Rationale: Transmembrane 4 superfamily member 4 (TM4SF4) has been identified as a key regulator of epithelial-mesenchymal transition (EMT)-associated stemness in non-small cell lung cancer (NSCLC) cells through autocrine signaling involving insulin-like growth factor 1 (IGF1) and osteopontin (OPN). Given its pivotal role in tumor progression and therapy resistance, TM4SF4 represents a promising therapeutic target. Methods: To develop a therapeutic antibody against TM4SF4, we generated anti-TM4SF4 monoclonal antibodies in mice by targeting the large extracellular loop (LEL) of human TM4SF4 using a 15-mer peptide, hTM4SF4 (T126-E140). Among the generated clones, the 2B7 antibody exhibited high specificity and reactivity to TM4SF4. Mechanistic studies were conducted to evaluate the effects of 2B7 on key signaling pathways, EMT-associated stemness, immune checkpoint ligand (ICL) expression, and immune responses. To facilitate clinical translation, 2B7 was humanized, generating the Hz2B7-1.1 antibody, which underwent affinity maturation to select the lead candidate, Hz2B7-1.2. Functional assays, including antibody-dependent cellular cytotoxicity (ADCC) and preclinical evaluations in xenograft models, were performed to assess its therapeutic potential. Results: The 2B7 antibody demonstrated significant antitumor efficacy in both A549 xenograft and patient-derived xenograft (PDX) models. Mechanistically, 2B7 inhibited key signaling pathways, including PI3K/AKT/GSK3β/β-catenin and JAK2/STAT3, leading to a reduction in EMT-associated stemness and therapy resistance. Additionally, 2B7 downregulated the expression of ICLs, such as PD-L1 and B7-H4, promoting T-cell activation and mitigating immune evasion. Furthermore, 2B7 reduced the secretion of exosomal ICLs by tumor cells and enhanced antitumor immune responses. The humanized antibody Hz2B7-1.2 retained binding properties and antitumor activity comparable to the parental 2B7 antibody and effectively induced ADCC as an IgG1-type antibody. Conclusions: The humanized anti-TM4SF4 antibody, Hz2B7-1.2, exhibits strong antitumor activity through multiple mechanisms, including inhibition of oncogenic signaling pathways, reduction of EMT-associated stemness, and modulation of immune responses. These findings support Hz2B7-1.2 as a promising therapeutic candidate for TM4SF4-positive cancers, warranting further clinical investigation.

Abstract Transmembrane 4 super family member 4(TM4SF4) protein has been shown to be involved in EMT-associated stemness through autocrine of insulin-like growth factor 1(IGF1) and osteopontin(OPN) in non-small cell lung cancer(NSCLC) cells. However, its potential as a therapeutic target has not been evaluated. In this study, anti-human TM4SF4 monoclonal antibodies(anti-hTM4SF4 mAbs) were prepared in mouse as part of a strategy for developing anticancer drug using humanized antibody. A synthetic peptide(15 mer, 126-140th AA) derived from the extracellular loop(ECL) 2 of human TM4SF4 exposed on outer cell surface was used as an immunization antigen. Among the selected anti-hTM4SF4 mAbs, ECL-2B7 and ECL-12A8 showed the best affinity for the antigen(Kd), approximately 2.66 nM and 8.34 nM, as determined by surface plasmon resonance analysis. Using mouse xenograft of NSCLC cell line, it was confirmed that ECL-2B7 was superior to ECL-12A8 in producing humanized antibody. ECL-2B7 inhibited EMT-associated stemness of TM4SF4 positive cancer cells by blocking cellular signaling events such as IGF1Rβ and CD44 and their downstream signals, PI3K/AKT/GSK3β/NF-κB and JAK2/STAT3 pathways. ECL-2B7 also enhanced antitumor activity by downregulating cellular and exosomal level of PD-L1 and B7-H4, immune-checkpoint ligands that elicit immunosuppression of T cell, and by mediating antibody-dependent cellular cytotoxicity. Treatment with OPN- and IGF1-neutralizing antibodies suppressed intracellular PD-L1 and B7-H4 level. It means that the regulation of PD-L1 and B7-H4 levels by TM4SF4 is closely related to the autocrine action of OPN and IGF1 induced by TM4SF4. Using patient-derived xenograft model, it was confirmed that the ECL-2B7 has excellent antitumor activity that effectively inhibits tumor progress. These results strongly suggest that it is necessary to construct humanized antibody based on ECL-2B7 to confirm its potential as a novel anticancer drug. Citation Format: Rae Kwon Kim, Chang-Kyu Heo, Yeon-jee Kahm, Uhee Jung, Byung-Chul Shin, Won-Yong Kim, Bum-Jin Kim, Sung-Chul Kim, Chun-jeih Ryu, Eun-Wie Cho, In-Gyu Kim. A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3189.

