CXCL10 upregulation in radiation-exposed human peripheral blood mononuclear cells as a candidate biomarker for rapid triage after radiation exposure.

PURPOSE: In case of a nuclear accident, individuals with high-dose radiation exposure (>1-2 Gy) should be rapidly identified. While ferredoxin reductase (FDXR) was recently suggested as a radiation-responsive gene, the use of a single gene biomarker limits radiation dose assessment. To overcome this limitation, we sought to identify reliable radiation-responsive gene biomarkers. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from mice after total body irradiation, and gene expression was analyzed using a microarray approach to identify radiation-responsive genes. RESULTS: In light of the essential role of the immune response following radiation exposure, we selected several immune-related candidate genes upregulated by radiation exposure in both mouse and human PBMCs. In particular, the expression of ACOD1 and CXCL10 increased in a radiation dose-dependent manner, while remaining unchanged following lipopolysaccharide (LPS) stimulation in human PBMCs. The expression of both genes was further evaluated in the blood of cancer patients before and after radiotherapy. CXCL10 expression exhibited a distinct increase after radiotherapy and was positively correlated with FDXR expression. CONCLUSIONS: CXCL10 expression in irradiated PBMCs represents a potential biomarker for radiation exposure.

Centella asiatica-Derived Endothelial Paracrine Restores Epithelial Barrier Dysfunction in Radiation-Induced Enteritis.

Radiation-induced enteritis is frequently observed following radiotherapy for cancer or occurs due to radiation exposure in a nuclear accident. The loss of the epithelial integrity leads to 'leaky gut', so recovery of damaged epithelium is an important strategy in therapeutic trials. Centella asiatica (CA), a traditional herbal medicine, is widely used for wound healing by protecting against endothelial damage. In this study, we investigated the radio-mitigating effect of CA, focusing on the crosstalk between endothelial and epithelial cells. CA treatment relieved radiation-induced endothelial dysfunction and mitigated radiation-induced enteritis. In particular, treatment of the conditioned media from CA-treated irradiated endothelial cells recovered radiation-induced epithelial barrier damage. We also determined that epidermal growth factor (EGF) is a critical factor secreted by CA-treated irradiated endothelial cells. Treatment with EGF effectively improved the radiation-induced epithelial barrier dysfunction. We also identified the therapeutic effects of CA-induced endothelial paracrine in a radiation-induced enteritis mouse model with epithelial barrier restoration. Otherwise, CA treatment did not show radioprotective effects on colorectal tumors in vivo. We showed therapeutic effects of CA on radiation-induced enteritis, with the recovery of endothelial and epithelial dysfunction. Thus, our findings suggest that CA is an effective radio-mitigator against radiation-induced enteritis.

Zileuton Alleviates Radiation-Induced Cutaneous Ulcers via Inhibition of Senescence-Associated Secretory Phenotype in Rodents.

Radiation-induced cutaneous ulcers are a challenging medical problem for patients receiving radiation therapy. The inhibition of cell senescence has been suggested as a prospective strategy to prevent radiation ulcers. However, there is no effective treatment for senescent cells in radiation ulcers. In this study, we investigated whether zileuton alleviated radiation-induced cutaneous ulcer by focusing on cell senescence. We demonstrate increased cell senescence and senescence-associated secretory phenotype (SASP) in irradiated dermal fibroblasts and skin tissue. The SASP secreted from senescent cells induces senescence in adjacent cells. In addition, 5-lipoxygenase (5-LO) expression increased in irradiated dermal fibroblasts and skin tissue, and SASP and cell senescence were regulated by 5-LO through p38 phosphorylation. Finally, the inhibition of 5-LO following treatment with zileuton inhibited SASP and mitigated radiation ulcers in animal models. Our results demonstrate that inhibition of SASP from senescent cells by zileuton can effectively mitigate radiation-induced cutaneous ulcers, indicating that inhibition of 5-LO might be a viable strategy for patients with this condition.

Metformin Protects the Intestinal Barrier by Activating Goblet Cell Maturation and Epithelial Proliferation in Radiation-Induced Enteropathy.

Radiotherapy or accidental exposure to high-dose radiation can cause severe damage to healthy organs. The gastrointestinal (GI) tract is a radiation-sensitive organ of the body. The intestinal barrier is the first line of defense in the GI tract, and consists of mucus secreted by goblet cells and a monolayer of epithelium. Intestinal stem cells (ISCs) help in barrier maintenance and intestinal function after injury by regulating efficient regeneration of the epithelium. The Wnt/ß-catenin pathway plays a critical role in maintaining the intestinal epithelium and regulates ISC self-renewal. Metformin is the most widely used antidiabetic drug in clinical practice, and its anti-inflammatory, antioxidative, and antiapoptotic effects have also been widely studied. In this study, we investigated whether metformin alleviated radiation-induced enteropathy by focusing on its role in protecting the epithelial barrier. We found that metformin alleviated radiation-induced enteropathy, with increased villi length and crypt numbers, and restored the intestinal barrier function in the irradiated intestine. In a radiation-induced enteropathy mouse model, metformin treatment increased tight-junction expression in the epithelium and inhibited bacterial translocation to mesenteric lymph nodes. Metformin increased the number of ISCs from radiation toxicity and enhanced epithelial repair by activating Wnt/ß-catenin signaling. These data suggested that metformin may be a potential therapeutic agent for radiation-induced enteropathy.

Metallothionein 2 activation by pravastatin reinforces epithelial integrity and ameliorates radiation-induced enteropathy.

BACKGROUND: Radiotherapy or accidental exposure to ionizing radiation causes severe damage of healthy intestinal tissues. Intestinal barrier function is highly sensitive to ionizing radiation, and loss of epithelial integrity results in mucosal inflammation, bacterial translocation, and endotoxemia. Few studies have of epithelial integrity as a therapeutic target to treat radiation toxicity. Here, we examined the effects of pravastatin (PS) and the molecular mechanisms underlying epithelial integrity on radiation-induced enteropathy. METHODS: The radio-mitigative effects of PS were evaluated in a minipig model by quantifying clinical symptoms, and performing histological and serological analyses and mRNA sequencing in intestinal tissues. To evaluate the role of intercellular junctions on radiation damage, we used tight junction regulator and metallothionein 2 (MT2) as treatments in a mouse model of radiation-induced enteropathy. Caco-2 monolayers were used to examine functional epithelial integrityand intercellular junction expression. FINDING: Using a minipig model of pharmaceutical oral bioavailability, we found that PS mitigated acute radiation-induced enteropathy. PS-treated irradiated minipigs had mild clinical symptoms, lower intestinal inflammation and endotoxin levels, and improved gastrointestinal integrity, compared with control group animals. The results of mRNA sequencing analysis indicated that PS treatment markedly influenced intercellular junctions by inhibiting p38 MAPK signaling in the irradiated intestinal epithelium. The PS-regulated gene MT2 improved the epithelial barrier via enhancement of intercellular junctions in radiation-induced enteropathy. INTERPRETATION: PS regulated epithelial integrity by modulating MT2 in radiation-damaged epithelial cells. These findings suggested that maintenance of epithelial integrity is a novel therapeutic target for treatment of radiation-induced gastrointestinal damage. FUNDING: As stated in the Acknowledgments.

Atorvastatin Inhibits Endothelial PAI-1-Mediated Monocyte Migration and Alleviates Radiation-Induced Enteropathy.

Intestinal injury is observed in cancer patients after radiotherapy and in individuals exposed to radiation after a nuclear accident. Radiation disrupts normal vascular homeostasis in the gastrointestinal system by inducing endothelial damage and senescence. Despite advances in medical technology, the toxicity of radiation to healthy tissue remains an issue. To address this issue, we investigated the effect of atorvastatin, a commonly prescribed hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor of cholesterol synthesis, on radiation-induced enteropathy and inflammatory responses. We selected atorvastatin based on its pleiotropic anti-fibrotic and anti-inflammatory effects. We found that atorvastatin mitigated radiation-induced endothelial damage by regulating plasminogen activator inhibitor-1 (PAI-1) using human umbilical vein endothelial cells (HUVECs) and mouse model. PAI-1 secreted by HUVECs contributed to endothelial dysfunction and trans-endothelial monocyte migration after radiation exposure. We observed that PAI-1 production and secretion was inhibited by atorvastatin in irradiated HUVECs and radiation-induced enteropathy mouse model. More specifically, atorvastatin inhibited PAI-1 production following radiation through the JNK/c-Jun signaling pathway. Together, our findings suggest that atorvastatin alleviates radiation-induced enteropathy and supports the investigation of atorvastatin as a radio-mitigator in patients receiving radiotherapy.

