Cultured Tumor Cell Lines Research Articles

Piperazirum, a Novel Bioactive Alkaloid from Arum palaestinum Boiss

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Triterpenoids Display Single Agent Anti-tumor Activity in a Transgenic Mouse Model of Chronic Lymphocytic Leukemia and Small B Cell Lymphoma

BackgroundThe synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) and derivatives display anti-tumor activity against a variety of cultured tumor cell lines and in mouse xenografts. In this report, we have studied the effects of CDDO and its imidazolide derivative (CDDO-Im) on chronic lymphocytic leukemia (CLL), using patients' CLL cells and a mouse model of CLL and small B cell lymphoma (SBL).Principal FindingsCDDO and CDDO-Im efficiently induced apoptosis of malignant human and mouse B-cells ex vivo, although CDDO-Im was over 10-fold more potent than CDDO. Treating mice with CLL/SBL with liposome-formulated CDDO or CDDO-Im resulted in significant reductions of B cells in blood, spleen and lung. CDDO-Im was shown to be more potent than CDDO, while treatment with empty liposomes had no impact on disease. CDDO-Im treatment initially resulted in an increase of circulating B cells, which correlates with a reduction in resident lymphocytes in spleen, and lungs, suggesting that CDDO-Im induces mobilization of tumor cells from lymphoid organs and infiltrated tissues into the circulation. Analysis of blood cells recovered from treated mice also showed that CDDO-Im is a potent inducer of tumor cells death in vivo. Furthermore, CDDO-Im efficiently eradicated mouse CLL/SBL cells but had little effect on the viability of normal B and T cells in vivo.SignificanceThe presented data demonstrate that triterpenoids CDDO and CDDO-Im reduce leukemia and lymphoma burden in vivo in a transgenic mouse model of CLL/SBL, and support the clinical testing of CDDO-based synthetic triterpenoids in patients with CLL.

Synthesis and evaluation of new 6-hydroximinosteroid analogs as cytotoxic agents

Taking into account the structural requirements for cytotoxicity, several new hydroximinosteroid derivatives have been prepared and evaluated for their cytotoxic activity against A-549, H116, PSN1, and T98G cultured tumor cell lines in order to obtain further information on the potential pharmacophoric core of this type of compound. The influence of the oxygenated position in the A ring, the presence of an additional oxygenated position at C-7 and C-16, and a fluorinated position at C-5 were considered in order to study the structure–activity relationships. The results reveal the importance of oxygenated positions in the A ring (e.g., 4,5-epoxide showed an IC 50 value against HCT-116 under micromolar level) for an increase in cytotoxic activity in this type of compound. Furthermore, they showed an important selectivity toward colon tumor line (HCT-116).

Piperazirum, a novel bioactive alkaloid from Arum palaestinum Boiss.

A novel alkylated piperazine (3α,5α-diisobutyryl-6α-isopropyl-piperazine-2-one, 1) has been isolated from the n-butanol soluble part of the leaves extract of Arum palaestinum Boiss., the chemical structure and the relative stereochemistry of 1 were elucidated on the basis of 1D and 2D NMR spectroscopic data. Piperazirum showed a significant cytotoxicity against cultured tumor cell lines in vitro.

Antibacterial, Antifungal, and Cytotoxic Activities of Distictis buccinatoria.

The current study was undertaken to evaluate the in vitro. antimicrobial and cytotoxic activity of six crude extracts obtained from the leaves and flowers of Distictis buccinatoria. (DC.) A.H. Gentry (Bignoniaceae). Antimicrobial activity was tested against the Gram-positive bacteria Staphylococcus aureus., Streptococcus pyogenes., and Streptococcus faecalis.; the Gram-negative bacteria Escherichia coli., Klebsiella pneumoniae., and Salmonella typhi.; and the fungi Candida albicans., Trichophyton mentagrophytes., Trichophyton rubrum., and Aspergillus niger.. The cytotoxic activity of each extract was determined using two human tumor cell lines in culture, nasopharyngeal carcinoma (KB) and colon carcinoma (HCT-15). The results showed that extracts from D. buccinatoria. possess antimicrobial activity against the Gram-positive bacteria and against both dermatophyte fungal species. The strongest antibacterial activity observed was that of the dichloromethane extract prepared from flowers, and the best antifungal activity was demonstrated by the dichloromethane extract from the leaves. The hexane and dichloromethane extracts from the flowers exhibited cytotoxicity against KB cells. These results support the traditional folk medicinal uses of this plant.

