Arrest Of Tumor Cells Research Articles

Synthesis and antitumor activity of novel 3,4-diaryl squaric acid analogs

A series of novel 3,4-diaryl squaric acid analogs 4a–r related to combretastatin A-4 (CA4) using squaric acid as the cis-restricted linker were prepared and studied for their anticancer activity against selected human cancer cell lines. New compounds 4g, 4k, 4m, 4n, 4p, 4q and 4r exhibit strong activities against human leukemia cells with IC50 values of ≤20 nM and compounds 4k, 4n, 4p, 4q and 4r showed potent activities against a panel of human tumor cell lines. Compounds 4n and 4p arrest tumor cell cycle in G2-M phase. Computational modeling analysis suggests that the binding mechanism of compound 4n to the colchicine binding site on the microtubules is similar to that of CA4.

β-Ionone Induces Cell Cycle Arrest and Apoptosis in Human Prostate Tumor Cells

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is the rate-limiting activity in the mevalonate pathway that provides essential intermediates for posttranslational modification of growth-associated proteins. Assorted dietary isoprenoids found in plant foods suppress HMG CoA reductase and have cancer chemopreventive activity. β-Ionone, a cyclic sesquiterpene and an end-ring analog of β-carotene, induced concentration-dependent inhibition of the proliferation of human DU145 (IC50 = 210 μmol/L) and LNCaP (IC50 = 130 μmol/L) prostate carcinoma cells and PC-3 prostate adenocarcinoma cells (IC50 = 130 μmol/L). Concomitantly, β-ionone-induced apoptosis and cell cycle arrest at the G1 phase in DU145 and PC-3 cells were shown by fluorescence microscopy, flow cytometry, and TUNEL reaction, and downregulation of cyclin-dependent kinase 4 (Cdk4) and cyclin D1 proteins. Growth suppression was accompanied by β-ionone-induced downregulation of reductase protein. A blend of β-ionone (150 μmol/L) and trans, trans-farnesol (25 μmol/L), an acyclic sesquiterpene that putatively initiates the degradation of reductase, suppressed the net growth of DU145 cells by 73%, an impact exceeding the sum of those of β-ionone (36%) and farnesol (22%), suggesting a synergistic effect. β-ionone, individually or in combination with other HMG CoA reductase suppressors, may have potential in prostate cancer chemoprevention and/or therapy.

Knockdown of interferon-induced transmembrane protein 3 expression suppresses breast cancer cell growth and colony formation and affects the cell cycle

Interferon-induced transmembrane protein 3 (IFITM3) is an important anti-virus protein and has been recently shown to play a role in human cancer development. Thus, the present study aimed to assess the expression of the IFITM3 protein in breast cancer tissues and to investigate the in vitro effects of IFITM3 knockdown in the regulation of breast cancer cell growth and cell cycle distributions. A total of 64 patients of breast cancer and the matched normal tissue specimens were obtained for immunohistochemical analysis of IFITM3 expression. Lentivirus-carrying IFITM3 shRNA was used to knock down IFITM3 expression in breast cancer cell lines. Phenotypic changes of cell viability, growth, colony formation and cell cycle distribution was also assayed using flow cytometry, MTT, BrdU incorporation and colony formation assays. The IFITM3 protein was highly expressed in invasive breast cancer compared to normal tissues and was significantly associated with estrogen receptor and progesterone receptor status. The lentivirus-carried IFITM3 shRNA significantly reduced the expression of IFITM3 mRNA and protein in breast cancer cells, inhibiting tumor cell viability, growth and colony formation, arrested tumor cells at the G0/G1 phase of the cell cycle and reduced the number of cells in the S phase of the cell cycle. Expression of IFITM3 protein could be a potential therapeutic target in future treatment of breast cancer.

Abstract 3326: TRAIL induces apoptosis preferentially in cell cycle arrested tumor cells, e.g., in tumor cells from children with acute lymphoblastic leukemia upon knockdown of cyclinE.