Kinesin family member 4A (KIF4A) belongs to the kinesin 4 subfamily of kinesin-related proteins and is involved in the regulation of chromosome condensation and segregation during mitotic cell division. The expression of KIF4A in various types of cancer, including lung, breast, and colon cancer, has been found to be associated with poor prognosis in cancer patients. However, the exact mechanism by which it promotes tumorigenesis is not yet understood. In osteosarcoma, the expression of KIF4A has been shown to be associated with cancer stem cells (CSCs), whereas in breast cancer, it is not associated with the maintenance of CSCs but regulates the migratory ability of cells. In this light, we identified phenotypic phenomena affecting the malignancy of cancer in lung cancer and glioma, and investigated the mechanisms promoting tumorigenesis. As a result, we demonstrated that KIF4A affected lung cancer stem cells (LCSCs) and glioma stem cells (GSCs) and regulated CSC signaling mechanisms. In addition, the migratory ability of cells was regulated by KIF4A, and epithelial-to-mesenchymal transition (EMT) marker proteins were controlled. KIF4A regulated the expression of the secretory factor plasminogen activator inhibitor-1 (PAI-1), demonstrating that it sustains cancer malignancy through an autocrine loop. Taken together, these findings suggest that KIF4A regulates CSCs and EMT, which are involved in cancer recurrence and metastasis, indicating its potential value as a novel therapeutic target and prognostic marker in lung cancer and glioma.

CTNNAL1 is a protein known to be involved in cell–cell adhesion and cell adhesion. Alterations in the expression or function of CTNNAL1 have been reported to contribute to the development and progression of various types of cancer. In breast cancer, CTNNAL1 has been reported as a cancer suppressor gene, and in melanoma and lung cancer, it has been reported as a cancer driver gene. However, due to a lack of research, its function remains unclear. In this study, it is shown that CTNNAL1 regulates cancer stem cells (CSCs) in lung cancer and glioblastoma and modulates their migration and invasion abilities. CSCs are known to play an important role in the malignant transformation of cancer. They have the ability to resist chemotherapeutic drugs and irradiation, which is a known obstacle to cancer treatment. We found that CTNNAL1 regulates the ability to resist irradiation. In addition, we observed that CTNNAL1 regulates the ability of cells to migrate and invade, a key feature of the epithelial to mesenchymal transition phenomenon associated with cancer metastasis. CTNNAL1 was also involved in the secretion of C–C motif chemokine ligand 2 (CCL2), one of the chemokines. CCL2 plays a role in the recruitment of immune cells to the tumor microenvironment, but in cancer, it is known to influence malignancy and metastasis. CTNNAL1 may be a novel target for treating lung CSCs and glioma stem cells and may be used as a marker of malignancy.

Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) is also known as survivin. BIRC5 is overexpressed in various carcinomas and is involved in cell growth and apoptosis. BIRC5, a member of the apoptosis inhibitor (IAP) family, negatively regulates apoptosis or programmed cell death by inhibiting caspase activation. Due to these properties, overexpression of BIRC5 enables specific survival and division associated with cancer malignancies. In addition, BIRC5 is highly expressed in stem cells, but not present at all in terminally differentiated cells. On this basis, there is speculation that BIRC5 may be involved in the regulation of cancer stem cells (CSCs), but few study results have been reported. In addition, the molecular mechanisms of BIRC5 regulation are not yet well understood. Through the present study, it was confirmed that BIRC5 is a key factor regulating CSCs and epithelial to mesenchymal transition (EMT) phenomena. BIRC5 was simultaneously overexpressed in lung cancer stem cells (LCSCs) and glioma stem cells (GSCs), and when the expression was suppressed, the characteristics of CSCs disappeared. In addition, IGFBP-3, a secreted factor regulated by BIRC5, is involved in signaling mechanisms that regulate cancer stem cells and EMT, and IGFBP-3 forms an autocrine chain. Based on these results, BIRC5 is proposed as a novel therapeutic target protein for LCSCs and GSCs.

Background: Ionizing radiation is a very powerful genetic mutagenic agent. Although immune cells are very sensitive to radiation, their sensitivity varies between different types of immune cell. We hypothesized that radiation-resistant immune cells survive after irradiation and then play a role in removing mutant cells. Materials and Methods: Splenic lymphocytes and mice were irradiated with γ-rays. Cell populations were analyzed using flow cytometry after dyeing with antibodies and expression of B-cell lymphoma 2 (BCL2) was measured by western blot analysis. To deplete natural killer (NK) cells, anti-asialo GM1 antiserum was used. Micronuclei in polychromatic erythrocytes were measured by May-Grunwald/Giemsa staining. H-2Kb loss variant in T-cells induced by irradiation of B6C3F1 mice were detected by flow cytometry. Results: When splenic lymphocytes were irradiated in vitro, B cells notably died, while NK cells did not. In vivo, on the third day after whole-body irradiation, the total number of lymphocytes in the spleen decreased rapidly, but the proportion of NK cells was approximately three times higher than that of the normal control group. In addition, it was confirmed that high expression of BCL2 in NK cells was maintained after irradiation, whereas that of B-cells was not. Removal of NK cells by injection with anti-asialo GM1 antiserum immediately after irradiation increased the micronuclei of polychromatic erythrocytes in the bone marrow and the variant fraction with H-2kb loss in the spleen. Conclusion: These results provide important evidence that radioresistant NK cells apparently survive by escaping apoptosis in the early stages after irradiation, and work to eliminate mutant cells resulting from γ-ray irradiation. Future studies are needed to reveal why NK cells are resistant to radiation and the in-depth mechanisms involved in the elimination of radiation-induced mutant cells.

Epithelial membrane protein 3 (EMP3) is a transmembrane glycoprotein that contains a peripheral myelin protein 22 domain. EMP3 first received attention as a tumor suppressor, but accumulating evidence has since suggested that it may exhibit a tumor‑promoting function. Nonetheless, the biological function of EMP3 remains largely unclear with regards to its role in cancer. Herein, it was shown that EMP3 expression is upregulated in non‑small cell lung cancer (NSCLC) cells overexpressing aldehyde dehydrogenase 1 (ALDH1). EMP3 was shown to be involved in cell proliferation, the formation of cancer stem cells (CSCs) and in epithelial‑mesenchymal transition (EMT). The ability to resist irradiation, one of the characteristics of CSCs, decreased when the EMP3 mRNA expression was knocked down using small interfering RNA. In addition, when EMP3 knockdown reduced the migratory ability of cells, a characteristic of EMT. Additionally, it was shown that the TGF‑β/Smad signaling axis was a target of EMP3. EMP3 was found to interact with TGF‑β receptor type 2 (TGFBR2) upon TGF‑β stimulation in lung CSCs (LCSC). As a result, binding of EMP3‑TGFBR2 regulates TGF‑β/Smad signaling activation and consequently affects CSCs and EMT. Kaplan‑Meier analysis results confirmed that patients with high expression of EMP3 had poor survival rates. Taken together, these findings showed that EMP3 may be a potential target for management of LCSCs with high expression of ALDH1, and that EMP3 is involved in TGF‑β/Smad signaling activation where it promotes acquisition of cancerous properties in tumors.

