Cultured Tumor Cell Lines Research Articles

HSP32 (HO-1) inhibitor, copoly(styrene-maleic acid)-zinc protoporphyrin IX, a water-soluble micelle as anticancer agent: In vitro and in vivo anticancer effect

We reported previously the antitumor effect of heme oxygenase-1 (HO-1) inhibition by zinc protoporphyrin IX (ZnPP). ZnPP per se is poorly water soluble and thus cannot be used as anticancer chemotherapeutic. Subsequently, we developed water-soluble micelles of ZnPP using styrene-maleic acid copolymer (SMA), which encapsulated ZnPP (SMA–ZnPP). In this report, the in vitro and in vivo therapeutic effects of SMA–ZnPP are described. In vitro experiments using 11 cultured tumor cell lines and six normal cell lines revealed a remarkable cytotoxicity of SMA–ZnPP against various tumor cells; average IC50 is about 11.1μM, whereas the IC50 to various normal cells is significantly higher, that is, more than 50μM. In the pharmacokinetic study, we found that SMA–ZnPP predominantly accumulated in the liver tissue after i.v. injection, suggesting its applicability for liver cancer. As expected, a remarkable antitumor effect was achieved in the VX-2 tumor model in the liver of rabbit that is known as one the most difficult tumor models to cure. Antitumor effect was also observed in murine tumor xenograft, that is, B16 melanoma and Meth A fibrosarcoma. Meanwhile, no apparent side effects were found even at the dose of ∼7 times higher concentration of therapeutics dose. These findings suggest a potential of SMA–ZnPP as a tool for anticancer therapy toward clinical development, whereas further investigations are warranted.

Abstract 4765: Synthesis and evaluation of nonclassical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates: Role of terminal amino acid moiety in membrane transport and antitumor activity.

Abstract Reduced folates are essential cofactors for biosynthesis of purines and pyrimidines. Since humans do not synthesize folate, it is necessary to obtain these cofactors from dietary sources. In mammals, three specialized systems exist that mediate membrane transport of folates and antifolates across biological membranes. These include the reduced folate carrier (RFC), the primary route for the uptake of folates and antifolates in mammalian cells, folate receptors (FRs) ≤ and β, and the proton-coupled folate transporter (PCFT). Whereas RFC is ubiquitously expressed, FRs and PCFT show a more narrow pattern of tissue expression. Toxicity of clinically used antifolates is due to their lack of selectivity for tumor cells over normal cells, since these drugs are transported by RFC. Antifolates with selective substrate activity for FRs and/or PCFT over RFC can potentially target tumors that express these non-RFC transporters relative to normal tissues. Thus, antifolates with tumor-specific FR and/or PCFT drug uptake would circumvent major toxicities of currently used antifolates. 6-Substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four carbon bridge length and with L-aspartic acid (AG170), aminomalonic acid (AG173) and α-glutamic acid (AG172) were designed and synthesized in place of the normal L-glutamic acid (AG118) of this series to evaluate the importance of L-glutamic acid for RFC, FRα, and PCFT transport and inhibition of cell proliferation. AG170 was slightly less active than AG118 in inhibiting growth of a CHO cell line (RT16) expressing human FRα (IC50s of 18.7 and 2.6 nM, respectively) but similarly active in inhibiting PCFT-expressing CHO cells (R2/PCFT4) (IC50s of 81.7 and 63.8, nM, respectively). AG173 and AG172 were inactive toward both RT16 and R2/PCFT4 cells (IC50>1000nM). All four analogues were inactive toward a CHO cell line (PC43-10) expressing human RFC but not FRα or PCFT. These analogues were further evaluated in KB and IGROV-1 human tumor cell lines in culture. Whereas AG118 was potently inhibitory toward KB and IGROV1 tumor cells (IC50s of 0.27 and 1.4 nM, respectively), AG170, AG173, and AG172 all were inert toward these tumor cells at concentrations up to 1000 nM. Collectively, these results establish an important role for the terminal amino acid in the anti-proliferative effects of this series of 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates. The disparate inhibitions of AG170 toward FRα-expressing CHO versus human tumor cells implies that there may be differences in metabolic pathways and/or target enzymes in CHO cell lines compared to human cancer cell lines that are responsible. 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 4765. doi:1538-7445.AM2012-4765

