Effects of small interfering RNA targeting thymidylate synthase on survival of ACC3 cells from salivary adenoid cystic carcinoma

<p>Abstract</p> <p>Background</p> <p>Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer and high expression of TS has been associated with poor prognosis or refractory disease in several cancers including colorectal and head and neck cancer. Although TS is known to regulate cell cycles and transcription factors, its potency as a therapeutic target has not been fully explored in adenoid cystic carcinoma (ACC).</p> <p>Methods</p> <p>An ACC cell line (ACC3) was transfected with siRNA targeting the TS gene and inhibition of cell growth and induction of apoptosis-associated molecules were evaluated <it>in vitro</it>. In addition, the <it>in vivo </it>effect of TS siRNA on tumor progression was assessed using a xenograft model.</p> <p>Results</p> <p>Our results demonstrated that ACC3 cells showed significantly higher TS expression than non-cancer cell lines and the induction of TS siRNA led to inhibition of cell proliferation. The effect was associated with an increase in p53, p21, and active caspase-3 and S-phase accumulation. We also found up-regulation of spermidine/spermine N1-acetyltransferase (SSAT), a polyamine metabolic enzyme. Furthermore, treatment with TS siRNA delivered by atelocollagen showed a significant cytostatic effect through the induction of apoptosis in a xenograft model.</p> <p>Conclusion</p> <p>TS may be an important therapeutic target and siRNA targeting TS may be of potential therapeutic value in ACC.</p

Evidence for Two Modes of Synergistic Induction of Apoptosis by Mapatumumab and Oxaliplatin in Combination with Hyperthermia in Human Colon Cancer Cells

Colorectal cancer is the third leading cause of cancer-related mortality in the world-- the main cause of death from colorectal cancer is hepatic metastases, which can be treated with isolated hepatic perfusion (IHP). Searching for the most clinically relevant approaches for treating colorectal metastatic disease by isolated hepatic perfusion (IHP), we developed the application of oxaliplatin concomitantly with hyperthermia and humanized death receptor 4 (DR4) antibody mapatumumab (Mapa), and investigated the molecular mechanisms of this multimodality treatment in human colon cancer cell lines CX-1 and HCT116 as well as human colon cancer stem cells Tu-12, Tu-21 and Tu-22. We showed here, in this study, that the synergistic effect of the multimodality treatment-induced apoptosis was caspase dependent and activated death signaling via both the extrinsic apoptotic pathway and the intrinsic pathway. Death signaling was activated by c-Jun N-terminal kinase (JNK) signaling which led to Bcl-xL phosphorylation at serine 62, decreasing the anti-apoptotic activity of Bcl-xL, which contributed to the intrinsic pathway. The downregulation of cellular FLICE inhibitory protein long isoform (c-FLIPL) in the extrinsic pathway was accomplished through ubiquitination at lysine residue (K) 195 and protein synthesis inhibition. Overexpression of c-FLIPL mutant (K195R) and Bcl-xL mutant (S62A) completely abrogated the synergistic effect. The successful outcome of this study supports the application of multimodality strategy to patients with colorectal hepatic metastases who fail to respond to standard chemoradiotherapy that predominantly targets the mitochondrial apoptotic pathway. © 2013 Song et al

Constitutive and Treatment-Induced CXCL8-Signalling Selectively Modulates the Efficacy of Anti-Metabolite Therapeutics in Metastatic Prostate Cancer

<div><h3>Background</h3><p>The current study was undertaken to characterize the effect of anti-metabolites on inducing CXCL8 signaling and determining whether the constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer (CaP) cells modulates their sensitivity to this class of agent.</p> <h3>Methods</h3><p>The response of metastatic CaP cells to 5-Fluorouracil (5-FU), Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to determine effects of drugs or CXCL8 administration on target gene/protein expression.</p> <h3>Results</h3><p>Administration of 5-FU but not pemetrexed potentiated CXCL8 secretion and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells. Consistent with this, the inhibition of CXCL8 signaling using a CXCR2 antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P<0.001), and increased 5-FU-induced apoptosis in PC3 cells (P<0.01). In contrast, while administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent experiments confirmed that administration of recombinant human CXCL8 increased TS expression, a response mediated in part by the CXCR2 receptor. Moreover, siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the sensitivity of PC3 cells to 5-FU.</p> <h3>Conclusions</h3><p>CXCL8 signaling provides a selective resistance of metastatic prostate cancer cells to specific anti-metabolites by promoting a target-associated resistance, in addition to underpinning an evasion of treatment-induced apoptosis.</p> </div

Pyrimidine biosynthesis is not an essential function for trypanosoma brucei bloodstream forms

&lt;p&gt;Background: African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite.&lt;/p&gt; &lt;p&gt;Methodology/Principal Findings: Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5−/− trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line.&lt;/p&gt; &lt;p&gt;Conclusions/Significance: Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.&lt;/p&gt

