Growth Of Human Tumor Cells Research Articles

Potential small-molecule activators of caspase-7 identified using yeast-based caspase-3 and -7 screening assays

Caspases-3 and -7 are at the core of the execution phase of apoptosis. The search for activators of these proteases has therefore deserved particular attention in the field of anticancer drug discovery.Here, a simplified yeast-based screening approach was developed and used to search for activators of caspases-3 and -7, followed by evaluation of the activity of the selected compounds in the human tumor cell lines HL-60 (acute promyelocytic leukemia) and MCF-7 (breast adenocarcinoma). By using the yeast approach, two potential activators of caspase-7, 5,6-dihydroxy-7-prenyloxyflavone (1a) and 3-hydroxy-7-geranyloxyflavone (2a), were identified. Unlike the known caspases-3 and -7 activator, the procaspase activating compound-1 (PAC-1), these flavonoids did not interfere with the caspase-3 activity in yeast. Moreover, flavonoids 1a and 2a processed procaspase-7 to the active caspase-7 both in yeast and in vitro processing assays, and inhibited the growth of HL-60 and MCF-7 human tumor cells with higher potencies than PAC-1, particularly in the absence of caspase-3 (MCF-7 cells). In MCF-7 cells, the flavonoids processed procaspase-7, increased its activity and sensitized these cells to the effects of the cytotoxic drug, etoposide.In conclusion, the developed yeast target-based screening assays led to the identification of potential caspase-7 activators. A proof of concept is therefore provided for the effectiveness of the yeast assays in the discovery of caspase activators. Additionally, the identified compounds may pave the way for a new class of caspase activators with improved anticancer properties.

Antimicrobial and Antiproliferative Potential of Anadenanthera colubrina (Vell.) Brenan.

The aim of the present study was to perform an in vitro analysis of the antimicrobial and antiproliferative potential of an extract from Anadenanthera colubrina (Vell.) Brenan (angico) and chemically characterize the crude extract. Antimicrobial action was evaluated based on the minimum inhibitory concentration (MIC), minimum bactericidal/fungicidal concentration, and the inhibition of formation to oral biofilm. Cell morphology was determined through scanning electron microscopy (SEM). Six strains of tumor cells were used for the determination of antiproliferative potential. The extract demonstrated strong antifungal activity against Candida albicans ATCC 18804 (MIC = 0.031 mg/mL), with similar activity found regarding the ethyl acetate fraction. The extract and active fraction also demonstrated the capacity to inhibit the formation of Candida albicans to oral biofilm after 48 hours, with median values equal to or greater than the control group, but the difference did not achieve statistical significance (P > 0.05). SEM revealed alterations in the cell morphology of the yeast. Regarding antiproliferative activity, the extract demonstrated cytostatic potential in all strains tested. The present findings suggest strong antifungal potential for Anadenanthera colubrina (Vell.) Brenan as well as a tendency toward diminishing the growth of human tumor cells.

Abstract A243: Identification of a target for collismycin A, a cytotoxic microbial product, by proteomic profiling.

