Annexin V-binding Assay Research Articles

Abstract 3272: Differential sensitivities to heat shock protein 90(HSP90) inhibitors in anaplastic lymphoma kinase(ALK)-positive non-small cell ling cancer(NSCLC) cells.

Abstract Objectives: HSP90 is an abundant cellular chaperone protein and its inhibition results in the instability and degradation of client proteins such as HER2, mutant KIT, mutant EGFR, and ALK. Recently, HSP90 inhibitors showed clinical activity against echinoderm microtubule-associated protein-like 4 (EML4)-ALK NSCLC. However, it is unknown whether HSP90 inhibitors remain sensitive against ALK-positive NSCLC cells that show acquired resistance to ALK inhibitors, such as crizotinib. Here, we evaluated the activity of two clinical stage HSP90 inhibitors in ALK-positive NSCLC cells that were sensitive and resistant to crizotinib. Methods: ALK-positive NSCLC cells that were crizotinib-sensitive (SNU-2292 and NCI-H3122) and crizotinib-resistant (SNU-2535 and H3122CR1) were used. In vitro cytotoxicities of HSP90 inhibitors alone (ganetespib and NVP-AUY922), or in combination with crizotinib, were evaluated against ALK-positive NSCLC cells using a modified MTT assay. Cell cycle and Annexin-V binding assays were evaluated by flow cytometry. Expression of ALK and its downstream effectors were detected by Western blot assay after exposure to HSP90 inhibitors ± crizotinib. Results: HSP90 inhibitors were highly potent against crizotinib sensitive SNU-2292 and NCI-H3122 cell lines and displayed enhanced activity when combined with 0.1 μM crizotinib. Consistent with previous reports, crizotinib resistant H3122 cells were ∼5x fold less sensitive to HSP90 inhibitors compared to parental H3122 cells, with IC50 still in the low nanomolar range. In a concentration dependent manner, the HSP90 inhibitors did not suppress ALK downstream signals in H3122CR1 and SNU-2535 cell lines compared to effects on NCI-H3122 and SNU-2292 cell lines. In addition, phospho-ALK signals were significantly diminished in Ba/F3 cells expressing EML4-ALK compared to cell lines expressing L1196M or G1269A after exposure to HSP90 inhibitors. Cell cycle analysis demonstrated HSP90 inhibitors and/or crizotinib significantly increased the sub-G1 populations of NCI-H3122 and SNU-2292 cells compared with those of H3122CR1 and SNU-2535. Similarly, apoptotic cells significantly increased in crizotinib-sensitive cells than in crizotinib-resistant cells treated with HSP90 inhibitors and/or crizotinib. Conclusions: HSP90 inhibitors showed differential sensitivities in ALK-positive NSCLC cells. Our results suggest that HSP90 inhibitors alone or in combination treatment with crizotinib are effective against crizotinib-sensitive, ALK-positive NSCLC cells. Citation Format: SunHwa Lee, Soyeon Kim, Tae Min Kim, Dong-Wan Kim, Dae Seog Heo. Differential sensitivities to heat shock protein 90(HSP90) inhibitors in anaplastic lymphoma kinase(ALK)-positive non-small cell ling cancer(NSCLC) cells. [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 3272. doi:10.1158/1538-7445.AM2013-3272

Abstract 5646: Overcoming acquired resistance to cetuximab by dual targeting of HER family members using antibody based therapy.

