Human Solid Tumor Cell Research Articles

Bioactivity screening of five Centaurea species and in vivo anti-inflammatory activity of C. athoa

Context: Centaurea L. (Asteraceae) species used as herbal remedies in Turkish traditional medicine have shown several biological properties.Objective: Extracts obtained from the aerial parts of Centaurea aphrodisea Boiss., Centaurea athoa DC., Centaurea hyalolepis Boiss., Centaurea iberica Trev. and Centaurea polyclada DC. were evaluated for their antioxidant, cytotoxic and anti-inflammatory activities.Materials and methods: Extracts of Centaurea species were tested for their antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) screening assays and for in vitro anti-inflammatory activity by Nf-κB and iNOS inhibition assays. The extracts were tested for their in vitro cytotoxicities against a panel of human solid tumor cell lines (SK-MEL: malignant melanoma, KB: oral epidermal carcinoma, BT-549: breast ductal carcinoma and SK-OV-3: ovary carcinoma) as well as non-cancerous kidney fibroblast (Vero) and kidney epithelial cells (LLC-PK1) by Neutral Red assay. In vivo anti-inflammatory activity of C. athoa was evaluated by the carrageenan-induced paw edema test in rats.Results: Antioxidant activities were observed for methanol extracts of plants. C. polyclada had the strongest effect on BT-549, KB and SK-OV-3 cell lines (30, 33 and 47 µg/ml, respectively). Nf-κB inhibition of chloroform extract of C. athoa was determined equivalent to positive control parthenolide (IC50: 6 µg/ml). This extract also showed anti-inflammatory activity by the carrageenan-induced paw edema test in rats, in all hours at a dose of 50 mg/kg compared to the control group.Discussion and conclusion: C. athoa is suggested to be a potential source of lead compounds for inflammatory diseases due to the significant in vitro and in vivo anti-inflammatory results.

Cell Surface-Associated Anti-MUC1-Derived Signal Peptide Antibodies: Implications for Cancer Diagnostics and Therapy

The MUC1 tumor associated antigen is highly expressed on a range of tumors. Its broad distribution on primary tumors and metastases renders it an attractive target for immunotherapy. After synthesis MUC1 is cleaved, yielding a large soluble extracellular alpha subunit containing the tandem repeats array (TRA) domain specifically bound, via non-covalent interaction, to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Thus far, inconclusive efficacy has been reported for anti-MUC1 antibodies directed against the soluble alpha subunit. Targeting the cell bound beta subunit, may bypass limitations posed by circulating TRA domains. MUC1’s signal peptide (SP) domain promiscuously binds multiple MHC class II and Class I alleles, which upon vaccination, generated robust T-cell immunity against MUC1-positive tumors. This is a first demonstration of non-MHC associated, MUC1 specific, cell surfaces presence for MUC1 SP domain. Polyclonal and monoclonal antibodies generated against MUC1 SP domain specifically bind a large variety of MUC1-positive human solid and haematological tumor cell lines; MUC1-positive bone marrow derived plasma cells obtained from multiple myeloma (MM)-patients, but not MUC1 negative tumors cells, and normal naive primary blood and epithelial cells. Membranal MUC1 SP appears mainly as an independent entity but also co-localized with the full MUC1 molecule. MUC1-SP specific binding in BM-derived plasma cells can assist in selecting patients to be treated with anti-MUC1 SP therapeutic vaccine, ImMucin. A therapeutic potential of the anti-MUC1 SP antibodies was suggested by their ability to support of complement-mediated lysis of MUC1-positive tumor cells but not MUC1 negative tumor cells and normal naive primary epithelial cells. These findings suggest a novel cell surface presence of MUC1 SP domain, a potential therapeutic benefit for anti-MUC1 SP antibodies in MUC1-positive tumors and a selection tool for MM patients to be treated with the anti-MUC1 SP vaccine, ImMucin.

Technical Aspects of 3-Dimentional Culture of Mammalian Cells

Three dimensional-tissue culture systems of human solid tumor cells has been implemented for effective chemotherapeutic screening and to avoid the disadvantages of the over simplified monolayer tissue culture system. The influence of tumor microenvironment to tumor therapeutics and screening of new anticancer drugs has been found to significant. The complex tumor microenvironment is poorly represented in the simple monolayer tissue culture system. The 3D culture models closely imitate several in vivo conditions of solid tumors in terms of geometry and expression of microenvironment. . However, several technical obstacles might discourage the use of this culture model. In the current mini-review, we are presenting the exact protocol for culturing mammalian cell as three dimensional tissue culture systems (multicellular spheroids and multicellular layers) and how to utilize these models for anti-cancer assessment.