Ghrelin reverts intestinal stem cell loss associated with radiation-induced enteropathy by activating Notch signaling.

BACKGROUD: Exposure to high-dose radiation, such as after a nuclear accident or radiotherapy, elicits severe intestinal damage and is associated with a high mortality rate. In treating patients exhibiting radiation-induced intestinal dysfunction, countermeasures to radiation are required. In principle, the cellular event underlying radiation-induced gastrointestinal syndrome is intestinal stem cell (ISC) apoptosis in the crypts. High-dose irradiation induces the loss of ISCs and impairs intestinal barrier function, including epithelial regeneration and integrity. Notch signaling plays a critical role in the maintenance of the intestinal epithelium and regulates ISC self-renewal. Ghrelin, a hormone produced mainly by enteroendocrine cells in the gastrointestinal tract, has diverse physiological and biological functions. PURPOSE: We investigate whether ghrelin mitigates radiation-induced enteropathy, focusing on its role in maintaining epithelial function. METHODS: To investigate the effect of ghrelin in radiation-induced epithelial damage, we analyzed proliferation and Notch signaling in human intestinal epithelial cell. And we performed histological analysis, inflammatory response, barrier functional assays, and expression of notch related gene and epithelial stem cell using a mouse model of radiation-induced enteritis. RESULTS: In this study, we found that ghrelin treatment accelerated the reversal of radiation-induced epithelial damage including barrier dysfunction and defective self-renewing property of ISCs by activating Notch signaling. Exogenous injection of ghrelin also attenuated the severity of radiation-induced intestinal injury in a mouse model. CONCLUSION: These data suggest that ghrelin may be used as a potential therapeutic agent for radiation-induced enteropathy.

Pravastatin Alleviates Radiation Proctitis by Regulating Thrombomodulin in Irradiated Endothelial Cells.

Although radiotherapy plays a crucial in the management of pelvic tumors, its toxicity on surrounding healthy tissues such as the small intestine, colon, and rectum is one of the major limitations associated with its use. In particular, proctitis is a major clinical complication of pelvic radiotherapy. Recent evidence suggests that endothelial injury significantly affects the initiation of radiation-induced inflammation. The damaged endothelial cells accelerate immune cell recruitment by activating the expression of endothelial adhesive molecules, which participate in the development of tissue damage. Pravastatin, a cholesterol lowering drug, exerts persistent anti-inflammatory and anti-thrombotic effects on irradiated endothelial cells and inhibits the interaction of leukocytes and damaged endothelial cells. Here, we aimed to investigate the effects of pravastatin on radiation-induced endothelial damage in human umbilical vein endothelial cell and a murine proctitis model. Pravastatin attenuated epithelial damage and inflammatory response in irradiated colorectal lesions. In particular, pravastatin improved radiation-induced endothelial damage by regulating thrombomodulin (TM) expression. In addition, exogenous TM inhibited leukocyte adhesion to the irradiated endothelial cells. Thus, pravastatin can inhibit endothelial damage by inducing TM, thereby alleviating radiation proctitis. Therefore, we suggest that pharmacological modulation of endothelial TM may limit intestinal inflammation after irradiation.

PLGA Nanoparticles Codelivering siRNAs against Programmed Cell Death Protein-1 and Its Ligand Gene for Suppression of Colon Tumor Growth.

