Single-strand DNA Damage Research Articles

Rapid Quantification of Oxidation and UV Induced DNA Damage by Repair Assisted Damage Detection-(Rapid RADD).

Knowing the amount and type of DNA damage is of great significance for a broad range of clinical and research applications. However, existing methods are either lacking in their ability to distinguish between types of DNA damage or limited in their sensitivity and reproducibility. The method described herein enables rapid and robust quantification of type-specific single-strand DNA damage. The method is based on repair-assisted damage detection (RADD) by which fluorescent nucleotides are incorporated into DNA damage sites using type-specific repair enzymes. Up to 90 DNA samples are then deposited on a multiwell glass slide, and analyzed by a conventional slide scanner for quantification of DNA damage levels. Accurate and sensitive measurements of oxidative or UV-induced DNA damage levels and repair kinetics are presented for both in vitro and in vivo models.

Induction of Oxidative DNA Damage and Epithelial Mesenchymal Transitions in Small Airway Epithelial Cells Exposed to Cosmetic Aerosols.

Engineered metal nanoparticles (ENPs) are frequently incorporated into aerosolized consumer products, known as nano-enabled products (NEPs). Concern for consumer pulmonary exposures grows as NEPs produce high concentrations of chemically modified ENPs. A significant knowledge gap still exists surrounding NEP aerosol respiratory effects as previous research focuses on pristine/unmodified ENPs. Our research evaluated metal-containing aerosols emitted from nano-enabled cosmetics and their induction of oxidative stress and DNA damage, which may contribute to epithelial mesenchymal transitions (EMT) within primary human small airway epithelial cells. We utilized an automated NEP generation system to monitor and gravimetrically collect aerosols from two aerosolized cosmetic lines. Aerosol monitoring data were inputted into modeling software to determine potential inhaled dose and in vitro concentrations. Toxicological profiles of aerosols and comparable pristine ENPs (TiO2 and Fe2O3) were used to assess reactive oxygen species and oxidative stress by fluorescent-based assays. Single-stranded DNA (ssDNA) damage and 8-oxoguanine were detected using the CometChip assay after 24-h exposure. Western blots were conducted after 21-day exposure to evaluate modulation of EMT markers. Results indicated aerosols possessed primarily ultrafine particles largely depositing in tracheobronchial lung regions. Significant increases in oxidative stress, ssDNA damage, and 8-oxoguanine were detected post-exposure to aerosols versus pristine ENPs. Western blots revealed statistically significant decreases in E-cadherin and increases in vimentin, fascin, and CD44 for two aerosols, indicating EMT. This work suggests certain prolonged NEP inhalation exposures cause oxidative DNA damage, which may play a role in cellular changes associated with reduced respiratory function and should be of concern.

Microsphere: a promising approach for drug delivery

This work was carried out to show the effects of phenylhydrazine (PHZ) induced anaemic condition. Anaemic condition is defined as reduction in red blood cells (RBC) than normal number of red blood cells. The anti-anaemic activity can be studied using the changes in haematological parameters (PCV, RBC & Haemoglobin) influenced by PHZ [(40mg/kg p.o.)] in rats. PHZ, a potent chemical that causes different effects on different tissues at several levels. Administration of PHZ causes haemolytic anaemia, genotoxic effects and rose in iron absorption in spleen, liver and duodenum & causes change in iron metabolism. PHZ acts by activating immune response which triggers phagocytosis and also interfere with the binding of erythropoietin (EPO) receptors and further JAK-STAT pathway. PHZ also causes genotoxic effect by forming single strand DNA damage. In view of lipid peroxidation along with the formation of Thiobarbituric acid (TBA)-reactive malonyldialdehyde, it is recommend that PHZ induces anaemia as an outcome of peroxidation of RBC membrane lipids and this effect may be a upshot of the autoxidation of the drug and the interaction of membrane lipids and oxygen radicals

Abstract B103: Torin2 and related analogs exploit replicative and checkpoint vulnerabilities to induce death of triple-negative breast cancer cells

Abstract Frequent mutation of genes in the PI3K/AKT/mTOR signaling pathway in human cancers has stimulated large investments in over 40 small molecule therapeutic drugs but most have low efficacy in patients. As a result, cancers with high PI3K pathway activity such as triple-negative breast cancer (TNBC) are still treated primarily with conventional chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors with varying degrees of polyselectivity, we find that the preclinical compound Torin2 and related analogs are unusually effective because they inhibit both mTORC1/2 and structurally related PI3K-like kinases (PIKKs). The activity of Torin2 in TNBC cells is most closely mimicked by combining selective inhibitors of mTORC1/2 and Chk1, a kinase that acts downstream of the PIKKs ATR and DNA-PK in the signaling response to replication stress. Torin2 is more cytotoxic in TNBC/basal-like cell lines (N=19) than clinical grade mTORC1/2 inhibitors such as sapanisertib, but produces no cytotoxicity in non-transformed cells with basal-like gene expression (N=2). The high activity of Torin2 in TNBC is associated with greater activation of caspase-dependent apoptosis and stronger inhibition of cell proliferation than conventional PI3K/AKT/mTOR inhibitors, which do not inhibit PIKKs. Pulse-labeling studies with thymidine analogs and live-cell imaging of cells expressing fluorescent cell cycle reporters show that unlike other PI3K pathway drugs, Torin2 blocks progression of S phase, thereby causing accumulation of single-stranded DNA, DNA damage and death by replication catastrophe or mitotic failure. These phenotypes appear to result from co-targeting distinct pathways required during S phase in TNBC cells: inhibiting mTOR perturbs de novo biosynthesis and salvage of pyrimidines and purines, and inhibiting PIKKs causes high levels of replication stress and DNA damage. Moreover, computational models developed to better understand the complex cell cycle mechanism of action of Torin2 demonstrate the importance of co-targeting S-phase vulnerabilities for cytotoxicity. Thus, Torin2 and related analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC and possibly to other cancer cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors. Citation Format: Sameer S. Chopra, Annie Jenney, Adam Palmer, Mario Niepel, Mirra Chung, Caitlin Mills, Sindhu Carmen Sivakumaren, Qingsong Liu, Jia-Yun Chen, Clarence Yapp, Nathanael S. Gray, Peter K. Sorger. Torin2 and related analogs exploit replicative and checkpoint vulnerabilities to induce death of triple-negative breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B103. doi:10.1158/1535-7163.TARG-19-B103

Oxidative stress-mediated photoactivation of carbazole inhibits human skin cell physiology.

Prolonged exposure of the earth's surface to the sun's ultraviolet radiation may result in various skin diseases and cataract. Carbazole (CBZ), as a polycyclic-aromatic hydrocarbon (PAH), is blended with a five-member nitrogen-containing ring. It is found in cigarette smoke, coal, eye kohl, tattoo ink, and wood combustion and affects various types of flora and fauna. Our findings suggest that CBZ generates reactive oxygen species (ROS) like through type-I photodynamic reaction and causes phototoxicity in the human keratinocyte cell line (HaCaT), which has been proved by mitochondrial dehydrogenase and neutral red uptake assays. CBZ induces single strand DNA damage. We have investigated the involvement of the apoptotic pattern of cell death and confirmed it by cytochrome C release from mitochondria and caspase-9 activation. Similarly, photo-micronuclei formation was associated to CBZ-induced phototoxicity. The results of this study strongly support that the upregulation of bax, cyto-C, apaf-1, casp-9 and down regulation of bcl2, keap-1, nrf-2, and hmox-1 genes cause apoptopic cell death. Downregulation of antioxidant genes showed a significant amount of ROS generation by photosensitized CBZ. Therefore, the current study will be a step forward to safeguard human beings from sunlight-induced photosensitive CBZ prolonged exposure.

Glycogen Synthase Kinase-3 Inhibition Sensitizes Pancreatic Cancer Cells to Chemotherapy by Abrogating the TopBP1/ATR-Mediated DNA Damage Response.

Pancreatic ductal adenocarcinoma (PDAC) is a predominantly fatal common malignancy with inadequate treatment options. Glycogen synthase kinase 3β (GSK-3β) is an emerging target in human malignancies including PDAC.Experimental Design: Pancreatic cancer cell lines and patient-derived xenografts were treated with a novel GSK-3 inhibitor 9-ING-41 alone or in combination with chemotherapy. Activation of the DNA damage response pathway and S-phase arrest induced by gemcitabine were assessed in pancreatic tumor cells with pharmacologic inhibition or siRNA depletion of GSK-3 kinases by immunoblotting, flow cytometry, and immunofluorescence. 9-ING-41 treatment significantly increased pancreatic tumor cell killing when combined with chemotherapy. Inhibition of GSK-3 by 9-ING-41 prevented gemcitabine-induced S-phase arrest suggesting an impact on the ATR-mediated DNA damage response. Both 9-ING-41 and siRNA depletion of GSK-3 kinases impaired the activation of ATR leading to the phosphorylation and activation of Chk1. Mechanistically, depletion or knockdown of GSK-3 kinases resulted in the degradation of the ATR-interacting protein TopBP1, thus limiting the activation of ATR in response to single-strand DNA damage. These data identify a previously unknown role for GSK-3 kinases in the regulation of the TopBP1/ATR/Chk1 DNA damage response pathway. The data also support the inclusion of patients with PDAC in clinical studies of 9-ING-41 alone and in combination with gemcitabine.

Identification and Characterization of a New Class of (6–4) Photolyase from Vibrio cholerae

Light is crucial for many biological activities of most organisms, including vision, resetting of circadian rhythm, photosynthesis, and DNA repair. The cryptochrome/photolyase family (CPF) represents an ancient group of UV-A/blue light sensitive proteins that perform different functions such as DNA repair, circadian photoreception, and transcriptional regulation. The CPF is widely distributed throughout all organisms, including marine prokaryotes. The bacterium Vibrio cholerae was previously shown to have a CPD photolyase that repairs UV-induced thymine dimers and two CRY-DASHs that repair UV-induced single-stranded DNA damage. Here, we characterize a hypothetical gene Vca0809 encoding a new member of CPF in this organism. The spectroscopic analysis of the purified protein indicated that this enzyme possessed a catalytic cofactor, FAD, and photoantenna chromophore 6,7-dimethyl 8-ribityl-lumazin. With a slot blot-based DNA repair assay, we showed that it possessed (6-4) photolyase activity. Further phylogenetic and computational analyses enabled us to classify this gene as a member of the family of iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP). Therefore, we named this gene Vc(6-4) FeS-BCP.

Latest development of PARP inhibitors in ovarian cancer

Abstract Ovarian cancer is a highly lethal gynecologic malignancy. Approximately 10000 new cases of ovarian cancers are diagnosed annually in Japan and 5,000 patients die per year. Almost half of patients are diagnosed as stage III or IV at the time of initial diagnosis, and despite the first-line aggressive treatment with surgery and platinum-based combination chemotherapy, the 5-year survival rate of stage III and IV is less than 25%. The improvement of new treatment for advanced ovarian cancer is of paramount importance. BRCA 1/2 gene is engaged in homologous recombination (HR), which is a major double-stranded DNA repair pathway. About 20% of high-grade serous ovarian carcinoma (HGSOC) harbor germline BRCA1/2 mutations, and about 50% of high-grade serous adenocarcinoma have somatic or germline mutations or epigenetic change in genes of HR DNA repair such as RAD51C, ATM, ATR, and FANCD2. Olaparib is an oral poly (ADP-ribose) polymerase inhibitor (PARPi) that inhibits single-stranded DNA damage repair. Olaparib has a significant clinical activity in BRCA1/2 mutation-associated breast and ovarian cancer, and also has activity in ovarian cancer. In the Study 19, maintenance therapy with olaparib significantly prolonged progression-free survival (PFS) versus placebo in patients with platinum-sensitive recurrent serous ovarian cancer. Moreover, subsequent phase 3 clinical trials show major improvement of prognosis in maintenance therapy by incorporating olaparib in a recurrent setting and recently in a first-line setting among patients with BRCA1/BRCA2 mutations. Furthermore, various combination treatment strategies such as other DNA damage response inhibitors such as ATR inhibitors, immune checkpoint inhibitors and molecular-targeted therapies such as VEGF inhibitors, has been examined to enhance the efficacy of PARP inhibitors. In this symposium, we discuss the current status and future perspectives of PARP inhibitors for ovarian cancer.

Withdrawn: In vitro single-strand DNA damage and cancer cell cytotoxicity of temozolomide.

Temozolomide (TMZ) has been approved against certain tumors, but substantial evidence is lacking to demonstrate TMZ as a generator of single-strand break in vitro DNA and cell-based assay. Presently, we have attempted to elucidate detailed evidence that TMZ is able to generate extensive single-strand break on DNA substrates. The cell-based cytotoxicity and DNA content was evaluated for TMZ to corroborate its single-strand break activity. We employed plasmid and genomic DNA as a substrate during assessment of in vitro single-strand break potential of TMZ. Further, we tried to evaluate DNA fragmentation, DNA content, and overall cytotoxicity, and DNA ligase III protein level in HeLa and MCF-7 using MTT, Trypan blue exclusion, apoptotic DNA fragmentation, fluorescent microscopy, and immunoblot assay. Our observations indicate that TMZ is able to generate extensive single-strand breaks on different DNA substrate. For cytotoxicity assay, L189 augmented the effects of TMZ against HeLa cells from 36% to 50% cell death, while MCF-7 was resistant. However, we did not notice any apoptotic DNA fragments due to toxicity effects of TMZ in both cell types. Furthermore, we observed higher DNA ligase III among HeLa and MCF-7 in comparison with normal epithelial cells. We suggest differential response of HeLa and MCF-7 against the combinatorial effects of TMZ and L189, which may be due to their distinct single-strand break repair. The findings support combinatorial treatment using TMZ and repair protein inhibitors to enhance cytotoxicity.