Abstract Background: Collismycin A, a microbial product, has antiproliferative activity against tumor cells, the mechanism of which yet remains unknown. Recently, we reported an indirect drug target identification approach, termed “ChemProteoBase," which is based on specific changes in the intracellular proteome induced by chemical manipulations. Here, we report on the identification of a molecular target for collismycin A by using ChemProteoBase. Materials and Methods: HeLa cells were treated with collismycin A for 18 h, and the resulting cell lysates were subjected to two-dimensional fluorescence differential gel electrophoresis (2D-DIGE). The gel images were linked to our database containing images that were generated by 134 well-characterized small molecule drugs and probes. The 296 spots that matched on all gel images were quantified, and then hierarchical cluster analysis and similarity ranking analysis were performed. Results: Collismycin A inhibited cell growth of various human tumor cells with IC50 values ranging from 100 to 400 nM. Flow cytometry and western blot analyses showed that collismycin A arrested cell cycle at G1 phase with the marked decrease of cyclin D1. To predict the mechanism of action of collismycin A, we performed ChemProteoBase profiling. As a result, collismycin A was clustered with Dp44mT, an iron chelator, and Dp44mT was also ranked in the highest position of a similarity ranking among 134 compounds contained in the ChemProteoBase. HR-ESI-MS analysis revealed that collismycin A forms 2 : 1 chelator-iron complex. In addition, the antiproliferative activity of collismycin A was completely abolished by the addition of excessive amounts of Fe(II) or Fe(III) ion but not other metal ions including Cu(II), Zn(II), Mn(II) and Mg(II). Conclusions: These results strongly suggest that collismycin A acts as an iron chelator, resulting in the inhibition of tumor cell growth. Recent studies have shown that iron contributes to tumor initiation, growth and metastasis, and several iron chelators are under preclinical or early clinical investigation as anticancer therapeutics. Therefore, a new iron chelator, collismycin A, might be useful for the treatment of cancers in which iron has been strongly implicated. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A243. Citation Format: Makoto Kawatani, Gyo Inoue, Makoto Muroi, Yushi Futamura, Harumi Aono, Masakazu Uramoto, Yayoi Hongo, Hiroyuki Koshino, Yasuhiro Igarashi, Naoko Takahashi-Ando, Hiroyuki Osada. Identification of a target for collismycin A, a cytotoxic microbial product, by proteomic profiling. [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 A243.

Escherichia coli Producing Colibactin Triggers Premature and Transmissible Senescence in Mammalian Cells

Cellular senescence is an irreversible state of proliferation arrest evoked by a myriad of stresses including oncogene activation, telomere shortening/dysfunction and genotoxic insults. It has been associated with tumor activation, immune suppression and aging, owing to the secretion of proinflammatory mediators. The bacterial genotoxin colibactin, encoded by the pks genomic island is frequently harboured by Escherichia coli strains of the B2 phylogenetic group. Mammalian cells exposed to live pks+ bacteria exhibit DNA-double strand breaks (DSB) and undergo cell-cycle arrest and death. Here we show that cells that survive the acute bacterial infection with pks+ E. coli display hallmarks of cellular senescence: chronic DSB, prolonged cell-cycle arrest, enhanced senescence-associated β-galactosidase (SA-β-Gal) activity, expansion of promyelocytic leukemia nuclear foci and senescence-associated heterochromatin foci. This was accompanied by reactive oxygen species production and pro-inflammatory cytokines, chemokines and proteases secretion. These mediators were able to trigger DSB and enhanced SA-β-Gal activity in bystander recipient cells treated with conditioned medium from senescent cells. Furthermore, these senescent cells promoted the growth of human tumor cells. In conclusion, the present data demonstrated that the E. coli genotoxin colibactin induces cellular senescence and subsequently propel bystander genotoxic and oncogenic effects.

Abstract B82: G-protein coupled estrogen receptor (GPER) agonist G-1 inhibits growth of human granulosa cell tumor cells via blocking microtubule assembly

