ABSTRACT
Combination of MEK inhibitor and 5-FU had showed limited efficacy in clinical trials. We previously reported that acquired resistance to 5-FU was related with continued activation of salvage pathway. Here we investigated whether combination of 5-FU and a MEK inhibitor had treatment sequence-dependent synergistic effects in KRAS or BRAF mutant colon cancer models. Treatment with 5-FU followed by selumetinib (FS) induced highest cell death and synergy compared with reverse (SF) and concomitant (cFS) treatment in six cell lines. SF or cFS combination induced synergy in 1 or 2 cell lines, respectively, in which the synergy was less than that by FS combination. FS enhanced apoptosis and decreased anchorage-independent growth. Induction of thymidine kinase 1, a rate-limiting enzyme in salvage pathway, by 5-FU was abrogated by subsequent treatment with selumetinib, and ERK reactivation after selumetinib was prohibited by pretreatment with 5-FU. FS altered mRNA expression in groups of genes distinct from SF. Administration of 5-FU (10 or 30â¯mg/kg/day) for 7â¯days, followed by selumetinib (10 or 25â¯mg/kg/day) for another 7â¯days, in colo205 and HCT8 xenograft models significantly decreased tumor growth compared with a single agent. However, co-administration in the reverse sequence did not show the difference in tumor size compared with the treatment of single agent. Decreased expression of Ki67 was observed in tumors from mice treated with FS. Our results suggest that sequential administration of 5-FU plus selumetinib would be a promising strategy for patients having KRAS or BRAF mutant colon cancers.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Colonic Neoplasms/drug therapy , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Benzimidazoles/administration & dosage , Cell Line, Tumor , Colonic Neoplasms/genetics , Drug Administration Schedule , Fluorouracil/administration & dosage , Humans , Male , Mice, Inbred BALB C , Mutation , Thymidine Kinase/antagonists & inhibitors , Thymidine Kinase/metabolism , Xenograft Model Antitumor AssaysABSTRACT
In cancer therapy, enhanced thymidine uptake by the salvage pathway can bypass dTMP depletion, thereby conferring resistance to thymidylate synthase inhibition. We investigated whether sequential combination therapy of capecitabine and trifluridine/tipiracil (TAS-102) could synergistically enhance antitumor efficacy in colon cancer xenograft models. We also examined 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) PET as a means to predict therapeutic response to a sequential combination of capecitabine and trifluridine/tipiracil. [3H]FLT uptake after 5-fluorouracil treatment in vitro and [18F]FLT uptake after capecitabine (360 mg/kg/day) in athymic nude mice (Balb/c-nu) with xenografts (n = 10-12 per group) were measured using eight human colon cancer cell lines. We determined the synergistic effects of sequential combinations of 5-fluorouracil and trifluridine in vitro as well as the sequential combination of oral capecitabine (30-360 mg/kg) and trifluridine/tipiracil (trifluridine 75 or 150 mg/kg with tipiracil) in six xenograft models (n = 6-10 per group). We observed significant increases in [3H]FLT uptake in all cell lines and [18F]FLT uptake in five xenograft models after 5-fluorouracil and capecitabine treatment, respectively. Increased [18F]FLT uptake after capecitabine followed by extinction of uptake correlated strongly with tumor growth inhibition (ρ = -0.81, P = 0.02). The effects of these combinations were synergistic in vitro A synergy for sequential capecitabine and trifluridine/tipiracil was found only in mouse xenograft models showing increased [18F]FLT uptake after capecitabine. Our results suggest that the sequential combination of capecitabine and trifluridine/tipiracil is synergistic in tumors with an activated salvage pathway after capecitabine treatment in mice, and [18F]FLT PET imaging may predict the response to capecitabine and the synergistic antitumor efficacy of a sequential combination of capecitabine and trifluridine/tipiracil. Cancer Res; 77(24); 7120-30. ©2017 AACR.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colonic Neoplasms/diagnosis , Colonic Neoplasms/drug therapy , Dideoxynucleosides , Fluorine Radioisotopes , Positron-Emission Tomography/methods , Animals , Capecitabine/administration & dosage , Cell Line, Tumor , Colonic Neoplasms/pathology , Dideoxynucleosides/chemistry , Drug Combinations , Drug Synergism , Female , Fluorine Radioisotopes/chemistry , HCT116 Cells , HT29 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Pyrrolidines/administration & dosage , Thymine/administration & dosage , Treatment Outcome , Trifluridine/administration & dosage , Uracil/administration & dosage , Uracil/analogs & derivatives , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: TAS-102 is an orally administered anticancer agent composed of α,α,α-trifluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). This study assessed 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) uptake after TAS-102 administration. METHODS: The human colorectal carcinoma cell lines HCT116, HT29, HCT8 and SW620 were exposed to FTD for 2 h, further incubated for 0, 2 and 24 h, and assayed for [(3)H]FLT uptake, nucleoside transport, thymidine kinase 1 (TK1) expression and TK1 activity. Static and 2-h dynamic [(18)F]FLT positron emission tomography (PET) was performed in mice bearing HT29 or SW620 tumours orally administered with vehicle or TAS-102. RESULTS: FTD decreased the viability of all cell lines, whereas increased [(3)H]FLT uptake (P < 0.05). Increased nucleoside transport and/or TK1 expression were observed 24 h after FTD, but not in 0-2 h. Static [(18)F]FLT PET in mice bearing HT29 tumours showed accumulation of [(18)F]FLT in tumours 1 h (day 1) after TAS-102. Two-hour dynamic PET in mice bearing SW620 tumours showed increased influx constant and volume of distribution of phosphorylated [(18)F]FLT on days 1 and 8 (P < 0.05) after TAS-102 with decreased dephosphorylation on day 1 (P < 0.001). Ex vivo studies showed that SW620 tumours after TAS-102 had higher TK1 expression than those with vehicle on days 8 and 15. CONCLUSION: TAS-102 administration induces an increase in [(18)F]FLT uptake. Mechanisms may involve decreased dephosphorylation of [(18)F]FLT phosphate early after TAS-102 administration. Increased TK1 expression and/or nucleoside transporter may be related to increased [(18)F]FLT uptake at a later time. [(18)F]FLT PET has a potential to assess the pharmacodynamics of TAS-102 in cancer patients.
