CellTiter-Glo Luminescent Cell Viability Research Articles

Abstract 5430: Inhibition of de novo and salvage pathways for dNTP synthesis enhances sensitivity to ionizing radiation in pancreatic neuroendocrine tumor cells

Abstract Introduction: The treatment of gastroenteropancreatic neuroendocrine tumors (GEP-NET) with ionizing radiation (IR) is a promising treatment modality for a disease that carries a poor prognosis. However, these tumors confer high levels of radioresistance based on their ability to repair DNA damaged by IR. Deoxynucleoside triphosphates (dNTPs) are needed for DNA repair and are produced by two pathways: de novo and salvage, which depend on ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), respectively. Ataxia telangiectasia and Rad3-related protein (ATR) activates dCK and can be targeted with inhibitors. We hypothesized that blocking both pathways for dNTP synthesis with an ATR inhibitor (ATRi) and the RNR inhibitor (RNRi), triapine, will sensitize GEP-NETs to IR in vitro. Methods: (i) The CellTiter-Glo (Promega) luminescent cell viability assay established drug sensitivities of two pancreatic NET cell lines (BON and QGP-1) for triapine and three ATRi (AZD6738, VX-970, BAY1895344). Assays were performed with at least three replicates. Data were fit using a four-parameter log-logistic model, and IC50 values were calculated with R statistical software. (ii) Loewe synergy models were used to calculate the two-drug combination effects in both cell lines with synergy scores greater than 0 indicating synergistic effects and scores less than 0 representative of antagonistic effects. (iii) Clonogenic assays were performed and analyzed via colorimetry with sulforhodamine B to assess the effect of each drug alone and in combination with IR. (iv) Immunoblots were used to assess ATR activation following IR, RNRi, and ATRi treatments. Results: (i) Cell viability assays showed lower IC50 values in the BON cell line compared to the QGP-1 cell line (BON cell IC50 : triapine 2.6µM ± 0.6µM, AZD6738 1.0µM ± 0.2µM, VX-970 204nM ± 20nM, BAY1895344 139nM ± 15nM; IC50 in QGP-1 cells: triapine 6.1µM ± 2.7µM, AZD6738 7.9µM ± 2.9µM, VX-970 1.7µM ± 0.5µM, BAY1895344 1.1µM ± 2.8µM). (ii) Loewe synergy models estimated the combination of BAY1895344 and triapine to be synergistic with scores greater than 0 in both cell lines (BON 9.89, p-value 4.07e-11; QGP-1 15.91, p-value 1.96e-7). (iii) Clonogenic assays showed an increase in efficacy of BAY1895344 when combined with IR in both BON (IC50 s: 150nM ± 8nM without radiation versus 18nM ± 35nM with 2Gy) and QGP-1 cells (IC50 values: 277nM ± 48nM without radiation versus 93nM ± 4nM with 2Gy). (iv) Western blot analysis showed strong activation of the ATR pathway with IR and triapine alone with subsequent inhibition by an ATRi. Conclusion: Our findings show that treatment of GEP-NET cell lines with inhibitors that block the de novo and salvage pathways for dNTP production markedly sensitize these cells to subsequent IR. The combination of IR with both an ATRi and an RNRi has the potential to be an effective treatment modality for GEP-NETs. Citation Format: Jennifer Castle, J. Robert McCorkle, Jeremy Johnson, Aman Chauhan, Percy Ivy, Susanne Arnold, William Carson, B. Mark Evers, Piotr Rychahou, Jill Kolesar. Inhibition of de novo and salvage pathways for dNTP synthesis enhances sensitivity to ionizing radiation in pancreatic neuroendocrine tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5430.

