γH2AX Expression Research Articles

Hexavalent chromium induces γH2AX and RAD51 involved in DNA damage repair in BEAS-2B cells by modulating LNC-DHFR-4:1

Hexavalent chromium [Cr(VI)] is rarely found in nature. Its occurrence in the environment is mainly due to anthropogenic sources. Our previous studies have shown that Cr(VI) exposure could change the expression profile of long noncoding RNAs (lncRNAs). However, the relationship between lncRNAs and genetic damage induced by Cr(VI) remains unclear. In this study, RT–qPCR was used to verify the expression of genes and lncRNAs involved in DNA damage repair in BEAS-2B cells exposed to different Cr(VI) concentrations. After screening out LNC-DHFR-4:1, overexpression and knockdown models of BEAS-2B cells were used to further identify the relationship between the lncRNA and RAD51. RT–qPCR and indirect immunofluorescence were used to detect expression. Our results revealed that with increasing Cr(VI) concentration, γH2AX expression was increased, while the expression of RAD51 was decreased. Meanwhile, LNC-DHFR-4:1 acted as a competitive endogenous RNA to regulate the expression of γH2AX and RAD51, which further affected DNA damage repair. The overexpression of LNC-DHFR-4:1 induced a twofold decrease in γH2AX and a onefold increase in RAD51, and its knockdown showed the opposite results. These results suggested that LNC-DHFR-4:1 might be a potential biomarker of Cr(VI)-induced DNA damage repair in BEAS-2B cells.

UBA1 inhibition contributes radiosensitization of glioblastoma cells via blocking DNA damage repair.

Glioblastoma multiforme (GBM) is a brain tumor with high mortality and recurrence rate. Radiotherapy and chemotherapy after surgery are the main treatment options available for GBM. However, patients with glioblastoma have a grave prognosis. The major reason is that most GBM patients are resistant to radiotherapy. UBA1 is considered an attractive potential anti-tumor therapeutic target and a key regulator of DNA double-strand break repair and genome replication in human cells. Therefore, we hypothesized that TAK-243, the first-in-class UBA1 inhibitor, might increase GBM sensitivity to radiation. The combined effect of TAK-243 and ionizing radiation on GBM cell proliferation, and colony formation ability was detected using CCK-8, colony formation, and EdU assays. The efficacy of TAK-243 combined with ionizing radiation for GBM was further evaluated in vivo, and the mechanism of TAK-243 sensitizing radiotherapy was preliminarily discussed. The results showed that TAK-243, in combination with ionizing radiation, significantly inhibited GBM cell proliferation, colony formation, cell cycle arrest in the G2/M phase, and increased the proportion of apoptosis. In addition, UBA1 inhibition by TAK-243 substantially increased the radiation-induced γ-H2AX expression and impaired the recruitment of the downstream effector molecule 53BP1. Therefore, TAK-243 inhibited the radiation-induced DNA double-strand break repair and thus inhibited the growth of GBM cells. Our results provided a new therapeutic strategy for improving the radiation sensitivity of GBM and laid a theoretical foundation and experimental basis for further clinical trials.

Acrylamide-induced meiotic arrest of spermatocytes in adolescent mice by triggering excessive DNA strand breaks: Potential therapeutic effects of resveratrol.

Background: Baked carbohydrate-rich foods are the main source of acrylamide (AA) in the general population and are widely consumed by teenagers. Considering the crucial development of the reproductive system during puberty, the health risks posed by AA in adolescent males have raised public concern.Methods: In this study, we exposed 3-week-old male pubertal mice to AA for 4weeks to evaluate its effect on spermatogenesis using computer-assisted sperm analysis (CASA) and historical analysis. Flow cytometric analysis and meiocyte spreading assay were conducted to assess meiosis in mice. The expression of meiosis-related proteins and double-strand break (DSB) proteins were evaluated by immunoblot analyses. Additionally, isolated spermatocytes were used to explore the role of resveratrol in AA-induced damages of meiosis.Results: Our results showed that AA decreased the testicular and epididymal indexes, reduced sperm count and motility, and induced morphological disruption of the testes in pubertal mice. Subsequent meiotic analysis revealed that AA increased the proportion of 4C spermatocytes and decreased the proportion of 1C spermatids. The expression levels of meiosis-related proteins (SYCP3, Cyclin A1 and CDK2) were downregulated, and signaling proteins (γH2AX, p-CHK2 and p-ATM) expression levels were upregulated in AA-treated mice testes. Similar expression patterns were observed in primary spermatocytes treated with AA and these effects were reversed significantly by resveratrol.Conclusions: Our results indicate that AA induces meiotic arrest via persistent activation of DSBs, which may contribute to AA-compromised spermatogenesis. Resveratrol could serve as a potential therapeutic agent against AA-induced meiotic toxicity. These data highlight the importance of natural product supplementation for treating AA-related reproductive toxicity.

DNA‑PKcs phosphorylation specific inhibitor, NU7441, enhances the radiosensitivity of clinically relevant radioresistant oral squamous cell carcinoma cells.

Radioresistant cancer cells lead to poor prognosis after radiotherapy. However, the mechanisms underlying cancer cell radioresistance have not been fully elucidated. Thus, the DNA damage response of clinically relevant radioresistant oral squamous cell carcinoma HSC2-R cells, established by long-term exposure of parental HSC2 cells to fractionated radiation, was investigated. The DNA double-strand break (DSB) repair protein-specific inhibitor, NU7441, which targets DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation, and IBR2, which targets Rad51, were administered to HSC2 and HSC2-R cells. NU7441 administration eliminated colony formation in both cell lines under 6 Gy X-ray irradiation, whereas IBR2 did not affect colony formation. NU7441 and IBR2 significantly enhanced 6 Gy X-ray irradiation-induced apoptosis in HSC2-R cells. In HSC2-R cells, cell cycle arrest released earlier than in HSC2 cells, and phosphorylated-H2A histone family member X (γH2AX) expression rapidly decreased. Following NU7441 administration, γH2AX expression and the cell percentages of the G2/M phase were not decreased at 48 h after treatment in HSC2-R cells. DNA-PKcs has been demonstrated to regulate non-homologous end-joining (NHEJ) and homologous recombination (HR) repair, and the later phase of DSB repair is dominated by HR. Therefore, the results of the present study indicated that the DSB repair mechanism in HSC2-R cells strongly depends on NHEJ and loss of HR repair function. The present study revealed a potential mechanism underlying the acquired radioresistance and therapeutic targets in radioresistant cancer cells.

Carcinogenic risk in the biliary epithelium of children with congenital biliary dilatation via the DNA damage repair pathway.

Congenital biliary dilatation (CBD) is a high-risk factor for biliary tract cancer (BTC). We previously reported the potential for carcinogenesis in the biliary epithelium of patients with CBD. In this study, we investigated potential carcinogenetic pathways, focusing on the DNA damage repair response, in children with CBD and compared the findings with those in adults. We enrolled 6 children with CBD and 10 adults with CBD without BTC who underwent extrahepatic bile duct resections, plus 4 control patients who underwent pancreaticoduodenectomy for non-biliary cancer. Levels of phosphorylated histone H2AX (γH2AX), MRE11, and Ku-70 in the biliary tract epithelium were evaluated by immunohistochemistry. The levels of γH2AX, MRE11, and Ku-70 were significantly higher in the gallbladder epithelium and bile duct epithelium of both children and adults than in controls. Children and adults with CBD might develop BTC via the DNA damage repair pathway, as evidenced by increased γH2AX, MRE11, and Ku-70 expression.

Development of Clioquinol Platinum(IV) Conjugates as Autophagy-Targeted Antimetastatic Agents.

A series of autophagy-targeted antimetastatic clioquinol (CLQ) platinum(IV) conjugates were designed and prepared by incorporating an autophagy activator CLQ into the platinum(IV) system. Complex 5 with the cisplatin core bearing dual CLQ ligands with potent antitumor properties was screened out as a candidate. More importantly, it displayed potent antimetastatic properties both in vitro and in vivo as expected. Mechanism investigation manifested that complex 5 induced serious DNA damage to increase γ-H2AX and P53 expression and caused mitochondria-mediated apoptosis through the Bcl-2/Bax/caspase3 pathway. Then, it promoted prodeath autophagy by suppressing PI3K/AKT/mTOR signaling and activating the HIF-1α/Beclin1 pathway. The T-cell immunity was elevated by restraining the PD-L1 expression and subsequently increasing CD3+ and CD8+ T cells. Ultimately, metastasis of tumor cells was suppressed by the synergistic effects of DNA damage, autophagy promotion, and immune activation aroused by CLQ platinum(IV) complexes. Key proteins VEGFA, MMP-9, and CD34 tightly associated with angiogenesis and metastasis were downregulated.

Anti-tumour and radiosensitising effects of PARP inhibitor on cervical cancer xenografts

This study evaluated the radiosensitising effect of niraparib; a poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor on HeLa cervical cancer cells in nude mice and explored its possible mechanism. Twenty-four 3–5-week-old female BALB/c nude mice, inoculated with HeLa cells into the right hind leg, were randomly assigned into eight groups with three mice per group and treated. The tumour volume was significantly reduced under niraparib + radiotherapy combination as compared to monotherapy and untreated mice. The tumour growth was significantly delayed by 23.33–39 days when treated with combination therapy (p<.05). Further, univariate analysis revealed prolonged time for tumour growth when radiotherapy was followed by niraparib (I.G.) rather than niraparib (I.P.) (p=.003). Combination therapy reduced levels of PARP-1 precursor, PARP-1 splicer, PAR and RAD51 protein with high expression of γ-H2AX/CC3 and low expression of Ki-67. Niraparib in combination with radiotherapy can enhance the formation of DNA double strand breaks in HeLa cells and up regulate the expression of γ-H2AX/CC3. IMPACT STATEMENT What is already known on this subject? Asia has the highest incidence of cervical cancer (58.2%). Poly(adenosine diphosphate-ribose) polymerases (PARPs) are family of enzymes involved in single-strand break (SSB) and double-strand break (DSB) repair pathways. Niraparib is an effective inhibitor of both PARP-1 and PARP-2 and has the ability to cross the blood–brain barrier. What the results of this study add? Our study demonstrated that the combination of niraparib and radiotherapy can significantly enhance the cytotoxicity induced by radiotherapy. The inhibition effect of radiotherapy combined with niraparib on the tumour growth of mice was prominent, thereby establishing the radio-sensitisation activity of niraparib. What are the implications of these findings for clinical practice and/or further research? Niraparib can improve the cytotoxic effect of radiotherapy by increasing the formation of DSBs and up regulating the expression of apoptotic protein in HeLa cells.

Synthetic lethal targeting of TET2-mutant haematopoietic stem and progenitor cells by XPO1 inhibitors.

TET2 inactivating mutations serve as initiating genetic lesions in the transformation of haematopoietic stem and progenitor cells (HSPCs). In this study, we analysed known drugs in zebrafish embryos for their ability to selectively kill tet2-mutant HSPCs in vivo. We found that the exportin 1 (XPO1) inhibitors, selinexor and eltanexor, selectively kill tet2-mutant HSPCs. In serial replating colony assays, these small molecules were selectively active in killing murine Tet2-deficient Lineage-, Sca1+, Kit+ (LSK) cells, and also TET2-inactivated human acute myeloid leukaemia (AML) cells. Selective killing of TET2-mutant HSPCs and human AML cells by these inhibitors was due to increased levels of apoptosis, without evidence of DNA damage based on increased γH2AX expression. The finding that TET2 loss renders HSPCs and AML cells selectively susceptible to cell death induced by XPO1 inhibitors provides preclinical evidence of the selective activity of these drugs, justifying further clinical studies of these small molecules for the treatment of TET2-mutant haematopoietic malignancies, and to suppress clonal expansion in age-related TET2-mutant clonal haematopoiesis.

Growth Hormone Promotes Oocyte Maturation In Vitro by Protecting Mitochondrial Function and Reducing Apoptosis.

Some studies have been conducted to explore the influence of growth hormone (GH) on oocytes in in vitro maturation (IVM); however, previous studies reporting showed different results, and the specific mechanisms were not clear. In the present study, GH supplementation improved oocyte maturation rate. The rate of germinal vesicle breakdown (GVBD) in the GH group was 83.9%, which was significantly higher than that (72.1%) in the control group (p = 0.001). The maturation rate of the GH group (79.2%) was significantly higher than that (65.4%) of the control group (p = 0.000). The fertilization (68.6 vs. 59.3%) and blastocyst (30 vs. 25.3%) rates showed an increasing trend in the GH group compared to those in controls. The dynamic parameters of nuclear maturation of oocytes were recorded by time-lapse monitoring system; oocytes in the GH group completed nuclear maturation earlier than did those in the control group. GH reduced cAMP levels to promote oocyte maturation. Single-cell RNA sequencing analysis revealed that the majority of differentially expressed genes (DEGs) involved in mitochondrial oxidative phosphorylation was upregulated in the GH group. Furthermore, the mitochondrial membrane potential of oocytes significantly increased, and the levels of intracellular reactive oxygen species (ROS) and Ca2+ largely decreased in the GH group. Finally, single-oocyte transcriptome analysis indicated that GH decreased the expression of apoptosis-related genes in oocytes. GH treatment reduced the expression of γH2AX and caspase-3. Therefore, GH improves the developmental potential of immature oocytes by reducing cAMP levels more rapidly within 0.5h, protecting mitochondrial function, and reducing DNA damage and apoptosis.

Spermine oxidase induces DNA damage and sensitizes fusion negative rhabdomyosarcoma cells to irradiation.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma that includes fusion-positive (FP) and fusion-negative (FN) molecular subtypes. FP-RMS expresses PAX3-FOXO1 fusion protein and often shows dismal prognosis. FN-RMS shows cytogenetic abnormalities and frequently harbors RAS pathway mutations. Despite the multimodal heavy chemo and radiation therapeutic regimens, high risk metastatic/recurrent FN-RMS shows a 5-year survival less than 30% due to poor sensitivity to chemo-radiotherapy. Therefore, the identification of novel targets is needed. Polyamines (PAs) such as putrescine (PUT), spermidine (SPD) and spermine (SPM) are low-molecular-mass highly charged molecules whose intracellular levels are strictly modulated by specific enzymes. Among the latter, spermine oxidase (SMOX) regulates polyamine catabolism oxidizing SPM to SPD, which impacts cellular processes such as apoptosis and DNA damage response. Here we report that low SMOX levels are associated with a worse outcome in FN-RMS, but not in FP-RMS, patients. Consistently, SMOX expression is downregulated in FN-RMS cell lines as compared to normal myoblasts. Moreover, SMOX transcript levels are reduced FN-RMS cells differentiation, being indirectly downregulated by the muscle transcription factor MYOD. Noteworthy, forced expression of SMOX in two cell lines derived from high-risk FN-RMS: 1) reduces SPM and upregulates SPD levels; 2) induces G0/G1 cell cycle arrest followed by apoptosis; 3) impairs anchorage-independent and tumor spheroids growth; 4) inhibits cell migration; 5) increases γH2AX levels and foci formation indicative of DNA damage. In addition, forced expression of SMOX and irradiation synergize at activating ATM and DNA-PKCs, and at inducing γH2AX expression and foci formation, which suggests an enhancement in DNA damage response. Irradiated SMOX-overexpressing FN-RMS cells also show significant decrease in both colony formation capacity and spheroids growth with respect to single approaches. Thus, our results unveil a role for SMOX as inhibitor of tumorigenicity of FN-RMS cells in vitro. In conclusion, our in vitro results suggest that SMOX induction could be a potential combinatorial approach to sensitize FN-RMS to ionizing radiation and deserve further in-depth studies.

Effects of plasma-activated Ringer’s lactate solution on cancer cells: evaluation of genotoxicity

BackgroundNon-thermal atmospheric pressure plasma technologies form the core of many scientific advances, including in the electronic, industrial, and biotechnological fields. The use of plasma as a cancer therapy has recently attracted significant attention due to its cancer cell killing activity. Plasma-activated Ringer’s lactate solution (PAL) exhibits such activity. In addition to ROS, PAL contains active compounds or species that cause cancer cell death, but the potential mutagenic risks of PAL have not been studied.ResultsPAL has a low pH value and a high concentration of H2O2. H2O2 was removed from PAL using catalase and catalase-treated PAL with a pH of 5.9 retained a killing effect on HeLa cells whereas this effect was not observed if the PAL was adjusted to pH 7.2. Catalase-treated PAL at pH 5.9 had no significant effect on mutation frequency, the expression of γH2AX, or G2 arrest in HeLa cells.ConclusionPAL contains one or more active compounds or species in addition to H2O2 that have a killing effect on HeLa cells. The compound(s) is active at lower pH conditions and apparently exhibits no genotoxicity. This study suggested that identification of the active compound(s) in PAL could lead to the development of novel anticancer drugs for future cancer therapy.

