The effect of Schizophyllan on the differentiation of osteoclasts and osteoblasts.

Schizophyllan (SPG), a ß-glucan from Schizophyllum commune, is recognized for its antioxidant, immunoregulatory, and anticancer activities. In this study, its effects on bone cells, particularly osteoclasts and osteoblasts, were examined. We demonstrated that SPG dose-dependently inhibited osteoclastogenesis and reduced gene expression associated with osteoclast differentiation. SPG also decreased bone resorption and F-actin ring formation. This inhibition could have been due to the downregulation of transcription factors c-Fos and nuclear factor of activated T cells 1 (NFATc1) via the MAPKs (JNK and p38), IκBα, and PGC1ß/PPARγ pathways. In coculture, SPG lowered osteoclastogenic activity in calvaria-derived osteoblasts by reducing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) expression. In addition, SPG slightly enhanced osteoblast differentiation, as evidenced by increased differentiation marker gene expression and alizarin red staining. It also exhibited antiresorptive effects in a lipopolysaccharide-induced calvarial bone loss model. These results indicated a dual role of SPG in bone cell regulation by suppressing osteoclastogenesis and promoting osteoblast differentiation. Thus, SPG could be a therapeutic agent for bone resorption-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Complex interplay of oral health, muscle and bone metabolism, and frailty in older individuals.

OBJECTIVES: To investigate molecular and clinical background of associations among oral health, muscle and bone metabolism, and frailty incidence in patients with fall and fracture history. MATERIALS AND METHODS: In total, 88 elderly participants (mean age 71.9 ± 5.8 years) with the distal radius fractures were included. Participants were divided into three groups based on an Oral Health Assessment Tool score. Fried criteria and Mini-nutritional assessments were adopted to diagnose frailty and malnutrition, respectively. Blood samples were collected and analyzed for serum levels of bone turnover markers, proteins, insulin-like growth factor-1, 25-hydroxyvitamin D, and inflammatory cytokines. The mRNA levels of markers of inflammation, muscle synthesis and wasting, and muscle homeostasis regulator in the pronator quadratus muscle were analyzed. RESULTS: Patients with deteriorated oral health demonstrated a higher prevalence of frailty and malnutrition. Significantly lower serum levels of total protein and higher concentrations of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were detected in patients with poor oral health. Significant interaction effects between oral health and frailty level in gait speed, serum TNF-α, IL-1ß, and total protein levels were exhibited. Significantly different mRNA expression levels in the pronator quadratus muscle of TNF-α, IL-1ß, NF kB, MYOG, and FOXO1 following the oral health were detected. CONCLUSION: This study highlights relationship between oral health, nutritional uptake, systemic inflammation, and their combined impact on muscle and bone metabolism, ultimately affecting frailty development in the aging populations. CLINICAL RELEVANCE: A comprehensive understanding of mutual interactions among oral health, nutrition, and inflammation is essential for managing frailty.

Transcriptional Profiling of Muscle in Females With Distal Radius Fracture and Functional Sarcopenia.

Skeletal muscle and bone interact with each other in mechanical and biochemical ways. This study aimed to investigate the molecular mechanisms of interaction between muscle and bone by analyzing the transcriptional profiles of total RNA from the muscle tissue of females with distal radius fractures. A total of 30 female participants (mean age 71.1 ±â€…8.9 years) with distal radius fractures were recruited. Participants were categorized into 2 groups: the NORM group consisted of participants with T score of the areal bone mineral density (aBMD) of the femoral neck higher than -1.0, handgrip strength greater than 18 kg, and gait speed faster than 1.0 m/s (n = 10). Otherwise, participants with T score of the aBMD of the femoral neck equal to or less than -1.0, handgrip strength lower than 18 kg, and gait speed slower than 1.0 m/s (n = 20) were categorized into EXP group. Pronator quadratus muscle samples were obtained from all participants. Total RNA was extracted from frozen muscle samples and sequenced. The gene ontology analysis demonstrated that the potential interactions between attached muscle function and the density of the associated bone would be linked with collagen biosynthetic activity and maintenance of extracellular matrix structures. The analysis of the pathway, network, and protein class exhibited that integrin signaling, inflammatory reactions, matrix metalloproteinase (MMP) activity, and extracellular matrix protein structure had possible associations with the molecular background of muscle-bone interaction. Through integrin signaling, MMP activity, inflammatory reactions, and collagen biosynthesis, muscle and bone may mutually interact with one another.

