Induced DNA Damage Research Articles

Polystyrene nanoplastics promote colitis-associated cancer by disrupting lipid metabolism and inducing DNA damage.

Nanoplastics (NPs) have attracted widespread attention owing to their presence in the body. Recent studies highlighted the detrimental effects of NPs on the digestive tract. However, no studies have reported an association between NPs exposure and colitis-associated cancer (CAC). An azoxymethane/dextran sodium sulfate-induced CAC model was used, and polystyrene nanoparticles (PS-NPs) were selected for long-term exposure. Non-targeted metabolomics and 16S rRNA sequencing were used to detect changes in colonic metabolites and gut microbes following PS-NPs exposure. A lipopolysaccharide (LPS)-treated cancer cell model (Caco-2) exposed to PS-NPs was used to investigate the underlying molecular mechanism. Compared to the normal control group, mice in the PS-NPs group exhibited more tumor nodes and reactive oxygen species (ROS), higher expression of pan-CK and Ki-67, and more severe DNA damage. 16S rRNA sequencing revealed that exposure to PS-NPs altered the abundance of Allobaculum and Lactobacillus, whereas metabolic analysis showed that the most significant metabolites were enriched mostly in fatty acid metabolism. Experiments in LPS intervened Caco-2 cells showed that exposure to PS-NPs led to lipid peroxidation, oxidative stress, and DNA damage in Caco-2. Exposure to PS-NPs activated the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway both in the AOM/DSS mouse model and cellular model. Key proteins involved in fatty acid metabolism were downregulated in Caco-2 cells exposed to PS-NPs. The metabolic effects of cancer cells exposed to PS-NPs were significantly inhibited by the activation of the fatty acid metabolism pathway by fenofibrate. PS-NPs exposure disturbed lipid metabolism and induced DNA damage via the activation of PI3K/AKT/mTOR to promote CAC progression. Inhibition of fatty acid metabolism is a therapeutic target for controlling PS-NP-induced CAC. Our study provides an important reference for the prevention and treatment of CAC from the perspective of the environment and enhances awareness of the necessity of plastic control.

Sulforaphane Induces Transient Reactive Oxygen Species-Mediated DNA Damage in HeLa Cells.

Sulforaphane (SFN), an isothiocyanate found in plants of the Brassicaceae family, possesses antioxidant, apoptosis-inducing, and radiosensitizing effects. As one of the mechanisms of cytotoxicity by SFN, SFN has been suggested to be involved in the induction of DNA damage and inhibition of DNA repair. Recently, we reported on the potency of SFN in inducing single-ended double-strand breaks (DSBs) that are caused by the collision of replication forks with single-strand breaks (SSBs). However, the mechanism of SSB accumulation by SFN remains unclear. In this study, we examined the effect of SFN on SSB-inducing factors in HeLa cells. Although the inhibitory effect of SFN on DNA topoisomerase I was not observed, we found that the reduced form of glutathione (GSH; an antioxidant) level was decreased in an SFN concentration-dependent manner. Furthermore, the addition of ascorbic acid partially increased the viability of SFN-treated HeLa cells. We subsequently observed that poly(ADP-ribose) accumulated in SFN-treated HeLa cells, which occurs during early SSB repair. Collectively, these findings suggest that SFN may transiently induce SSBs via reactive oxygen species in HeLa cells.

KLF4 activates LATS2 to promote cisplatin sensitivity in ovarian cancer through DNA damage.

We aimed to investigate the role of large tumor suppressor kinase 2 (LATS2) in cisplatin (DDP) sensitivity in ovarian cancer. Bioinformatic analysis explored LATS2 expression, pathways, and regulators. Quantitative reverse transcription-PCR measured LATS2 and KLF4 mRNA levels. Dual-luciferase and chromatin immunoprecipitation assays confirmed their binding relationship. Cell viability, half maximal inhibitory concentration (IC 50 ) values, cell cycle, and DNA damage were assessed using CCK-8, flow cytometry, and comet assays. Western blot analyzed protein expression. The effect of LATS2 on the sensitivity of ovarian cancer to DDP was verified in vivo . LATS2 and KLF4 were downregulated in ovarian cancer, with LATS2 enriched in cell cycle, DNA replication, and mismatch repair pathways. KLF4, an upstream regulator of LATS2, bound to its promoter. Overexpressing LATS2 increased G1-phase cells, reduced cell viability and IC 50 values, and induced DNA damage. Silencing KLF4 alone showed the opposite effect on LATS2 overexpression. Knocking out LATS2 reversed the effects of KLF4 overexpression on cell viability, cell cycle, IC 50 values, and DNA damage in ovarian cancer cells. Inhibiting LATS2 inactivated the Hippo-YAP signaling pathway. In vivo experiments showed that overexpression of LATS2 enhanced the sensitivity of ovarian cancer to DDP. KLF4 activates LATS2 via DNA damage to enhance DDP sensitivity in ovarian cancer, providing a potential target for improving treatment outcomes.

Nano versus bulk: Evaluating the toxicity of lanthanum, yttrium, and cerium oxides on Enchytraeus crypticus.

Considering the increase in demand for rare earth elements (REEs) and their accumulation in soil ecosystems, it is crucial to understand their toxicity. However, the impact of lanthanum, yttrium and cerium oxides (La2O3, Y2O3 and CeO2, respectively) on soil organisms remains insufficiently studied. This study aims to unravel the effects of La2O3, Y2O3 and CeO2 nanoparticles (NPs) and their corresponding bulk forms (0, 156, 313, 625, 1250 and 2500 mg/kg) on the terrestrial species Enchytraeus crypticus. The effects on survival, reproduction (21 days (d)), avoidance behavior (2 d) and DNA integrity (2 and 7 d) of E. crypticus were evaluated. No significant effects on survival were observed. For La2O3, the bulk form affected more endpoints than the NPs, inducing avoidance behavior (1250 mg/kg) and DNA damage (1250 mg/kg - 2 d; 2500 mg/kg - 7 d). The Y2O3 NPs demonstrated higher toxicity than the bulk form: decreased reproduction (≥ 1250 mg/kg); induced avoidance behavior (≥ 625 mg/kg) and DNA damage (≥ 156 mg/kg - 2 d; 2500 mg/kg - 7 d). For CeO2, both forms exhibited similar toxicity, decreasing reproduction (625 mg/kg for bulk and 2500 mg/kg for NPs) and inducing DNA damage at all tested concentrations for both forms. REEs oxides toxicity was influenced by the REEs type and concentration, exposure time and material form, suggesting different modes of action. This study highlights the distinct responses of E. crypticus after exposure to REEs oxides and shows that REEs exposure may differently affect soil organisms, emphasizing the importance of risk assessment.

Unveiling the impact of polystyrene and low-density polyethylene microplastics on arsenic toxicity in earthworms.

The high global production combined with low recycling rates of polystyrene (PS) and low-density polyethylene (LDPE) contributes to the abundance of these commonly used plastics in soil, including as microplastics (MPs). However, the combined effects of MPs and heavy metals, such as arsenic (As) on earthworms are poorly understood. Here, we show that neither PS nor LDPE altered the effects of As on the survival, growth, and reproduction of the earthworm Eisenia fetida. As stress, both alone and in combination with the MPs, induced DNA damage in coelomocytes. In As-exposed earthworms, PS and LDPE increased the accumulation of reactive oxygen species while the activities of the antioxidant enzymes peroxidase, superoxide dismutase, and catalase were significantly lower under combined PS/LDPE+As exposure than under As exposure alone. As stress alone reduced cocoon production and the mRNA level of the reproduction-related gene ANN whereas As combined with PS/LDPE reduced the mRNA levels of CYP450, an enzyme involved in detoxification. Integrated biomarker response analysis revealed that PS/LDPE did not significantly impact the overall ecotoxicological effects of As exposure on earthworms. This study provides important insights into the potential ecological risks of MPs in heavy-metal-contaminated soil.

Comparative Investigation of In Vitro Induction of DNA Damage and Micronuclei by Pro-Mutagens in Human-Derived Hepatoma HepG2 Cells

It is important to know whether the DNA initial lesions are repaired or derive into chromosome aberration, gene mutation and/or cell death to discuss chemical carcinogenicity. To know whether DNA damage detected by the comet assay is repaired or fixed, the results of micronucleus (MN) test was compared to those of the comet assay in HepG2 cells. HepG2 cells were exposed to 38 pro-mutagens for 4 h followed by the cultivation for 24 h with cytochalasin B and the results were compared to those of the comet assay shown in our previous study where HepG2 cells were exposed to same 38 pro-mutagens for 4 h followed by immediate sampling. Among 38 pro-mutagens studied, 35 were positive in the comet assay and 22 were positive in the MN test. Since for 7 pro-mutagens, the lowest comet-positive concentration was higher than MN-positive concentration range, it is difficult to declare simply that the detecting ability of the comet assay is superior to that of the MN test. Comet assay-detected DNA damages that were induced by aromatic nitro compounds, azo dyes, heterocyclic amines, and aromatic hydrocarbons having bay structure, and aromatic amines with condensed multiple ring tended to result in cytotoxicity rather than MNs. It was considered that one important factor to decide the fate to produce MNs of comet-detected DNA damages might be whether detected DNA damages are bulky base adducts at non-cytotoxic concentration range and that chemical structures of pro-mutagens are important factor to decide whether induced base adducts are bulky.

Insecticide chlorfenapyr confers induced toxicity in human cells through mitochondria-dependent pathways of apoptosis.

Pesticides are always used in the environment, the unexpected effects of pesticides on the environment and non-target organisms need to be continuously studied. Insecticide chlorfenapyr (Chl) is widely used in agriculture and also recommended for public health use (e.g., providing protection from malaria). Here we study toxic effects of Chl on human alveolar carcinoma cells (A549) and human normal liver cells (L02) in vitro. Chl's ability to induce DNA damage and apoptosis in human cells was confirmed through alkaline comet assay, immunofluorescence assay, and flow cytometric analysis. Further research showed that Chl induced mitochondrial damage (the collapse of mitochondrial membrane potential and the opening of mitochondrial permeability transition pore) with up-regulated expression of Bax/Bcl-2 leads to the release of cytochrome c from mitochondria which in turn activated the apoptotic pathway. Meanwhile, the key protein PARP is cleaved during apoptosis, resulting in the inhibition of DNA damage repair. In short, human A549 and L02 cells exposed to Chl were experiencing DNA damage and apoptosis linked to mitochondria. The results of this study supply theoretical understanding of Chl's toxicity on human cells, and can attract attention on the potential threat of insecticide Chl to human health.

Improved bioavailability of polydatin and its protective effect against cisplatin induced nephrotoxicity through self-assembled fucoidan and carboxymethyl chitosan delivery system.

Cisplatin induced acute kidney injury (AKI) is clinically prevalent, with a complex pathogenesis and a lack of effective therapeutic drugs. Polydatin (Po) has excellent biological activity, but its low solubility and bioavailability limit its application. In this study, fucoidan (Fu) and carboxymethyl chitosan (Cs) self-assembled into nanoparticles through electrostatic interactions/hydrogen bonding and loaded Po (Fu/Cs Po NPs). In vitro studies found that Fu/Cs Po NPs protected human renal tubular epithelial (HK-2) cells from cisplatin induced damage and accumulation of reactive oxygen species (ROS). Mechanistic studies showed that Fu/Cs Po NPs inhibited cisplatin induced DNA damage and activation of cyclic guanosine monophosphate synthase (cGAS) and intron gene stimulator (STING) pathways. In vivo studies showed that Fu/Cs Po NPs treatment alleviated cisplatin induced AKI symptoms, including elevated blood urea nitrogen (BUN) and serum creatinine (SCr), as well as pathological damage to kidney tissues. In vivo mechanism studies also showed that Fu/Cs Po NPs treatment inhibited cisplatin induced DNA damage and activation of the cGAS-STING pathway. The pharmacokinetic and tissue distribution results demonstrated that the Fu/Cs delivery system enhanced the bioavailability and kidney accumulation of Po in vivo. In summary, our study provided potential drugs for the treatment of cisplatin induced AKI.

Polystyrene nanoparticles induce DNA damage and apoptosis in HeLa cells.

Nanoplastics (NPs) are plastic particles, typically less than 100nm in size, that result from daily life products as well as the degradation of larger plastic debris. Due to their small size and chemical composition, they can interact with biological systems in ways that larger plastic particles cannot. Humans are continuously exposed to NPs and several studies showed the potentially toxic effects of these latter on health. Polystyrene nanoplastics (PS-NPs) are the prevalent form of nanoparticles found in the environment and their cellular uptake can cause cytotoxicity and structural alteration of biomolecules. Thus, there is an urgent need for evaluation of the genotoxic effects of PS-NPs on human cell models. Through different and complementary experimental approaches, we investigated the potential genotoxic and cytotoxic effects of PS-NPs exposure on HeLa cell lines. We highlighted the genotoxic effects of polystyrene nanoplastics by showing the formation of multinuclei and micronuclei in all the studied concentrations and time points, also at short incubation time (6h) and low concentration. At higher concentrations, we demonstrate the presence of apoptotic and necrotic cells outlining the acute cytotoxic effects of nanoplastics. The genotoxic potential is further highlighted by the presence of low molecular weight DNA fragments in PS-NPs treated cells, and by the relationship between polystyrene nanoplastics and γ-H2AX. Thus, our data provide important insights at a cellular level into the possible risks produced by these nanoparticles and recommend further deeper research studies to address the impacts of nanoplastics on human health.

Influence of macrophages and neutrophilic granulocyte-like cells on crystalline silica-induced toxicity in human lung epithelial cells

Abstract In many industrial activities, workers may be exposed by inhalation to particles that are aerosolized, To predict the human health hazard of these materials, we propose to develop a co-culture model (macrophages, granulocytes, and alveolar epithelial cells) designed to be more representative of the inflammatory pulmonary response occurring in vivo. Phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells were used as macrophages, All-trans retinoic acid (ATRA)-differentiated HL60 were used as granulocytes and A549 were used as epithelial alveolar type II cells. A crystalline silica sample DQ12 was used as a prototypical particle for its capabilities to induce DNA damage, inflammatory response, and oxidative stress in epithelial cells; its polyvinylpyridine-N-oxide (PVNO)-surface modified counterpart was also used as a negative particulate control. Cells in mono-, bi- or tri-culture were exposed to DQ12 or DQ12-PVNO for 24 h. DQ12 but not DQ12-PVNO induced a significant increase in DNA damage in A549 cells. The presence of differentiated THP-1 reduced the genotoxic effects of this crystalline silica sample. The exposure of A549 to DQ12 but not DQ12-PVNO induced a significant change in interleukin-8 (IL-8) protein levels which was exacerbated when differentiated THP-1, and HL-60, were added. In addition, while no production of TNFα was detected in the A549 monoculture, elevated levels of this cytokine were observed in the co-culture systems. This work shows that a cell culture model that takes into consideration the complexity of the pulmonary inflammatory response might be more dependable to study the toxicological properties of particles than “simple” monoculture models.

The Significance of the Response: Beyond the Mechanics of DNA Damage and Repair-Physiological, Genetic, and Systemic Aspects of Radiosensitivity in Higher Organisms.

Classical radiation biology as we understand it clearly identifies genomic DNA as the primary target of ionizing radiation. The evidence appears rock-solid: ionizing radiation typically induces DSBs with a yield of ~30 per cell per Gy, and unrepaired DSBs are a very cytotoxic lesion. We know very well the kinetics of induction and repair of different types of DNA damage in different organisms and cell lines. And yet, higher organisms differ in their radiation sensitivity-humans can be unpredictably radiosensitive during radiotherapy; this can be due to genetic defects (e.g., ataxia telangiectasia (AT), Fanconi anemia, Nijmegen breakage syndrome (NBS), and the xeroderma pigmentosum spectrum, among others) but most often is unexplained. Among other mammals, goats (Capra hircus) appear to be very radiosensitive (LD50 = 2.4 Gy), while Mongolian gerbils (Meriones unguiculatus) are radioresistant and withstand quadruple that dose (LD50 = 10 Gy). Primary radiation lethality in mammals is due most often to hematopoietic insufficiency, which is, in the words of Dr. Theodor Fliedner, one of the pioneers of radiation hematology, "a disturbance in cellular kinetics". And yet, what makes one cell type, or one particular organism, more sensitive to ionizing radiation? The origins of radiosensitivity go above and beyond the empirical evidence and models of DNA damage and repair-as scientists, we must consider other phenomena: the radiation-induced bystander effect (RIBE), abscopal effects, and, of course, genomic instability and immunomodulation. It seems that radiosensitivity is not entirely determined by the mathematics of DNA damage and repair, and it is conceivable that radiation biology may benefit from an informed enquiry into physiology and organism-level signaling affecting radiation responses. The current article is a review of several key aspects of radiosensitivity beyond DNA damage induction and repair; it presents evidence supporting new potential venues of research for radiation biologists.

Genetic instability of a single exposure to sevoflurane at different concentrations in monitored mice.

Sevoflurane is an inhalation anesthetic widely used for general anesthesia, but its genotoxic potential is controversial in clinical studies. It is unknown whether the effects are due to surgery or the anesthetic. Thus, for the first time, the present study investigated genotoxicity in peripheral blood cells and in target organs (liver, lung, and kidney) and micronucleus (MN) in the bone marrow of a single exposure to sevoflurane at three different concentrations in monitored mice. Ninety Swiss mice were distributed into the following groups: exposure to sevoflurane at 3.3% (low), 4.5% (intermediate), and 6.0% (high) in 40% oxygen (O2) for 2 h; negative control (no exposure); negative control with O2; and positive control. The exposed animals were heated, monitored for vital signs (temperature, O2 saturation, heart rate/pulse, and respiratory rate), and anesthetized via a modern low-flow digital system. Mice were euthanized 2 and 24 h after exposure for evaluation by the comet assay and MN test, respectively. No DNA damage occurred in the 3.3% group for any of the organs evaluated, and no genotoxic or mutagenic effects were observed at any sevoflurane concentration in the peripheral blood or liver cells. However, a significant increase in DNA damage was observed at higher concentrations in kidney (4.5%) and lung cells (6.0%) and in the MN frequency (groups 4.5% and 6.0%). No cytotoxicity or histological alterations were observed. In conclusion, high concentrations of sevoflurane induce DNA damage, but concentrations equivalent to those used in clinical practice do not demonstrate genotoxic or mutagenic effects.

Particulate hexavalent chromium exposure induces DNA double-strand breaks and inhibits homologous recombination repair in rat and human lung tissues.

Lung cancer is an important human health concern because of its high mortality rate, with many cases caused by environmental chemicals other than tobacco. Particulate hexavalent chromium [Cr(VI)] is a well-established human lung carcinogen, but how Cr(VI) induces lung cancer is poorly understood. Chromosome instability, a hallmark of lung cancer, is considered a major driving factor in Cr(VI)-induced lung cancer. Our previous studies in cultured human lung cells showed that particulate Cr(VI) induces DNA double-strand breaks during the late S and G2 phases of the cell cycle, which are repaired by homologous recombination, one of the main repair pathways of DNA double-strand breaks. Our previous data showed that prolonged exposure to Cr(VI) inhibits homologous recombination repair by targeting RAD51, a key protein that mediates homologous recombination. Therefore, particulate Cr(VI)-induced DNA damage combined with failure of DNA repair can lead to chromosome instability. In this study we translated these results to rat lung tissue and lung tumor tissue from Cr(VI)-exposed workers. Wistar rats were exposed to zinc chromate in a saline solution or saline alone by oropharyngeal aspiration with a single dose repeated weekly for 90 days. We observed DNA double-strand breaks increased in a concentration-dependent manner, but homologous recombination repair decreased in rat lungs after 90 days of exposure. Notably, these effects were more pronounced in bronchioles than alveoli. We also considered these effects in Cr(VI)-associated human lung tumors and observed increased DNA double-strand breaks and reduced RAD51 levels in lung tumor tissue compared with adjacent normal lung tissue. Thus, Cr(VI)-induced induction of DNA double-strand breaks, and inhibition of homologous recombination repair translates from cultured cells to experimental animals, normal lung tissue adjacent to the tumor, and Cr(VI)-associated human lung tumors.

Aldo-keto reductase family 1 member B10 prevents esophageal squamous cell carcinoma from reactive carbonyl species-induced cell death and promotes its progression