Parviflorene J, a Cytotoxic Sesquiterpene Dimer with a New Rearranged Skeleton from Curcuma parviflora

A new sesquiterpenoid dimer, parviflorene J, was isolated from Curcuma parviflora. The structure was elucidated using spectroscopic methods. Parviflorene J possessed an unprecedented backbone-rearranged skeleton and showed cytotoxicity against cultured tumor cell lines.

Polyamine-Substituted Gadolinium Chelates: A New Class of Intracellular Contrast Agents for Magnetic Resonance Imaging of Tumors

A new class of intracellular contrast agents (CA) for magnetic resonance imaging has been developed, based on Gd(DTPA) with two positively charged amide-linked substituents. Uptake of Gd(DTPA) into cultured tumor cell lines (B16 mouse melanoma, MH3924A Morris hepatoma) was below the detection limit while CA with the melanin-binding pharmacophore 2-(diethylamino)ethylamine reached intracellular concentrations of ca. 0.03 fmol/cell (ca. 20 microM) for melanoma and 0.02 fmol/cell for hepatoma (24 h at 10 microM CA). With the polyamine substituents bis(2-aminoethyl)amine or spermidine, CA uptake increased up to 3-fold for melanoma (0.083 fmol/cell) and 9-fold for hepatoma (0.18 fmol/cell). Uptake of polyamine-substituted CA was reduced by the polyamine transport inhibitor benzyl viologen. Molar relaxivities for three Gd-DTPA-polyamine complexes were in the range 5.6-6.9 for the free complex in solution and 7.7-23.5 s-1 mM-1 for Morris hepatoma cell pellets. T1-weighted magnetic resonance imaging at 2.35 T of rats with MH3924A tumors showed contrast enhancement in tumor at 1 and 24 h postinjection of polyamine-substituted CA.

CRA-026440: a potent, broad-spectrum, hydroxamic histone deacetylase inhibitor with antiproliferative and antiangiogenic activity in vitro and in vivo

CRA-026440 is a novel, broad-spectrum, hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor and antiangiogenic activities in vitro and in vivo preclinically. CRA-026440 inhibited pure recombinant isozymes HDAC1, HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-026440 resulted in the accumulation of acetylated histone and acetylated tubulin, leading to an inhibition of tumor cell growth and the induction of apoptosis. CRA-026440 inhibited ex vivo angiogenesis in a dose-dependent manner. CRA-026440 parenterally given to mice harboring HCT116 or U937 human tumor xenografts resulted in a statistically significant reduction in tumor growth. CRA-026440, when used in combination with Avastin, achieved greater preclinical efficacy in HCT 116 colorectal tumor model. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells and an alteration in the expression of many genes in the tumors, including several involved in angiogenesis, apoptosis, and cell growth. These results reveal CRA-026440 to be a novel HDAC inhibitor with potent antitumor activity.

Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment

Expression profiling using microarrays has become an essential tool for interrogating tumor biology. However, profiling of whole tumor RNA reflects both tumor and host cells, making it difficult to dissect molecular events within specific cellular compartments in the tumor microenvironment. We developed and optimized a simple, rapid technique combining immunohistochemistry and laser-capture microdissection (immuno-LCM) to purify specific cell populations from the tumor microenvironment followed by RNA isolation and amplification for microarray analysis. Using this methodology, we were able to elucidate the in situ expression profile of pure tumor cells and tumor endothelial cells from ovarian tumors with brisk immune infiltrates. This technique not only increased the specificity of profiling isolated cell populations, eliminating genes expressed by surrounding cells, but also increased the sensitivity of analysis, allowing for the detection of low expression genes that were not detected in whole tumor arrays. Pathway analysis of tumor cells in situ identified distinct activation of signaling pathways converging on NF-κB, as compared to pathways identified in cultured tumor cell lines, which were primarily metabolic. Profiling of tumor vascular endothelial cells revealed most known panendothelial and tumor endothelial-specific markers, and unveiled genes specific to the myeloid-monocytic lineage. We propose that immuno-LCM coupled with transcriptional profiling is a convenient tool to dissecting molecular and cellular events in complex biological systems such as the tumor microenvironment.

CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo

CRA-024781 is a novel, broad spectrum hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and is under evaluation in phase I clinical trials for cancer. CRA-024781 inhibited pure recombinant HDAC1 with a K(i) of 0.007 mumol/L, and also inhibited the other HDAC isozymes HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-024781 resulted in the accumulation of acetylated histone and acetylated tubulin, resulting in an inhibition of tumor cell growth and the induction of apoptosis. CRA-024781 parenterally administered to mice harboring HCT116 or DLD-1 colon tumor xenografts resulted in a statistically significant reduction in tumor growth at doses that were well tolerated as measured by body weight. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells, and an alteration in the expression of many genes in the tumors, including several involved in apoptosis and cell growth. These results reveal CRA-024781 to be a novel HDAC inhibitor with potent antitumor activity.

Dietary resistant starch type 3 prevents tumor induction by 1,2-dimethylhydrazine and alters proliferation, apoptosis and dedifferentiation in rat colon

Some epidemiological and experimental studies suggest that consumption of resistant starch is preventive against colon cancer. Resistant starch leads to a fermentation-mediated increase in the formation of short-chain fatty acids, with a particularly high butyrate fraction in large bowel. Butyrate is considered to be protective against colon cancer because it causes growth arrest and apoptosis and regulates expression of proteins involved in cellular dedifferentiation in various tumor cell lines in culture. We sought to investigate these processes under conditions of a carcinogenicity experiment in vivo. In the present study, 1,2-dimethylhydrazine-treated Sprague-Dawley rats were fed standard diet (n=12) or diet with 10% hydrothermally modified Novelose 330, a resistant starch type 3 (RS3), replacing digestible starch (n=8). After 20 weeks tumor number, epithelial proliferation, apoptosis, immunoreactivity of carcinogenesis-related proteins [protein kinase C-delta (PKC-delta), heat shock protein 25 (HSP25) and gastrointestinal glutathione peroxidase (GI-GPx)], as well as mucin properties were evaluated in proximal and distal colon in situ. No tumors developed under RS3 diet, compared to a tumor incidence of 0.6+/-0.6 (P<0.05) under the standard diet. RS3 decreased the number of proliferating cells, the length of the proliferation zone and the total length of the crypt in the distal colon, but not proximal colon, and enhanced apoptosis in both colonic segments. It induced PKC-delta and HSP25 expression, but inhibited GI-GPx expression in the epithelium of distal colon. RS3 increased the number of predominantly acidic mucin containing goblet cells in the distal colon, but had no effect on the goblet cell count. We conclude that hydrothermally treated RS3 prevented colon carcinogenesis, and that this effect was mediated by enhanced apoptosis of damaged cells accompanied by changes in parameters of dedifferentiation in colonic mucosa.

Intracellular localization of matrix metalloproteinases and their inhibitors in cultured tumor cell lines: Flow cytometric analysis

Coordinate cell-surface expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) on cancer cells, tumor-infiltrating lymphocytes (TIL) and cancer-associated myofibroblasts has been shown to play an important role in tumor invasion and metastasis in organ tissues. However, precise localization of tumor cell lines in vitro has remained obscure. The aim of this study was to investigate the spatial localization of these proteolytic enzymes in 21 tumor cell lines consisting of 8 gastric, 4 pancreatic, 2 gallbladder, 1 extrahepatic bile duct cancer and 6 T- or B-cell tumors. The cell-surface and/or intracellular localization of MMP-2, -7, and -9, membrane type-1 (MT1)-MMP and TIMP-2 and -4 was quantitatively determined by flow cytometry in living (non-permeabilized) and permeabilized tumor cell lines. As a result, all of the tumor cell lines showed a high level of intracellular expression, but a low or undetectable level of cell-surface expression of MMPs and TIMPs. The findings revealed that intracellular MMPs and TIMPs in tumor cell lines were preserved as latent pro-enzymes.

