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To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000., Genomics, Proteomics and Bioinformatics, 18 (2)

Thymidine phosphorylase (TPase) is also known as the platelet-derived endothelial cell growth factor (PD-ECGF) and plays a role in angiogenesis. Deoxyribose (dR; a downstream TPase-product) addition to endothelial cells may stimulate FAK and p70/S6k signaling, which can be inhibited by rapamycin. Rapamycin is a specific mammalian target of the rapamycin (mTOR) inhibitor, a kinase that lies directly upstream of p70/S6k. This suggests a role for TPase in the mTOR/p70/S6k pathway. In order to study this in more detail, we exposed cells with and without TPase expression to dR and rapamycin and determined the effect on cell growth. We observed protection in cytotoxicity in Colo320 cells, but not Colo320 TP1 cells. This was in part mediated by activation of p70/S6k and inhibition of autophagy. Further studies are recommended to elucidate the mechanism behind the protective effect of dR.

BACKGROUND: Thymidine phosphorylase (TP) is often overexpressed in tumours and has a role in tumour aggressiveness and angiogenesis. Here, we determined whether TP increased tumour invasion and whether TP-expressing cancer cells stimulated angiogenesis.METHODS: Angiogenesis was studied by exposing endothelial cells (HUVECs) to conditioned medium (CM) derived from cancer cells with high (Colo320TP1 = CT-CM, RT112/TP = RT-CM) and no TP expression after which migration (wound-healing-assay) and invasion (transwell-assay) were determined. The involvement of several angiogenic factors were examined by RT-PCR, ELISA and blocking antibodies.RESULTS: Tumour invasion was not dependent on intrinsic TP expression. The CT-CM and RT-CM stimulated HUVEC-migration and invasion by about 15 and 40%, respectively. Inhibition by 10 mu M TPI and 100 mu M L-dR, blocked migration and reduced the invasion by 50-70%. Thymidine phosphorylase activity in HUVECs was increased by CT-CM. Reverse transcription-polymerase chain reaction revealed a higher mRNA expression of bFGF (Colo320TP1), IL-8 (RT112/TP) and TNF-alpha, but not VEGF. Blocking antibodies targeting these factors decreased the migration and invasion that was induced by the CT-CM and RT-CM, except for IL-8 in CT-CM and bFGF in RT-CM.CONCLUSION: In our cell line panels, TP did not increase the tumour invasion, but stimulated the migration and invasion of HUVECs by two different mechanisms. Hence, TP targeting seems to provide a potential additional strategy in the field of anti-angiogenic therapy.

Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2′-deoxy-5-fluorouridine (>170-fold). Exposure to IC75-TFT increased the S/G2-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047–57. ©2010 AACR.

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0–6andnbsp;Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24–72andnbsp;h exposure to 250–750 μM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24andnbsp;h for all cell lines. Exposure to 750andnbsp;μM meloxicam for 24 h increased the fraction of cells in the radiosensitive G2/M cell cycle phase in D384 (18–27%) and U251 (17–41%) cells. 750 μM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.

The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its contribution of centrosome amplification (CA) and early development of aggressive cell behaviour. However its regulation in metastatic cells remains unclear. Here we identified miR-129-3p as a novel metastatic microRNA. CP110 was identified as its target protein. In PCa cells that have metastatic capacity, CP110 expression was repressed by miR-129-3p. High miR-129-3p expression levels increased cell invasion, while increasing CP110 levels decreased cell invasion. Overexpression of CP110 in metastatic PCa cells resulted in a decrease in the number of metastasis. In tissues of PCa patients, low CP110 and high miR-129-3p expression levels correlated with metastasis, but not with the expression of genes related to EMT. Furthermore, overexpression of CP110 in metastatic PCa cells resulted in excessive-CA (E-CA), and a change in F-actin distribution which is in agreement with their reduced metastatic capacity. Our data demonstrate that miR-129-3p functions as a CA gatekeeper in metastatic PCa cells by maintaining pro-metastatic centrosome amplification (CA) and preventing anti-metastatic E-CA.