Biological dosimetry dose-response curves for residents living near nuclear power plants in South Korea.

The purpose of this study was to establish the dose-response curves for biological dosimetry of the Dong Nam Institute of Radiological and Medical Sciences to monitor radiation exposure of local residents in the vicinity of the nuclear power plant. The blood samples of five healthy volunteers were irradiated with gamma ray, and each sample was divided equally for analysis of chromosomal aberrations by Giemsa staining and three-color fluorescence in situ hybridization painting of the triplet (chromosomes #1, #2, and #4). The results of chromosomal aberrations followed the Poisson distribution in all individual and averaged data which include inter-individual variation in radiation susceptibility. Cytogenetics Dose Estimate Software version 5.2 was used to fit the dose-response curve and to determine the coefficients of linear-quadratic equations. The goodness of fit of the curves and statistical significance of fitted α and ß-coefficients were confirmed in both Giemsa-based dicentric analysis and FISH-based translocation analysis. The coefficients calculated from the five-donor average data were almost identical in both of the analyses. We also present the results that the dose-response curve for dicentric chromosomes plus fragments could be more effective for dose estimation following low-dose radiation accidents.

Undercarboxylated, But Not Carboxylated, Osteocalcin Suppresses TNF-α-Induced Inflammatory Signaling Pathway in Myoblasts.

Undercarboxylated osteocalcin (ucOCN) has been considered to be an important endocrine factor, especially to regulate bone and energy metabolism. Even with the mounting evidence showing the consistent inverse correlation of ucOCN levels in chronic inflammatory diseases, however, the mechanism underlying the involvement of ucOCN in the muscular inflammation has not been fully understood. In the present study, we explored 1) the endocrine role of ucOCN in the regulation of inflammation in C2C12 myoblasts and primary myoblasts and the underlying intracellular signaling mechanisms, and 2) whether G protein-coupled receptor family C group 6 member A (GPRC6A) is the ucOCN-sensing receptor associated with the ucOCN-mediated anti-inflammatory signaling pathway in myoblasts. ucOCN suppressed the tumor necrosis factor-α (TNF-α)-induced expressions of major inflammatory cytokines, including interleukin-1ß (IL-1ß) and inhibited the TNF-α-stimulated activities of transcription factors, including NF-κB, in C2C12 and primary myoblasts. Both knockdown and knockout of GPRC6A, by using siRNA or a CRISPR/CAS9 system, respectively, did not reverse the effect of ucOCN on IL-1ß expression in myoblasts. Interestingly, TNF-α-induced IL-1ß expression was inhibited by knockdown or deletion of GPRC6A itself, regardless of the ucOCN treatment. ucOCN was rapidly internalized into the cytoplasmic region via caveolae-mediated endocytosis, suggesting the presence of new target proteins in the cell membrane and/or in the cytoplasm for interaction with ucOCN in myoblasts. Taken together, these findings indicate that ucOCN suppresses the TNF-α-induced inflammatory signaling pathway in myoblasts. GPRC6A is not a sensing receptor associated with the ucOCN-mediated anti-inflammatory signaling pathway in myoblasts.

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.

Differential Effects of Low and High Radiation Dose Rates on Mouse Spermatogenesis.

