Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38±9 and 67±8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

Spontaneous canine transmissible venereal tumor: cell morphology and influence on P-glycoprotein expression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antiproliferative activity of Eremanthus crotonoides extracts and centratherin demonstrated in brain tumor cell lines

The genus Eremanthus is recognized by the predominance of sesquiterpene lactones from the furanoheliangolide type, a class of substances extensively tested against cancer cell lines. Thus, the species E. crotonoides (DC.) Sch. Bip., Asteraceae, obtained on "restinga" vegetation was evaluated against U251 and U87-MG glioma cell lines using the MTT colorimetric assay. Dichloromethane fraction was cytotoxic to both glioblastoma multiforme cell lines. We then conducted UPLC-PDA-ESI-MS/MS analysis of the dichloromethane fraction, which allowed the identification of the sesquiterpene lactones centratherin and goyazensolide. The isolation of centratherin was performed using chromatographic techniques and the identification of this substance was confirmed according to NMR data. Cytotoxic activity of centratherin alone was also evaluated against both U251 and U87-MG cells, which showed IC50 values comparable with those obtained for the commercial anticancer drug doxorubicin. All the tested samples showed cytotoxic activity against glioblastoma multiforme cells which suggests that E. crotonoides extracts may be important sources of antiproliferative substances and that the centratherin may serve as prototype for developing new antiglioblastoma drugs.

T cell stimulation by dendritic cell-tumor cell hybrids is enhanced in the presence of free dendritic cells

CNPq, FAPESP (2009/54599-5 and 2012/10939-0).

Inhibition of hepatic tumor cell proliferation in vitro and tumor growth in vivo by taltobulin, a synthetic analogue of the tripeptide hemiasterlin

AIM: To investigate the inhibitory effects of taltobulin (HTI-286), a synthetic analogue of natural hemiasterlin derived from marine sponges, on hepatic tumor growth in vitro and in vivo. METHODS: The potential anti-proliferative effects of HTI-286 on different hepatic tumor cell lines in vitro and in vivo were examined. RESULTS: HTI-286 significantly inhibited proliferation of all three hepatic tumor cell lines (mean IC50 = 2 nmol/L +/- 1 nmol/L) in vitro. Interestingly, no decrease in viable primary human hepatocytes (PHH) was detected under HTI-286 exposure. Moreover, intravenous administration of HTI-286 significantly inhibited tumor growth in vivo (rat allograft model). CONCLUSION: HTI-286 might be considered a potent promising drug in treatment of liver malignancies. HTI-286 is currently undergoing clinical evaluation in cancer patients.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death

HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.

Inhibition of Deubiquitinase Activity and Ubiquitination of Jak2 Blocks Cytokine Signaling and Induces Tumor Cell Apoptosis

The Jak-stat pathway is critical for cellular proliferation and is commonly found to be deregulated in many solid tumors as well as hematological malignancies. Such findings have spurred the development of novel therapeutic agents that specifically inhibit Jak2 kinase, thereby suppressing tumor cell growth. Tyrphostin AG490, the first described Jak2 inhibitor, displays poor pharmacology and requires high concentrations for anti-tumor activities. Our research group screened a small library of AG490 structural analogues and identified WP1130 as a potent inhibitor of Jak2 signaling. However, unlike AG490, WP1130 did not directly inhibit Jak2 kinase activity. Our results show that WP1130 induces rapid ubiquitination and subsequent re-localization of Jak2 into signaling incompetent aggresomes. In addition to Jak2, WP1130 also induces accumulation of other ubiquitinated proteins without inhibiting 20S proteasome activity. Further analysis of the mechanism of action of WP1130 revealed that WP1130 acts as a partly selective DUB inhibitor. It specifically inhibits the deubiquitinase activity of USP9x, USP5, USP14 and UCH37. WP1130 mediated inhibition of tumor-associated DUBs resulted in down-regulation of anti-apoptotic and up-regulation of pro-apoptotic proteins, such as MCL-1 and p53 respectively. Our results demonstrate that chemical modification of a previously described Jak2 inhibitor results in the unexpected discovery of a novel compound which acts as a DUB inhibitor, suppressing Jak-Stat signaling by a novel mechanism.