Abstract The EGFR is recognized as a key mediator of proliferation and progression in many human tumors and strategies to inhibit EGFR signaling have emerged as highly promising cancer therapy approaches. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to treatment. In an effort to examine mechanisms of acquired resistance to anti-EGFR monoclonal antibody we developed a series of cetuximab-resistant clones using the NSCLC line NCI-H226. During efforts to elucidate molecular pathways that are involved in acquired resistance to cetuximab we found that 1) EGFR ubiquitination and degradation is altered in cetuximab-resistant cells resulting in increased steady-state expression of the EGFR and 2) enhanced signaling from the EGFR to HER3 and the PI(3)K/Akt pathway indicating that activation of HER3 may lead to escape from cetuximab therapy. To investigate the role of HER3 in acquired resistance to cetuximab we used both RNAi and targeted approaches using the anit-HER3 antibody U3-1287/AMG888. siRNA directed against HER3 indicated that resistant cells had a strong dependency on HER3. Further, targeting HER3 using U3-1287/AMG888 showed a dose-dependent anti-proliferative response in resistant cells. In studies using siRNA against the EGFR and HER3 resulted in robust inhibition of cell proliferation than did silencing of either receptor alone in resistant cells. Comparable effects on cell proliferation were observed after combinatorial treatment of cells by the EGFR antibody cetuximab and the HER3 antibody U3-1287/AMG888. In addition, combined EGFR and HER3 inhibition by either siRNAs or antibodies decreased the phosphorylation of downstream signaling pathways AKT and MAPK. Annexin-V binding assays showed a significant increase in apoptosis in U3-1287/AMG888 plus cetuximab treated cells as compared to either agent alone. To determine if U3-1287/AMG888 could have therapeutic benefit as a combination treatment with cetuximab in vivo, mice harboring NCI-H226 xenografts will be treated with cetuximab for approximately 70-100 days until cetuximab-resistant tumors develop. At this time mice with resistant tumors will be divided into two groups that will be treated with both cetuximab and U3-1287/AMG888 or cetuximab alone. The work presented herein suggests that cells with acquired resistance to cetuximab maintain their dependence on EGFR and HER3 signaling via the AKT and MAPK pathways. Citation Format: Mari Iida, Toni M. Brand, Megan M. Starr, Neha Luthar, Matthew J. Wleklinski, Deric L. Wheeler. Overcoming acquired resistance to cetuximab by dual targeting of HER family members using antibody based therapy. [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 5646. doi:10.1158/1538-7445.AM2013-5646

Targeting Metabolic Pathways for Leukemia Treatment

AbstractAbstract 1371Aberrant signal transduction pathways influence metabolic changes in neoplastic cells. It is well established, in fact, that cancer cells are characterized by a pro-glycolytic metabolic phenotype and recent evidences confirmed that also leukemia cells may switch to a non-oxidative metabolism. Among the different substrates used to sustain the anabolic processes and to keep the tricarboxylic acid cycle (TCA) active, the fatty acid oxidation (FAO) may represent an alternative carbon source. The carnitine palmitoyl transferase 1a (CPT1a) catalyzes the first step of FAO, by loading long chain fatty acyl groups onto carnitine, transporting them through the mitochondrial membrane. Moreover, CPT1a has been previously demonstrated to interact with members of the apoptotic machinery, such as Bcl-2 and t-Bid, and its inhibition can cause an accumulation of the toxic metabolite palmitate, resulting in mitochondrial damage and cell death. In diabetic patients the use of a recently discovered antihyperglycemic agent characterized by the selective and reversible inhibition of CPT1a has been reported. Therefore, we aimed in this study to investigate the in vitro anti-leukemic effect of the CPT1a inhibition. Particularly, we evaluated the activity of two CPT1a-inhibitors, the novel ST1326 (kindly provided by Sigma-Tau) and the previously known Etomoxir on the proliferation and apoptosis of leukemia cell lines and primary cells obtained from patients affected by acute leukemias. The drug concentration inducing a 50% cell killing (IC50) was calculated from the dose-response curve. The cytotoxic drug effects on leukemia cell lines (HL-60, HL-60/MX2, U937, K562, CEM S, CEM R, MOLT-4) and primary cells of acute myeloid (AML) and lymphoid (ALL) leukemia were evaluated using the MTT test. Flow cytometry techniques Acridine-Orange staining, AnnexinV binding assay and Chloromethyl-X-Rosamine (CMXRos)/MitoTracker Green (MTGreen) staining were used to examine cell cycle changes, apoptosis and the loss of mitochondrial membrane potential (ΔΨm). We evaluated the activity of ST1326 (1–50 μM) on leukemia models, demonstrating a dose- and time-dependent cell growth arrest, caused by mitochondrial damage and apoptosis induction. In fact, 6 hours of ST1326 exposure were able to induce a dramatic loss of ΔΨm, as observed in MOLT-4 cells (cells affected increased from 15% in the control to 59.7% at 50 μM). Following 72 hours of ST1326 exposure, the same cells showed then an increase in the subG1 peak from a baseline value of 10% to 8.1%, 7.3%, 10.5%, 37% and 95.8% at 1, 5, 10, 20 and 50 μM, respectively (IC50= 39.2 μM). Etomoxir failed to show any activity (data not shown). The myeloid cell lines HL60, HL60/MX2 and U937 were more sensitive to ST1326, showing an IC50 of 11.8, 8.2 and 8.8 μM, respectively. A milder activity was found in the other lymphoid-derived model, the CEM S cell lines (IC50= 71.1 μM). Instead, both the lymphoid CEM R and the myeloid K562 cell lines proved resistant (IC50= n.d.). We then exposed in vitro (72 hours) primary cells from 12 AML and found in all a significant pro-apoptotic activity (AnnexinV positive cells): from 23.27% ± 13.63 (control) to 36.59% ± 19.97 (p=0.23), 40.51% ± 18.66 (p=0.0074), 43.43% ± 19.81 (p=0.0071) and 75.30% ± 11.52 (p=0.00018) following 72 hours of exposure to 5, 10, 20 and 50 μM ST1326, respectively. ALL primary samples (6/8 samples), instead, showed a significant (p<0.005) increase of the sub-G1 DNA apoptotic cells only at the higher ST1326 dose (from 38.9% ± 23.7 to 68.9% ± 23.4 of cells at 50 μM). In conclusion, ST1326 shows a high in vitro pro-apoptotic activity on acute leukemia models and on primary cells, especially in AML, prompting further studies to better define this novel approach based on the targeted inhibition of metabolic pathways in leukemia treatment. Disclosures:Petrucci:Jansse-Cilag, Celgene: Honoraria. Nicolai:Sigma Tau Pharmaceutical Industries SpA: Employment. Tafuri:Sigma Tau Pharmaceuticals: Research Funding.

