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Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-Prkdcscid Il2rγtm1Sug/Jic) and the NSG (NOD/SCID-IL2Rγ−/−, NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ) mouse, and their human-cytokine-producing (interleukin-3, granulocyte-macrophage colony-stimulating factor, and stem cell factor) counterparts (huNOG and NSGS), rely partly on a mutation in the DNA repair protein PRKDC, causing a severe combined immune deficiency (SCID) phenotype and rendering the mice less tolerant to DNA-damaging therapeutics, thereby limiting their usefulness in the investigation of novel acute myeloid leukemia (AML) therapeutics. NRG (NOD/RAG1/2−/−IL2Rγ−/−) mice show equivalent immune ablation through a defective recombination activation gene (RAG), leaving DNA damage repair intact, and human-cytokine-producing NRGS (NRG-SGM3) mice were generated, improving myeloid engraftment. Our findings indicate that unconditioned NRG and NRGS mice can harbor established AML CDXs and can tolerate aggressive induction chemotherapy at higher doses than NSG mice without overt toxicity. However, unconditioned NRGS mice developed less clinically relevant disease, with CDXs forming solid tumors throughout the body, whereas unconditioned NRG mice were incapable of efficiently supporting PDX or human hematopoietic stem cell engraftment. These findings emphasize the contextually dependent utility of each of these powerful new strains in the study of normal and malignant human hematopoiesis. Therefore, the choice of mouse strain cannot be random, but must be based on the experimental outcomes and questions to be addressed.

The tumor microenvironment (TME) is composed of a heterogeneous biological ecosystem of cellular and non-cellular elements including transformed tumor cells, endothelial cells, immune cells, activated fibroblasts or myofibroblasts, stem and progenitor cells, as well as the cytokines and matrix that they produce. The constituents of the TME stroma are multiple and varied, however cancer associated fibroblasts (CAF) and their contribution to the TME are important in tumor progression. CAF are hypothesized to arise from multiple progenitor cell types, including mesenchymal stem cells. Currently, isolation of TME stroma from patients is complicated by issues such as limited availability of biopsy material and cell stress incurred during lengthy adaptation to atmospheric oxygen (20% O2) in cell culture, limiting pre-clinical studies of patient tumor stromal interactions. Here we describe a microenvironment mimetic in vitro cell culturing system that incorporates elements of the in vivo lung environment, including lung fibroblast derived extracellular matrix and physiological hypoxia (5% O2). Using this system, we easily isolated and rapidly expanded stromal progenitors from patient lung tumor resections without complex sorting methods or growth supplements. These progenitor populations retained expression of pluripotency markers, secreted factors associated with cancer progression, and enhanced tumor cell growth and metastasis. An understanding of the biology of these progenitor cell populations in a TME-like environment may advance our ability to target these cells and limit their effects on promoting cancer metastasis.

Determining mechanistic details about how drugs kill cancer cells is critical for predicting which cancers will respond to given therapeutic regimens and for identifying effective combinations of drugs that more potently kill cancer cells while sparing normal cells. The BCL2 family of proteins and bioactive sphingolipids are intricately linked during apoptotic cell death. In fact, many chemotherapeutic drugs are known to cause accumulation of the pro-apoptotic sphingolipid ceramide; however, the mechanism by which this occurs is not completely understood. In the present study we demonstrate that direct inhibition of anti-apoptotic BCL2 proteins with ABT-263 is sufficient to induce C16-ceramide synthesis in multiple cell lines, including human leukaemia and myeloma cells. ABT-263 activates CerS (ceramide synthase) activity only in cells expressing BAK or in cells capable of activating BAK. Importantly, recombinant BAK is sufficient to increase in vitro CerS activity in microsomes purified from Bak-KO (knockout) cells and activated BAK more potently activates CerS than inactive BAK. Likewise, ABT-263 addition to wild-type, but not Bak-deficient, microsomes increases CerS in vitro activity. Furthermore, we present a feed-forward model by which BAK activation of CerS by chemotherapeutic drugs leads to elevated ceramide levels that result in synergistic channel formation by ceramide (or one of its metabolites) and BAX/BAK.