Synthesis and biological activity of polyalthenol and pentacyclindole analogues

A series of indole sesquiterpenes analogues of polyalthenol and pentacyclindole have been synthesized starting from ent-halimic acid in order to test their biological activity. These analogues include diverse oxidation levels at the sesquiterpenyl moiety and different functionalization on the indole ring. All synthetic derivatives were tested against a representative panel of Gram positive and Gram negative bacterial strains, and the human solid tumour cell lines A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast) and WiDr (colon). Overall, the compounds presented activity against the cancer cell lines. The resulting lead, displaying a polyalthenol scaffold, showed GI50 values in the range 1.2–5.7 μM against all cell lines tested.

Novel Antimitotic Activity of 2-Hydroxy-4-methoxy-2′,3′-benzochalcone (HymnPro) through the Inhibition of Tubulin Polymerization

The natural chalcones and their derivatives exhibit many biological activities, such as anti-inflammatory and antitumoral. However, the precise mechanisms of action of benzochalcone derivatives are currently unknown. Here, a set of benzochalcones was synthesized, and the molecular mechanisms underlying inhibition of tumor growth were investigated. Colony-forming assays revealed that among tested compounds, 2-hydroxy-4-methoxy-2',3'-benzochalcone (HymnPro) most effectively inhibited the clonogenicity of Capan-1 human pancreatic cancer cells. HymnPro inhibited cell proliferation in several human solid tumor cell lines and suppressed xenografted tumor growth in nude mice. Mechanistically, HymnPro induced cell cycle arrest at the G2/M phase, followed by an increase in apoptotic cell death. These events were associated with the inhibition of tubulin polymerization through binding of HymnPro to tubulin, leading to the formation of abnormal mono- or multipolar mitotic microtubule structures accompanied by spherical arrangement of multinucleated chromosomes. Furthermore, HymnPro activated caspase-2, caspase-9, caspase-3, and caspase-7 and increased the cleavage of poly(ADP-ribose) polymerase (PARP). HymnPro increased the phosphorylation of JNK1/2, Erk1/2, and p38 kinase. Pretreatment with SP600125, U0126, or SB600125 abrogated HymnPro-induced activation of caspases-3 and caspase-7 and the cleavage of PARP, suggesting that MAPK signalings are involved in HymnPro-induced apoptosis. It was concluded that a novel HymnPro compound exerts antitumor activity by disrupting microtubule assembly, which leads to mitotic arrest and sequential activation of the caspase pathway, resulting in apoptosis.

Abstract B253: Novel water-soluble phenyl N-mustard-benzenealkylamide conjugate, BO-2094, potent agent for treatment of human colorectal cancer.

Abstract Colorectal cancer (CRC) is the second leading cause of cancer related death in the United States and Europe. Surgery, radiotherapy, and chemotherapy are the main strategies for cure of CRC patient. However, the mortality risk associated with CRC is metastasis, which may be diminished by systemic treatments. Therefore, there is an urgent need of finding a better agent to treat CRC. Recently, we have synthesized a series of novel water soluble and chemically stable phenyl N-mustard- benzenealkylamide conjugates, which is consisted of phenyl N-mustard pharmacophore and benzenealkylamide moiety bearing a variety of hydrophilic alkylamide side chains. Of these conjugates, BO-2094 exhibits a broad spectrum of antitumor activity against a panel of human leukemia and solid tumor cell lines in vitro and potent therapeutic efficacy in various tumor xenograft-moles. This agent is able to induce DNA interstrand cross-linking, cell cycle arrest at sub-G1 and G2/M phase, and cell death via apoptosis. Particularly, BO-2094 displays potent antitumor activity in nude mice bearing human colon cancer HCT-116 and H334 xenografts. More than 95% tumor suppression was observes when BO-2094 [30 mg/kg, once per day for 6 consecutive days (QD×6), via intravenous injection (iv. inj.)] was used alone. The combination of BO-2094 and 5-FU at ratios of 1:3 induced synergistic cytotoxicity to HCT-116 cells in vitro. Notable, the growth of HCT-116 xenografts in nude mice was almost completely suppressed (>98% suppression) when co-treated compound BO-2094 (30 mg/kg, QD×6, iv. inj.) with 5-FU [75 mg/kg, every 7 days for 2 doses (Q7D×2), intraperitoneal injection (ip. inj.)]; all mice (n=5) survived on day 35. The results revealed that the combination of BO-2094+5-FU is superior to that of oxaliplatin [15 mg/kg every 3 days for 3 doses (Q3D×3), iv. inj.] and 5-FU [75 mg/kg, Q7D×2, ip. inj.]. It should be noted that 2 of 5 mice died on day 28 when mice were co-treated with oxaliplatin+5-FU, indicating that the combination of BO-2094+5-FU is less toxic to the host. The early preclinical studies of BO-2094 showed that this agent is likely to have no cardiac arrhythmic side effect based on hERG assay and has low toxicity to the drug treated mice based on a 14-day acute iv injection study. The present studies indicate that the combination of compound BO-2094+5-FU has great potential benefit for the treatment of advanced colon cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B253. Citation Format: Tung-Hu Tsai, Satishkumar D. Tala, Tai-Hsin Ou, Yi-Wen Lin, Kiranben S. Tala, Ming-Hsi Wu, Te-Chang Lee, Tsann-Long Su. Novel water-soluble phenyl N-mustard-benzenealkylamide conjugate, BO-2094, potent agent for treatment of human colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B253.

