Impairing the division of cancer cells with genotoxic small molecules has been a primary goal to develop chemotherapeutic agents. However, DNA mismatch repair (MMR)-deficient cancer cells are resistant to most conventional chemotherapeutic agents. Here we have identified baicalein as a small molecule that selectively kills MutSα-deficient cancer cells. Baicalein binds preferentially to mismatched DNA and induces a DNA damage response in a MMR-dependent manner. In MutSα-proficient cells, baicalein binds to MutSα to dissociate CHK2 from MutSα leading to S-phase arrest and cell survival. In contrast, continued replication in the presence of baicalein in MutSα-deficient cells results in a high number of DNA double-strand breaks and ultimately leads to apoptosis. Consistently, baicalein specifically shrinks MutSα-deficient xenograft tumors and inhibits the growth of AOM-DSS–induced colon tumors in colon-specific MSH2 knockout mice. Collectively, baicalein offers the potential of an improved treatment option for patients with tumors with a DNA MMR deficiency. Cancer Res; 76(14); 4183–91. ©2016 AACR.
The DNA mismatch repair (MMR) pathway maintains genome stability by removing base–base mismatches and insertion/deletion loops that arise during DNA replication or as a result of DNA damage (1, 2). These DNA lesions are recognized by MutSα (a heterodimer of MSH2/MSH6) or MutSβ (a heterodimer of MSH2/MSH3), which recruit the MutLα (MLH1/PMS2) or MutLβ (MLH1/PMS1) proteins. The MutS/MutL complex then loads exonuclease I to degrade the strand containing the mispaired nucleotide, and the resulting gap is filled in by polymerase δ (3, 4). The MMR proteins can also affect cell-cycle control and apoptosis in response to certain types of DNA damage (4, 5). However, the precise mechanism by which MMR proteins regulate the cell cycle is not clearly understood.
Germline loss-of-function mutations in genes involved in the DNA MMR pathway cause the autosomal dominant Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC). Individuals with Lynch syndrome have an 80% risk of developing colorectal cancer in their lifetime, with the average age of onset being 45 years (6). Lynch syndrome accounts for approximately 2% of all colorectal cancers diagnosed each year (7), and patients with Lynch syndrome are at an increased risk of developing a range of other tumor types, including gastric, endometrial, ovarian, and bladder cancers (8–10). Although some standard chemotherapeutic regiments as well as immunotherapy PD-1 may be beneficial to Lynch syndrome patients (11–13), the underlying mechanisms of these treatment options for Lynch syndrome tumors are not clear. Some studies have also pointed out that MMR-deficient cells may be resistant to the standard chemotherapeutic regimens for colorectal cancer (14).
We have recently developed an ATAD5 (ATPase family AAA domain containing protein 5)-luciferase assay that allows high-throughput screening for potential chemotherapeutic compounds that act by inducing DNA replication stress or DNA damage. In this assay, the stabilization of the ATAD5-luciferase protein, which is measured by an increase in luciferase activity, serves as a biomarker of DNA replication stress or DNA damage (15). Using the ATAD5-luciferase assay, we screened 344,385 small molecules from the NIH Molecular Library Probe production Centers Network (MLPCN) collection, and selected 289 compounds that induced an increase in luciferase activity, indicating that they caused DNA damage. Hypothesizing that these compounds could provide a better treatment option for Lynch syndrome tumors, we tested the ability of the compounds to selectively kill cells with mutations in the MSH2 gene. From this screen, we identified baicalein as a small molecule capable of inducing apoptosis in MSH2-deficient cells both in vitro and in vivo. Baicalein is a flavone derived from the roots of Scutellaria baicalensis and Scutellaria lateriflora. Previously, baicalein was used in some Asian countries as an herbal supplement to enhance liver health. In the recent years, increasing studies of baicalein have proceeded on many human disease-related areas, such as cancer and diabetes (16–20). Although baicalein has previously been reported to have antitumor activities (18, 19, 21), this article identifies a novel mechanism of action for baicalein in which the compound acts on the MutSα/CHK2/ATM pathway as well as preferentially binds to the mismatched DNA.