Subject(s)
Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/drug therapy , Dideoxynucleosides , Trifluridine/pharmacology , Uracil/analogs & derivatives , Animals , Antineoplastic Agents/pharmacology , Cell Growth Processes/drug effects , Cell Line, Tumor , Colonic Neoplasms/metabolism , Dideoxynucleosides/pharmacology , Drug Combinations , Female , Fluorine Radioisotopes , HCT116 Cells , HT29 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Positron-Emission Tomography , Pyrrolidines , Radiopharmaceuticals/pharmacology , Random Allocation , Thymidine Kinase/metabolism , Thymine , Uracil/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Thymidine phosphorylase (TP), a key enzyme in the pyrimidine nucleoside salvage pathway, catalyses the reversible phosphorylation of thymidine, thereby generating thymine and 2-deoxy-D-ribose-1-phosphate. By regulating the levels of endogenous thymidine, TP may influence [(18)F]fluorothymidine ([(18)F]FLT) uptake. We investigated the effect of TP activity on [(18)F]FLT uptake by tumours. METHODS: Uptake of [(3)H]FLT and [(3)H]thymidine ([(3)H]Thd) and the activities of TP, thymidine kinase 1 (TK1), and equilibrative nucleoside transporter 1 (ENT1) were determined in exponentially growing A431, A549, HT29, HOP92, ACHN, and SKOV3 cells in the presence or absence of tipiracil hydrochloride, a TP inhibitor. Eighty-five non-small cell lung cancer tissues from a patient cohort that was previously studied with [(18)F]FLT positron emission tomography (PET) were retrieved and subjected to immunohistochemical analysis of TP expression. Factors that affected the maximum standardised uptake value (SUVmax) of [(18)F]FLT-PET were identified by multiple linear regression analysis. RESULTS: A431 cells had the highest TP activity; A549 and HT29 cells had moderate TP activity; and ACHN, SKOV3, and HOP92 cells had little detectable TP activity. Cell lines with high TP activity took up more [(3)H]FLT than [(3)H]Thd, whereas cells with little TP activity took up more [(3)H]Thd than [(3)H]FLT. In cells with high TP activity, TP inhibition decreased [(3)H]FLT uptake and increased [(3)H]Thd uptake. However, TP inhibition had no effect on ACHN, SKOV3, and HOP92 cells. TP inhibition did not change TK1 or ENT1 activity, but did increase the intracellular level of thymidine. The SUVmax of [(18)F]FLT was affected by three independent factors: Ki-67 expression (P < 0.001), immunohistochemical TP score (P < 0.001), and tumour size (P = 0.015). CONCLUSIONS: TP activity influences [(18)F]FLT uptake, and may explain preferential uptake of [(18)F]FLT over [(3)H]Thd. These results provide important insights into the biology of [(18)F]FLT as a proliferation marker.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Dideoxynucleosides/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Thymidine Phosphorylase/metabolism , Adult , Aged , Biological Transport/drug effects , Carcinoma, Non-Small-Cell Lung/enzymology , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Lung Neoplasms/enzymology , Male , Middle Aged , Pyrrolidines/metabolism , Thymidine/metabolism , Thymidine Phosphorylase/antagonists & inhibitors , Thymine/metabolism , Time FactorsABSTRACT
BMS-754807 is an inhibitor of insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor that also represses aurora kinase. Cancers that express high levels of IGF-1/IGF-1R are sensitive to BMS-754807; however, it shows limited efficacy in non-small cell lung cancer (NSCLC) in which IGF-1R-driven signals may not be dominant factors in cell proliferation. In this study, we investigated whether a combination of BMS-754807 and gefitinib would be synergistic in H292 NSCLC and whether [(18)F]fluorothymidine ([(18)F]FLT)-positron emission tomography (PET) could predict the effects. We found that BMS-754807 synergized with gefitinib in reducing cell viability (combination index=0.38) and Akt phosphorylation, and increasing the subG1 fraction in H292 cells. BMS-754807 alone and in combination with gefitinib increased the cells in G2M phase and polyploid cells and decreased the phosphorylation of IGF-1R and histone H3. The inhibition of tumor growth by gefitinib was increased by BMS-754807 (%T/C, 17.5 % vs. 58.0 % for gefitinib alone and combined treatment, respectively), although BMS-754807 alone had little effect. The standardized uptake value by [(18)F]FLT-PET were increased in vehicle-treated mice by 73 %, minimally changed in gefitinib- or BMS-754807-treated mice, whereas decreased in co-treated mice by -48.8 % between day 0 and day 3. The combination therapy with BMS-754807 and gefitinib might be a more effective anticancer strategy than BMS-754807 alone in tumors that are less IGF-1R-dependent and that [(18)F]FLT-PET can be used to assess early therapeutic responses.