Abstract A121: Preclinical evaluation of OTX015 a novel BET-BRD inhibitor across a panel of solid tumor cell lines of different lineages.

Abstract Background: The human BET family bromodomains which consists of BRD2, BRD3, BRD4 and BRDT proteins has become a druggable target for the development of specific gene transcription inhibitors. Here, we report anti-proliferative activity of OTX015, an orally bioavailable small-molecule BRD-inhibitor which displays high potency and specificity to BRDs 2, 3, and 4, across a large panel of human solid tumor cell lines. Material and Methods: Established 20 human cancer cell lines derived from head and neck, lung, liver, colorectal, renal and pancreatic cancers, were treated with increasing doses of OTX015 (OncoEthix SA, Switzerland). MTT assays were performed after 72 hours exposure. GI50 (CI95%) values were estimated using GraphPad Prism 3.0. Cell lines displaying GI50 values500nM were considered insensitive. Genomic DNA from the cell lines was PCR-amplified, sequenced, and assessed for potential sequence alterations (ABI BigDye Terminator Sequencing kit) in the genes KRAS (exon 2 and 3); BRAF (exon 11 and 15); EGFR (exon 20) and PI3KCA (exon 20). Protein levels were analyzed by Western Blot using commercial antibodies. For cell cycle analysis, cells were stained with propidium iodide and analyzed for DNA content using a FACScan flow cytometer. RNA was extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit following manufacturer's instructions. RT- PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. Results: Twenty cell lines derived from a wide range of solid tumor types, belonging to head and neck squamous cell carcinoma (HNSCC), lung adenocarcinoma (LA), hepatocarcinoma (HCC), colorectal carcinoma (CRC), renal (RC), pancreatic (PC) and triple-negative breast cancer (BC) cells were exposed to various concentrations of OTX015 (from 3µM to 9 nM) for 72 h. Eight cell lines displayed GI50 values lower than 500 nM, ranging from 45.9 (15.5-135.1)nM in HT-29 cells (CRC) to 432.0 (38.0-618.7)nM in A-549 (LA) cells. Baseline BRD4/2, c-MYC, BCL-2, CyclinD1 m-RNA leves and protein were characterized in our panel of cell lines and no difference in BRD4/2, c-MYC or BCL-2 basal levels was observed between OTX015 sensitive or resistant cell lines. In addition, no correlation was found between OTX015 anti-proliferative activity and the most prevalent mutations seen in cancer cells (KRAS, BRAF, EGFR and PI3K). In sensitive cell lines, OTX015 caused a cell cycle arrest in G1 in a dose-dependent manner without an increase in cell death (sub-G0 increased). No c-MYC protein level down-regulation was observed after treatment (500nM; 72h) in most of the OTX015 sensitive cell lines, suggesting that alternative pathways can be affected by BRD-inhibition. Noteworthy, after 72 h exposure, significant changes in the cell morphology such as expanded cytoplasm, cytoskeleton reorganization and pyknotic nuclei observed in OTX015 treated cells compared to vehicle treated group, further studies are being conducted to characterize these findings. Conclusion: Together, these findings suggest that OTX015 is active across a wide range of tumor types but this activity could not be restricted only cancers with genomic alterations resulting in MYC overexpression. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A121. Citation Format: Maria Eugenia Riveiro, Lucile Astorgues-Xerri, Elodie Odore, Mohamed Bekradda, Esteban Cvitkovic, Kay Noel, Eric Raymond. Preclinical evaluation of OTX015 a novel BET-BRD inhibitor across a panel of solid tumor cell lines of different lineages. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A121.

