Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their ...correlation.
Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients.
Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine.
This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors.
Because the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and the multitargeted antifolate pemetrexed
are registered in the treatment of second-line non-small-cell lung ...cancer (NSCLC), empirical combinations of these drugs are
being tested. This study investigated molecular mechanisms underlying their combination in six NSCLC cell lines. Cells were
characterized by heterogeneous expression of pemetrexed determinants, including thymidylate synthase (TS) and dihydrofolate
reductase (DHFR), and mutations potentially affecting chemosensitivity. Pharmacological interaction was studied using the
combination index (CI) method, whereas cell cycle, apoptosis induction, and EGFR, extracellular signal-regulated kinases 1
and 2, and Akt phosphorylation were studied by flow cytometry, fluorescence microscopy, and enzyme-linked immunosorbent assays.
Reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and activity assays were performed to assess whether
erlotinib influenced TS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays demonstrated that EGFR and k-Ras mutations were related to erlotinib sensitivity, whereas TS and DHFR expression were related to pemetrexed sensitivity. Synergistic
cytotoxicity was found in all cells, most pronounced with pemetrexed + erlotinib (24 h) â erlotinib (48 h) sequence (CI, 0.09-0.40),
which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt
phosphorylation, which was additionally reduced by drug combination (-70.6% in H1650). Erlotinib significantly reduced TS
expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and Western blot, and the combination decreased
TS in situ activity in all cells. Erlotinib and pemetrexed showed a strong synergism in NSCLC cells, regardless of their genetic
characteristics. Induction of apoptosis, modulation of EGFR and Akt phosphorylation, and changes in the expression of critical
genes involved in pemetrexed activity contribute to this synergistic interaction and support the clinical investigation of
these markers.
Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including ...gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth ...through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.
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 G1 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 2009)
5-Fluorouracil
(5FU)-based therapy is given to patients with advanced colorectal
cancer and as adjuvant treatment. Thymidylate synthase (TS) is the
target for 5FU, and may have a prognostic role for ...the outcome of
5FU-based therapy together with proliferation markers such as p53 and
Ki67. Thymidine phosphorylase (TP, also known as platelet-derived
endothelial cell growth factor) may be of importance both in the
5FU drug activation pathway and in tumor angiogenesis, similar to
vascular endothelial growth factor (VEGF). TS and TP levels were
determined biochemically in fresh-frozen tumor specimens of 32
untreated patients with colorectal cancer, whereas in paraffin-embedded
tissue samples, immunohistochemistry was performed for TS, TP, and
additional prognostic markers such as p53, Ki67, and VEGF as well as
microvessel density. All factors were correlated with patient
characteristics such as age, gender, Dukes’ stage, angio-invasion, and
differentiation grade. TS and TP as measured by various assays were
correlated with overall and disease-free survival in this patient
group. TP enzyme activity and protein expression correlated with each
other. A significant correlation was found between TP enzyme activity
and 5-fluoro-2′-deoxyuridine-5′-monophosphate binding activity. VEGF
expression correlated significantly with TP immunostaining and Ki67
index. Survival analysis revealed a significant relation of TS levels
to the overall survival in this small patient group and a significant
correlation between TP activity and disease-free survival. TS and TP
both were of prognostic significance in these patients with colorectal
cancer. The interesting relationship of TS and TP with angiogenesis and
proliferation needs further investigation.
For decades 5-fluorouracil (5-FU) has remained the treatment of choice in the adjuvant and palliative setting of colorectal cancer (CRC). The combinations of 5-FU or its oral prodrug capecitabine ...with irinotecan/oxaliplatin and the novel agents bevacizumab/cetuximab increased responses. However, the overall prognosis is poor, and predictive biomarkers of cytotoxic drugs activity are missing. Pharmacogenetic studies focused on candidate determinants of drug activity/metabolism, such as thymidylate synthase or dihydropyrimidine dehydrogenase, but reported controversial results. Given the heterogeneous and complex nature of CRC, it is likely that many aberrations underlying its progression can also affect therapeutic response. Therefore, high-throughput arrays for genome-wide-DNA aberrations play a pivotal role for new markers discovery by moving from hypothesis-driven, targeted-research to unbiased screening of the whole genetic spectrum. Chromosomal aberrations are critical events in tumorigenesis, and genomic regions harbouring DNA gains/losses have been identified in 85% of CRC patients. These aberrations change the expression of many genes, which might explain the differential effects of specific chemotherapeutic agents. In particular, recent studies reported correlations between DNA copy-number profiles and response to fluoropyrimidine-based regimens, such as leucovorin-modulated-5-FU+irinotecan (FOLFIRI), capecitabine+irinotecan (CAPIRI) and pemetrexed+irinotecan (ALIRI). Genome-wide profiling by oligonucleotide-based array-comparative-genomic-hybridization (aCGH) revealed genomic loci, of which the copy-number status may serve as marker for outcome after FOLFIRI and CAPIRI. Larger randomized and prospective trials of these aCGH platforms in CRC patients treated with fluoropyrimidine-based regimens are ongoing, and will ultimately demonstrate whether these findings can be of actual value to predict clinical outcome and direct the choice of therapy.
Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. In a panel of 13 nonselected
human ...colon cancer cell lines, we evaluated the role of TS levels in sensitivity to 5-fluorouracil (5FU) and four folate-based
TS inhibitors that have been introduced recently into the clinic: ZD1694 (Tomudex, Raltitrexed, TDX), GW1843U89 (GW), LY231514
(LY), and AG337 (Thymitaq, AG). Because the latter compounds have different transport and polyglutamylation characteristics,
we also related these parameters with drug sensitivity, measured by the sulforhodamine B assay after 72 h of drug exposure.
For 5FU, the IC 50 s varied from 0.8 to 43.0 μ m . Leucovorin (LV) potentiated the activity of 5FU in only 4 of 13 cell lines. Sensitivity to folate-based TS inhibitors was
variable; IC 50 s were in the range of: 5.3–59.0 n m TDX; 11.0–1570 n m LY; and 0.5–8.9 n m GW. Eleven of 13 cell lines had an IC 50 for AG between 1.3 and 5.3 μ m . Two cell lines were resistant to AG, Colo201 and SW1116, with IC 50 s of 27 and 29 μ m , respectively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 to 777 pmol/h/10 6 cells. The number of FdUMP binding sites varied from 32 to 231 fmol/10 6 cells. Regression analysis showed a significant relation between TS catalytic activity and IC 50 s for 5FU and 5FU/LV. Ki s for FdUMP showed a significant Spearman rank correlation with the IC 50 s of AG and GW. The role of antifolate transport, accumulation, and polyglutamylation was determined with 3 Hmethotrexate (MTX) as a reference compound. 3 HMTX influx via the reduced folate carrier varied from 18.6 to 150 fmol/10 6 cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range from 47 to 429 pmol/10 6 cells/h. A total of 24 h of 3 HMTX accumulation showed a 20-fold variation, from 1.2 to 21.8 pmol/10 6 cells. FPGS levels showed a Spearman rank positive correlation with cytotoxicity to TDX.
In conclusion, in a heterogeneous nonselected human colon cancer cell line panel, the best predictor for sensitivity to 5FU
and 5FU/LV was TS activity. Multiple sensitivity determinants were of importance for antifolate TS inhibitors, including FPGS
activity and TS enzyme kinetics.
2′,2′-Difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a ...complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2′,2′-difluoro-2′-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of 5-3H-2′-deoxyuridine or 5-3H-2′-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4h exposure to 1μM dFdC tritium release was inhibited by 50% but did not increase after 24h, Inhibition was also observed following dFdU at 100μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2′-C-cyano-2′-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase.
Inhibition of the enzyme resulted in a relative increase of mis-incorporation of 5-3H-2′-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130μM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.
Purpose
Many patients with Crohn’s disease (CD) and ulcerative colitis (UC) who have a high 6-methylmercaptopurine/6-thioguanine (6-MMP/6-TGN) ratio receive allopurinol 100 mg in addition to ...thiopurines to optimize metabolite concentrations. However, some patients do not tolerate allopurinol at this dosage.
The aim of this study was to determine the intra-patient effect of reducing the allopurinol dosage from 100 to 50 mg, in terms of metabolite concentrations, enzyme activities, efficacy, and tolerability.
Methods
A prospective non-inferiority one-way crossover study was performed. CD and UC patients with stable disease using a thiopurine and allopurinol 100 mg were switched to 50 mg for 1 month. Primary outcomes were thiopurine metabolite concentrations. Secondary outcomes were enzyme activities of xanthine oxidase, thiopurine methyltransferase and hypoxanthine-guanine phosphoribosyltransferase, disease activity, and tolerability.
Results
Twenty-two patients were included. Treatment with allopurinol 50 mg compared with 100 mg resulted in a significant decrease in mean 6-TGN levels (761 to 625 pmol/8 × 10
8
RBC;
p
= 0.005) and a significant increase in mean 6-MMP levels (451 to 665 pmol/8 × 10
8
RBC;
p
= 0.01). However, the mean metabolite concentrations were still therapeutic. Enzyme activities, disease activity scores, and patient experiences did not alter significantly. Generally, UC patients were more positive about their improved treatment than CD patients.
Conclusion
Combination therapy with 50 mg allopurinol led to a decrease of 6-TGN levels compared with 100 mg allopurinol. Disease activity, side effects, and patient experience, however, were similar between allopurinol 100 and 50 mg. UC patients seem to benefit and prefer lower doses whereas the contrary is seen in CD patients.
Trial registration
EudraCT trial registry - number
2016-001638-84
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