Fatty acid synthase inhibition induces apoptosis independent of lipid metabolism

Fatty acid synthase (FAS) is the sole eukaryotic enzyme responsible for condensing acetyl-CoA with malonyl-CoA to form palmitate, the precursor of long chain fatty acids. FAS expression is upregulated in a broad range of cancers and its inhibition has been shown to induce tumor cell cycle arrest and cell death. The decrease in palmitate and the accumulation of malonyl-CoA accompanying FAS inhibition have both been hypothesized as mediators of these antitumorigenic effects. In the present study, inhibition of FAS activity by FAS siRNA and Orlistat led to apoptosis in various cultured tumor cell lines as shown by activation of caspase 8 and subsequent DNA fragmentation. Co-transfection with caspase 8 siRNA, but not caspase 9 siRNA, rescued tumor cells from undergoing FAS induced cell death. Interestingly, transfection with either acetyl-CoA carboxylase (ACACA) siRNA to block malonyl-CoA production or ATP citrate lyase (ACLY) siRNA to block acetyl-CoA production failed to activate caspase 8 and did not lead to apoptosis. Knockdown of ACACA led to an inhibition in palmitate similar to that observed with FAS siRNA indicating that suppression of fatty acid production on its own is not sufficient to promote cell death. Co-transfection of ACACA or ACLY siRNA with FAS siRNA to block accumulation of acetyl-CoA or malonyl-CoA failed to prevent cells from undergoing FAS induced apoptosis. Collectively, these data show that FAS inhibition induces apoptosis through caspase 8. Suppression of palmitate does not account for the ability of FAS inhibition to induce tumor cell death. Research was funded by DoD Grant DAMD17-02-0693 and CBCRP Grants CA6982713, CA69306 and 8FB-0107.

Absence of Radioadaptive Responses in Four Cell-lines in Vitro as Determined by Colony Formation Assay

The purpose of this study was to investigate radioadaptive response in 4 cell-lines under identical conditions using a colony assay. First, 4 cell-lines (V79, HeLa S3, EMT6 and SCCVII) were exposed to 8 Gy at various intervals after pretreatment with an adapting dose of 50 mGy or without it. Second, V79 cells were exposed to 8 Gy at 4.5 hrs after an adapting dose of 0 to 400 mGy. Third, V79 cells were exposed to 2, 4 or 6 Gy at 6 hrs after an adapting dose of 0 or 50 mGy. In the last experiment, an adapting dose was given either immediately after cell plating or 24 hrs later. Cell survival was assessed by a standard colony assay. Adaptive response was not observed in any of the 4 lines tested. In V79 cells, no adaptive response was seen even by changing the adapting dose, challenging dose, and timing of adapting radiation after cell plating. Although radioadaptive response has been reported for the V79 cell-line, we could not reproduce the result. We also failed to demonstrate the phenomenon in the other 3 tumor cell-lines in culture.

Distinct Pathways Regulate Proapoptotic Nix and BNip3 in Cardiac Stress

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 microM), angiotensin II (100 nM), or isoproterenol (10 microM). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC alpha, but not PKC epsilon or dominant negative PKC alpha, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKC alpha-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G(q) overexpression model of hypertrophy. Luciferase activity increased in G alpha(q)-NixP hearts 3.2 +/- 0.4-fold and in TAC hearts 2.8 +/- 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxia-inducible, whereas Nix expression was induced by G alpha(q)-mediated hypertrophic stimuli. PKC alpha, a G(q) effector, transduced Nix transcriptional induction via Sp1.

Analysis of Expanded Human Cytokine Induced Killer Cells for Cellular Therapy.

