Predictive DNA damage signaling for low­dose ionizing radiation.

Numerous studies have attempted to develop biological markers for the response to radiation for broad and straightforward application in the field of radiation. Based on a public database, the present study selected several molecules involved in the DNA damage repair response, cell cycle regulation and cytokine signaling as promising candidates for low­dose radiation­sensitive markers. The HuT 78 and IM­9 cell lines were irradiated in a concentration­dependent manner, and the expression of these molecules was analyzed using western blot analysis. Notably, the activation of ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), p53 and H2A histone family member X (H2AX) significantly increased in a concentration­dependent manner, which was also observed in human peripheral blood mononuclear cells. To determine the radioprotective effects of cinobufagin, as an ATM and CHK2 activator, an in vivo model was employed using sub­lethal and lethal doses in irradiated mice. Treatment with cinobufagin increased the number of bone marrow cells in sub­lethal irradiated mice, and slightly elongated the survival of lethally irradiated mice, although the difference was not statistically significant. Therefore, KU60019, BML­277, pifithrin­α, and nutlin­3a were evaluated for their ability to modulate radiation­induced cell death. The use of BML­277 led to a decrease in radiation­induced p­CHK2 and γH2AX levels and mitigated radiation­induced apoptosis. On the whole, the present study provides a novel approach for developing drug candidates based on the profiling of biological radiation­sensitive markers. These markers hold promise for predicting radiation exposure and assessing the associated human risk.

Poliovirus receptor inhibition in breast cancer cells induces antitumor immunity via T cell activation.

Radiotherapy (RT) is a commonly used treatment option for patients with cancer because it can effectively control tumor growth and kill tumor cells. However, the impact of RT goes beyond direct tumor cell killing because it can change the tumor microenvironment by altering surrounding tissues and infiltrating cells and modulating the expression of immune checkpoints. Poliovirus receptor (PVR, cluster of differentiation (CD)155), a member of the nectin-like molecule family, is overexpressed in many human cancers. However, its role in the tumor growth and T-cell immune responses of triple-negative breast cancer (TNBC) remains unclear. In the present study, we observe that radiation exposure increases PVR expression in MDA-MB-231 and BT549 cells. Silencing PVR not only inhibited the proliferation of breast cancer cells but also significantly enhanced the cytotoxicity of cytotoxic T lymphocytes (CTLs) compared with the control or RT groups. Treatment of T cells with PVR decreased CD8+ T cells, increased CD4+ T cells, and induced PVR ligands such as T cell immunoreceptor with immunoglobulin and ITIM domain, CD226, and CD96. However, after treatment with PVR, CTL responses decreased and secretion of interferon-γ, tumor necrosis factor-α, interleukin (IL)-2, IL-6, and IL-10 was significantly inhibited. In contrast, PVR knockdown increased the production of these cytokines, illustrating the immunosuppressive function of PVR. Suppression of PVR using an anti-PVR antibody inhibited 4T1 tumor growth by increasing immune cell infiltration. These results provide new insights into the role of PVR in TNBC and highlight its potential as a target for T cell-mediated immunotherapy in breast cancer.

Receptor interacting protein 1 knockdown induces cell death in liver cancer by suppressing STAT3/ATR activation in a p53-dependent manner.

The survival and death of eukaryotic cells are tightly controlled by a variety of proteins in response to the cellular environment. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a receptor-interacting Ser/Thr kinase that has recently been reported as an important regulator of cell survival, apoptosis, and necroptosis; however, its role in liver cancer remains unclear. In this study, we examined the effect of siRNA-mediated RIPK1 knockdown on the survival and death of liver cancer cells. Treatment with siRIPK1 decreased the growth rate of liver cancer cells and increased apoptotic, but not necrotic cell death, which was higher in wild-type p53 (wt-p53) cells than in mutant-type p53 (mt-p53) cells. In addition, RIPK1 knockdown increased p53 expression and G1 phase arrest in wt-p53 cells. Although suppressing p53 did not alter RIPK1 expression, it did attenuate siRIPK1-induced cell death. Interestingly, RIPK1 knockdown also increased the generation of reactive oxygen species and DNA damage by inhibiting signal transduced and activator of transcription 3 (STAT3) and ATM and RAD3-related (ATR) in wt-p53 cells but not in mt-p53 cells. Moreover, STAT3 or ATR inhibition in p53 mutant cells restored siRIPK1-mediated cell death. Together, the results of this study suggest that RIPK1 suppression induces apoptotic cell death by inhibiting the STAT3/ATR axis in a p53-dependent manner. Furthermore, these findings suggest that RIPK1, alone or in combination, may be a promising target for treating liver cancer.

BHMPS Inhibits Breast Cancer Migration and Invasion by Disrupting Rab27a-Mediated EGFR and Fibronectin Secretion.

Our previous work demonstrated that (E)-N-benzyl-6-(2-(3, 4-dihydroxybenzylidene) hydrazinyl)-N-methylpyridine-3-sulfonamide (BHMPS), a novel synthetic inhibitor of Rab27aSlp(s) interaction, suppresses tumor cell invasion and metastasis. Here, we aimed to further investigate the mechanisms of action and biological significance of BHMPS. BHMPS decreased the expression of epithelial-mesenchymal transition transcription factors through inhibition of focal adhesion kinase and c-Jun N-terminal kinase activation, thereby reducing the migration and invasion of breast cancer. Additionally, knockdown of Rab27a inhibited tumor migration, with changes in related signaling molecules, whereas overexpression of Rab27a reversed this phenomenon. BHMPS effectively prevented the interaction of Rab27a and its effector Slp4, which was verified by co-localization, immunoprecipitation, and in situ proximity ligation assays. BHMPS decreased the secretion of epidermal growth factor receptor and fibronectin by interfering with vesicle trafficking, as indicated by increased perinuclear accumulation of CD63-positive vesicles. Moreover, administration of BHMPS suppressed tumor growth in Rab27a-overexpressing MDA-MB-231 xenograft mice. These findings suggest that BHMPS may be a promising candidate for attenuating tumor migration and invasion by blocking Rab27a-mediated exocytosis.

Evaluation of Anti-Tumor Effects of Whole-Body Low-Dose Irradiation in Metastatic Mouse Models.

Low-dose irradiation (LDI) has recently been shown to have various beneficial effects on human health, such as on cellular metabolic activities, DNA repair, antioxidant activity, homeostasis potency, and immune activation. Although studies on the immunogenic effects of LDI are rapidly accumulating, clinical trials for cancer treatment are considered premature owing to the lack of available preclinical results and protocols. Here, we aim to investigate anti-tumor and anti-metastatic effects of whole-body LDI in several tumor-bearing mouse models. Mice were exposed to single or fractionated whole-body LDI prior to tumor transplantation, and tumor growth and metastatic potential were determined, along with analysis of immune cell populations and expression of epithelial-mesenchymal transition (EMT) markers. Whole-body fractionated-LDI decreased tumor development and lung metastasis not only by infiltration of CD4+, CD8+ T-cells, and dendritic cells (DCs) but also by attenuating EMT. Moreover, a combination of whole-body LDI with localized high-dose radiation therapy reduced the non-irradiated abscopal tumor growth and increased infiltration of effector T cells and DCs. Therefore, whole-body LDI in combination with high-dose radiation therapy could be a potential therapeutic strategy for treating cancer.

A new FGFR inhibitor disrupts the TGF-ß1-induced fibrotic process.

Pulmonary fibrosis (PF) is chronic and irreversible damage to the lung characterized by fibroblast activation and matrix deposition. Although recently approved novel anti-fibrotic agents can improve the lung function and survival of patients with PF, the overall outcomes remain poor. In this study, a novel imidazopurine compound, 3-(2-chloro-6-fluorobenzyl)-1,6,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (IM-1918), markedly inhibited transforming growth factor (TGF)-ß-stimulated reporter activity and reduced the expression of representative fibrotic markers, such as connective tissue growth factor, fibronectin, collagen and α-smooth muscle actin, on human lung fibroblasts. However, IM-1918 neither decreased Smad-2 and Smad-3 nor affected p38MAPK and JNK. Instead, IM-1918 reduced Akt and extracellular signal-regulated kinase 1/2 phosphorylation increased by TGF-ß. Additionally, IM-1918 inhibited the phosphorylation of fibroblast growth factor receptors 1 and 3. In a bleomycin-induced murine lung fibrosis model, IM-1918 profoundly reduced fibrotic areas and decreased collagen and α-smooth muscle actin accumulation. These results suggest that IM-1918 can be applied to treat lung fibrosis.

Tumor-Treating Fields Induce RAW264.7 Macrophage Activation Via NK-κB/MAPK Signaling Pathways.