Abstract 1738: Assay development for detecting cMET expression in circulating tumor cells (CTC), a potential patient tailoring marker for evaluation of cMET inhibitors

Abstract Hepatocyte growth factor and its receptor c-MET have been implicated in tumor formation and progression as well as drug treatment resistance to targeted agents such as EGFR inhibitors. cMET over-expression or gene amplification has been reported in a wide variety of human malignancies correlating with poor patient prognosis. A reasonable hypothesis for experimental agents targeting the cMET receptor is that patients with tumors expressing high levels of cMET may respond better. CTC counts are prognostic of survival in metastatic breast, colorectal, and prostate cancers (NEJM 2004;351:781-91; J Clin Oncol 2008;26:3213-21; Clin Can Res 2008;14:6302-9). CTCs are shed from tumors and are believed to be representative of cells migrating to form distal metastasis. The presence of these cells in blood provides an attractive and easy source for serial monitoring of tumors without the limitations of traditional solid tumor biopsy. We describe here the development of an assay that measures cMET expression in CTCs. The CTC assay was developed using the Veridex CellSearch® System, and RUO reagents for enumeration of CTCs and a Lilly proprietary cMET antibody (Ab) optD11. For assay development, cultured tumor cell lines with differing cMET expression levels representing solid epithelial tumor types were spiked into whole blood drawn into a CellSave tube. For initial assay development, mouse whole blood was used and results reproduced subsequently with human whole blood from healthy subjects. The spiked tumor cells in blood samples were recovered using the CellCapture™ Mouse/Rat CTC kit (for mouse blood) or CellSearch® CXC kit (for human blood), supplemented with the fluorescent dye (R- phycoerythrin) conjugated anti-cMET Ab. cMET in the recovered tumor cells was detected in the open/fourth channel on the CellTrack® Analyzer II®. Several proprietary and commercial cMET antibodies were screened for specificity and sensitivity. A commonly used commercially available cMET Ab (Santa Cruz Biotechnology C28) was not able to discriminate between cMET negative and positive cell lines. Results were confirmed by flow cytometry and by western blot analyses. optD11 was selected for CTC cMET expression assay development for the following reasons: a) ability to detect different cMET levels among positive cell lines (H441, MKN45, SNU5, SKOV3) while identifying SKBR3 as cMET negative; b) high affinity for cMET to allow for an image acquisition time of 0.02-0.04 second in the open channel; c) tolerance to the presence of LY2875358 (LA480), an experimental therapeutic anti-cMET Ab, allowing evaluation of cMET levels in CTCs post-treatment. The CTC assay for cMET expression, as well as a CTC assay measuring cMET gene amplification are currently being implemented in the early phase clinical studies for Lilly cMET inhibitors (JSBA, NCT01285037 & JTBA, NCT01287546). 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 1738. doi:1538-7445.AM2012-1738

Drug resistance in the mouse cancer clinic

Drug resistance is one of the most pressing problems in treating cancer patients today. Local and regional disease can usually be adequately treated, but patients eventually die from distant metastases that have become resistant to all available chemotherapy. Although work on cultured tumor cell lines has yielded a lot of information on potential drug resistance mechanisms, it has proven difficult to translate these results to clinical drug resistance in patients. The controversy regarding the contribution of ABC transporters to drug resistance in patients is one example. The study of genetically engineered mouse models (GEMMs), which closely resemble cancer in human patients, can help to bridge this gap. In models for BRCA1- or BRCA2-associated breast cancer, we observed a substantial synergy between the defect in homology-directed DNA repair and sensitivity to DNA-targeting drugs. Nevertheless, tumors are not easily eradicated and eventually drug resistance develops. In this review we will discuss the use of the new generation mouse models to address major clinical problems, such as mechanisms of drug resistance, predicting chemotherapy response or characterizing the nature of residual tumor cells that escape eradication. Moreover, we will address the contribution of ABC transporters to drug resistance in our model.

Abstract A47: Circulating tumor cell (CTC) assay development for detection of phosphohistone H3 (pHH3) as a clinical pharmacodynamic (PD) marker for LY2523355, an Eg5 kinesin inhibitor.

