Evaluation of the antitumor activity of NOV202, a novel microtubule targeting and vascular disrupting agent

Purpose: Overall, similar to 65% of patients diagnosed with advanced ovarian cancer (OC) will relapse after primary surgery and adjuvant first-line platinum-and taxane-based chemotherapy. Significant improvements in the treatment of OC are expected from the development of novel compounds having combined cytotoxic and antiangiogenic properties that make them effective on refractory tumors.Methods: Permeability of NOV202 was determined with Caco-2 monolayer assay. The compound's pharmacokinetic profile and plasma: brain distribution were assessed in male C57Bl/6 mice. The compound's impacts on tubulin, microtubules and cell cycle were investigated by using in vitro tubulin polymerization assay, cell-based immunofluorescence and live cell microscopy. The IC50 concentrations of NOV202 were assessed in a panel of eight cancer cell lines. Impact of the compound on vascular tube formation was determined using the StemKit and Chick chorioallantoic membrane assays. The in vivo efficacy of the compound was analyzed with an OC xenograft mouse model.Results: NOV202 was found to suppress cancer cell proliferation at low nanomolar concentrations (IC50 2.3-12.0 nM) and showed equal efficacy between OC cell line A2780 (IC50 2.4 nM) and its multidrug-resistant subline A2780/Adr (IC50 2.3 nM). Mechanistically, NOV202 targeted tubulin polymerization in vitro in a dose-dependent manner and in cells induced an M phase arrest. In vivo, NOV202 caused a dose-dependent reduction of tumor mass in an A2780 xenograft model, which at the highest dose (40 mg/kg) was comparable to the effect of paclitaxel (24 mg/kg). Interestingly, NOV202 exhibited vascular disrupting properties that were similar to the effects of Combretastatin A4.Conclusion: NOV202 is a novel tubulin and vascular targeting agent that shows strong anticancer efficacy in cells and OC xenograft models. The finding that the compound induced significantly more cell death in Pgp/MDR1 overexpressing OC cells compared to vincristine and paclitaxel warrants further development of the compound as a new therapy for OC patients with treatment refractory tumors and/or relapsing disease

Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library

<p>Abstract</p> <p>Background</p> <p>Casein kinase 2 (CK2) is dysregulated in various human cancers and is a promising target for cancer therapy. To date, there is no small molecular CK2 inhibitor in clinical trial yet. With the aim to identify novel CK2 inhibitors, we screened a natural product library.</p> <p>Methods</p> <p>We adopted cell-based proliferation and CK2 kinase assays to screen CK2 inhibitors from a natural compound library. Dose-dependent response of CK2 inhibitors <it>in vitro </it>was determined by a radioisotope kinase assay. Western blot analysis was used to evaluate down stream Akt phosphorylation and apoptosis. Apoptosis was also evaluated by annexin-V/propidium iodide (PI) labeling method using flow cytometry. Inhibition effects of CK2 inhibitors on the growth of cancer and normal cells were evaluated by cell proliferation and viability assays.</p> <p>Results</p> <p>Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases. It appears to be an ATP non-competitive and partially reversible CK2 inhibitor with an IC₅₀value of 0.55 μM. In addition, hematein inhibited cancer cell growth partially through down-regulation of Akt phosphorylation and induced apoptosis in these cells. Furthermore, hematein exerted stronger inhibition effects on the growth of cancer cells than in normal cells.</p> <p>Conclusion</p> <p>In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor. Hematein showed stronger growth inhibition effects to cancer cells when compared to normal cells. This compound may represent a promising class of CK2 inhibitors.</p

STAT1-dependent expression of energy metabolic pathways links tumour growth and radioresistance to the Warburg effect