Chemistry of phosphorus ylides 31: Reaction of azidocoumarin with active phosphonium ylides, synthesis and antitumour activities of chromenones

The reaction of 4- azidochromen-2-one (1) with the nucleophilic phosphacumulene ylides 2, 8, and 12 afforded the new heterocyclic triazoles, triazepines, aziridine, pyrrolone containing a coumarin moiety. Cycloaddition reactions took place first to give triazoline 3 and 9. The triazolines rearranged to the triazepines 4, 10, and 13 accompanied by elimination of triphenylphosphine leading to the phosphorus-free triazepines 5, 11, and moreover, aziridine 6 was produced via nitrogen extrusion from the triazoline 3, followed by ring expansion to the pyrrolone 7. On the other hand, the reaction of the azidocoumarin 1 with the phosphallene yield 15 behaves differently and afforded the triazine 17 and azetone 18. The antitumour activity of compounds 3, 4, 6, and 11 was evaluated, in vitro, against (breast: MCF-7 and liver: HPEG2) human solid tumour cell lines. They showed values closed to that recorded by the reference drug doxorubicin. When the azidochromenone was allowed to react with active phosphacumulenes, it gave triazol-, triazepin-, aziridin-, and pyrrolchromenone derivatives. Furthermore, the reaction of azide towards Phosphallene ylide was, investigated. The newly synthesized compounds were screened for their in vitro cytotoxic and growth inhibitory activities against, human breast and liver carcinoma cell lines.

Synthesis and antiproliferative activity of α-branched α,β-unsaturated ketones

A series of α-branched α,β-unsaturated ketones were prepared in a straightforward manner by the acid catalyzed coupling between arylalkynes and carbaldehydes. The method also allows producing as side product chalcone analogs bearing an additional α,β-unsaturated arylketone in the molecular scaffold. The evaluation of the antiproliferative activity in the human solid tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast) and WiDr (colon) provided a structure–activity relationship. Overall, the compounds presented active against the resistant cancer cells T-47D. The resulting lead, displaying an unprecedented chalcone scaffold, showed GI50 values in the range 0.32–0.53 μM against all cell lines tested. The methoxy group present in the lead might play an important role in the activity.

The synthesis and anticancer activity of analogs of the indole phytoalexins brassinin, 1-methoxyspirobrassinol methyl ether and cyclobrassinin

An effective synthesis of analogs of the indole phytoalexin cyclobrassinin with NR1R2 group instead of SCH3 was developed starting from indole-3-carboxaldehyde. The target compounds were prepared by spirocyclization of 1-Boc-thioureas with the formation of isolable spiroindoline intermediates, followed by the trifluoroacetic acid-induced cascade reaction consisting of methanol elimination, deprotection and rearrangement of the iminium ion. The structures of novel products were elucided by the 1H and 13C NMR spectroscopy, including HMBC, HSQC, COSY, NOESY and DEPT measurements. Several newly synthesized compounds demonstrated significant antiproliferative/cytotoxic activity against human leukemia and solid tumor cell lines, as well as remarkable selectivity of these effects against cancer cells relative to the non-malignant HUVEC cells.

Molecular docking studies of the interaction between propargylic enol ethers and human DNA topoisomerase IIα

Having identified a novel human DNA topoisomerase IIα (TOP2) catalytic inhibitor from a small and structure-focused library of propargylic enol ethers, we decided to analyze if the chirality of these compounds plays a determinant role in their antiproliferative activity. In this study, we describe for the first time the synthesis of the corresponding enantiomers and the biological evaluation against a panel of representative human solid tumor cell lines. Experimental results show that chirality does not influence the reported antiproliferative activity of these compounds. Docking studies of corresponding enantiomers against TOP2 reinforce the finding that the biological effect is not chiral-dependent and that these family of compounds seem to act as TOP2 catalytic inhibitors.