The adverse effects of radiation are proportional to the total dose and dose rate. We aimed to investigate the effects of radiation dose rate on different organs in mice. The mice were subjected to low dose rate (LDR, ~3.4 mGy/h) and high dose rate (HDR, ~51 Gy/h) radiation. LDR radiation caused severe tissue toxicity, as observed in the histological analysis of testis. It adversely influenced sperm production, including sperm count and motility, and induced greater sperm abnormalities. The expression of markers of early stage spermatogonial stem cells, such as Plzf, c-Kit, and Oct4, decreased significantly after LDR irradiation, compared to that following exposure of HDR radiation, in qPCR analysis. The compositional ratios of all stages of spermatogonia and meiotic cells, except round spermatid, were considerably reduced by LDR in FACS analysis. Therefore, LDR radiation caused more adverse testicular damage than that by HDR radiation, contrary to the response observed in other organs. Therefore, the dose rate of radiation may have differential effects, depending on the organ; it is necessary to evaluate the effect of radiation in terms of radiation dose, dose rate, organ type, and other conditions.

Intratesticular Peptidyl Prolyl Isomerase 1 Protein Delivery Using Cationic Lipid-Coated Fibroin Nanoparticle Complexes Rescues Male Infertility in Mice.

Male infertility is a multifactorial condition. Unexplained male infertility is often caused by spermatogenesis dysfunction. Knockout of Pin1, an important regulator of cell proliferation and differentiation, produces male infertility phenotypes such as testicular immaturity and azoospermia with spermatogonia depletion and blood-testis barrier (BTB) dysfunction. Gene therapy has been clinically considered for the treatment of male infertility, but it is not preferred because of the risks of adverse effects in germ cells. Direct intracellular protein delivery using nanoparticles is considered an effective alternative to gene therapy; however, in vivo testicular protein delivery remains a pressing challenge. Here, we investigated the direct intracellular protein delivery strategy using a fibroin nanoparticle-encapsulated cationic lipid complex (Fibroplex) to restore intratesticular PIN1. Local intratesticular delivery of PIN1 via Fibroplex in Pin1 knockout testes produced fertile mice, achieving recovery from the infertile phenotypes. Mechanistically, PIN1-loaded Fibroplex was successfully delivered into testicular cells, including spermatogonial cells and Sertoli cells, and the sustained release of PIN1 restored the gene expression required for the proliferation of spermatogonial cells and BTB integrity in Pin1 knockout testes. Collectively, testicular PIN1 protein delivery using Fibroplex might be an effective strategy for treating male infertility.

Gene expression profiling of glioblastoma cell lines depending on TP53 status after tumor-treating fields (TTFields) treatment.

Glioblastoma is frequently associated with TP53 mutation, which is linked to a worse prognosis and response to conventional treatments (chemoradiotherapy). Therefore, targeting TP53 is a promising strategy to overcome this poor therapeutic response. Tumor-treating fields (TTFields) are a recently approved treatment for newly diagnosed glioblastoma, which involves direct application of low-intensity, intermediate-frequency alternating electric fields to the tumor, thereby offering a local tumor-killing effect. However, the influence of TP53 mutation status on the effectiveness of TTFields is controversial. Here, we identified the key gene signatures and pathways associated with TTFields in four glioblastoma cell lines varying in TP53 mutation status using gene profiling and functional annotation. Overall, genes associated with the cell cycle, cell death, and immune response were significantly altered by TTFields regardless of TP53 status. TTFields appeared to exert enhanced anti-cancer effects by altering the immune system in the inflammatory environment and regulating cell cycle- and cell death-related genes, but the precise genes influenced vary according to TP53 status. These results should facilitate detailed mechanistic studies on the molecular basis of TTFields to further develop this modality as combination therapy, which can improve the therapeutic effect and minimize side effects of chemoradiotherapy.

Kinesin light chain 4 as a new target for lung cancer chemoresistance via targeted inhibition of checkpoint kinases in the DNA repair network.

The poor therapeutic efficacy of non-small cell lung cancer (NSCLC) is partly attributed to the acquisition of chemoresistance. To investigate the mechanism underlying this resistance, we examined the potential link between kinesin light chain 4 (KLC4), which we have previously reported to be associated with radioresistance in NSCLC, and sensitivity to chemotherapy in human lung cancer cell lines. KLC4 protein levels in lung cancer cells correlated with the degree of chemoresistance to cisplatin treatment. Furthermore, KLC4 silencing enhanced the cytotoxic effect of cisplatin by promoting DNA double-strand breaks and apoptosis. These effects were mediated by interaction with the checkpoint kinase CHK2, as KLC4 knockdown increased CHK2 activation, which was further enhanced in combination with cisplatin treatment. In addition, KLC4 and CHEK2 expression levels showed negative correlation in lung tumor samples from patients, and KLC4 overexpression correlated negatively with survival. Our results indicate a novel link between the KLC4 and CHK2 pathways regulating DNA damage response in chemoresistance, and highlight KLC4 as a candidate for developing lung cancer-specific drugs and customized targeted molecular therapy.

GDF11 promotes osteogenesis as opposed to MSTN, and follistatin, a MSTN/GDF11 inhibitor, increases muscle mass but weakens bone.

Growth and differentiation factor 11 (GDF11) and myostatin (MSTN) are closely related transforming growth factor ß (TGF-ß) family members, but their biological functions are quite distinct. While MSTN has been widely shown to inhibit muscle growth, GDF11 regulates skeletal patterning and organ development during embryogenesis. Postnatal functions of GDF11, however, remain less clear and controversial. Due to the perinatal lethality of Gdf11 null mice, previous studies used recombinant GDF11 protein to prove its postnatal function. However, recombinant GDF11 and MSTN proteins share nearly identical biochemical properties, and most GDF11-binding molecules have also been shown to bind MSTN, generating the possibility that the effects mediated by recombinant GDF11 protein actually reproduce the endogenous functions of MSTN. To clarify the endogenous functions of GDF11, here, we focus on genetic studies and show that Gdf11 null mice, despite significantly down-regulating Mstn expression, exhibit reduced bone mass through impaired osteoblast (OB) and chondrocyte (CH) maturations and increased osteoclastogenesis, while the opposite is observed in Mstn null mice that display enhanced bone mass. Mechanistically, Mstn deletion up-regulates Gdf11 expression, which activates bone morphogenetic protein (BMP) signaling pathway to enhance osteogenesis. Also, mice overexpressing follistatin (FST), a MSTN/GDF11 inhibitor, exhibit increased muscle mass accompanied by bone fractures, unlike Mstn null mice that display increased muscle mass without fractures, indicating that inhibition of GDF11 impairs bone strength. Together, our findings suggest that GDF11 promotes osteogenesis in contrast to MSTN, and these opposing roles of GDF11 and MSTN must be considered to avoid the detrimental effect of GDF11 inhibition when developing MSTN/GDF11 inhibitors for therapeutic purposes.

Antimicrobial photodynamic therapy efficacy against specific pathogenic periodontitis bacterial species.

BACKGROUND: To determine the safety and efficacy of antimicrobial photodynamic therapy (aPDT) combination of 0.33 mM Toluidine Blue O (TBO) with 60 mW/cm2 LED irradiation for 5 min that we had established, this study investigated the cytotoxic effect of aPDT combination on mammalian oral cells (gingival fibroblast and periodontal ligament cells) and compared the antimicrobial efficacy of antibiotics (the combination of amoxicillin (AMX) and metronidazole (MTZ)) against representative periodontitis pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans) versus our aPDT combination. RESULT: aPDT combination did not show any detectable effect on the viability of Streptococcus sanguinis or Streptococcus mitis, the most common resident species in the oral flora. However, it significantly reduced CFU values of P. gingivalis, F. nucleatum, and A. actinomycetemcomitans. The cytotoxicity of the present aPDT combination to mammalian oral cells was comparable to that of standard antiseptics used in oral cavity. In antimicrobial efficacy test, the present aPDT combination showed equivalent bactericidal rate compared to the combination of AMX + MTZ, the most widely used antibiotics in the periodontitis treatment. The bactericidal ability of the AMX + MTZ combination was effective against all five bacteria tested regardless of the bacterial species, whereas the bactericidal ability of the aPDT combination was effective only against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, the representative periodontitis pathogenic bacterial species. CONCLUSION: The present study demonstrated the safety and efficacy of the present aPDT combination in periodontitis treatment. TBO-mediated aPDT with LED irradiation has the potential to serve as a safe single or adjunctive antimicrobial procedure for nonsurgical periodontal treatment without damaging adjacent normal oral tissue or resident flora.