<p>Abstract</p> <p>Background</p> <p>The Signal Transducer and Activator of Transcription 1 (STAT1) has traditionally been regarded as a transmitter of interferon signaling and a pro-apoptotic tumour suppressor. Recent data have identified new functions of STAT1 associated with tumourigenesis and resistance to genotoxic stress, including ionizing radiation (IR) and chemotherapy. To investigate the mechanisms contributing to the tumourigenic functions of STAT1, we performed a combined transcriptomic-proteomic expressional analysis and found that STAT1 is associated with regulation of energy metabolism with potential implication in the Warburg effect.</p> <p>Methods</p> <p>We generated a stable knockdown of STAT1 in the SCC61 human squamous cell carcinoma cell line, established tumour xenografts in athymic mice, and compared transcriptomic and proteomic profiles of STAT1 wild-type (WT) and knockdown (KD) untreated or irradiated (IR) tumours. Transcriptional profiling was based on Affymetrix Human GeneChip^®Gene 1.0 ST microarrays. Proteomes were determined from the tandem mass spectrometry (MS/MS) data by searching against the human subset of the UniProt database. Data were analysed using Significance Analysis of Microarrays for ribonucleic acid and Visualize software for proteins. Functional analysis was performed with Ingenuity Pathway Analysis with statistical significance measured by Fisher's exact test.</p> <p>Results</p> <p>Knockdown of STAT1 led to significant growth suppression in untreated tumours and radio sensitization of irradiated tumours. These changes were accompanied by alterations in the expression of genes and proteins of glycolysis/gluconeogenesis (GG), the citrate cycle (CC) and oxidative phosphorylation (OP). Of these pathways, GG had the most concordant changes in gene and protein expression and demonstrated a STAT1-dependent expression of genes and proteins consistent with tumour-specific glycolysis. In addition, IR drastically suppressed the GG pathway in STAT1 KD tumours without significant change in STAT1 WT tumours.</p> <p>Conclusion</p> <p>Our results identify a previously uncharacterized function of STAT1 in tumours: expressional regulation of genes encoding proteins involved in glycolysis, the citrate cycle and mitochondrial oxidative phosphorylation, with predominant regulation of glycolytic genes. STAT1-dependent expressional regulation of glycolysis suggests a potential role for STAT1 as a transcriptional modulator of genes responsible for the Warburg effect.</p

Identification of molecular mechanisms for cellular drug resistance by combining drug activity and gene expression profiles

Acquired drug resistance is a major problem in cancer treatment. To explore the genes involved in chemosensitivity and resistance, 10 human tumour cell lines, including parental cells and resistant subtypes selected for resistance against doxorubicin, melphalan, teniposide and vincristine, were profiled for mRNA expression of 7400 genes using cDNA microarray technology. The drug activity of 66 cancer agents was evaluated on the cell lines, and correlations between drug activity and gene expression were calculated and ranked. Hierarchical clustering of drugs based on their drug–gene correlations yielded clusters of drugs with similar mechanism of action. Genes correlated with drug sensitivity and resistance were imported into the PathwayAssist software to identify putative molecular pathways involved. A substantial number of both proapoptotic and antiapoptotic genes such as signal transducer and activator of transcription 1, mitogen-activated protein kinase 1 and focal adhesion kinase were found to be associated to drug resistance, whereas genes linked to cell cycle control and proliferation, such as cell division cycle 25A and signal transducer of activator of transcription 5A, were associated to general drug sensitivity. The results indicate that combined information from drug activity and gene expression in a resistance-based cell line panel may provide new knowledge of the genes involved in anticancer drug resistance and become a useful tool in drug development

Evaluation of the antitumor activity of NOV202, a novel microtubule targeting and vascular disrupting agent