Abstract Accumulating evidence indicates that the G-protein coupled estrogen receptor (GPER) mediates the non-genomic actions of estrogen in many different cells and tissues. The selective GPER agonist G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro- 3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed to distinguish estrogen actions mediated by GPER from those mediated by the traditional nuclear estrogen receptors. Our recent data showed that G-1 suppresses the growth of breast and ovarian cancer cells in a GPER-independent manner. However, the mechanisms underlying the suppressive actions of G-1 on cancer cell growth are still unclear. The aim of the present study was to explore potential mechanisms of G-1 action in human ovarian granulosa tumor cells. The KGN cell line, a cell line derived from a metastatic adult ovarian granulosa cell tumor (aGCT), and the COV434 cell line, a cell line derived from a juvenile granulosa cell tumor (jGCT), were used as cellular models. Cell proliferation and viability were determined by cell number counting and MTT assay, respectively. Cell cycle analysis was monitored by flow cytometry. Key proteins associated with cell cycle progression were analyzed with Western blot. Cell apoptosis was detected by analysis of caspase activity and DNA integrity. Cellular cytoskeleton changes were monitored by fluorescent immunocytochemistry, confocal microscopy and in vitro microtubule assembly assays. Consistent with our previous results, the putative GPER agonist G-1 suppressed proliferation in a concentration- and time-dependent manner in both KGN and COV434 cells. G-1 inhibited GCT cell growth via suppressing cell viability, which was indicated by a significant decrease in the MTT metabolism, an increase in DNA fragmentation, and an increase in PARP cleavage. G-1 treatment did not affect the expression of cyclin D1, cyclin D2 and cyclin E, suggesting that G-1 treatment had no effect on the early stage cell cycle progression, a result further confirmed by flow cytometry. However, flow cytometry showed that G-1 treatment arrested the cell cycle in the G2/M phase. G2/M phase arrest of KGN cells was confirmed by a significant increase in the phosphorylation of histone H3 after G-1 treatment. Surprisingly, the Western blot analysis showed that G-1 treatment did not affect the activation of mitotic promoting factors (MPF) and had no effect on G2/M entry checkpoint proteins and kinases. Fluorescent immunocytochemistry and confocal microscopy showed that G-1 treatment disrupted the segregation of chromosomes during mitosis, an effect that may be attributed to the abnormal assembly of microtubules. In vitro microtubule assembly assays demonstrated that G-1 treatment indeed suppressed tubulin polymerization and thereby inhibited the assembly of the microtubules. In conclusion, our results from the present study demonstrate that G-1, the putative GPER agonist, suppresses GCT cell proliferation and induces tumor cell death via blocking microtubule assembly. The ability of G-1 to suppress tumor cell proliferation and induce tumor cell apoptosis by targeting microtubules makes it a promising candidate drug for the treatment of GCT. Citation Format: Xiangmin Lv, Guohua Hua, Chunbo He, John S Davis, Cheng Wang. G-protein coupled estrogen receptor (GPER) agonist G-1 inhibits growth of human granulosa cell tumor cells via blocking microtubule assembly. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B82.

Effects of estradiol and progestogens on human breast cells: Regulation of sex steroid receptors

ObjectivesTo examine the effects of 17β-estradiol (E2) and progestogens, used in hormone therapy, on estrogen receptors (ER), progesterone receptors (PR), and human breast tumor cell growth. Materials and methodsMCF-7 cells were incubated in pure E2 (1 nM and 10 nM) as well as in E2 in conjunction with 10 nM progestogens, including progesterone (P4), medroxyprogesterone acetate (MPA), norethisterone acetate (NET), and cyproterone acetate (CPA). Cell proliferation, apoptosis, expression of caspase-3, and both ER and PR isoforms were evaluated. ResultsCaspase-3 was significantly diminished in cultures with only E2, whereas ERα significantly increased. A significant increase of caspase-3 in addition to the entire abolishment of E2-induced augmentation of ERα was observed in 1 nM E2 plus MPA and 10 nM E2 plus NET, whereas PR isoform B (PRB) was significantly increased. The ratios of apoptosis: proliferation significantly increased in 1 nM E2 plus progestogens (except P4) and 10 nM E2 plus NET. The changes of the PRA/PRB ratio were inversely related to the changes of the apoptosis to proliferation ratio. Significant increase of ERβ and PRB was noted in the E2 plus MPA or NET, in addition to a significant increase of ERα and decrease of PRA in the E2 plus CPA, as well as an increase of ERα and decrease of PRA and PRB in the E2 plus P4. ConclusionsThe combination of E2 and various progestogens resulted in diverging effects on ERs and PRs expressions, which induced different effects on MCF-7 cell growth. Compared with P4, aberrant hormone and biological activity of synthetic progestin, by way of altered receptor expression, may be an important factor in affecting breast cell growth.

Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells

IntroductionMalignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy.MethodsTo evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice.ResultsFAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice.ConclusionFAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells.

Identification and Characterization of a Small-Molecule Inhibitor of Wnt Signaling in Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased β-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics.

