Synergistic Anticancer Effects of the 9.2.27PE Immunotoxin and ABT-737 in Melanoma

In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE) - based immunotoxin, the 9.2.27PE, and the BH-3 mimetic compound ABT-737 in a panel of melanoma cell lines. The drug combination resulted in synergistic cytotoxicity, and the cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose) polymerase (PARP) and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. We further show that ABT-737 caused endoplasmic reticulum (ER) stress with increased GRP78 and phosphorylated eIF2α protein levels. Moreover, treatment with ABT-737 increased the intracellular calcium levels, an effect which was enhanced by 9.2.27PE, which as a single entity drug had minimal effect on calcium release from the ER. In addition, silencing of Mcl-1 by short hairpin RNA (shRNA) enhanced the intracellular calcium levels and cytotoxicity caused by ABT-737. Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model, supporting further investigations of this particular drug combination

Assessing conventionalized language in English learner essays by applying a method of "warming up" in Swedish L1

The aim of this study is to look at the use of formulaic language, i.e. memorized and conventionalized combinations of words, in essays written by Swedish intermediate level students of English. Drawing on previous research (Cohen and Brooks -Carson 2001) this study will apply a method of “warming up” in Swedish (L1) before writing in English (L2). The primary material includes thirty essays written directly in English, and thirty essays starting from an outline in Swedish and then written in English, by the same students but on a different topic. Since there is evidence that native speakers always score the highest on amount of formulaic language in written discourse, the main hypothesis of this study is that by starting from an outline in Swedish and then writing in English the students will make use of more formulaic language than when writing directly in English. The first research question involves the quantity of formulaic language in the students’ essays. The second question concerns the distribution and use of the different categories of FSs (NPs, VPs etc) in the direct and indirect modes of writing. The last question addresses the erroneous attempts made by the students in forming FSs and the possible influence of transfer from L1 in the essays starting from an outline in Swedish compared to the essays written directly in English. The findings of this study lend support to the main hypothesis. Moreover, the analysis shows that there are significant differences in the distribution and use of the different categories of formulaic language in the direct and indirect modes of writing. Results are suggestive of a more conscious and less uncertain approach in the essays starting from an outline in Swedish, however the findings may be circumstantial. Furthermore, the results indicate that the method of “warming up” in Swedish may encourage the use of combinations of words that are more conventionalized and do not always interact with syntax in a regular way. The findings also indicate that transfer is not more salient in the essays starting from an outline in Swedish if compared to the essays written directly in English, at least not in relation to the erroneous FSs found in the students’ essays

Pro-survival responses to the dual inhibition of anti-apoptotic Bcl-2 family proteins and mTOR-mediated signaling in hypoxic colorectal carcinoma cells

Background The use of targeted agents to impel dual inhibition of anti-apoptotic mechanisms and mTOR-mediated pro-survival signaling in colorectal carcinoma (CRC) cell lines with KRAS or BRAF mutation has been shown to induce apoptosis, a timely result given CRC entities harboring such mutations are in need of new therapies. Since CRC comprises heterogeneous tumors with predominant hypoxic components, we investigated effects of an inhibitor of anti-apoptotic Bcl-2 family proteins (ABT-737) in combination with an mTOR inhibitor (AZD8055)—collectively referred to as combo-Rx, in hypoxic CRC cell lines. Methods Cell viability measures, expression of proteins implicated in apoptosis and MAPK/PI3K-AKT/mTOR pathway signaling, and profiling of composite kinase activities were undertaken in a panel of 14 cell lines. Results In hypoxic conditions, combo-Rx suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. Hypoxic KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis but with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. In contrast, in hypoxic KRAS-mutant LoVo (devoid of PIK3CA mutation), BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines (all with high endogenous Mcl-1 expression and being resistant to the additional effect of ABT-737 to AZD8055), combo-Rx did not elicit apoptotic or pro-survival responses. Conclusions The concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic KRAS/PIK3CA-mutant CRC cell lines resulted in pro-survival responses in parallel with the intended anti-proliferative effects, a finding that should be of note if considering combinatory targeting of multiple pathways in this CRC entity

An experimental strategy unveiling exosomal microRNAs 486-5p, 181a-5p and 30d-5p from hypoxic tumour cells as circulating indicators of high-risk rectal cancer

Tumour hypoxia contributes to poor treatment outcome in locally advanced rectal cancer (LARC) and circulating extracellular vesicles (EVs) as potential biomarkers of tumour hypoxia and adverse prognosis have not been fully explored. We examined EV miRNAs from hypoxic colorectal cancer cell lines as template for relevant miRNAs in LARC patients participating in a prospective biomarker study (NCT01816607). Five cell lines were cultured under normoxia (21% O2) or hypoxia (0.2% O2) for 24 h, and exosomes were isolated by differential ultracentrifugation. Using a commercial kit, exosomes were precipitated from 24 patient plasma samples collected at the time of diagnosis. Exosome size distribution and protein cargo were determined by cryo-electron microscopy, nanoparticle tracking analysis, immunoblotting and flow cytometry. The vesicles harboured strong cell line-specific miRNA profiles with 35 unique miRNAs differentially expressed between hypoxic and normoxic cells. Six of these miRNAs were considered candidate-circulating markers of tumour hypoxia in the patients based on the frequency or magnitude of variance in hypoxic versus normoxic cell line experiments and prevalence in patient plasma. Of these, low plasma levels of exosomal miR-486-5p and miR-181a-5p were associated with organ-invasive primary tumour (p = 0.029) and lymph node metastases (p = 0.024), respectively, both attributes of adverse LARC prognosis. In line with this, the plasma level of exosomal miR-30d-5p was elevated in patients who experienced metastatic progression (p = 0.036). Our strategy confirmed that EVs from colorectal cancer cell lines were exosomes containing the oxygen-sensitive miRNAs 486-5p, 181a-5p and 30d-5p, which were retrieved as circulating markers of high-risk LARC