Abstract Resting tumor cells represent a huge challenge during anti-cancer therapy due to their increased treatment resistance. Example given, during minimal residual disease remaining tumor cells might be non-cycling. TNF-related apoptosis-inducing ligand (TRAIL) is a putative future anti-cancer drug currently in phase I and II clinical studies. We recently showed that TRAIL is able to target leukemia stem cell surrogates (Blood 2012,119,4224). Here we tested the ability of TRAIL to target cell cycle arrested tumor cells. Primary tumor cells from children with acute lymphoblastic leukemia were amplified in immuno-compromised mice and used for in vitro knockdown studies, besides tumor cell lines. Cell cycle arrest was induced in G0, G1 or G2 using cytotoxic drugs, phase-specific inhibitors or RNA interference against cyclinB and E. Cell cycle was visualized by flowcytometry analysis including phosphorylation of Serine 10 in Histone H3 to discriminate G2 from M phase. Biochemical or molecular arrest at any point of the cell cycle increased TRAIL-induced apoptosis, independently from the tumor cell type, the cell cycle inhibitor used or the check point addressed. In contrast, when cell cycle arrest was disabled by addition of caffeine, the anti-tumor activity of TRAIL was reduced. Most importantly for clinical translation, tumor cells from three children with B-precursor or T-cell acute lymphoblastic leukemia showed increased TRAIL-induced apoptosis upon knockdown of either cyclinB or cyclinE arresting the cell cycle in G2 or G1, respectively. Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts enhanced anti-tumor activity against cell cycle arrested tumor cells. Therefore, TRAIL might represent an interesting drug to treat static tumor disease, e.g., during minimal residual disease. Citation Format: Harald Ehrhardt, Franziska Wachter, Michaela Grunert, Irmela Jeremias. TRAIL induces apoptosis preferentially in cell cycle arrested tumor cells, e.g., in tumor cells from children with acute lymphoblastic leukemia upon knockdown of cyclinE. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3326. doi:10.1158/1538-7445.AM2013-3326 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

Local bystander effect induces dormancy in human medullary thyroid carcinoma model in vivo

The extent of local bystander effect induced by fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD) in combination with 5-fluorocytosine (5FC) was evaluated in xenogeneic model of human medullary thyroid carcinoma (MTC). This approach to gene-directed enzyme/prodrug therapy (GDEPT) induces strong bystander cytotoxicity. Effector yCD-TT mixed with target EGFP-TT cells in a ratio 2:9 could achieve significant tumor regression and 14-fold decrease in serum marker calcitonin upon 5FC administration. Histopathological analysis unraveled that antitumor effect resulted in tumor dormancy and proliferation arrest of remaining tumor cell clusters in vivo. yCD/5FC combination represents another GDEPT approach to achieve tumor growth control in MTC.

DFMO/eflornithine inhibits migration and invasion downstream of MYCN and involves p27Kip1 activity in neuroblastoma

Neuroblastoma (NB) is the most common extracranial pediatric tumor. NB patients over 18 months of age at the time of diagnosis are often in the later stages of the disease, present with widespread dissemination, and often possess MYCN tumor gene amplification. MYCN is a transcription factor that regulates the expression of a number of genes including ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Inhibiting ODC in NB cells produces many deleterious effects including G1 cell cycle arrest, inhibition of cell proliferation, and decreased tumor growth, making ODC a promising target for drug interference. DFMO treatment leads to the accumulation of the cyclin-dependent kinase inhibitor p27Kip1 protein and causes p27Kip1/Rb-coupled G1 cell cycle arrest in MYCN-amplified NB tumor cells through a process that involves p27Kip1 phosphorylation at residues Ser10 and Thr198. While p27Kip1 is well known for its role as a cyclin-dependent kinase inhibitor, recent studies have revealed a novel function of p27Kip1 as a regulator of cell migration and invasion. In the present study we found that p27Kip1 regulates the migration and invasion in NB and that these events are dependent on the state of phosphorylation of p27Kip1. DFMO treatments induced MYCN protein downregulation and phosphorylation of Akt/PKB (Ser473) and GSK3-β (Ser9), and polyamine supplementation alleviated the DFMO-induced effects. Importantly, we provide strong evidence that p27Kip1 mRNA correlates with clinical features and the survival probability of NB patients.

Effect of annatto-tocotrienols supplementation on the development of mammary tumors in HER-2/neu transgenic mice

Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% δ-T3 and 10% γ-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified δ-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.

Neo-endocrinochemotherapy: A novel approach for enhancing chemotherapeutic efficacy in clinic?