Synthesis, in Vitro, and in Cell Studies of a New Series of [Indoline-3,2′-thiazolidine]-Based p53 Modulators

Analogues of the previously described spiro[imidazo[1,5-c]thiazole-3,3'-indoline]-2',5,7(6H,7aH)-trione p53 modulators were prepared to explore new structural requirements at the thiazolidine domain for the antiproliferative activity and p53 modulation. In cell, antiproliferative activity was evaluated against two human tumor cell lines. Derivative 5-bromo-3'-(cyclohexane carbonyl)-1-methyl-2-oxospiro[indoline-3,2'-thiazolidine] (4n) emerged as the most potent compound of this series, inhibiting in vitro 30% of p53-MDM2 interaction at 5 μM and the cell growth of different human tumor cells at nanomolar concentrations. Docking studies confirmed the interactions of 4n with the well-known Trp23 and Phe19 clefts, explaining the reasons for its binding affinity for MDM2. 4n at 50 nM is capable of inducing the accumulation of p53 protein, inducing significant apoptotic cell death without affecting the cell cycle progression. Comparative studies using nutlin in the same cellular system confirm the potential of 4n as a tool for increasing understanding of the process involved in the nontranscriptional proapoptotic activities of p53.

Abstract 5554: PBI-05204, a supercritical CO2 extract of Nerium oleander, inhibits the growth of human pancreatic cancer in a Panc-1 orthotopic model by down-regulation of PI3k/mTOR pathways.

Abstract Recent studies have suggested that cardiac glycosides (CGs), such as oleandrin, have an ability to inhibit the proliferation of various cancer cells, including those from pancreatic, non-small cell lung cancer, prostate, colon and breast cancers. However, the inhibitory effect of oleandrin or extracts containing this CG on the growth of human pancreatic cancer xenografts has not been assessed. In this study, the anticancer activity and related pharmacology of the botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, was examined in a human pancreatic Panc-1 orthotopic mouse model. Five out of 7 (70%) of control mice exhibited tumor growth measurable by imaging. In contrast, only one out of seven mice treated for 6 weeks with PBI-05204 (40 mg/kg) exhibited a measureable orthotropic tumor. At 20 mg/kg, the average tumor weight (51 ± 58 mg) in mice treated with PBI-05204 was markedly reduced from that in control (134 ± 99 mg). In contrast, gemcitabine (80 mg/kg) did not appear to inhibit the proliferation of this particular tumor. Histopathological examination of serial sections from each pancreas showed that the pancreas of 3/6 mice treated with PBI-05204 (40 mg/kg) were normal while the other 3/6 had tumor sizes less than 4 mm3 (control range: 55 to 500 mm3). Ki-67 staining was substantially reduced in pancreatic tumors treated with PBI-05204 (20 mg/kg) compared to that of control, suggesting that PBI-05204 indeed inhibited the proliferation of Panc-1 tumor cells. Mechanistically, tissue proteomic array analyses suggest that PBI-05204 suppresses the expression of phosphorylation of Src, Akt, S6, 4EPB1, and VEGF in a conc.-dependent manner. The effects of PBI-05204 on suppression of these proteins were further confirmed by immunohistochemical staining of pancreatic tumor tissues and Western blotting of tissue lysates. To delineate whether PBI-05204 also regulated these important oncogenic pathways in Panc-1 cells in vitro, Panc-1 cells were treated with PBI-05204 (0.3 to 1.2 μg/ml) for 24 hrs and subjected to examination of signaling protein expression. The cellular expression of these important signaling proteins was also markedly reduced by PBI-05204 in a conc.-dependent manner while proliferation of Panc-1 cells was notably reduced. Taken together, these results suggest that PBI-05204 exerts potent anticancer activity in this human pancreatic cancer Panc-1 orthotropic mouse model that may, in turn, be mediated by down-regulation of PI3k/Akt and mTOR pathways. Given the observed PBI-05204 mediated reduction in tumor growth of human Panc-1 tumor cells, we are currently determining whether pancreatic tumor cells are able to continue growing when drug treatment is discontinued. The results will be presented at the annual meeting. This study was supported in part by Phoenix Biotechnology, Inc. Citation Format: Yong Pan, Sun-Jin Kim, Patrea Rhea, Carrie Cartwright, Ho Jeong Lee, Crandell Addington, Mihai Gagea, Robert A. Newman, Peiying Yang. PBI-05204, a supercritical CO2 extract of Nerium oleander, inhibits the growth of human pancreatic cancer in a Panc-1 orthotopic model by down-regulation of PI3k/mTOR pathways. [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 5554. doi:10.1158/1538-7445.AM2013-5554

Abstract 1531: Macrophage FLT1 signaling activates an inflammatory response that promotes breast cancer distal metastasis.