Materials and Methods
Baicalein, netropsin, colochicine, CHK2 inhibitor hydrate (2-(4-(4-Chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide hydrate), azoxymethane (AOM), and dextran sodium sulfate (DSS; MW 40,000–50,000) were purchased from Sigma-Aldrich and USB Affymetrix, respectively.
Control and baicalein-supplemented diets
All diets were made by Harlan Laboratories using baicalein purchased from Sigma. The control mice were fed a global 18% protein rodent diet, and the experimental group was fed a global 18% protein rodent diet containing 0.05% baicalein.
HEC59, HEC59-2, LoVo [gift from Dr. Peggy Hsieh's (NIH) laboratory], HeLa, HEK293T [gift from Dr. Pamela Schwartzberg's (NIH) laboratory], and MRC5 [gift from Dr. Roger Woodgate's (NIH) laboratory] cells were cultured in DMEM + GlutaMAX (Invitrogen) supplemented with 10% FBS (Hyclone Laboratories) and 50 U/mL penicillin and 50 μg/mL streptomycin (Invitrogen). HEC59-2 cells were also maintained in 400 μg/mL G418 (Invitrogen). HT-29 cells (purchased from ATCC) were cultured in McCoy 5A medium (Invitrogen) supplemented with 10% FBS and 50 U/mL penicillin and 50 μg/mL streptomycin (Invitrogen). All cells were maintained at 37°C under a humidified atmosphere and 5% CO2. To authenticate cells used in the study, we frequently checked the expression of DNA repair proteins, especially MMR-related proteins, to make sure cells used in the study did (HEC59-2, HeLa, MRC5, HEK293T, HCT116-3) or did not (HEC59, HCT116, LoVo) express proteins having mutations. In addition, we checked the morphology of cell lines with microscopy to make sure they did not change their original shapes. We checked all cells for mycoplasma to make sure that there was no contamination. Finally, we checked chromosome numbers by karyotyping of cell lines used to make sure cells did not produce abnormal chromosome numbers. We did not use cell lines more than a month from original frozen stocks in culture to avoid potential accumulation of unnecessary mutations.
Cell viability assay
Cells were plated in white, solid-bottom 96-well plates at a final density of 15,000 cells/well. After the cells had attached to the plate, 1:2 dilutions of the indicated compounds were added. Twenty-four hours after treatment, cell viability was determined using Cell Titer-Glo (Promega) according to the manufacturer's protocol. Viability was quantified on a Fluoroskan Ascent Luminometer (Thermo Scientific). Dose–response curves and EC50 values were generated using GraphPad Prism.
Western blot and coimmunoprecipitation
Anti-PARP was purchased from BD Biosciences; anti-caspase 3 and anti-p-CHK2 (Thr68) were purchased from Cell Signaling Technology; anti-CHK2, anti-ATM, HRP-conjugated-anti-PCNA, and anti-MSH2 were purchased from Santa Cruz Biotechnology; anti-MSH2, anti-MSH6, and HRP-conjugated–anti-β-tubulin were purchased from Abcam; anti-histone H3 was purchased from Upstate; anti-phospho-histone H2AX was purchased from GeneTex; anti-CDC25A was purchased from Thermo Scientific; anti-SHPRH was purchased from Origene; and anti-digoxigenin was purchased from Roche.
For Western immunoblotting, proteins from cells or mice colon were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane filter using Bio-Rad mini, midi transfer packs and Transblot turbo. After incubation with the desired antibodies, the blots were developed with Pierce ECL plus Western blotting substrate. For coimmunoprecipitation, cells lysed with the lysis/wash buffer [0.025 mol/L Tris, 0.15 mol/L NaCl, 0.001 mol/L EDTA, 1% NP-40, 5% glycerol, pH 7.4 with Protease Inhibitor Cocktail (Roche)] for 0.5 hour at 4°C. After incubation with the desired primary antibody for 18 hours at 4°C via gentle rocking, immune complexes were captured by mixing with a final concentration of 2.5% protein G Sepharose beads (GE) for 2 hours at 4°C on a rotator. Beads were subsequently washed three times with the lysis/wash buffer, followed by SDS-PAGE and immunoblotting analysis after elution by boiling in 2× SDS-loading buffer.
Human wild-type MutSα were expressed in baculovirus and purified as described previously (22). SHPRH protein was expressed in baculovirus and purified (unpublished data).
Hematoxylin and eosin staining, TUNEL, and IHC
Hematoxylin and eosin (H&E) staining, TUNEL, and IHC were commercially performed by the Histoserv Company (tumors or tissues were collected, fixed in formalin, and then were picked up by Histoserv Company). Detailed procedures can be found on the company's website (http://www.histoservinc.com/). To quantify the images, the number of positive signals was manually counted in multiple fields of view. The number of total positive signals was then divided by the number of fields visualized for counting.
Baicalein selectively kills MutSα-deficient cells
The human endometrial adenocarcinoma cell line HEC59 carrying two different loss-of-function MSH2 nonsense mutations (23) and HEC59-2 cells, which are HEC59 cells into which human chromosome 2 was transferred, restoring wild-type MSH2 expression and functional MMR (24), were treated with serial dilutions of the 289 compounds, and dose–response viability curves were used to calculate the concentration of each compound required to induce 50% cell death (EC50). The screen revealed 9 compounds that were at least 2-fold more potent in the HEC59 cells than in the HEC59-2 cells (Fig. 1A). A secondary viability screen using a luciferase-based ATP assay and a colony formation assay showed that one of these compounds, baicalein (Fig. 1A black dot, B), generated the most reproducible results (Fig. 1C and Supplementary Fig. S1A). Baicalein treatment resulted in PARP1 and caspase-3 cleavages in HEC59 cells, but not in the MMR-proficient HEC59-2 cells (Fig. 1E), indicating that the loss of viability observed upon MSH2 deficiency occurs via apoptosis. We confirmed the specificity of baicalein's activity for MMR deficiency by reducing MSH2 protein levels by siRNA in HT29 (colorectal adenocarcinoma) cells and HeLa (cervical cancer) cells (Fig. 1D and Supplementary Fig. S1B–S1D). Furthermore, we measured the cell viability of LoVo cell, which is a colorectal carcinoma cell defective in MSH2. Because of the lack of LoVo WT cells, we used MMR-proficient MRC5 (human lung fibroblast) cells and HT29 cells for comparison. Consistent with other cell viability results, LoVo cells were more sensitive to baicalein than MRC5 cells and HT29 cells (Supplementary Fig. S1E). In addition, we tested baicalein in another pair of cell lines, HCT116, which harbors a homozygous mutation of the MLH1 gene, and HCT116-3 cell line, where the transfer of human chromosome 3 into HCT116 cells restores wild-type MLH1 expression (Supplementary Fig. S1F; ref. 25). Baicalein also exhibited selective sensitivity on HCT116 cells, which suggests a general activity of baicalein targeting MMR-deficient cells. In contrast to MMR deficiency, there was no selective killing of other DNA repair–deficient cells including PARP1, p53, RAD54b, FANCA, FANCG, FANCD2, ATM, ATR, NBS, Ku70/Rad54, FANCC, XPA, PolB, UBC13, FEN1, ATG5, Pol η/ζ, and Rev3 by baicalein (data not shown).
Baicalein-treated HEC59 cells are deficient in CHK2-regulated S-phase checkpoint arrest
Given the influence of MutSα on S-phase cell-cycle checkpoint activation (5), we tested the effect of baicalein on cell-cycle progression in cells synchronized with hydroxyurea. We found that baicalein-treated HEC59-2 cells remained largely in the S-phase of the cell cycle, whereas a large percentage of baicalein-treated HEC59 cells were able to progress to the G2–M phase (Fig. 2A). In addition, we monitored DNA replication using an EdU incorporation assay. Baicalein inhibited DNA replication in MutSα-proficient HEC59-2 cells, but not in the MutSα-deficient HEC59 cells (Supplementary Fig. S2A and S2B). We hypothesized that there would be MMR-dependent checkpoint activation in response to baicalein. Consistent with this hypothesis, we found an induction of CHK2 phosphorylation and subsequent CDC25A degradation predominately in baicalein-treated MutSα-proficient HEC59-2 cells, although baicalein treatment resulted in similar chromatin-bound monoubiquitylated PCNA regardless of MMR activity (Fig. 2B). Low levels of CHK2 phosphorylation were also observed in baicalein-treated HEC59 cells, most likely due to the MutSα-independent activity of ATM. Consistent with the activation of CHK2 by baicalein in a MutSα-dependent manner, the MutSα-proficient cells became sensitive to baicalein after treatment with a CHK2 inhibitor (Fig. 2C).
Baicalein binds to MutSα
It has been demonstrated that MutSα interacts with CHK2, and that it forms a complex with ATM (5, 26). We hypothesized that baicalein would affect the interaction between MutSα and CHK2/ATM that is required for cell-cycle arrest. In MutSα-proficient HEC59-2 cells, immunoprecipitation of MutSα with a MSH6 antibody also pulled down ATM and CHK2 (Fig. 3A). Similarly, immunoprecipitation of CHK2 coprecipitated MutSα and ATM (Fig. 3B). Baicalein treatment inhibited the interaction between MutSα and CHK2/ATM (Fig. 3A and B). The interactions were mediated by MutSα, because cells not expressing MSH2 (either by mutation or silencing by siRNA) had significantly reduced interactions between MutSα and CHK2/ATM (Fig. 3A and B and Supplementary Fig. S3A) and between ATM and CHK2 (Fig. 3B and Supplementary Fig. S3A). The MutSα/CHK2/ATM interactions were not mediated through DNA because the interactions between proteins remained intact even when cell extracts were pretreated with ethidium bromide (EB), which disrupts the interaction between proteins and DNA (Supplementary Fig. S3B). Thus, MutSα, acting as a docking platform for ATM and CHK2 is dissociated from ATM and CHK2 by baicalein, which in turn, activates the S-phase checkpoint through CHK2 phosphorylation by ATM. To study the dynamics of the interaction among these proteins, we used siRNA targeting CHK2 or ATM. The absence of CHK2 had little effect on the interaction between MutSα and ATM in untreated cells and in cells treated with baicalein, and absence of ATM also had little effect on the interaction between MutSα and CHK2 in untreated cells and in cells treated with baicalein (Supplementary Fig. S3C). In addition, we used a double-thymidine block to synchronize HeLa cells to compare the interaction pattern in different cell-cycle phases. Immunoprecipitation of MSH6 coprecipitated a similar amount of ATM or CHK2 in both G1–S and G2 phases, suggesting the interaction patterns are similar in G1–S and G2 (Supplementary Fig. S3D).
The baicalein-induced dissociation of MutSα from CHK2/ATM suggested that baicalein could directly bind to MutSα and inhibit its interaction with CHK2/ATM. To test this hypothesis, we used the drug affinity responsive target stability (DARTS) assay that detects the interaction between the target protein and a small molecule by measuring resistance to proteolysis (Supplementary Fig. S4A; refs. 27, 28). As a control, we used SHPRH, a nuclear protein with a molecular weight similar to MSH6. DARTS analysis revealed that baicalein protects MutSα, but not SHPRH, from degradation by pronase, suggesting that MutSα interacts with baicalein (Supplementary Fig. S4B and S4C). To confirm the direct interaction, biotin-tagged baicalein (baicalein-biotin; Supplementary Fig. S4D), which was shown to be as active in MMR-dependent cell viability assays as the untagged molecule (Supplementary Fig. S4E), was incubated with MutSα or SHPRH and pulled down with streptavidin beads. MutSα, but not SHPRH, was coprecipitated, suggesting that baicalein directly binds to MutSα (Fig. 3C). In addition, we used a Biacore assay to measure the interaction between baicalein and MutSα as well as baicalein and SHPRH, and calculate the Kd between baicalein and MutSα, which was about 32.2 μmol/L. In contrast, SHPRH did not show interaction with baicalein (Supplementary Fig. S4F and S4G). Collectively, these data suggest that baicalein directly binds to MutSα, which in turn dissociates CHK2/ATM from MutSα.
Baicalein preferentially binds to mismatched DNA
As CHK2 activation is often linked to DNA double-strand break (DSB) damage, we hypothesized that baicalein would generate DSBs, and that MutSα deficiency would further increase the level of DSBs due to the absence of proper checkpoint activation through CHK2. Consistent with the hypothesis, pulsed-field gel electrophoresis (PFGE) revealed that there were more DSBs produced in MutSα-mutant cells than wild-type cells after baicalein treatment (Fig. 4A and B). To determine whether baicalein intercalates in DNA, we conducted an assay that measures the decrease in fluorescence due to the displacement of DNA-bound EB by a DNA-binding compound (29). Similar to netropsin, which is known to bind to the minor groove of DNA, but in contrast to colchicine, which does not interact with DNA, the incubation of baicalein with EB-bound DNA resulted in a dose-dependent decrease in fluorescence (Supplementary Fig. S5A). However, baicalein does not form interstrand crosslinks as denatured baicalein-bound DNA only produced single-stranded DNA (Supplementary Fig. S5B). To determine whether baicalein has a preference for binding to matched or mismatched double-stranded DNA, we compared one dimensional proton NMR spectra of both free and baicalein-bound matched and mismatched DNA. Baicalein resulted in much higher chemical shift perturbation of mismatched DNA compared with the matched DNA (Fig. 4C). The resonances of the imino group generated by the mismatched bases disappeared upon baicalein binding, suggesting that baicalein binds to the mismatched bases. Consistently, baicalein–biotin preferentially pulled down mismatched DNA (Supplementary Fig. S5C). In addition, we used a Biacore assay to measure the interaction between baicalein and DNA, and calculate the Kd between baicalein and DNA, which was about 210 μmol/L for matched DNA and about 31.7 μmol/L for mismatched DNA (Supplementary Fig. S5D). To elucidate how baicalein interacts with both MutSα and mismatched DNA, mismatched DNA and baicalein–biotin were mixed together and baicalein was pulled down with streptavidin beads. The double bindings were examined separately (Supplementary Fig. S5E and S5F), and revealed that baicalein interacts with both mismatched DNA and MutSα. Interestingly, the binding of baicalein to mismatched DNA reduced the interaction of MutSα with mismatched DNA (Supplementary Fig. S5G). Consistently, the dose-dependent interaction between MutSα and mismatched DNA measured by the biacore assay (Supplementary Fig. S5H) was blocked when baicalein was preincubated with MutSα (Supplementary Fig. S5I). Such inhibition of the interaction between MutSα and mismatched DNA by baicalein was baicalein dose dependent (Supplementary Fig. S5J). Collectively, these data indicate that baicalein binds to mismatched DNA and MutSα, which releases MutSα from DNA and dissociates it from CHK2/ATM.
We hypothesized that the baicalein-induced DSBs would be generated by endonuclease(s). We silenced the expression of APE1, XPF, XPG, and MUS81 endonucleases with siRNA to determine their role in DSB formation upon baicalein treatment. Only XPF expression was required for baicalein-induced H2AX phosphorylation (γ-H2AX) in the MMR-deficient HEC59 cells (Fig. 5A). Moreover, baicalein-induced cell death in HEC59 cells was partially rescued by silencing XPF expression (Fig. 5B). To further confirm this observation, we transfected HT29 cells with negative control siRNA, siRNA targeting MSH2, and/or siRNA targeting XPF. Upon the silencing of XPF expression, baicalein-induced γ-H2AX was reduced in MSH2-deficient HT29 cells (Supplementary Fig. S6A). In addition, baicalein-induced cell death was rescued by silencing XPF expression in MSH2-deficient HT29 cells (Supplementary Fig. S6B).
Baicalein reduces the size of tumors formed by MutSα-deficient cells
MutSα-deficient cancer cells are not sensitive to radiation, alkylating agents, or most chemotherapeutic treatments (14). Thus, we investigated whether baicalein would be an improved chemotherapy option for MutSα-deficient tumors. We generated xenograft tumors with MutSα-deficient HEC59 cells and MMR-proficient HT29 cells in nude mice. When tumors grew to approximately 1 cm, matrix-driven delivery pellets were implanted (day 0) to deliver baicalein at a constant dose for two weeks (Fig. 6A and Supplementary Fig. S7A–S7C). During this process, the body weights of these mice were not significantly changed in either the placebo- or baicalein-treated groups (Supplementary Fig. S7D and S7E). Baicalein significantly shrank the MutSα-deficient xenografted HEC59 tumors (Fig. 6B and Supplementary Fig. S7F). Apoptotic cell death and DNA damage, as measured by the TUNEL assay and by γ-H2AX, respectively, were induced approximately 2-fold by baicalein in MutSα-deficient HEC59–xenografted tumors (Fig. 6D–F). However, CHK2 phosphorylation was not changed by baicalein (Fig. 6D and G). Baicalein had no significant effect on the growth of MutSα-expressing HT29-xenografted tumors (Fig. 6C and Supplementary Fig. S7G) and there was no induction of the TUNEL signal or γ-H2AX (Fig. 6H–J). Consistent with in vitro results, CHK2 phosphorylation was induced about 2-fold by baicalein in MutSα-expressing HT29–xenografted tumors (Fig. 6H and K). Thus, baicalein can selectively inhibit the growth of MutSα-deficient tumors in vivo.
To assess the effect of baicalein on tumor size over the course of time, we repeated the xenograft experiment, implanting the pellets for one week or two weeks before harvest. The tumor size before implantation was similar in all treatment groups as shown in Supplementary Fig. S8A and the body weights of the mice in both the placebo- and baicalein-treated groups remained relatively constant throughout the study (Supplementary Fig. S8B). Again, baicalein significantly shrank the MutSα-deficient xenografted HEC59 tumors in a time-dependent manner (Supplementary Fig. S8C and S8D).
To further confirm the ability of baicalein to shrink MSH2-deficient tumors in vivo, we used another MSH2-deficient colorectal carcinoma cell line, LoVo. The tumor size before implantation was similar in all treatment groups (Supplementary Fig. S9A) and the body weight of the mice did not change during the course of the study (Supplementary Fig. S9B). Consistent with what we observed with the HEC59 tumors, baicalein significantly shrank the MutSα-deficient LoVo tumors (Supplementary Fig. S9C and S9D).
Baicalein prevents the growth of AOM-DSS–induced colon tumors in Msh2LoxP/LoxPVilCre mice
Msh2LoxP/LoxPVilCre mice display a strong predisposition to intestinal cancers at 10 months of age (30) making them a good model to study the effects of baicalein treatment on the growth of Msh2-deficient tumors in the colon (Supplementary Fig. S10A and S10B). AOM and DSS can be used to accelerate and enhance the onset of colon tumors in Msh2 knockout mice (31). To study the effect of baicalein on the chronic-AOM-DSS–induced colon tumors, male and female Msh2LoxP/LoxPVilCre mice were fed a control or baicalein-supplemented diet for 4 weeks and then treated with AOM and DSS as shown in Supplementary Fig. S10C. Both male and female mice in the baicalein-supplemented diet group gained slightly more weight than mice in the control diet group (Supplementary Fig. S10D and S10E). In male mice, the number of tumors in the baicalein-supplemented diet group was significantly less than that in control diet group (Fig. 7A and C and Supplementary Fig. S10F). The female mice also showed a similar trend toward reduced tumor formation when fed baicalein-containing diet (Figs. 7B and C and Supplementary Fig. S10F). To study the role of baicalein on the acute-AOM-DSS–induced colon tumors, female Msh2LoxP/LoxPVilCre mice were fed a control or baicalein-supplemented diet for 4 weeks and then treated with AOM and DSS as shown in Supplementary Fig. S11A. The mice in the baicalein-supplemented diet group gained slightly more weight than mice in the control diet group (Supplementary Fig. S11B). The colon condition of both diet groups is shown in Supplementary Fig. S11C. The mice showed a trend toward reduced tumor formation when fed a baicalein-containing diet, compared with the control diet group (Supplementary Fig. S11D and S11E). Male Msh2LoxP/LoxPVilCre mice were fed a control or baicalein-supplemented diet for 4 weeks and then treated with AOM and DSS as shown in Supplementary Fig. S11F. The mice in the baicalein-supplemented diet group gained slightly more weight than mice in control diet group (Supplementary Fig. S11G). The colon condition of both diet groups is shown in Supplementary Fig. S11H. The number of tumors in the baicalein-supplemented diet group was significantly less than that in control diet group (Supplementary Fig. S11I and S11J).
To further evaluate the effect of baicalein on the AOM-DSS–induced colon tumors, WT male mice were fed a control or baicalein-supplemented diet for 4 weeks and then treated with AOM and DSS as shown in Supplementary Fig. S12A. The mice in baicalein-supplemented diet group gained slightly more weight than mice in control diet group (Supplementary Fig. S12B). The number of severe colon tumors in the WT mice was lower compared with Msh2LoxP/LoxPVilCre mice as indicated by the lack of tumors more than 2 mm in WT mice. In the WT mice, there was not much difference in the number of tumors between the two groups (Supplementary Fig. S12C–S12E).
In this article, we provide a molecular mechanism for the activation of the MutSα-dependent damage checkpoint that allows for cell survival in the presence of baicalein, as well as an alternative pathway explaining the baicalein-induced cell death observed in the absence of MutSα both in vitro and in vivo. Baicalein simultaneously binds directly to mismatched DNA and to MutSα. The interaction between baicalein and MutSα dissociates MutSα from CHK2, resulting in the activation of CHK2 by ATM. The activated checkpoint allows the DNA repair machinery to remove damage, resulting in cell survival. In the absence of MutSα, baicalein-bound mismatched DNA is converted to DSBs by XPF more frequently and without activation of CHK2, resulting in cell death (Supplementary Fig. S13). Although MutSα has previously been implicated in damage signaling following DNA methylation and exposure to ionizing radiation (5, 32), our data provide the first direct evidence that MutSα plays an important role in signaling DSBs to the checkpoint machinery.
Baicalein has been reported to have some anti-inflammatory (33) and antiproliferative (16) effects. Given these properties, it is no surprise that baicalein previously has been reported to kill several different cancer cell types, including colon cancer. Many mechanisms of action have been proposed to explain these effects, including reduction of reactive oxygen species (ROS), attenuation of NFκB signaling, suppression of COX-2 gene expression, upregulation of death receptor 5, and inhibition of cell-cycle checkpoints (18, 19, 21). Although the current study also provides strong evidence that the regulation of cell-cycle progression is involved in the baicalein-induced death of MutSα-deficient cells, our study differs from previous reports describing the antitumor effects of baicalein in that it implicates CHK2/ATM–mediated G2–M phase arrest. Additional studies in our laboratory have shown a decrease in ROS following treatment with the concentrations of baicalein used in this article (data not shown). However, further studies are needed to investigate the contribution of baicalein's antioxidant activity, as well as the potential contributions of NFκB, COX-2, and death receptor 5, in the baicalein-induced death of MutSα-deficient cells.
Although baicalein is not the only small molecule that has been reported to kill MutSα-deficient cancer cells in vitro, baicalein offers several advantages over other MSH2-mutant–sensitizing drugs such as methotrexate (34) and psoralen (35). A common critique of therapeutic strategies that target DNA repair deficiencies is that drugs that cause DNA damage often increase malignancy in the long term by inducing mutation rates. Indeed, methotrexate tested positive in many of the standard genotoxicity assays (Chemical Carcinogenesis Research Information System http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CCRIS), and the UV radiation required to activate psoralen is also a mutagen. Baicalein, on the other hand, does not increase mutagenesis in HEK293T due to the ability of the DNA repair polymerase Polη to bypass baicalein-induced lesions in an error-free manner (15). However, due to MutSα interaction of baicalein, it is possible that baicalein would affect MMR pathway for reducing mutagenesis. In addition, whereas the majority of chemotherapeutic agents have no clear targets, here we showed that baicalein targets both MutSα and mismatched DNA, providing the molecular mechanisms by which baicalein would target the MMR-deficient tumor population and serve as a personalized cancer therapy. Although the EC50 values of baicalein in MutSα-deficient cancer cells are within the range of those reported for other types of cancers (10–264 μmol/L; ref. 21), they far exceed the nanomolar values required for the successful use of baicalein as a human therapeutic. In addition, high Kd of baicalein for mismatched DNA also raises the possibility of side and off-target effects. Thus, future studies will involve collaborations with structural biologists and synthetic chemists to optimize the structure of this lead compound and improve its potency.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: Y. Zhang, J.T. Fox, M. Xia, K. Myung
Development of methodology: Y. Zhang, J.T. Fox, G. Elliott, G. Rai, S. Sakamuru, R. Huang, M. Xia, S.Y. Hong, K. Myung
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.T. Fox, Y.-U. Park, G. Elliott, G. Rai, M. Cai, S. Sakamuru, K. Lee, H.D. Park, W. Edelmann, S.Y. Hong
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Zhang, J.T. Fox, Y.-U. Park, G. Rai, M. Cai, R. Huang, K. Myung
Writing, review, and/or revision of the manuscript: Y. Zhang, J.T. Fox, G. Rai, R. Huang, M. Xia, W. Edelmann, K. Myung
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Huang, M.H. Jeon, B.P. Mathew, C.Y. Park, K. Myung
Study supervision: K. Myung
Other (synthesis of target molecule): M.H. Jeon
Other (chemical synthesis and scale up of target molecule): B.P. Mathew
Other (provided reagents that were used in the study): D. Maloney
The authors thank P. Hsieh for providing MutSα expression baculovirus constructs; S. Anderson (National Human Genome Research Institute; NHGRI) for flow cytometry; Dr. D. Bodine (NHGRI), P. Hsieh (National Institute of Diabetes and Digestive and Kidney Diseases; NIDDK), W. Yang (NIDDK), G. Li (University of Kentucky), and members in the Myung laboratory for helpful discussions and comments on the manuscript. K. Myung thanks E. Cho.
This research was supported in part by the intramural research program of the National Center for Advancing Translational Sciences (R. Huang, M. Xia, D. Maloney) and the National Human Genome Research Institute and NIH grant (MH092164-01 to K. Myung). This work was also supported by the Institute for Basic Science (IBS-R022-D1-2015 to K.Myung).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.