CellTiter-Glo Luminescent Cell Viability Assay Research Articles

Local anaesthetics or their combination with morphine and/or magnesium sulphate are toxic for equine chondrocytes and synoviocytes in vitro

BackgroundChondrotoxic effects of local anaesthetics are well reported in humans and some animal species but knowledge on their toxic effects on synoviocytes or equine chondrocytes or the effects on cellular production of inflammatory cytokines is limited. The purpose of this study was to evaluate the in vitro effects of local anaesthetics, morphine, magnesium sulphate (MgSO4) or their combinations on cell viability and pro-inflammatory cytokine gene expression of equine synoviocytes and chondrocytes.Equine synoviocytes and cartilage explants harvested from normal joints in a co-culture system were exposed to mepivacaine (4.4 mg/ml), bupivacaine (2.2 mg/ml), morphine (2.85 mg/ml) and MgSO4 (37 mg/ml) alone or each local anaesthetic plus morphine or MgSO4 or both together. Chondrocyte and synoviocyte cell viability was assessed by CellTiter-Glo Luminescent Cell Viability Assay. Synoviocyte gene expression of IL-1β, IL-6 or TNF-α was measured and compared using the ∆∆CT method.ResultsMorphine alone, MgSO4 alone or their combination did not alter cell viability or the expression of IL-1β, IL-6 or TNF-α. However, local anaesthetics alone or in combination with morphine and/or MgSO4 reduced cell viability and increased the gene expression of IL-1β, IL-6 or TNF-α. Single short exposure to local anaesthetics is toxic to both chondrocytes and synoviocytes and their combination with morphine and/or MgSO4 enhanced the cytotoxic effects.ConclusionsThis in vitro study gives further evidence of the absence of cytotoxic effects of morphine alone, MgSO4 alone or their combination on normal articular tissues. However, local anaesthetics alone or in combination with morphine and/or MgSO4 have cytotoxic effects on equine articular tissues.

The Differentiation-Inducing Effect of a Retinoic Acid Derivative on a Pulmonary Carcinoma Cell Line

Pulmonary carcinoma is a main cause of cancer death internationally. Chemotherapeutic regimens in most solid tumors have limited effect. And in the targeted inhibition of carcinogenic driver mutations with molecular therapies, in a large number of cases, patients acquire resistance to drugs. Therefore, the development of new therapeutic approaches is highly desirable. In the present study, we evaluated the applicability of 4[(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl- 2-naphthalenyl)carbamoyl]benzoic acid (Am80), a synthetic retinoid, as a differentiation inducer in pulmonary carcinoma and examined its effect on the viability and differentiation of Calu-6 cells (a human nonsmall cell lung cancer cell line), with the final goal of establishing a new approach to pulmonary carcinoma therapy. The cell viability of Calu-6 cells after Am80 treatment was assessed by the CellTiter-Glo Luminescent Cell Viability Assay. The early apoptosis of cells was detected by Terminal deoxynucleotidyl Transferase-mediated deoxyuridine triphosphate Nick End Labeling (TUNEL) staining. The differentiation-inducing effect of Am80 on Calu-6 cells was investigated by immunostaining. Am80 induced the differentiation of Calu-6 to alveolar type I and II cells. However, Am80 reduced the viability of Calu-6 cells without inducing apoptosis. In addition, in order to investigate whether Am80 inhibits tumor growth in vivo, we conducted an in vivo experiment to determine he effect of the intratumoral administration of Am80 in mice harboring sc tumors composed of Calu-6 cells. Am80 significantly influenced the suppression of tumor growth.

Abstract 1487: ONC201 kills breast cancer cells by inhibiting mitochondrial respiration

Abstract Background: ONC201 is a small molecule originally identified as a TRAIL inducing compound currently being tested in phase1/2 clinical trials in multiple cancer types. Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP. Methods: ONC201 was obtained from Oncoceutics, Inc. Recombinant GST-TRAIL was prepared in the laboratory. Cell viability was tested with MTS assay and CellTiter-Glo luminescent cell viability assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNAseq and western blotting were performed to investigate change of gene expression. Mitochondrial respiration was monitored by Seahorse XF analyzer. Live cell imaging was performed to examine the mode of cell death. Confocal microscopy and electron microscopy analysis were performed to study mitochondrial morphology. Results: We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2 amplified, TNBC basal A and TNBC basal B breast cancer. ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 μM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 toxicity was not dependent on TRAIL receptors or caspases and live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture. By contrast, GST-TRAIL induced TRAIL-receptor dependent caspase mediated death and classic apoptosis morphology. These results suggest that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism distinct from TRAIL-induced apoptosis. Western blots revealed that ONC201 induces the stress pathway proteins ATF4 and CHOP, consistent with the recently published observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Seahorse XF analysis found that ONC201 inhibited mitochondrial oxygen consumption rate but did not inhibit glycolysis as measured by the extracellular acidification rate. Both ONC201-induced toxicity and ATP depletion were enhanced when cells were cultured in non-glucose (galactose) medium. Supplementing glucose to cells grown in galactose medium partially prevented ONC201-dependent ATP depletion, induction of phospho-AMPK, ATF4 and CHOP, and cell death. These data are consistent with an inhibition of oxidative phosphorylation (OxPhos) by ONC201. RNAseq revealed ONC201 inhibits expression of multiple mitochondrial genes involved in OxPhos and other mitochondrial functions, and western blot confirmed those findings. Confocal and electron microscopic evaluation revealed abnormal mitochondrial morphology. Conclusion: Our data demonstrate that ONC201 can kill breast cancer cells by a novel mechanism involving disruption of mitochondrial morphology and inhibition of mitochondrial respiration. Citation Format: Yoshimi Greer, Samuel Gilbert, Celia Islam, Yun Ji, Luca Gattinoni, Christina Stuelten, Natalie Porat-Shliom, Roberto Weigert, Xiantao Wang, Markus Hafner, Kunio Nagashima, Donna Voeller, Stanley Lipkowitz. ONC201 kills breast cancer cells by inhibiting mitochondrial respiration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2017-1487