Abstract A21: Utilizing endoscopic-derived gastric cancer organoids for personalized neoadjuvant chemotherapy

Abstract Introduction: Patient-derived organoids (PDOs) have become attractive tools for genetic studies, biomarker identification, drug screening, and preclinical evaluation of personalized medicine strategies. Previously, we created gastric cancer PDOs from esophagogastroduodenoscopy (EGD) biopsy specimens. As a continuation of our previous work, we sought to optimize methods for creating EGD-derived PDOs for drug sensitivity testing within clinically actionable time constraints. Methods: We previously established a standard operating protocol for creating PDOs from EGD specimens of patients with gastric cancer. To optimize the development of PDOs, we enrolled patients with gastric adenocarcinoma undergoing EGD at Markey Cancer Center. During diagnostic EGDs, additional research biopsies were collected for creation of gastric cancer PDOs and placed in ice-cold organoid medium. We employed different dissociation methods based on the texture of the biopsy tissues. Briefly, soft tissues were washed and isolated in a chelating solution to release the glands, while the hard tissues were digested with collagenase and dispase to release epithelium. Glands or epithelia cells were collected, resuspended in BME (basement membrane extract), and plated in 2 wells of 24-well chambers. On day 4-6, PDOs were dissociated and passaged using manual pipetting. For passage 2 on day 9-11, PDOs were isolated into smaller and uniform-sized PDOs or single cells with both mechanical and enzymatic (TripLE Express) dissociation and plated in a 96-well plate at 10uL (500-1000 cells)/well. To test drug sensitivities, PDOs were treated on day 12 with current standard-of-care cytotoxic combination chemotherapies (e.g., FLOT, ECF, FOLFIRI, and FOLFOX) or solvent control. We tested concentrations determined by the Cmax and AUC of each drug associated with a single highest recommended dose in drug product label. We performed LIVE/DEAD assay on CellInsight CX7 High-Content Screening (HCS) Platform (ThermoFisher) and CellTiter Glo luminescent cell viability assay consecutively to measure response of PDOs to chemotherapy regimens. Result: Consistent with our prior experience, we successfully developed EGD-derived gastric cancer PDOs from patients undergoing diagnostic EGD. Using our modified technique for PDO creation and expansion, we obtained sufficient and uniform-sized PDOs at the second passage on day 10 and then treated these PDOs with current standard-of-care chemotherapy regimens to predict in vivo drug response. Creation of EGD-derived PDOs and drug sensitivity testing was feasible within two weeks of tissue collection. Conclusions: We have optimized methods to create, grow, and test EGD-derived PDOs within a clinically actionable time period. Our modified technique yielded higher concentration and more uniform-sized PDOs, providing sufficient biologic material for future implementation of rapid and comprehensive drug testing for neoadjuvant chemotherapy trials. Citation Format: Mei Gao, Miranda Lin, Wesam M Frandah, Moamen Gabr, Houssam E. Mardini, Michael Cavnar, Joseph Kim. Utilizing endoscopic-derived gastric cancer organoids for personalized neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A21.

Abstract P5-21-16: Preclinical studies of RX-5902, a beta-catenin modulator in triple negative breast cancer