DNA repair activation and apoptosis suppression in keratinocytes as a possible mechanism of cytoprotective action of plant polyphenols under conditions of UV-irradiation

The work investigated the responses of cultured human keratinocytes to ultraviolet radiation in the C range (UVC) with and without a number of plant polyphenolic compounds. The experimental data obtained in this work indicate a cytoprotective effect of the PPs added immediately after UVC exposure. The cytoprotective activity of plant polyphenolic compounds was reduced in the series: acacetin, silybin, baikalein, leontopodium acid, quercetin, cyanidine chloride, taxifolin, trans-ferulic acid. The effect of UVC irradiation with and without of acacetin on the process of phosphorylation of histones H2AX, the triggering mechanism for the repair of single-stranded DNA damage, was also investigated in keratinocytes. It has been established that, H2AX phosphorylation is activated in response to UVC radiation and acacetin has a significant effect on the kinetics of this process. It is concluded that the PPs can reduce the destructive effect of UVR on the skin cells, activating the process of repairing genetic damage.

Double-stranded sperm DNA damage is a cause of delay in embryo development and can impair implantation rates

To analyze the effect of single- and double-stranded sperm DNA fragmentation (ssSDF and dsSDF) on human embryo kinetics monitored under a time-lapse system. Observational, double blind, prospective cohort study. University spin-off and private center. One hundred ninety-six embryos from 43 infertile couples were included prospectively. None. SsSDF and dsSDF were analyzed in the same semen sample used for intracytoplasmic sperm injection. Embryo kinetics was then monitored using time-lapse technology, and the timing of each embryo division was obtained. When comparing embryos obtained from semen samples with low dsSDF and high dsSDF, splitting data using a statistically significant delay in high dsSDF was observed in second polar body extrusion, T4, T8, morula, and starting blastocyst and embryo implantation rates were impaired. Embryo kinetics and implantation rates are not significantly affected when high values of ssSDF are present. Different patterns of delay in embryo kinetics were observed for these different types of DNA damage: dsSDF caused a delay along all stages of embryo development; however, its major effect was observed at the second polar body extrusion and morula stages, coinciding with embryo DNA damage checkpoint activation as described before; ssSDF had its major effect at the pronucleus stage, but embryo kinetics was then restored at all following stages. The results show that dsSDF could be the main type of DNA damage that affects embryo development in intracytoplasmic sperm injection cycles, probably due to motility-based sperm selection in this assisted reproduction procedure. Double-stranded sperm DNA damage caused a delay in embryo development and impaired implantation, while single-stranded DNA damage did not significantly affect embryo kinetics and implantation.

Histone H4K20 methylation mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. Mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase. Upon mitotic exit, chromatin relaxation is controlled by SET8-dependent methylation of histone H4 on lysine 20. In the absence of either SET8 or H4K20 residue, substantial genome-wide chromatin decompaction occurs allowing excessive loading of the origin recognition complex (ORC) in the daughter cells. ORC overloading stimulates aberrant recruitment of the MCM2-7 complex that promotes single-stranded DNA formation and DNA damage. Restoring chromatin compaction restrains excess replication licensing and loss of genome integrity. Our findings identify a cell cycle-specific mechanism whereby fine-tuned chromatin relaxation suppresses excessive detrimental replication licensing and maintains genome integrity at the cellular transition from mitosis to G1 phase.

Rucaparib: a new treatment option for ovarian cancer

ABSTRACTIntroduction: Approximately 50% of high-grade serous ovarian cancers present a deficiency in the pathways involved in homologous recombination (HR). PARP inhibitors prevent single-strand DNA damage repair and determine a progression of the defect towards double-strand breaks, which results in a process known as ‘synthetic lethality’.Areas covered: In this review, the authors discuss the efficacy and toxicity of rucaparib either as a single agent or as a maintenance treatment for ovarian cancer. This includes the NGS Foundation Medicine evaluation of the role of this drug in the treatment algorithm of ovarian cancer. Moreover, perspectives on the future development of this drug are presented.Expert opinion: The FDA has approved this drug for the treatment of recurrent BRCA-mutated ovarian cancers, which were previously treated with at least two chemotherapies and has accepted the supplemental new drug application for maintenance use in patients who respond to platinum-based chemotherapy via the Prescription Drug User Fee Act (PDUFA) on 6 April 2018. European Medicines Agency (EMA) approval in the same setting is awaited. The possibility of using PARP inhibitors as a maintenance therapy, as a front-line therapy to combat recurrence, and in combination with anti-angiogenic agents and immune-therapies appears to be of particular interest.