The objective of the present study is to determine whether uptake of [(18)F]fluoromethylcholine ([(18)F]FCH) in comparison with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) accurately reflects chemotherapy efficacy at the tumor cell level in prostate cancer (PC).The effects of docetaxel and cabazitaxel on viable tumor cell number were explored in four PC cell lines. Cellular uptake of [(18)F]FDG and [(18)F]FCH was compared with the effects measured using sulforhodamine B (SRB) assay, cell counting and colony formation assay (CFA), as proximators of viable tumor cell number. Agreement between uptake and cell numbers was assessed by Bland-Altman plots.[(18)F]FCH uptake in all PC cell lines significantly correlated to the cell numbers surviving the respective drug concentrations. Bland-Altman analysis showed that [(18)F]FDG uptake resulted in signal overestimation and higher variability after chemotherapy.[(18)F]FCH uptake correlates well with viable tumor cell numbers remaining after docetaxel and cabazitaxel exposure. Radiolabeled choline is a potential response monitoring biomarker after chemotherapy for PC.

TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL.Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms.The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression.TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.

TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 μM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 μM TPI did not or slightly affect radiosensitivity, but 100 μM TPI alone enhanced the radiation response (p.05). TFT alone at 1 μM and in combination with 10 μM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ϒH2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.

This study aimed to investigate cabazitaxel efficacy in a model for docetaxel-resistant prostate cancer cells and to evaluate the involvement of ATP-cassette binding protein 4 (ABCC4) with regard to multidrug resistance.Docetaxel and cabazitaxel sensitivity was measured in PC3 and R3327-MATLyLu (MLL) cell lines, using the sulforhodamine B (SRB) assay. ABCC4 expression was examined by western blotting and its functional involvement in drug sensitivity by blocking with MK571 inhibitor.The docetaxel-resistant MLL cells (4.5-fold compared to cabazitaxel; p0.001) were shown to express high levels of ABCC4, while non-resistant PC3 cells had no detectable ABCC4 expression. Functional inhibition of ABCC4 in MLL cells resulted in a two-fold decrease in effective concentration of docetaxel and had no effect on toxicity of cabazitaxel.Cabazitaxel showed an improved therapeutic efficacy over docetaxel in ABCC4-expressing prostate cancer cells. ABCC4 appears to be an important determinant of docetaxel resistance, since its inhibition almost completely reversed resistance.

Prostate cells are dependent on androgens for growth and proliferation. Androgen deprivation therapy is the recommended treatment for advanced/metastatic prostate cancer. Under this therapy, prostate cancer will inevitably progress to castration resistant prostate cancer (CRPC). Despite putative castration resistance, testosterone might still play a crucial role in the progression of CRPC. The goal of this study was to determine the role of testosterone in the formation of metastases of CRPC in both in vitro and in vivo settings. In vitro, the effect of testosterone and the non-aromatizable androgen methyltrienolone on migration, invasion and proliferation of a castration-resistant prostate cancer rat cell line (Dunning R3327-MATLyLu) was assessed using a transwell assay and a sulforhodamine B assay and immunohistochemical detection of ki67. Androgen receptor status was determined using Western blot. In vivo, Copenhagen rats were divided in four groups (males, females, castrated males and females with testosterone suppletion) and inoculated with MATLyLu cells. Tumor size was assessed daily. Testosterone increased cell migration and invasion in a concentration-dependent manner in vitro. Testosterone did not affect in vitro cell proliferation. No difference was shown between the effect of testosterone and methyltrienolone. In vivo, in groups with higher levels of circulating testosterone, more rats had (micro)metastases compared with groups with low levels of testosterone. No effect was observed on primary tumor size/growth. Despite assumed castration resistance, progression of prostate cancer is still influenced by androgens. Therefore, continuous suppression of serum testosterone in patients who show disease progression during castration therapy is still warranted.