Abstract Circulating tumor cells have been shown to be prognostic of survival in metastatic breast, colon, and prostate cancers. Additionally, CTCs are of interest because they may be representative of the phenotype/genotype of the primary and metastatic tumors, and may be a useful tool (i.e. patient stratification) for drug development. CTCs, as “liquid biopsies” are potentially useful as a PD marker as it allows easy repeat sampling before and after drug treatment. We describe the development of a CTC assay that measures the induction of pHH3 by LY2523355, a selective small molecule inhibitor of the human Eg5 kinesin. pHH3 is a biomarker of mitosis. In tumor xenograft mouse models, LY2523355 treatment was shown to reduce tumor volume with elevation of pHH3 in tumors. The CTC assay was developed using the FDA cleared Veridex/CellSearch™ instrument and reagents for enumeration of CTCs from blood. CellSearch defines CTCs as EpCam+/DAPI+/CK 8, 18, 19+/CD45−. For assay development, cells from tumor cell lines representing major solid tumor types were chosen, cultured, treated with LY2523355, or with various standard-of-care chemotherapeutics, and spiked into whole blood drawn into CellSave™ (preservative+EDTA). For initial assay development, mouse whole blood was used and results reproduced subsequently with human whole blood from healthy subjects. The spiked tumor cells in blood samples were recovered using the CellSearch Mouse/Rat CTC kit (for mouse blood) or human CXC kit (for human blood), supplemented with the R-PE conjugated anti-pHH3 antibody. pHH3 in the recovered tumor cells was detected in the open/fourth channel on the CellSearch instrument. About 1–4% of all cultured tumor cells were positive for pHH3 without drug treatment. Significant induction of pHH3 (30–50% cells) in sensitive tumor cell lines was observed with 24 hour treatment with only drugs interfering with mitosis (paclitaxel, taxotere, and LY2523355). The magnitude of the induction of pHH3 in the cells after treatment was dose and cell-line dependent. A dose response of pHH3 induction was observed with 0.5nM-25nM of LY2523355 with several cultured tumor cell lines (Colo205, HCT116, H441, SKBR3, MDA-MB-468), spiked into blood, and assayed using CellSearch. The concentrations of the dose response are similar to the drug exposure levels of that of preclinical tumor xenograft studies. LY2523355 is currently in early phase clinical development, and monitoring CTCs with pHH3 expression post-treatment may be a useful PD marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A47.

Chidamide (CS055/HBI-8000): a new histone deacetylase inhibitor of the benzamide class with antitumor activity and the ability to enhance immune cell-mediated tumor cell cytotoxicity

Chidamide (CS055/HBI-8000) is a new histone deacetylase (HDAC) inhibitor of the benzamide class currently under clinical development in cancer indications. This study reports the in vitro and in vivo antitumor characteristics of the compound. Selectivity and potency of chidamide in inhibition of HDAC isotypes were analyzed by using a panel of human recombinant HDAC proteins. Tumor cell lines either in culture or inoculated in nude mice were used for the evaluation of the compound's antitumor activity. To investigate the immune cell-mediated antitumor effect, isolated peripheral blood mononuclear cells from healthy donors were treated with chidamide, and cytotoxicity and expression of relevant surface proteins were analyzed. Microarray gene expression studies were performed on peripheral white blood cells from two T-cell lymphoma patients treated with chidamide. Chidamide was found to be a low nanomolar inhibitor of HDAC1, 2, 3, and 10, the HDAC isotypes well documented to be associated with the malignant phenotype. Significant and broad spectrum in vitro and in vivo antitumor activity, including a wide therapeutic index, was observed. Chidamide was also shown to enhance the cytotoxic effect of human peripheral mononuclear cells ex vivo on K562 target cells, accompanied by the upregulation of proteins involved in NK cell functions. Furthermore, the expression of a number of genes involved in immune cell-mediated antitumor activity was observed to be upregulated in peripheral white blood cells from two T-cell lymphoma patients who responded to chidamide administration. The results presented in this study provide evidence that chidamide has potential applicability for the treatment of a variety of tumor types, either as a single agent or in combination therapies.