Selective Induction of Apoptosis and Autophagy Through Treatment With Dandelion Root Extract in Human Pancreatic Cancer Cells

Pancreatic cancer has a 100% mortality rate; the aim of this study is to evaluate the efficacy of dandelion root extract (DRE) in inducing apoptosis and autophagy in aggressive and resistant pancreatic cancer cells. The effect of DRE was evaluated using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. Apoptotic cell death was confirmed by nuclear condensation by Hoechst staining and externalization of phosphatidylserine to the outer leaflet of the plasma membrane by Annexin-V binding assay. Loss of mitochondrial membrane potential was observed using the JC-1 (5,5',6, 6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) dye. The induction of autophagy was detected using a monodansylcadaverine assay and this was confirmed by immunofluorescence for light chain 3-II. BxPC-3 and PANC-1 pancreatic cells were sensitive to aqueous DRE. This extract induces selective apoptosis in a dose- and time-dependent manner. Dandelion root extract caused the collapse of the mitochondrial membrane potential, leading to prodeath autophagy. Normal human fibroblasts were resistant at similar doses. We demonstrate that DRE has the potential to induce apoptosis and autophagy in human pancreatic cancer cells with no significant effect on noncancerous cells. This will provide a basis on which further research in cancer treatment through DRE can be executed.

Excitotoxicity Through NMDA Receptors Mediates Cerebellar Granule Neuron Apoptosis Induced by Prion Protein 90-231 Fragment

Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrP(C)) into an aberrant isoform (PrP(Sc)). In this work, we characterize the in vitro toxicity of amino-terminally truncated recombinant PrP fragment (amino acids 90-231, PrP90-231), on rat cerebellar granule neurons (CGN), focusing on glutamatergic receptor activation and Ca(2+) homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231(TOX)) after controlled thermal denaturation (1h at 53°C) acquiring structural characteristics identified in PrP(Sc) (enrichment in β-structures, increased hydrophobicity, partial resistance to proteinase K, and aggregation in amyloid fibrils). By annexin-V binding assay, and evaluation of the percentage of fragmented and condensed nuclei, we show that treatment with PrP90-231(TOX), used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca(2+)]i. Both CGN apoptosis and [Ca(2+)]i increase were not observed using PrP90-231 in PrP(C)-like conformation. PrP90-231(TOX) effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca(2+)]i increase were largely reduced, although not fully abolished, by pre-treatment with the NMDA antagonists APV and memantine, while the AMPA antagonist CNQX produced a lower, although still significant, effect. In conclusion, we report that CGN apoptosis induced by PrP90-231(TOX) correlates with a sustained elevation of [Ca(2+)]i mediated by the activation of NMDA and AMPA receptors.

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or two-photon microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format.

Flotillin-1 Promotes Tumor Necrosis Factor-α Receptor Signaling and Activation of NF-κB in Esophageal Squamous Cell Carcinoma Cells

The flotillin family of proteins, including flotillin-1 (FLOT1 or Reggie-2), are lipid raft proteins that initiate receptor kinase signaling and are up-regulated in several tumor types. We investigated the role of FLOT1 signaling and activation of the transcription factor nuclear factor (NF)-κB in esophageal squamous cell carcinoma (ESCC) cells. We used immunoblot and immunochemical analyses to determine levels of the lipid raft-associated protein FLOT1 in ESCC cell lines and 432 ESSC samples from patients; primary normal esophageal epithelial cells and matched adjacent nontumor tissues were used as controls. We determined the ability of FLOT1 to activate NF-κB using kinase, electrophoretic mobility shift, and luciferase reporter assays. We measured the effects of FLOT1 overexpression and knockdown with short hairpin RNAs in ESCC cell lines using colony formation, anchorage-independent growth, chicken chorioallantoic membrane, transwell matrix penetration, and Annexin V-binding assays. We analyzed growth of ESCC xenograft tumors in nude mice. Levels of FLOT1 were increased in ESCC cell lines and samples from patients, compared with controls; protein levels correlated with disease stage and survival time. Overexpression of FLOT1 in Kyse30 and Kyse510 ESCC cell lines increased proliferation, anchorage-independent growth, and invasive activity and protected them from apoptosis. FLOT1-transduced ESCC cells formed larger tumors in nude mice than control cells (transduced with only the vector). FLOT1 facilitated recruitment of the tumor necrosis factor-α receptor to lipid rafts; promoted K63-linked polyubiquitination of the signaling intermediaries tumor necrosis factor receptor associated factor 2, receptor interacting protein, and NEMO; and sustained the activation of NF-κB. Levels of FLOT1 correlated with activation of NF-κB in ESCC samples from patients. The lipid raft protein FLOT1 is up-regulated in ESCC cell lines and samples from patients and promotes ESCC cell proliferation and tumor growth in mice. FLOT1 activates tumor necrosis factor-α receptor signaling and sustains activation of NF-κB in ESCC cells.

Abstract 5726: Human epidermal growth factor 3 (HER3) blockade with U3-1287/AMG888 modulates radiosensitivity in the lung and head and neck carcinomas