The pyrimidine trifluorothymidine (TFT) inhibits thymidylate synthase (TS) and can be incorporated into the DNA. TFT, as part of TAS-102, is clinically evaluated in phase II studies as an oral chemotherapeutic agent. Erlotinib is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) that is often deregulated in colorectal cancer. This study investigated molecular mechanisms underlying the cytotoxic actions of the combination of an EGFR-tyrosine kinase inhibitor with TFT in colorectal cancer cells Caco2, WiDR, Lovo92, and Colo320. Drug interactions were examined by the sulforhodamine B assay and subsequent combination index (CI) analyses, cell cycle effects by FACS analysis of propidium iodide stained cells, Akt, MAPK and EGFR phosphorylation and expression levels by Western blotting and TS activity by the TS in situ assay. All combination schedules were synergistic in wt-EGFR expressing (but with mutated downstream pathways) WiDR and Lovo92 (CI 0.4-0.8) and very synergistic in Caco2 cells (with wt-EGFR and functional downstream pathways; CI 0.1-0.3), but in EGFR-lacking Colo320 cells, no additional activity was found (CI 1.0-1.2). Synergism was mostly related to the induction of cell cycle arrest and an erlotinib-mediated inhibition of the pro-survival signaling through Akt and MAPK that was activated (phosphorylated) by TFT. Erlotinib inhibited TS activity in EGFR-expressing cell lines, probably due to cell cycle arrest in the G(1) phase. TS activity was slightly lower in the combinations, probably due to cell cycle interference. Taken together, the combination of erlotinib with TFT seems to present a potential strategy in the field of molecular therapeutics. (Cancer Sci 2010; 101: 440-447)

Trifluorothymidine (TFT) is part of the oral drug formulation TAS-102. Both 5-fluorouracil (5-FU) and TFT can inhibit thymidylate synthase and be incorporated into DNA. TFT shows only moderate cross-resistance to 5-FU. Therefore, we examined whether mechanistic differences in cell death could underlie their different modes of action in colorectal cancer cell lines (WiDR, Lovo92 and Colo320). Drug cytotoxicity was determined by SRB- and clonogenic assays, cell death by flow cytometry (PI and annexin V), caspase cleavage by Western blotting and activity assays and in vivo activity in the hollow fiber assay. The IC50 values of TFT were 1-6 fold lower than for 5-FU, and clonogenic survival was less than 0.9% at 3 mu M TFT, while 2-20% of the cells still survived after 20 mu M 5-FU. In general, TFT was a more potent inducer of apoptosis than 5-FU, although the contribution of caspases varied between the used cell lines and necrosis-like cell death was detected. Accordingly, both drugs induced caspase (Z-VAD) independent cell death and lysosomal cathepsin B was involved. Activation of autophagy recovery mechanisms was only triggered by 5-FU, but not by TFT as determined by LC3B expression and cleavage. Inhibition of autophagy by 3-MA in 5-FU exposed cells reduced cell survival. Also, in vivo TFT (as TAS-102) caused more cell death than a 5-FU formulation. We conclude that TFT and 5-FU induce cell death via both caspase-dependent and independent mechanisms. The TFT was more potent than 5-FU, because it induces higher levels of cell death and does not elicit an autophagic survival response in the cancer cell lines. This provides a strong molecular basis for further application of TFT in cancer therapy.

Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis The exact mechanism of angiogenesis induction is unclear, but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to thymine and deoxynbose-1-phosphate (dR-1-P), which can be converted to dR-5-P, glyceraldehyde-3-phosphate (G3P) or deoxyribose (dR) However, it is unclear which sugar accumulates in this reaction Therefore, in the TP overexpressing Colo320 TP1 and RT112/TP cells we determined by LC-MS/MS which sugars accumulated, their subcellular localization (using H-3-TdR) and whether dR was secreted from the cells In both TP-overexpressing cell lines, dR-1-P and dR-5-P accumulated intracellularly at high levels and dR was secreted extensively by the cells. A specific inhibitor of TP completely blocked TdR conversion, and thus no sugars were formed To examine whether these sugars may be used for the production of angiogenic factors or other products, we determined with H-3-TdR in which subcellular location these sugars accumulated. TdR-derived sugars accumulated in the cytoskeleton and to some extent in the cell membrane, while incorporation into the DNA was responsible for trapping in the nucleus. In conclusion, various metabolic routes were entered, of which the TdR-derived sugars accumulated in the cytoskeleton and membrane Future studies should focus on which exact metabolic pathway is involved in the induction of angiogenesis (C) 2010 Elsevier Inc All rights reserved.