Cancer stem cells (CSCs) play an important role in cancer recurrence and metastasis. It is suggested that the CSC properties in heterogeneous cancer cells can be induced by ionizing radiation (IR). This study investigated the role of DLX2 in the radioresistance and CSC properties induced by IR in NSCLC cancer cells. Here, A549 cells were exposed to fractionated irradiation at a cumulative dose of 52 Gy (4 Gy × 13 times) for a generation of radioresistant cells. After fractionated irradiation, surviving A549 cells exhibited resistance to IR and enhanced expression of various cancer stem cell markers. They also showed upregulation of mesenchymal molecular markers and downregulation of epithelial molecular markers, correlating with an increase in the migration and invasion. Fractionated irradiation triggered the secretion of TGF-β1 and DLX2 expression. Interestingly, the increased DLX2 following fractionated irradiation seemed to induce the expression of the gene for the EGFR-ligand betacellulin via Smad2/3 signaling. To contrast, DLX2 knockdown dramatically decreased the expression of CSC markers, migration, and proliferation. Moreover, A549 cells expressing DLX2 shRNA formed tumors with a significantly smaller volume compared to those expressing control shDNA in a mouse xenograft assay. These results suggest that DLX2 overexpression in surviving NSCLC cancer cells after fractionated IR exposure is involved in the cancer stemness, radioresistance, EMT, tumor survival, and tumorigenic capability.

Background/Aim: Although cisplatin is an effective anticancer drug, its toxic effects on normal tissues limit its use. We developed a herbal formula, MH-30, with increased fat-soluble polyphenols by improving the manufacturing method of HemoHIM. In this study, we examined whether the combination of MH-30 with cisplatin exerts synergistic antitumor effect while it reduces cisplatin-induced toxicities. Materials and Methods: MH-30 was produced by adding the ethanol-insoluble fraction to its extract after decocting herbs in 30% ethanol and water. We used a melanoma-bearing mice model to investigate synergistic anticancer effects. The NK cell activity and cytokine levels were measured by Cr51-release assay and ELISA. The AST, ALT, BUN, and creatinine levels were estimated in the serum. Results: MH-30 effectively inhibited melanoma growth in vitro. Furthermore, MH-30 had a synergistic effect in combination with cisplatin on melanoma growth inhibition in vitro and in vivo. In melanoma-bearing mice, cisplatin alone decreased the activity of NK cells and the levels of IL-2 and IFN-γ, which were effectively restored by the combination of MH-30 with cisplatin. Combined treatment with MH-30 and cisplatin significantly inhibited the cisplatin-induced increase in the levels of AST, ALT, BUN, and creatinine. Conclusion: Combination of MH-30 with cisplatin may be a beneficial anticancer treatment with reduced adverse effects.

Studies have shown that cancer stem cells (CSCs) are involved in resistance and metastasis of cancer; thus, therapies targeting CSCs have been proposed. Here, we report that heat shock 70-kDa protein 1-like (HSPA1L) is partly involved in enhancing epithelial–mesenchymal transition (EMT) and CSC-like properties in non-small cell lung cancer (NSCLC) cells. Aldehyde dehydrogenase 1 (ALDH1) is considered a CSC marker in some lung cancers. Here, we analyzed transcriptional changes in genes between ALDH1high and ALDH1low cells sorted from A549 NSCLC cells and found that HSPA1L was highly expressed in ALDH1high cells. HSPA1L played two important roles in enhancing CSC-like properties. First, HSPA1L interacts directly with IGF1Rβ and integrin αV to form a triple complex that is involved in IGF1Rβ activation. HSPA1L/integrin αV complex-associated IGF1Rβ activation intensified the EMT-associated cancer stemness and γ-radiation resistance through its downstream AKT/NF-κB or AKT/GSK3β/β-catenin activation pathway. Secondly, HSPA1L was also present in the nucleus and could bind directly to the promoter region of β-catenin to function as a transcription activator of β-catenin, an important signaling protein characterizing CSCs by regulating ALDH1 expression. HSPA1L may be a novel potential target for cancer treatment because it both enhances IGF1Rβ activation and regulates γβ-catenin transcription, accumulating CSC-like properties.

