contacting small ligands, making FireDB a more complete database and providing the scientific community with easy access to the pharmacological annotations of PDB ligands. In addition to the manual curation of ligands, FireDB also provides insights into the biological relevance of individual binding sites. Here, biological relevance is calculated from the multiple sequence alignments of related binding sites that are generated from all-against-all comparison of each FireDB binding site. The database can be accessed by RESTful web services and is available for download via MySQL. The Spanish Ministry of Economy and Competitiveness (BIO2012-40205); technical assistance was provided by the Spanish Bioinformatics Institute (INB), a platform of the ISCII; European Union [FP7-REGPOT-2012-CT2012-31637-IMBRAIN to L.G.L.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [grant: BIO2012-40205] Sí

Two new and five known withanolides (jaborosalactones 2, 3, 4, 5, and 24) were isolated from the leaves of Jaborosa runcinata Lam. We also obtained some derivatives from jaborosalactone 5, which resulted to be the major isolated metabolite. The natural compounds as well as derivatives were evaluated for their antiproliferative activity and the induction of quinone reductase 1 (QR1; NQ01) activity. Structureeactivity relationships revealed valuable information on the pharmacophore of withanolide-type compounds. Three compounds of this series showed significantly higher antiproliferative activity than jaborosalactone 5. The effect of these compounds on the cell cycle was determined. Furthermore, the ability of major compounds to induce QR1 was evaluated. It was found that all the active test compounds are monofunctional inducers that interact with Keap1. The most promising derivatives prepared from jaborosalactone 5 include (23R)-4b,12b,21-trihydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen- 26,23-olide (18) and (23R)-21-acetoxy-12b-hydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen- 26,23-lactame (20). Fil: García, Manuela Emila. Universidad Nacional de Cordoba. Facultad de Ciencias Quimicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Nicotra, Viviana Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Cordoba. Facultad de Ciencias Quimicas; Argentina Fil: Oberti, Juan Carlos María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Cordoba. Facultad de Ciencias Quimicas; Argentina Fil: Ríos Luci, Carla. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "antonio Gonzalez"; España Fil: Leon, Leticia G.. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "antonio Gonzalez"; España Fil: Marler, Laura. University of Hawaii at Hilo. The Daniel K. Inouye College of Pharmacy; Estados Unidos Fil: Li, Guannan. University of Illinois at Chicago. College of Pharmacy. Department of Medicinal Chemistry and Pharmacognosy; Estados Unidos Fil: Pezzuto, John. College Of Pharmacy, University Of Hawaii At Hilo; Fil: Van Breemen, Richard B.. University of Illinois at Chicago. College of Pharmacy. Department of Medicinal Chemistry and Pharmacognosy; Estados Unidos Fil: Padron, Jose. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "antonio Gonzalez"; España Fil: Hueso Falcon, Idaira. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "antonio Gonzalez"; España Fil: Estevez Braun, Ana. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-orgánica "antonio Gonzalez"; España

A series of functionalized N-sulfonyl-piperidines and N-sulfonyl-tetrahydropyridines were evaluated for their antiproliferative activity against the representative panel of human solid tumor cells A2780 (ovarian), SW1573 (non-small cell lung) and WiDr (colon). The SAR study showed for WiDr cells a correlation between the biological activity and the length of the N-sulfonyl group, the nature of the substituents and the type of alkyl side chain. Further QSAR studies indicate that the size and nature of the N-sulfonyl group, the atomic polarizability (MP) and the partition coefficient are the most important descriptors for the activity. The major contribution is the size (F05C-S) of the N-sulfonyl group.

Selective acid-promoted cyclization of lapachols (I) affords the α- (III) and β-lapachones (II), respectively.

In this study, we describe the synthesis of a series of α- and β-lapachone containing hydroxyl or methoxyl groups on the benzene ring, by means of the selective acid promoted cyclization of the appropriate lapachol analog. The evaluation of the antiproliferative activity in human solid tumor cell lines provided 7-hydroxy-β-lapachone as lead with enhanced activity over the parent drug β-lapachone. Cell cycle studies, protein expression experiments, and reactive oxygen species analysis revealed that, similarly to β-lapachone, ROS formation and DNA damage are critical factors in the cellular toxicity of 7-hydroxy-β-lapachone., Co-financed by the European Social Fund (FEDER), the Spanish Instituto de Salud Carlos III (PI11/00840), the Spanish MSC (RTICC RD06/0020/1046 and RD06/0020/0041), the Canary Islands ACIISI (PI 2007/021), the Canary Islands FUNCIS (PI 43/ 09), and by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET PIP 0447), and Universidad de Buenos Aires (UBACyT 20020090200697). E.L.B. thanks CONICET for a fellowship. L.G.L. thanks ACIISI for a postdoctoral contract (SE- 10/19).