Abstract 691: DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid pediatric tumor and is associated with MYCN amplification. MYCN is a regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis. Inhibition of this pathway in MYCN-amplified NB tumors has been shown to be a target for treatment. Alpha-difluoromethylornithine (DFMO) inhibits ODC and is currently being used in a Phase II clinical trial for NB. BET inhibitors JQ1 and OTX-015 have been shown to be effective against MYCN-amplified cancers; it is hypothesized that they downregulate MYCN as well as cancer stem cell (CSC) signaling. We propose that inhibiting MYCN with a BET inhibitor, coupled with inhibition of ODC with DFMO, will result in enhanced inhibition of NB growth and tumor-initiating properties. Methods: Single and combination drug treatments were conducted on BE(2)-C, SMS-KCNR, CHLA90, and one patient-derived cell line. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability in 96-well plates. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. Drug combination studies were conducted in MYCN amplified tumors BE(2)-C and SMS-KCNR using ray designs to evaluate for synergism. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity and apoptosis in NB cells. Western blots measured protein levels of apoptosis markers (cleaved caspase 3 and cleaved PARP) and CSC markers (Nanog, Sox2, NF-kB, CXCR4, Lin28B, and MYCN). Neurosphere assays were used to evaluate tumor initiation via sphere formation. Results: Cell viability of MYCN amplified NB showed an IC50 range of 1.48-1.69 μM for JQ1 and 839.3 nM-2.36 μM for OTX-015; MYCN non-amplified NB showed an IC50 range of 4.22-11.58 μM for JQ1 and 2.99-11.03 uM for OTX-015. Combination treatment in MYCN NB had a synergistic effect, based on Loewe-additivity as the null hypothesis, for cell viability suppression by ray design experiments for BE(2)-C and SMS-KCNR. Western blots showed greater expression of apoptosis markers and decreased expression of CSC markers in combination relative to single drug treatments. A greater than 50% decrease in neurosphere formation with combination treatment in BE(2)-C, SMS-KCNR, and the patient cell line provides evidence for reduced CSC activity. IncuCyte imaging showed an increase in cell death with time post-treatment. Conclusion: The combination of DFMO with BET inhibitors has a synergistic effect in treating MYCN amplified NBs as shown by a decrease in cell viability. This combination targets CSC pathways and decreases tumor-initiating ability. Given the lack of effective treatment options for children with relapsed or refractory high-risk NB, this combination may be a promising novel therapy. Citation Format: Sarah DeCou, Ping Zhao, Tracey Avequin, Abhinav Nagulapally, Jeffrey Bond, Giselle Saulnier Sholler. DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 691. doi:10.1158/1538-7445.AM2017-691

Abstract 173: Synthesis of substituted tetrahydroisoquinoline derivatives as anticancer agents

Abstract Breast cancer is the second leading cause of cancer-related deaths in women today and is the most common cancer among women, excluding non-melanoma skin cancers. Over 246,000 women will be diagnosed with breast cancer and 40,450 are expected to die of it in 2016. Estrogen receptors ERα, ERβ and their associated steroid hormones play vital role in breast cancer development and progression. Estrogen binds and activates the estrogen receptors in certain breast cancer cells. Endocrine therapy which aims to block ER action (antagonism) on breast tumor cells and thereby stopping the proliferation of cancer cells is a very useful strategy in treating breast cancer. Tamoxifen is the first selective estrogen receptor modulator (SERM) and is the leading drug used in treating breast cancer. Although Tamoxifen has shown great benefit in treating breast cancer, its agonistic effect on the uterus is said to be associated with an increased risk of developing endometrial cancer. We are interested in designing a single chemical entity which acts at specific multiple biomolecular targets. This strategy may exert favorable advantages in improving efficacy at desired multiple targets and lower incidence of side effects. Thus, alternative chemical entities, preferably non-steroidal molecules which act as estrogen receptor modulators and microtubule disruptors are sought. The tetrahydroisoquinoline (THIQ) core structure is an important pharmacophore in natural products and small molecules which act as drug molecules. The steroidomimetic tetrahydroisoquinoline moieties were reported to be selective SERMs and microtubule disruptors. Based on these considerations and in continuation with our previous research, herein we report in vitro cytotoxic and in silico docking studies of substituted THIQs. Novel substituted THIQ derivatives were designed and synthesized. N-amination of substituted isoquinolines by the aminating agent, O-mesytelene sulfnylhydroxylamine led to the formation of ylides. The ylides were reduced using sodium borohydride to yield the desired substituted tetrahydroisoquinolines in moderate to good yields. These compounds were evaluated for their cytotoxic effects using MCF-7, MDA-MB-231 and Ishikawa cells using the CellTiter-Glo luminescent cell viability assay. Among all the compounds screened, 4-ethyl-N-(8-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)benzamide showed IC50 values of 0.61, 1.36 and 0.09 µg/ml on MCF-7, MDA-MB-231 and Ishikawa cells respectively. This research was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28. Citation Format: Kinfe Ken Redda, Madhavi Gangapuram, Suresh Eyunni. Synthesis of substituted tetrahydroisoquinoline derivatives as anticancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 173. doi:10.1158/1538-7445.AM2017-173

Abstract 3252: Synthesis of N-substituted [Benzoylamino]-3-Ethyl-1,2,3,6-tetrahydropyridines as potential anti-cancer agents