Abstract Macrophages are abundantly found in the tumor microenvironment enhance malignancy. At distal metastatic sites a distinct population of metastasis associated macrophages (MAMs) promotes tumor cell extravasation, seeding and persistent growth. Compared with lung resident macrophages, MAMs associated with breast cancer lung metastasis in pre-clinical models express high levels of cell surface FMS-like tyrosine kinase 1 (Flt1). Blockade of FLT1 signaling using specific inhibitory antibodies significantly inhibited the metastatic seeding and persistent growth of both human and murine breast tumor cell. This was confirmed using a genetic model of Flt1 tyrosine kinase domain (TK) ablation. Using bone marrow transplantation techniques, we further showed that this metastasis inhibitory effect was due to the lack of FLT1 signaling in hematopoietic cells. Furthermore, macrophage specific ablation of the tyrosine kinase domain of Flt1 by crossing Flt1 flox/flox mice with macrophage restricted Cre mice (Csf1r-Cre) also significantly inhibited tumor cell metastatic potential. Since Flt1 is not expressed by other hematopoietic cells and FLT1 inhibition did not affect the recruitment of MAMs, it indicated that specific FLT1 signaling in MAMs are important for their metastasis promoting functions. FLT1 regulated genes were identified by comparing gene expression profiles using microarray techniques of FACS sorted MAMs from mice treated with control and anti-FLT1 inhibitory antibody. Bioinformatics analysis showed that inflammatory response genes were highly enriched in these genes. Together, our studies indicated that FLT1 signaling is required for the activation of metastasis associated macrophages and regulates a set of inflammatory genes that promote breast tumor distal metastasis. Citation Format: Bin-Zhi Qian, Richard A. Lang, Jeffrey W. Pollard. Macrophage FLT1 signaling activates an inflammatory response that promotes breast cancer distal metastasis. [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 1531. doi:10.1158/1538-7445.AM2013-1531

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

Palladin promotes invasion of pancreatic cancer cells by enhancing invadopodia formation in cancer-associated fibroblasts

The stromal compartment surrounding epithelial-derived pancreatic tumors is thought to have a key role in the aggressive phenotype of this malignancy. Emerging evidence suggests that cancer-associated fibroblasts (CAFs), the most abundant cells in the stroma of pancreatic tumors, contribute to the tumor's invasion, metastasis and resistance to therapy, but the precise molecular mechanisms that regulate CAFs behavior are poorly understood. In this study, we utilized immortalized human pancreatic CAFs to investigate molecular pathways that control the matrix-remodeling and invasion-promoting activity of CAFs. We showed previously that palladin, an actin-associated protein, is expressed at high levels in CAFs of pancreatic tumors and other solid tumors, and also in an immortalized line of human CAFs. In this study, we found that short-term exposure of CAFs to phorbol esters reduced the number of stress fibers and triggered the appearance of individual invadopodia and invadopodial rosettes in CAFs. Molecular analysis of invadopodia revealed that their composition resembled that of similar structures (that is, invadopodia and podosomes) described in other cell types. Pharmacological inhibition and small interfering RNA knockdown experiments demonstrated that protein kinase C, the small GTPase Cdc42 and palladin were necessary for the efficient assembly of invadopodia by CAFs. In addition, GTPase activity assays showed that palladin contributes to the activation of Cdc42. In mouse xenograft experiments using a mixture of CAFs and tumor cells, palladin expression in CAFs promoted the rapid growth and metastasis of human pancreatic tumor cells. Overall, these results indicate that high levels of palladin expression in CAFs enhance their ability to remodel the extracellular matrix by regulating the activity of Cdc42, which in turn promotes the assembly of matrix-degrading invadopodia in CAFs and tumor cell invasion. Together, these results identify a novel molecular signaling pathway that may provide new molecular targets for the inhibition of pancreatic cancer metastasis.