Chemosensitization means enhancing the sensitivity of tumor cells to chemotherapy with certain methods for better efficacy. Tumor progression depends on stimulation of multiple hormones, decrease in hormones during chemotherapy induces G0/G1 arrest of tumor cells, which may be the main cause for chemoresistance. Some of the choriocarcinoma and testicular tumors are curable with chemotherapy only, underlying mechanism may refer high level of human chorionic gonadotropin, which has thyroid stimulating hormone like effect and even induce hyperthyroidism in these patients. Some of these patients usually have high levels of thyroid hormones or suffer thyroid crisis during chemotherapy. Possibly owning to the proliferative or metabolic promotion effect of thyroid hormones and/or other endocrine hormones, which can promote tumor cells in G0 phase metabolizing actively or stepping into division cycle again, tumors are more sensitive to chemotherapy. Both endocrinotherapy and chemotherapy are major therapies for tumor, traditional endocrinotherapy suppresses tumor progression through decreasing tumor-dependent hormones or competitively combining and blocking hormone receptors. While, chemotherapy mostly killed tumor cells that proliferate actively, because tumor cells retardant in G0 phase by endocrinotherapy are no more sensitive to chemotherapy, endocrinotherapy cannot be concurrently used with chemotherapy. Nevertheless, decrease in hormones during chemotherapy is similar to concurrently using endocrinotherapy and chemotherapy. It has been found in some basic researches that some chemotherapeutics concurrently combined with endocrine hormones can achieve better efficacy compared with chemotherapy only. It is therefore hypothesized in this article that decrease in hormones during chemotherapy causes cell cycle arrest and renders tumor cells insensitive to chemotherapy; addition of endocrine hormones to mimic the hormones and microenvironment changes during chemotherapy for patients with choriocarcinoma or testicular tumor-curable with chemotherapy only, will rescue tumor cells from cell cycle arrest rendering them sensitive to chemotherapy. This new combinative therapy of concurrently using endocrine hormones and chemotherapy is defined as choriocarcinoma-mimic chemotherapy or neo-endocrinochemotherapy to distinguish the routine term of endocrinochemotherapy and is expected to be a novel approach to enhance chemotherapeutic efficacy in clinic.

The Initial Hours of Metastasis: The Importance of Cooperative Host–Tumor Cell Interactions during Hematogenous Dissemination

Tumor cells transit from the primary tumor via the blood circulation to form metastases in distant organs. During this process, tumor cells encounter a number of environmental challenges and stimuli that profoundly impact their metastatic potential. Here, we review the cooperative and dynamic host-tumor cell interactions that support and promote the hematogenous dissemination of cancer cells to sites of distant metastasis. In particular, we discuss what is known about the cross-talk occurring among tumor cells, platelets, leukocytes, and endothelial cells and how these cell-cell interactions are organized both temporally and spatially at sites of extravasation and in the early metastatic niche. Metastasis is a function not only of tumor cells but also involves cooperative interactions of those cells with normal cells of the body, in particular platelets and leukocytes. These other cell types alter the behavior of the tumor cells themselves and of endothelial cells lining the vasculature and assist in tumor cell arrest and extravasation at sites of metastasis and subsequently in the establishment of tumor cells in the early metastatic niche. A better understanding of the important role that these contact and paracrine interactions play during metastasis will offer new opportunities for therapeutic intervention.

Expression of the Nuclear Corepressor Ski Predicts Response to Histone Deacetylase Inhibitor Valproic Acid in Cells of Acute Myeloid Leukemia.