Abstract Chronic inflammation is believed to promote cancer through cellular and genomic damage. Inflammation creates a microenvironment within the tissue that enhances cell proliferation. In 2016, American Cancer Society estimates that 1,685,210 new cancer cases are expected to be diagnosed in the United States. Cyclooxygenase (COX) is an enzyme found to play an essential role in inflammation and converts arachidonic acid to prostaglandin-like molecules. The three isomers of COX are COX-1, COX-2, and COX-3. COX-2 has been found to be up-regulated in inflammation and some cancers, such as breast cancer and prostate cancer. Tetrahydropyridines (THPs) developed by our research group were effective in inhibiting inflammation, COX, and the growth of some breast cancer cells. Previous research has shown that pharmacological activities of the THP derivatives depend greatly on the nature and position of the substituents on the THP ring structure. Here, we present the synthesis of a series of novel THP derivatives. 3-Ethylpyridine is reacted with O-mesitylenesulfonylhydroxylamine (O-MSH) to furnish N-amino-3-ethylpyridinium mesitylenesulfonate. The reaction of N-amino-3-ethylpyridinium mesitylenesulfonate with substituted acid chlorides gives the stable crystalline pyridinium ylides. A sodium borohydride reduction of ylides furnishes the target compounds, N-substituted [benzoylamino]-3-ethyl-1,2,3,6-tetrahydropyridines. Five novel N-substituted [benzoylamino]-3-ethyl-1,2,3,6-tetrahydropyridines were synthesized, purified, and characterized. The evaluation of these compounds cytotoxicity studies on MCF-7 estrogen receptor positive breast cancer cells, MDA-MB-231 estrogen receptor negative breast cancer cell lines, and Ishikawa cells, using the CellTiter-Glo (CTG) luminescent cell viability assay is underway. This research was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28. Citation Format: Elizabeth Henderson, Madhavi Gangapuram, Suresh Eyunni, Kinfe Redda, Tiffany Ardley. Synthesis of N-substituted [Benzoylamino]-3-Ethyl-1,2,3,6-tetrahydropyridines as potential anti-cancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3252. doi:10.1158/1538-7445.AM2017-3252

Synthesis and Biological Evaluations of Ring Substituted Tetrahydroisoquinolines (THIQs) as Anti-Breast Cancer Agents.

Breast cancer is a leading cause of mortality among women, resulting in more than half a million deaths worldwide every year. Although chemotherapeutic drugs remain the main stay of cancer treatment, it is observed that toxicity to normal cells poses a limitation to their therapeutic values. Moreover, the patient recovery rate from advanced breast cancer by chemotherapy is still unacceptably low. Tetrahydroisoqinoline derivatives (THIQs) were reported to act as selective subtype estrogen receptor antagonists/agonists and may serve as potential therapeutic agents for breast cancer. In continuation of previous work we systematically synthesized and characterized the tetrahydroisoquinoline (THIQs) analogs. In-vitro antiproliferative activity of new substituted tetrahydroisoquinoline analogs were evaluated against human ER (+) MCF-7 (breast), ER (−) MDA-MB-231 (breast) and Ishikawa (endometrial) cancer cell lines using the CellTiter-Glo luminescent cell viability assay. The most active compounds obtained in this study were 2b, 2i, and 3 g as demonstrated by their activity (IC50=0.2 μg/mL, 0.08 μg/mL; 0.61 μg/mL, 0.09 μg/mL; 0.25 μg/mL, 0.11 μg/mL) against MCF-7 and Ishikawa cell lines respectively, in comparison to Tamoxifen activity (IC50=3.99 μg/mL, 7.87 μg/ml). The newly synthesized molecules were docked in the active sites of the ER-α (PDB: 3ERT), ER-β (PDB: 1QKN) and alpha-beta tubulin taxol complex (1JFF) crystal structures to determine the probable binding modes (bioactive conformations) of the active compounds.

Targeting Oxphos Pathway Against Ibrutinib Resistance to Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses to frontline therapy. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) has achieved 68% of overall response rate in relapsed/refractory mantle cell lymphoma (MCL) patients. However, the vast majority of MCL patients experience disease progression, demonstrating that standard-of-care approaches are failing and that a means for targeting ibrutinib resistant MCL is clinically needed. Our hypothesis is that the ibrutinib-resistant MCL may rely on the mitochondrial oxidative phosphorylation (OXPHOS) pathway to produce energy for tumor growth. In this study, we investigated the effects of IACS-010759, a small molecule mitochondrial complex I inhibitor discovered in MD Anderson Cancer Center which can block the OXPHOS pathway, to overcome ibrutinib resistance in MCL in vitro and in a patient-derived xenograft (PDX) model.Methods: The OXPHOS metabolic pathways were investigated by RNASeq in a panel of ibrutinib-sensitive and -resistant MCL samples. Cell growth inhibition assays were tested after 72-hour treatment with IACS-010759 in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, and ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, by CellTiter-Glo luminescent cell viability assay (Promega). Furthermore, an IBN-resistant MCL PDX model was established and the therapeutic effects and tolerability of IACS-010759 were investigated in the primary MCL-bearing PDX model.Results: We have done RNA sequencing (RNASeq) in 7 primary ibrutinib-resistant and 16 ibrutinib-sensitive MCL patient samples, and analyzed the data using Gene Set Enrichment Analysis (GSEA) software. The results demonstrated that the OXPHOS pathway was activated in the primary ibrutinib-resistant MCL cells but not ibrutinib-sensitive MCL cells. Based on the RNASeq data, we selected an OXPHOS inhibitor IACS-010759 to investigate its effects on both primary ibrutinib-resistant and ibrutinib-sensitive MCL cells in vitroand in PDX mice. IACS-010759 significantly inhibited cell proliferation in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, but not in ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, during a 72-hour incubation. Furthermore, the primary ibrutinib-resistant MCL PDX mice were administrated with 10 mg/kg IACS-10759 by oral gavage, for 28 days using a 5 on/2 off dosing schedule. Our data showed that IACS-010759 completely eradicated tumor growth in ibrutinib-resistant MCL PDX mice (n=5, p=0.045). All mice tolerated the treatment dose and no toxicity was found during 28 days of IACS-010759 treatment.Conclusions: The OXPHOS inhibitor IACS-010759 overcomes ibrutinib resistance both in vitro and in the PDX mouse model. The investigation of its mechanism-of-action is ongoing. IACS-010759 could have the potential for clinical use in ibrutinib-resistant relapsed/refractory MCL patients. DisclosuresWang:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Juno Therapeutics: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Dava Oncology: Honoraria; BeiGene: Research Funding; Acerta: Consultancy, Research Funding.