Tumor-infiltrating T lymphocytes highly express programmed cell death protein-1 (PD-1) that interacts with its ligand, programmed cell death protein ligand-1 (PD-L1) on tumors. PD-1/PD-L1 interactions cause functional exhaustion of effector T cells and impair antitumor immunity, allowing tumors to escape immune surveillance. In addition to such extrinsic interactions, tumors proliferate by transmitting intrinsic PD-L1 signals via the mTOR pathway. Here, we simultaneously silenced PD-1 and PD-L1 expressions on CTLs and colon tumors using PD-1 siRNA/PD-L1 siRNA-loaded PLGA nanoparticles and investigated functional activation of tumor-specific CTLs. When compared to a single PD-1 silencing on CTLs or a single PD-L1 silencing on tumors, cosilencing of PD-1/PD-L1 on CTLs and tumors more efficiently promoted effector functions of tumor-specific CTLs. Moreover, PD-L1-silenced tumors inhibited mTOR signaling and showed an antiproliferative response independent of the adaptive immune response. Ultimately, systemic administration of PD-1 and PD-L1 siRNA via PLGA nanoparticles restored the effector functions of tumor-specific CTLs in MC38 tumor-bearing mice. Compared with antitumor effects of single silencing of PD-1 or PD-L1 alone, cosilencing of PD-1 and PD-L1 showed more significant tumor growth suppression and long-term tumor inhibition in colon cancer. Thus, this study provides an efficient therapeutic strategy for achieving immunotherapy in colon cancer.

Tumor-treating fields induce autophagy by blocking the Akt2/miR29b axis in glioblastoma cells.

Tumor-treating fields (TTFs) - a type of electromagnetic field-based therapy using low-intensity electrical fields - has recently been characterized as a potential anticancer therapy for glioblastoma multiforme (GBM). However, the molecular mechanisms involved remain poorly understood. Our results show that the activation of autophagy contributes to the TTF-induced anti-GBM activity in vitro or in vivo and GBM patient stem cells or primary in vivo culture systems. TTF-treatment upregulated several autophagy-related genes (~2-fold) and induced cytomorphological changes. TTF-induced autophagy in GBM was associated with decreased Akt2 expression, not Akt1 or Akt3, via the mTOR/p70S6K pathway. An Affymetrix GeneChip miRNA 4.0 Array analysis revealed that TTFs altered the expression of many microRNAs (miRNAs). TTF-induced autophagy upregulated miR-29b, which subsequently suppressed the Akt signaling pathway. A luciferase reporter assay confirmed that TTFs induced miR-29b to target Akt2, negatively affecting Akt2 expression thereby triggering autophagy. TTF-induced autophagy suppressed tumor growth in GBM mouse models subjected to TTFs as determined by positron emission tomography and computed tomography (PET-CT). GBM patient stem cells and a primary in vivo culture system with high Akt2 levels also showed TTF-induced inhibition. Taken together, our results identified autophagy as a critical cell death pathway triggered by TTFs in GBM and indicate that TTF is a potential treatment option for GBM.

A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry.

The transient silencing effects currently demonstrated by nonviral siRNA delivery systems limit the therapeutic utility of RNAi, but it remains a technical challenge to prolong duration of gene silencing. We have developed a T7 autogene-based hybrid mRNA/DNA system to enable long-term expression of shRNA in cytoplasm in vitro and in vivo. This hybrid mRNA/DNA system consists of T7 polymerase (T7pol) mRNA, pT7/shRNA-encoding DNA fragment and T7 autogene plasmid, and it can generate higher levels of T7pol proteins, compared to pCMV-triggering T7 autogene system, especially without the need of nuclear entry of any gene. A large amount of T7pol proteins produced are used to induce pT7-driven expression of shRNA in cytoplasm, and through cellular processing of RNA hairpins, mature siRNAs are generated for more than 13 days. We here demonstrate that a single liposomal delivery of this hybrid system leads to the long-term silencing effects in vitro and in vivo, in contrast to the conventional siRNA methods relying on the repeated administrations every 2 or 3 days. These sustainable shRNA expression properties in cytoplasm can provide an efficient strategy to address the limitations caused by shRNA-encoding plasmid DNA systems such as low nuclear entry efficiency and short-term silencing effect. The development of long-term shRNA expression system in vivo could scale down administration frequency of RNAi therapeutics in the treatment of chronic diseases, thereby increasing its clinical utility.

Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

MicroRNAs (miRNAs) play pivotal roles in tumorigenesis as either tumor suppressors or oncogenes. In the present study, we discovered and demonstrated the tumor suppressive function of a novel miRNA miR-5582-5p. miR-5582-5p induced apoptosis and cell cycle arrest in cancer cells, but not in normal cells. GAB1, SHC1, and CDK2 were identified as direct targets of miR-5582-5p. Knockdown of GAB1/SHC1 or CDK2 phenocopied the apoptotic or cell cycle arrest-inducing function of miR-5582-5p, respectively. The expression of miR-5582-5p was lower in tumor tissues than in adjacent normal tissues of colorectal cancer patients, while the expression of the target proteins exhibited patterns opposite to that of miR-5582-5p. Intratumoral injection of a miR-5582-5p mimic or induced expression of miR-5582-5p in tumor cells suppressed tumor growth in HCT116 xenografts. Collectively, our results suggest a novel tumor suppressive function for miR-5582-5p and its potential applicability for tumor control.

miR-5003-3p promotes epithelial-mesenchymal transition in breast cancer cells through Snail stabilization and direct targeting of E-cadherin.

One of the initial steps in metastatic dissemination is the epithelial-mesenchymal transition (EMT). Along this line, microRNAs (miRNAs) have been shown to function as important regulators of tumor progression at various stages. Therefore, we performed a functional screening for EMT-regulating miRNAs and identified several candidate miRNAs. Among these, we demonstrated that miR-5003-3p induces cellular features characteristic of EMT. miR-5003-3p induced upregulation of Snail, a key EMT-promoting transcription factor and transcriptional repressor of E-cadherin, through protein stabilization. MDM2 was identified as a direct target of miR-5003-3p, the downregulation of which induced Snail stabilization. E-cadherin was also demonstrated to be a direct target of miR-5003-3p, reinforcing the EMT-promoting function of miR-5003-3p. In situ hybridization and immunohistochemical analyses using tissue microarrays revealed that miR-5003-3p expression was higher in paired metastatic breast carcinoma tissues than in primary ductal carcinoma tissues, and was inversely correlated with the expression of MDM2 and E-cadherin. Furthermore, miR-5003-3p enhanced the formation of metastatic nodules in the lungs of mice in a tail vein injection experiment. Collectively, our results suggest that miR-5003-3p functions as a metastasis activator by promoting EMT through dual regulation of Snail stability and E-cadherin, and may therefore be a potential therapeutic target in metastatic cancers.

miR-181b-3p promotes epithelial-mesenchymal transition in breast cancer cells through Snail stabilization by directly targeting YWHAG.

Epithelial-mesenchymal transition (EMT) is essential for increased invasion and metastasis during cancer progression. Among the candidate EMT-regulating microRNAs that we previously identified, miR-181b-3p was found to induce EMT in MCF7 breast cancer cells, as indicated by an EMT-characteristic morphological change, increased invasiveness, and altered expression of an EMT marker. Transfection with a miR-181b-3p inhibitor reduced the expression of mesenchymal markers and the migration and invasion of highly invasive breast cancer cells. miR-181b-3p induced the upregulation of Snail, a master EMT inducer and transcriptional repressor of E-cadherin, through protein stabilization. YWHAG was identified as a direct target of miR-181b-3p, downregulation of which induced Snail stabilization and EMT phenotypes. Ectopic expression of YWHAG abrogated the effect of miR-181b-3p, including Snail stabilization and the promotion of invasion. In situ hybridization and immunohistochemical analyses indicated that YWHAG expression was inversely correlated with the expression of miR-181b-3p and Snail in human breast cancer tissues. Furthermore, transfection with miR-181b-3p increased the frequency of metastatic nodule formation in the lungs of mice in experimental metastasis assays using MDA-MB-231 cells. Taken together, our data suggest that miR-181b-3p functions as a metastasis activator by promoting Snail-induced EMT, and may therefore be a therapeutic target in metastatic cancers.

Ionizing radiation-inducible miR-30e promotes glioma cell invasion through EGFR stabilization by directly targeting CBL-B.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the transcriptional and post-transcriptional levels. Here we show that miR-30e, which was previously identified as an ionizing radiation-inducible miRNA, enhances cellular invasion by promoting secretion of the matrix metalloproteinase MMP-2. The enhancement of cellular invasion by miR-30e involved up-regulation of the epidermal growth factor receptor (EGFR) and subsequent activation of its downstream signaling mediators, AKT and extracellular signal-regulated kinase. EGFR up-regulation by miR-30e occurred due to stabilization of the EGFR protein. The E3 ubiquitin ligase casitas B-lineage lymphoma B (CBL-B) was down-regulated by miR-30e, and this led to increased EGFR abundance. A 3' UTR reporter assay confirmed that CBL-B is a direct target of miR-30e. Knocking down CBL-B expression phenocopied the effects of miR-30e, whereas ectopic expression of CBL-B suppressed miR-30e-induced EGFR up-regulation and invasion. Collectively, our results suggest that targeting miR-30e may limit the invasiveness induced during glioma radiotherapy.

Association of p21-activated kinase-1 activity with aggressive tumor behavior and poor prognosis of head and neck cancer.

BACKGROUND: This study investigated the role of p21-activated kinase (PAK)-1 in progression of head and neck squamous cell carcinoma (HNSCC). METHODS: We examined PAK isoforms and explored whether PAK activation enhanced in vitro invasion of the HNSCC cell line. We analyzed the relationship between PAK1 expression and various clinicopathological features and investigated the effect of PAK1 overexpression on survival in 119 patients with HNSCC. RESULTS: PAK1 and PAK2 are predominantly expressed in HNSCC cells and patient tissues. Particularly, PAK1 makes the dominant contribution to increase in cell migration and invasion. There was a statistically significant correlation between PAK1 overexpression and aggressive cancer behavior. Moreover, PAK1 seemed to be a prognostic factor for overall and disease-specific survival in patients. Interestingly, enhancement of PAK1 expression was found in the invasive front of cancer. CONCLUSION: PAK1 is associated with the aggressive tumor behavior and poor prognosis of head and neck cancer.

Role of mineralization inhibitors in the regulation of hard tissue biomineralization: relevance to initial enamel formation and maturation.

Vertebrate mineralized tissues, i.e., enamel, dentin, cementum, and bone, have unique hierarchical structures and chemical compositions. Although these tissues are similarly comprised of a crystalline calcium apatite mineral phase and a protein component, they differ with respect to crystal size and shape, level and distribution of trace mineral ions, the nature of the proteins present, and their relative proportions of mineral and protein components. Despite apparent differences, mineralized tissues are similarly derived by highly concerted extracellular processes involving matrix proteins, proteases, and mineral ion fluxes that collectively regulate the nucleation, growth and organization of forming mineral crystals. Nature, however, provides multiple ways to control the onset, rate, location, and organization of mineral deposits in developing mineralized tissues. Although our knowledge is quite limited in some of these areas, recent evidence suggests that hard tissue formation is, in part, controlled through the regulation of specific molecules that inhibit the mineralization process. This paper addresses the role of mineralization inhibitors in the regulation of biological mineralization with emphasis on the relevance of current findings to the process of amelogenesis. Mineralization inhibitors can also serve to maintain driving forces for calcium phosphate precipitation and prevent unwanted mineralization. Recent evidence shows that native phosphorylated amelogenins have the capacity to prevent mineralization through the stabilization of an amorphous calcium phosphate precursor phase, as observed in vitro and in developing teeth. Based on present findings, the authors propose that the transformation of initially formed amorphous mineral deposits to enamel crystals is an active process associated with the enzymatic processing of amelogenins. Such processing may serve to control both initial enamel crystal formation and subsequent maturation.

Regulation of calcium phosphate formation by native amelogenins in vitro.