AbstractAbstract 2410Acute myeloid leukemia (AML) with deletion of chromosome 7 (−7) or 7q (del7q) has a poor prognosis. Using gene expression analysis, we previously identified the nuclear oncogene Ski as being up-regulated in AML, especially in AML with −7/del7q. We demonstrated that the transcriptional corepressor Ski acts as an inhibitor of vitamin A induced myeloid differentiation through interaction with N-CoR recruiting histone deacetylases (HDAC) (Ritter et al., Leukemia 2006). HDAC inhibitors such as valproic acid (VPA) promote histone acetylation, induce apoptosis and cell growth arrest in tumor cells (e.g. Kraemer et al., Trends Endocrinol Metab 2001). As Ski interacts with HDACs the aim of our investigation was to test the effect of HDAC inhibitors to cellular differentiation, apoptosis and Ski expression in primary AML cells. Treatment of the AML cell line HL60 expressing Ski with the HDAC inhibitor VPA enhances expression of the myeloid differentiation markers CD11b and CD11c as well as apoptosis. To address whether this effect is also observed in primary AML cells, we isolated mononuclear cells from blood or bone marrow of 12 AML patients (first diagnosis or relapse) and treated these cells with the HDAC inhibitor VPA. After harvesting, Ski protein expression was determined by Western blot. Flow cytometry was used to analyse expression of the differentiation markers CD11b and CD11c and apoptosis after propidium iodide staining. Of six AMLs with Ski protein expression, four responded either with differentiation or apoptosis, whereas none of six primary AMLs without Ski expression showed an effect to VPA compared to untreated control cells. To test whether other HDAC inhibitors would also reveal this effect we treated primary AML cells with further HDAC inhibitors (TSA, SAHA, LBH589) and confirmed our observation that HDAC inhibitors renders AML cells expressing Ski sensitive to differentiation.In parallel, we observed that VPA down regulates Ski in AML cells as well as in melanoma cell lines with high Ski protein levels. Our goal was to elucidate the molecular background of Ski reduction by VPA. Treatment of melanoma cells expressing Ski with VPA and/or the proteasomal inhibitor MG132 revealed that decrease of Ski depends on proteasomal degradation. The ring finger protein Arkadia (RNF111) is an E3-ligase of Ski (Nagano et al., J Biol Chem 2007) and we tested whether Arkadia is involved in Ski reduction after VPA addition. First we demonstrated that Arkadia expression is inversely associated with Ski expression in several AML cell lines. Furthermore the expression of Arkadia is induced by VPA on protein and RNA level while Ski protein is down regulated in melanoma cells. We also found that knockdown of Arkadia using RNAi impairs reduction of Ski by VPA in melanoma cells.Taken together our data suggest that high Ski expression in AML cells could be a molecular marker for VPA therapy. Thereby, VPA reduces the expression of the oncogene Ski as VPA induces expression of the E3-ligase Arkadia which abolish Ski by proteasomal degradation. Disclosures:No relevant conflicts of interest to declare.

Retinoblastoma gene‐independent G1 phase arrest by flavone, phosphatidylinositol 3‐kinase inhibitor, and histone deacetylase inhibitor

In most human malignant tumors, retinoblastoma tumor-suppressor gene (RB) product is inactivated by phosphorylation. Therefore, cancer preventive agents or molecular-targeting agents can inhibit the tumor growth at G(1) phase through RB reactivation. However, little is known about the effectiveness of RB reactivating agents against malignancies with mutated RB. We report here that chemopreventive agent flavone, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) also induce G(1) phase arrest in malignant tumor cells with mutated RB. In human prostate cancer DU145 cells with mutated RB, flavone increased cyclin-dependent kinase (CDK) inhibitors p21 and p27, and reduced cdk4 and cdk6, resulting in decrement of phosphorylated RB family proteins p130 and p107. LY294002 also dephosphorylated p107 and p130 proteins, whereas TSA dephosphorylated p130, but not p107. Furthermore, flavone induced G(1) phase arrest in both mouse embryo fibroblast (MEF) wild-type and MEF RB(-/-) cells, but did not do so in RB, p107, and p130 triple-knockout MEF cells. These results suggested that p130 and p107 contributed to G(1) phase arrest by flavone in RB-mutated cells. However, flavone induced tumor suppressor microRNA miR-34a with reduction of E2F1 and E2F3, known to be downregulated by miR-34a, raising the possibility that miR-34a might partially contribute to G(1) arrest by flavone. These results raise the possibility that RB reactivating chemopreventive agents or molecular targeting agents might also be effective against a variety of malignant tumor cells with mutant RB.

4-Methylumbelliferone leads to growth arrest and apoptosis in canine mammary tumor cells

Hyaluronan (HA), a major component of the extracellular matrix (ECM), is synthesized by HA synthase (HAS) 1, HAS2 and HAS3 and is intricately involved in cell growth and metastasis. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) has been reported to exhibit anticancer properties in various types of malignant tumors. However, the underlying mechanisms at the molecular and cellular levels remain unclear. In this study, to establish an animal model for studying the function of HA in human breast cancer, we investigated the antitumor effects of 4-MU using canine mammary tumor (CF33) cells. First, we investigated the effects of 4-MU on HA production in CF33 cells. Quantitative analysis of HA in culture media showed that 4-MU inhibited HA synthesis, accompanied by downregulation of HAS2 mRNA levels, in a dose-dependent manner at 24-72 h. Additionally, we observed a 4-MU-mediated decrease in the extent of the cell-associated HA matrix. We examined the effect of 4-MU on cell growth and apoptosis in CF33 cells. 4-MU markedly inhibited cell proliferation and induced apoptosis in CF33 cells. In particular, our experiments showed that the mechanism of 4-MU-induced apoptosis in CF33 cells involved increased levels of expression of pro-apoptotic BAX mRNA and protein molecules. These data suggest that 4-MU may be a candidate therapeutic agent for the treatment of canine mammary tumors. Furthermore, this study provides the first indication that the canine mammary tumor may be a suitable model for comparative study of the function of HA in human breast cancer.