Subject(s)
Antineoplastic Agents/administration & dosage , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/administration & dosage , Pyrazoles/administration & dosage , Quinazolines/administration & dosage , Triazines/administration & dosage , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Fluorine Radioisotopes , Gefitinib , Humans , Lung Neoplasms/pathology , Male , Mice , Mice, Nude , Pilot Projects , Positron-Emission Tomography/methods , Receptor, IGF Type 1/metabolism , Thymidine/analogs & derivatives , Tumor Burden/drug effectsABSTRACT
BACKGROUND: Thymidine kinase 1 (TK1) and 5'(3')-deoxyribonucleotidase (dNT1) regulate the metabolism of thymidine and its analogs. However, the expression patterns and roles of these enzymes in fluorothymidine (FLT) uptake have not been systemically studied. MATERIALS AND METHODS: TK1 and dNT1 mRNAs were determined by quantitative PCR in 20 asynchronously growing lung and colon cancer cell lines. [3H]FLT uptake and doubling time were measured. RESULTS: The TK1/GAPDH values varied from 3.09×10(-3)±6.62×10(-4) to 2.44×10(-2)±8.49×10(-4) and dNT1/GAPDH values from 5.84×10(-4)±4.88×10(-5) to 1.59×10(-2)±1.20×10(-3). The correlation coefficient of TK1/GAPDH versus dNT1/GAPDH was 0.669 (p<0.001). [3H]FLT uptake showed negative moderate correlation with dNT1/GAPDH levels (r=-0.563; p<0.01), but no significance with TK1/GAPDH levels. Doubling time had no relationship with the TK1 or dNT1 expression. CONCLUSION: This study profiled correlative expression of TK1 and dNT1 in cancer cell lines, and showed that dNT1 expression determines the lower uptake of FLT, providing a basis for understanding deoxyribonucleotide metabolism.
Subject(s)
5'-Nucleotidase/genetics , Colonic Neoplasms/metabolism , Dideoxynucleosides/pharmacokinetics , Lung Neoplasms/metabolism , RNA, Messenger/biosynthesis , Thymidine Kinase/genetics , 5'-Nucleotidase/biosynthesis , Cell Line, Tumor , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , HCT116 Cells , HT29 Cells , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , RNA, Messenger/genetics , Thymidine Kinase/biosynthesis , TritiumABSTRACT
In this study the diagnostic potential of Rickettsia conorii recombinant antigens was analyzed. For this, site-specific PCR primers were used to clone the OmpA and OmpB genes of R. conorii into pMAL-c2X plasmids. Six fragments of OmpA and four of OmpB were expressed as fusion proteins with maltose-binding protein in Escherichia coli. OmpA(1350-1784), OmpB(801-1269,) and OmpB(1227-1634) regions from truncated proteins were selected as diagnostic candidate antigens by ELISA using control sera. ELISA results of three antigens were compared to the results obtained by using a commercial ELISA kit which contained whole OmpA and OmpB antigens from R. conorii. For this analysis, 40 serum samples taken from febrile patients and uninfected controls were tested. Of the 20 R. conorii test results which were positive with the commercial kit, 18 were shown to be positive by ELISA using OmpA(1350-1784) (a sensitivity of 90%). The specificity of the ELISA was 100%; all of the 20 samples shown to be negative using the commercial kit were also negative in our assay. The sensitivities of the ELISA using the OmpB(801-1269) and OmpB(1227-1634) were 90% and 95%, respectively. The specificities of the OmpB(801-1269) and the OmpB(1227-1634) were 100% and 95%, respectively. These results suggest that specific regions of OmpA and OmpB effectively detect antibodies against R. conorii, and the truncated recombinant antigens could be used for development of diagnostic tools for rickettsial disease.