Abstract The epidermal growth factor receptor (EGFR) family consists of four members including HER1 (EGFR), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Collectively this family of receptor tyrosine kinases (RTKs) play critical roles in the etiology of several human cancers as well as a role in resistance to radiation therapy. In 2006, a study in head and neck squamous cell carcinoma (HNSCC) indicated that using the anti-EGFR antibody cetuximab in combination with radiation resulted in a 10% advantage of 3-year overall survival rate when compared to radiotherapy alone. This study highlighted the therapeutic utility of RTK blockade in combination with radiation. Studies investigating the role of HER3 in radiation response are limited. In the present study, we investigated whether or not HER3 blockade could enhance radiation therapy using the HER3 antibody U3-1287/AMG888. We screened a battery of cell lines from lung, HNSCC and colorectal tumor lines for HER3 expression. The results indicated that all 15 cell lines tested showed expression of HER3. In addition, U3-1287/AMG888 was able to block basal HER3 activity and radiation induced HER3 activation. Further, proliferation assays, using U3-1287/AMG888, indicated that HER3 blockade could block proliferation in SCC6, SCC1483, H226 cell lines, highlighting the importance of HER3 in these tumor cells. Clonogenic assays showed that U3-1287/AMG888 could sensitize these lines to radiation. Furthermore, γ-H2AX analyses, detected by immunofluorescence, lead to a statistically significant increase in apoptotic cells. Cell cycle analysis, 24 and 48 hours after U3-1287/AMG888 and radiation treatments resulted in a significant G1 and G2 cell cycle arrest. Annexin-V binding assays showed a significant increase in apoptosis in the U3-1287/AMG888 plus radiation group as compared to either agent alone. To determine if HER3 blockade using U3-1287/AMG888 could enhance radiation therapy in vivo we performed tumor growth delay experiments using SCC6, SCC1483 and H226 xenografts in nude mice. Mice were inoculated and treated with 1) IgG, 2) U3-1287/AMG888, 3) radiation or 4) the combination. The results of these experiments indicated that the combination of U3-1287/AMG888 and radiation had a strong impact on tumor growth in studies using single dose or fractionated dosing. Tumor analysis indicated that radiation treatment activated HER3 in vivo and U3-1287/AMG888 could abrogate this activation. Collectively our findings in vitro and in vivo suggest that U3-1287/AMG888 in combination with radiation has an impact on cell and tumor growth by impacting cell cycle progression, increasing apoptosis and increasing DNA damage. These findings suggest that HER3 may play a critical role in response to radiation therapy and blocking its activity may be of strong therapeutic benefit in human tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5726. doi:1538-7445.AM2012-5726

Efficient Induction of Extrinsic Cell Death by Dandelion Root Extract in Human Chronic Myelomonocytic Leukemia (CMML) Cells

BackgroundChronic Myelomonocytic Leukemia (CMML) is a heterogeneous disease that is not only hard to diagnose and classify, but is also highly resistant to treatment. Available forms of therapy for this disease have not shown significant effects and patients rapidly develop resistance early on in therapy. These factors lead to the very poor prognosis observed with CMML patients, with median survival duration between 12 and 24 months after diagnosis. This study is therefore centered around evaluating the selective efficacy of a natural extract from dandelion roots, in inducing programmed cell death in aggressive and resistant CMML cell lines.Methodology/Principal FindingsTo confirm the induction of programmed cell death in three human CMML cell lines, nuclear condensation and externalization of the phosphatidylserine, two main characteristics of apoptosis, were detected using Hoechst staining and annexin-V binding assay. The induction of another mode of cell death, autophagy, was determined using a monodansylcadaverine (MDC) stain, to detect the formation of autophagy vacuoles. The results from this study indicate that Dandelion Root Extract (DRE) is able to efficiently and selectively induce apoptosis and autophagy in these cell lines in a dose and time dependent manner, with no significant toxicity on non-cancerous peripheral blood mononuclear cells. More importantly, we observed early activation of initiator caspase-8, which led to mitochondrial destabilization and the induction of autophagy, suggesting that DRE acts through the extrinsic pathway of apoptosis. The inability of DRE to induce apoptosis in dominant-negative FADD cells, confirms the mechanism of action of DRE in in vitro models of CMML.ConclusionThe results from this study indicate that natural products, in particular Dandelion Root Extract, have great potential, as non-toxic and effective alternatives to conventional modes of chemotherapy available today.

Enzymatic measurement of phosphatidylserine in cultured cells

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0-50 µM) and high (50-1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.