The identification of novel combinations of effective cancer drugs is required for the successful treatment of cancer patients for a number of reasons. First, many “cancer specific” therapeutics display detrimental patient side-effects and second, there are almost no examples of single agent therapeutics that lead to cures. One strategy to decrease both the effective dose of individual drugs and the potential for therapeutic resistance is to combine drugs that regulate independent pathways that converge on cell death. BCL2-like family members are key proteins that regulate apoptosis. We conducted a screen to identify drugs that could be combined with an inhibitor of anti-apoptotic BCL2-like proteins, ABT-263, to kill human leukemia cells lines. We found that the combination of D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) hydrochloride, an inhibitor of glucosylceramide synthase, potently synergized with ABT-263 in the killing of multiple human leukemia cell lines. Treatment of cells with PDMP and ABT-263 led to dramatic elevation of two pro-apoptotic sphingolipids, namely ceramide and sphingosine. Furthermore, treatment of cells with the sphingosine kinase inhibitor, SKi-II, also dramatically synergized with ABT-263 to kill leukemia cells and similarly increased ceramides and sphingosine. Data suggest that synergism with ABT-263 requires accumulation of ceramides and sphingosine, as AMP-deoxynojirimycin, (an inhibitor of the glycosphingolipid pathway) did not elevate ceramides or sphingosine and importantly did not sensitize cells to ABT-263 treatment. Taken together, our data suggest that combining inhibitors of anti-apoptotic BCL2-like proteins with drugs that alter the balance of bioactive sphingolipids will be a powerful combination for the treatment of human cancers.

A pharmacological screening of the ethanol extract and fractions of two Peruvian medicinal plants, Plagiochila disticha and Ambrosia peruviana, led to the isolation and characterization of three ENT-2,3-secoaromadendrane-type sesquiterpenoids, named plagiochiline A ( 1), I ( 2), and R ( 3), as well as of two pseudoguaianolids, damsin ( 4) and confertin ( 5), which exhibited significant cytotoxic activity against a panel of human tumor cell lines. Compounds 1, 4, and 5 were also investigated for their in vitro antileishmanial, trypanocidal, and antituberculosis activity against Leishmania amazonensis axenic amastigotes and Trypanosoma cruzi trypomastigotes, as well as against MDR and sensitive strains of Mycobacterium tuberculosis, respectively.

AS1411 is a quadruplex-forming oligonucleotide aptamer that targets nucleolin. It is currently in clinical trials as a treatment for various cancers. We have proposed that AS1411 inhibits cancer cell proliferation by affecting the activities of certain nucleolin-containing complexes. Here, we report that protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes the formation of symmetrical dimethylarginine (sDMA), is a nucleolin-associated protein whose localization and activity are altered by AS1411. Levels of PRMT5 were found to be decreased in the nucleus of AS1411-treated DU145 human prostate cancer cells, but increased in the cytoplasm. These changes were dependent on nucleolin and were not observed in cells pretreated with nucleolin-specific small interfering RNA. Treatment with AS1411 altered levels of PRMT5 activity (assessed by sDMA levels) in accord with changes in its localization. In addition, our data indicate that nucleolin itself is a substrate for PRMT5 and that distribution of sDMA-modified nucleolin is altered by AS1411. Because histone arginine methylation by PRMT5 causes transcriptional repression, we also examined expression of selected PRMT5 target genes in AS1411-treated cells. For some genes, including cyclin E2 and tumor suppressor ST7, a significant up-regulation was noted, which corresponded with decreased PRMT5 association with the gene promoter. We conclude that nucleolin is a novel binding partner and substrate for PRMT5, and that AS1411 causes relocalization of the nucleolin-PRMT5 complex from the nucleus to the cytoplasm. Consequently, the nuclear activity of PRMT5 is decreased, leading to derepression of some PRMT5 target genes, which may contribute to the biological effects of AS1411.