Aberrant Overexpression of the Cell Polarity Module Scribble in Human Cancer

Human Scribble (Scrib) is an evolutionary-conserved cell polarity protein, but its potential role in human cancer is controversial. Herein, we show that Scrib is nearly universally overexpressed in cultured tumor cell lines and genetically disparate cancer patient series compared with matched normal tissues in vivo. Instead of a membrane association seen in normal epithelia, tumor-associated Scrib is mislocalized and found predominantly in the cytosol. Small-interfering RNA silencing of Scrib in model lung adenocarcinoma A549 cells inhibited cell migration in wound-healing assays, suppressed tumor cell invasion across Matrigel-coated inserts, and down-regulated the expression of cell motility markers and mediators of epithelial-mesenchymal transition. These data uncover a previously unrecognized exploitation of Scrib for aberrant tumor cell motility and invasion, thus potentially contributing to disease progression in humans.

Abstract SY26-01: Mitochondrial metabolism in the survival and growth of cancer cells

Abstract Interest in tumor metabolism stems from the assumption that metabolic differences between normal and malignant cells can be exploited to improve imaging, therapy and clinical outcomes in cancer patients. Mutations in tumor suppressors and oncogenes influence nutrient utilization in cancer cells, presumably to allow malignant cells to meet the metabolic demands of survival, growth and proliferation. Many of these effects converge on glucose utilization, reinforcing the notion first proposed by Otto Warburg that malignant cells have high rates of glycolysis but suppressed or impaired mitochondrial metabolism (the “Warburg effect”). However this simple model fails to explain the most fundamental property of cancer cells: their uncontrolled growth in culture and in vivo. Cell growth requires an abundant supply of macromolecular precursors, and many of these key molecules are formed as byproducts of mitochondrial metabolism. Thus, a more realistic alternative to Warburg's model is that the high glycolytic flux observed in tumor cells must be complemented by mitochondrial systems optimized to generate precursors for biosynthesis; the former pathway helps maintain cellular bioenergetics and the latter pathways support maximal cell growth and proliferation. We used metabolic flux analysis to determine the effects of oncogenic signaling on glycolysis and mitochondrial metabolism in normal human diploid cells. The data demonstrate that oncogenes and enhanced growth factor signaling stimulate the delivery of carbon from glucose and other nutrients into pools of mitochondrial metabolites. The effects on these mitochondrial activities are even more dramatic than the effects on glycolysis, implying that enhanced mitochondrial fluxes are essential components of malignant transformation. We identified three distinct modes of mitochondrial metabolism that differed from each other by the allocation of carbon from glucose and glutamine into specific mitochondrial pathways. Strikingly, the choice of pathway used to supply carbon to the tricarboxylic acid (TCA) cycle determines cellular sensitivity to perturbations of glucose and glutamine metabolism in vitro and in vivo. We applied similar methods in flux analysis to interrogate the metabolism in situ of brain tumors in mice and humans. These studies confirmed the presence of robust mitochondrial activity, along with the Warburg effect, in all tumors analyzed. In particular, all tumors used the TCA cycle and other mitochondrial pathways to produce macromolecular precursors from glucose and/or glutamine. Together, the findings underscore the importance and complexity of mitochondrial metabolism in tumor cell growth. They also emphasize the feasibility of using stable isotope infusions to obtain metabolic information directly from intact tumors in human patients and mouse models of cancer, rather than relying exclusively on the behavior of tumor cell lines in culture. We anticipate that studies like these will provide an accurate picture of the metabolic basis of tumor cell growth, and will support the rational design of novel metabolic strategies to image and treat cancer. 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 SY26-01. doi:10.1158/1538-7445.AM2011-SY26-01

Abstract 2389: Efficacy of ABT-737 against c-Myc-induced mammary adenocarcinomas in transgenic and syngrafted mouse tumor models