Abstract Background: RX-5902 (Supinoxin) is a novel anti-cancer compound that targets phosphorylated p68 RNA helicase, a member of the DEAD box family of helicases, affecting upstream and downstream molecules in the Wnt canonical pathway. As a single agent, RX-5902 exhibits strong growth inhibition in both in vitro and in vivo settings. Specifically, RX-5902 enhances survival and tumor growth inhibition in numerous xenograft models, including ovarian, renal and breast. We have previously shown RX-5902 inhibits cell growth in a dose-dependent fashion in the triple-negative breast cancer (TNBC) xenograft MDA-MB231. In the current study, we have expanded our investigation of the therapeutic potential of RX-5902 against TNBC using both in vitro and in vivo preclinical models. Methods: RX-5902 was provided by Rexahn, Inc. (Rockville, MD). Cell proliferation was measured using the Cell-Titer Glo luminescent cell viability assay (Promega). Apoptosis was assessed using Incucyte Caspase 3/7 Green apoptosis assay (Essenbioscience). Immunoblots of MDA-MB-231 cell line were probed for ß-catenin (Cell Signaling). Syngeneic 4T1 murine TNBC mice were obtained from Sippr-BK Laboratory Animal Co (Shanghai, China) and tumor volumes were measured twice a week. When the mean tumor volumes reached ˜90 mm3, mice were randomized and treated with vehicle or RX-5902 PO daily alone or in combination with anti-CTLA4 or anti-PD-1 BIW for 3 weeks. Tumor growth inhibition (TGI) was calculated at Day 25. Results: A panel of 18 TNBC cell lines were treated with RX-5902 and effects on cell proliferation were measured by the Cell Titer-Glo assay. Using 100nM as a cutoff, 14 sensitive lines and 4 resistant lines were identified, with an average IC50 of 56 nM in the sensitive lines. Of these, we chose 2 sensitive lines (MDA-MB-231, HCC1806) and 2 resistant lines (MDA-MB-436 and CAL-120) and assessed induction of apoptosis by the Incucyte caspase activity assay. Robust induction of apoptosis was observed in both sensitive lines (N=3). These lines were then subjected to cell cycle analysis by flow cytometry, which revealed a pronounced G2/M cell cycle arrest and aneuploidy following exposure to RX-5902. Western blot analysis of the MDA-MB-231 cell line showed decreases in the Wnt pathway-related protein nuclear ß-catenin in doses ranging from 20 nM to 200 nM. Finally, the therapeutic efficacy of RX-5902 was assessed as a single agent and in combination with two immune-oncology agents in the treatment of the TNBC 4T1 animal model. RX-5902 as a single agent showed dose dependency in the 4T1 model, and when given in combination with either anti-CTLA4 or anti-PD1 showed an additive effect (p<0.001). All the treatments were well-tolerated and no severe body weight loss was observed in this study. Conclusions: RX-5902 showed efficacy against several in vitro and in vivo preclinical models of TNBC. RX-5902 resulted in G2/M arrest and induced apoptosis in sensitive TNBC cell lines and decreases in nuclear beta-catenin. In vivo, RX-5902 demonstrated additive anti-tumor effects when combined with either anti-CTLA4 or anti-PD1 immunotherapies. Together, these finding indicate that RX-5902 may have important clinical implications for the treatment of TNBC. A phase 2a clinical study in metastatic TNBC is ongoing..training_cert Citation Format: Tentler JJ, Frank JG, Kim DJ, George C, Lee YB, Ely B, Tan AC, Kim J, Pitts TM, Capasso A, Dailey KL, Eckhardt G, Diamond JR. Preclinical studies of RX-5902, a beta-catenin modulator in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-16.

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

Abstract 2229: Synthesis of heterocyclic compounds as anticancer agents.

Abstract Cancer is a very complex disease, linked with different initiating causes, cofactors and promoters, and several types of cellular damages. There is a strong correlation between chronic inflammatory conditions in a particular organ and cancer specific to that organ. The longer the inflammation persists, the higher the risk of associated carcinogenesis. Drug discovery process has changed dramatically over the past decade as there is an increasing demand to obtain more drug candidates and decrease attrition during drug development. Chromone scaffold [(4H)-1-benzopyran-4-one] has been recognized as a pharmacophore of a large number of bioactive molecules of either natural or synthetic origin. Until now, numerous biological effects such as anti-inflammatory, antitumoral, and antimicrobial activities especially in popular medicine, have been attributed to this benzo-γ-pyrone nucleus. Compounds of this type also possess inhibition properties towards different enzymes such as oxidoreductases, kinases, tyrosinases, lipoxygenases and cyclooxygenases. Chemotherapeutic agents like tetrahydropyridine (THP) analogs exhibit diverse biological activities like analgesic and anti-inflammatory effects. Our earlier research results indicated that some of the THP derivatives showed COX-2 inhibition and anti-inflammatory activities on rat paw edema assay studies. Incorporation of THP moiety might enhance biological activity of the chromone derivatives. Herein, we report the synthesis of substituted N-(5,6-dihydropyridin-1-(2H)-yl)-4-oxo-4H-chromene-2-carboxamide as anticancer and anti-inflammatory agents. The starting material 4-oxo-4H-chromene-2-carbonyl chloride was obtained by the reaction of 4-oxo-4H-chromene-2-carboxylic acid, dissolved in 1,2-dichloroethane and SOCl2 containing few drops of DMF under reflux for 4h. The crude acid chloride obtained by removing the excess of SOCl2 and solvent under vaccum, was used immediately for next step. Reaction of the acid chloride with substituted N-amino pyridinium salt in anhydrous tetrahydrofuran gave stable ylides. This was followed by reduction with sodim borohydride in absolute ethanol, which furnished the target compound. These compounds were evaluated for their cytotoxic effects on MCF-7 ER positive breast cancer cells, MDA-MB ER receptor negative breast cancer cells, and Ishikawa cell line using the CellTiter-Glo (CTG) luminescent cell viability assay. N-(4-tert-butyl-5,6-dihydropyridin-1(2H)-yl)-6-chloro-7-methyl-4-oxo-4H-chromene-2-carboxamide showed the cytotoxicity with an IC50 values of 73.28 μM on MDA-MB-231 cell lines. 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, Mohammad A. Ghaffari, Suresh Eyunni, Nelly Mateeva, Bereket Mochona. Synthesis of heterocyclic compounds as anticancer agents. [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 2229. doi:10.1158/1538-7445.AM2013-2229