AGRO100, also known as AS1411, is an experimental anticancer drug that recently entered human clinical trials. It is a member of a novel class of antiproliferative agents known as G-rich oligonucleotides (GRO), which are non-antisense, guanosine-rich phosphodiester oligodeoxynucleotides that form stable G-quadruplex structures. The biological activity of GROs results from their binding to specific cellular proteins as aptamers. One important target protein of GROs has been previously identified as nucleolin, a multifunctional protein expressed at high levels by cancer cells. Here, we report that AGRO100 also associates with nuclear factor-κB (NF-κB) essential modulator (NEMO), which is a regulatory subunit of the inhibitor of κB (IκB) kinase (IKK) complex, and also called IKKγ. In the classic NF-κB pathway, the IKK complex is required for phosphorylation of IκBα and subsequent activation of the transcription factor NF-κB. We found that treatment of cancer cells with AGRO100 inhibits IKK activity and reduces phosphorylation of IκBα in response to tumor necrosis factor-α stimulation. Using a reporter gene assay, we showed that AGRO100 blocks both tumor necrosis factor-α-induced and constitutive NF-κB activity in human cancer cell lines derived from cervical, prostate, breast, and lung carcinomas. In addition, we showed that, in AGRO100-treated cancer cells, NEMO is coprecipitated by nucleolin, indicating that both proteins are present in the same complex. Our studies suggest that abrogation of NF-κB activity may contribute to the anticancer effects of AGRO100 and that nucleolin may play a previously unknown role in regulating the NF-κB pathway. [Mol Cancer Ther 2006;5(7):1790–9]

Molecular defects in apoptotic pathways are thought to often contribute to the abnormal expansion of malignant cells and their resistance to chemotherapy. Therefore, a comprehensive knowledge of the mechanisms controlling induction of apoptosis and subsequent cellular disintegration could result in improved methods for prognosis and treatment of cancer. In this study, we have examined apoptosis-induced alterations in two proteins, nucleolin and poly(ADP-ribose) polymerase-1 (PARP-1), in U937 leukemia cells. Nucleolin is expressed at high levels in malignant cells, and it is a multifunctional and mobile protein that can shuttle among the nucleolus, nucleoplasm, cytoplasm, and plasma membrane. Here, we report our findings that UV irradiation or camptothecin treatment of U937 cells induced apoptosis and caused a significant change in the levels and localization of nucleolin within the nucleus. Additionally, nucleolin levels were dramatically decreased in extracts containing the cytoplasm and plasma membrane. These alterations could be abrogated by pre-incubation with an inhibitor of PARP-1 (3-aminobenzamide), and our data support a potential role for nucleolin in removing cleaved PARP-1 from dying cells. Furthermore, both nucleolin and cleaved PARP-1 were detected in the culture medium of cells undergoing apoptosis, associated with particles of a size consistent with apoptotic bodies. These results indicate that nucleolin plays an important role in apoptosis, and could be a useful marker for assessing apoptosis or detecting apoptotic bodies. In addition, the data provide a possible explanation for the appearance of nucleolin and PARP-1 autoantibodies in some autoimmune diseases.

Background. MYCN (N-myc) amplification in neuroblastoma is associated with poor clinical outcome. Factors that regulate MYCN expression have not been elucidated. MYCN is considered a TATA-less promoter, whereas significant promoter activity resides within 160 bp 5′ of the major transcription start site. This region contains two GC-rich motifs and a CT box, regions for potential transcription factor interaction. Methods. To characterize DNA-protein interactions in this region of the MYCN promoter, electrophoretic mobility shift assays, and promoter-reporter were used. Results. A MYCN promoter fragment was incubated with HeLa nuclear extract, with or without competitors. Three major protein/DNA complexes were formed. Formation of 2 complexes could be inhibited by unlabeled Sp1 consensus duplex and by the Sp1 site-specific drug WP631. Purified Sp1 protein produced a complex similar to that formed with HeLa extract. To determine whether these DNA/protein interactions could be blocked in a sequence-specific fashion, a triplex forming oligonucleotide (TFO) was used. This TFO was designed to bind in the major groove of the promoter, covering the CT-box (putative Sp1 binding) motif. When triplex formation was followed by addition of nuclear extract, protein binding was indeed inhibited. Functional significance of this inhibition was tested with pE/Bnmyc-luc, a promoter-reporter plasmid containing the human MYCN promoter driving luciferase expression. Incubation with TFO, but not control oligodeoxynucleotides, completely inhibited luciferase activity. Conclusions. These data suggest that protein binding does occur in regions of the MYCN promoter containing GC and CT box elements and that this interaction is important for MYCN promoter activity. By inference, these data also suggest that the proteins that bind in this region are Sp1 family members. (Surgery 2002;132:232-8.)

