Synthetic TGF-ß Signaling Agonist-Treated Dendritic Cells Induce Tolerogenicity and Antirheumatic Effects.

The newly synthesized compound TGF-ß signaling agonist (T74) is a small molecule associated with the TGF-ß receptor signaling pathway. Tolerogenic dendritic cells (tDCs) have been used to examine immunosuppressive and anti-inflammatory effects in multiple autoimmune disease models. The aim of this study was to investigate whether treatment of DCs with T74 has an antirheumatic effect in a mouse model of collagen-induced arthritis (CIA). Bone marrow-derived cells were obtained from DBA/1J mice and differentiated into DCs. T74-treated DCs (T74-DCs) were generated by treating bone marrow-derived DCs with LPS, type II collagen, and T74. T74-DCs expressed lower levels of surface molecules and inflammatory cytokines associated with antigen presentation and T cell stimulation. The ability of T74-DCs to differentiate effector T cells was lower than that of T74-untreated DCs (NT-DCs), but T74-DCs increased the regulatory T (Treg) cell differentiation in vitro. DBA/1J mice received two subcutaneous (s.c.) injections of type II collagen to establish CIA. Mice then received two s.c. injections of T74-DCs or NT-DCs. Joint inflammation was ameliorated in the paws of T74-DC-treated mice. Additionally, Treg populations in T74-DC-treated mice were higher than in NT-DC-treated or PBS-treated CIA mice. Taken together, these results demonstrate that T74 induces tolerance in DCs, and that T74-mediated DCs exert antirheumatic effects via induction of Tregs.

A novel function of HRP-3 in regulating cell cycle progression via the HDAC-E2F1-Cyclin E pathway in lung cancer.

To improve the poor survival rate of lung cancer patients, we investigated the role of HDGF-related protein 3 (HRP-3) as a potential biomarker for lung cancer. The expression of endogenous HRP-3 in human lung cancer tissues and xenograft tumor models is indicative of its clinical relevance in lung cancer. Additionally, we demonstrated that HRP-3 directly binds to the E2F1 promoter on chromatin. Interestingly, HRP-3 depletion in A549 cells impedes the binding of HRP-3 to the E2F1 promoter; this in turn hampers the interaction between Histone H3/H4 and HDAC1/2 on the E2F1 promoter, while concomitantly inducing Histone H3/H4 acetylation around the E2F1 promoter. The enhanced Histone H3/H4 acetylation on the E2F1 promoter through HRP-3 depletion increases the transcription level of E2F1. Furthermore, the increased E2F1 transcription levels lead to the enhanced transcription of Cyclin E, known as the E2F1-responsive gene, thus inducing S-phase accumulation. Therefore, our study provides evidence for the utility of HRP-3 as a biomarker for the prognosis and treatment of lung cancer. Furthermore, we delineated the capacity of HRP-3 to regulate the E2F1 transcription level via histone deacetylation.

Overexpression of VEGF in the MOPC 315 Plasmacytoma Induces Tumor Immunity in Mice.

Vascular endothelial growth factor (VEGF) has important effects on hematopoietic and immune cells. A link between VEGF expression, tumor progression, and metastasis has been established in various solid tumors; however, the impact of VEGF expression by hematopoietic neoplasias remains unclear. Here, we investigated the role of VEGF in plasma cell neoplasia. Overexpression of VEGF in MOPC 315 tumor cells (MOPCSVm) had no effect on their growth in vitro. However, constitutive ectopic expression of VEGF dramatically reduced tumorigenicity of MOPC 315 when implanted subcutaneously into BALB/c mice. Mice implanted with MOPCSVm effectively rejected tumor grafts and showed strong cytotoxic T lymphocyte (CTL) activity against parental MOPC 315 cells. MOPCSVm implants were not rejected in nude mice, suggesting the process is T-cell-dependent. Adoptive transfer of splenocytes from recipients inoculated with MOPCSVm cells conferred immunity to naïve BALB/c mice, and mice surviving inoculation with MOPCSVm rejected the parental MOPC 315 tumor cells following a second inoculation. Immunohistochemical analysis showed that MOPCSVm induced a massive infiltration of CD3+ cells and MHC class II+ cells in vivo. In addition, exogenous VEGF induced the expression of CCR3 in T cells in vitro. Together, these data are the first to demonstrate that overexpression of VEGF in plasmacytoma inhibits tumor growth and enhances T-cell-mediated antitumor immune response.

