Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Constituintes químicos de Parmotrema lichexanthonicum Eliasaro & Adler: isolamento, modificações estruturais e avaliação das atividades antibiótica e citotóxica

From the lichen Parmotrema lichexantonicum were isolated the depsidone salazinic acid, the xanthone lichexanthone, and the depside atranorin. The two major compounds, salazinic acid and lichexanthone, were selected for structure modifications. Salazinic acid afforded O-alkyl salazinic acids, some of them potentially cytotoxic against tumor cell lines (HCT-8, SF-295 and MDA/ MB - 435). From lichexanthone were obtained norlichexanthone, 3-O-methylnorlichexanthone, 3-O-methyl-6-O-prenylnorlichexanthone, 3,6-di-O-prenyl-norlichexanthone, 3,6-bis[(3,3-dimethyloxyran-2-il)methoxy]-1-hydroxy-8-methyl-9H-xanten-9-one and 3,6-bis[3-(dimethylamine)propoxy]-1-hydroxy-8-methyl-9H-xanten-9-one. The last compound was the most active against S. aureus.

Trachylobane and kaurane diterpenes from Croton floribundus spreng

A new trachylobane diterpene ent-trachyloban-18,19-diol (1) was isolated from root bark of Croton floribundus, along with known diterpenes ent-trachyloban-19-oic acid (2), 15b-hydroxy-ent-trachyloban-19-oic acid (3), ent-trachyloban-19-ol (4), ent-kaur-16-en-19-oic acid (5), ent-kaur-16-ene-6a,19-diol (6) and ent-16a-hydroxykaur-11-en-19-oic acid (7). ent-trachyloban-18,19-diol (1) was submitted to derivatization reactions affording four new compounds (8-11). Cytotoxic activity of diterpenes 1, 3, 4, 7-11 against three human cancer cell lines was evaluated. No compounds showed cytotoxic potential with IC50 values greater than 25 mg/mL. Compound 6 was evaluated against five human cancer cell lines, showing moderate effect against three cancer cell lines, MDA-MB-435, HCT-8 and HCT-116, with IC50 values of 14.32, 13.47 and 12.1 mg/mL, respectively.

The Role of Integrins in Enterovirus Infections and in Metastasis of Cancer

Integrins are a family of transmembrane glycoproteins, composed of two different subunits (alpha and beta). Altered expression of integrins in tumor cells contributes to metastasis tendency by influencing on the cells‟ attachment to adjacent cells and their migration. Viral pathogens, including certain enteroviruses, use integrins as receptors. Enteroviruses have also been suggested to be involved in the etiopathogenesis of type 1 diabetes. The study focuses on the role of integrins in the pathogenesis of metastasis to cortical bone and on type 1 diabetes (T1D) and echovirus 1 infection. In the first part of the thesis, the role of different integrins in the initial attachment of MDA-MD-231 breast cancer cells to bovine cortical bone disks was studied. A close correlation between alpha2beta1 and alpha3beta1 integrin receptor expression and the capability of the tumor to attach to bone were observed. In the second part, a possible correlation between susceptibility to enterovirus infections in diabetic children and differences in enterovirus receptor genes, including certain integrins, was investigated. In parallel, virus-specific neutralizing antibodies and diabetic risk alleles were studied. In the diabetic group, an amino acid change was detected in the polio virus receptor and the neutralizing antibody titers against echovirus 30 were lower. However, to obtain statistically sustainable results, a larger number of individuals should be analyzed. Echovirus 1 (EV1) enters cells by attaching to the alpha2I domain of the alpha2beta1 integrin. In the third part EV1 was shown to attach to a chimeric receptor construct of the transferrin receptor and the alpha2I domain and to enter cells through clathrin-mediated endocytosis that is normally not used by the virus. The chimeric receptor was recycled to the plasma membrane, whereas the virus remained in intracellular vesicles. The virus replication cycle was initiated in these cells, suggesting that evolution pressure could possibly cause the virus to evolve to use a different entry mechanism. Moreover, a cDNA microarray analysis of host gene expression during EV1 replication showed that 0.53% of the total genes, including several immediate early genes, were differently expressed.

RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

Oestrogenic activity of benzylparaben

Previous work has demonstrated that the alkyl esters of p-hydroxybenzoic acid (parabens) possess oestrogenic activity, which increases with length of alkyl chain from methylparaben to n-butylparaben and with branching in the alkyl chain from n-butylparaben to isobutylparaben. This study reports on the oestrogenic activity of benzylparaben in a variety of assays in vitro and in vivo. Benzylparaben was able to displace [H-3]oestradiol from cytosolic oestrogen receptor (ER) of MCF7 human breast cancer cells by 22% at 1000-fold molar excess, by 40% at 10000-fold molar excess, by 57% at 100000-fold molar excess and by 100% at 1000000-fold molar excess. It was able to increase expression of a stably transfected oestrogen responsive reporter gene (ERE-CAT) in MCF7 cells after 24 h at 10(-5)M/10(-4)M and after 7 days at 10(-6)M/10(-5)M/10(-4)M. Proliferation of MCF7 cells could be increased by 10(-6)M/10(-5)M benzylparaben and this could be inhibited by 10(-7)M pure anti-oestrogen ICI 182,780, indicating that growth effects were ER mediated. Further evidence for ER-mediation was provided from the ability of benzylparaben to increase the growth of a second oestrogen-dependent human breast cancer cell line ZR-75-1, but not the oestrogen-insensitive NIDA-MB-231 cell line. When tested in the presence of 10(-10)M 17beta-oestradiol, benzylparaben gave no antagonist response on the growth of either MCF7 or ZR-75-1 cells. Finally, benzylparaben could increase uterine weight in the immature mouse following topical application of three daily doses of 33 mg to dorsal skin. These results demonstrate that the oestrogenicity of methylparaben can be increased by the addition of an aryl group as well as by lengthening or branching the alkyl grouping. Copyright (C) 2003 John Wiley Sons, Ltd.

(+)- and (-)-Mutisianthol: First Total Synthesis, Absolute Configuration, and Antitumor Activity

The first synthesis of the natural product (+)-mutisianthol was accomplished in 11 steps and in 21% overall yield from 2-methylanisole. The synthesis of its enantiomer was also performed in a similar overall yield. The absolute configuration of the sesquiterpene (+)-mutisianthol was assigned as (1S,3R). Key steps in the route are the asymmetric hydrogenation of a nonfunctionalized olefin using chiral iridium catalysts and the ring contraction of 1,2-dihydronaphthalenes using thallium(III) or iodine(III). The target molecules show moderate activity against the human tumor cell lines SF-295, HCT-8, and MDA-MB-435.

In vitro cytotoxic activity of Brazilian Middle West plant extracts

Cytotoxic activity of eight plant extracts, native from the Mid-West of Brazil comprising Cerrado, Pantanal and semideciduous forest, was evaluated for MDA-MB-435, SF-295, and HCT-8 cancer cell strains. A single 100 µg.mL-1 dose of each extract was employed with 72 h of incubation for all tests. Doxorubicin (1 µg.mL-1) was used as the positive control and the MTT method was used to detect the activity. Cytotoxicity of distinct polarities was observed in thirty extracts (46%), from different parts of the following species: Tabebuia heptaphylla (Vell.) Toledo, Bignoniaceae, Tapirira guianensis Aubl., Anacardiaceae, Myracrodruon urundeuva Allemão, Anacardiaceae, Schinus terebinthifolius Raddi, Anacardiaceae, Gomphrena elegans Mart., Amaranthaceae, Attalea phalerata Mart. ex Spreng., Arecaceae, Eugenia uniflora L., Myrtaceae, and Annona dioica A. St.-Hil., Annonaceae. Extracts of at least two tested cell strains were considered to be highly active since their inhibition rate was over 75%.

Cytotoxic Clerodane Diterpenes from Casearia rupestris

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Involvement of microRNAs in Androgen Receptor-dependent Breast Cancers