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

Characterization of the cytochrome P-450 drug metabolism system of LS174T human colon tumor cell line

The human colon tumor cell line, LS174T, has been shown to have four major components of the drug metabolizing system; cytochrome b$\sb5$ reductase, cytochrome b$\sb5$, cytochrome P450 reductase and cytochrome P450, by activity measurements, spectral studies and antibody cross-reactivity. Cytochrome P450IA1 is induced by benzanthracene in these cells as shown by activity with the specific substrate, ethoxyresorufin, cross-reactivity with rabbit antibodies to rat IA1, and by a hybridizing band on a Northern blot to a rat IA1 probe.^ Further, this system has proven responsive to various inducers and conditions of growth. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 $\mu$mol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b$\sb5$ per milligram and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone treatment showed a consistent, but not always significant increase in the NADPH and NADH cyt c reducing activity and benzanthracene treatment an increase in the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5mM) caused a significant decrease in the specific activity of all enzyme contents and activities tested.^ Finally, the cytochrome b$\sb5$ to cytochrome P450, by the coordinate induction of the cytochrome b$\sb5$ pathway by P450 inducers, by the high ratio of NADH to NADPH ethoxycoumarin deethylase activity in uninduced cell microsomes, and by the increase in NADH and NADPH ethoxycoumarin deethylase activity when the microsomes were treated with potassium cyanide, a desaturase inhibitor. ^

Tumor cell-associated epitopes recognized by lymphokine-activated killers (LAK)

Interleukin-2 activated lymphocytes, designated lymphokine-activated killers (LAK), acquire the unique capacity to express potent cytologic activity against a broad spectrum of abnormal and/or transformed NK-sensitive and NK-resistant target cells while sparing normal cell types. Investigations into the target spectra exhibited by cloned effector cells indicate that LAK cells express a polyspecific recognition mechanism that identifies an undefined class of cell surface-associated molecules expressed on susceptible targets. This report extends our previous investigations into the biochemical nature of these molecules by characterizing the functional role of two tumor cell-surface-associated epitopes implicated in conferring target cells with susceptibility to LAK-mediated cytotoxicity. The first moiety is implicated in the formation of effector/target cell conjugates. This binding ligand is preferentially expressed on tumor cells relative to LAK-resistant PBL target cells, sensitive to trypsin treatment, resistant to functional inactivation by heat- and detergent-induced conformational changes, and does not require N-linked glycosylation to maintain binding activity. In contrast, a carbohydrate-associated epitope represents the second tumor-associated molecule required for target cell susceptibility to LAK cells. Specifically, N-linked glyoprotein synthesis represents an absolute requirement for post-trypsin recovery of target cell susceptibility. The minimal N-linked oligosaccharide residue capable of restoring this second signal has been identified as a high mannose structure. Although ultimately required for tumor cell susceptibility, as measured in $\sp{51}$Cr-release assays, this N-glycan-associated molecule is not required for the differential tumor cell binding expressed by LAK cells. Furthermore, N-glycan expression is not adequate in itself to confer target cell susceptibility. Additional categories of cell surface components have been investigated, including O-linked oligosaccharides, and glycosaminoglycans, without identifying additional moieties relevant to target cell recognition. Collectively, these data suggest that tumor cell recognition by LAK cells is dependent on cell surface presentation of two epitopes: a trypsin-sensitive molecule that participates in the initial conjugate formation and an N-glycan-associated moiety that is involved in a post-binding event required for target cell killing. ^

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

COMPARISON OF DNA TRANSFECTION AND MICROCELL-MEDIATED CHROMOSOME TRANSFER FOR DETECTING TRANSFORMING GENES IN HUMAN TUMOR CELL LINES

DNA mediated gene transfection is an important tool for moving and isolating genes from one cell type and putting them into a foreign genetic background. DNA transfection studies have been done routinely in many laboratories to identify and isolate transforming sequences in human tumors and tumor cell lines. A second technique, microcell-mediated chromosome transfer, allows the transfer of small numbers of intact human chromosome from one cell to another. This work was done to compare the efficiency of these two techniques in the transformation of NIH 3T3 mouse fibroblast cells.^ My intent in comparing these two techniques was to see if there was a difference in the transforming capability of DNA which has been purified of all associated protein and RNAs, and that of DNA which is introduced into a cell in its native form, the chromosome. If chromosomal sequences were capable of transforming the 3T3 cells in culture, the method could then be used as a way to isolate the relevant tumorigenic chromosomes from human tumors.^ The study shows, however, that even for those cell lines that contain transforming sequences identified by DNA-mediated gene transfer, those same sequences were unable to transform 3T3 cells when introduced to the cells by somatic fusion of human tumor microcells. I believe that the human transforming sequences in their original genetic conformation are not recognized by the mouse cell as genes which should be expressed; therefore, no noticeable transformation event was selected by this technique. ^