Biomimetic synthesis of an antitumour indole sesquiterpene alkaloid, 12-epi-ent-pentacyclindole

Biomimetic synthesis of 12-epi-ent-pentacyclindole 7, using as key step the cyclization of 12-epi-ent-polyalthenol acetate has been carried out. This way, the structure and absolute configuration of the natural product pentacyclindole 1 has been confirmed. The synthesized indole sesquiterpenes 7, 11 and 12 show cellular proliferation inhibition of a number of human leukaemic and solid tumour cell lines.

Synthesis and in vitro antitumor activity of novel iridium(III) complexes with enantiopure C2-symmetrical vicinal diamine ligands

Four novel iridium(III) complexes with enantiopure C2-symmetrical vicinal diamine ligands were designed, synthesized, and characterized by FT-IR, NMR, and MS. The cytotoxicities of all of the complexes against the human solid tumor cell lines A2780, A549, KB, and MDA-MB-231 were evaluated. Both R,R-configured complexes (R,R)-5a and (R,R)-5b exhibited more potent or similar activity compared with oxaliplatin, whereas their corresponding (S,S)-isomers (S,S)-5a and (S,S)-5b were found to be mostly inactive. As indicated by the activation of caspase-3, the cleavage of PARP, and the upregulation of p53, the preliminary mechanism studies revealed that the mode of cell death initiated by (R,R)-5a in A2780 cells was predominantly p53-mediated apoptosis. In addition, the structure of (R,R)-5a was unambiguously confirmed through single crystal X-ray structure determination.

Derivatives of grindelic acid: From a non-active natural diterpene to synthetic antitumor derivatives

Using several reactions that include homologations and asymmetric epoxidations as well as Ugi and Huisgen couplings, we generated a small focused library of new derivatives from the labdane-type diterpene grindelic acid. These compounds were evaluated as cytotoxic agents against a panel of five human solid tumor cell lines (HBL-100, HeLa, SW1573, T-47D, and WiDr). The presence of the diamide functionalizations enhanced the cytotoxic effect. N-Benzyl-N-(1-(benzylamino)-2-methyl-1-oxopropan-2-yl)grindelicamide, proved to be the most active product in all cell lines tested, with values of 0.95 (±0.38) μM against HBL-100 cells.

Abstract 2185: Development and characterization of the fully human chimeric protein GrB-TWEAK targeting Fn14-positive solid tumor cells.

Abstract The TNFR superfamily member Fn14, the receptor for TNF-like weak inducer of apoptosis (TWEAK), has emerged as a potentially clinically valuable target for cancer therapy. Specifically, Fn14 expression is relatively low in normal tissues but elevated in various human solid tumor types, including brain, breast, lung and melanoma, and can be a negative prognostic indicator. To target Fn14-overexpressing cancer cells, we constructed via PCR a novel chimeric protein designated granzyme B (GrB)-TWEAK, using the human TWEAK receptor-binding domain as the targeting moiety and the human pro-apoptotic serine protease GrB as the cytotoxic agent. The fusion construct was expressed in mammalian cells and purified to homogeneity from conditioned media. BiaCore analysis of TWEAK and GrB-TWEAK binding to the Fn14 extracellular domain indicated that these proteins bound to Fn14 with similar affinity, with Kds of ∼ 3 nM and ∼ 8 nM, respectively. Confocal immunofluoresence studies showed that GrB-TWEAK specifically and rapidly (within 3 hrs) internalized into Fn14-expressing MDA-MB-231 breast cancer and HT-29 colorectal adenocarcinoma cells. GrB-TWEAK activity was assessed using a panel of 26 human cancer cell lines and it demonstrated impressive and selective cytotoxicity to Fn14-expressing cells in the low nanomolar range (IC50 ranged from 0.4 nM-330 nM), which was 6- to 1117-fold more potent than free GrB. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion construct in vitro. Mechanistic studies demonstrated that GrB-TWEAK activated caspase cascades and cytochrome C-related pro-apoptotic mechanisms consistent with the known intracellular functions of GrB in target cells. Preliminary mouse xenograft tumor model studies are ongoing. This work was conducted, in part, by the Clayton Foundation for Research (MGR) and supported by NIH grant NS055126 (JAW). Citation Format: Hong Zhou, Mary Migliorini, Yu Cao, Lawrence H. Cheung, Walter N. Hittelman, Jeffrey A. Winkles, Michael G. Rosenblum. Development and characterization of the fully human chimeric protein GrB-TWEAK targeting Fn14-positive solid tumor 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 2185. doi:10.1158/1538-7445.AM2013-2185

Abstract 2424: CD40 expression in human solid tumors and NCI60 tumor cell lines.