Background/Aim: Ionizing radiation induces pulmonary fibrosis, which is a common dose-limiting complication in patients receiving radiotherapy. Fibrosis occurs through the accumulation of large amounts of ECM components, synthesized by myofibroblasts in damaged lung tissue. Epithelial cells serve as one of the cellular sources of myofibroblasts via the epithelial–to–mesenchymal transition (EMT) process. In this study, we investigated the role of TGF-β-secreting M2 macrophages in association with ionizing radiation-induced EMT. Materials and Methods: The lung epithelial cell line MLE12, was irradiated and the expression of EMT markers and chemokines was examined. Moreover, the mouse lung macrophage MH-S cell line was cultured with conditioned media from irradiated MLE12 cells, to examine the effects of the secreted factors on the migration ability of macrophages. For the murine pulmonary fibrosis model, mice were locally irradiated and the levels of M1 or M2 macrophage-related markers and cytokines were measured in bronchoalvelolar lavage (BAL) fluid and lung tissue. Results: In MLE12 cells, irradiation directly induced expression of EMT-related markers and secretion of various chemokines, which lead to macrophage migration. Interestingly, the sub-population of macrophages recruited in the lung of mice after thoracic irradiation was M2 macrophages that expressed Arg-1 and CD206. M2 macrophages induced the MLE12 to undergo phenotypic conversion to form fibroblast-like cells, which leads to a down-regulation of epithelial markers and an up-regulation of new EMT-related markers. In thoracic irradiated mice, pro-inflammatory cytokines such as IL-1β, IL-4 and IL-10 were increased at 2 weeks, but returned to normal levels from 16 weeks or 24 weeks after irradiation. However, thoracic irradiation led to a rapid increase of TGF-β and IGF-1 levels, which lasted up to 24 weeks. It was confirmed that M2 macrophages secreted the high levels of TGF-β. Moreover, the elimination of TGF-β from M2 macrophages attenuated mesenchymal transition of MLE12. Conclusion: TGF-β-secreting M2 macrophages play an important regulatory role in mesenchymal transition of epithelial cells in the lung of irradiated mice, thus contributing to radiation-induced pulmonary fibrosis.

Context: HemoHIM is a medicinal herbal preparation of Angelica gigas Nakai (Apiaceae), Cnidium officinale Makino (Umbelliferae), and Paeonia japonica Miyabe (Paeoniaceae) developed for immune regulation. HemoHIM has been investigated for its ability to enhance tissue self-renewal and stimulate immune systems. To date, studies on the protective effects of HemoHIM against gastritis and gastric ulcers have not been conducted.Objective: The protective effects of HemoHIM using models of indomethacin and ethanol/hydrochloric acid (EtOH/HCl)-induced gastric mucosal injury were investigated.Materials and methods: Rats were divided into five groups (n = 10): control, indomethacin, or EtOH/HCl groups, HemoHIM 250, 500 mg kg−1, and cimetidine 100 mg kg−1, respectively. Indomethacin (80 mg kg−1) and 60% EtOH/150 mM HCl were administered orally 1 h after the administration of samples and rats were anesthetized 3 h after induction. The lesion area (%), inhibition ratio (%), and total acidity were investigated, and tissues were histopathologically analyzed using hematoxylin and-eosin (H&E) staining.Results: HemoHIM significantly reduced gastric injury in indomethacin-induced model (250 and 500 mg kg−1; 64.30% and 67.75%, p < 0.001) compared to indomethacin group. In the EtOH/HCl-induced model, HemoHIM reduced gastric lesion (250 and 500 mg kg−1; 61.05% and 73.37%, p < 0.001) and gastric acidity (250 and 500 mg kg−1; 37.80 and 45.20 meq L−1, p < 0.001) compared to EtOH/HCl group. H&E staining of the gastric mucosa showed decreased erosion and hemorrhage in HemoHIM group compared to EtOH/HCl group.Discussion and conclusions: Based on the results, HemoHIM is potential candidate for the treatment of gastritis and gastric ulcers.