Abstract 3816: DNA variation at BARD1 predicts response to aurora kinase inhibition in neuroblastoma.

Abstract Background: Neuroblastoma remains responsible for a disproportionately high number of pediatric cancer deaths, necessitating novel treatment approaches. Our lab has previously reported DNA variations in the BRCA1-associated RING domain 1 (BARD1) gene to be significantly associated with high-risk neuroblastoma in an ongoing genome-wide association study (Capasso Nat Gen 2009). The expression of an oncogenic isoform, BARD1β, correlated with this disease associated common variation, and isoform specific depletion showed differential growth inhibition in a risk haplotype dependent manner (Bosse Can Res 2012). Furthermore, this isoform was shown to bind to and stabilized aurora kinase A and B in neuroblastoma cells, suggesting a mechanism for its oncogenicity and a potential therapeutic strategy. Methods: Short interfering RNAs (siRNAs) targeting AURKA, AURKB and BARD1β were tested individually in a panel of neuroblastoma cell lines (n=8) stratified by risk haplotype. Cell growth was measured by real time cell electronic sensing (RT-CES), and mRNA knockdown was confirmed by quantitative PCR. The pharmacologic inhibitors MLN8237, a selective aurora kinase A inhibitor; AZD1152, a selective aurora kinase B inhibitor; and CCT137690, a pan-aurora kinase A and B inhibitor were tested in vitro in an expanded neuroblastoma cell line panel (n=10), and cell viability was measured with a CellTiter-Glo(R) Luminescent Cell Viability Assay. Results: AURKB knockdown resulted in a trend of greater growth inhibition in neuroblastoma cell lines harboring a BARD1 risk haplotype, directly correlating with growth inhibition following BARD1β knockdown (R2=0.57, p=0.03). AURKA depletion showed significant growth inhibition (mean inhibition=45%, p<0.0001), but no correlation with BARD1 genotype was observed. Aurora A kinase inhibition with MLN8237 resulted in significant cytotoxicity (IC50 30 -750nM), and further analysis of published studies using MLN8237 in vivo on murine xenograft models of neuroblastoma showed that the cell lines with the BARD1 risk haplotype had significantly greater tumor reduction with this single agent therapy. AZD1152 and CCT137690 were less potent inhibitors of growth (IC50 30nM-10mM and 250nM-3mM, respectively). Conclusion: Taken together, these data support a model of BARD1 acting in a pathway involving the aurora kinase proteins in neuroblastoma cells, suggesting that BARD1 genotypes have the potential to be a biomarker of aurora kinase inhibitor activity, but the associations are complex and unlikely to be binary predictors of sensitivity or insensitivity. These data support the continued investigation of MLN8237 in clinical trials for high-risk neuroblastoma and further exploration of the mechanisms by which DNA variation at BARD1 influences neuroblastoma oncogenicity and therapeutic vulnerabilities. Citation Format: Vanessa M. Pineros, Kris R. Bosse, Lori S. Hart, Sharon J. Diskin, JulieAnn Rader, John M. Maris. DNA variation at BARD1 predicts response to aurora kinase inhibition in neuroblastoma. [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 3816. doi:10.1158/1538-7445.AM2013-3816