DTA0100 is a new catalytic inhibitor of the human DNA topoisomerase IIα that induces G2/M phase cell cycle arrest in human solid tumor cells lines from various malignancies. In our study, we investigated the effectiveness of the combined treatment of ionizing radiation with DTA0100 on the survival of three representative human solid tumor cell lines: HeLa (cervix), WiDr (colon) and SW1573 (non-small cell lung cancer). The concomitant treatment of DTA0100 and irradiation showed a synergistic and antagonistic effect in the three cell lines tested. A synergistic cytotoxic effect of the combination of DTA0100 and radiation was confirmed by the median drug effect analysis method. It was found that in those protocols where the drug was administered after radiation the most synergistic effect was achieved. Our study constitutes the first in vitro evidence for synergistic effects between DTA0100 and radiation. This combination therapy might thus be expected to be more effective than either treatment alone in patients with cervical, colon and non-small cell lung cancer cells.

Non-small-cell-lung cancer (NSCLC) is the leading cause of cancer-related deaths. However, chemotherapy has reached a therapeutic plateau and deals with significant toxicity. Novel anticancer treatments to neutralize specific molecules or genes involved in cancer development (" targeted- therapy") are being developed to reduce side-effects and improve outcome. The epidermalgrowth-factor receptor (EGFR) is over-expressed in NSCLC and emerged as an attractive target. Two classes of anti-EGFR agents (tyrosine-kinase-inhibitors and monoclonal antibodies) have shown clinical activity, depending on EGFR mutations and expression. However, clinical outcome, including tolerability, can not always be explained by these biomarkers. Thus, the identification of novel biomarkers is a viable area of research. Germline polymorphisms can be easily assessed, and polymorphisms in EGFR, AKT1 and ABCG2 have been correlated with outcome and toxicity in NSCLC patients given anti-EGFR therapies. However, there is lack of unanimity in findings, influenced by differences in study design/analysis, and the prognostic/predictive role of these polymorphisms needs to be evaluated within prospective studies. Finally, there is a critical need to conduct more studies on the relation of genotype with drug concentration/activity.

Aim: This multicenter study evaluated the association of 11 candidate polymorphisms in eight genes with outcome of pancreatic cancer patients treated with the equivalent polychemotherapeutic regimens: cisplatin/epirubicin/capecitabine/gemcitabine, cisplatin/docetaxel/capecitabine/gemcitabine and gemcitabine/capecitabine plus epirubicin/cisplatin intra-arterial infusion. Patients & methods: Towards this end, polymorphisms were assessed in DNA from 122 pancreatic cancer stage-III/IV patients, and their associations with toxicity/response and progression-free survival (PFS) and overall survival were evaluated using Pearson-χ2 and log-rank test. Results: Patients harboring XPD Gln751Gln, XPD Asp312Asn + Asn312Asn or XRCC1 Arg399Gln + Gln399Gln genotypes had a worse prognosis. XPD Gln751Gln (hazard ratio: 1.9; p = 0.003), as well as a combination of over two risk genotypes (hazard ratio: 2.7; p < 0.001), emerged as independent predictors for death risk at multivariate analysis. No correlations were observed with toxicity. Conversely, XPD Gln751Gln was associated with shorter PFS, while the lack of association with overall survival/PFS in gemcitabine monotherapy-treated patients suggested its role only for platinum-based regimens. Conclusion: Polymorphisms of DNA-repair genes appear to be candidate biomarkers of primary resistance to gemcitabine/cisplatin-based polychemotherapeutic regimens. The relatively small sample size, coupled with the retrospective and exploratory design of the present study, imply that these results should be considered as hypothesis generators, and should be further evaluated in larger and adequately designed retrospective/prospective studies. Original submitted 26 April 2011; Revision submitted 20 July 2011

Conventional chemotherapeutic regimens have limited impact against most solid tumors and deal with significant toxicity. During the last years novel anticancer treatments targeting specific molecules or genes involved in cancer development are being developed to improve outcome and reduce side-effects. In particular several tyrosine-kinase inhibitors (TKIs, gefitinib, erlotinib, sorafenib and sunitinib) have been approved for the treatment of different solid tumors. Their clinical activity has been related to different clinical and biological parameters, such as the EGFR-activating mutations for gefitinib and erlotinib. However, not all clinical outcomes, including tolerability, are explained, and the identification/ validation of novel biomarkers is a viable area of research. Germline polymorphisms can be easily assessed in blood samples, and polymorphisms in EGFR, AKT1 and ABCG2 have been correlated with outcome and toxicity in lung cancer patients given EGFR-TKIs therapies. However, there are several controversial findings, influenced by differences in study design/analysis, while the prognostic/predictive role of these polymorphisms still needs to be evaluated within prospective studies. More studies on the relationship of the genotype with drug pharmacokinetics and mechanism of action are also warranted. All these studies, as well as further development and application of novel technologies to decipher genetic alterations, might contribute to the validation of selected polymorphisms as molecular markers predictive of drug activity and help in the selection of TKIs best suited to the individual patient.