Pathogenic role of DNA single-strand break accumulation and DNA damage response in pressure overload-induced heart failure

Pathogenic role of DNA single-strand break accumulation and DNA damage response in pressure overload-induced heart failure

SsDNA damage dependence from singlet oxygen concentration at photodynamic interaction

Single stranded DNA damage at photodynamic treatment with Radachlorin photosensitizer was investigated. Chemical trap method was used to evaluate generation of singlet oxygen in water solution. Interaction of singlet oxygen with ssDNA resulted into decrease of the replication activity of ssDNA. DNA stopped replicating during PCR at irradiation doses greater than 15 J/cm2 and concentration of photosensitizer [PS] = 3.8 μM. The dependence of replication activity of ssDNA on generated singlet oxygen concentration was identified.

Novel technology identifies significant rise in sublethal single strand DNA damage in oocytes following chemotherapy exposure which correlates with increased fetal malformations and pregnancy failure

Novel technology identifies significant rise in sublethal single strand DNA damage in oocytes following chemotherapy exposure which correlates with increased fetal malformations and pregnancy failure

Comparative performance of the BGISEQ-500 vs Illumina HiSeq2500 sequencing platforms for palaeogenomic sequencing.

Ancient DNA research has been revolutionized following development of next-generation sequencing platforms. Although a number of such platforms have been applied to ancient DNA samples, the Illumina series are the dominant choice today, mainly because of high production capacities and short read production. Recently a potentially attractive alternative platform for palaeogenomic data generation has been developed, the BGISEQ-500, whose sequence output are comparable with the Illumina series. In this study, we modified the standard BGISEQ-500 library preparation specifically for use on degraded DNA, then directly compared the sequencing performance and data quality of the BGISEQ-500 to the Illumina HiSeq2500 platform on DNA extracted from 8 historic and ancient dog and wolf samples. The data generated were largely comparable between sequencing platforms, with no statistically significant difference observed for parameters including level (P = 0.371) and average sequence length (P = 0718) of endogenous nuclear DNA, sequence GC content (P = 0.311), double-stranded DNA damage rate (v. 0.309), and sequence clonality (P = 0.093). Small significant differences were found in single-strand DNA damage rate (δS; slightly lower for the BGISEQ-500, P = 0.011) and the background rate of difference from the reference genome (θ; slightly higher for BGISEQ-500, P = 0.012). This may result from the differences in amplification cycles used to polymerase chain reaction–amplify the libraries. A significant difference was also observed in the mitochondrial DNA percentages recovered (P = 0.018), although we believe this is likely a stochastic effect relating to the extremely low levels of mitochondria that were sequenced from 3 of the samples with overall very low levels of endogenous DNA. Although we acknowledge that our analyses were limited to animal material, our observations suggest that the BGISEQ-500 holds the potential to represent a valid and potentially valuable alternative platform for palaeogenomic data generation that is worthy of future exploration by those interested in the sequencing and analysis of degraded DNA.

SSRP1 Cooperates with PARP and XRCC1 to Facilitate Single-Strand DNA Break Repair by Chromatin Priming

DNA single-strand breaks (SSB) are the most common form of DNA damage, requiring repair processes that to initiate must overcome chromatin barriers. The FACT complex comprised of the SSRP1 and SPT16 proteins is important for maintaining chromatin integrity, with SSRP1 acting as an histone H2A/H2B chaperone in chromatin disassembly during DNA transcription, replication, and repair. In this study, we show that SSRP1, but not SPT16, is critical for cell survival after ionizing radiation or methyl methanesulfonate-induced single-strand DNA damage. SSRP1 is recruited to SSB in a PARP-dependent manner and retained at DNA damage sites by N-terminal interactions with the DNA repair protein XRCC1. Mutational analyses showed how SSRP1 function is essential for chromatin decondensation and histone H2B exchange at sites of DNA strand breaks, which are both critical to prime chromatin for efficient SSB repair and cell survival. By establishing how SSRP1 facilitates SSB repair, our findings provide a mechanistic rationale to target SSRP1 as a general approach to selectively attack cancer cells. Cancer Res; 77(10); 2674-85. ©2017 AACR.

DNA single-strand break-induced DNA damage response causes heart failure

The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure.

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

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.