Induction of Apoptosis and Growth Arrest in Human Breast Carcinoma Cells by a Snake (Walterinnesia aegyptia) Venom Combined With Silica Nanoparticles: Crosstalk Between Bcl2 and Caspase 3

We recently demonstrated that the snake venom extracted from Walterinnesia aegyptia (WEV) either alone or combined with silica nanoparticles (WEV+NP) enhanced the proliferation of mice immune cells and simultaneously decreased the proliferation of human breast carcinoma cell line (MDA-MB-231). However, the molecular mechanism of how this venom induced growth arrest of breast cancer cells has not been studied. In this context, we extended our study to evaluate the anti-tumor potential of WEV and WEV+NP on the human breast carcinoma cell lines MDA-MB-231 and MCF-7, as well as their effects on non-tumorigenic normal breast epithelial cells (MCF-10). The IC₅₀values of WEV alone and WEV+NP in these cell lines were determined to be 50 ng/ml and 20 ng/ml, respectively. Interestingly, at these concentrations, the venom did not affect the viability of normal MCF-10 cells and treatment of all these cell lines with NP alone did not affect their viability. Using annexin-V binding assay followed by flow cytometry analysis, we found that combination of WEV with NP strongly induced apoptosis in MDA-MB-231 and MCF-7 cancer cells without significant effect on normal MCF-10 cells. Furthermore, we found that WEV+NP decreased the expression of Bcl2 and enhanced the activation of caspase 3 in MDA-MB-231 and MCF-7 cells. Most importantly, WEV+NP-treated breast cancer cells, but not normal MCF-10 cells, exhibited a significant (P<0.05) reduction in actin polymerization and cytoskeletal rearrangement in response to CXCL12. Our data reveal biological effects of WEV or WEV+NP and the underlying mechanisms to fight breast cancer cells.

Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

BackgroundEpidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. MethodsIn this study, using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. ResultsAmong the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. ConclusionOur studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.

Abstract 2506: Preclinical evaluation of sorafenib in combination with ionizing radiation in metastatic breast cancer model

Abstract Sorafenib tosylate (Bay 54-9085) is an oral, small molecule multikinase inhibitor of several targets including RAF/MEK/ERK signaling, vascular endothelial growth factor receptor-2 (VEGFR-2), VEGFR-3, and platelet derived growth factor receptor-beta (PDGFR-b). Sorafenib has shown clinical efficacy in solid tumors such as renal cell and hepatocellular carcinomas. Retrospective analysis of breast cancer patients has shown an unfavorable prognosis in patients with high expression level of VEGF, indicating that VEGF could be associated with efficacy of chemotherapy and radiotherapy. It has also been shown that radiation resistance is partially due to tumor cell production of angiogenic cytokines, particularly VEGF that protects endothelial cells through survival pathways. The aim of this study is to investigate if radiation response is enhanced through inhibition of p-VEGFR2/PDGFR-b by Sorafenib and if the combination of Sorafenib and radiation will increase the treatment response in a metastatic breast cancer model. Mouse metastatic mammary cancer cells, 4T1 were used in this study. Clonogenic assay was performed to assess the radiomodulating effect of Sorafenib. In addition, cell cycle analysis and annexin-V binding assay were performed 24 and 48 hrs post treatment respectively. To confirm our in vitro data, a tumor growth delay assay was completed in a syngeneic mouse model. Western blot analysis showed strong dose dependent inhibition of ERK1/2 phosphorylation by sorafenib. Our clonogenic assay showed a supra-additive effect of Sorafenib (7.5 μM) and radiation (4 Gy) with a dose enhancement factor (DEF) of 1.81. Sorafenib in combination with radiation demonstrated a significant G2/M cell cycle arrest (p&amp;lt;0.0001). Moreover, annexin-V staining assay showed a significant increase in the level of apoptosis in cells treated with both sorafenib and radiation (p&amp;lt;0.0004). Our in vivo results strongly suggest that radiation prolongs the anti tumor activity of sorafenib. In addition, tumor growth was delayed when radiation was combined with sorafenib concurrently (tumor growth delay was increased from 10.5 days in irradiated group to 18 days in combined treatment group). Our results demonstrate that sorafenib increases both the level of apoptosis and the sensitivity of 4T1 cancer cells to radiation. The higher potency of sorafenib combined with radiation can be partially due to the strong cell cycle arrest at G2/M phase. Moreover, our in vivo results show that sorafenib in combination with radiation exhibit a superior tumor growth control. Overall, our findings suggest that greater treatment response may be achieved when sorafenib is combined with radiation. More studies are being conducted to investigate the optimal schedule for this combination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2506. doi:10.1158/1538-7445.AM2011-2506

Circulating cycloxygenase-2 in patients with tobacco-related intraoral squamous cell carcinoma and evaluation of its peptide inhibitors as potential antitumor agent

The aim of this study was to quantitate circulating COX-2 levels in patients with tobacco-related intraoral cancer and to evaluate antitumor activities of COX-2 peptide inhibitors in vitro on KB cell lines. We used a novel biosensor-based surface plasmon resonance (SPR) technique for estimation of circulating COX-2 levels in 76 patients with oral cancer and 43 normal individuals. Antitumor activities of five COX-2 inhibitory peptides were evaluated using propidium iodide labeling and flow cytometry, alamar blue, MTS, and annexin-V binding assays. Patients with oral cancer showed threefold increase in serum COX-2 level when compared to normal controls (P < 0.0001). Further, late-stage tumors and lymph node metastasis were associated with significant increase in serum COX-2 levels. Patients with higher circulating COX-2 also showed higher immunoreactivity to anti-COX-2 antibody in the lesions. The peptides significantly reduced viability and inhibited growth/proliferation, induced cytotoxicity and apoptosis in tumor cells. However, no such effect was observed either on normal human leukocytes or on MCF-7 cell line that did not over express COX-2. Our results indicate that SPR may be a useful proteomic technique for quantitative assessment of COX-2 and to identify patients with high-risk oral premalignant or occult cancer, as well as in monitoring response to novel COX-2 targeting strategies. Furthermore, COX-2 peptide inhibitors appear to be a new class of potent anticancer agent for human oral carcinoma.

Specific Accumulation and Antitumor Effects of Hybrid Liposomes on the Growth of Lung Tumor Cells

In general, chemotherapeutic effects were low for non-small cell lung cancer (NSCLC) in the lung tumor. We examined the accumulation and antitumor effects of hybrid liposomes (HL-23) composed of phospholipid (L-α-dimyristoylphosphatidylcholine: DMPC) and PEG surfactant [polyoxyethylene(23)dodecyl ether: C₁₂(EO)₂₃] on NSCLC cells in vitro. Accumulation of HL-23 including a fluorescence probe [1-Palmitoyl-2-[12(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phosphocholine: NBDPC] was observed for NSCLC cells using a confocal laser microscope, but no accumulation of HL-23 in normal lung cells was observed. Furthermore, inhibitory effects of HL-23 on the growth of NSCLC cells were obtained on the basis of a WST-1 assay. It was also clarified that HL-23 induced apoptosis for NSCLC cells on the basis of Annexin-V binding and TUNEL assay. These results suggest that HL-23 could be applied in effective chemotherapies for NSCLC.

Antigenotoxic potential of Gymnema montanum leaves on DNA damage in human peripheral blood lymphocytes and HL‐60 cell line

In this study we have evaluated the genoprotective effect of the ethanol extract of Gymnema montanum (GLEt) leaves in human peripheral blood lymphocytes and HL-60 cell line in vitro using the comet assay. DNA damage was induced by treating the cells with H(2)O(2) and methyl methane sulphonate (MMS). GLEt treatment effectively protected the lymphocytes and HL-60 cell line from H(2)O(2)-induced oxidative DNA damage in a dose-dependent manner whereas it was not effective against alkylative DNA damage caused by MMS. The global percent repair efficiency also showed that both pre- and post- GLEt treatment provided effective protection against H(2)O(2) induced DNA damage but not as effective against MMS. At 200 microg ml(-1) level, its repair capacity against H(2)O(2) induced DNA damage was comparable to that of vitamin-C (100 microM). Furthermore, exposure to GLEt reduced the formation of apoptotic cells caused by H(2)O(2), which was demonstrated by the decreased sub-G1-DNA content in cell cycle analysis and apoptotic frequencies of lymphocytes in an annexin-V binding assay. In addition, GLEt was found to have effective peroxide scavenging ability in dose-dependent manner. The protective efficiency of the extract was found to be directly proportional to its total phenolic content. The present study indicates that G. montanum leaves are a significant source of phytochemicals with antigenotoxic and antioxidant activity, and thus has potential therapeutic use.