Additional file 4: Table S4. of Pro-survival responses to the dual inhibition of anti-apoptotic Bcl-2 family proteins and mTOR-mediated signaling in hypoxic colorectal carcinoma cells

Tyrosine Kinase PamChip® Array substrates—phosphorylation levels. The color map visualizes normalized log2-transformed signal intensities from kinase substrate arrays incubated with lysates from the three colorectal carcinoma cell lines treated for 24 h with ABT-737 (inhibitor of anti-apoptotic Bcl-2 family proteins; 10 μM), AZD8055 (mTOR inhibitor; 10 μM), or combo-Rx (10 μM of both compounds in combination). Red corresponds to higher and blue to lower substrate phosphorylation levels relative to levels from the corresponding control cells. a Retrieved from UniProtKB/SwissProt ( http://www.uniprot.org/ ). b Position(s) of the tyrosine phosphorylation site(s) within the protein. c Retrieved from PathCard ( http://pathcards.genecards.org/ ). Super-pathway definitions were ‘PI3K-AKT signaling pathway’ and ‘MAPK signaling pathway’. (DOC 536 kb

Additional file 1: Fig. S1. of Pro-survival responses to the dual inhibition of anti-apoptotic Bcl-2 family proteins and mTOR-mediated signaling in hypoxic colorectal carcinoma cells

Cell viability of hypoxic colorectal carcinoma (CRC) cell lines. a. Four CRC cell lines were treated for 24 h with the indicated concentrations of ABT-737 (inhibitor of anti-apoptotic Bcl-2 family proteins) or AZD8055 (mTOR inhibitor) under normoxic or hypoxic culture conditions. Cell viability (measured by the MTS assay) value for each condition relative to the corresponding control cell (vehicle-treated) value is shown as mean ± SD from at least three independent experiments, each plated at least in triplicate. Statistically significant changes are indicated (asterisk, p < 0.05; cross, p < 0.01; circle, p < 0.001). b. Cultures of the CRC cell line were left in the hypoxic chamber at 0.2 % O2 and harvested after the indicated time periods. Expression of the hypoxia-inducible factor type 1α (HIF-1α) and its target gene carbonic anhydrase IX (CAIX) was examined by Western blot analysis, with α-tubulin expression as loading control, and with 24 h of normoxia and 4 h of 100 μM CoCl2 exposure in normoxia to generate negative and positive biological controls for HIF-1α expression, respectively. (DOCX 369 kb

Induction of Apoptosis in Intestinal Toxicity to a Histone Deacetylase Inhibitor in a Phase I Study with Pelvic Radiotherapy

Purpose When integrating molecularly targeted compounds in radiotherapy, synergistic effects of the systemic agent and radiation may extend the limits of patient tolerance, increasing the demand for understanding the pathophysiological mechanisms of treatment toxicity. In this Pelvic Radiation and Vorinostat (PRAVO) study, we investigated mechanisms of adverse effects in response to the histone deacetylase (HDAC) inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) when administered as a potential radiosensitiser. Materials and Methods This phase I study for advanced gastrointestinal carcinoma was conducted in sequential patient cohorts exposed to escalating doses of vorinostat combined with standard-fractionated palliative radiotherapy to pelvic target volumes. Gene expression microarray analysis of the study patient peripheral blood mononuclear cells (PBMC) was followed by functional validation in cultured cell lines and mice treated with SAHA. Results PBMC transcriptional responses to vorinostat, including induction of apoptosis, were confined to the patient cohort reporting dose-limiting intestinal toxicities. At relevant SAHA concentrations, apoptotic features (annexin V staining and caspase 3/7 activation, but not poly-(ADP-ribose)-polymerase cleavage) were observed in cultured intestinal epithelial cells. Moreover, SAHA-treated mice displayed significant weight loss. Conclusion The PRAVO study design implemented a strategy to explore treatment toxicity caused by an HDAC inhibitor when combined with radiotherapy and enabled the identification of apoptosis as a potential mechanism responsible for the dose-limiting effects of vorinostat. To the best of our knowledge, this is the first report deciphering mechanisms of normal tissue adverse effects in response to an HDAC inhibitor within a combined-modality treatment regimen

Differential Inhibition of <i>Ex-Vivo</i> Tumor Kinase Activity by Vemurafenib in <i>BRAF</i>(V600E) and <i>BRAF</i> Wild-Type Metastatic Malignant Melanoma

<div><p>Background</p><p>Treatment of metastatic malignant melanoma patients harboring <i>BRAF</i>(V600E) has improved drastically after the discovery of the <i>BRAF</i> inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring <i>BRAF</i> wild-type. Better markers for targeted therapy are therefore urgently needed.</p><p>Methodology</p><p>In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall <i>ex-vivo</i> inhibitory effects of vemurafenib and sunitinib on kinase activity status.</p><p>Results</p><p>Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, <i>ex-vivo</i> incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in <i>BRAF</i> wild-type and <i>BRAF</i>(V600E) tumors, analysis with <i>ex-vivo</i> vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in <i>BRAF</i>(V600E) tumor lysates, distinguishing the <i>BRAF</i> wild-type and <i>BRAF</i>(V600E) tumors. Interestingly, a few <i>BRAF</i> wild-type tumors showed inhibition profiles similar to <i>BRAF</i>(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different <i>BRAF</i> mutational status with various vemurafenib sensitivity <i>in-vitro</i>.</p><p>Conclusions</p><p>Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.</p></div