CUDC-907: An Oral HDAC/PI3K Dual Inhibitor with Strong Preclinical Efficacy in MCL Model

IntroductionMantle cell lymphoma (MCL) is an aggressive lymphoma with elevated B-cell receptor activity. Ibrutinib (IBN), a Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have a response rate of 68% in relapsed or refractory MCL patients. However, with the emergence of IBN-resistant lymphomas, new therapies are needed. It is suspected that upregulation of the PI3K-Akt-mTOR pathway allows survival in the presence of IBN. CUDC-907 is a next generation PI3K and HDAC dual-inhibitor currently in phase II clinical trials. Our objective is to investigate the effects of CUDC-907 in IBN-resistant MCL cells in vitro and in PDX model.MethodsMCL cells were seeded at 10,000 cells per well in 96-well plate and were treated with various doses of compounds at the following concentrations: CUDC-907/Ibrutinib 0.015, 0.05, 0.15, 0.5, 1.5, 5, and 15 uM. Cell viability was tested by CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation. Next, MCL cells were incubated with IBN at varying doses (0.39 uM, 1.56 uM, 6.25 uM), CUDC-907 (0.39 uM, 1.56 uM, 6.25 uM), and IBN+CUDC-907 (0.39 uM, 1.56 M, 6.25 uM) for 24 hours. Apoptosis was detected by Annexin V-binding assay. In patient derived xenograft (PDX) model: CUDC-907 was administered at a dose of 50 mg/kg in ibrutinib-resistant MCL-bearing PDX mice daily. Tumor volumes were measured as the length X width2 X 0.5 weekly. Toxicity was also observed every week. Mice were sacrificed once diameter of tumor mass reached 15 mm size.ResultsCUDC-907 inhibited the growth of both ibrutinib-sensitive and resistant MCL cells in vitro. Sensitive cell lines include: Rec-1, Mino, and JVM-13 with IC50 values of 1.1 nM, 1.0 nM, and 5 nM respectively. Resistant cell lines include: Granta-519, Maver-1, and Z-138 with IC50 values of 2 nM, 3 nM, and 1.5 nM respectively. All tested cell lines were more sensitive to CUDC-907 than to ibrutinib. In addition, combination treatments of CUDC-907 and IBN increased cell death in comparison to single agent treatments. Next, one pair of MCL cell lines, Jeko-1 (sensitive to IBN) and Jeko-R (resistant to IBN) were treated for 24 hours with varying doses of CUDC-907 or ibrutinib either as single agent inhibitors or in combination therapies. The results demonstrated that CUDC-907 induced apoptosis in both Jeko-1 and Jeko-R cell lines in a dose-dependent manner. Combination therapies increased cell death in a dose-dependent manner as well. In PDX model, tumor volume in treated mice of ibrutinib-resistant PDX decreased significantly compared with vehicle control (pvalue = 0.032). Control mice also weighed considerably more than treated mice (p value = 0.073). Common toxicities included a decrease in body mass for first 28 days of treatment.ConclusionCUDC-907, a dual inhibitor of PI3K-Akt-mTOR and HDAC, inhibits tumor growth of ibrutinib-resistant MCL in vitro and in PDX model. It would be a potential drug for the patients with ibrutinib-resistant/relapsed MCL. DisclosuresWang:Celgene: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Research Funding; Juno Therapeutics: Research Funding; Asana BioSciences: Research Funding.

Boehmenan, a Lignan From the Chinese Medicinal Plant Clematis armandii, Inhibits A431 Cell Growth via Blocking p70S6/S6 Kinase Pathway.

Previously, we have shown that boehmenan, a natural product isolated from the dried stem of Caulis clematidis armandii, exhibits various biological activities. The current study investigated the effects of boehmenan on the growth of human epidermoid carcinoma A431 cells. Cell viability and 50% inhibiting concentration (IC50) were assessed by CellTiter-Glo luminescent cell viability assay. Cell cycle arrest was measured by flow cytometry. Intracellular reactive oxygen species production and mitochondrial membrane potential (ΔΨm) collapse were analyzed by a fluorescence spectrophotometer. The activation of epidermal growth factor receptor signaling pathway was evaluated by Western blot. The results showed that boehmenan significantly inhibited the growth of A431 cells (IC50 = 1.6 µM) in a concentration- and time-dependent manner. This compound also blocked cell cycle progression at G2/M phase and modulated mitochondrial apoptosis-related proteins, as evidenced by upregulating p21, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase protein levels and by downregulating Bcl-2, pro-caspase-9 levels. In addition, boehmenan also markedly induced intracellular reactive oxygen species production and ΔΨm depolarization in a concentration-dependent manner. Furthermore, boehmenan-attenuated epidermal growth factor mediated the phosphorylation of signal transducer and activator of transcription 3 (STAT3), p70 ribosomal protein S6 kinase (p70S6)/S6 in a concentration-dependent manner. Taken together, our results suggest that boehmenan-mediated antiproliferative property in A431 cells was mediated partially by modulation of mitochondrial function and inhibition of STAT3 and p70S6 signal pathways.

Effect of visible laser light on ATP level of anaemic red blood cell

In this work we present influence of visible laser light on ATP level and viability of anaemic red blood cell (RBC). The visible laser lights used in this work are 460nm and 532nm. The responses of ATP level in anaemic and normal RBC before and after laser irradiation at different exposure time (30, 40, 50 and 60s) were observed. Three aliquots were prepared from the ethylenediaminetetraacetic acid (EDTA) blood sample. One served as a control (untreated) and another two were irradiated with 460nm and 560nm lasers. Packed RBC was prepared to study ATP level in the RBC using CellTiter-GloLuminescent cell Viability Assay kit. The assay generates a glow type signal produced by luciferase reaction, which is proportional to the amount of ATP present in RBCs. Paired t-test were done to analyse ATP level before and after laser irradiation. The results revealed laser irradiation improve level of ATP in anaemic RBC. Effect of laser light on anaemic RBCs were significant over different exposure time for both 460nm (p=0.000) and 532nm (p=0.003). The result of ATP level is further used as marker for RBC viability. The influence of ATP level and viability were studied. Optical densities obtained from the data were used to determine cell viability of the samples. Results showed that laser irradiation increased viability of anaemic RBC compared to normal RBC.

