IBC and BKC synergistically inhibit proliferation of cancer cell lines.

A. Chemical structures of isobavachalcone (IBC) and bakuchiol (BKC). B. BJ, MCF-7 and A549 cells were treated with DMSO, 25 μg/ml crude extract (PR7), 15 μM IBC, 40 μM BKC or the combination 15 μM IBC and 40 μM BKC for 72 hours. These concentrations were used throughout the study. Cell number was quantified by using the WST-1 assay. Data are means +/- SD of three independent experiments. The p-values were calculated by two-tailed unpaired t-test. C. Concentration matrix analyses of a panel of eight cancer cell lines treated with IBC and BKC at the indicated doses for 72 hours. Cell viability was measured by using the sulforhodamine B colorimetric assay. Antagonist combinations (green), synergistic combinations (red) and additive effects (black) were calculated. A representative analysis of three independent experiments is shown.

IBC and BKC induce replication stress and impede fork progression.

A. MCF-7 and BJ cells were treated with either 15 μM IBC, 40 μM BKC or both (IBC + BKC) for 24 hours, then 10 μM EdU was added for 10 minutes and γ-H2AX foci in EdU-positive cells were detected by using Click chemistry and immunofluorescence microscopy. Representative immunofluorescence images of MCF-7 cells are shown. Bar: 5 μm. B. Mean fluorescence intensity (MFI) of γ-H2AX foci was quantified by CellProfiler. One of three independent experiments is shown (n=3). ****, p<0.0001; ns, not significant, Mann-Whitney rank sum test. C. MCF-7 cells were treated with IBC/BKC for 24 hours, as in panel A, and CHK1 phosphorylated on S345 (pCHK1) was detected by western blotting. The ratio of pCHK1 to total CHK1, relative to the DMSO control, is indicated. A representative example of two independent experiments is shown. D. BJ and MCF-7 cells were treated with IBC/BKC for 24 hours, as in panel A, then IdU and CldU were added sequentially each for 15 minutes. Replication fork progression was determined by measuring CldU track lengths in DNA fiber spreads. The median length of CldU tracks is indicated in red. At least 150 fibers were measured for each condition. Median of two independent experiments is indicated in red. E. MCF-7 cells were treated with indicated concentrations of BKC or 1 µM Aphidicolin for 2 hours then IdU and CldU were added sequentially each for 20 minutes. Replication fork progression was determined as in panel D. The length of IdU and CldU was measured. Median of three independent experiments is indicated in red. The p-values were determined by two-tailed unpaired t-test (n=3). F. MCF-7 cells were treated with DMSO or 20 μM BKC for 2 hours then IdU and CldU were added sequentially each for 20 minutes. Replication fork progression was determined as indicated in panel D. The ratio of the CldU signal of two sister forks was calculated. At least 80 sister forks were measured in each biological replicate. The ratio of two sister forks between 0.8-1.2 was considered as symmetric forks. Mean + SEM of three independent experiments are shown. The p-values were determined by two-tailed unpaired t-test. G. MCF-7 cells were treated with 40 µM BKC, aphidicolin (Aph, 10 μM) or both (BKC + Aph) for 2 hours, prior to DNA fiber spreading assay. The p-values were determined by two-tailed unpaired t-test (n=3). H. Xenopus egg extracts were incubated with demembranated sperm nuclei and treated immediately (0 min) or after 40 minutes (40 min) with DMSO, BKC (100 μM) or aphidicolin (Aph, 60 μM). Samples were collected at the indicated time points after addition of the sperm nuclei. The percentage of replicated DNA was calculated as described in the Materials and Methods.

BKC directly binds to DNA polymerases.

A-B. In silico molecular docking of bakuchiol in the predicted active site structures of human DNA Pol δ and ε, respectively. C-D. MCF-7 cells were treated with DMSO, 40 µM BKC or 10 µM Aphidicolin for 2 hours prior to the cellular thermal sensitivity shift assay (CETSA) at indicated temperature, as described in the Materials and Methods. Levels of POLD1 (Pol δ; panel C) and POLE (Polε; panel D) catalytic subunits were detected by western blotting. Mean and SEM of three independent experiments are shown. The p-values were determined by two-tailed paired t-test.

IBC inhibits the CHK2 kinase.

A. Selected protein kinases were incubated with the indicated range of IBC concentrations and kinase activity in vitro was determined by using a radiometric assay. The IC50 of IBC for each kinase is indicated in the panel on the right. B. MCF-7 cells were pre-treated with DMSO, 15 µM IBC, or 20 µM BML-277 for 2 hours, then camptothecin (CPT, 1 μM) was added for 2 hours. Phosphorylation of CHK2 on S516 (pCHK2) was detected by western blotting. The ratio of pCHK2-S516 induction, relative to the DMSO+CPT control, is indicated. (n=3) C. MCF-7 cells were treated as indicated in panel B. Phosphorylation of chromatin-bound BRCA1 at residue S988 was detected by western blotting. The relative ratio of pBRCA1-S988 signal, after normalization to ponceau signal, is indicated. TBP was used as a marker of chromatin fraction D. MCF-7 cells were treated with 15 μM IBC for 2 hours, then 4 mM HU was added for 2 hours. CHK1 auto-phosphorylation on S296 (pCHK1) was detected by western blotting. E-F. In silico molecular docking of IBC in the active sites of CHK2 and CHK1, respectively. G-H. Cellular Thermal Shift Assay (CETSA) of IBC on the thermal stability of CHK2 and CHK1. MCF-7 cells were treated with 15 µM IBC or 20 μM BML-277 for 2 hours. Cells were proceeded to CETSA as described in the Materials and Methods. The amount of CHK2 and CHK1 present in the supernatant was detected by western blotting. The relative CHK2 and CHK1 signal was quantified. The p-values were determined by two-tailed paired t-test (n=3).

IBC impedes DNA end resection and RAD51 foci formation.

A. MCF-7 cells were treated with DMSO or 15 μM IBC for 1 hour, then camptothecin (CPT, 1 μM) was added for 2 hours. The cells were fixed immediately or washed and allowed to recover in medium containing DMSO or IBC for 24 hours before fixation. 53BP1 foci in p27-positive nuclei were detected by immunofluorescence microscopy. Representative images are shown. Bar: 5 μm. B. 53BP1 foci number was quantified by using CellProfiler. Representative data from three independent experiments are shown. ****, p<0.0001; ns, not significant, Mann-Whitney rank sum test. C. MCF-7 cells were treated with DMSO or 15 μM IBC for 2 hours, then camptothecin (CPT, 1 μM) was added for 2 hours, as indicated. Cells were fractionated into cytosol and nuclei (chromatin) and RPA in both fractions was detected by western blotting. Actin and TBP were used as markers of cytosol and chromatin fractions, respectively. The fold change of the chromatin-bound RPA signal relative to the DMSO control was quantified. The p-value was determined by unpaired t-test. (n=3) D. MCF-7 cells were incubated with 10 µM BrdU for 24 hours to label genomic DNA, then DMSO, 15 µM IBC or the CHK2 inhibitor BML-277 (20 μM) were added for 2 hours followed by addition of 5 μg/ ml bleomycin for 1 hour. DNA fibers were spread on glass slides and BrdU was detected by immunofluorescence microscopy without DNA denaturation. The length of BrdU the tracks was measured and the median for each condition is indicated in red. At least 250 fibers were measured for each condition. Median of two independent experiments is indicated in red (n=2). E. MCF-7 cells were incubated with 10 µM BrdU for 24 hours to label genomic DNA in the presence of DMSO or IBC then exposed to ionizing radiations (8 Gy). Cells were collected 60 or 120 minutes after irradiation and BrdU tracks measured as in part B. ****, p<0.0001, Mann-Whitney rank sum test. F. DIvA cells were treated with either DMSO or IBC for 2 hours then DNA breaks were induced by treatment with 300 nM 4-hydroxytamoxifen (4-OHT) for 4 hours. Resection at two break sites, DSB-II and DSB-V, was determined as the percentage of ssDNA at these sites, calculated as indicated in the Material and Methods. Data are means +/- SD (n=3). The p-values are indicated (two-tailed paired t-test). G. MCF-7 cells were treated with DMSO, IBC or BML-277 for 2 hours, then irradiated as described above. After 1 hour, RAD51 foci were detected by CSK-immunofluorescence microscopy and foci number was quantified using CellProfiler. Representative data (left) and immunofluorescence images (right) from two independent experiments are shown. Bar, 10 μm. ****, p<0.0001, Mann-Whitney rank sum test.

IBC and BKC synergistically inhibit tumor development and induce DNA damage in a xenograft mouse model.

A. Schematic description of the protocol. MCF-7/Luc cells (1×104) were injected into the fat pads of female NOD/SCID mice at Day 0. Two days later, randomized mice were injected intraperitoneally with PBS, Taxol, IBC, BKC, or IBC+BKC at the indicated doses. Tumor sizes were measured weekly thereafter. Tumor tissues were collected 28 days after grafting and analyzed by immunohistochemistry. Mice survival was also evaluated. B. Tumor size in the fat pads was measured once per week by using the IVIS bioluminescence system. The number of xenografted mice receiving each treatment is indicated. ****, p<0.0001 ***, p<0.001; **, p<0.01; *, p<0.05, Mann-Whitney rank sum test. C. Survival (left) and median survival time (days, right) is shown for xenografted mice receiving each treatment. ****, p<0.0001 ***, p<0.001; **, P<0.01; *, P<0.05, Mann-Whitney rank sum test. D. Tumor tissues collected from xenograft mice were analyzed immunohistochemically for the cell proliferation maker Ki67; E. broken DNA and apoptosis marker, TUNEL staining; F. pCHK1 (S345) and g. pCHK2 (Thr68). ****, P<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant, Mann-Whitney rank sum test.

IBC potentiates the effect of chemotherapeutic agents on lymphoma cells.

A. A panel of eight diffuse large B-cell lymphoma (DLBCL) cell lines were incubated with the indicated concentrations of IBC for 72 hours (left) and growth inhibition was measured to calculate the IC50 in each cell line (right). Data are presented as means +/- SD (n=3). B. U2932 cells were treated with the indicated concentrations of etoposide (Eto), doxorubicin (Doxo) or 4-OH-cyclophosphamide (4-OH-Cyclo) without (black points) or with 1.5 μg/ ml (the IC20 concentration for this cell line) IBC (red points) for 72 hours. Cell viability was measured. Data are presented means +/- SD (n=3). C. Full-concentration matrix analyses of U2932 cells treated with IBC and Eto (left), Doxo (middle) or 4-OH-Cyclo at the indicated concentrations for 72 hours. Synergy and antagonism were calculated as described in Fig. 1.

Model of the synergistic action of BKC and IBC that kills cancer cells.

By inhibiting the DNA polymerases, BKC enhances the endogenous RS in cancer cells, leading to increased DNA damage. IBC specifically inhibits CHK2 to prevent DNA repair. The combined use of BKC and IBC enhances DNA damage to a level that triggers cancer cell death.

IBC and BKC synergistically inhibit proliferation of cancer cell lines.

A. MCF10A and MCF-7 cells were treated with DMSO, or indicated concentrations of IBC and BKC for 72 hours. Cell number was quantified by using the WST-1 assay. Mean +/- SD of three independent experiments are shown. The p-values were calculated by two-tailed unpaired t-test. B. Human telomerase-immortalized RPE-1 cells and MCF-7 and A549 cells were treated with DMSO, indicated concentrations of IBC or BKC or the combination 7.5 μM IBC and 20 μM BKC for 72 hours. Cell number was quantified by using the WST-1 assay. Mean +/- SD of three independent experiments are shown. The p-values were calculated by two-tailed unpaired t-test. C. A panel of cancer cell lines were treated with different concentrations of IBC or BKC for 72 hours. Cell viability was measured and IC50 for each cell line was calculated from 3 independent experiments. Mean +/- SD is shown. D. Viability matrix for the concentrations of IBC and BKC combinations tested in a panel of cancer cell lines. Percentage of cell viability is indicated by the blue gradient. A representative analysis of three independent experiments is shown.

BKC inhibits DNA replication and induces S-phase accumulation in the cell cycle.

A. BJ and MCF-7 cells were treated for 24 hours with IBC, BKC or the combination IBC + BKC for 24 hours, then labelled with 10 μM EdU for 30 minutes. Cell cycle distribution was analyzed by flow cytometry. Data are presented as mean +/- SD from three independent experiments. B. Densitometric quantification of the pCHK1-S345 signal for Fig. 2C. C. Xenopus High Speed (HSS) egg extracts were treated with DMSO, Bakuchiol (BKC, 100 μM, 25 μg/ml) or aphidicolin (Aph, 60 μM) before the addition of ssDNA. Samples were collected at 30, 60 or 90 minutes after the addition of sperm DNA. The percentage of replicated DNA was calculated as described in materials and methods. Representative figure from two independent experiments is shown.

BKC does not interact with PCNA.

A-C. Xenopus egg extracts were incubated with DMSO or 40 μM BKC for 2 hours prior to the cellular thermal sensitivity shift assay (CETSA) at indicated temperature, as described in the Materials and Methods. The residual amount of the DNA Polδ catalytic subunit POLD1 (p125, A) and the accessory subunit POLD3 (p66, B) or PCNA (C) in the supernatant was detected by western blotting. Mean of two independent experiments is shown.

IBC does not inhibit AKT activity in MCF-7 cells.

A. MCF-7 cells treated with DMSO, 15 μM IBC, 20 μM BKC, the combination IBC + BKC or AKT inhibitor, MK-2206, (AKTi, 10 μM) for 24 hours. Auto-phosphorylation of AKT was detected by immunoblotting analysis. Densitometric quantification of phosphor-AKT signal is shown. (n=2) B. MCF-7 cells treated overnight with DMSO, IBC or AKT inhibitor (AKTi). Total RNA was isolated. Reverse transcription and quantitative PCR was performed with specific primers targeting the gene bodies of PCNA, E2F1 and E2F2. (n=2) C. MCF-7 cells were treated with IBC or AKT inhibitor (AKTi) for 24h. They were then sequentially labelled with IdU and CldU for 20 minutes. Replication fork progression was measured using DNA fiber spreading. The median length of CldU tracks of two biological replicates is indicated in red. D. In vitro kinase assay of 43 cell cycle-related kinases following treatment with 30 µM IBC. The CHK2(I157T) mutation is linked to an increased risk of breast and colorectal cancers. CHK2(R145W) is associated with Li-Fraumeni Syndrome. Both mutations do not affect the basal kinase activity of CHK2. E. Percentage inhibition of kinase activity by IBC treatment is shown. F. AKT kinase peptides were incubated with a half-log range dilution series of Isobavachalcone and in-vitro kinase activity was measured using a radiometric assay. Data are presented as mean +/- SD with a technical triplicate. G. Densitometric quantification of the pCHK2-S516 induction by CPT for Fig. 4B. The relative induction of pCHK2-S516 by CPT after IBC or BML-277 treatment compared to DMSO control is indicated. Mean +/- SD of 3 independent experiments for DMSO+CPT and IBC+CPT are shown. The p-value was determined using two-tailed unpaired t-test. H. Densitometric quantification of the pCHK2-S516 induction by CPT for Fig. 4C. The relative induction of pBRCA1-S988 by CPT after IBC or BML-277 treatment compared to DMSO control is indicated. Mean +/- SD of 3 independent experiments for DMSO+CPT and IBC+CPT are shown. The p-value was determined using two-tailed unpaired t-test. I. Densitometric quantification of the pCHK1-S296 induction by HU for Fig. 3D. The relative induction of pCHK1-S296 by HU after IBC or BML-277 treatment compared to DMSO control is indicated (n=2).

IBC results in the persistence of DNA breaks and impairs DNA end resection.

A. MCF-7 cells were pre-treated with DMSO or IBC for 1 hour, followed by incubation with or without camptothecin (CPT, 1 μM) for another 2 hours. Cells were recovered immediately or washed and let recover in the presence of DMSO or IBC for 24 or 48 hours. The amount of broken DNA was detected by PFGE. Fold increase of broken DNA was normalized by the control sample at t0. The p-values are indicated (two-tailed paired t-test). Representative gel image from 4 independent experiments is shown. B. MCF-7 cells were pre-treated with DMSO or IBC for 1 hours, followed by incubation with or without camptothecin (CPT) for another 2 hours. Cells were fixed and the formation of RPA foci was detected by CSK-immunofluorescence microscopy. Mean fluorescence intensity (MFI) of RPA signal was quantified by CellProfiler. **** p<0.0001, Mann-Whitney rank sum test. Representative data from 3 independent experiments are shown. Bar: 5 μm. C. DIvA cells were treated with either DMSO, IBC or CHK2 inhibitor (BML-277) for 2 hours before induction of DNA breaks by 4-hydroxytamoxifen (4-OHT) for 4 hours. Resection at two break sites is evaluated. Percentage of ssDNA was calculated as indicated in the Material and Methods. D. MCF-7 cells were pre-treated with DMSO, IBC for 2 hours, followed by bleomycin treatment for 1 hour. Cells were allowed to recover for 1 hour and the formation of RAD51 foci was detected by CSK-immunofluorescence microscopy. RAD51 foci number was quantified using CellProfiler. **** p<0.0001, Mann-Whitney rank sum test. A representative example of three independent experiments is shown (n=3). E. MCF-7 cells were pre-treated with DMSO, IBC for 2 hours, followed by bleomycin treatment for one hour. The amount of broken DNA was detected by PFGE. Fold increase of broken DNA was normalized by the control sample.

IBC and BKC extend survival and induce replication stress and DNA damage in a xenograft mouse model.

A. Luminometry images of MCF-7/Luc xenograft mice treated with PBS, Taxol, Isobavachalcone (IBC), Bakuchiol (BKC) or the combination IBC + BKC during treatments. B. Immunohistochemistry images of Ki67, TUNEL staining, pCHK1 (S345) and pCHK2 (Thr68) in xenografted tumor tissues.

IBC potentiates the anti-cancer of chemotherapeutic agents in DLBCL.

U2932 cells were treated with indicated concentrations of etoposide, doxorubicin, or 4-OH-cyclophosphamide in combination with indicated concentrations of IBC for 72 hours. Cell viability was measured. Percentage of cell viability is indicated by the blue gradient in the matrix.