Differential effect of adenosine receptors on growth of human colon cancer HCT 116 and HT-29 cell lines

The study aimed to evaluate the impact of adenosine receptors (ARs) on human colon tumor cells (HCT 116, HT-29) growth and sensitivity to 5-Fluorouracil (5-FU) an anticancer chemotherapeutic drug. The exposure of cancer cells to a selective A3-AR agonist (IB-MECA) resulted in an increase in HT-29 cells number, whereas the number of HCT 116 cells decreased significantly. In the presence of IB-MECA (1μM) the percentage of apoptotic HT-29 cells significantly decreased, whereas the number of apoptotic and necrotic HCT 116 cells increased by 3- and 2,5-fold, respectively. The application of a selective A2A-AR agonist resulted in the increased survival of HCT 116 cells, but not HT-29 cells. The blockade of A2A-AR with ZM 241385 (0.1μM) significantly increased the cytotoxicity of 5-FU (1μM) in HCT 116 cells but not in HT-29 cells. The suppression of A3-AR with MRS 1523 (1μM) increased the sensitivity of HT-29 cells to 5-FU while response of HCT 116 cells to 5-FU decreased. The growth promoting effect of IB-MECA in HT-29 cells was associated with the decreased intracellular cAMP level, whereas IB-MECA growth inhibitory effect in HCT 116 cells was abolished by okadaic acid (2nM) indicating the involvement of protein phosphatase PP2A.

The anti-mitotic drug griseofulvin induces apoptosis of human germ cell tumor cells through a connexin 43-dependent molecular mechanism

Griseofulvin, a widely used antifungal antimitotic drug has been proposed as an anti-tumoral treatment by way of in vitro experiments. Recently, in vivo demonstration of griseofulvin efficacy against multiple myeloma in mice argues for its potential as therapeutics for cancer. Nevertheless, the molecular mechanisms by which griseofulvin disrupts cancerous cell progression are far from being understood. In the present study, we found that griseofulvin inhibits human germ cell tumor cell growth through activation of mitochondrial caspase pathway (caspase 9 and 3) leading to the activation of apoptosis rather than an alteration of cell proliferation. Strikingly, we demonstrated that griseofulvin triggered the expression level of connexin 43 (mRNA and protein), a well described tumor-suppressor gene, known to participate in apoptosis regulation. Consistently, together with microtubule instability, a mechanism classically associated with cell death in response to griseofulvin, we observed a disruption of connexin 43/tubulin association concomitant of an enhanced translocation of connexin 43, or an immunoreactive fragment of the protein, from the cytoplasm to the nucleus. Finally, by using siRNA approaches we demonstrated the requirement of connexin 43 in the apoptotic induction of griseofulvin on our tumor cell model. Altogether, these results described a new molecular mechanism connexin 43-dependent targeted by griseofulvin leading to apoptosis of human germ cell tumor cells.

Anticancer activity of oxovanadium compounds

Cytotoxic and antitumor activity of the biligand vanadyl derivative of L-malic acid (bis(L-malato)oxovanadium(IV) (VO(mal)2) was investigated in comparison with inorganic vanadium(IV) compound--vanadyl sulfate (VOSO4) and also with oxovanadium monocomplex with L-malic acid (VO(mal)) and vanadyl biscomplex with acetylacetonate. In this purpose the effect of vanadyl compounds on growth of normal human skin fibroblasts and tumor cells of different lines: mouse fibrosarcoma (L929), rat pheochromocytome (PC12), human liver carcinoma (HepG2), virus transformated mouse fibroblast (NIN 3T3), virus transformated cells of human kidney (293) were investigated. The results showed that VO(mal)2 was not toxic for normal human skin fibroblasts but considerably inhibited growth of cancer cells in culture. Cytotoxic antitumor effect of vanadium complexes was found to be dependent on incubation time and concentration and on type of cells and nature of ligands of the central group of the complex (VO2+). These studies provide evidence that VO(mal)2 may be considered as a potential antitumor agent due to its low toxicity in non-tumor cells and significant anticancer activity.