Effect of annatto-tocotrienols supplementation on the Development of Mammary Tumors in HER-2/neu Transgenic Mice

Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% δ-T3 and 10% γ-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified δ-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.

The Synergistic Effects of DNA-Targeted Chemotherapeutics and Histone Deacetylase Inhibitors As Therapeutic Strategies for Cancer Treatment

Histone deacetylase (HDAC) inhibitors are a group of anticancer drugs which cause growth arrest and apoptosis of several tumor cells. HDAC inhibitors have been also found to increase the anticancer efficacy of several treatment modalities i.e. chemotherapy or radiotherapy. Here, we review the literature on combinations of HDAC inhibitors both with ionizing radiation and with other drugs, highlighting DNA-damaging compounds. The results of numerous studies with several types of cancer cells discussed in this review demonstrate that HDAC inhibitors enhance the effect of DNA damaging agents, such as inhibitors of topoisomerases, inhibitors of DNA synthesis, DNA-intercalators and agents covalently modifying DNA (i.e. doxorubicin, etoposid, 5-fluorouracil, cisplatin, melphalan, temozolomide and ellipticine) or of irradiation. Hence, the use of HDAC inhibitors combined with these antitumor drugs or ionizing radiation is a promising tool which may make treatment of patients suffering from many types of cancer more efficient. Several molecular mechanisms are responsible for the observed higher sensitivity of tumor cells towards therapeutic agents elicited by HDAC inhibitors. These mechanisms are discussed also in this review.

Antagonism between curcumin and the topoisomerase II inhibitor etoposide

The use of combinations of chemotherapy and natural products has recently emerged as a new method of cancer therapy, relying on the capacity of certain natural compounds to trigger cell death with low doses of chemotherapeutic agents and few side effects. The current study aims to evaluate the modulatory effects of curcumin (CUR), Nigella sativa (NS) and taurine on etoposide (ETP) cytotoxicity in a panel of cancer cell lines and to identify their underlying mechanisms. CUR alone showed potent antitumor activity, but surprisingly, its interaction with ETP was antagonistic in four out of five cancer cell lines. Neither taurine nor Nigella sativa affect the sensitivity of cancer cells to ETP. Examination of the DNA damage response machinery (DDR) showed that both ETP and CUR elicited DNA double-strand breaks (DSB) and evoked γ-H2AX foci formation at doses as low as 1 µg/ml. Cell cycle analysis revealed S phase arrest after ETP or CUR application, whereas co-treatment with ETP and CUR led to increased arrest of the cell cycle in S phase (MCF-7 cells) or the accumulation of cells in G2/M phases (HCT116, and HeLa cells). Furthermore, cotreatment with ETP and CUR resulted in modulation of the level of DNA damage induction and repair compared with either agent alone. Electron microscopic examination demonstrated that different modalities of cell death occurred with each treatment. CUR alone induced autophagy, apoptosis and necrosis, whereas ETP alone or in combination with CUR led to apoptosis and necrosis. Conclusions: Cotreatment with ETP and CUR resulted in an antagonistic interaction. This antagonism is related, in part, to the enhanced arrest of tumor cells in both S and G2/M phases, which prevents the cells from entering M-phase with damaged DNA and, consequently, prevents cell death from occurring. This arrest allows time for the cells to repair DNA damage so that cell cycle -arrested cells can eventually resume cell cycle progression and continue their physiological program.

Metabolic Shifts Induced by Fatty Acid Synthase Inhibitor Orlistat in Non-small Cell Lung Carcinoma Cells Provide Novel Pharmacodynamic Biomarkers for Positron Emission Tomography and Magnetic Resonance Spectroscopy