We have previously reported on the ex vivo expansion of cytotoxic effector T cells termed Cytokine Induced Killer (CIK) cells. CIK cells demonstrate both in vivo and in vitro anti-tumor activity in murine models with limited potential for GVHD induction. Human CIK cells are readily expanded from peripheral blood mononuclear cells (PBMC) when cultured with IFNg, CD3 and IL-2 and show cytotoxic activity against a variety of tumor cell lines and fresh autologous tumor cells through an NKG2D dependent mechanism. Clinical application of CIK cells requires expansion of effector cells in GMP-compliant manner. In this study, PBMC were collected by apheresis from 9 healthy non-mobilized donors and placed in closed, continuous perfusion cultures using Aastrom RepliCell@biochambers for 21–28 days. Cultures were inoculated with 3x108–7.5x108 nucleated cells containing a median of 46% CD3+ cells (range 29%–71%). At culture harvest, the median cell count was > 4x109 (range 2.6x109–8.7x109) with 97% CD3+ (86%–99%) and an average cell viability of 84.8% (range, 75%–99.6%). The average expansion of CD3+ cells in culture was > 30-fold (range13–91). CD3+CD56+ are the subpopulation of cells that demonstrated the highest cytotoxicity against tumor cell lines in culture. The mean percentage of these cells was 1.8% however at harvest, the mean CD3+CD56+ was 14% representing an average 34-fold expansion (range 19–515). The majority of CD3+ cells were CD8+ (range, 33–82%), NKG2D was predominately expressed by cells recovered from most cultures (range 47–76%). CD56+ was expressed by 1% – 36% of cells in different cultures and CD8+NKG2D+ expression ranged from 21%–50% of harvested cells. There was a moderate up-regulation of the activation markers CD25 and CD69 and the NK markers CD16, CD161 and CD94. In all cultures, the majority of CD8+ and CD56+ cells co-expressed the selectin marker a4b7. Furthermore the majority of the CD3+CD8+ cells were effector memory cells expressing CD62L−CD44+ (50%–71%) while the central memory cells CD62L+CD44+ ranged from 10%–40%, almost no naïve cells were detected in time of harvest. Cytotoxicity assays against a panel of tumor cell lines (B and T cell lymphomas, uterine cancer, erythroleukemia, ovarian cancer and plasmacytoma) showed significant yet variable killing. This system allows for the rapid expansion of cytotoxic cell in GMP-compliant manner suitable for clinical application.

Synthesis of N-{4-[(2,4-Diamino-5-methyl-4,7-dihydro-3H- pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-l-glutamic Acid and N-{4-[(2-Amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin- 6-yl)thio]benzoyl}-l-glutamic Acid as Dual Inhibitors of Dihydrofolate Reductase and Thymidylate Synthase and as Potential Antitumor Agents

Two novel classical antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR-TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the growth of several human tumor cell lines in culture with GI50 < 10(-8) M. This study shows that the pyrrolo[2,3-d]pyrimidine scaffold is conducive to dual DHFR-TS and tumor inhibitory activity, and the potency is determined by the 4-position substituent.

14 INVITED Expression profiling of peripheral blood cells (PBC) for early detection

The mammalian target of rapamycin (mTOR), an atypical serine/threonine kinase, plays a central role in the regulation of cell proliferation, growth, differentiation, migration and survival. Dysregulation of mTOR signaling occurs in diverse human tumours, and can confer higher susceptibility to inhibitors of mTOR. Rapamycin and its derivatives, CCI-779 and RAD001 (designated rapamycins), specifically inhibit the function of mTOR, leading to inactivation of ribosomal S6K1 and inhibition of cap-dependent translation initiation through the 4E–BP1/eIF4E pathway. The overall effect is an accumulation of cells in the G1 phase of the cell-cycle, and potential apoptosis. Preclinical studies indicate that rapamycins are potent inhibitors of the proliferation of numerous tumour cell lines in culture and of murine syngeneic tumour models or human xenografts. RAD001 and CCI-779 are in phase I and II trials, respectively, as anti-cancer agents. These trials have demonstrated promising anti-cancer activity and relatively mild side effects of CCI-779. Emerging results suggest that inhibition of mTOR signaling can be exploited as a potential tumour-selective therapeutic strategy.

New Bisindole Alkaloids Isolated from Myxomycetes Arcyria cinerea and Lycogala epidendrum.

AbstractFor Abstract see ChemInform Abstract in Full Text.

8- O-Azeloyl-14-benzoylaconine: A new alkaloid from the roots of Aconitum karacolicum Rapcs and its antiproliferative activities

A new alkaloid of Aconitum karacolicum Rapcs, from the Ranunculaceae family, collected in Kirghizstan, was isolated from the roots of this plant, using a purification scheme based upon its in vitro antiproliferative properties against three human tumour cell lines in culture. Structural identification was performed using high resolution MS–MS mass spectrometry and 1H, 13C, 2D NOESY NMR spectroscopy analysis. This compound consists of a 14-benzoylaconine moiety substituted on C-8 by an azeloyl chain. It presents in vitro cytotoxicity with an IC 50 of about 10–20 μM, which warrants further investigation on its possible interest in cancer chemotherapy.