Evaluating predictive pharmacogenetic signatures of adverse events in colorectal cancer patients treated with fluoropyrimidines

The potential clinical utility of genetic markers associated with response to fluoropyrimidine treatment in colorectal cancer patients remains controversial despite extensive study. Our aim was to test the clinical validity of both novel and previously identified markers of adverse events in a broad clinical setting. We have conducted an observational pharmacogenetic study of early adverse events in a cohort study of 254 colorectal cancer patients treated with 5-fluorouracil or capecitabine. Sixteen variants of nine key folate (pharmacodynamic) and drug metabolising (pharmacokinetic) enzymes have been analysed as individual markers and/or signatures of markers. We found a significant association between TYMP S471L (rs11479) and early dose modifications and/or severe adverse events (adjusted OR = 2.02 [1.03; 4.00], p = 0.042, adjusted OR = 2.70 [1.23; 5.92], p = 0.01 respectively). There was also a significant association between these phenotypes and a signature of DPYD mutations (Adjusted OR = 3.96 [1.17; 13.33], p = 0.03, adjusted OR = 6.76 [1.99; 22.96], p = 0.002 respectively). We did not identify any significant associations between the individual candidate pharmacodynamic markers and toxicity. If a predictive test for early adverse events analysed the TYMP and DPYD variants as a signature, the sensitivity would be 45.5 %, with a positive predictive value of just 33.9 % and thus poor clinical validity. Most studies to date have been under-powered to consider multiple pharmacokinetic and pharmacodynamic variants simultaneously but this and similar individualised data sets could be pooled in meta-analyses to resolve uncertainties about the potential clinical utility of these markers

In-depth clinical and biological exploration of DNA Damage Immune Response (DDIR) as a biomarker for oxaliplatin use in colorectal cancer

PURPOSE: The DNA Damage Immune Response (DDIR) assay was developed in breast cancer (BC) based on biology associated with deficiencies in homologous recombination and Fanconi Anemia (HR/FA) pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and oesophageal cancers. In colorectal cancer (CRC) there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in CRC and characterised the biology in DDIR-positive CRC. METHODS: Samples and clinical data were assessed according to DDIR status from patients who received either 5FU or FOLFOX within the FOCUS trial (n=361, stage 4), or neo-adjuvant FOLFOX in the FOxTROT trial (n=97, stage 2/3). Whole transcriptome, mutation and immunohistochemistry data of these samples were used to interrogate the biology of DDIR in CRC. RESULTS: Contrary to our hypothesis, DDIR negative patients displayed a trend towards improved outcome for oxaliplatin-based chemotherapy compared to DDIR positive patients. DDIR positivity was associated with Microsatellite Instability (MSI) and Colorectal Molecular Subtype 1 (CMS1). Refinement of the DDIR signature, based on overlapping interferon-related chemokine signalling associated with DDIR positivity across CRC and BC cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in CRC. CONCLUSIONS: DDIR positivity does not predict improved response following oxaliplatin treatment in CRC. However, data presented here suggests the potential of the DDIR assay in identifying immune-rich tumours that may benefit from immune checkpoint blockade, beyond current use of MSI status

A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs

The quiescent cell population of tumours poses a barrier to the success of many cancer therapies. Most chemotherapeutic drugs target proliferating cells, but the growth fraction of many tumours is low. Based on the multicellular tumour spheroid model, a system was developed using human colon adenocarcinoma (DLD-1) cells to mimic the microenvironment of quiescent microregions of solid tumours. The quiescent tumour spheroids (TSQ) showed decreased expression of the proliferation marker Ki-67 and increased expression of the quiescence marker p27kip1 compared to proliferating spheroids (TSP). The quiescent status of the TSQ was confirmed by long-term growth assessment. The quiescence was completely reversible demonstrating that the TSQ retained the ability to proliferate and morphological assessment by light microscopy confirmed the absence of significant apoptosis. When the efficacy of widely used chemotherapeutic drugs was determined, vinblastine, doxorubicin, cisplatin and 5-fluorouracil (5-FU) all produced significant cell death in the TSP. However, while still effective, the potencies of doxorubicin and cisplatin were significantly reduced in TSQ. In contrast, 5-FU and vinblastine did not produce cell death in the TSQ. In summary, TSQ show considerable resistance to a panel of established chemotherapeutic agents and represent a useful model for evaluating the efficacy of drugs and other cancer therapies in quiescent tumours

Evidence That Aberrant Expression of Tissue Transglutaminase Promotes Stem Cell Characteristics in Mammary Epithelial Cells

Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population, but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44high/CD24low/- subpopulation, increased ability of cells to form mammospheres, and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells, which express high basal levels of TG2, shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together, these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells

Role of RecA and the SOS Response in Thymineless Death in Escherichia coli

Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans