In carboplatin-resistant Ov4Carbo cells, chloroxine co-treatment enhanced carboplatin-associated DNA-damage markers and reduced later RAD51 recruitment. The response depended on dose and readout: additional γH2AX foci were observed at 50 μM, but not 100 μM carboplatin. Total RAD51 protein remained stable. [2]
MMP-14 enzyme inhibition
Biochemical assay · Isolated MMP-14 fluorogenic peptide-cleavage assay [1]
Chloroxine reduced MMP-14 activity with a reported IC50 of 25.6 ± 4.2 μM.
Literature-Reported Treatment Conditions
- Treatment concentration
- Dose response; Figure 3C spans approximately 1–100 μM
- Exposure duration
- 10 min preincubation; fluorescence monitored up to 50 min
- Method
- 10 nM final MMP-14; 7 μM VEGFR1-derived fluorogenic substrate; initial-rate analysis
- Vehicle
- DMSO for inhibitor solutions
- Reported limits
- Exact dose list and definition of ± are not specified in the retrieved main-text IC50 description; two to four independent assays are reported globally.
Methods and source locations
Compound reported in the paper: Chloroxine
Evidence concerns the tested research material; no Adooq lot or formulation validation.
Section 2.2; Figure 3C; sections 3.1–3.4
Inhibition of MMP-14 peptide cleavage: Fluorogenic initial-rate assay, Hill fit; ± definition not established in read scope — Figure 3C annotation; section 2.2; section 3.4
Direct MMP-14 binding by surface plasmon resonance
Biochemical assay · Immobilized catalytic domain of MMP-14 on a CM5 sensor [1]
Chloroxine bound the MMP-14 catalytic domain with a reported KD of 2.4 ± 0.9 μM.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0.78–25 μM, twofold series
- Method
- SPR at 25 °C; steady-state affinity fit
- Vehicle
- PBSP (PBS plus 0.05% P20)
- Reported limits
- Exact contact duration, experiment-specific n and meaning of ± are not established in the retrieved description.
Methods and source locations
Compound reported in the paper: Chloroxine
Evidence concerns the tested research material; no Adooq lot or formulation validation.
Section 2.3; Figure 4A–B; section 3.5
Binding to MMP-14 catalytic domain: SPR steady-state affinity; blank-channel and zero-concentration subtraction — Figure 4B annotation and caption; section 3.5
MMP counterscreen limits target selectivity
Biochemical assay · Fluorometric enzyme assays for MMP-2, MMP-7, MMP-9 and MMP-13 [1]
Chloroxine also inhibited MMP-9 and MMP-13. MMP-2 and MMP-7 showed partial inhibition without reaching 50% in the plotted range.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response series shown in Figure 8A; exact dose list not reconstructed
- Exposure duration
- 10 min preincubation; 25 min fluorescence monitoring
- Method
- 10 μM fluorogenic substrate; triplicate assays
- Vehicle
- DMSO stock solutions diluted in assay buffer
- Reported limits
- Meaning of ± for the fitted IC50 values is not defined in the retrieved main text. Do not assume selectivity-assay enzyme concentrations match MMP-14 conditions.
Methods and source locations
Compound reported in the paper: Chloroxine
Evidence concerns the tested research material; no Adooq lot or formulation validation.
Section 2.5; Figure 8A axis and annotations; section 3.9
MMP-9 inhibition: Fluorometric concentration response; triplicates — Figure 8A annotation; sections 2.5 and 3.9
MMP-13 inhibition: Fluorometric concentration response; triplicates — Figure 8A annotation; sections 2.5 and 3.9
MMP-2 and MMP-7 inhibition: Visual review; no exact percentages inferred from curves — Figure 8A; section 2.5
DNA-damage and repair responses during carboplatin co-treatment
Cell-based study · Ov4Carbo carboplatin-resistant human ovarian cancer cells; OVCAR4 comparator for immunoblots [2]
With 50 μM carboplatin, 10 μM chloroxine increased γH2AX foci at 4 h and was followed by reduced RAD51 recruitment at 24 h. At 100 μM carboplatin, immunofluorescence did not show an additional γH2AX increase; immunoblotting detected an increase at both doses. Total RAD51 protein remained stable.
Literature-Reported Treatment Conditions
- Treatment concentration
- Chloroxine 10 μM; carboplatin 50 or 100 μM
- Exposure duration
- 4 and 24 h for immunofluorescence
- Method
- γH2AX/RAD51 immunofluorescence; cells with >5 foci/nucleus scored positive; immunoblot corroboration
- Co-treatment
- Carboplatin; chloroxine-only, carboplatin-only and untreated controls included
- Reported limits
- Vehicle is not specified for this mechanistic experiment; do not import the screening vehicle. Figure 6D blot harvest timing is not explicit in the viewed caption/methods.
Methods and source locations
Compound reported in the paper: Chloroxine
Evidence concerns the tested research material; no Adooq lot or formulation validation.
Results: γ-H2AX–RAD51 axis section; Figure 6A–D/caption; Methods: Immunofluorescence for DNA damage and Western blot
γH2AX and RAD51 foci in Ov4Carbo cells: Immunofluorescence; untreated and individual-drug controls — Figure 6A–C; DNA-damage Results paragraphs 1–3 and Discussion
γH2AX and total RAD51 protein: Immunoblot with tubulin/GAPDH normalization — Figure 6D; DNA-damage Results final paragraph