OBJECTIVE: Tumor-treating fields are currently used to successfully treat various cancers; however, the specific pathways associated with its efficacy remain unknown in the immune responses. Here, we evaluated tumor-treating fields-mediated initiation of the macrophage-specific immune response. MATERIALS AND METHODS: We subjected RAW 264.7 mouse macrophages to clinically relevant levels of tumor-treating fields (0.9 V/cm, 150 kHz) and evaluated alterations in cytokine expression and release, as well as cell viability. Additionally, we investigated the status of immunomodulatory pathways to determine their roles in tumor-treating fields-mediated immune activation. RESULTS AND DISCUSSION: Our results indicated that tumor-treating fields treatment at 0.9 V/cm decreased cell viability and increased cytokine messenger RNA/protein levels, as well as levels of nitric oxide and reactive oxygen species, relative to controls. The levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 were markedly increased in tumor-treating fields-treated RAW 264.7 cells cocultured with 4T1 murine mammary carcinoma cells compared with those in 4T1 or RAW 264.7 cells with or without tumor-treating fields treatment. Moreover, the viability of 4T1 cells treated with the conditioned medium of tumor-treating fields-stimulated RAW 264.7 cells decreased, indicating that macrophage activation by tumor-treating fields effectively killed the tumor cells. Moreover, tumor-treating fields treatment activated the nuclear factor κB and mitogen-activated protein kinase pathways involved in immunomodulatory signaling. CONCLUSION: These results provide critical insights into the mechanisms through which tumor-treating fields affect macrophage-specific immune responses and the efficacy of this method for cancer treatment.

Inhibition of Karyopherin-α2 Augments Radiation-Induced Cell Death by Perturbing BRCA1-Mediated DNA Repair.

Ionizing radiation (IR) has been widely used in the treatment of cancer. Radiation-induced DNA damage triggers the DNA damage response (DDR), which can confer radioresistance and early local recurrence by activating DNA repair pathways. Since karyopherin-α2 (KPNA2), playing an important role in nucleocytoplasmic transport, was significantly increased by IR in our previous study, we aimed to determine the function of KPNA2 with regard to DDR. Exposure to radiation upregulated KPNA2 expression in human colorectal cancer HT29 and HCT116 cells and breast carcinoma MDA-MB-231 cells together with the increased expression of DNA repair protein BRCA1. The knockdown of KPNA2 effectively increased apoptotic cell death via inhibition of BRCA1 nuclear import following IR. Therefore, we propose that KPNA2 is a potential target for overcoming radioresistance via interruption to DDR.

Radiosensitizing Effect of Novel Phenylpyrimidine Derivatives on Human Lung Cancer Cells via Cell Cycle Perturbation.

Radiotherapy is one of the most common treatments for cancer, but radioresistance and injury to normal tissue are considered major obstacles to successful radiotherapy. Thus, there is an urgent need to develop radiosensitizers to improve the therapeutic outcomes of radiotherapy in cancer patients. Our previous efforts to identify novel radiosensitizers, using high-throughput screening targeting p53 and Nrf2 revealed a promising N-phenylpyrimidin-2-amine (PPA) lead compound. In the present study, 17 derivatives of this lead compound were examined, and it was found that 4-(4-fluorophenyl)-N-(4-nitrophenyl)-6-phenylpyrimidin-2-amine (PPA5), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-3-methoxy-N-methyl -benzamide (PPA13), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA14), 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA15), and 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)-N-methylbenzamide (PPA17) inhibited cell viability by more than 50%, with a marked increase in the proportion of cells arrested at the G2/M phase of cell cycle. Among these compounds, PPA15 markedly increased the sub-G1 cell population and increased the levels of cyclin B1 and the phosphorylation levels of cyclin-dependent kinase (CDK) 1. Combined treatment with radiation and PPA14 or PPA15 significantly decreased clonogenic survival. An in vitro kinase assay revealed that PPA15 inhibited multiple CDKs involved in cell cycle regulation. Compared with drug or radiation treatment alone, combined treatment with PPA15 and radiation resulted in the suppression of A549 tumor growth in mice by 59.5% and 52.7%, respectively. Treatment with PPA15 alone directly inhibited tumor growth by 25.7%. These findings suggest that the novel pan CDK inhibitor, PPA15, may be a promising treatment to improve the effectiveness of radiotherapy for the treatment of cancer. SIGNIFICANCE STATEMENT: Several inhibitors of CDK have been successfully evaluated in combination with other chemotherapeutics in clinical trials, but negative side effects have partially restricted their clinical use. In this study, we identified a novel pan-CDK inhibitor to increase radiosensitivity, and we hope this work will encourage the development of promising small-molecule radiosensitizers.