Abstract 3197: BKM120 promotes apoptosis and suppresses tumor growth in medulloblastoma by targeting the phosphoinositide 3-kinase pathway

Abstract Background: Medulloblastoma (MB) is the most common malignant brain tumor in children with poor survival outcome. New treatment strategies are needed for control of MB. The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) intracellular signaling pathway plays a key role in cellular metabolism, proliferation, survival and angiogenesis. This pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. BKM120 (Buparlisib) is an oral pan-class I PI3K inhibitor that targets all 4 isoforms of class I PI3K. BKM120 is currently being clinically evaluated for the treatment of different adult cancers including breast cancer, glioblastoma, prostate cancer, advanced non-small cell lung cancer, and colorectal cancer. In this study, we screened our MB established and patient primary cell lines by genomic profiling analysis, and validated the targeted therapy both in vitro and in vivo in xenograft mouse model. Methods: RNA expression profiling analysis was performed with Affymetrix GeneChip U133 Plus 2.0 genome wide expression cDNA microarray. Analysis was done using R/Bioconductor packages and Partek Genomics Suite. Eleven MB cell lines were treated with increasing concentrations (0-4 μM) of BKM120 for 48 hours. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity of BKM120 and apoptosis in MB cells. Western blot analysis was used to measure phospho-Akt, phospho-mTOR, and cleaved caspase 3 protein levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with CellTiter-Glo luminescent cell viability assay. Xenograft study was performed with DAOY cells implanted in the flank of nude mice and treated with vehicle, BKM120 at 30 mg/kg and 60 mg/kg via oral gavage daily. Results: BKM120 exhibited cytotoxicity in MB cells in a dose-dependent manner by inhibiting activation of downstream signaling molecules Akt and mTOR, and activating apoptotic pathways and inducing cell death in the eleven cell lines tested. IC50s of BKM120 in the MB cell lines ranged from 0.456 to 2.9 μM determined by cell viability assay. Furthermore, BKM120 decreased cellular glycolytic metabolic activity in MB cell lines in a dose-dependent manner. In MB xenograft mouse study, BKM120 significantly suppressed tumor growth and prolonged mouse survival at 30 mg/kg and 60 mg/kg. Conclusion: This study indicates that BKM120 promotes apoptosis and suppresses medulloblastoma tumor growth both in vitro and in vivo. Additional investigation of BKM120 for the treatment of pediatric medulloblastoma is warranted. Citation Format: Ping Zhao, Jacob Hall, Austin Voydanoff, Mary Durston, Elizabeth VanSickle, Abhinav B. Nagulapally, Jeffery Bond, Giselle Saulnier Sholler. BKM120 promotes apoptosis and suppresses tumor growth in medulloblastoma by targeting the phosphoinositide 3-kinase pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3197.

Abstract 3072: Combination of ibrutinib and corticosteroids in B-cell non-Hodgkin lymphomas (NHL)

Abstract Introduction: The B-cell receptor (BCR) signaling pathway is a major driver in the pathogenesis of B-cell malignancies. A vast array of BCR-associated kinases have emerged as rational therapeutic targets, including Bruton's tyrosine kinase (BTK), which plays a pivotal role in BCR signaling. Ibrutinib is a first-in-class, oral, covalent BTK inhibitor approved in the US for patients with mantle cell lymphoma and chronic lymphocytic leukemia (CLL) who have received at least 1 prior therapy, CLL patients with 17p deletion, and patients with Waldenström's macroglobulinemia. While ibrutinib is efficacious as a single agent, combinations with other drugs may further enhance efficacy and increase response rates in patients with NHLs. Corticosteroids are included in nearly all combination regimens for NHL treatment, showing promising results when combined with chemotherapy and antibodies (Cunningham, Lancet 2013). We therefore evaluated corticosteroids in combination with ibrutinib in preclinical models of activated B cell-like (ABC) and germinal center B cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma (FL). Methods: ABC-DLBCL, GCB-DLBCL, and FL cell lines were used in this study. Drug effect on cell growth was evaluated with CellTiter-Glo luminescent cell viability assay (Promega) following treatment with ibrutinib or the combinations for 3 days. Combination index (CI) was determined using CalcuSyn. Synergy score (SS) was calculated using the Chalice Analyzer (Horizon CombinatoRx). Mutation profiles were extracted from the Catalogue of Somatic Mutations in Cancer database. Results: Synergistic growth suppression of dexamethasone and ibrutinib was identified in 7 of 14 GCB-DLBCL cell lines. Dexamethasone had a stronger effect on cells with lower EC50 on ibrutinib treatment. Intriguingly, 4 of these 7 cell lines that showed synergy of the 2 compounds had BCL-2 nonsynonymous mutations, whereas only 1 of the cell lines with no combination effect had BCL-2 silent mutation. In addition to dexamethasone, other corticosteroids, including hydrocortisone, prednisolone, and mometasone, also showed synergy with ibrutinib in GCB-DLBCL (SU-DHL-4, CI = 0.219-0.668, SS = 5.08-15.15; SU-DHL-10, CI = 0.008-0.224, SS = 7.27-11.24), ABC-DLBCL (TMD-8, CI = 0.001-0.016, SS = 21.1), and FL cell lines (DoHH2, CI = 0.020-0.467, SS = 12.3-30.86; WSU-FSCCL, CI = 0.017-0.022, SS = 18.24). The in vivo effect and underlying mechanisms of the combinations are currently under investigation. Conclusions: We identified synergistic inhibitory effects of ibrutinib and corticosteroids on ABC-DLBCL, GCB-DLBCL, and FL cell growth, providing a rationale for the design of combination clinical trials in these NHL types. Further understanding of the mechanisms contributing to the synergy may help to stratify patient populations and expand treatment to other lymphoid neoplasms. Citation Format: Hsu-Ping Kuo, Sidney Hsieh, Karl J. Schweighofer, Leo WK Cheung, Mutiah Apatira, Mint Sirisawad, Shiquan Wu, Karl Eckert, Yu Liang, Jeff Hsu, Chun-Te Chen, Darrin Beaupre, Betty Y. Chang. Combination of ibrutinib and corticosteroids in B-cell non-Hodgkin lymphomas (NHL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3072.