Triple negative breast cancer (TNBC) is a very aggressive tumor subtype characterized by the lack of expression of estrogen receptor 1 (ESR1), due in the most of cases to an increased expression of DNA methyltransferases (DNMTs) and hypermethylation in CpG islands, resulting in gene silencing. Furthermore, in ESR1- negative breast cancers, androgen receptor (AR) is highly expressed and some studies suggest that it can drive tumor progression and might represent a therapeutic target. A correlation between microRNAs, small non-coding RNAs that regulate gene expression, and DNMTs was investigated in a TNBC cell line to restore a normal methylation pattern of ESR1, leading to its re-expression and conferring again sensitivity to selective estrogen receptor modulators (SERMs). miR-148A and miR-29B were found to be involved in the reduction of the expression of DNMT1 and DNMT3A and in a slight increase of ESR1 expression, but not at protein level. Then, we found a down-regulation of AR by miRs-7, -9, -27a, -27b, -29a, -29b, -29c, -127-3p, -127-5p and -376 at 48h post transfection and an up-regulation by miR-15a and miR-16 at every time considered. We concomitantly investigated a possible increase of Tamoxifen, Herceptin and Metformin sensitivity after AR silencing in MDA-MB 453 and T-47D cell lines. Cells seemed more sensitive when silenced for AR only in MDA-MB-453 at 24h post Tamoxifen treatment. Studies on Metformin have basically confirmed an increase of drug sensitivity due to AR silencing in both cell lines. Analysis of Herceptin showed how MDA-MB 453 samples silenced for AR have a slight decrease in the percentage of proliferating cells, demonstrating a possible increase in the response to treatment. These preliminary data provide the basis for further study of the modulation of the expression of AR by microRNAs and it will be interesting to understand the molecular mechanisms underlying these interactions.

DEFINING THE ROLE OF EGFR ACETYLATION IN CELLULAR PROCESSES: CLINICAL IMPLICATIONS

Epidermal growth factor receptor (EGFR) is a cell membrane tyrosine kinase receptor and plays a pivotal role in regulating cell growth, differentiation, cell cycle, and tumorigenesis. Deregulation of EGFR causes many diseases including cancers. Intensive investigation of EGFR alteration in human cancers has led to profound progress in developing drugs to target EGFR-mediated cancers. While exploring possible synergistic enhancement of therapeutic efficacy by combining EGFR tyrosine kinase inhibitors (TKI) with other anti-cancer agents, we observed that suberoylanilide hydroxamic acid (SAHA, a deacetylase inhibitor) enhanced TKI-induced cancer cell death, which further led us to question whether SAHA-mediated sensitization to TKI was associated with EGFR acetylation. What we know so far is that SAHA can inhibit class I and II histone deacetylases (HDACs), which could possibly preserve acetylation of underlying HDAC-targeted proteins including both histone and non-histone proteins. In addition, it has been reported that an HDAC inhibitor, TSA, enhanced EGFR phosphorylation in ovarian cancer cells. EGFR acetylation has also been reported to play a role in the regulation of EGFR endocytosis recently. These observations indicate that there might be an intrinsic correlation between acetylation and phosphorylation of EGFR. In other words, the interplay between EGFR acetylation and phosphorylation may contribute to HDAC inhibitors (HDACi)-augmented EGFR phosphorylation. In this investigation, we showed that CBP acetyltransferase acetylated EGFR in vivo. In response to EGF stimulation, CBP rapidly translocated from the nucleus to the cytoplasm. We also demonstrated protein-protein interaction between CBP and EGFR as well as the enhancement of EGFR acetylation by CBP. Moreover, EGFR acetylation enhanced EGFR tyrosine phosphorylation and augmented its association with Src kinase. Acetylation-deficient EGFR mutant (EGFR-K3R) significantly reduced the function and activity of EGFR. Furthermore, ectopic expression of EGFR-K3R mutant abrogated its ability to respond to EGF-induced cell proliferation, DNA synthesis, and anchorage-independent growth using cell-based assays and tumor growth in nude mice. In addition, we demonstrated that EGFR expression was associated with SAHA resistance in the treatment of cancer cells that overexpress EGFR. The knockdown of EGFR in MDA-MB-468 breast cancer cells could sensitize the cells to respond to SAHA. The overexpression of EGFR in SAHA-sensitive MDA-MB-453 breast cancer cells rendered the cells resistant to SAHA. Together, these findings suggest that EGFR plays an important role in SAHA resistance in breast carcinoma cells that we tested. The combination therapy of HDACi with TKI has been proposed for treating cancers with aberrant expression of EGFR. The evidence from pre-clinical or clinical trials demonstrated significant enhancement of therapeutic efficacy by using such a combination therapy. Our in vivo study also demonstrated that the combination of SAHA and TKI for the treatment of breast cancer significantly reduced tumor burden compared with either SAHA or TKI alone. The significance of our study elucidated another possible underlying molecular mechanism by which HDACi mediated sensitization to TKI. Our results unveiled a critical role of EGFR acetylation that regulates EGFR tyrosine phosphorylation and may further provide an experiment-based rationale for combinatorial targeted therapy.