Our previous in vitro studies have shown that recombinant full-length porcine amelogenin rP172 can transiently stabilize amorphous calcium phosphate (ACP) and uniquely guide the formation of well-aligned bundles of hydroxyapatite (HA) crystals, as seen in the secretory stage of amelogenesis. This functional capacity is dependent on the hydrophilic C-terminal domain of full-length amelogenin. However, we have also found that native phosphorylated (single S-16 site) forms of full-length (P173) and C-terminal cleaved (P148) amelogenins can stabilize ACP for > 2 d and prevent HA formation. The present study was carried out to test the hypothesis that, at reduced concentrations, native full-length P173 also has the capacity to guide ordered HA formation. The effect of P148 and P173 concentrations (0.2-2.0 mg/ml) on the rate of spontaneous calcium phosphate precipitation was monitored via changes in solution pH, while mineral phases formed were assessed using TEM. At higher P173 concentrations (1.0-2.0 mg/ml), limited mineral formation occurred and only ACP nanoparticles were observed during a 48 h period. However, at 0.4 mg/ml P173, a predominance of organized bundles of linear, needle-like HA crystals were observed. At 0.2 mg/ml of P173, limited quantities of less organized HA crystals were found. Although P148 similarly stabilized ACP, it did not guide ordered HA formation, like P173. Hence, the establishment of the hierarchical enamel structure during secretory stage amelogenesis may be regulated by the partial removal of full-length amelogenin via MMP20 proteolysis, while predominant amelogenin degradation products, like P148, serve to prevent uncontrolled mineral formation.

ß,ß-Dimethylacrylshikonin sensitizes human colon cancer cells to ionizing radiation through the upregulation of reactive oxygen species.

Shikonin, a naphthoquinone derivative, has been shown to possess antitumor activity. In the present study, the effects of shikonin and its analog, ß,ß-dimethylacrylshikonin, were investigated as radiosensitizers on the human colon cancer cell line, HCT-116. Shikonin and, to a greater extent, its analog-induced apoptosis of HCT-116 cells further synergistically potentiated the induction of apoptosis when combined with ionizing radiation (IR) treatment. Shikonins also stimulated an increase in reactive oxygen species (ROS) production and IR-induced DNA damage. Pre-treatment with the ROS scavenger, N-acetylcysteine, suppressed the enhancement of IR-induced DNA damage and apoptosis stimulated by shikonins, indicating that shikonins exert their radiosensitizing effects through ROS upregulation. The radiosensitizing effect of shikonins was also examined in vivo using the xenograft mouse model. Consistent with the in vitro results, injection of ß,ß-dimethylacrylshikonin combined with IR treatment significantly suppressed tumor growth of the HCT-116 xenograft. Taken together, the results show that ß,ß-dimethylacrylshikonin is a promising agent for developing an improved strategy for radiotherapy against tumors.

Ionizing radiation-inducible miR-494 promotes glioma cell invasion through EGFR stabilization by targeting p190B rhoGAP.

MicroRNAs (miRNAs) play an important role in various stages of tumor progression. miR-494, which we had previously identified as a miRNA induced by ionizing radiation (IR) in the glioma cell line U-251, was observed to enhance invasion of U-251 cells by activating MMP-2. The miR-494-induced invasive potential was accompanied by, and dependent on, epidermal growth factor receptor (EGFR) upregulation and the activation of its downstream signaling constituents, Akt and ERK. The upregulation of EGFR by miR-494 involved the suppression of lysosomal protein turnover. Among the putative target proteins tested, p190B RhoGAP (p190B) was downregulated by miR-494, and its reduced expression was responsible for the increase in EGFR expression. A reporter assay using a luciferase construct containing p190B 3'-untranslated region (3'UTR) confirmed that p190B is a direct target of miR-494. Downregulation of p190B by small interfering RNA (siRNA) transfection closely mimicked the outcomes of miR-494 transfection, and showed increased EGFR expression, MMP-2 secretion, and invasion. Ectopic expression of p190B suppressed the miR-494-induced EGFR upregulation and invasion promotion, thereby suggesting that p190B depletion is critical for the invasion-promoting action of miR-494. Collectively, our results suggest a novel function for miR-494 and its potential application as a target to control invasiveness in cancer therapy.