Abstract 592: Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation

Abstract Death receptor 5 (DR5) is a highly interesting tumor target based on the enhanced sensitivity of cancer cells for DR5-dependent apoptosis. In recent years, multiple therapeutic DR5 antibodies have been evaluated in the clinic for which results however have been disappointing. IgG molecules against membrane-bound targets have shown an ability to form ordered hexameric structures upon antigen binding, a process that is dependent on Fc-Fc interactions between IgG molecules. We identified specific mutations in the human IgG1 Fc domain that enhance such antigen-dependent hexamerization while retaining solution-monomericity and developability characteristics of regular IgG1 molecules (HexaBody technology). We hypothesized that antibody-mediated hexamerization, when applied to DR5-specific antibodies, would enhance DR5 signaling and apoptosis, resulting in strongly improved therapeutic potential. The technology was applied to two non-crossblocking DR5-specific IgG1 antibodies, IgG1-DR5-01 and IgG1-DR5-05, by mutating a glutamic acid residue at position 430 in the Fc domain to glycine (the HexaBody mutants were designated Hx-DR5-01 and Hx-DR5-05). Cytotoxicity of the DR5 antibodies was explored in vitro using the CellTiter-Glo luminescent cell viability assay and the Caspase-Glo 3/7 assay in a broad panel of cancer cell lines, and in vivo in xenograft models. Both Hx-DR5-01 and Hx-DR5-05 induced increased cytotoxicity compared to their wild type (WT) IgG1 counterparts. Moreover, the combination of Hx-DR5-01 and Hx-DR5-05 (referred to as Hx-DR5-01/05) was found to be more potent than either Hx-DR5-01 or Hx-DR5-05 alone, or than the combination of the WT antibodies (IC50 in BxPC3 cells 0.5 and 1.5 μg/ml; maximal cytotoxicity 91% and 25% for Hx-DR5-01/05 and WT IgG1-DR5-01/05 respectively). In contrast to wild type agonistic DR5 antibodies, tumor cell killing by Hx-DR5-01/05 was independent of secondary crosslinking. Potent anti-tumor activity was observed in seven xenograft models for multiple indications, with Hx-DR5-01/05 consistently showing significantly better efficacy than the WT DR5 comparator antibody conatumumab. The cytotoxic activity of DR5 antibodies was significantly enhanced by the introduction of a hexamer-enhancing mutation in the IgG1 Fc domain. Maximal killing activity was obtained by combining two non-crossblocking DR5 antibodies carrying this mutation (Hx-DR5-01 and Hx-DR5-05). The strong cytotoxicity of Hx-DR5-01/05 was completely dependent on target binding but, in contrast to WT antibodies, did not require secondary crosslinking. These promising pre-clinical results support the selection of Hx-DR5-01/05 for clinical development for the treatment of cancer. Citation Format: Marije B. Overdijk, Kristin Strumane, Antonio Ortiz Buijsse, Claudine Vermot-Desroches, Andreas Lingnau, Esther C.W. Breij, Janine Schuurman, Paul W.H.I. Parren. Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 592.

Abstract 3198: HDAC inhibitor panobinostat as a selective agent, synergizes with chemotherapeutics in medulloblastoma and neuroblastoma

Abstract Background: Embryonal tumors of the central nervous system continue to present therapeutic challenges with high rates of relapse and poor prognosis at advanced stages. Medulloblastoma (MB) accounts for over 15% of pediatric brain tumors, with a five-year survival rate of 62%. Neuroblastoma (NB) is the most common extracranial solid tumor in children, accounting for 15% of pediatric cancer deaths. Among many pathways that contribute to MB and NB potency, several histone deacetylases (HDACs) have been shown to prevent cell differentiation. In cells expressing wt-p53, HDAC inhibitors induce nuclear relocalization of p53 to the nucleus to induce expression of the cell cycle inhibitor p21/Waf1/Cip1. As most NB tumors express wt-p53, HDAC inhibitors are promising candidates for therapy. Panobinostat has been shown to induce differentiation, cell cycle arrest, and apoptosis in NB cells. This effect is partially due to down-regulation of CHK1, a pathway by which cancer cells can develop resistance to conventional chemotherapeutic drugs. This study proposes panobinostat coupled with conventional chemotherapeutics such as doxorubicin, etoposide, and velcade, as an effective target against the HDAC pathway in MB and NB. Methods: Established MB and NB cell lines and patient derived cell lines (MB: DAOY, D283, D341, 384MED, 458MED, 487MED, 556MED, 581MED, 721MED; NB: BE(2)-C, CHLA-90, SMS-KCNR, SH-SY5Y, MGT8-117-08, BIO-036-08) were used to quantify panobinostat's effects. Cell viability was measured using Calcein AM fluorescent assay at doses of 0.39-50 nM. Isobologram analysis of panobinostat in combination with doxorubicin, etoposide, and velcade were generated using Calcein AM. Western blot was used to measure HIF1 alpha, CHK1, and acetyl H4, Caspase 3 and PARP levels. ATP level per cell was measured using CyQuant fluorescent DNA assay with CellTiter-Glo luminescent cell viability assay. Results: Cell viability assays show cytotoxicity of panobinostat in MB and NB cell lines, with IC50 values from 2-10 nM in MB cells, 5-20 nM in established NB cell lines and 23-91 nM in patient derived NB lines. ATP/cell activity was inhibited in MB and NB cells following treatment with panobinostat alone. Isobologram experiments suggest synergistic cytotoxicity of panobinostat in combination with doxorubicin, etoposide, and velcade in NB cell lines BE(2)-C and SMS-KCNR. Western blot analysis indicates caspase-mediated apoptosis occurs, with inhibition of overexpressed HDAC proteins among multiple classes in MB and NB cells. Conclusions: This study indicates that panobinostat targets the HDAC inhibition pathway of MB and NB cells. Additionally, panobinostat synergizes with doxorubicin, etoposide, and velcade in inducing apoptosis in MB and NB cells. This study provides rationale for initiation of a clinical trial in treating MB and NB patients with panobinostat in combination with conventional chemotherapeutics. Citation Format: Amanda B. Witte, Mary Durston, Ping Zhao, Abhinav B. Nagulapally, Jeffrey Bond, Giselle L. Saulnier Sholler. HDAC inhibitor panobinostat as a selective agent, synergizes with chemotherapeutics in medulloblastoma and neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3198.