Abstract 3903: Synthesis of substituted N-{4-[(2-hydroxyethyl)sulfanyl]-3,6-dihydropyridin-1(2h)-yl} benzamide/benzenesulfonamide as antiinflamatory and anticancer agents

Abstract Breast cancer is the most common type of cancer among women in the industrialized world, accounting for nearly one of every three cancers diagnosed. There is a strong association between chronic inflammatory conditions in a particular organ and cancer specific to that organ. The longer the inflammation persists, the higher the risk of associated carcinogenesis. There is abundant epidemiological and experimental evidence that NSAID's might inhibit tumor development in a number of organs. COX-2 expression may be a fundamental step in breast cancer pathogenesis acting through prostaglandin-dependent mechanisms. Tetrahydropyridine (THP) analogs are compounds of diverse biological activities like analgesics, anti-inflammatory and chemotherapeutic agents. Our earlier research results indicated that some of the THP derivatives showed COX-2 inhibition and anti-inflammatory activities on rat paw edema assay studies. There has been remarkable effort in developing new classes of compounds to minimize the side effects of the NSAIDs and increase the selectivity of COX-2 inhibitors. We now report the synthesis of substituted N-{4-[(2-hydroxyethyl)sulfanyl]-3,6-dihydropyridin-1(2H)-yl} benzamide/benzenesulfonamide as anti-inflammatory and anticancer agents to continue the development of new classes of anti-inflammatory and anticancer agents. The starting material ethyl (pyridin-4-ylsulfanyl)acetate was obtained by the reaction of (4-pyridylthio)acetic acid dissolved in ethanol and several drops of 98% H2SO4 and the mixture was heated to reflux for 12 h. Mesitylene sulfonyl chloride was added with stirring to a solution of ethylacetohydroxymate and triethylamine in dimethylformamide at 0o C. Hydrolysis of this compound with the mixture of p-dioxane-70% perchloric acid and allowing them to react for 45 min gave a white solid of O-mesitylene sulfonyl hydroxylamine (MSH). Ethyl (pyridin-4-ylsulfanyl)acetate was reacted with MSH in dichloromethane to produce 1-amino-4-(2-ethoxy-2-oxoethylthio)pyridinium mesitylenesulfonate. Reaction of the amino salt with substituted benzoyl chloride/benzenesulfonyl chloride in anhydrous tetrahydrofuran gave stable benzoyl/benzene sulfonyl imino ylides. This was followed by reduction with sodium borohydride in absolute ethanol, which furnished the target THP compounds. These compounds were evaluated for their cytotoxic effects on MCF-7 estrogen receptor positive breast cancer cells, MDA-MB-231 estrogen receptor negative breast cancer cell line, and Ishikawa cells, using the CellTiter-Glo (CTG) luminescent cell viability assay. Two compounds showed potent anticancer activities using these cell lines. This research was supported by NIH/ RCMI Grant # G12 RR 03020 and Pharmaceutical Research Center NIH/NCRR Grant 1 C06 RR12512-01. 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 3903. doi:1538-7445.AM2012-3903

Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

Abstract 3263: Synthesis of substituted N-(4-(6-methyl-1H-benzo[d]imidazol-2-yl)-5,6-dihydropyridin-1(2H)-yl) benzamide/benzenesulfonamide as anticancer agents

