UDG depletion increased floxuridine-associated loss of clonogenic survival in DLD1 and HEC1A cancer cells. Comparator experiments showed that this sensitization did not extend to cisplatin, doxorubicin or temozolomide under the tested conditions. In DLD1 cells, concurrent thymidine largely rescued colony survival, whereas supplementation after 24 h did not fully rescue UDG-depleted cells. These responses depend on repair status and treatment timing. [2]
Floxuridine caused greater viability loss in ARID1A-knockout HCT116 colorectal cancer cells than in wild-type cells. ARID1A overexpression reduced sensitivity in an independent RKO background. These isogenic-cell findings support ARID1A-dependent response in the tested models. [3]
Intracellular thymidylate synthase engagement in K562 cells
Cell-based study · Human K562 myelogenous leukemia cells [1]
Floxuridine stabilized endogenous TS after cellular exposure. A dedicated confirmation titration gave an apparent CETSA EC50; orthogonal western blots corroborated stabilization without a detectable increase in total TS under the tested conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- Dose-response titration; 15 μM floxuridine for Figure 1b thermal aggregation validation
- Exposure duration
- 2 h pre-incubation; 3 min heat challenge at 50 °C for isothermal dose-response
- Method
- AlphaScreen-based cellular thermal shift assay (CETSA)
- Vehicle
- 0.5% DMSO
- Reported limits
- The exact concentration series of the Figure 2b floxuridine titration is not enumerated in the caption; Methods give the general titration design. No purified-enzyme IC50 is assigned.
Methods and source locations
Compound reported in the paper: Floxuridine; Compound label in the paper: floxuridine (FdU)
Applies to the named literature compound under these conditions; does not establish Adooq sourcing, lot purity or analytical stereochemical validation.
Figure 2b and caption; Figure 1b–c; Results: assay validation and screening/hit confirmation; Methods: dose-response/analysis; SI Figure 7 and Table 3
Half-maximal thermal stabilization of endogenous TS at 50 °C: 2 h cellular exposure; AlphaScreen. Figure 2b reports mean and s.e.m. from one independent confirmation experiment in quadruplicate. — Figure 2b caption; Methods: Screening and dose-response characterization; Screen and ITDRFCETSA data analysis
Intracellular FdUMP formation accompanies TS engagement
Cell-based study · Human K562 cells; separate CETSA titrations and LC–MS/MS metabolite time courses [1]
Intracellular FdUMP was detectable after 10 min of floxuridine exposure and accumulated during the initial hours. The time course coincided with increasing potency of TS thermal stabilization, supporting intracellular activation of floxuridine.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 μM floxuridine for LC–MS/MS (SI Figure 14c–d); concentration titrations for CETSA
- Exposure duration
- 10 min, 30 min, 2 h and 6 h for the combined Figure 3b–c comparison
- Method
- LC–MS/MS quantitation of intracellular FdUMP, assessed alongside cellular TS stabilization
- Vehicle
- 0.5% DMSO for CETSA; vehicle not specified for the LC–MS/MS measurements
- Reported limits
- No exact metabolite amount or fitted EC50 is digitized from the time-course plots. The LC–MS/MS dose is not substituted for the CETSA titration doses.
Methods and source locations
Compound reported in the paper: Floxuridine; Compound label in the paper: floxuridine (FdU)
Applies to the named literature compound under these conditions; does not establish Adooq sourcing, lot purity or analytical stereochemical validation.
Figure 3b–c and caption; Results: Addressing kinetics of compound transport and metabolism; Methods: Intracellular compound and metabolite concentrations; Table 2; SI Figure 14c–d and caption (PDF pp. 19, 21)
Intracellular FdUMP formation and corresponding TS stabilization: Figure 3c: CETSA mean and range of two independent experiments; LC–MS/MS mean and s.e.m. from experiments on three occasions. SI Figure 14 specifies 1 μM floxuridine exposure. — Figure 3b–c and caption; SI Figure 14c–d, caption; Methods: Intracellular compound and metabolite concentrations
UDG depletion increases floxuridine sensitivity in two cancer cell lines
Cell-based study · DLD1 colon cancer and HEC1A endometrial cancer cells: UDG-depleted shUDG versus scrambled shSCR controls [2]
Floxuridine reduced clonogenic survival more strongly in UDG-depleted cells. Parallel comparator assays showed sensitization to pemetrexed, but no additional sensitization to cisplatin, doxorubicin or temozolomide.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0–200 nM 5-FdU in Figure 2a–b
- Exposure duration
- 24 h drug exposure; subsequent colony growth for at least 10 days
- Method
- Colony survival after washout; DMEM with 10% dialyzed fetal bovine serum
- Reported limits
- Drug vehicle not specified in the read cell-lines/drugs or colony-survival methods. The caption uses >50 cells per colony; Methods uses ≥50.