Abstract 196: The oncogenic role of PRL-3 in multiple myeloma through regulation of Src kinase family members

Abstract Introduction: Phosphatase of regenerating liver 3 (PRL-3) is a dual specificity phosphatase and its up-regulation in cancer cells is related to poor prognosis. We have previously described PRL-3 as a downstream target of IL-6 in multiple myeloma (MM) by demonstrating that it was upregulated in response to this cytokine. The Src kinase family (SFK) is composed of 8 members and regulates proto-oncogenic cellular pathways. As it has been reported that the SFK members Lyn, Fyn and Hck are important in signal transduction of IL-6 signals in MM cells and that Src is reported to be the major kinase regulated by PRL-3 in colon cancer, we evaluated effects of PRL-3 on activation of Fyn, Lyn, Hck and Src in MM. Methods: By retroviral transduction in the IL-6-dependent MM cell line INA-6,we generated functional PRL-3(INA-PRL-3) overexpressing cells, and control cells expressing catalytically inactive mutant PRL-3 (C104S) or empty vector (Mock). We measured global tyrosine (Y) phosphorylation (P) by immunoblotting using (P-Y1000) antibody. We measured the expression of 4 SFK members by qRT PCR and immunoblotting. Activity of SFK members was evaluated using MILLIPLEX MAP8-plex Human SFK kit. We confirmed MILLIPLEX result for Src by immunoblotting with antibody against P-Y416 Src (activating phosphorylation site). We also measured Src activity after inhibiting PRL-3 by PRL-3 inhibitor I or shRNA. Finally, we showed the influence of PRL-3 on cell growth and cell sensitivity to two Src inhibitors (PP2 and Su6656) by using CellTiter-Glo Luminescent Cell Viability Assay. Results: INA-PRL-3 had more global P-Y than C104S and Mock cells in the absence of IL-6 and showed a pattern of P-Y more similar to that of the parental INA-6 cells grown in the presence of IL-6. C104S and INA-PRL-3 cells showed lowest and highest activity, respectively, of Lyn and Src. Stimulation of cells with IL-6 increased active Lyn and Src but still C104S had lowest activity. We did not observe significant difference in activity of Fyn and Hck. Measuring total amount of 4 SFK members showed up-regulation of total Hck and Fyn in C104S cells in mRNA and protein level. This indicated that inactive PRL-3 in C104S cells leads to less SFK activity compared to both INA-PRL-3 and Mock cells. Increased total amount of Hck and Fyn in C104S could be the result of diminished negative feedback regulation of expression of SFK members by their active forms. INA-PRL-3 showed more P-Y416 Src also by immunoblotting and its inhibition by PRL-3 inhibitor or shRNA decreased Src activity. INA-PRL-3 had higher growth rate than both C104S and Mock cells. This difference was more prominent in the absence of IL-6 and high SFK activity in INA-PRL-3 could be one of the possible reasons for this. INA-PRL-3 also showed high sensitivity to both PP2 and Su6656. Conclusion: Inhibition of PRL-3 and SFK members could be considered as a possible treatment for MM. Citation Format: Pegah Abdollahi, Esten Nymoen Vandsemb, Magnus Aassved Hjort, Kristine Misund, Toril Holien, Torstein Baade Rø, Tobias Schmidt Slørdahl, Magne Børset. The oncogenic role of PRL-3 in multiple myeloma through regulation of Src kinase family members. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 196.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.

Abstract P3-14-07: Intra-cellular dsRNA receptor RIG-I: A ubiquitous novel target for treatment of chemotherapy drug resistant breast cancer

Abstract INTRODUCTION: Toll-like receptor 3 (TLR3), the cell surface receptor for double stranded RNA (dsRNA), expressed in ∼40% of advanced primary breast cancers (T2/T3/N+) has been shown to be an effective therapeutic target for synthetic dsRNA poly A:U in combination with radiation and adjuvant chemotherapy, producing a significant decrease in risk of metastatic relapse (HR 1.85-2.0; 1.03-3.89; Cancer Res;71:1607). AIM: The present pre-clinical study aimed to explore the therapeutic potential of a ubiquitously expressed intracellular dsRNA receptor, retinoic-acid-inducible gene-I (RIG-I) - a cytoplasmic pathogen recognition receptor directed at pathogen-associated molecular pattern (PAMP) motifs to differentiate viral from cellular dsRNAs. We have shown Ampligen, poly I:C12U (a synthetic dsRNA polymer designed to rapidly degrade in vivo to prevent the toxicity of long dsRNA polymers such as poly I:C) is capable of entering cells in its fragmented dsRNA oligomeric form (<1–2 kb) optimal for activation of intra-cellular RIG-I (J Exp Med 2008;205:1601–1610). METHODOLOGY & RESULTS: Preliminary experiments with Ampligen on several human breast cancer cell lines (MCF-7 & MDA-MB 453) and normal human mammary epithelial & fibroblast cell lines (HMEC & HFC) unexpectedly showed it to consistently cause a significant loss of cell viability (CellTiter-Glo Luminescent Cell Viability Assay) in p53-deficient drug resistant (5-FU/doxorubicin) MDA-MB 453 cell line in contrast to cell growth arrest (Guava cell Cycle Assay) in p53 wild type MCF-7 cancer and the two non-neoplastic cells lines. This selective effect was confirmed using syngeneic clones of MCF-7 breast cancer cells stably transfected with a dominant negative p53 construct or vector alone: p53-function blocked (DD1) vs p53-function active (EV1) MCF-7 cell lines, respectively (J Nucl Med 2006; 47:1525–1530), and shown to be associated with RIG-I specific mRNA induction (RT-PCR) and Type I interferon pathway activation both inhibited by BX795 (selective inhibitor of IRF3 activation and IFN-b production). Decitabine (DNA demethylating drug capable of intra-cellular generation of dsRNA through transcriptional activation of Alu retrotransposons - PNAS 2012; December 10: E89–E98 ) was next tested as an alternative source of intra-cellular dsRNA, and found to produce results similar to Ampligen on DD1 and EV1. Work is in progress to examine dose response and time-course relationships of the effects Ampligen or decitabine added singly or in combination with chemotherapeutic drug (e.g. doxorubicin) on DD1 and triple negative breast cancer cell lines such as MDA-MB 453 to explore potential therapeutically most effective protocols. PROVISIONAL CONCLUSIONS: Intra-cellular dsRNA receptor RIG-I constitutively expressed in all cells offers a more ubiquitous target compared to TLR3 for the treatment of breast cancer. Ampligen and decitabine with their selective growth inhibitory effect on drug resistant p53-deficient breast cancer cell lines, merit testing as novel drugs for treatment of p53-deficient drug resistant breast cancer e.g. triple negative breast cancer frequently (∼70%) associated with drug resistance and altered p53 status. Citation Format: Jasani B, Navabi N, Barrett-Lee P, Thompson A, Chester J, Mason M. Intra-cellular dsRNA receptor RIG-I: A ubiquitous novel target for treatment of chemotherapy drug resistant breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-07.

The Effect of a Retinoic Acid Derivative on Cell-Growth Inhibition in a Pulmonary Carcinoma Cell Line.

Pulmonary carcinoma is a major cause of cancer-related death worldwide. Because the prognosis remains poor, the development of novel therapeutic approaches is highly desirable. In this study, we investigated the effect of Tamibarotene (Am80), a retinoic acid derivative, on the growth of human lung adenocarcinoma cell line A549. Our ultimate goal in this study is to provide pulmonary carcinoma therapy with a new approach. First, we treated A549 cells with Am80 to clarify the effect of cell-growth inhibition. Am80 significantly reduced the viability of A549 cells in a dose- and time-dependent manner. The IC50 value, which was determined using CellTiter-Glo Luminescent Cell Viability assay, of Am80 and all-trans retinoic acid (ATRA) against A549 cells at 6 d was 49.1±8.1 µM and 92.3±8.0 µM, respectively. Furthermore, Am80 reduced the anchorage-independent cell-growth ability of A549 cells. However, it was not an apoptosis-mediated mechanism. These results suggest that Am80 can be used as an effective, novel cell-growth inhibitor in lung adenocarcinoma.

ABT199 and ONC201 in Diffuse Large B Cell Lymphoma Cell Lines

Background:Diffuse Large B cell lymphoma, the most commonly diagnosed type of non-Hodgkin lymphoma is curable in many cases, despite this, up to 30% of patients will relapse after initial therapy, necessitating salvage chemotherapy and transplantation if feasible. There is a pressing need for novel treatment strategies in highly chemo refractory cases. ONC201 is a small molecule that induces p53-independent cell death in tumor cells while sparing normal cells through a number of putative mechanisms, including inactivation of the pro-survival kinases Akt and ERK. ONC201 is currently entering Phase I/II clinical trials as a monoagent in adult advanced cancers. ONC201 promotes up-regulation of TRAIL gene transcription by inactivating AKT and ERK kinases which leads to translocation of transcription factor Foxo3a into the nucleus. It appears to act on a p53 independent pathway.ABT-199 is a selective, potent and orally bioavailable small molecule that selectively inhibits BCL-2 and triggers apoptosis. The Bcl-2 family of proteins is key regulator of the apoptotic process, comprising proapoptotic and prosurvival proteins. BH3-only proteins (such as Bim) bind to pro-survival proteins and cause increased permeability of mitochondrial membrane, release of cytochrome c and activation of caspases through release of Bax and others.Methods:Cell lines Pfeiffer and Toledo were purchased from ATCC and one patient DLBCL cell specimen from the ascitic fluid was cultured for use in experiments. ABT-199 was purchased from MedKoo Biosciences and ONC201 was provided from Oncoceutics.Cytotoxicity was evaluated by using the CellTiter-Glo Luminescent Cell Viability Assay as per the manufacturerÕs instructions. Cell viability was measured over time in response to treatment with ONC201(1-64μM) and ABT-199(128 nM-4μM).Western Blotting was performed on treated cells with well-established methodologies, with antibodies to c-Myc, Bcl-2, pAKT, pERK.Results:Immunohistochemical Staining Pattern of Cell LinesTable 1PatientToledoPfeifferBcl-2++++++Bcl-6+++++c-Myc++++++p-ERK++++p-AKT++++Bax++NANABim++NANAMean IC50 Calculated for Cell LinesTable 2Cell LineTherapeutic Agent ABT199 ONC201Patient Sample8 μM5 nMToledo9 μM28 nMPfeiffer6 μM2 μMSHAPEConclusion/Discussion:The patient cell line, an ascitic fluid sample of DLBCL was sensitive to both ONC201 and ABT-199 and manifested bright Bcl-2 expression, the target of ABT-199. In this series there was a higher sensitivity to ABT199 in DLBCL cells with higher Bcl-2 expression. ONC201 down regulated pAKT expression, as seen in Western Blots in treated cells, consistent with prior investigation with the agent.We further found that ONC201 synergizes to potentiate cytotoxicity with ABT199, as demonstrated in the cell viability assay for Toledo cell lines (at the 24 hour time point), which were the least sensitive to ONC201 (highest IC50) when given as a single agent. Yet, when combined with increasing doses of ABT199, there was synergistic lymphoma cell kill with a fixed dose of ONC201. Together these results suggest that ONC201 has potential as an antitumor agent in NHL as monoagent and in combination with ABT-199, which may be further explored in phase Ib/II trials. Further analysis in larger patient sample series may elucidate the biomarkers that predict for greater therapeutic sensitivity to these highly potent lymphoma agents. [Display omitted] DisclosuresAllen:Oncoceutics, Inc: Employment, Equity Ownership. Eldeiry:Oncoceutics, Inc: Equity Ownership.