The human 1‐8D gene (IFITM2) is a novel p53 independent pro‐apoptotic gene

The human 1-8 interferon inducible gene family consists of at least 3 functional genes; 9-27, 1-8D and 1-8U, which are all linked on an 18-kb fragment of chromosome 11 and are highly homologous. It has recently been shown by us and others that the 1-8D gene is overexpressed in colon carcinoma. Here, we show, by sequence comparison of the 1-8D in pairs of tumor/normal colon tissues, the existence of 6 different alleles, containing single-nucleotide polymorphisms with no mutations. Transformation assays revealed a possible role for the 1-8D gene as a transformation inhibitor. Further, transient expression of the human 1-8D gene in multiple mammalian cell lines showed accumulation of cells in the G1 phase followed by elevation in the subG1 phase. SubG1 elevation was confirmed as apoptosis by Annexin-V binding assays and transferase-mediated dUTP nick end labeling assays. Moreover, knock-down of 1-8D provided partial protection from Etoposide and UV-induced apoptosis. The induction of apoptosis by 1-8D is dependent on caspase activities but not on p53 expression. Although 1-8D induces apoptosis independently of p53, p53 expression downregulates 1-8D protein expression. Our data suggest a role for the 1-8D gene as a novel pro-apoptotic gene that will provide new insights into the regulated cellular pathways to death.

Inhibitory Effects of Hybrid Liposomes on the Growth of Gastric Tumor Cells Established from Cotton Rats

We established cell-line (CoRa 622 G6) of gastric carcinoma using cotton rats with spontaneous malignant gastric carcinoma with hypergastrinaemia. Inhibitory effects of hybrid liposomes (HL) composed of dimyristoylphosphatidylcoline (DMPC) and polyoxyethylene (n) dodecyl ether (C(12)(EO)(n): n=21, 23, 25) on the growth of CoRa 622 G6 cells were clarified on the basis of WST-1 assay. Fusion and accumulation of HL including fluorescence probe into CoRa 622 G6 cell membrane were clarified using confocal laser microscopy and total internal reflection fluorescence microscopy. Induction of apoptosis of CoRa 622 G6 cells after the treatment with HL was observed in fluorescence micrographs on the basis of Annexin-V binding assay and TUNEL method using confocal laser microscopy. The results in this study could contribute to the chemotherapy for patients with gastric carcinoma.

Interactions between Shiga toxins and human polymorphonuclear leukocytes

Human intestinal infections by Shiga toxin (Stx)-producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G-CSF-treated donors. The use of the human myeloid leukemia cell (HL-60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V-binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx-positive and Stx-negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.

Effect of combining anti-epidermal growth factor receptor antibody C225 and radiation on DU145 prostate cancer

The epidermal growth factor receptor (EGFR) network has rich targets for prostate cancer killing. Herein we evaluated the effects of combining the EGFR inhibition and radiation on DU145 prostate cancer. We treated DU145 prostate cancer cells with various doses of anti-EGFR antibody (C225) and gamma-irradiation (RAD). The effects of the treatment on cell viability and growth were assessed with cell counting, XTT and clonogenic assays. In vivo treatment effects were assessed using a subcutaneous tumor xenograft in mice. Cell cycle distribution and progression were assessed with flow cytometry. The apoptotic components of cell death were quantified using Annexin-V binding assays. The results demonstrated that when combined with radiation, C225 augmented the inhibition of cell viability and growth in the DU145 cell line and EGFR inhibition appeared to have some interaction with RAD. C225 inhibited the growth of implanted DU145 tumors and increased the efficacy of radiation treatment. Flow cytometric analysis suggested that mostly necrotic cell death resulted from the EGFR inhibition or irradiation, although there may be some apoptosis. We drew the conclusion that the inhibition of EGFR augments the radiation killing of DU145 prostate cancer via a combination of cytostatic, necrotic and apoptotic mechanisms.