Abstract Cell-surface CD40 is a member of the tumor necrosis factor receptor superfamily (TNFRSF) expressed and functionally well characterized in immune and hematopoietic cells. However, its expression and role in human solid tumors remain to be evaluated. As a molecular target currently with the antibody-based cancer therapy, it is important to characterize expression of CD40 across a range of human solid tumors and cancer cell lines. This could help improve our understanding of biology of CD40 molecule in human cancers especially epithelial tumors, and in turn increase the odds of success of targeting CD40 pathway. Expression of CD40 in the NCI60 human tumor cell lines and common types of human solid tumors were examined by immunohistochemistry in formalin-fixed and paraffin-embedded tumor specimens. CD40, predominantly membranous signal, was constitutively expressed in 15 lines, respectively derived from carcinomas of the breast, colorectum, non-small cell lung, ovary, prostate, and renal cell, of the NCI60 tumor cell line panel, and confirmed by Western blotting and gene expression microarray data available at http://dtp.nci.nih.gov/mtweb/search.jsp. CD40 expression was largely in accordance with the mRNA expression data measured by gene expression profiling. The status of CD40 was verified in MDA-MB-231, T-47D and HCT-116 cells (CD40-positive), and MCF-7 and HT-29 cells (CD40-negative) by Western blot. Moreover, CD40 was detected in tumor cells in 17% (6/34) of breast cancer, 24% (8/34) colorectal cancer, 54% (23/43) non-small cell lung cancer, and 47% (17/36) ovarian cancer. CD40 was also spotted in infiltrating lymphocytes in the tumor stroma. The data indicate that CD40 is expressed in human solid tumors with varying expression frequencies and one quarter of cancer cell lines examined. In addition, we have established a ready-for-use immunohistochemistry assay with an antibody that specifically detects CD40 in formalin-fixed and paraffin-embedded human tumor samples. Its application holds promise to evaluate the prognostic role of CD40 in human cancers. Citation Format: Dat Nguyen, Joseph E. Tomaszewski, Stephen M. Hewitt, James H. Doroshow, Sherry X. Yang. CD40 expression in human solid tumors and NCI60 tumor cell lines. [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 2424. doi:10.1158/1538-7445.AM2013-2424

Abstract 4475: New water-soluble and stable N-mustard-benzeneconjugates with potent antitumor activity, BO-2094, generated via leadoptimization by bioisostere approach.

Abstract The water solubility of drug candidate plays an important determinant of the bioavailability and supports whether a drug candidate can be successfully developed for clinical application. Previously, we have synthesized a series of water-soluble N-mustards, in which the phenyl N-mustard pharmacophore is linked to a benzamide moiety containing a hydrophilic side-chain at the meta- or para-position of the carboxamide function via a urea spacer. Of these derivatives, the water-soluble BO-1055 HCl exhibits a broad spectrum of antitumor activity and potent therapeutic efficacy against various human solid tumor (such as breast MX-1, colon HCT-116, and prostate PC-3) xenografts. To optimize the antitumor activity of BO-1055 via structural modification by bioisostere approach, we synthesized a series of new N-mustard-benzene conjugates, in which the phenyl N-mustard pharmacophore is linked to a benzene moiety via a urea linker, which can reduce the reactivity of the reactive N-mustard moiety. The benzene ring contains a variety of ω-N,N-dialkylaminoalkylamide or ω-cyclicaminoalkylamide side-chains at the meta- or para-position of the ureido linker. The tertiary amino function on the side-chain can be converted into a variety of water-soluble salts with inorganic or organic acids. The newly synthesized conjugates were subject to antitumor studies both in vitro and in tumor xenograft model. The results showed that these water-soluble conjugates exhibit a broad spectrum of antitumor activity against a panel of human leukemia and solid tumor cell growth in culture. Among these derivatives, BO-2094 [N-(4-(3-(4-(bis(2-chloroethyl)amino)phenyl)ureido)phenyl)-2-(diethylamino)acetamide], was revealed to be more cytotoxic than BO-1055 in inhibiting various tumor cell growth in vitro. It also showed that this agent is more potent than BO-1055 against human colon HCT-116, prostate PC3, and lung cancer H460 xenografts in mice. Studies on the mechanism of action revealed that BO-2094 is able to induce DNA cross-linking and cell arrest at G2/M phase. The present investigations conclude that BO-2094, a new water-soluble N-mustard-benzene conjugated with more potent therapeutic effect than BO-1055, has high potential to be selected as a candidate for preclinical antitumor studies. Citation Format: Tsann-Long Su, Satishkumar Tala, Tai-Hsin Ou, Kiranben Tala, Rajesh Kakadiya, Yi-wen Lin, Chi-Wei Chen, Te-Chang Lee. New water-soluble and stable N-mustard-benzeneconjugates with potent antitumor activity, BO-2094, generated via leadoptimization by bioisostere approach. [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 4475. doi:10.1158/1538-7445.AM2013-4475