Gemcitabine, a deoxycytidine analog, active against non-small cell lung cancer, is phosphorylated by deoxycytidine kinase (dCK) to active nucleotides. Earlier, we found increased sensitivity to gemcitabine in P-glycoprotein (SW-2R160) and multidrug resistance-associated protein (SW-2R120), overexpressing variants of the human SW1573 non-small cell lung cancer cells. This was related to increased dCK activity. As protein kinase C (PKC) is higher in 2R120 and 2R160 cells and may control the dCK activity, we investigated whether gemcitabine sensitivity was affected by the protein kinase C inhibitor, staurosporine, which also modulates the cell cycle. Ten nmol/l staurosporine enhanced the sensitivity of SW1573, 2R120 and 2R160 cells 10-fold, 50-fold and 270-fold, respectively. Staurosporine increased dCK activity about two-fold and the activity of thymidine kinase 2, which may also activate gemcitabine. Staurosporine also directly increased dCK in cell free extracts. Staurosporine decreased expression of the free transcription factor E2F and of ribonucleotide reductase (RNR), a target for gemcitabine inhibition. In conclusion, staurosporine may potentiate gemcitabine by increasing dCK and decreasing E2F and RNR, which will lead to a more pronounced RNR inhibition. Anti-Cancer Drugs 21:591-599 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

A small structure-focused library of propargylic enol ethers was prepared by means of a modular and efficient chemodifferentiating organocatalyzed multicomponent reaction. The most active compound (GI50 0.25 μM) against solid tumor cells was selected as lead. Cell cycle analysis and immunoblotting demonstrated arrest at the metaphase, pointing out human topoisomerase II as plausible molecular target. In vitro assays were carried out, showing that the lead behaves as a catalytic inhibitor of the enzyme., Financial support cofinanced by the EU-FEDER: the Spanish MICIIN (CTQ2008-06806-C02-01/BQU, CTQ2008-06806-C02-02/BQU, and BFU2006-01813/BMC), MSC (RTICC RD06/0020/1046 and RD06/0020/0041); Canary Islands’ ACIISI (PI 2007/021) and FUNCIS (REDESFAC PI 01/06 and 35/06). L.G.L and E.P.R.: Spanish MSC-FIS Sara Borrell contracts. J.M.P.: SpanishMEC-FSE Ramón y Cajal contract.

A series of 5-hydroxy-1,4-naphthoquinones analogues was synthesized from juglone (6) and their antiproliferative activity against a representative panel of six human solid tumor cell lines has been investigated. The 2,5-dihydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-dione (4) and 2,3-dihydro-5-hydroxy-2-(prop-1-en-2-yl)naphtho[2,3-b]furan-4,9-dione (27) were the most potent antiproliferative agents with GI50 values of 0.42-8.1 and 0.80-2.2 μM, respectively. The results provide insight into the correlation between some structural properties of 5-hydroxynaphthoquinones and their antiproliferative activity. Fil: Bonifazi, Evelyn Lucia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina Fil: Ríos Luci, Carla. Universidad de La Laguna; España. Instituto Canario de Investigación del Cáncer; España Fil: Leon, Leticia G.. Universidad de La Laguna; España. Instituto Canario de Investigación del Cáncer; España Fil: Burton, Gerardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina Fil: Padron, Jose. Universidad de La Laguna; España. Instituto Canario de Investigación del Cáncer; España Fil: Misico, Rosana Isabel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina

Two iridoid scaffolds were synthesized enantioselectively using as key step an l-proline-catalyzed α-formyl oxidation. The in vitro antiproliferative activities were evaluated against a representative panel of human solid tumor cell lines. Both iridoids induced considerably growth inhibition in the range 0.38–1.86 μM. Cell cycle studies for these compounds showed the induction of cell cycle arrest at the G1 phase. This result was consistent with a decrease in the expression of cyclin D1. Damaged cells underwent apoptosis as indicated by specific Annexin V staining.

A set of γ-lactones α,β-fused and β,γ-fused to carbocycles have been synthesized and evaluated for their in vitro antiproliferative activities using the human cancer cell lines SW1573 (lung), T-47D (breast) and WiDr (colon). The compounds are obtained by intramolecular ring closing metathesis of the corresponding dienes. Active compounds exhibited GI 50 values in the range 8–18 μM. A structure–activity relationship is also discussed.

The Prins reaction was the basis to synthesize functionalized alkyl chlorodihydropyran derivatives. The inexpensive, stable, and environmentally friendly FeCl3 promotes the cyclization. The method represents an efficient and regioselective manner to obtain in a single step chlorovinyl–TMS oxacycles. The in vitro antiproliferative activities of the compounds were examined in the human solid tumor cell lines A2780 (ovarian cancer), SW1573 (non-small cell lung cancer), and WiDr (colon cancer). Overall, the results show an enhancement in the cytotoxicity exhibited by the new analogs when compared to their parental compounds.