Honokiol Inhibits DNA Polymerases β and λ and Increases Bleomycin Sensitivity of Human Cancer Cells

Abstract

A major concept to sensitize cancer cells to DNA damaging agents is by inhibiting proteins in the DNA repair pathways. X-family DNA polymerases play critical roles in both base excision repair (BER) and nonhomologous end joining (NHEJ). In this study, we examined the effectiveness of honokiol to inhibit human DNA polymerase β (pol β), which is involved in BER, and DNA polymerase λ (pol λ), which is involved in NHEJ. Kinetic analysis with purified polymerases showed that honokiol inhibited DNA polymerase activity. The inhibition mode for the polymerases was a mixed-function noncompetitive inhibition with respect to the substrate, dCTP. The X-family polymerases, pol β and pol λ, were slightly more sensitive to inhibition by honokiol based on the Ki value of 4.0 μM for pol β, and 8.3 μM for pol λ, while the Ki values for pol η and Kf were 20 and 26 μM, respectively. Next we extended our studies to determine the effect of honokiol on the cytotoxicity of bleomycin and temozolomide in human cancer cell lines A549, MCF7, PANC-1, UACC903, and normal blood lymphocytes (GM12878). Bleomycin causes both single strand DNA damage that is repaired by BER and double strand breaks that are repaired by NHEJ, while temozolomide causes methylation damage repaired by BER and O6-alkylguanine-DNA alkyltransferase. The greatest effects were found with the honokiol and bleomycin combination in MCF7, PANC-1, and UACC903 cells, in which the EC50 values were decreased 10-fold. The temozolomide and honokiol combination was less effective; the EC50 values decreased three-fold due to the combination. It is hypothesized that the greater effect of honokiol on bleomycin is due to inhibition of the repair of the single strand and double strand damage. The synergistic activity shown by the combination of bleomycin and honokiol suggests that they can be used as combination therapy for treatment of cancer, which will decrease the therapeutic dosage and side effects of bleomycin.

Synergistic Effects of an Irreversible DNA Polymerase Inhibitor and DNA Damaging Agents on HeLa Cells

“…A structurally diverse group of Pol β inhibitors have been identified. , Some of these molecules target the polymerase active site, while others such as the molecule examined here inhibit the enzyme’s lyase activity. Comparison of inhibitors from independent studies is challenging due to different cell types, DNA damaging agents employed, and inhibition mechanism. ,, Nucleic acid lesions produced by highly cytotoxic DNA damaging agents that inactivate Pol β (and Pol λ) have provided the inspiration for the design of the small libraries of irreversible inhibitors used in our research. Inhibitors that target the lyase activity of Pol β are less common than those that affect the polymerase site .…”

“…6–12 There are an increasing number of examples of BER enzyme inhibitors that potentiate the effects of cytotoxic DNA damaging agents. 13–17 We have identified an irreversible inhibitor of DNA polymerase β (Pol β) that works synergistically with methyl methanesulfonate (MMS) to kill HeLa cells.…”

“…The structural modifications include single strand breaks, modified nucleotides, and abasic sites. , The base excision repair (BER) pathway mitigates the cytotoxicity of these agents by removing the damage and restoring the DNA to its original structure. , While BER is critical for maintaining the genome integrity of healthy cells, it is a hindrance for anticancer modalities that target DNA . Consequently, the enzymes involved in BER are inhibition targets. − There are an increasing number of examples of BER enzyme inhibitors that potentiate the effects of cytotoxic DNA damaging agents. − We have identified an irreversible inhibitor of DNA polymerase β (Pol β) that works synergistically with methyl methanesulfonate (MMS) to kill HeLa cells.…”

Natural Compounds That Target DNA Repair Pathways and Their Therapeutic Potential to Counteract Cancer Cells

“…Honokiol is a biphenolic compound with a powerful antineoplastic activity which is obtained from the Magnolia officinalis plant. It is more toxic in tumor cells than in normal cells and has been reported to inhibit the activity of the X family polymerases (b and l), affecting the base excision repair pathway and making various cancer cells more susceptible to the effect of bleomycin and temozolomide (94). Ellagic acid obtained from various fruits and vegetables is a polyphenolic compound that can reduce MGMT expression in glioblastoma cells and together with antiangiogenic therapy with bevacizumab (which also affects DNA repair by reducing the expression of ERCC-1 and XRCC-1) improves the radiosensitivity of tumors (95,96).…”

RanBP2-mediated SUMOylation promotes human DNA polymerase lambda nuclear localization and DNA repair

“…Interestingly, the suppression of Polλ activity induces synthetic lethality when combined with Chk1 inhibitors [49], what turns Polλ and its nucleocytoplasmic shuttling as additional potential chemotherapeutic target to be analyzed in more detail. Importantly, targeting the N-terminal region of Polλ would solve undesired effects of many of Polλ inhibitors identified so far, that can also affect other polymerases, in particular those from PolX family, due to high similarity in their catalytic center [50,51].…”