Intracellular calprotectin (S100A8/A9) facilitates DNA damage responses and promotes apoptosis in head and neck squamous cell carcinoma

ObjectivesIn head and neck squamous cell carcinoma (HNSCC), poor prognosis and low survival rates are associated with downregulated calprotectin. Calprotectin (S100A8/A9) inhibits cancer cell migration and invasion and facilitates G2/M cell cycle arrest. We investigated whether S100A8/A9 regulates DNA damage responses (DDR) and apoptosis in HNSCC after chemoradiation. Materials and methodsHuman HNSCC cases in TCGA were analyzed for relationships between S100A8/A9 and expression of apoptosis-related genes. Next, S100A8/A9-expressing and non-expressing carcinoma lines (two different lineages) were exposed to genotoxic agents and assessed for 53BP1 and γH2AX expression and percent of viable/dead cells. Finally, S100A8/A9-wild-type and S100A8/A9null C57BL/6j mice were treated with 4-NQO to induce oral dysplastic and carcinomatous lesions, which were compared for levels of 53BP1. ResultsIn S100A8/A9-high HNSCC tumors, apoptosis-related caspase family member genes were upregulated, whereas genes limiting apoptosis were significantly downregulated based on TCGA analyses. After X-irradiation or camptothecin treatment, S100A8/A9-expressing carcinoma cells (i.e., TR146 and KB-S100A8/A9) showed significantly higher 53BP1 and γH2AX expression, DNA fragmentation, proportions of dead cells, and greater sensitivity to cisplatin than wild-type KB or TR146-S100A8/A9-KD cells. Interestingly, KB-S100A8/A9Δ113-114 cells showed similar 53BP1 and γH2AX levels to S100A8/A9-negative KB and KB-EGFP cells. After 4-NQO treatment, 53BP1 expression in oral lesions was significantly greater in calprotectin+/+ than S100A8/A9null mice. ConclusionsIn HNSCC cells, intracellular calprotectin is strongly suggested to potentiate DDR and promote apoptosis in response to genotoxic agents. Hence, patients with S100A8/A9-high HNSCC may encounter more favorable outcomes because more tumor cells enter apoptosis with increased sensitivity to chemoradiation therapy.

The Detection of DNA Damage Response in MCF7 and MDA-MB-231 Breast Cancer Cell Lines after X-ray Exposure.

Radiotherapy is one of the main options to cure and control breast cancer. The aim of this study was to investigate the sensitivity of two human breast cancer cell lines, MCF7 and MDA-MD-231, to radiation exposure at timepoints 4 h and 24 h after radiation. MCF7 and MDA-MD-231 were irradiated with different radiation doses using a Gilardoni CHF 320 G X-ray generator (Mandello del Lario, Italy) at 250 kVp, 15 mA [with half-value layer (HVL) = 1.6 mm copper]. The ApoTox-Glo triplex assay combines three assays used to assess viability, cytotoxicity, and apoptosis. The expression of γH2AX and BAX was analyzed by Western blotting. Viability and cytotoxicity did not change 4 h and 24 h after irradiation in either cell line, but we found a significant increase in the expression of cleaved caspase-3/7 at 24 h after irradiation with 8.5 Gy in MDA-MB231. The expression of γH2AX and BAX was low in MCF7, whereas the expression of γH2AX and BAX increased with radiation dose in a dose-dependent manner in MDA-MB231. The results show that the MCF7 cell line is more radioresistant than the MDA-MB 231 cell line at 4 h and 24 h after X-ray irradiation. In contrast, MDA-MB-231 cells were radiosensitive at a high radiation dose of 8.5 Gy at 24 h after irradiation. γH2AX and BAX indicated the radiosensitivity in both cell lines. These results open the possibility of using these cancer cell lines as models for testing new therapeutic strategies to improve radiation therapy.

Early detection of hepatocarcinogens in rats by immunohistochemistry of γ-H2AX.

We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

A series of ligustrazine platinum(IV) complexes with potent anti-proliferative and anti-metastatic properties that exert chemotherapeutic and immunotherapeutic effects.

The development of novel anticancer drugs with antiproliferative and antimetastatic activities is of great importance in the pharmaceutical field. Herein, a series of ligustrazine (LSZ) platinum(IV) complexes with chemotherapeutic and immunotherapeutic effects were designed, prepared and evaluated as antitumor agents for the first time. Complex 4 with potent antitumor activities both in vitro and in vivo was screened out as a candidate. Notably, it displays significantly more effective anti-metastatic activities than the platinum(II) drugs cisplatin and oxaliplatin. Mechanism detection discloses that it causes serious DNA damage and increases the expression of γ-H2AX and P53. Then, the apoptosis of tumor cells is promoted by activating the mitochondrial apoptotic pathway Bcl-2/Bax/caspase-3 and causing autophagy via modulating LC3-I/II and P62 expression. Furthermore, the immune therapeutic responses are significantly elevated by blocking HIF-1α, ERK 1/2 and COX-2 pathways to reduce PD-L1 expression, and further increasing CD3+ and CD8+ T cells to elevate T cell immunity in tumors. Tumor metastasis is blocked by the synergistic functions of DNA damage, hypoxia modulation and immune activation.

The PARP inhibitor Rucaparib synergizes with radiation to attenuate atypical teratoid rhabdoid tumor growth.

Atypical teratoid rhabdoid tumors (ATRT) are highly aggressive pediatric brain tumors. The available treatments rely on toxic chemotherapy and radiotherapy, which themselves can cause poor outcomes in young patients. Poly (ADP-ribose) polymerases (PARP), multifunctional enzymes which play an important role in DNA damage repair and genome stability have emerged as a new target in cancer therapy. An FDA-approved drug screen revealed that Rucaparib, a PARP inhibitor, is important for ATRT cell growth. This study aims to investigate the effect of Rucaparib treatment in ATRT. This study utilized cell viability, colony formation, flow cytometry, western blot, immunofluorescence, and immunohistochemistry assays to investigate Rucaparib's effectiveness in BT16 and MAF737 ATRT cell lines. In vivo, intracranial orthotopic xenograft model of ATRT was used. BT16 cell line was transduced with a luciferase-expressing vector and injected into the cerebellum of athymic nude mice. Animals were treated with Rucaparib by oral gavaging and irradiated with 2 Gy of radiation for 3 consecutive days. Tumor growth was monitored using In Vivo Imaging System. Rucaparib treatment decreased ATRT cell growth, inhibited clonogenic potential of ATRT cells, induced cell cycle arrest and apoptosis, and led to DNA damage accumulation as shown by increased expression of γH2AX. In vivo, Rucaparib treatment decreased tumor growth, sensitized ATRT cells to radiation and significantly increased mice survival. We demonstrated that Rucaparib has potential to be a new therapeutic strategy for ATRT as seen by its ability to decrease ATRT tumor growth both in vitro and in vivo.

Berbamine Exerts an Anti-oncogenic Effect on Pancreatic Cancer by Regulating Wnt and DNA Damage-related Pathways.

This study aimed to determine the effects of berbamine on pancreatic cancer as well as the underlying mechanisms. The pancreatic cancer cells were treated with different concentrations of berbamine and then subjected to cell viability assay, colony formation assay, cell cycle analysis, and apoptosis detection. Western blotting and immunofluorescence analyses were performed to investigate the mechanisms underlying the biological effects of berbamine on the pancreatic cancer cells. Furthermore, the in vivo anti-pancreatic cancer effect of berbamine was examined using a mouse xenograft model. Berbamine significantly inhibited the proliferation and colony-forming ability of BxPC3 and PANC-1 pancreatic cancer cells while inducing a cell cycle arrest and apoptosis. Moreover, berbamine decreased the expression of β- catenin and phosphorylation of GSK3β but increased the expression of γ-H2AX and 53BP1. Meanwhile, in vivo studies revealed that berbamine attenuated the growth of xenograft tumors derived from PANC-1 cells. Notably, berbamine treatment led to an increase in the expression of Cleaved Caspase 3 and γ-H2AX, as well as a decrease in the expression of Ki-67 and β-catenin in the tumor xenografts. Berbamine exerts an anti-pancreatic cancer effect, possibly by regulating Wnt and DNA damage-related pathways, suggestive of its therapeutic potential for pancreatic cancer.

Hepatocyte-specific damage in acute toxicity of sodium ferrous citrate: Presentation of a human autopsy case and experimental results in mice.

Acute iron overload is known to exert deleterious effects in the liver, but detailed pathology has yet to be documented. Here, we report pathological findings in an autopsy case of acute iron toxicity and validation of the findings in mouse experiments. In a 39-year-old woman who intentionally ingested a large amount of sodium ferrous citrate (equivalent to 7.5g of iron), severe disturbance of consciousness and fulminant hepatic failure rapidly developed. Liver failure was refractory to treatment and the patient died on Day 13. Autopsy revealed almost complete loss of hepatocytes, while bile ducts were spared. To examine the detailed pathologic processes induced by excessive iron, mice were orally administered equivalent doses of ferrous citrate. Plasma aminotransferase levels markedly increased after 6h, which was preceded by increased plasma iron levels. Hepatocytes were selectively damaged, with more prominent damage in the periportal area. Phosphorylated c-Jun was detected in hepatocyte nuclei after 3h, which was followed by the appearance of γ-H2AX expression. Hepatocyte injury in mice was associated with the expression of Myc and p53 after 12 and 24h, respectively. Even at lethal doses, the bile ducts were morphologically intact and fully viable. Our findings indicate that acute iron overload induces hepatocyte-specific liver injury, most likely through hydroxyl radical-mediated DNA damage and subsequent stress responses.

Synergistic effects of radiotherapy with JNK inhibitor-incorporated nanoparticle in an intracranial Lewis Lung Carcinoma Mouse Models.

Radiosurgery has been recognized as a reasonable treatment for metastatic brain tumors. Increasing the radiosensitivity and synergistic effects are possible ways to improve the therapeutic efficacy of specific regions of tumors. c-Jun-N-terminal kinase (JNK) signaling regulates H2AX phosphorylation to repair radiation-induced DNA breakage. We previously showed that blocking JNK signaling influenced radiosensitivity in vitro and in an in vivo mouse tumor model. Drugs can be incorporated into nanoparticles to produce a slow-release effect. This study assessed JNK radiosensitivity following the slow release of the JNK inhibitor SP600125 from a poly (DL-lactide-co-glycolide) (LGEsese) block copolymer in a brain tumor model. A LGEsese block copolymer was synthesized to fabricate SP600125-incorporated nanoparticles by nanoprecipitation and dialysis methods. The chemical structure of the LGEsese block copolymer was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy. The physicochemical and morphological properties were observed by transmission electron microscopy (TEM) imaging and measured with particle size analyzer. The blood-brain barrier (BBB) permeability to the JNK inhibitor was estimated by BBBflammaTM 440-dye-labeled SP600125. The effects of the JNK inhibitor were investigated using SP600125-incorporated nanoparticles and by optical bioluminescence, magnetic resonance imaging (MRI), and a survival assay in a mouse brain tumor model for Lewis lung cancer (LLC)-Fluc cells. DNA damage was estimated by histone γH2AX expression and apoptosis was assessed by the immunohistochemical examination of cleaved caspase 3. The SP600125-incorporated nanoparticles of the LGEsese block copolymer were spherical and released SP600125 continuously for 24h. The use of BBBflammaTM 440-dye-labeled SP600125 demonstrated the ability of SP600125 to cross the BBB. The blockade of JNK signaling with SP600125-incorporated nanoparticles significantly delayed mouse brain tumor growth and prolonged mouse survival after radiotherapy. γH2AX, which mediates DNA repair protein, was reduced and the apoptotic protein cleaved-caspase 3 was increased by the combination of radiation and SP600125-incorporated nanoparticles.

Lestaurtinib induces DNA damage that is related to estrogen receptor activation

A number of chemicals in the environment pose a threat to human health. Recent studies indicate estradiol induces DNA damage through the activation of the estrogen receptor alpha (ERα). Given that many environmental chemical compounds act like hormones once they enter the human body, it is possible that they induce DNA damage in the same way as estradiol, which is of great concern to females with the BRCA1 mutation. In this study, we developed an antibody-based high content method measuring γH2AX, a biomarker for DNA damage, to test a subset of 907 chemical compounds in MCF7 cells. The assay was optimized for a 1536 well plate format and had a satisfactory assay performance with Z-factor of 0.67. From the screening, we identified 128 compounds that induce γH2AX expression in the cells. These compounds were further examined for their γH2AX induction in the presence of an ER inhibitor, tamoxifen. After tamoxifen treatment, four compounds induced less γH2AX expression compared to those without tamoxifen treatment, suggesting these compounds induced γH2AX that is related to ERα activation. These four compounds were chosen for further studies to assess their ERα activating capability and c-MYC induction. Only lestaurtinib, a selective tyrosine kinase inhibitor, induced ERα activation, which was confirmed by both ERα beta-lactamase reporter gene assay and molecular docking analysis. Lestaurtinib also increased c-MYC expression, a target gene of ERα signaling, measured by the quantitative PCR method. This data suggests that lestaurtinib acts as a DNA damage inducer that is related to ERα activation.