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

HRGbeta1 inhibits metastasis of breast cancer cell lines, one of which exhibits lineage infidelity

Recent publications have questioned the origin of the MDA-MB-435 breast cancer cell line and have suggested that it is of melanocyte origin rather than breast epithelial origin. The data presented herein show unequivocally that MDA-MB-435 does express breast epithelial markers and produces milk-specific lipids. The data also indicated that MDA-MB-435 does express some melanocyte proteins but this expression occurs in the same MDA-MB-435 cells that express breast epithelial proteins. Although MDA-MB-435 does not strictly adhere to a breast lineage, it does retain breast specific markers and is thus valid as an experimental cell line in breast cancer studies. ^ Heregulinβ1 (HRGβ1) has been shown to both stimulate and inhibit breast tumorigenic and metasastasic phenotypes. Some studies used only the EGF-like domain of the extracellular domain of HRGβ1 while others used bacterially-expressed HRGβ1. Our in vitro data demonstrated that the full-length extracellular domain of human HRGβ1 reduced clonal growth of MDA-MB-435 breast cancer cells but stimulated apoptosis in MDA-MB-435 and MCF-7 breast cancer cells. In addition, mammalian-expressed HRGβ1 did not dramatically affect matrix metalloproteinase-9 activity but did inhibit cell motility of MDA-MB-435 and MCF-7 cells. Taken together, the in vitro data indicated that HRGβ1 inhibits metastasis-associated properties. ^ The in vivo data demonstrated that inducible expression of the full-length extracellular domain of human HRGβ1 in MDA-MB-435 cells reduced tumor volume and cell proliferation but increased apoptosis of cells injected at the mammary fat pad in nude mice. More importantly, HRGβ1 reduced the number of metastases observed by a spontaneous metastasis assay. Taken together, these data indicate that the full-length extracellular domain of human HRGβ1 has the net effect of inhibiting breast cancer metastasis. ^

Tackling ErbB2: ErbB2-targeting peptide therapy and combination therapy with trastuzumab plus PI3K inhibitors

ErbB2 is an excellent target for cancer therapies because its overexpression was found in about 30% of breast cancers and correlated with poor prognosis of the patients. Unfortunately, current therapies for ErbB2-positive breast cancers remain unsatisfying due to side effects and resistance, and new therapies for ErbB2 overexpressing breast cancers are needed. Peptide/protein therapy using cell-penetrating peptides (CPPs) as carriers is promising because the internalization is highly efficient and the cargos can be bioactive. The major obstacle in using CPPs for therapy is their lack of specificity. We sought to develop a peptide carrier specifically introducing therapeutics to ErbB2-overexpressing breast cancer cells. By modifying the TAT-derived CPP, and attaching anti-HER2/neu peptide mimetic (AHNP), we developed the peptide carrier (P3-AHNP) specifically targeted ErbB2-overexpressing breast cancers in vitro and in vivo. A STAT3 SH2 domain-binding peptide conjugated to this peptide carrier (P3-AHNP-STAT3BP) was delivered preferentially into ErbB2-overexpressing breast cancer cells in vitro and in vivo. P3-AHNP-STAT3BP inhibited growth and induced apoptosis in vitro, with ErbB2-overexpressing 435.eB cells being more sensitive than the ErbB2-lowexpressing MDA-MB-435 cells. P3-AHNP-STAT3BP preferentially accumulated and inhibited growth in 435.eB xenografts, comparing with MDA-MB-435 xenografts or normal tissues with low levels of ErbB2. This ErbB2-targeting peptide delivery system provided the basis for future development of novel cancer target-specific treatments with low toxicity to normal cells. ^ Another urgent issue in treating ErbB2-positive breast cancers is trastuzumab resistance. Trastuzumab is the only FDA-approved ErbB2-targeting antibody for treatment of metastatic breast cancers overexpressing ErbB2, and has remarkable therapeutic efficacy in certain patients. The overall trastuzumab response rate, however, is limited, and understanding the mechanisms of trastuzumab resistance is needed to overcome this problem. We report that PTEN activation contributes to trastuzumab's anti-tumor activity. Trastuzumab treatment quickly inactivated Src, which reduced PTEN tyrosine phosphorylation, increased PTEN membrane localization and its phosphatase activity in cancer cells. Reducing PTEN expression in breast cancer cells by antisense oligonucleotides conferred trastuzumab resistance in vitro and in vivo. Importantly, PI3K inhibitors sensitized PTEN-deficient breast cancers to the growth inhibition by trastuzumab in vitro and in vivo, suggesting that combination therapies with PI3K inhibitors plus trastuzumab could overcome trastuzumab resistance. ^