TuBG1 promotes hepatocellular carcinoma via ATR/P53-apoptosis and cycling pathways.

BACKGROUND: As reported, γ-tubulin (TuBG1) is related to the occurrence and development of various types of malignant tumors. However, its role in hepatocellular cancer (HCC) is not clear. The present study was to investigate the relationship between TuBG1 and clinical parameters and survival in HCC patients. METHODS: The correlation between TuBG1 and clinical parameters and survival in HCC patients was explored by bioinformatics analysis. Immunohistochemistry was used for the verification. The molecular function of TuBG1 was measured using colony formation, scratch assay, trans-well assay and flow cytometry. Gene set enrichment analysis (GSEA) was used to pick up the enriched pathways, followed by investigating the target pathways using Western blotting. The tumor-immune system interactions and drug bank database (TISIDB) was used to evaluate TuBG1 and immunity. Based on the TuBG1-related immune genes, a prognostic model was constructed and was further validated internally and externally. RESULTS: The bioinformatic analysis found high expressed TuBG1 in HCC tissue, which was confirmed using immunohistochemistry and Western blotting. After silencing the TuBG1 in HCC cell lines, more G1 arrested cells were found, cell proliferation and invasion were inhibited, and apoptosis was promoted. Furthermore, the silence of TuBG1 increased the expressions of Ataxia-Telangiectasia and Rad-3 (ATR), phospho-P38 mitogen-activated protein kinase (P-P38MAPK), phospho-P53 (P-P53), B-cell lymphoma-2 associated X protein (Bax), cleaved caspase 3 and P21; decreased the expressions of B-cell lymphoma-2 (Bcl-2), cyclin D1, cyclin E2, cyclin-dependent kinase 2 (CDK2) and CDK4. The correlation analysis of immunohistochemistry and clinical parameters and survival data revealed that TuBG1 was negatively correlated with the overall survival. The constructed immune prognosis model could effectively evaluate the prognosis. CONCLUSIONS: The increased expression of TuBG1 in HCC is associated with poor prognosis, which might be involved in the occurrence and development of HCC.

Veratramine Inhibits the Cell Cycle Progression, Migration, and Invasion via ATM/ATR Pathway in Androgen-Independent Prostate Cancer.

Prostate cancer (PC) is the second leading cause of cancer-related death among men. Treatment of PC becomes difficult after progression because PC that used to be androgen-dependent becomes androgen-independent prostate cancer (AIPC). Veratramine, an alkaloid extracted from the root of the Veratrum genus, has recently been reported to have anticancer effects that work against various cancers; however, its anticancer effects and the underlying mechanism of action in PC remain unknown. We investigated the anticancer effects of veratramine on AIPC using PC3 and DU145 cell lines, as well as a xenograft mouse model. The antitumor effects of veratramine were evaluated using the CCK-8, anchorage-independent colony formation, trans-well, wound healing assays, and flow cytometry in AIPC cell lines. Microarray and proteomics analyses were performed to investigate the differentially expressed genes and proteins induced by veratramine in AIPC cells. A xenograft mouse model was used to confirm the therapeutic response and in vivo efficacy of veratramine. Veratramine dose dependently reduced the proliferation of cancer cells both in vitro and in vivo. Moreover, veratramine treatment effectively suppressed the migration and invasion of PC cells. The immunoblot analysis revealed that veratramine significantly downregulated Cdk4/6 and cyclin D1 via the ATM/ATR and Akt pathways, both of which induce a DNA damage response that eventually leads to G1 phase arrest. In this study, we discovered that veratramine exerted antitumor effects on AIPC cells. We demonstrated that veratramine significantly inhibited the proliferation of cancer cells via G0/G1 phase arrest induced by the ATM/ATR and Akt pathways. These results suggest that veratramine is a promising natural therapeutic agent for AIPC.

Studying the effect of cocaine on the structure of cosmetically treated human hair using micro-Attenuated Total Reflection spectroscopy (ATR).

It has been demonstrated that hair can accumulate and trap drugs over time with exposure and use, thus being able to provide a useful indication of long-term exposure. Studying changes in the structural components of hair, which may occur as a result of drug binding processes, can be a useful tool in understanding the issues involving false positive results from hair drug testing. Furthermore, the effect of cosmetic treatments, such as bleaching and straightening on hair structural changes, may help to understand the effect of drugs on human hair structure with pre-treatment history. In this investigation, changes resulting from exposure of bleached, unbleached, and straightened, neat blonde Caucasian hair to external cocaine were examined. Cocaine was chosen as the test drug as it is among the most widely abused substances. The results from infrared spectra and their second derivatives with aid of multivariate statistical method, PCA analysis demonstrated that changes in the hair structure occurred as a result of these treatments.

The potential efficacy and mechanism of bendamustine in entra-nodal NK/T cell lymphoma.

Bendamustine has been shown to have anti-tumor activities in hematological malignancies, but the role of bendamustine in natural killer (NK)/T cell lymphoma (NKTCL) treatment is unclear. Our study has shown that bendamustine had potent growth-inhibitory and apoptosis-inducing effects on NKTCL cells. Interestingly, we noticed that the combination of either gemcitabine or etoposide results in additive or synergistic cytotoxicity. Bendamustine induced mitochondria-mediated apoptosis in concentration- and time-dependent manners in NKTCL cells, shown as down-regulation of Bcl-2 and activation of cleavage of caspases 3, 7, 9 and poly adenosinediphosphate-ribose polymerase (PARP). Bendamustine arrested NKTCL cells in G2/M phase, with downregulation of expression of cyclin B1 and upregulation of expression of p-cdc2, p-cdc25c and p-P53. Furthermore, we confirmed that bendamustine activated DNA damage response (DDR) directly or through Ataxia Telangiectasia Mutated Protein (ATM)/Chk2 and ATR/Chk1 pathway and increased the intracellular reactive oxygen species (ROS) level in NKTCL cells, which caused G2/M phase arrest and apoptosis. Bendamustine also inhibited phosphorylation of transcriptional factor STAT3, contributing to cell apoptosis and proliferation inhibition. Finally, we verified the effect of bendamustine on NKTCL cells in vivo. It showed that bendamustine dramatically inhibited the growth of the subcutaneous tumor, with no obvious impact on mice weight. These findings demonstrate that bendamustine activates DDR pathway, induces the accumulation of intracellularROS level as well as inhibition of STAT3, leading to cell apoptosis and G2/M cell cycle arrest in NKTCL cells, which indicates that bendamustine dramatically suppressed NKTCL both in vitro and in vivo and provides a potential therapeutic strategy in the treatment of NK/T lymphoma.

Positive regulation of ataxia-telangiectasia-mutated protein (ATM) by E2F transcription Factor 1 (E2F-1) in cisplatin-resistant nasopharyngeal carcinoma cells.

OBJECTIVE: To explore the mechanism of E2F transcription Factor 1 (E2F-1)-mediated ataxia-telangiectasia-mutated protein (ATM) in cisplatin (DDP)-resistant nasopharyngeal carcinoma (NPC). METHODS: E2F-1 and ATM expression was assessed in DDP-resistant NPC cell lines (CNE2/DDP and HNE1/DDP) and parental cells. Then, DDP-resistant NPC cells were transfected with control shRNA (short hairpin RNA) or E2F-1 shRNAs with or without ATM lentiviral activation particles. The half maximal inhibitory concentration (IC50) was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, and the cell cycle and cell proliferation were measured by flow cytometry and EdU staining, respectively. In addition, the expression of genes and proteins was quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. RESULTS: Both E2F-1 and ATM expression in DDP-resistant NPC cells was much higher than that in parental cells. E2F-1 shRNA reduced ATM expression in DDP-resistant NPC cells, but ATM overexpression had no significant effect on E2F-1. ATM overexpression enhanced DDP resistance in DDP-resistant NPC cells with increased IC50 values, which was reversed by E2F-1 inhibition. Meanwhile, ATM overexpression resulted in upregulation of ABCA2 and ABCA5 in DDP-resistant NPC cells, induced elevations in the transition of the cells into S-phase, and increased cell proliferation with enhanced expression of cyclin E1, CDK2, and Ki67, which was reversed by E2F-1 shRNAs. CONCLUSION: Downregulation of E2F-1, possibly by regulating ATM, could block the cell cycle in the G1 phase and reduce the proliferation of CNE2/DDP cells, thereby reversing the resistance of human NPC cells to DDP.

DNA repair inhibitors sensitize cells differently to high and low LET radiation.

The aim of this study was to investigate effects of high LET α-radiation in combination with inhibitors of DDR (DNA-PK and ATM) and to compare the effect with the radiosensitizing effect of low LET X-ray radiation. The various cell lines were irradiated with α-radiation and with X-ray. Clonogenic survival, the formation of micronuclei and cell cycle distribution were studied after combining of radiation with DDR inhibitors. The inhibitors sensitized different cancer cell lines to radiation. DNA-PKi affected survival rates in combination with α-radiation in selected cell lines. The sensitization enhancement ratios were in the range of 1.6-1.85 in cancer cells. ATMi sensitized H460 cells and significantly increased the micronucleus frequency for both radiation qualities. ATMi in combination with α-radiation reduced survival of HEK293. A significantly elicited cell cycle arrest in G2/M phase after co-treatment of ATMi with α-radiation and X-ray. The most prominent treatment effect was observed in the HEK293 by combining α-radiation and inhibitions. ATMi preferentially sensitized cancer cells and normal HEK293 cells to α-radiation. DNA-PKi and ATMi can sensitize cancer cells to X-ray, but the effectiveness was dependent on cancer cells itself. α-radiation reduced proliferation in primary fibroblast without G2/M arrest.

A dual-targeting ruthenium nanodrug that inhibits primary tumor growth and lung metastasis via the PARP/ATM pathway.

BACKGROUND: Many studies have found that ruthenium complexes possess unique biochemical characteristics and inhibit tumor growth or metastasis. RESULTS: Here, we report the novel dual-targeting ruthenium candidate 2b, which has both antitumor and antimetastatic properties and targets tumor sites through the enhanced permeability and retention (EPR) effect and transferrin/transferrin receptor (TF/TFR) interaction. The candidate 2b is composed of ruthenium-complexed carboline acid and four chloride ions. In vitro, 2b triggered DNA cleavage and thus blocked cell cycle progression and induced apoptosis via the PARP/ATM pathway. In vivo, 2b inhibited not only Lewis lung cancer (LLC) tumor growth but also lung metastasis. We detected apoptosis and decreased CD31 expression in tumor tissues, and ruthenium accumulated in the primary tumor tissue of C57BL/6 mice implanted with LLC cells. CONCLUSIONS: Thus, we conclude that 2b targets tumors, inhibits tumor growth and prevents lung metastasis.

Pharmacology of the ATM Inhibitor AZD0156: Potentiation of Irradiation and Olaparib Responses Preclinically.

AZD0156 is a potent and selective, bioavailable inhibitor of ataxia-telangiectasia mutated (ATM) protein, a signaling kinase involved in the DNA damage response. We present preclinical data demonstrating abrogation of irradiation-induced ATM signaling by low doses of AZD0156, as measured by phosphorylation of ATM substrates. AZD0156 is a strong radiosensitizer in vitro, and using a lung xenograft model, we show that systemic delivery of AZD0156 enhances the tumor growth inhibitory effects of radiation treatment in vivo Because ATM deficiency contributes to PARP inhibitor sensitivity, preclinically, we evaluated the effect of combining AZD0156 with the PARP inhibitor olaparib. Using ATM isogenic FaDu cells, we demonstrate that AZD0156 impedes the repair of olaparib-induced DNA damage, resulting in elevated DNA double-strand break signaling, cell-cycle arrest, and apoptosis. Preclinically, AZD0156 potentiated the effects of olaparib across a panel of lung, gastric, and breast cancer cell lines in vitro, and improved the efficacy of olaparib in two patient-derived triple-negative breast cancer xenograft models. AZD0156 is currently being evaluated in phase I studies (NCT02588105).

The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway.

Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4(+) T lymphocytes and inhibits the PVR downregulating activity of the viral Nef protein.