IntroductionChronic alcohol consumption and tobacco usage are major risk factors for esophageal squamous cell carcinoma (ESCC). Excessive tobacco and alcohol consumption lead to oxidative stress and the generation of reactive carbonyl species (RCS) which induce DNA damage and cell apoptosis. This phenomenon contributes to cell damage and carcinogenesis in various organs including ESCC. However, it also raises an important question on how ESCC cells evade RCS-induced apoptosis and grow rapidly under these conditions. Therefore, we hypothesize that some enzymes produced by ESCC cells are capable of catabolizing RCS, preventing ESCC neoplastic cells from undergoing RCS-induced apoptosis, potentially contributing to ESCC progression.MethodsTo identify significant gene clusters involved in the metabolism of RCS in ESCC, we used an Agilent SurePrint G3 Human V2 GE 8 × 60 K microarray kit to analyze differentially expressed genes between nine paired ESCC tissues and adjacent normal esophageal tissues taken from areas distant from the tumor site. Bioinformatics analysis using gene ontology (GO) was performed to categorize these genes. To validate the findings, immunohistochemical staining in specimens from 169 surgically resected ESCC patients was performed and then correlated with treatment outcomes. Furthermore, the identified signaling pathway and its biological effects were investigated in ESCC cell lines in vitro and 4-nitroquinoline 1-oxide (4-NQO)-induced-ESCC murine model in vivo.ResultsInterestingly, we found that one of the significantly altered 57 GO molecular function domain terms (GO:0004033 aldo-keto reductase activity; P = 0.021) between nine paired ESCC tumors and adjacent normal tissue specimens was associated with the RCS metabolism. Among significant genes within this domain, AKR1B10 (aldo-keto reductase family 1 member B10; P = 0.006) was identified as the most significantly altered gene. Immunohistochemical analysis revealed that AKR1B10 expression was higher in ESCC cells than in adjacent normal esophageal epithelium. In addition, AKR1B10 expression was independently significantly associated with a poorer prognosis in 169 ESCC patients. Enzyme-linked immunosorbent assay results further demonstrated that blood AKR1B10 concentrations were significantly higher in 72 ESCC patients than in 24 healthy controls. In vitro experiments revealed that inhibiting endogenous AKR1B10 enhanced the cytotoxicity of 4-hydroxy trans-2-nonenal, a type of RCS. In a 4-NQO-induced-ESCC murine model, oleanolic acid, an AKR1B10 inhibitor, significantly reduced the incidence of esophageal tumors.ConclusionsOur findings suggested that AKR1B10 is an independent adverse prognosticator for patients with ESCC, and could prevent ESCC neoplastic cells from undergoing RCS-induced apoptosis, and promote ESCC progression. Therefore, AKR1B10 signaling could be a potential therapeutic strategy for ESCC.

Fertility potential in patients with ovarian cancer

Malignant neoplasms of the reproductive system are the most common form of oncological morbidity in women, accounting for over 30% of all cancer cases. Most antineoplastic agents act by inducing DNA damage in highly proliferating cancer cells, resulting in oocyte death. Ovarian toxicity is the most common side effect of cancer treatment in young women. Both chemotherapy and radiotherapy have been shown to be toxic to the ovaries, increasing the risk of premature ovarian failure, early menopause, endocrine disorders, and infertility. Patients who have undergone cancer treatment have severe follicular atresia, even if they have a regular menstrual cycle. Currently, the most effective methods of preserving fertility in cancer patients include cryopreservation of oocytes and embryos after ovarian hyperstimulation. Other fertility preservation methods include ovarian tissue cryopreservation, follicle or embryo maturation in vitro, ovarian transposition, ovarian suppression, and adjuvant therapy. Despite promising fertility prospects, iatrogenic infertility is one of the most undesirable adverse effects of cancer therapy for young women. Timely referral to a gynecologist prior to chemotherapy or radiation therapy is key to successful fertility preservation. Women should be aware of the available opportunities of assisted reproductive technologies, along with potential risks and failures with regard to their age, stage of disease, and treatment method. At this stage, it is necessary to develop well-defined and effective algorithms for oncologists, obstetrician-gynecologists, fertility specialists, and embryologists.

Regorafenib induces DNA damage and enhances PARP inhibitor efficacy in pancreatic ductal carcinoma

BackgroundThere is increasing interest in enhancing the response of the PARP inhibitor olaparib, which is currently approved for pancreatic ductal adenocarcinoma (PDAC) patients with defects in DNA damage repair associated with germline BRCA1/2 mutations. Moreover, agents that can mimic these defects in the absence of germline BRCA1/2 mutations are an area of active research in hopes of increasing the number of patients eligible for treatment with PARP inhibitors. The extent to which regorafenib, an FDA-approved tyrosine kinase inhibitor, can be used to enhance the efficacy of PARP inhibitors in PDAC cells without known BRCA1/2 mutations remains to be investigated.MethodsComet assay, cell cycle analysis, western blotting, and immunofluorescent detection of H2AXS139 were used to evaluate the extent to which regorafenib induces DNA damage in PDAC cell lines. The effects of regorafenib, either alone or in combination with PARPi inhibitors, on PDAC cell death were assessed by Annexin V/PI co-staining assay in cell lines and by immunohistochemistry staining for cleaved caspase-3 in mouse tumors and in ex vivo slice cultures of human PDAC tumors. Flow cytometry-based analysis was used to evaluate the ability of regorafenib to reprogram PDAC tumor microenvironment.ResultsWe now show that regorafenib, a tyrosine-kinase inhibitor with efficacy in several gastrointestinal malignancies, can enhance the response of olaparib in pancreatic cancer. While regorafenib induces DNA damage and limits the ability of PDAC cells to resolve the damage, regorafenib by itself does not induce apoptosis. However, regorafenib in combination with olaparib further induces DNA damage in vitro, in tumor-bearing mice, and in ex vivo slice cultures of human PDAC tumors, resulting in increased apoptosis compared to olaparib alone. Notably, we show that the efficacy of the combination treatment is not dependent on cytolytic T cells.ConclusionsTogether, these findings demonstrate that regorafenib can attenuate DNA damage response and potentiate the efficacy of PARP inhibitors in PDAC tumors.

Artesunate disrupts ribosome RNA biogenesis and inhibits ovarian cancer growth by targeting FANCA.

The dysregulation of ribosome biogenesis has been extensively identified in various cancers, making it emerge as a hallmark of malignant cells. This highlights the potential of targeting ribosome biogenesis as an effective approach for treating cancer patients. Although chemotherapy drugs including doxorubicin and cisplatin often target ribosome biogenesis to induce DNA damage or inhibit tumor cell proliferation, they are associated with significant side effects. This study aims to reveal the novel role of artesunate (ART), a well-known antimalarial drug, in suppressing ribosome RNA biogenesis in ovarian cancer. In this study, the inhibitory effects of ART on ovarian cancer were studied both in vitro and in vivo. The effects of ART on ribosome RNA biogenesis were detected by 5-ethynyl uridine staining, RT-qPCR, and western blotting. Drug affinity responsive target stability, mass spectrometry, molecular docking and western blotting were combined to identify ART molecular targets. Ovarian cancer cells treated with ART exhibited significant reduction in nascent rRNA synthesis, accompanied by a remarkable down-regulation of pre-rRNA and mature rRNA expression. The inhibitory effect of ART on ribosome biogenesis subsequently impaired cell proliferation, cell migration and invasion, and induced apoptosis. In eukaryotes, ribosome RNA synthesis primarily occurs in the nucleus, involving processes such as rDNA transcription, pre-rRNA splicing and the assembly of ribosome precursors with ribosomal proteins, other closely-related proteins and small nucleolar RNAs. We observed that ART inhibited the nuclear translocation of FANCA through binding to FANCA protein, consequently leading to the inhibition of ribosome RNA synthesis. Moreover, knockdown of FANCA in ovarian tumor cells resulted in reduced rRNA transcription, suppressed cell proliferation and migration, and induced apoptosis which might be mediated through the inhibition of mTOR/RPS6 activity. In vivo studies using xenograft tumors in nude mice demonstrated that ART repressed the growth of established ovarian cancer tumors. Additionally, ART treatment significantly altered FANCA protein level in these tumors, especially suppressed its nuclear localization. These findings establish ART as a potent inhibitor of ribosome biogenesis, presenting a promising therapeutic avenue for ovarian tumors with high FANCA expression or for cancer patients exhibiting abnormal activation of the mTOR-RPS6 pathway.

The interplay of the translocase activity and protein recruitment function of PICH in ultrafine anaphase bridge resolution and genomic stability.

Incomplete sister centromere decatenation results in centromeric ultrafine anaphase bridges (UFBs). PICH (PLK1-interacting checkpoint helicase), a DNA translocase, plays a crucial role in UFB resolution by recruiting UFB-binding proteins and stimulating topoisomerase IIα. However, the involvement of distinct PICH functions in UFB resolution remains ambiguous. Here, we demonstrate that PICH depletion in non-transformed diploid cells induces DNA damage, micronuclei formation, p53 activation, G1-phase delay and cell death. Whole-genome sequencing reveals that segregation defects induced by PICH depletion cause chromosomal rearrangements, including translocations and inversions, emphasizing its significance in preserving genomic integrity. Furthermore, a PICH mutant that impairs UFB recruitment of BLM and RIF1 partially inhibits UFB resolution while a translocase-inactive mutant (PICHK128A) fails to resolve UFBs. Notably, expression of PICHK128A inhibits single-stranded UFB formation and induces hypocondensed chromosomes. We propose that PICH's translocase activity plays a dual role in promoting UFB resolution by facilitating the generation of single-stranded UFBs and stimulating topoisomerase IIα.

Olaparib and Radiotherapy Induce Type I Interferon- and CD8+ T Cell-Dependent Sensitization to Immunotherapy in Pancreatic Cancer.

PARP inhibitors sensitize pancreatic ductal adenocarcinoma (PDAC) to radiation by inducing DNA damage and replication stress. These mechanisms also have the potential to enhance radiation-induced type I interferon (T1IFN)-mediated antitumoral immune responses. We hypothesized that the PARP inhibitor olaparib would also potentiate radiation-induced T1IFN to promote antitumor immune responses and sensitization of otherwise resistant PDAC to immunotherapy. To test this hypothesis, we assessed the effects of olaparib and radiation on T1IFN production and sensitivity to αPD-L1 immunotherapy, as well as on the tumor microenvironment by single-cell RNA sequencing. We found that olaparib enhanced T1IFN production after radiation and had superior therapeutic efficacy in immunocompetent models. Olaparib and radiation treatment sensitized PDAC tumors to αPD-L1, resulting in decreased tumor burden and a 33% complete response rate. Combination treatment provided durable immune responses as shown by tumor rejection upon tumor rechallenge of previously cured mice. Furthermore, single-cell RNA sequencing analysis revealed that combination treatment induced an immunogenic tumor microenvironment characterized by interferon (IFN) responses in both PDAC and myeloid cell populations, macrophage polarization, and increased CD8+ terminal effector T-cell frequency and activity, findings which were confirmed by IHC and flow cytometry. Furthermore, CD8+ T cells and T1IFN signaling were required for therapeutic efficacy as host depletion of CD8+ T cells or the T1IFN receptor diminished treatment responses. Overall, our results indicate that olaparib enhances radiation-induced T1IFN-mediated immune signaling and subsequently an adaptive immune response, thus sensitizing pancreatic cancer to αPD-L1 therapy, supporting an ongoing clinical trial of this therapy in patients with PDAC.

Metabolic reprogramming in sepsis-associated acute kidney injury: insights from lipopolysaccharide-induced oxidative stress and amino acid dysregulation.

Sepsis-associated acute kidney injury (SA-AKI) stands out as a critical health issue due to its high mortality and morbidity rates. This study aimed to comprehensively investigate the biochemical and metabolic alterations induced by lipopolysaccharide (LPS) in human embryonic kidney cells (HEK-293) using an in vitro model. The study investigated the impact of LPS on HEK-293 cells by evaluating cytotoxicity using the MTT assay, analyzing apoptosis, cell cycle progression, and oxidative stress via flow cytometry, measuring TNF-α levels through ELISA, and assessing amino acid metabolism with LC-MS/MS. The findings demonstrated that LPS significantly reduced cell viability in a dose-dependent manner, increased apoptotic cell populations, induced DNA damage by arresting the cell cycle in the Sub-G1 phase, and activated oxidative stress pathways. Notably, elevated reactive oxygen species (ROS) production and increased secretion of the pro-inflammatory cytokine TNF-α highlighted LPS's inflammatory and cytotoxic effects. Furthermore, systematic analysis revealed LPS-induced disruptions in amino acid metabolism, including marked reductions in alanine, arginine, and aspartic acid levels. KEGG pathway analysis identified significant metabolic alterations in pathways such as the urea cycle, TCA cycle, and glutathione metabolism. Interestingly, elevated citrulline levels suggested a potential adaptive mechanism to counteract LPS-induced inflammation and oxidative stress. Additionally, ROC analysis identified cystine as a highly reliable biomarker, with an AUC value of 1.00, emphasizing its critical role in metabolic reprogramming associated with SA-AKI. This study provides critical insights into the molecular pathophysiology of SA-AKI and emphasizes the promise of metabolomic approaches in the early diagnosis of sepsis-related complications and the development of targeted therapies.