Walleye dermal sarcoma virus: expression of a full-length clone or the rv-cyclin (orf a) gene is cytopathic to the host and human tumor cells

Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated from infected tumor cells was used to construct a full-length WDSV provirus clone pWDSV, while orf a was cloned into pSVK3 to construct the expression vector porfA. Stable co-transfection of a walleye cell line (W12) with pWDSV and pcDNA3 generated fewer and smaller G418-resistant colonies compared to the control. By Northern blot analysis, several small transcripts (2.8, 1.8, 1.2, and 0.8kb) were detected using a WDSV LTR-specific probe. By RT-PCR and Southern blot analysis, three cDNAs (2.4, 1.6 and 0.8kb) were identified, including both orf a and orf b messenger. Furthermore stable co-transfection of both a human lung adenocarcinoma cell line (SPC-A-1) and a cervical cancer cell line (HeLa) with pcDNA3 and ether porfA or pWDSV also generated fewer and smaller G418-resistant colonies. We conclude that expression of the full-length WDSV clone or the orf a gene inhibits the host fish and human tumor cell growth, and Orf A protein maybe a potential factor which contributes to the seasonal tumor development and regression. This is the first fish provirus clone that has been expressed in cell culture system, which will provide a new in vitro model for tumor research and oncotherapy study.

Design and one-pot synthesis of new 7-acyl camptothecin derivatives as potent cytotoxic agents

New 7-acyl camptothecin derivatives were designed and synthesized from camptothecin in a one-pot reaction through a Minisci type-reaction and were evaluated for cytotoxicity against four tumor cell lines, A-549, DU-145, KB, and KB-vin. All of the new compounds showed significant inhibition of human tumor cell growth, with IC50 values ranging from 0.01538 to 13.342μM. Most of the derivatives were more cytotoxic than irinotecan, and the (7a) and 7-propionyl (7b) analogs exhibited the highest cytotoxic activity against the tumor cell lines tested. This compound class merits further development as anticancer clinical trial candidates.

An adaptive Src–PDGFRA–Raf axis in rhabdomyosarcoma

Alveolar rhabdomyosarcoma (aRMS) is a very aggressive sarcoma of children and young adults. Our previous studies have shown that small molecule inhibition of Pdgfra is initially very effective in an aRMS mouse model. However, slowly evolving, acquired resistance to a narrow-spectrum kinase inhibitor (imatinib) was common. We identified Src family kinases (SFKs) to be potentiators of Pdgfra in murine aRMS primary cell cultures from mouse tumors with evolved resistance in vivo in comparison to untreated cultures. Treating the resistant primary cell cultures with a combination of Pdgfra and Src inhibitors had a strong additive effect on cell viability. In Pdgfra knockout tumors, however, the Src inhibitor had no effect on tumor cell viability. Sorafenib, whose targets include not only PDGFRA but also the Src downstream target Raf, was effective at inhibiting mouse and human tumor cell growth and halted progression of mouse aRMS tumors in vivo. These results suggest that an adaptive Src–Pdgfra–Raf–Mapk axis is relevant to PDGFRA inhibition in rhabdomyosarcoma.

Crataegus monogyna buds and fruits phenolic extracts: Growth inhibitory activity on human tumor cell lines and chemical characterization by HPLC–DAD–ESI/MS

Crataegus monogyna has been extensively studied due to its various alleged health benefits. This study aimed to determine the human tumor cells growth inhibitory activity of phenolic extracts of its flower buds and fruits in three phenological stages, and further characterize the extracts by HPLC–DAD–ESI/MS. Flower bud extract showed the highest antiproliferative activity as indicated by the lowest GI50 values obtained in all the tested cell lines: MCF-7, breast adenocarcinoma; NCI-H460, non-small cell lung cancer; HeLa, cervical carcinoma; HepG2, hepatocellular carcinoma. Furthermore, porcine liver primary cell culture (PLP2) was used to evaluate toxicity to non-tumor cells. Flavonoids, particularly flavonols and flavones (higher in flower buds) and proanthocyanidins (higher in unripe fruits) were the main classes in the studied samples. Phenolic acids (mainly hydroxycinnamic acid derivatives) were also detected in significant amounts, especially in flower bud extract. Regarding anthocyanins, over ripened fruits gave the highest content. The higher bioactivity observed in flower buds might be related with its higher content in phenolic compounds.