Characterization of small spheres derived from various solid tumor cell lines: are they suitable targets for T cells?

T cell based immunotherapy has been investigated in a variety of malignancies and analyses have been mostly founded on in vitro data with tumor cell monolayers. However, three-dimensional (3D) culture models might mimic more closely the 'in vivo' conditions than 2D monolayers. Therefore, we analyzed the expression of tumor-associated antigens (TAA) and of molecules involved in antigen processing and presentation (APM) in tumor spheres, which served as an in vitro model for micrometastasis which might be enriched in tumor propagating cancer stem cells. For enrichment of sphere cells 12 human solid tumor cell lines were cultured in serum-free medium. Expression of a variety of TAA and APM were analyzed by RT-PCR and/or flow cytometry and compared to expression in corresponding adherent bulk cells grown in regular growth medium. Compared to adherent cells, spheres showed equal or higher mRNA expression levels of LMP2, LMP7 and MECL-1, of TAP1 and TAP2 transporters and, surprisingly, also of TAA including differentiation antigens. However, downregulation or loss of HLA-I and HLA-II molecules in spheres was observed in 8 of 10 and 1 of 2 cell lines, respectively, and was unresponsive to stimulation with IFN-γ. Although tumor spheres express TAA and molecules of intracellular antigen processing, they are defective in antigen presentation due to downregulation of HLA surface expression which may lead to immune evasion.

Amphidinolide B: total synthesis, structural investigation, and biological evaluation.

The total syntheses of amphidinolide B1 and the proposed structure of amphidinolide B2 have been accomplished. Key aspects of this work include the development of a practical, non-transition-metal-mediated method for the construction of the C13-C15 diene, the identification of α-chelation and dipole minimization models for diastereoselective methyl ketone aldol reactions, the discovery of a spontaneous Horner-Wadsworth-Emmons macrocyclization strategy, and the development of a novel late stage method for construction of an allylic epoxide moiety. The originally proposed structure for amphidinolide B2 and diastereomers thereof display potent antitumor activities with IC50 values ranging from 3.3 to 94.5 nM against human solid and blood tumor cells. Of the different stereoisomers, the proposed structure of amphidinolide B2 is over 12-fold more potent than the C8,9-epimer and C18-epimer in human DU145 prostate cancer cells. These data suggest that the epoxide stereochemistry is a significant factor for anticancer activity.

Induction of Caspase-8 and Death Receptors by a New Dammarane Skeleton from the Dried Fruits of Forsythia koreana

A new naturally occurring compound based on the dammarane skeleton, i.e. cabralealactone 3-acetate-24-methyl ether, was isolated from the aqueous methanolic extract of Forsythia koreana fruits, along with eight known compounds: cabralealactone 3-acetate, ursolic acid, arctigenin, arctiin, phillyrin, rutin, caffeic acid, and rosmarinic acid. The identification of the isolated compounds was based on their spectral analysis including: HREI-MS, 1D and 2D NMR spectroscopy. The selected compounds and the aqueous methanolic extract were evaluated for their cytotoxic activity against human solid tumour cell lines. Cabralealactone 3-acetate-24-methyl ether and ursolic acid were found to be active against human breast cancer cells (MCF-7). The cytotoxicity was associated with the activation of caspase-8, the induction of the death receptors DR4 and DR5, as well as DNA fragmentation, and was thus due to apoptosis rather than necrosis.