Abstract Traditional preclinical discovery methods and models only poorly predict drug efficacy in clinical trials. It has been reasoned that present cultured tumor cell line and xenotransplantation-based in vivo models are too reductionist, failing to recapitulate tumour heterogeneity, tumor-microenvironment interactions and tumor microevolutionary trajectories. The genetically engineered mouse models (GEMMs) hold promise as next generation preclinical models, but the caveat is that GEMMs are not straightforward suitable for preclinical cohort studies. Here we hypothesized that mammary tumors from tumor-prone WAP-Myc mice retain their molecular characteristics and authentic drug responses when recreated as syngrafts in the mammary fat pads of recipient mouse. This platform would allow rapid and non-laborious pilot cohort studies with GEMM tumors. We are testing the hypothesis by determining tumor markers and apoptotic efficacy of BH3-mimetic ABT-737 against c-Myc-induced mammary adenocarcinomas and pulmonary metastases in both transgenic and syngraft models. In the WAP-Myc mice c-Myc is induced in the luminal mammary epithelial cells during late pregnancy resulting in development of endogeneous mammary adenocarcinomas in nearly 100% of multiparous animals. We observe that the tumors are transplantable as cells suspensions (25 000 cells/gland) with approximately 90% success rate. While the transgenic WAP-Myc tumors develop with an average of 3.5 months latency, the syngrafted tumors develop within 3-4 weeks post-transplantation. However, in contrast to 20% rate of pulmonary metastases in the transgenic WAP-Myc mice, syngrafted animals need to be sacrificed prior to metastasis formation. Our preliminary results suggest that the tumor histopathology does not significantly change and the tumor heterogeneity is preserved in the syngrafted tumors. To investigate the response of tumors to ABT-737, animals bearing either endogenous or transplanted tumors were treated with 100mg/kg of ABT-737 for 21 days. ABT-737 reduced primary tumor growth and lung metastases as well as enhanced caspase-3 activity in the transgenic WAP-Myc tumors. Also the syngrafted tumors respond to ABT-737 and the data comparing effects in transgenic versus syngrafted tumors will be presented. Our results demonstrating feasibility of the syngraft platform may help to alleviate many shortcomings of GEMMs in preclinical studies. 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 2389. doi:10.1158/1538-7445.AM2011-2389

Sudemycins, Novel Small Molecule Analogues of FR901464, Induce Alternative Gene Splicing

Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant antitumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, which allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity toward human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells, and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumors, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents.

Studies on antitumor activities of triterpene glycoside colochiroside A from sea cucumber Colochirus anceps

To study the antitumor activities of the triterpene glycoside colochiroside A (CA) from the sea cucumber Colochirus anceps. The tests of antitumor activities in vitro and in vivo were applied to demonstrate the effect of CA. The preliminary cytotoxic assay of CA exhibited significant cytotoxic activity against 6 types cultured tumor cell lines of p388, HL60, A-549, SpC-A4, MKN-28, and SGC-7901, the mean of IC50 were (3.61 +/- 0.55) mg x L(-1). The preliminary antitumor assay of CA indicated that this saponin exhibited high inhibiting activity against the H22 live cancer and the S180 sarcoma cells in mouse. The inhibition ratio to H22 liver cancer were 34.8%, 43.9% and 52.2%, while the ratio to S180 sarcoma were 36.4%, 70.0%, the immunoregulatory founction study indicated CA has not significant effect on the developments of thymus and spleen. The saponin CA exhibited remarkable antineoplastic activities in vitro and in vivo, and could not reduce the immunoregulatory founction of mice.

MK-5108, a Highly Selective Aurora-A Kinase Inhibitor, Shows Antitumor Activity Alone and in Combination with Docetaxel

Aurora-A kinase is a one of the key regulators during mitosis progression. Aurora-A kinase is a potential target for anticancer therapies because overexpression of Aurora-A, which is frequently observed in some human cancers, results in aberrant mitosis leading to chromosomal instability and possibly tumorigenesis. MK-5108 is a novel small molecule with potent inhibitory activity against Aurora-A kinase. Although most of the Aurora-kinase inhibitors target both Aurora-A and Aurora-B, MK-5108 specifically inhibited Aurora-A kinase in a panel of protein kinase assays. Inhibition of Aurora-A by MK-5108 in cultured cells induced cell cycle arrest at the G(2)-M phase in flow cytometry analysis. The effect was confirmed by the accumulation of cells with expression of phosphorylated Histone H3 and inhibition of Aurora-A autophosphorylation by immunostaining assays. MK-5108 also induced phosphorylated Histone H3 in skin and xenograft tumor tissues in a nude rat xenograft model. MK-5108 inhibited growth of human tumor cell lines in culture and in different xenograft models. Furthermore, the combination of MK-5108 and docetaxel showed enhanced antitumor activities compared with control and docetaxel alone-treated animals without exacerbating the adverse effects of docetaxel. MK-5108 is currently tested in clinical trials and offers a new therapeutic approach to combat human cancers as a single agent or in combination with existing taxane therapies.