Linda Rickardson,1 Emma Kutvonen,2 Satu Orasniemi,2 Marita H&ouml;gberg,1 Marko J Kallio,2,3 Stefan Rehnmark1 1Noviga Research AB, S&ouml;dert&auml;lje, Sweden; 2Department of Physiology, Institute of Biomedicine, University of Turku, 3Centre for Biotechnology, University of Turku, Turku, Finland Purpose: Overall, ~65% of patients diagnosed with advanced ovarian cancer (OC) will relapse after primary surgery and adjuvant first-line platinum- and taxane-based chemotherapy. Significant improvements in the treatment of OC are expected from the development of novel compounds having combined cytotoxic and antiangiogenic properties that make them effective on refractory tumors. Methods: Permeability of NOV202 was determined with Caco-2 monolayer assay. The compound&rsquo;s pharmacokinetic profile and plasma:brain distribution were assessed in male C57Bl/6 mice. The compound&rsquo;s impacts on tubulin, microtubules and cell cycle were investigated by using in&nbsp;vitro tubulin polymerization assay, cell-based immunofluorescence and live cell microscopy. The IC50 concentrations of NOV202 were assessed in a panel of eight cancer cell lines. Impact of the compound on vascular tube formation was determined using the StemKit and Chick chorioallantoic membrane assays. The in vivo efficacy of the compound was analyzed with an OC xenograft mouse model. Results: NOV202 was found to suppress cancer cell proliferation at low nanomolar concentrations (IC50 2.3&ndash;12.0 nM) and showed equal efficacy between OC cell line A2780 (IC50 2.4&nbsp;nM) and its multidrug-resistant subline A2780/Adr (IC50 2.3 nM). Mechanistically, NOV202 targeted tubulin polymerization in vitro in a dose-dependent manner and in cells induced an M phase arrest. In vivo, NOV202 caused a dose-dependent reduction of tumor mass in an A2780 xenograft model, which at the highest dose (40 mg/kg) was comparable to the effect of paclitaxel (24 mg/kg). Interestingly, NOV202 exhibited vascular disrupting properties that were similar to the effects of Combretastatin A4. Conclusion: NOV202 is a novel tubulin and vascular targeting agent that shows strong anticancer efficacy in cells and OC xenograft models. The finding that the compound induced significantly more cell death in Pgp/MDR1 overexpressing OC cells compared to vincristine and paclitaxel warrants further development of the compound as a new therapy for OC patients with treatment refractory tumors and/or relapsing disease. Keywords: ovarian cancer, NOV202, microtubule-targeting agent, vascular disruption, M&nbsp;phase arrest, multidrug resistanc

In Vitro Drug Sensitivity-Gene Expression Correlations Involve a Tissue of Origin Dependency

A major concern of chemogenomics is to associate drug activity with biological variables. Several reports have clustered cell line drug activity profiles as well as drug activity-gene expression correlation profiles and noted that the resulting groupings differ but still reflect mechanism of action. The present paper shows that these discrepancies can be viewed as a weighting of drug-drug distances, the weights depending on which cell lines the two drugs differ in. 1

Phenotype-based drug screening in primary ovarian carcinoma cultures identifies intracellular iron depletion as a promising strategy for cancer treatment

Primary cultures of patient tumor cells (PCPTC) have been used for prediction of diagnosis-specific activity and individual patient response to anticancer drugs, but have not been utilized as a model for identification of novel drugs in high throughput screening. In the present study, ovarian carcinoma cells from three patients were tested in response to a library of 3000 chemically diverse compounds. Eight hits were retrieved after counter screening using normal epithelial cells, and one of the two structurally related hit compounds was selected for further preclinical evaluation. This compound, designated VLX 50, demonstrated a broad spectrum of activity when tested in a panel of PCPTCs representing different forms of leukemia and solid tumors and displayed a high tumor to normal cell activity. VLX 50 induced delayed cell death with some features of classical apoptosis. Significant in vivo activity was confirmed on primary cultures of human ovarian carcinoma cells in mice using the hollow fiber model. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. This query signature was analyzed using the Gene Set Enrichment Analysis and the Connectivity Map database. Strong connections to hypoxia inducible factor 1 and iron chelators were retrieved. The mechanistic hypothesis of intracellular iron depletion leading to hypoxia signaling was confirmed by a series of experiments. The results indicate the feasibility of using PCPTC for cancer drug screening and that intracellular iron depletion could be a potentially important strategy for cancer therapy

Characterization of the cytotoxic activity of the indoloquinoline alkaloid cryptolepine in human tumour cell lines and primary cultures of tumour cells from patients.

noThe plant derived indoloquinoline alkaloid cryptolepine was investigated for its cytotoxic properties in 12 human tumour cell lines and in primary cultures of tumour cells from patients. The fluorometric microculture cytotoxicity assay was used to assess cytotoxicity and DNA mocro-array analysis to evaluate gene expression. Cryptolepine mean IC50 in the cell line panel was 0.9 microM compared with 1.0 and 2.8 microM in haemaotological and solid tumour malignancies respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as sensitive as haematological malignancies, respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as senstive as haematological malignancies. Cryptolepine activity showed highest correlations to topoisomerase II and microtubule targetting drugs. In the cell lines cryptolepine activity was essentially unaffected by established mechanisms of drug resistance. A number of genes were identified as associated with cryptolepine activity. In conclusion, cryptolepine shows interesting in vitro cytotoxic properties and its further evaluation as an anticancer drug seems warranted