We previously reported the discovery of XB05, a synthetic small molecule with antiproliferative activity. XB05 was found to be a strong inhibitor of DNMT1 activity in cell-free and cell-based assays. However, screening in the NCI 60 tumor cell lines and analysis using COMPARE indicated a novel mechanism of action for XB05, different from standard demethylating agents such as 5-azacytidine (azaC) and decitabine. Here, we report on new research to further characterize XB05 activity. We have examined the effects of XB05 on clonogenicity, promoter methylation, gene expression, and DNMT1 protein. XB05 was found to inhibit colony formation of HCT116 colon carcinoma cells in a dose-dependent manner with a sub-micromolar IC50. Western blots of nuclear extracts from HCT116 cells treated for 72 h with 100 nM XB05 or 10 µM azaC (as positive control) showed greatly decreased levels of DNMT1, suggesting that binding to XB05 can induce degradation of DNMT1 (previous research indicates direct drug-enzyme binding). Analysis of CDKN2A promoter methylation by bisulfite modification and sequencing demonstrated heavy methylation in untreated HCT116 cells. This was reversed by treatment of with 100 nM XB05 or 10 µM azaC, resulting in re-expression of the gene (which encodes tumor suppressor, p16), as shown by qRT-PCR. Experiments to assess promoter methylation using multiplex arrays and to examine gene expression changes using DNA microarrays were recently completed and data analysis is ongoing. To evaluate in vivo activity, we treated mice bearing subcutaneous HCT116 xenografts by IP injection of XB05 with multiple doses of up to 10 mg/kg. There was no evidence of toxicity as judged by body weights and gross necropsies. The tumors of mice treated with XB05, although not significantly smaller than in control animals, were characterized by extensive necrosis in the center of the tumor, leading to a “hollow” tumor in the majority (12/20) of mice. No similar necrosis was observed in vehicle-treated or azaC-treated mice, suggesting that this effect is treatment related. Because induced necrosis is seen with vascular disrupting agents, we also examined the effect of XB05 on endothelial cells in an in vitro assay, and observed reproducible inhibition of endothelial tube formation at 400 nM or higher. In summary, our new data confirm that XB05 is a potent inhibitor of DNA methylation in cultured cancer cells, and provide the first evidence of in vivo activity, which may involve effects on the tumor vasculature, as well as the tumor itself. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5447.

We recently reported on the isolation of plagiochiline A, a 2,3-secoaromadendrane-type sesquiterpenoid, from the Peruvian medicinal plant Plagiochila distichia. This compound was found to have antiproliferative activity against a variety of solid tumor cell lines, including those derived from prostate, colon, breast, lung, and cervical cancers, as well as several leukemia cell lines. Other researchers have also noted the cytotoxicity of plagiochiline A (isolated from other plant species), but the mechanism for this activity was unknown. Here, we describe new research where we have investigated the mechanism of plagiochiline A using a variety of experimental techniques. First of all, we evaluated cell cycle parameters in DU145 prostate cancer cells treated for 24 h with 4 µg/mL (11.4 µM) plagiochiline A using flow cytometry based analysis of propidium iodide-stained cells. We found that plagiochiline A caused a statistically significant increase in the percentage of cells in the G2/M phase compared to vehicle-treated cells (41.7% compared to 24.8%, based on three independent experiments). We next examined similarly treated cells stained with DAPI (a DNA intercalator) and anti-tubulin antibodies (microtubule marker) using fluorescence microscopy to evaluate the mitotic status of the cells. These experiments revealed a large number of binucleate cells, many of which had intercellular bridges evident, specifically in the plagiochiline A-treated samples. Only a few cells were observed in earlier stages of mitosis, suggesting that the accumulation of cells in G2/M is due to a defect in cytokinesis. We are currently carrying out additional research in order to test this hypothesis. We also plan to determine the effects on cells following longer treatments with plagiochiline A and to investigate possible molecular target(s) of plagiochiline A. Although many anti-mitotic compounds are in current use as anticancer agents, there are apparently only a few agents in development that specifically target cytokinesis. Therefore, we anticipate that this research may ultimately lead to novel anticancer agents and new tools to study cytokinesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2486.