Small-cell neuroendocrine carcinoma of the prostate: are heterotransplants a better experimental model?

Small-cell neuroendocrine carcinoma of the prostate (SCNCP) is an uncommon type of prostate cancer. However, it is of clinical importance because it is one of the most aggressive tumors of the prostate with a very poor prognosis. There exist few artificially cultured tumor cell lines to study SCNCP. Then, another approach to that study consists in the use of fresh tumor tissue obtained from patients and its heterotransplantation into host mice. The purpose of this review is to integrate data from more than 20 years of heterotransplantation research in the study of small-cell neuroendocrine carcinoma of the prostate (SCNCP). Heterotransplantation has provided data regarding the histopathology, karyotype, DNA content, cell cycle frequency, tumor markers, androgen receptor expression, metastasis and take rate of this prostate disease. When possible, comparisons between original in situ specimens removed from patients and heterotransplanted tissue from host mice have been made. There are advantages, as well as limitations, that have been identified for SCNCP heterotransplants versus xenotransplantation of cultured cells. Overall, heterotransplanted tumors are better than conventional tumor xenografts at retaining tumor morphology, pathology, secretory activity and expression of tumor markers of the patient's original specimen. Furthermore, heterotransplanted tissue preserves the three-dimensional tumor architecture of the prostate to maintain critical stromal-epithelial cell interactions.

Synthesis of a new cytotoxic cephalostatin/ritterazine analogue from hecogenin and 22- epi-hippuristanol

A new cephalostatin/ritterazine analogue was prepared from the commercially available hecogenin acetate and the natural cytotoxic steroid 22- epi-hippuristanol. The method involved the reductive dimerization of enaminoketones (condensation of α-aminoketones) and condensation between an enaminoketone and an α-hydroxyketone. The new analogue showed higher cytotoxic activity than the cytotoxic 22- epi-hippuristanol against MDA-MB-231, A-549 and HT-29 cultured tumor cell lines.

338 Genetic susceptibility to breast cancer – new developments and clinical application

Drug resistance is one of the most pressing problems in treating cancer patients today. Local and regional disease can usually be adequately treated, but patients eventually die from distant metastases that have become resistant to all available chemotherapy. Although work on cultured tumor cell lines has yielded a lot of information on potential drug resistance mechanisms, it has proven difficult to translate these results to clinical drug resistance in patients. The controversy regarding the contribution of ABC transporters to drug resistance in patients is one example. The study of genetically engineered mouse models (GEMMs), which closely resemble cancer in human patients, can help to bridge this gap. In models for BRCA1- or BRCA2-associated breast cancer, we observed a substantial synergy between the defect in homology-directed DNA repair and sensitivity to DNA-targeting drugs. Nevertheless, tumors are not easily eradicated and eventually drug resistance develops. In this review we will discuss the use of the new generation mouse models to address major clinical problems, such as mechanisms of drug resistance, predicting chemotherapy response or characterizing the nature of residual tumor cells that escape eradication. Moreover, we will address the contribution of ABC transporters to drug resistance in our model.

Phytochemical Investigation and Cytotoxic Evaluation of the Components of the Medicinal Plant Ligularia atroviolacea