Trifluorothymidine (TFT), a potent anticancer agent, inhibits thymidylate synthase (TS) and is incorporated into the DNA, both events resulting in cell death. Cell death induction related to DNA damage often involves activation of p53. We determined the role of p53 in TFT cytotoxicity and cell death induction, using, respectively, the sulforhodamine B-assay and FACS analysis, in a panel of cell lines with either wild type, inactive, or mutated p53. Neither TFT cytotoxicity nor cell death induction changed with TFT exposure in cell lines with wt, inactive or mutated p53. Conclusion: sensitivity to TFT is not dependent on the expression of wt p53.

Docetaxel is a microtubule inhibitor that has actions in the S and G(2)-M phase of the cell cycle. The pyrimidine trifluorothymidine (TFT) induces DNA damage and an arrest in the G(2)-M phase. TFT, as part of TAS-102, has been clinically evaluated as an oral chemotherapeutic agent in colon and gastric cancer. The aim of the present study was to determine the optimal administration sequence of TFT and docetaxel and to investigate the underlying mechanism of cytotoxicity. Drug interactions were examined by sulforhodamine B assays and subsequent combination index analyses, and for long-term effects the clonogenic assay was used. A preincubation with docetaxel was synergistic in sulforhodamine B (combination index 0.6-0.8) and clonogenic assays, and was accompanied by a time-dependent cell death induction (17-36%), the occurrence of polynucleation (22%), and mitotic spindle inhibition as determined by flow cytometry and immunostaining. Interestingly, administration of TFT followed by the combination displayed strong antagonistic activity, and was accompanied by less polynucleation and cell death induction than the synergistic combinations. Western blotting showed that the G(2)-M-phase arrest (25-50%) was accompanied by phosphorylation of Chk2 and dephosphorylation of cdc25c in the synergistic combinations. Together, this indicates that synergistic activity requires docetaxel to initiate mitotic failure prior to the activation of TFT damage signaling, whereas antagonism is a result of TFT cell cycle-arrested cells being less susceptible to docetaxel. Caspase 3 activation was low after docetaxel, suggestive of caspase-independent mechanisms of cell death. Taken together, our models indicate that combination treatment with docetaxel and TFT displays strong synergy when docetaxel is given first, thus providing clues for possible clinical studies.

Multidrugs have the potential to bypass resistance. We investigated the in vitro activity and resistance circumvention of the multidrug cytarabine-L-fluorodeoxyuridine (AraC-L-5FdU), linked via a glycerophospholipid linkage. Cytotoxicity was determined using sensitive (A2780, FM3A/0) and resistant (AG6000, AraC resistant, deoxycytidine kinase deficient; FM3A/TK-, 5FdU resistant, thymidine kinase deficient) cell lines. Circumvention of nucleoside transporter and activating enzymes was determined using specific inhibitors, HPLC analysis and standard radioactivity assays. AraC-L-5FdU was active (IC50: 0.03 microM in both A2780 and FM3A/0), had some activity in AG6000 (IC50: 0.28 microM), but no activity in FM3A/TK(-) (IC50: 18.3 microM). AraC-nucleotides were not detected in AG6000. 5FdU-nucleotides were detected in all cell lines. AraC-L-5FdU did not inhibit TS in FM3A/TK(-) (5%). Since phosphatase/nucleotidase-inhibition reduced cytotoxicity 7-70-fold, cleavage seems to be outside the cell, presumably to nucleotides, and then to nucleosides. The multidrug was orally active in the HT-29 colon carcinoma xenografts which are resistant toward the single drugs.

Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2'deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK(-). ETC-FdUrd was active (IC(50) of 2.2 and 79 nM) in FM3A/0 and TK(-) cells, respectively. ETC-L-FdUrd was less active (IC(50): 7 nM in FM3A/0 vs 4500 nM in FM3A/TK(-)). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80-90%), but to a lower extent in FM3A/TK(-) (10-50%). FdUMP was hardly detected in FM3A/TK(-) cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.