TNF-α Induces Mitophagy in Rheumatoid Arthritis Synovial Fibroblasts, and Mitophagy Inhibition Alleviates Synovitis in Collagen Antibody-Induced Arthritis.

Mitophagy is a selective form of autophagy that removes damaged mitochondria. Increasing evidence indicates that dysregulated mitophagy is implicated in numerous autoimmune diseases, but the role of mitophagy in rheumatoid arthritis (RA) has not yet been reported. The aim of the present study was to determine the roles of mitophagy in patient-derived RA synovial fibroblasts (RASFs) and in the collagen antibody-induced arthritis mouse model. We measured the mitophagy marker PTEN-induced putative kinase 1 (PINK1) in RASFs treated with tumor necrosis factor-α (TNF-α) using Western blotting and immunofluorescence. Arthritis was induced in PINK1-/- mice by intraperitoneal injection of an anti-type II collagen antibody cocktail and lipopolysaccharide. RA severity was assessed by histopathology. PINK1 expression and damaged mitochondria increased in TNF-α treated RASFs via increased intracellular levels of reactive oxygen species. PINK1 knockdown RASFs decreased cellular migration and invasion functions. In addition, PINK1-/- mice with arthritis exhibited markedly reduced swelling and inflammation relative to wild-type mice with arthritis. Taken together, these findings suggest that regulation of PINK1 expression in RA could represent a potential therapeutic and diagnostic target for RA.

JNC-1043, a Novel Podophyllotoxin Derivative, Exerts Anticancer Drug and Radiosensitizer Effects in Colorectal Cancer Cells.

The objective of this study was to determine whether (5S)-5-(4-benzyloxy-3,5-dimethoxy-phenyl)-5,9-dihydro-8H-furo [3',4':6,7] naphtho [2,3-d] [1,3]dioxol-6-one (JNC-1043), which is a novel chemical derivative of ß-apopicropodophyllin, acts as a novel potential anticancer reagent and radiosensitizer in colorectal cancer (CRC) cells. Firstly, we used MTT assays to assess whether JNC-1043 could inhibit the cell proliferation of HCT116 and DLD-1 cells. The IC50 values of these cell lines were calculated as 114.5 and 157 nM, respectively, at 72 h of treatment. Using doses approximating the IC50 values, we tested whether JNC-1043 had a radiosensitizing effect in the CRC cell lines. Clonogenic assays revealed that the dose-enhancement ratios (DER) of HCT116 and DLD-1 cells were 1.53 and 1.25, respectively. Cell-counting assays showed that the combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. Treatment with JNC-1043 or IR alone induced cell death by 50~60%, whereas the combination of JNC-1043 and IR increased this cell death by more than 20~30%. Annexin V-propidium iodide assays showed that the combination of JNC-1043 and IR increased apoptosis by more 30~40% compared to that induced by JNC-1043 or IR alone. DCFDA- and MitoSOX-based assays revealed that mitochondrial ROS production was enhanced by the combination of JNC-1043 and IR. Finally, we found that suppression of ROS by N-acetylcysteine (NAC) blocked the apoptotic cell death induced by the combination of JNC-1043 and IR. The xenograft model also indicated that the combination of JNC-1043 and IR increased apoptotic cell death in tumor mass. These results collectively suggest that JNC-1043 acts as a radiosensitizer and exerts anticancer effects against CRC cells by promoting apoptosis mediated by mitochondrial ROS.

Radiation-induced IL-1ß expression and secretion promote cancer cell migration/invasion via activation of the NF-κB-RIP1 pathway.

Here, we demonstrate that interleukin-1ß (IL-1ß) contributes to the γ-ionizing radiation (IR)-induced increase of migration/invasion in A549 lung cancer cells, and that this occurs via RIP1 upregulation. We initially observed that the protein expression and secreted concentration of IL-1ß were increased upon exposure of A549 cells to IR. We then demonstrated that IR-induced IL-1ß is located downstream of the NF-κB-RIP1 signaling pathway. Treatments with siRNA and specific pharmaceutical inhibitors of RIP1 and NF-κB suppressed the IR-induced increases in the protein expression and secreted concentration of IL-1ß. IL-1Ra, an antagonist of IL-1ß, treatment suppressed the IR-induced epithelial-mesenchymal transition (EMT) and IR-induced invasion/migration in vitro. These results suggest that IL-1ß could regulate IR-induced EMT. We also found that IR could induce the expression of IL-1ß expression in vivo and that of IL-1 receptor (R) I/II in vitro and in vivo. The IR-induced increases in the protein levels of IL-1 RI/II and IL-1ß suggest that an autocrine loop between IL-1ß and IL-1 RI/II might play important roles in IR-induced EMT and migration/invasion. Based on these collective results, we propose that IR concomitantly activates NF-κB and RIP1 to trigger the NF-κB-RIP1-IL-1ß-IL-1RI/II-EMT pathway, ultimately promoting metastasis.

Discovery of a Novel Triazolopyridine Derivative as a Tankyrase Inhibitor.

More than 80% of colorectal cancer patients have adenomatous polyposis coli (APC) mutations, which induce abnormal WNT/ß-catenin activation. Tankyrase (TNKS) mediates the release of active ß-catenin, which occurs regardless of the ligand that translocates into the nucleus by AXIN degradation via the ubiquitin-proteasome pathway. Therefore, TNKS inhibition has emerged as an attractive strategy for cancer therapy. In this study, we identified pyridine derivatives by evaluating in vitro TNKS enzyme activity and investigated N-([1,2,4]triazolo[4,3-a]pyridin-3-yl)-1-(2-cyanophenyl)piperidine-4-carboxamide (TI-12403) as a novel TNKS inhibitor. TI-12403 stabilized AXIN2, reduced active ß-catenin, and downregulated ß-catenin target genes in COLO320DM and DLD-1 cells. The antitumor activities of TI-12403 were confirmed by the viability of the colorectal cancer cells and its lack of visible toxicity in DLD-1 xenograft mouse model. In addition, combined 5-FU and TI-12403 treatment synergistically inhibited proliferation to a greater extent than that in a single drug treatment. Our observations suggest that TI-12403, a novel selective TNKS1 inhibitor, may be a suitable compound for anticancer drug development.

Functional Ambivalence of Dendritic Cells: Tolerogenicity and Immunogenicity.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) and inducers of T cell-mediated immunity. Although DCs play a central role in promoting adaptive immune responses against growing tumors, they also establish and maintain peripheral tolerance. DC activity depends on the method of induction and/or the presence of immunosuppressive agents. Tolerogenic dendritic cells (tDCs) induce immune tolerance by activating CD4+CD25+Foxp3+ regulatory T (Treg) cells and/or by producing cytokines that inhibit T cell activation. These findings suggest that tDCs may be an effective treatment for autoimmune diseases, inflammatory diseases, and infertility.

TGF-ß/IL-7 Chimeric Switch Receptor-Expressing CAR-T Cells Inhibit Recurrence of CD19-Positive B Cell Lymphoma.

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-ß/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-ß signaling into immune-activating IL-7 signaling. The effect of TGF-ß on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ-/-) mice were treated with CD19 CAR-tTRII-I7R-T cells to analyze the in vivo anti-tumor effect. In vitro, CD19 CAR-tTRII-I7R-T cells had a lower level of phosphorylated SMAD2 and a higher level of target-specific cytotoxicity than controls in the presence of rhTGF-ß1. In the animal model, the overall survival and recurrence-free survival of mice that received CD19 CAR-tTRII-I7R-T cells were significantly longer than in control mice. These findings strongly suggest that CD19 CAR-tTRII-I7R-T cell therapy provides a new strategy for long-lasting, TGF-ß-resistant anti-tumor effects against B cell lymphoma, which may lead ultimately to increased clinical efficacy.

Radiosensitizer Effect of ß-Apopicropodophyllin against Colorectal Cancer via Induction of Reactive Oxygen Species and Apoptosis.

ß-apopicropodophyllin (APP), a derivative of podophyllotoxin (PPT), has been identified as a potential anti-cancer drug. This study tested whether APP acts as an anti-cancer drug and can sensitize colorectal cancer (CRC) cells to radiation treatment. APP exerted an anti-cancer effect against the CRC cell lines HCT116, DLD-1, SW480, and COLO320DM, with IC50 values of 7.88 nM, 8.22 nM, 9.84 nM, and 7.757 nM, respectively, for the induction of DNA damage. Clonogenic and cell counting assays indicated that the combined treatment of APP and γ-ionizing radiation (IR) showed greater retardation of cell growth than either treatment alone, suggesting that APP sensitized CRC cells to IR. Annexin V-propidium iodide (PI) assays and immunoblot analysis showed that the combined treatment of APP and IR increased apoptosis in CRC cells compared with either APP or IR alone. Results obtained from the xenograft experiments also indicated that the combination of APP and IR enhanced apoptosis in the in vivo animal model. Apoptosis induction by the combined treatment of APP and IR resulted from reactive oxygen species (ROS). Inhibition of ROS by N-acetylcysteine (NAC) restored cell viability and decreased the induction of apoptosis by APP and IR in CRC cells. Taken together, these results indicate that a combined treatment of APP and IR might promote apoptosis by inducing ROS in CRC cells.

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.

Pdlim4 is essential for CCR7-JNK-mediated dendritic cell migration and F-actin-related dendrite formation.

Dendritic cells (DCs) are the most potent professional antigen (Ag)-presenting cells and inducers of T cell-mediated immunity. A previous microarray analysis identified PDZ and LIM domain protein 4 (Pdlim4) as a candidate marker for DC maturation. The aim of this study was to investigate whether Pdlim4 influences DC migration and maturation. Mouse bone marrow-derived DCs were transduced lentivirally with Pdlim4 short hairpin RNA and examined by confocal microscopy, flow cytometry, ELISA, and Western blotting. Pdlim4 was highly induced in LPS-stimulated mature DCs (mDCs). Pdlim4-knockdown mDCs showed reduced expression of molecules associated with Ag presentation and T-cell costimulation, reduced cytokine production, and functional defects in their ability to activate T cells. Moreover, Pdlim4 was necessary for mDC migration via C-C chemokine receptor type 7 (CCR7)-JNK in in vitro Transwell assays. The importance of Pdlim4 in DC migration was confirmed with an in vivo migration model in which C57BL/6 mice were injected with fluorescently labeled DCs in the footpad and migration to the popliteal lymph nodes was assessed by flow cytometry. Moreover, dendrite formation in mDCs was remarkably attenuated under Pdlim4 knockdown. Taken together, these results demonstrate that Pdlim4 is necessary for DC migration via CCR7-JNK, dendrite formation, and subsequent development of functional T-cell responses.-Yoo, J.-Y., Jung, N.-C., Lee, J.-H., Choi, S.-Y., Choi, H.-J., Park, S.-Y., Jang, J.-S., Byun, S.-H., Hwang, S.-U., Noh, K.-E., Park, Y., Lee, J., Song, J.-Y., Seo, H. G., Lee, H. S., Lim, D.-S. Pdlim4 is essential for CCR7-JNK-mediated dendritic cell migration and F-actin-related dendrite formation.

RIP1 Is a Novel Component of γ-ionizing Radiation-Induced Invasion of Non-Small Cell Lung Cancer Cells.

Abstract: Previously, we demonstrated that γ-ionizing radiation (IR) triggers the invasion/migration of A549 cells via activation of an EGFR-p38/ERK-STAT3/CREB-1-EMT pathway. Here, we have demonstrated the involvement of a novel intracellular signaling mechanism in γ-ionizing radiation (IR)-induced migration/invasion. Expression of receptor-interacting protein (RIP) 1 was initially increased upon exposure of A549, a non-small cell lung cancer (NSCLC) cell line, to IR. IR-induced RIP1 is located downstream of EGFR and involved in the expression/activity of matrix metalloproteases (MMP-2 and MMP-9) and vimentin, suggesting a role in epithelial-mesenchymal transition (EMT). Our experiments showed that IR-induced RIP1 sequentially induces Src-STAT3-EMT to promote invasion/migration. Inhibition of RIP1 kinase activity and expression blocked induction of EMT by IR and suppressed the levels and activities of MMP-2, MMP-9 and vimentin. IR-induced RIP1 activation was additionally associated with stimulation of the transcriptional factor NF-κB. Specifically, exposure to IR triggered NF-κB activation and inhibition of NF-κB suppressed IR-induced RIP1 expression, followed by a decrease in invasion/migration as well as EMT. Based on the collective results, we propose that IR concomitantly activates EGFR and NF-κB and subsequently triggers the RIP1-Src/STAT3-EMT pathway, ultimately promoting metastasis.

Discovery of Oxazol-2-amine Derivatives as Potent Novel FLT3 Inhibitors.

Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) is the most common mutation in patients with acute myeloid leukemia (AML). FLT3-ITD+ induces constitutive activation of FLT3, causing an abnormally rapid proliferation of cancer cells. In this study, we identified novel FLT3 inhibitors and investigated 5-(4-fluorophenyl)-N-phenyloxazol-2-amine (compound 7; 7c) as candidates for the treatment of AML. The results showed that 7c inhibited the activities of FLT3 and mutated FLT3 in a cell-free kinase assay and Molm-13 and MV4-11 cells, as well as the proliferation of FLT3-ITD+ AML cells, increasing apoptosis. The anti-leukemic activity of 7c was confirmed by in vivo tumor growth inhibition in MV4-11 xenograft mice. Besides, 7c suppressed the expression of DNA damage repair genes. Combination treatment with 7c and olaparib (a poly (ADP-ribose) polymerase [PARP] inhibitor) synergistically inhibited cell proliferation in Molm-13 and MV4-11 cells. Our findings demonstrated that 7c is a therapeutic candidate targeting FLT3 for AML treatment and suggested that combination treatment with 7c and a PARP inhibitor may be an effective therapy regimen for FLT3-mutated AML.

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.

A Small Compound KJ-28d Enhances the Sensitivity of Non-Small Cell Lung Cancer to Radio- and Chemotherapy.

We previously reported on a poly (ADP-ribose) polymerase (PARP) 1/2 inhibitor N-(3-(hydroxycarbamoyl)phenyl)carboxamide (designated KJ-28d), which increased the death of human ovarian cancer BRCA1-deficient SNU-251 cells. In the present study, we further investigated the antitumor activities of KJ-28d in BRCA-proficient non-small cell lung cancer (NSCLC) cells to expand the use of PARP inhibitors. KJ-28d significantly inhibited the growth of NSCLC cells in vitro and in vivo, and induced DNA damage and reactive oxygen species in A549 and H1299 cells. Combined treatment with KJ-28d and ionizing radiation led to increased DNA damage responses in A549 and H1299 cells compared to KJ-28d or ionizing radiation alone, resulting in apoptotic cell death. Moreover, the combination of KJ-28d plus a DNA-damaging therapeutic agent (carboplatin, cisplatin, paclitaxel, or doxorubicin) synergistically inhibited cell proliferation, compared to either drug alone. Taken together, the findings demonstrate the potential of KJ-28d as an effective anti-cancer therapeutic agent for BRCA-deficient and -proficient cancer cells. KJ-28d might have potential as an adjuvant when used in combination with radiotherapy or DNA-damaging agents, pending further investigations.

Antitumor Activity of a Novel Tyrosine Kinase Inhibitor AIU2001 Due to Abrogation of the DNA Damage Repair in Non-Small Cell Lung Cancer Cells.

Class III receptor tyrosine kinase (RTK) inhibitors targeting mainly FLT3 or c-KIT have not been well studied in lung cancer. To identify a small molecule potentially targeting class III RTK, we synthesized novel small molecule compounds and identified 5-(4-bromophenyl)-N-(naphthalen-1-yl) oxazol-2-amine (AIU2001) as a novel class III RKT inhibitor. In an in vitro kinase profiling assay, AIU2001 inhibited the activities of FLT3, mutated FLT3, FLT4, and c-KIT of class III RTK, and the proliferation of NSCLC cells in vitro and in vivo. AIU2001 induced DNA damage, reactive oxygen species (ROS) generation, and cell cycle arrest in the G2/M phase. Furthermore, AIU2001 suppressed the DNA damage repair genes, resulting in the 'BRCAness'/'DNA-PKness' phenotype. The mRNA expression level of STAT5 was downregulated by AIU2001 treatment and knockdown of STAT5 inhibited the DNA repair genes. Our results show that compared to either drug alone, the combination of AIU2001 with a poly (ADP-ribose) polymerase (PARP) inhibitor olaparib or irradiation showed synergistic efficacy in H1299 and A549 cells. Hence, our findings demonstrate that AIU2001 is a candidate therapeutic agent for NSCLC and combination therapies with AIU2001 and a PARP inhibitor or radiotherapy may be used to increase the therapeutic efficacy of AIU2001 due to inhibition of DNA damage repair.

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.

The small molecule AU14022 promotes colorectal cancer cell death via p53-mediated G2/M-phase arrest and mitochondria-mediated apoptosis.

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death, with its activation capable of sensitizing cancer cells to radiotherapy or chemotherapy. To identify small molecules that induce apoptosis via increased p53 transcriptional activity, we used a novel in-house library containing 96 small-molecule compounds. Using a cell-based screening method with a p53-responsive luciferase-reporter assay system involving benzoxazole derivatives, we found that AU14022 administration significantly increased p53 transcriptional activity in a concentration-dependent manner. Treatment with AU14022 increased p53 protein expression, p53 Ser15 phosphorylation, p53-mediated expression of downstream target genes, and apoptosis in p53-wild-type HCT116 human colon cancer cells, but not in p53-knockout HCT116 cells. Additionally, p53-wild-type HCT116 cells treated with AU14022 exhibited mitochondrial dysfunction, including modulated expression of B-cell lymphoma-2 family proteins and cytochrome c release. Combination treatment with AU14022 and ionizing radiation (IR) synergistically induced apoptosis as compared with IR or AU14022 treatment alone, with further investigation demonstrating that cell cycle progression was significantly arrested at the G2/M phase following AU14022 treatment. Furthermore, in a mouse p53-wild-type HCT116 colon cancer xenograft model, combined treatment with AU14022 and IR inhibited tumor growth more effectively than radiation alone. Therefore, AU14022 treatment induced apoptosis through p53-mediated cell cycle arrest involving mitochondrial dysfunction, leading to enhanced radiosensitivity in colon cancer cells. These results provide a basis for further assessments of AU14022 as a promising anticancer agent.

Infarcted Myocardium-Primed Dendritic Cells Improve Remodeling and Cardiac Function After Myocardial Infarction by Modulating the Regulatory T Cell and Macrophage Polarization.

BACKGROUND: Inflammatory responses play a critical role in left ventricular remodeling after myocardial infarction (MI). Tolerogenic dendritic cells (tDCs) can modulate immune responses, inducing regulatory T cells in a number of inflammatory diseases. METHODS: We generated tDCs by treating bone marrow-derived dendritic cells with tumor necrosis factor-α and cardiac lysate from MI mice. We injected MI mice, induced by a ligation of the left anterior descending coronary artery in C57BL/6 mice, twice with tDCs within 24 hours and at 7 days after the ligation. RESULTS: In vivo cardiac magnetic resonance imaging and ex vivo histology confirmed the beneficial effect on postinfarct left ventricular remodeling in MI mice treated with tDCs. Subcutaneously administered infarct lysate-primed tDCs near the inguinal lymph node migrated to the regional lymph node and induced infarct tissue-specific regulatory T-cell populations in the inguinal and mediastinal lymph nodes, spleen, and infarcted myocardium, indicating that a local injection of tDCs induces a systemic activation of MI-specific regulatory T cells. These events elicited an inflammatory-to-reparative macrophage shift. The altered immune environment in the infarcted heart resulted in a better wound remodeling, preserved left ventricular systolic function after myocardial tissue damage, and improved survival. CONCLUSIONS: This study showed that tDC therapy in a preclinical model of MI was potentially translatable into an antiremodeling therapy for ischemic tissue repair.