Cisplatin-based chemotherapy often results in the development of chemoresistance when used to treat ovarian cancer, which is difficult to overcome. The present study investigated the cytotoxic and anti-migratory effects of shikonin, a naphthoquinone compound, on cisplatin-resistant human ovarian cancer A2780 cells (A2780-CR). Shikonin had a potent dose-dependent cytotoxic effect on A2780-CR cells, with 9 µM shikonin treatment reducing A2780-CR cell viability by 50%, validate using an MTT assay. Shikonin induced apoptosis, as evidenced by the increased number of apoptotic bodies, following staining with Hoechst 33342, and terminal deoxynucleotidyl cell transferase dUTP nick end labeling-positive cells following treatment. Flow cytometry and fluorescent microscope imaging, following JC-1 staining, revealed that shikonin increased mitochondrial membrane depolarization. Also it altered the levels of apoptosis-associated proteins, leading to diminished expression of B cell lymphoma-2 (Bcl-2), enhanced expression of Bcl-associated X, and cleavage of caspase-9 and -3, as revealed using western blot analysis. Shikonin activated mitogen-activated protein kinases; while treatment with specific inhibitors of these kinases attenuated the decline in cell viability induced by shikonin treatment. In addition, the cell migration assay and western blot analysis indicated that shikonin decreased the migratory capacity of A2780-CR cells via the upregulation of epithelial-cadherin and downregulation of neural-cadherin. Taken together, the results of the present study indicated that shikonin induces mitochondria-mediated apoptosis and attenuates the epithelial-mesenchymal transition in A2780-CR cells.

Background Radiation-induced pneumonitis and pulmonary fibrosis are common dose-limiting complications in patients receiving radiotherapy for lung, breast, and lymphoid cancers. In this study, we investigated the characteristics of effective immune cells related to pneumonitis and fibrosis after irradiation. Materials and Methods After anesthesia, the whole thorax of C57BL/6 mice was irradiated at 14 Gy. The lung tissue and bronchoalveolar lavage fluid were collected at defined time points post-irradiation for the determination of histological and immunohistochemical analysis and inflammatory cell population infiltrated into the lung. Results and Discussion Whole thoracic irradiation increased the deposition of extracellular matrix (ECM), lung weight, and pleural effusions, which started to die from 4 months later. At 4 months after irradiation, the numbers of macrophages and lymphocytes as well as neutrophils were increased dramatically in the lung. Interestingly, the macrophages that were recruited into the lung after irradiation had an enlarged foamy morphology. In addition, the expressions of chemokines (CCL-2, CCL-3, CXCL-10) for the attraction of macrophages and T cells were higher in the lung of irradiated mice. The high expressions of these chemokines were sustained up to 6 months following irradiation. In thoracic irradiated mice, infiltrated macrophages into the lung had the high levels of Mac-3 antigens on their surface and upregulated the hallmarks of alternatively activated macrophages such as arginase-1 and CD206. Furthermore, the levels of IL-4 and IL-13 were higher in a BAL fluid of irradiated mice. Conclusion All results show that thoracic irradiation induces to infiltrate various inflammation-related immune cells, especially alternatively activated macrophages, through enhancing the expression of chemokines, suggesting that alternatively activated macrophages are most likely important for leading to pulmonary fibrosis. Keywords: Thoracic irradiation, Pneumonitis, Fibrosis, Inflammatory immune cells, Alternatively activated macrophages