Methods and source locations
Compound reported in the paper: Floxuridine; Compound label in the paper: 5-FdU
Applies to the named literature compound under these conditions; does not establish Adooq sourcing, lot purity or analytical stereochemical validation.
Figure 2a–j and caption; Figure 1a–d (DLD1 UDG validation), Figure 2b inset (HEC1A); Results: Loss of UDG enhances cytotoxicity; Methods: Cell lines and drugs; Colony survival assay
Clonogenic survival relative to untreated control: Figure 2: mean and SEM from at least 3 independent experiments. No exact curve-derived survival percentage or IC50 extracted. — Figure 2a–b and caption; Methods: Colony survival assay
Specificity of sensitization across comparator drugs: Parallel colony-survival comparisons; doses differ by drug and cell line. — Figure 2c–j and caption; Results: Loss of UDG enhances cytotoxicity
Timing of thymidine supplementation changes clonogenic rescue
Cell-based study · DLD1 shSCR and UDG-depleted shUDG cancer cells [2]
Simultaneous thymidine supplementation largely prevented floxuridine-associated colony loss. Adding thymidine after 24 h did not fully rescue UDG-depleted cells, showing that the timing of rescue matters.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0–200 nM 5-FdU; 20 μM thymidine
- Exposure duration
- 24 h floxuridine exposure; thymidine given simultaneously or 24 h after exposure began; colony assay follow-up at least 10 days
- Method
- Colony survival with concurrent versus delayed supplementation; DMEM with 10% dialyzed serum
- Co-treatment
- Thymidine as a mechanistic rescue condition, not a proposed anticancer combination
- Reported limits
- Vehicle not specified in read methods; Figure 3a–b caption labels uncertainty as “error” without identifying SD or SEM.
Methods and source locations
Compound reported in the paper: Floxuridine; Compound label in the paper: 5-FdU
Applies to the named literature compound under these conditions; does not establish Adooq sourcing, lot purity or analytical stereochemical validation.
Figure 3a–b and caption; Results: Thymidine treatment after 5-FdU exposure; Methods: Colony survival assay
Rescue of clonogenic survival by thymidine: At least 3 independent experiments; Figure 3a–b caption says mean and error, error type unspecified. No exact survival percentages extracted. — Figure 3a–b and caption
ARID1A status modifies floxuridine response in colorectal cancer cells
Cell-based study · HCT116 ARID1A-WT and two ARID1A-KO clones; parental ARID1A-deficient RKO and two ARID1A-overexpressing clones [3]
HCT116 knockout clones showed greater viability loss after floxuridine than the wild-type cells. Restoring ARID1A expression in RKO shifted the response toward reduced sensitivity.
Literature-Reported Treatment Conditions
- Treatment concentration
- Floxuridine concentration-response series as displayed in Figures 1d and 2d; no fitted IC50 extracted
- Exposure duration
- 72 h for HCT116 confirmation curves (Figure 1 caption); RKO curve exposure duration not specified in Figure 2d caption
- Method
- Alamar Blue viability assay for the screening/confirmation workflow
- Reported limits
- The separate follow-up FUDR supplier, vehicle, assay-specific replicate counts and exact tabulated dose lists are not specified in the read material. Do not transfer the 20 μM screen dose to confirmation curves or assume the RKO exposure duration.
Methods and source locations
Compound reported in the paper: Floxuridine; Compound label in the paper: FUDR
Applies to the named literature compound under these conditions; does not establish Adooq sourcing, lot purity or analytical stereochemical validation.
Figure 1a,d and caption; Figure 2c–d and caption; Results: screening and growth response; Methods: FDA-approved drug library screening and cell viability measurement
Cell viability in ARID1A isogenic models: Figure 1d HCT116 dose-response; Figure 2d RKO dose-response with s.d. error bars per caption. No exact fitted potency reported here. — Figures 1a,d and 2c–d; associated captions