PurposeAbnormal fatty acid (FA) synthesis is one of the common features of cancer. Fatty acid synthase (FASN), a multifunctional enzyme playing a key role in biosynthesis of FA, is up-regulated in prostate, breast, and lung carcinomas. Orlistat is a FDA-approved anti-obesity drug that inhibits the thioesterase domain of FASN, interferes with cellular FA synthesis, can arrest tumor cell proliferation, and induces tumor cell apoptosis. The current study was aimed to investigate the metabolic changes associated with FASN inhibition by orlistat and to understand the molecular mechanisms behind the observed metabolic changes in non-small cell lung carcinoma (NSCLC) cell lines.ProceduresChanges in metabolite pools in four NSCLC cell lines (H441, H1975, H3255, and PC14) with different mutational profiles were studied using NMR spectroscopy before and after in vitro incubation with sub-toxic concentration of orlistat and [1-13C]d-glucose or [1,2-13C2]choline. In vitro radiotracer accumulation assays in cells were performed with [3H]acetate, [14C]fluoroacetate, and 2-deoxy-2-[18F]fluoro-d-glucose. In parallel, microarray profiling of genes involved in the regulation of carbohydrate and lipid metabolism was performed.ResultsIn orlistat-treated NSCLC cells, FASN inhibition results in characteristic changes in intermediary metabolites (FAs, choline, phospholipids, and TCA cycle metabolites) as observed by magnetic resonance spectroscopy. Further, FASN inhibition by orlistat induces multiple adaptive changes in FA synthetic pathway and associated metabolic pathways, including induction of ketone metabolism and glutaminolysis, as well as the up-regulation of 5' adenosine monophosphate-activated protein kinase.ConclusionsThese observed changes in metabolic pools in orlistat-treated cells demonstrate the critical role of fatty acid de novo synthesis and metabolism for cellular energy production, especially in tumor cells with low glycolytic activity, which goes beyond the widely accepted concept that FA synthesis is important for cell membrane biosynthesis in rapidly proliferating tumor cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-012-0587-6) contains supplementary material, which is available to authorized users.

Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A-Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A-Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A-Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A-Br in HT1080 cells are discussed.

Proteomic Analysis of Covalent Modifications of Tubulins by Isothiocyanates1–3

Although isothiocyanates (ITC), which are found in cruciferous vegetables, have been shown to inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in tumor cells, the biochemical mechanisms of cell growth inhibition by these compounds are not fully understood. Studies have reported that ITC binding to intracellular proteins may be an important event for initiating apoptosis. Specific protein target(s) and molecular mechanisms for ITC have been investigated in human lung cancer A549 cells using proteomic tools. Cells were treated with various amounts (1–100μmol/L) of radiolabeled phenethyl-ITC (PEITC) and sulforaphane (SFN) and the extracted proteins resolved using 2-dimensional gel electrophoresis. The results of mass spectrometric analyses suggested that tubulin may be an in vivo binding target for ITC. The binding of ITC to tubulin was associated with growth arrest. The proliferation of A549 cells was significantly reduced by ITC, with benzyl-ITC (BITC) having a greater relative activity than PEITC or SFN. Mitotic arrest and apoptosis as well as disruption of microtubule polymerization were induced in the order: BITC > PEITC > SFN. An analysis of tubulins isolated from BITC-treated A549 cells showed that Cys347, a conserved cysteine in all α-tubulin isoforms, was covalently modified by BITC. Taken together, these results suggest that tubulin is a binding target of ITC and that this interaction can lead to growth inhibition and apoptosis.

G2 checkpoint abrogator abates the antagonistic interaction between antimicrotubule drugs and radiation therapy

Background and purposeWe previously demonstrated that radiation may arrest tumor cells at G2 phase, which in turn prevents the cytotoxicity of antimicrotubule drugs and results in antagonistic interaction between these two modalities. Herein we tested whether G2 abrogators would attenuate the above antagonistic interaction and improve the therapeutic efficacy of combination therapy between radiation and antimicrotubule drugs. Materials and methodsBreast cancer BCap37 and epidermoid carcinoma KB cell lines were administered with radiation, UCN-01 (a model drug of G2 abrogator), paclitaxel or vincristine, alone or in combinations. The antitumor activities of single and combined treatments were analyzed by a series of cytotoxic, apoptotic, cell cycle, morphological and biochemical assays. ResultsUCN-01 significantly enhanced the cytotoxicity of radiation, antimitotic drugs, and their combined treatments in vitro. Further investigations demonstrated that UCN-01 attenuated radiation-induced G2 arrest, and subsequently repressed the inhibitory effect of radiation on drug-induced mitotic arrest and apoptosis. ConclusionsThis is the first report demonstrating that G2 checkpoint abrogation represses the inhibitory effect of radiation on antimicrotubule drugs, which may be implicated in cancer combination therapy. Considering that G2 abrogators are under extensive evaluation for cancer treatment, our findings provide valuable information for this class of promising compounds.