A phytochemical investigation of the roots of Ligularia atroviolacea resulted in the isolation of 24 compounds including seven new eremophilanoids named eremophila-3,7(11),8-triene-12,8;14,6alpha-diolide (1), 3beta-(angeloyloxy)eremophil-7(11)-en-12,8beta-olid-14-oic acid (2), 1alpha-chloro-10beta-hydroxy-6beta-(2-methylpropanoyloxy)-9-oxo-7,8-furoeremophilane (3), (10betaH)-8-oxoeremophila-3(4),6(7)-diene-12,14-dioic acid (4), (10alphaH)-8-oxoeremophila-3(4),6(7)-diene-12,14-dioic acid (5), 8beta-[eremophila-3',7'(11')-diene-12',8'alpha;14',6'alpha-diolide]eremophila-3,7(11)-diene-12,8alpha;14,6alpha-diolide (6), and ligulatrovine A (7), eleven known eremophilanoids, 8-18, four steroids, one glucose derivative, and one fatty acid. The structures of these compounds were elucidated by spectroscopic methods including 2D-NMR experiments. The structure of 3 was also established by an X-ray diffraction study. The in vitro cytotoxicity evaluation of selected compounds was performed on seven cultured tumor cell lines, i.e., KB, BEL-7404, A549, HL-60, HeLa, CNE, and P-388D1. The preliminary taxonomy of this species was also discussed, and the possible biogenesis of a dimer possessing a new noreremophilanoid type skeleton, 7, is presented in a preliminary form.

MicroRNA Expression Ratio Is Predictive of Head and Neck Squamous Cell Carcinoma

The involvement of microRNAs in cancer and their potential as biomarkers of diagnosis and prognosis are becoming increasingly appreciated. We sought to identify microRNAs altered in head and neck squamous cell carcinoma (HNSCC) and to determine whether microRNA expression is predictive of disease. RNA isolated from fresh-frozen primary tumors, fresh-frozen nondiseased head and neck epithelial tissues, and HNSCC cell lines was profiled for the expression of 662 microRNAs by microarray. The microRNAs that were both differentially expressed on the array and by quantitative reverse transcription-PCR were subsequently validated by quantitative reverse transcription-PCR using a total of 99 HNSCC samples and 14 normal epithelia. A marked difference in microRNA expression pattern was observed between tumors and cell lines. Eighteen microRNAs were significantly altered in their expression between normal tissues and tumors. Four of these microRNAs were validated in the larger sample series, and each showed significant differential expression (P < 0.0001). Furthermore, an expression ratio of miR-221:miR-375 showed a high sensitivity (0.92) and specificity (0.93) for disease prediction. These data suggest that cultured tumor cell lines are inappropriate for microRNA biomarker identification and that the pattern of microRNA expression in primary head and neck tissues is reflective of disease status, with certain microRNAs exhibiting strong predictive potential. These results indicate that miR-221 and miR-375 should be evaluated further as diagnostic biomarkers because they may hold utility in defining broadly responsive prevention and treatment strategies for HNSCC.

Cytotoxic activity of Brazilian Cerrado plants used in traditional medicine against cancer cell lines

The search for new anti-cancer drugs is one of the most prominent research areas of natural products. Numerous active compounds isolated from Brazilian Cerrado plant species have been studied with promising results. Aim of the study To investigate the cytotoxic potential of 412 extracts from Brazilian Cerrado plants used in traditional medicine belonging to 21 families against tumor cell lines in culture. Material and method Maceration of 50 plant species resulted in 412 hexane, dichloromethane, ethanol and hydroalcohol extracts. The cytotoxicity of the extracts was tested against human colon carcinoma (HCT-8), melanoma (MDA-MB-435), and brain (SF-295) tumor cell lines, using the thiazolyl blue test (MTT) assay. Bioassay-guided fractionation was performed for one active extract. Results and Conclusions Twenty-eight of the 412 tested extracts demonstrated a substantial antiproliferative effect, at least 85% inhibition of cell proliferation at 50 μg/mL against one or more cell lines. Those extracts are obtained from different parts of Anacardiaceae, Annonaceae, Apocynaceae, Clusiaceae, Flacourtiaceae, Sapindaceae, Sapotaceae, Simaroubaceae and Zingiberaceae. Complete dose-response curves were generated and IC 50 values were calculated for these active extracts against four cell lines HCT-8, MDA-MB-435, SF-295 and HL-60 (leukemia), and their direct cytotoxic effects were determined. In summary, 14 extracts of 13 species showed toxicity in all tested tumor cell lines, with IC 50 values ranging from 0.1 to 19.1 μg/mL. The strongest cytotoxic activity was found for the hexane extract of Casearia sylvestris var. lingua stem bark, with an IC 50 of 0.1 μg/mL for HCT-8, 0.9 μg/mL for SF-295, 1.2 μg/mL for MDA-MB-435, and 1.3 μg/mL for HL-60, and Simarouba versicolor root bark, with an IC 50 of 0.5 μg/mL for HCT-8, 0.7 μg/mL for SF-295, 1.5 μg/mL for MDA-MB-435, 1.1 μg/mL for HL-60. Bioassay-guided fractionation of the last extract led to the isolation of glaucarubinone, which showed pronounced activity against the four cell lines studied. Further studies of the active extracts are necessary for chemical characterization of the active compounds and more extensive biological evaluations.