Abstract There is a strong association between chronic inflammatory conditions in a particular organ and the incidence of cancer specific to that organ. The longer the inflammation persists, the higher is the risk of associated carcinogenesis. Our interest is to synthesis new, potent and safer anticancer agents. The functionalized tetrahydropyridine (THP) ring systems are widely found in biologically active natural products and pharmaceuticals. The pharmacological activities of the THP derivatives depended greatly on the position and nature of the substitutions on the THP ring structure. 2-Substituted benzimidazole derivatives have been found to possess anti-inflammatory, antihistaminic, antimicrobial, anticancer, and cycloxygenase inhibiting activities. It is believed that synthesizing new compounds that contain both the pharmacophores of THP and benzimidazole could have the potential of becoming effective anticancer agents. 6-Methyl-2-(pyridin-4-yl)-1H-benzo[d]imidazole was obtained by the reaction of isonicotinc acid, 4-methyl benzene-1,2-diamine and polyphosphoric acid, which were stirred in an oil bath at 180oC for 2h. Mesitylene sulfonyl chloride was added with stirring to a solution of ethylacetohydroxymate and triethylamine in dimethylformamide at 0o C. Hydrolysis of this compound with the mixture of p-dioxane and 70% perchloric acid for 45 min gave a white solid of MSH. The MSH reacted with 6-methyl-2-(pyridin-4-yl)-1H-benzo[d]imidazole to give 1-amino-4-(6-methyl-1H-benzo[d]- imidazol-2-yl) pyridinium mesitylenesulfonate, which further reacted with different substituted benzoyl chlorides/benzenesulfonyl chlorides in 10% NaOH solution to gave stable benzoyl/benzenesulfonyl imino ylides. These ylides were reduced using sodium borohydride in absolute ethanol and gave the target compounds, substituted N-(4-(6-methyl-1H-benzo[d]imidazol-2-yl)-5,6-dihydropyridin-1(2H)-yl)benzamide/benzenesulfonamides. The cytotoxic effects of these substituted THP derivatives were determined using the MCF-7 estrogen receptor positive breast cancer cells, MDA-MB-231 estrogen receptor negative breast cancer cells, and Ishikawa cells, using the CellTiter-Glo (CTG) luminescent cell viability assay. Two target molecules exhibited strong cytotoxicity activities with significant IC50 values on MCF-7, Ishikawa and MDA-MB-231 cell lines. This research was supported by the NIH/RCMI Grant G12RR03020 and Pharmaceutical Research Center NIH/NCRR Grant 1 C06 RR12512-01. 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 3263. doi:10.1158/1538-7445.AM2011-3263

Abstract 1369: Synthesis of substituted N-[(3-(1H-pyrrol-1yl)methyl]-5,6-dihydropyridin-1(2H)-yl) benzamide/benzene sulfonamide as anticancer agents