Histone acetylation together with DNA demethylation empowers higher plasticity in adipocytes to differentiate into osteoblasts.

Bone regeneration has been a challenge for both researchers and clinicians. In the field of tissue engineering, much effort has been made to identify cell sources including stem cells. The present study aimed to induce trans-differentiation from adipocytes to osteoblasts using epigenetic modifiers; 5-aza-dC and/or trichostatin-A (TSA). 3 T3-L1 preadipocytes were treated with TSA (100 nM) and then with Wnt3a (50 ng/ml). Microscopic observation showed trans-differentiated cell morphology. Methylation-specific PCR and immunoblotting were performed to analyze the DNA methylation and histone acetylation patterns. The gene expression was determined by real-time PCR. Based on these in vitro experiments, in vivo mouse experiments supplemented the possibility of trans-differentiation by epigenetic modification. TSA induced the acetylation of lysine9 on histone H3, and a sequential Wnt3a treatment stimulated the expression of bone marker genes in adipocytes, suppressing adipogenesis and stimulating osteogenesis. Furthermore, TSA induced DNA hypomethylation, and a combined treatment with TSA and 5-aza-dC showed a synergistic effect in epigenetic modifications. The number of adipocytes and DNA methylation patterns of old (15 months) and young (6 weeks) mice were significantly different, and TSA and sequential Wnt3a treatments increased bone formation in the old mice. Collectively, our results confirmed cell trans-differentiation via epigenetic modifications and osteogenic signaling from adipocytes to osteoblasts for the bone regeneration in vitro and in vivo, and indicated that histone acetylation could induce DNA hypomethylation, enhancing the chance of trans-differentiation.

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.

Growth differentiation factor 11 locally controls anterior-posterior patterning of the axial skeleton.

Growth and differentiation factor 11 (GDF11) is a transforming growth factor ß family member that has been identified as the central player of anterior-posterior (A-P) axial skeletal patterning. Mice homozygous for Gdf11 deletion exhibit severe anterior homeotic transformations of the vertebrae and craniofacial defects. During early embryogenesis, Gdf11 is expressed predominantly in the primitive streak and tail bud regions, where new mesodermal cells arise. On the basis of this expression pattern of Gdf11 and the phenotype of Gdf11 mutant mice, it has been suggested that GDF11 acts to specify positional identity along the A-P axis either by local changes in levels of signaling as development proceeds or by acting as a morphogen. To further investigate the mechanism of action of GDF11 in the vertebral specification, we used a Cdx2-Cre transgene to generate mosaic mice in which Gdf11 expression is removed in posterior regions including the tail bud, but not in anterior regions. The skeletal analysis revealed that these mosaic mice display patterning defects limited to posterior regions where Gdf11 expression is deficient, whereas displaying normal skeletal phenotype in anterior regions where Gdf11 is normally expressed. Specifically, the mosaic mice exhibited seven true ribs, a pattern observed in wild-type (wt) mice (vs. 10 true ribs in Gdf11-/- mice), in the anterior axis and nine lumbar vertebrae, a pattern observed in Gdf11 null mice (vs. six lumbar vertebrae in wt mice), in the posterior axis. Our findings suggest that GDF11, rather than globally acting as a morphogen secreted from the tail bud, locally regulates axial vertebral patterning.

Undercarboxylated osteocalcin downregulates pancreatic lipase expression in an ATF4-dependent manner in pancreatic acinar cells.

Osteocalcin is an osteoblast-specific secreted protein that has been associated with endocrine roles in multiple aspects of energy metabolism. We examined whether undercarboxylated osteocalcin (ucOC) downregulates pancreatic lipase (PNLIP) expression in pancreatic acinar cells and then identified the downstream signaling pathway involved. We previously demonstrated that ß adrenergic blockade attenuates body weight/fat mass gain in high-fat diet-fed mice and that this effect is associated with decreased PNLIP expression in pancreatic acinar cells. In the present study, we first confirmed that the serum ucOC level is inversely correlated with PNLIP expression, i.e., mice exhibiting high serum levels of ucOC showed low PNLIP levels in the pancreas. In in vitro experiments using primary pancreatic acinar and 266-6 cells, ucOC downregulated PNLIP expression. cAMP/PKA signaling inhibitors significantly reversed ucOC-induced downregulation of PNLIP expression. ucOC promoted the phosphorylation of cAMP response element-binding protein 2 (ATF4). Overexpression of ATF4 significantly suppressed PNLIP expression. Knockdown of ATF4 by siRNA reversed the ucOC-induced downregulation of PNLIP expression. A luciferase reporter assay showed that ucOC suppressed PNLIP promoter transactivation. Chromatin immunoprecipitation and a luciferase reporter assay demonstrated that ATF4 directly bound to the CRE on the mouse PNLIP promoter and suppressed PNLIP transactivation. Knockdown of G-protein coupled receptor 6A (Gprc6a), a candidate receptor for mediating the response to ucOC in the bone-pancreas endocrine loop, by siRNA reversed the downregulating effect of ucOC on PNLIP expression. Taken together, ucOC downregulates pancreatic lipase expression in a cAMP/protein kinase A/ATF4-dependent manner. Gprc6a is a potential osteocalcin-sensing receptor that regulates PNLIP expression in pancreatic acinar cells.

PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway.

Current lung cancer treatments are far from satisfactory; thus, finding novel treatment targets is crucial. We recently identified procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3), which is involved in fibrosis and tissue remodeling as a radioresistance-related protein in lung cancer cells; however, its mechanism is unclear. In this study, we designed human PLOD3-specific short interfering (si)RNAs and tested their effects on tumor growth inhibition in vitro and in vivo. PLOD3 knockdown overcame chemoresistance and decreased radioresistance by inducing caspase-3-dependent apoptosis in lung cancer cells. Furthermore, PLOD3 interacted with PKCδ to activate caspase-2,4-dependent apoptosis through ER-stress-induced IRE1α activation and the downstream unfolded-protein response pathway. In a mouse xenograft model, PLOD3 knockdown promoted radiation-induced tumor growth inhibition, without side effects. Moreover, lung cancer patients with high PLOD3 expression showed poorer prognosis than those with low PLOD3 expression upon radiotherapy, suggesting that PLOD3 promotes tumor growth. Therefore, PLOD3 siRNA suppresses radioresistance and chemoresistance by inducing apoptosis and renders PLOD3 as a candidate lung cancer biomarker. PLOD3 gene therapy might enhance the efficacy of radiotherapy or chemotherapy in lung cancer patients.

Distal-less homeobox 3, a negative regulator of myogenesis, is downregulated by microRNA-133.

Distal-less homeobox 3 (Dlx3), a member of the Dlx family of homeobox proteins, is a transcriptional activator of runt-related transcription factor 2 (Runx2) during osteogenic differentiation. It has been demonstrated that forced expression of Runx2 induces an osteogenic program and ectopic calcification in muscles. Therefore, it would be reasonable to predict that Dlx3 also affects myogenic differentiation. The relationship between Dlx3 and myogenesis, however, remains poorly understood. Therefore, in this study, the role and regulation of Dlx3 during myogenic differentiation were investigated. Expression level of Dlx3 was downregulated in human mesenchymal stem cells (MSCs), mouse MSCs, and C2C12 cells cultured in myogenic medium. Dlx3 level was inversely correlated with myogenic differentiation 1 and the muscle-specific microRNA, microRNA-133 (miR-133). The expression level of Runx2 was closely regulated by Dlx3 even under myogenic conditions. Overexpression of Dlx3 markedly downregulated expression levels of myogenic transcription factors and myotube formation in C2C12 cells, whereas Dlx3 knockdown enhanced myogenic differentiation. The Dlx3 3'-untranslated region (3'-UTR) has two potential binding sites for miR-133. Luciferase reporter assays demonstrated that Dlx3 is a direct target of miR-133a and miR-133b, and that the two target sites are redundantly active. Taken together, these results suggest that Dlx3 is a negative regulator of myogenic differentiation and that miR-133a and miR-133b enhance myogenic differentiation, partly through inhibition of Dlx3 expression via direct targeting of the Dlx3 3'-UTR.

Functional Biological Activity of Sorafenib as a Tumor-Treating Field Sensitizer for Glioblastoma Therapy.

Glioblastoma, the most common primary brain tumor in adults, is an incurable malignancy with poor short-term survival and is typically treated with radiotherapy along with temozolomide. While the development of tumor-treating fields (TTFields), electric fields with alternating low and intermediate intensity has facilitated glioblastoma treatment, clinical outcomes of TTFields are reportedly inconsistent. However, combinatorial administration of chemotherapy with TTFields has proven effective for glioblastoma patients. Sorafenib, an anti-proliferative and apoptogenic agent, is used as first-line treatment for glioblastoma. This study aimed to investigate the effect of sorafenib on TTFields-induced anti-tumor and anti-angiogenesis responses in glioblastoma cells in vitro and in vivo. Sorafenib sensitized glioblastoma cells to TTFields, as evident from significantly decreased post-TTFields cell viability (p < 0.05), and combinatorial treatment with sorafenib and TTFields accelerated apoptosis via reactive oxygen species (ROS) generation, as evident from Poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore, use of sorafenib plus TTFields increased autophagy, as evident from LC3 upregulation and autophagic vacuole formation. Cell cycle markers accumulated, and cells underwent a G2/M arrest, with an increased G0/G1 cell ratio. In addition, the combinatorial treatment significantly inhibited tumor cell motility and invasiveness, and angiogenesis. Our results suggest that combination therapy with sorafenib and TTFields is slightly better than each individual therapy and could potentially be used to treat glioblastoma in clinic, which requires further studies.

PLOD3 promotes lung metastasis via regulation of STAT3.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD3), a membrane-bound homodimeric enzyme, hydroxylates lysyl residues in collagen-like peptides; however, its role in lung cancer is unknown. This study aimed to investigate the role of PLOD3 as a pro-metastatic factor and to elucidate the underlying mechanism. First, we experimentally confirmed the release of PLOD3 in circulation in animal models, rendering it a potential serum biomarker for lung cancer in humans. Thereafter, we investigated the effects of PLOD3 overexpression and downregulation on cancer cell invasion and migration in vitro and in vivo, using human lung cancer cell lines and a mouse tumor xenograft model, respectively. Further, PLOD3 levels were determined in lung tissue samples from lung cancer patients. Functional analyses revealed that PLOD3 interacts with STAT3, thereby expressing matrix metalloproteinases (MMP-2 and MMP-9) and with urokinase plasminogen activator (uPA) to enhance tumor metastasis. PLOD3 and the STAT3 pathway were significantly correlated in the metastatic foci of lung cancer patients; PLOD3-STAT3 levels were highly correlated with a poor prognosis. These results indicate that PLOD3 promotes lung cancer metastasis in a RAS-MAP kinase pathway-independent manner. Therefore, secreted PLOD3 serves as a potent inducer of lung cancer metastasis and a potential therapeutic target to enhance survival in lung cancer.