Increased PADI4 expression in blood and tissues of patients with malignant tumors

BackgroundPeptidylarginine deiminase type 4 (PAD4/PADI4) post-translationally converts peptidylarginine to citrulline. Recent studies suggest that PADI4 represses expression of p53-regulated genes via citrullination of histones at gene promoters.MethodsExpression of PADI4 was investigated in various tumors and non-tumor tissues (n = 1673) as well as in A549, SKOV3 and U937 tumor cell lines by immunohistochemistry, real-time PCR, and western blot. Levels of PADI4 and citrullinated antithrombin (cAT) were investigated in the blood of patients with various tumors by ELISA (n = 1121).ResultsImmunohistochemistry detected significant PADI4 expression in various malignancies including breast carcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cancer cells, colorectal adenocarcinomas, renal cancer cells, ovarian adenocarcinomas, endometrial carcinomas, uterine adenocarcinomas, bladder carcinomas, chondromas, as well as other metastatic carcinomas. However, PADI4 expression was not observed in benign leiomyomas of stomach, uterine myomas, endometrial hyperplasias, cervical polyps, teratomas, hydatidiform moles, trophoblastic cell hyperplasias, hyroid adenomas, hemangiomas, lymph hyperplasias, schwannomas, neurofibromas, lipomas, and cavernous hemangiomas of the liver. Additionally, PADI4 expression was not detected in non-tumor tissues including cholecystitis, cervicitis and synovitis of osteoarthritis, except in certain acutely inflamed tissues such as in gastritis and appendicitis. Quantitative PCR and western blot analysis showed higher PADI4 expression in gastric adenocarcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cell cancers and breast cancers (n = 5 for each disease) than in the surrounding healthy tissues. Furthermore, western blot analysis detected PADI4 expression in cultured tumor cell lines. ELISA detected increased PADI4 and cAT levels in the blood of patients with various malignant tumors compared to those in patients with chronic inflammation and benign tumors. This was consistent with immunohistochemical results. Additionally, PADI4 and cAT levels were significantly associated with higher levels of known tumor markers.ConclusionOur results suggest that PADI4 expression is increased in the blood and tissues of many malignant tumors, a finding useful for further understanding of tumorigenesis.

PDGF-A promoter and enhancer elements provide efficient and selective antineoplastic gene therapy in multiple cancer types

Development of antineoplastic gene therapies is impaired by a paucity of transcription control elements with efficient, cancer cell-specific activity. We investigated the utility of promoter (AChP) and 5'-distal enhancer (ACE66) elements from the platelet-derived growth factor-A (PDGF-A) gene, which are hyperactive in many human cancers. Efficacy of these elements was tested in multiple tumor cell lines, both in cell culture and as tumor explants in athymic nude mice. Plasmid and viral vectors were constructed with the AChP promoter alone or in fusion with three copies of the ACE66 enhancer for expression of the prototype suicide gene, thymidine kinase (TK). ACE/AChP and AChP cassettes elicited ganciclovir (GCV)-induced cytotoxicity in multiple tumor cell lines. The ACE enhancer element also exhibited synergism with placental and liver-specific promoter elements. An adenovirus containing the AChP-TK cassette produced striking increases in GCV sensitivity in cultured tumor cell lines, as well as GCV-induced regression of U87 MG glioblastoma explants in vivo. TK expression was distributed throughout tumors receiving the therapeutic virus, whereas TdT-mediated dUTP nick end labeling (TUNEL) analysis revealed numerous regions undergoing apoptosis. Vascularization and reticulin fiber networks were less pronounced in virus-GCV-treated tumors, suggesting that both primary and stromal cell types may have been targeted. These studies provide proof-of-principle for utility of the PDGF-A promoter and ACE66 enhancer in antineoplastic gene therapy for a diverse group of human cancers.