Abstract Cancer has proven to be one of the most intractable human diseases, and as yet no anticancer agents that are highly effective and safe are available. Therefore, synthesis of novel, potent, selective and less toxic anticancer agents remains the most pressing goal of many investigators. Tetrahydropyridine (THP) ring moieties have attracted synthetic interests for being essential structures in many agrochemicals as well as medicinal agents. The anti-inflammatory and anticancer activities of compounds consisting of reduced pyridine ring systems were investigated in our laboratory. The pharmacological activities of the (THP) derivatives depended on the nature of the substituents on the THP ring moiety. In the current investigation, we synthesized many analogs maintaining the N-(3-[(1-H-pyrrol-1-yl)methyl]-5,6-dihydropyridin-1(2H)-yl) benzamide/benzene sulfonamides, at which the phenyl ring was substituted with groups having different electronic, steric and lipophilic properties. Mesitylene sulfonyl chloride was added with stirring to a solution of ethylacetohydroxymate and triethylamine in dimethylformamide at 0o C. Hydrolysis of this compound with the mixture of p-dioxane-70% perchloric acid and allowing them to react for 45 min gave a white solid of O-mesitylene sulfonyl hydroxylamine (MSH). MSH was used to prepare the N-amino salt since it served as an aminating agent. 3-[(1H-pyrrol-1-yl)methyl]pyridine was reacted with MSH in dichloromethane to produce 3-[(1H-pyrrol-1-yl)methyl]-1-aminopyridinium mesitylenesulfonate, the N-amino salt. Reaction of the amino salt with substituted benzoyl chloride/benzenesulfonyl chloride in anhydrous tetrahydrofuran containing triethylamine gave stable benzoyl/benzene sulfonyl imino ylides. This was followed by reduction with sodium borohydride in absolute ethanol, which furnished the target compounds substituted N-(3-[(1H-pyrrol-1-yl)methyl]-5,6-dihydropyridin-1(2H)-yl) benzamide/benzene sulfonamides. These compounds were evaluated for their cytotoxic effects on MCF-7 estrogen receptor positive breast cancer cells, MDA-MB-231 estrogen receptor negative breast cancer cell line, and Ishikawa cells, using the CellTiter-Glo (CTG) luminescent cell viability assay. N-(3-[(1H-pyrrol-1-yl)methyl]-5,6-dihydropyridin-1(2H)-yl)-4-methoxybenzene- sulfonamide showed the most potent cytotoxicity with an IC50 values of 47, 28, and 44 µM on MCF-7, Ishikawa and MDA-MB-231 cell lines, respectively. This research was supported by NIH/ RCMI Grant # G12 RR 03020 and Pharmaceutical Research Center NIH/NCRR Grant 1 C06 RR12512-01. 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 1369. doi:10.1158/1538-7445.AM2011-1369

Pharmacogenetics of thiopurine therapy: from thiopurine S-methyltransferase to S-adenosylmethionine

Background The efficiency and safety of the thiopurine therapy rely on the concentration of patient’s cytotoxic thioguanine nucleotides (TGN), which in turn depend on the deactivation of thiopurine drugs by thiopurine S-methyltransferase (TPMT). The activity of TPMT largely depends on the presence of genetic polymorphisms. Determination of mutations in the TPMT gene before starting 6-mercaptopurine (6-MP) therapy represents a quick, simple and cost-effective strategy to individualize thiopurine dosing. However, TPMT phenotype-to-genotype correlation is not complete, indicating a need for identification of novel biomarkers. The prime candidate is S-adenosylmethionine (SAM) which by binding into the active site of TPMT stabilizes its structure and consequently influences the metabolism and toxicity of thiopurine drugs [1]. Methods 6-MP-induced cytotoxicity was studied in MOLT cells. Metabolic activity of cells was determined by the CellTiter Aqueous One Proliferation Assay. Cytosolic TGN, SAM and methylthioinosine monophosphate (MeTIMP) levels as well as TPMT activity were measured by the modified reverse-phase HPLC method. Intracellular ATP levels were determined by the CellTiter Glo Luminescent Cell Viability Assay (Promega). Apoptotic cells were detected by the Annexin V-FITC Apoptosis Detection Kit (Sigma) and visualized by fluorescence microscopy. Caspase-3 activity was measured using labeled DEVD substrate. Results We herein present evidence of a novel TPMT-mediated mechanism of SAM-specific effects on 6-MP-induced cytotoxicity [2]. Our results show that exogenous SAM rescues cells from the toxic effects of 6-MP by restoring cell proliferation and delaying the onset of apoptosis. This is achieved by altering the dynamics of 6-MP metabolism, resulting in lower production of TGNs and MeTIMP. We prove that the extent of MeTIMP-induced inhibition of de novo purine synthesis (DNPS) determines the concentrations of intracellular ATP, and consequently SAM, which acts as a positive modulator of TPMT activity. This leads to a greater conversion of 6-MP to inactive 6-methylmercaptopurine, and lower availability of thioinosine monophosphate for the biotransformation to TGNs and MeTIMP. By acting as a TPMT-stabilizing factor, the availability of SAM contributes to the extent of 6-MP cytotoxicity. Conclusions

RNA Interference Screen to Identify Pathways That Enhance or Reduce Nonviral Gene Transfer During Lipofection

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.