Brain exposure to ionizing radiation can cause functional deficits in the hippocampus, including memory impairment. However, the specific molecular mechanisms underlying irradiation-induced cognitive impairments are largely unknown. Changes in DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), which are involved in DNA methylation and histone remodeling, may be associated with behavioral changes in learning and memory. We assessed changes in the levels of enzymes associated with the epigenetic modification of gene expression, including DNMT1, HDAC1, HDAC2, Sirtuin 1 (SIRT1), and acetylated histone H3 (Ace-H3) in the mouse hippocampus 1 and 30days after a single exposure to cranial irradiation (0 or 10Gy). mRNA levels of HDAC1 were significantly downregulated 1day after irradiation with 10Gy, and those of DNMT1, HDAC1, and HDAC2 were significantly downregulated 30days post-irradiation. Western blot analysis revealed significant decreases in DNMT1, HDAC1, and HDAC2 protein levels 1 and 30days after irradiation with 10Gy. Furthermore, protein levels of SIRT1 and Ace-H3 were significantly downregulated in the mouse hippocampus 1 and 30days after cranial irradiation. Our findings suggest that the reduction in epigenetic gene expression is associated with hippocampal dysfunction in mice exposed to cranial irradiation.

BACKGROUND:Hyperoside, a flavonoid which is mainly found in Hypericum perforatum L., has many biological effects. One of the most important effects is to prevent the oxidative stress induced by reactive oxygen species. However, the molecular mechanisms underlying its effect are not fully understood. Oxidative stress is implicated in the occurrence of various physical diseases. A wide array of enzymatic antioxidant defense systems include NADH: quinone oxidoreductase 1, superoxide dismutase, and heme oxygenase-1 (HO-1). In the present study, the protective effects of hyperoside against hydrogen peroxide-induced oxidative stress in human lens epithelial cells, HLE-B3, were investigated in terms of HO-1 induction.METHODS:The protein and mRNA expressions of HO-1 were examined by Western blotting and reverse transcriptase-PCR assays, respectively. To evaluate the ability of hyperoside to activate nuclear factor erythroid 2-related factor 2 (Nrf2), Western blotting and electrophoretic mobility shift assay were performed with nuclear extracts prepared from HLE-B3 cells treated with hyperoside. The activation of extracellular signal-regulated kinase (ERK), the upstream kinase of Nrf2 signaling, was monitored by Western blot analysis. The protective effect of hyperoside in HLE-B3 cells against hydrogen peroxide was performed by MTT assay.RESULTS:Hyperoside increased both the mRNA and protein expression of HO-1 in a time- and dose-dependent manner. In addition, hyperoside elevated the level of of Nrf2 and its antioxidant response element-binding activity, which was modulated by upstream of ERK. Moreover, it activated ERK and restored cell viability which was decreased by hydrogen peroxide.CONCLUSIONS:Hyperoside is an effective compound to protect cells against oxidative stress via HO-1 induction.

BACKGROUND:HemoHIM, which is an herbal preparation of three edible herbs (Angelicam gigas Nakai, Cnidium offinale Makino, and Peaonia japonica Miyabe), is known to have various biological and immunological activities, but the modulatory effects of this preparation on dendritic cells (DCs)-mediated immune responses have not been examined previously. DCs are a unique group of white blood cells that initiate primary immune responses by capturing, processing, and presenting antigens to T cells.RESULTS:In the present study, we investigated the effect of HemoHIM on the functional and phenotypic maturation of murine bone marrow-derived dendritic cells (BMDCs) both in vitro and in vivo. The expression of co-stimulatory molecules (CD40, CD80, CD86, MHC I, and MHC II) and the production of cytokines (IL-1β, IL-6, IL-12p70, and TNF-α) were increased by HemoHIM in BMDCs. Furthermore, the antigen-uptake ability of BMDCs was decreased by HemoHIM, and the antigen-presenting ability of HemoHIM-treated mature BMDCs increased TLR4-dependent CD4(+) and CD8(+) T cell responses.CONCLUSIONS:Our findings demonstrated that HemoHIM induces TLR4-mediated BMDCs functional and phenotypic maturation through in vivo and in vitro. And our study showed the antigen-presenting ability that HemoHIM-treated mature BMDCs increase CD4(+) and CD8(+) T cell responses by in vitro. These results suggest that HemoHIM has the potential to mediate DC immune responses.

