Dulaglutide reduces oxidative DNA damage and hypermethylation in the somatic cells of mice fed a high-energy diet by restoring redox balance, inflammatory responses, and DNA repair gene expressions.

Obesity is an established risk factor for numerous malignancies, although it remains uncertain whether the disease itself or weight-loss drugs are responsible for a greater predisposition to cancer. The objective of the current study was to determine the impact of dulaglutide on genetic and epigenetic DNA damage caused by obesity, which is a crucial factor in the development of cancer. Mice were administered a low-fat or high-fat diet for 12 weeks, followed by a 5-week treatment with dulaglutide. Following that, modifications of the DNA bases were examined using the comet assay. To clarify the underlying molecular mechanisms, oxidized and methylated DNA bases, changes in the redox status, levels of inflammatory cytokines, and the expression levels of some DNA repair genes were evaluated. Animals fed a high-fat diet exhibited increased body weights, elevated DNA damage, oxidation of DNA bases, and DNA hypermethylation. In addition, obese mice showed altered inflammatory responses, redox imbalances, and repair gene expressions. The findings demonstrated that dulaglutide does not exhibit genotoxicity in the investigated conditions. Following dulaglutide administration, animals fed a high-fat diet demonstrated low DNA damage, less oxidation and methylation of DNA bases, restored redox balance, and improved inflammatory responses. In addition, dulaglutide treatment restored the upregulated DNMT1, Ogg1, and p53 gene expression. Overall, dulaglutide effectively maintains DNA integrity in obese animals. It reduces oxidative DNA damage and hypermethylation by restoring redox balance, modulating inflammatory responses, and recovering altered gene expressions. These findings demonstrate dulaglutide's expediency in treating obesity and its associated complications.

Epigenetic regulation of DNA repair gene program by Hippo/YAP1-TET1 axis mediates sorafenib resistance in HCC.

Hepatocellular carcinoma (HCC) is a malignancy that occurs worldwide and is generally associated with poor prognosis. The development of resistance to targeted therapies such as sorafenib is a major challenge in clinical cancer treatment. In the present study, Ten-eleven translocation protein 1 (TET1) was found to be highly expressed in sorafenib-resistant HCC cells and knockdown of TET1 can substantially improve the therapeutic effect of sorafenib on HCC, indicating the potential important roles of TET1 in sorafenib resistance in HCC. Mechanistic studies determined that TET1 and Yes-associated protein 1 (YAP1) synergistically regulate the promoter methylation and gene expression of DNA repair-related genes in sorafenib-resistant HCC cells. RNA sequencing indicated the activation of DNA damage repair signaling was extensively suppressed by the TET1 inhibitor Bobcat339. We also identified TET1 as a direct transcriptional target of YAP1 by promoter analysis and chromatin-immunoprecipitation assays in sorafenib-resistant HCC cells. Furthermore, we showed that Bobcat339 can overcome sorafenib resistance and synergized with sorafenib to induce tumor eradication in HCC cells and mouse models. Finally, immunostaining showed a positive correlation between TET1 and YAP1 in clinical samples. Our findings have identified a previously unrecognized molecular pathway underlying HCC sorafenib resistance, thus revealing a promising strategy for cancer therapy.

Potential Benefits of Combining Proton or Carbon Ion Therapy with DNA Damage Repair Inhibitors.

The use of charged particle radiotherapy is currently increasing, but combination therapy with DNA repair inhibitors remains to be exploited in the clinic. The high-linear energy transfer (LET) radiation delivered by charged particles causes clustered DNA damage, which is particularly effective in destroying cancer cells. Whether the DNA damage response to this type of damage is different from that elicited in response to low-LET radiation, and if and how it can be targeted to increase treatment efficacy, is not fully understood. Although several preclinical studies have reported radiosensitizing effects when proton or carbon ion irradiation is combined with inhibitors of, e.g., PARP, ATR, ATM, or DNA-PKcs, further exploration is required to determine the most effective treatments. Here, we examine what is known about repair pathway choice in response to high- versus low-LET irradiation, and we discuss the effects of inhibitors of these pathways when combined with protons and carbon ions. Additionally, we explore the potential effects of DNA repair inhibitors on antitumor immune signaling upon proton and carbon ion irradiation. Due to the reduced effect on healthy tissue and better immune preservation, particle therapy may be particularly well suited for combination with DNA repair inhibitors.

Enhancing cisplatin drug sensitivity through PARP3 inhibition: The influence on PDGF and G-coupled signal pathways in cancer.

Drug resistance poses a significant challenge in cancer treatment despite the clinical efficacy of cisplatin. Identifying and targeting biomarkers open new ways to improve therapeutic outcomes. In this study, comprehensive bioinformatic analyses were employed, including a comparative analysis of multiple datasets, to evaluate overall survival and mutation hotspots in 27 base excision repair (BER) genes of more than 7,500 tumors across 23 cancer types. By using various parameters influencing patient survival, revealing that the overexpression of 15 distinct BER genes, particularly PARP3, NEIL3, and TDG, consistently correlated with poorer survival across multiple factors such as race, gender, and metastasis. Single nucleotide polymorphism (SNP) analyses within protein-coding regions highlighted the potential deleterious effects of mutations on protein structure and function. The investigation of mutation hotspots in BER proteins identified PARP3 due to its high mutation frequency. Moving from bioinformatics to wet lab experiments, cytotoxic experiments demonstrated that the absence of PARP3 by CRISPR/Cas9-mediated knockdown in MDA-MB-231 breast cancer cells increased drug activity towards cisplatin, carboplatin, and doxorubicin. Pathway analyses indicated the impact of PARP3 absence on the platelet-derived growth factor (PDGF) and G-coupled signal pathways on cisplatin exposure. PDGF, a critical regulator of various cellular functions, was downregulated in the absence of PARP3, suggesting a role in cancer progression. Moreover, the influence of PARP3 knockdown on G protein-coupled receptors (GPCRs) affects their function in the presence of cisplatin. In conclusion, the study demonstrated a synthetic lethal interaction between GPCRs, PDGF signaling pathways, and PARP3 gene silencing. PARP3 emerged as a promising target.

DNA-PKcs Inhibition Sensitizes Human Chondrosarcoma Cells to Carbon Ion Irradiation via Cell Cycle Arrest and Telomere Capping Disruption.

In order to overcome the resistance to radiotherapy in human chondrosarcoma cells, the prevention from efficient DNA repair with a combined treatment with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) inhibitor AZD7648 was explored for carbon ion (C-ion) as well as reference photon (X-ray) irradiation (IR) using gene expression analysis, flow cytometry, protein phosphorylation, and telomere length shortening. Proliferation markers and cell cycle distribution changed significantly after combined treatment, revealing a prominent G2/M arrest. The expression of the G2/M checkpoint genes cyclin B, CDK1, and WEE1 was significantly reduced by IR alone and the combined treatment. While IR alone showed no effects, additional AZD7648 treatment resulted in a dose-dependent reduction in AKT phosphorylation and an increase in Chk2 phosphorylation. Twenty-four hours after IR, the key genes of DNA repair mechanisms were reduced by the combined treatment, which led to impaired DNA repair and increased radiosensitivity. A time-dependent shortening of telomere length was observed in both cell lines after combined treatment with AZD7648 and 8 Gy X-ray/C-ion IR. Our data suggest that the inhibition of DNA-PKcs may increase sensitivity to X-rays and C-ion IR by impairing its functional role in DNA repair mechanisms and telomere end protection.

The type of DNA damage response after decitabine treatment depends on the level of DNMT activity.

Decitabine and azacytidine are considered as epigenetic drugs that induce DNA methyltransferase (DNMT)-DNA crosslinks, resulting in DNA hypomethylation and damage. Although they are already applied against myeloid cancers, important aspects of their mode of action remain unknown, highly limiting their clinical potential. Using a combinatorial approach, we reveal that the efficacy profile of both compounds primarily depends on the level of induced DNA damage. Under low DNMT activity, only decitabine has a substantial impact. Conversely, when DNMT activity is high, toxicity and cellular response to both compounds are dramatically increased, but do not primarily depend on DNA hypomethylation or RNA-associated processes. By investigating proteome dynamics on chromatin, we show that decitabine induces a strictly DNMT-dependent multifaceted DNA damage response based on chromatin recruitment, but not expression-level changes of repair-associated proteins. The choice of DNA repair pathway hereby depends on the severity of decitabine-induced DNA lesions. Although under moderate DNMT activity, mismatch (MMR), base excision (BER), and Fanconi anaemia-dependent DNA repair combined with homologous recombination are activated in response to decitabine, high DNMT activity and therefore immense replication stress induce activation of MMR and BER followed by non-homologous end joining.

ATM inhibition exploits checkpoint defects and ATM-dependent double strand break repair in TP53-mutant glioblastoma.

Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a method for simultaneously measuring non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). Using this method, we show that patient-derived glioblastoma (GBM) samples with acquired temozolomide (TMZ) resistance display elevated HR and MMEJ activity, suggesting that these pathways contribute to treatment resistance. We screen clinically relevant small molecules for DSBR inhibition with the aim of identifying improved GBM combination therapy regimens. We identify the ATM kinase inhibitor, AZD1390, as a potent dual HR/MMEJ inhibitor that suppresses radiation-induced phosphorylation of DSBR proteins, blocks DSB end resection, and enhances the cytotoxic effects of TMZ in treatment-naïve and treatment-resistant GBMs with TP53 mutation. We further show that a combination of G2/M checkpoint deficiency and reliance upon ATM-dependent DSBR renders TP53 mutant GBMs hypersensitive to TMZ/AZD1390 and radiation/AZD1390 combinations. This report identifies ATM-dependent HR and MMEJ as targetable resistance mechanisms in TP53-mutant GBM and establishes an approach for simultaneously measuring multiple DSBR pathways in treatment selection and oncology research.

Targeting mTOR and survivin concurrently potentiates radiation therapy in renal cell carcinoma by suppressing DNA damage repair and amplifying mitotic catastrophe.

BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Increasing DNA damage sensitivity through corylin-mediated inhibition of homologous recombination.

BACKGROUND: DNA repair allows the survival of cancer cells. Therefore, the development of DNA repair inhibitors is a critical need for sensitizing cancers to chemoradiation. Sae2CtIP has specific functions in initiating DNA end resection, as well as coordinating cell cycle checkpoints, and it also greatly interacts with the DDR at different levels. RESULTS: In this study, we demonstrated that corylin, a potential sensitizer, causes deficiencies in DNA repair and DNA damage checkpoints in yeast cells. More specifically, corylin increases DNA damage sensitivity through the Sae2-dependent pathway and impairs the activation of Mec1-Ddc2, Rad53-p and γ-H2A. In breast cancer cells, corylin increases apoptosis and reduces proliferation following Dox treatment by inhibiting CtIP. Xenograft assays showed that treatment with corylin combined with Dox significantly reduced tumor growth in vivo. CONCLUSIONS: Our findings herein delineate the mechanisms of action of corylin in regulating DNA repair and indicate that corylin has potential long-term clinical utility as a DDR inhibitor.

BET bromodomain inhibition potentiates radiosensitivity in models of H3K27-altered diffuse midline glioma.

Diffuse midline glioma (DMG) H3K27-altered is one of the most malignant childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation antitumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extraterminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-Seq and the CUT&RUN (cleavage under targets and release using nuclease) analysis showed that BET bromodomain inhibitors regulated the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as potential radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.

Thermal Proteome Profiling Reveals Insight to Antiproliferative and Pro-Apoptotic Effects of Lagunamide A in the Modulation of DNA Damage Repair.

Lagunamide A is a biologically active natural product with a yet unidentified molecular mode of action. Cellular studies revealed that lagunamide A is a potent inhibitor of cancer cell proliferation, promotes apoptosis and causes mitochondrial dysfunction. To decipher the cellular mechanism responsible for these effects, we utilized thermal protein profiling (TPP) and identified EYA3 as a stabilized protein in cells upon lagunamide A treatment. EYA3, involved in the DNA damage repair process, was functionally investigated via siRNA based knockdown studies and corresponding effects of lagunamide A on DNA repair were confirmed. Furthermore, we showed that lagunamide A sensitized tumor cells to treatment with the drug doxorubicin highlighting a putative therapeutic strategy.

Olaparib for childhood tumors harboring defects in DNA damage repair genes: arm H of the NCI-COG Pediatric MATCH trial.

BACKGROUND: The National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice (MATCH) precision oncology platform trial enrolled children aged 1-21 years with treatment-refractory solid tumors and predefined actionable genetic alterations. Patients with tumors harboring alterations in DNA damage repair (DDR) genes were assigned to receive olaparib. METHODS: Tumor and blood samples were submitted for centralized molecular testing. Tumor and germline sequencing were conducted in parallel. Olaparib was given twice daily for 28-day cycles starting at a dose 30% lower than the adult recommended phase 2 dose (RP2D). The primary endpoint was the objective response. RESULTS: Eighteen patients matched (1.5% of those screened) based on the presence of a deleterious gene alteration in BRCA1/2, RAD51C/D, or ATM detected by tumor sequencing without germline subtraction or analysis of loss of heterozygosity (LOH). Eleven (61%) harbored a germline mutation, with only one exhibiting LOH. Six patients enrolled and received the olaparib starting dose of 135 mg/m2/dose. Two participants were fully evaluable; 4 were inevaluable because <85% of the prescribed dose was administered during cycle 1. There were no dose-limiting toxicities or responses. Minimal hematologic toxicity was observed. CONCLUSION: Most DDR gene alterations detected in Pediatric MATCH were germline, monoallelic, and unlikely to confer homologous recombination deficiency predicting sensitivity to olaparib monotherapy. The study closed due to poor accrual. CLINICALTRIALS.GOV IDENTIFIER: NCT03233204. IRB approved: initial July 24, 2017.

The 5-WS of targeting DNA-damage repair (DDR) pathways in prostate cancer.

DNA-damage repair (DDR) pathways alterations, a growing area of interest in oncology, are detected in about 20% of patient with prostate cancer and are associated with improved sensitivity to poly(ADP ribose) polymerases (PARP) inhibitors. In May 2020, the Food and Drug Administration (FDA) approved two PARP inhibitors (olaparib and rucaparib) for prostate cancer treatment. Moreover, germline aberrations in DDR pathways genes have also been related to familial or hereditary prostate cancer, requiring tailored health-care programs. These emerging scenarios are rapidly changing diagnostic, prognostic and therapeutic approaches in prostate cancer management. The aim of this review is to highlight the five W-points of DDR pathways in prostate cancer: why targeting DDR pathways in prostate cancer; what we should test for genomic profiling in prostate cancer; "where" testing genetic assessment in prostate cancer (germline or somatic, solid or liquid biopsy); when genetic testing is appropriate in prostate cancer; who could get benefit from PARP inhibitors; how improve patients outcome with combinations strategies.

Combining inotuzumab ozogamicin with PARP inhibitors olaparib and talazoparib exerts synergistic cytotoxicity in acute lymphoblastic leukemia by inhibiting DNA strand break repair.

Inotuzumab ozogamicin (IO), a novel therapeutic drug for relapsed or refractory acute lymphoblastic leukemia (RR)­(ALL), is a humanized anti­cluster of differentiation (CD) 22 monoclonal antibody conjugated with calicheamicin that causes DNA single­ and double­strand breaks. Although the efficacy of IO is significantly improved compared with that of conventional chemotherapies, the prognosis for RR­ALL remains poor, highlighting the need for more effective treatment strategies. The present study examined the role of DNA damage repair inhibition using the poly (ADP­ribose) polymerase (PARP) inhibitors olaparib or talazoparib on the enhancement of the antitumor effects of IO on B­ALL cells in vitro. The Reh, Philadelphia (Ph)­B­ALL and the SUP­B15 Ph+ B­ALL cell lines were used for experiments. Both cell lines were ~90% CD22+. The half­maximal inhibitory concentration (IC50) values of IO were 5.3 and 49.7 ng/ml for Reh and SUP­B15 cells, respectively. The IC50 values of IO combined with minimally toxic concentrations of olaparib or talazoparib were 0.8 and 2.9 ng/ml for Reh cells, respectively, and 36.1 and 39.6 ng/ml for SUP­B15 cells, respectively. The combination index of IO with olaparib and talazoparib were 0.19 and 0.56 for Reh cells and 0.76 and 0.89 for SUP­B15 cells, demonstrating synergistic effects in all combinations. Moreover, the addition of minimally toxic concentrations of PARP inhibitors augmented IO­induced apoptosis. The alkaline comet assay, which quantitates the amount of DNA strand breaks, was used to investigate the degree to which DNA damage observed 1 h after IO administration was repaired 6 h later, reflecting successful repair of DNA strand breaks. However, DNA strand breaks persisted 6 h after IO administration combined with olaparib or talazoparib, suggesting inhibition of the repair processes by PARP inhibitors. Adding olaparib or talazoparib thus synergized the antitumor effects of IO by inhibiting DNA strand break repair via the inhibition of PARP.

Oncogenic functions and therapeutic potentials of targeted inhibition of SMARCAL1 in small cell lung cancer.

Small cell lung cancer (SCLC) is a recalcitrant cancer characterized by high frequency loss-of-function mutations in tumor suppressors with a lack of targeted therapy due to absence of high frequency gain-of-function abnormalities in oncogenes. SMARCAL1 is a member of the ATP-dependent chromatin remodeling protein SNF2 family that plays critical roles in DNA damage repair and genome stability maintenance. Here, we showed that SMARCAL1 was overexpressed in SCLC patient samples and was inversely associated with overall survival of the patients. SMARCAL1 was required for SCLC cell proliferation and genome integrity. Mass spectrometry revealed that PAR6B was a downstream SMARCAL1 signal molecule which rescued inhibitory effects caused by silencing of SMARCAL1. By screening of 36 FDA-approved clinically available agents related to DNA damage repair, we found that an aza-anthracenedione, pixantrone, was a potent SMARCAL1 inhibitor which suppressed the expression of SMARCAL1 and PAR6B at protein level. Pixantrone caused DNA damage and exhibited inhibitory effects on SCLC cells in vitro and in a patient-derived xenograft mouse model. These results indicated that SMARCAL1 functions as an oncogene in SCLC, and pixantrone as a SMARCAL1 inhibitor bears therapeutic potentials in this deadly disease.

Shaping DNA damage responses: Therapeutic potential of targeting telomeric proteins and DNA repair factors in cancer.

Shelterin proteins regulate genomic stability by preventing inappropriate DNA damage responses (DDRs) at telomeres. Unprotected telomeres lead to persistent DDR causing cell cycle inhibition, growth arrest, and apoptosis. Cancer cells rely on DDR to protect themselves from DNA lesions and exogenous DNA-damaging agents such as chemotherapy and radiotherapy. Therefore, targeting DDR machinery is a promising strategy to increase the sensitivity of cancer cells to existing cancer therapies. However, the success of these DDR inhibitors depends on other mutations, and over time, patients develop resistance to these therapies. This suggests the need for alternative approaches. One promising strategy is co-inhibiting shelterin proteins with DDR molecules, which would offset cellular fitness in DNA repair in a mutation-independent manner. This review highlights the associations and dependencies of the shelterin complex with the DDR proteins and discusses potential co-inhibition strategies that might improve the therapeutic potential of current inhibitors.

The novel SMYD3 inhibitor EM127 impairs DNA repair response to chemotherapy-induced DNA damage and reverses cancer chemoresistance.

BACKGROUND: SMYD3 has been found implicated in cancer progression. Its overexpression correlates with cancer growth and invasion, especially in gastrointestinal tumors. SMYD3 transactivates multiple oncogenic mechanisms, favoring cancer development. Moreover, it was recently shown that SMYD3 is required for DNA restoration by promoting homologous recombination (HR) repair. METHODS: In cellulo and in vivo models were employed to investigate the role of SMYD3 in cancer chemoresistance. Analyses of SMYD3-KO cells, drug-resistant cancer cell lines, patients' residual gastric or rectal tumors that were resected after neoadjuvant therapy and mice models were performed. In addition, the novel SMYD3 covalent inhibitor EM127 was used to evaluate the impact of manipulating SMYD3 activity on the sensitization of cancer cell lines, tumorspheres and cancer murine models to chemotherapeutics (CHTs). RESULTS: Here we report that SMYD3 mediates cancer cell sensitivity to CHTs. Indeed, cancer cells lacking SMYD3 functions showed increased responsiveness to CHTs, while restoring its expression promoted chemoresistance. Specifically, SMYD3 is essential for the repair of CHT-induced double-strand breaks as it methylates the upstream sensor ATM and allows HR cascade propagation through CHK2 and p53 phosphorylation, thereby promoting cancer cell survival. SMYD3 inhibition with the novel compound EM127 showed a synergistic effect with CHTs in colorectal, gastric, and breast cancer cells, tumorspheres, and preclinical colorectal cancer models. CONCLUSIONS: Overall, our results show that targeting SMYD3 may be an effective therapeutic strategy to overcome chemoresistance.

Glycyrrhizic acid inhibits DNA damage repair and enhances cisplatin-induced apoptosis of melanoma cells.

This research was designed to prospect the mechanism and impact of glycyrrhizic acid (GA) on DNA damage repair and cisplatin (CP)-induced apoptosis of melanoma cells. First, human melanoma cell SK-MEL-28 was stimulated using GA for 24, 48, and 72 h. Then, the optimal treatment time and dosage were selected. After that, cell counting kit-8 (CCK-8) was employed for testing the cell viability, flow cytometry for the apoptosis, comet assay for the DNA damage of cells, and western blot for the cleaved-Caspase3, Caspase3, Bcl-2, and γH2AX protein expression levels. The experimental outcomes exhibited that as the GA concentration climbed up, the SK-MEL-28 cell viability dropped largely, while the apoptosis level raised significantly, especially at the concentration of 100 µm. In addition, compared with GA or CPtreatment only, CP combined with GA notably suppressed the viability of melanoma cells and promoted cell apoptosis at the cytological level. At the protein level, the combined treatment notably downregulated the Bcl-2 and Caspase3 expression levels, while significantly upregulated the cleaved-Caspase3 and γH2AX expression levels. Besides, CP + GA treatment promoted DNA damage at the DNA molecular level. Collectively, both GA and CP can inhibit DNA damage repair and enhance the apoptosis of SK-MEL-28 cells, and the synergistic treatment of both exhibits better efficacy.

A unified mechanism for PARP inhibitor-induced PARP1 chromatin retention at DNA damage sites in living cells.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) not only suppress PARP1 catalytic activity but also prolong its association to damaged chromatin. Here, through live-cell imaging, we quantify the alterations in PARP1 dynamics and activity elicited by seven PARPis over a wide range of concentrations to deliver a unified mechanism of PARPi-induced PARP1 chromatin retention. We find that gross PARP1 retention at DNA damage sites is jointly governed by catalytic inhibition and allosteric trapping, albeit in a strictly independent manner-catalytic inhibition causes multiple unproductive binding-dissociation cycles of PARP1, while allosteric trapping prolongs the lesion-bound state of PARP1 to greatly increase overall retention. Importantly, stronger PARP1 retention produces greater temporal shifts in downstream DNA repair events and superior cytotoxicity, highlighting PARP1 retention, a complex but precisely quantifiable characteristic of PARPis, as a valuable biomarker for PARPi efficacy. Our approach can be promptly repurposed for interrogating the properties of DNA-repair-targeting compounds beyond PARPis.

Effect of oxidative stress induced by 2,3,7,8- tetrachlorodibenzo-p-dioxin on DNA damage.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic persistent organic pollutant (POP) that can induce DNA damage within cells. Although oxidative stress is one of the primary mechanisms causing DNA damage, its role in the process of TCDD-induced DNA damage remains unclear. In this study, the TCDD-induced production of reactive oxygen species (ROS) and the occurrence of DNA damage at the AP site were monitored simultaneously. Further investigation revealed that TCDD impaired the activities of superoxide dismutase (SOD) and catalase (CAT), compromising the cellular antioxidant defense system. Consequently, this led to an increase in the production of O2.- and NO, thus inducing DNA damage at the AP site under oxidative stress. Our findings were further substantiated by the upregulation of key genes in the base excision repair (BER) pathway and the absence of DNA AP site damage after inhibiting O2.- and NO. In addition, transcriptome sequencing revealed that TCDD induces DNA damage by upregulating genes associated with oxidative stress in the mitogen-activated protein kinase (MAPK), cyclic adenosine monophosphate (cAMP), and breast cancer pathways. This study provides important insights into the toxicity mechanisms of TCDD.