Most studies of IR effects on neural cells and tissues in the brain are still focused on loss of neural stem cells. On the other hand, the effects of IR on neuronal differentiation and its implication in IR-induced brain damage are not well defined. To investigate the effects of IR on C17.2 mouse neural stem-like cells and mouse primary neural stem cells, neurite outgrowth and expression of neuronal markers and neuronal function-related genes were examined. To understand this process, the signaling pathways including PI3K, STAT3, metabotrophic glutamate receptor 1 (mGluR1) and p53 were investigated. In C17.2 cells, irradiation significantly increased the neurite outgrowth, a morphological hallmark of neuronal differentiation, in a dose-dependent manner. Also, the expression levels of neuronal marker proteins, β-III tubulin were increased by IR. To investigate whether IR-induced differentiation is normal, the expression of neuronal function-related genes including synaptophysin, a synaptic vesicle forming proteins, synaptotagmin1, a calcium ion sensor, γ-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors and glutamate receptors, excitatory neurotransmitter receptors was examined and compared to that of neurotrophin-stimulated differentiation. IR increased the expression of synaptophysin, synaptotagmin1 and GABA receptors mRNA similarly to normal differentiation by stimulation of neurotrophin. Interestingly, the overall expression of glutamate receptors was significantly higher in irradiated group than normal differentiation group, suggesting that the IR-induced neuronal differentiation may cause altered neuronal function in C17.2 cells. Next, the molecular mechanism of the altered neuronal differentiation induced by IR was studied by investigating signaling pathways including p53, mGluR1, STAT3 and PI3K. Increases of neurite outgrowth, neuronal marker and neuronal function-related gene expressions by IR were abolished by inhibition of p53, mGluR-1, STAT3 or PI3K. The inhibition of PI3K blocked both p53 signaling and STAT3-mGluR1 signaling but inhibition of p53 did not affect STAT3-mGluR1 signaling in irradiated C17.2 cells. Finally, these results of the IR-induced altered differentiation in C17.2 cells were verified in ex vivo experiments using mouse primary neural stem cells. In conclusion, the results of this study demonstrated that IR is able to trigger the altered neuronal differentiation in undifferentiated neural stem-like cells through PI3K-STAT3-mGluR1 and PI3K-p53 signaling. It is suggested that the IR-induced altered neuronal differentiation may play a role in the brain dysfunction caused by IR.

Cranial irradiation can trigger adverse effects on brain functions, including cognitive ability. However, the cellular and molecular mechanisms underlying radiation-induced cognitive impairments remain still unknown. Immediate-early genes (IEGs) are implicated in neuronal plasticity and the related functions (i.e., memory formation) in the hippocampus. The present study quantitatively assessed changes in the mRNA and protein levels of the learning-induced IEGs, including Arc, c-fos, and zif268, in the mouse hippocampus after cranial irradiation using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry, respectively. Mice (male, 8-week-old C57BL/6) received whole-brain irradiation with 0 or 10 Gy of gamma-ray and, 2 weeks later, contextual fear conditioning (CFC) was used to induce IEGs. In the CFC task, mice evaluated 2 weeks after irradiation exhibited significant memory deficits compared with sham (0 Gy)-irradiated controls. The levels of mRNA encoding IEGs were significantly upregulated in the hippocampus 10 and 30 min after CFC training. The mRNA levels in the irradiated hippocampi were significantly lower than those in the sham-irradiated controls. The IEG protein levels were significantly increased in all hippocampal regions, including the hippocampal dentate gyrus, cornu ammonis (CA)1, and CA3, after CFC training. The CFC-induced upregulation of Arc and c-fos in 10 Gy-irradiated hippocampi was significantly lower than that in sham-irradiated controls, although there were no significant differences in the protein levels of the learning-induced zif268 between sham-irradiated and 10 Gy-irradiated hippocampi. Thus, cranial irradiation with 10 Gy of gamma-ray impairs the induction of hippocampal IEGs (particularly Arc and c-fos) via behavioral contextual fear memory, and this disturbance may be associated with the memory deficits evident in mice after cranial irradiation, possibly through the dysregulation of neuronal plasticity during memory formation. (C) 2016 Elsevier Inc. All rights reserved.