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Gold nanoparticles (GNP, AuNPs) have received much attention as a tool to improve the therapeutic index of radiation therapy. This study aimed to evaluate the normalUsing the same treatment protocol applied to tumor cell lines, hepatocytes were exposed to AuNPs and/or radiation at various time points.The combination of X-ray irradiation and AuNPs did not have any significant effect on cell survival and apoptosis in normal hepatocytes. Furthermore, the combination treatment resulted in no or little change in the level of gamma-H2A histone family member X (γ-H2AX), a marker for DNA double-strand breaks (DSB), nor on the proportion of cells in the G2/M phase. Additionally, interleukin-8 (IL-8) secretion was measured using an enzyme-linked immunosorbent assay (ELISA) to assess its role in tumor progression and angiogenesis. The combination of irradiation and AuNP treatment revealed no significant reduction in hepatocyte viability, proliferation, or secretory capacity compared to cells receiving either treatment alone. According to this study, AuNPs in combination with radiation do have potentially in the treatment of hepatocellular carcinoma (HCC) with no critical cytotoxicity on normal tissue.Therefore, it is postulated that radiation and AuNPs are an effective combination therapy against HCC with no little cytotoxic effects on normal tissue, a hypothesis which warrants further investigation in

Tumor-treating fields (TTFields) have been extensively used to treat various cancers as well as glioblastoma multiforme (GBM), owing to their antimitotic effects. Furthermore, sorafenib is also extensively used to treat hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) and is under phase II/III clinical trials for other solid tumors. Hence, this investigation aimed to assess the efficacy of combination therapy with TTFields and sorafenib for colorectal carcinoma (CRC).Human CRC HCT116 and SW480 cells were subjected to cell viability assay, followed by the assessment of their cell death using fluorescence-activated cell sorting (FACS) analysis. Furthermore, the expression of proteins involved in AKT/STAT3 signaling and apoptosis was assessed via western blotting.Combination treatment inhibited cell proliferation and induced apoptosis via Reactive oxygen species (ROS) generation, evident from caspase-3 cleavage in CRC cells and suppressed the AKT/STAT3 signaling pathway, as evident from downregulation of BCL-2 after post-treatment. The present results indicate that combination treatment with TTFields and sorafenib inactivates AKT/STAT3 signaling pathway, thus altering the expression of BCL-2, thus inducing apoptosis and inhibiting the growth of CRC cells.Thus, combination treatment with TTFields and sorafenib is clinically applicable for treating metastatic CRC, although safety examination in patients with CRC will required to be achieved before this protocol can be implemented clinically for TTFields-sensitizer.

Colorectal cancer is reported to have the highest mortality rate among human malignancies. Although many research results for the treatment of colorectal cancer have been reported, there is no suitable treatment when resistance has developed. Therefore, it is necessary to develop new therapeutic agents. Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling plays an essential role in cell differentiation, proliferation, and survival. Abnormal activation of the JAK/STAT signaling pathway, by gene mutation or amplification, may induce cancer development, and sustained JAK/STAT activation is involved in chemoresistance. While many therapeutic agents have been developed to treat colon cancer, there remains no drug to overcome resistance to chemotherapies. The purpose of this study was to determine the potential of CJ14939 as a novel JAK inhibitor for the treatment of colorectal cancer.In this study, cell culture, cell death assay, 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, colony formation assay, immunoblot analysis and tumor xenograft were applied.CJ14939 induced cell death, and inhibited phosphorylation of JAK1 and STAT3 in colorectal cancer cells. Furthermore, CJ14939 also promoted oxaliplatin-induced cell death, up-regulated expression of cleaved caspase-3, and down-regulated expression of phospho-JAK1 and phospho-STAT3. In vivo, co-treatment with CJ14939 and oxaliplatin notably reduced tumor growth when compared with CJ14939 or oxaliplatin treatment alone.This study identifies the important potential of CJ14939 in colorectal cancer treatment and suggests that combining CJ14939 with oxaliplatin might be a novel therapeutic strategy for patients with colorectal cancer.

Supplementary Figures 1-6 from Heat Shock Factor 1–Mediated Aneuploidy Requires a Defective Function of p53

Supplementary Figures 1-6 from HSF1 as a Mitotic Regulator: Phosphorylation of HSF1 by Plk1 Is Essential for Mitotic Progression

Because heat shock factor 1 (HSF1) phosphorylation by Plk1 has been previously reported to be involved in mitotic regulation and p53 function may be involved in this mitotic regulation, we have further examined HSF1 functions in mitotic regulation according to p53 status. Nocodazole-mediated aneuploidy was increased in p53-defective (p53Mut) cells; however, it was not increased in p53 wild-type (p53WT) cells. Phosphorylation of HSF1 at Ser216 was increased in p53Mut cells with increased stability of securin and cyclin B1 in mitosis compared with p53WT cells. The interaction of p53 with Plk1 that was shown in p53WT cells and that induced normal mitotic checkpoint function was not observed in p53Mut cells; instead, the binding of HSF1 with Plk1 and HSF1 phosphorylation at Ser216 were seen in p53Mut cells, which resulted in increased aneuploidy production. Moreover, the interaction affinity of Cdc20 with Mad2 was inhibited in p53Mut cells, whereas the interaction between Cdc20 and HSF1 was increased. From the data, it was suggested that HSF1-mediated aneuploidy was more facilitated in p53-defective cells, indicating the importance of novel mechanisms for p53 function in HSF1-mediated mitotic regulation and genomic instability. [Cancer Res 2009;69(24):9404–12]

Heat shock protein 27 (HSP27), which is highly expressed in human lung and breast cancer tissues, induced resistance to cell death against various stimuli. Treatment of NCI-H1299 cells, which express a high level of HSP27, with small interference RNA specifically targeting HSP27 resulted in inhibition of their resistance to radiation or cisplatin, suggesting that HSP27 contributed to cellular resistance in these lung cancer cells. Furthermore, because HSP27 interacts directly with the COOH terminus of the protein kinase Cδ (PKCδ)-V5 region with ensuing inhibition of PKCδ activity and PKCδ-mediated cell death, we wished to determine amino acid residues in the V5 region that mediate its interaction with HSP27. Investigation with various deletion mutants of the region revealed that amino acid residues 668 to 674 of the V5 region mediate its interaction with HSP27. When NCI-H1299 cells were treated with biotin or with FITC-tagged heptapeptide of the residues 668 to 674 (E-F-Q-F-L-D-I), the cells exhibited dramatically increased cisplatin or radiation-induced cell death with the heptapeptide having efficient interaction with HSP27, which in turn restored the PKCδ activity that had been inhibited by HSP27. In vivo nude mice grafting data also suggested that NCI-H1299 cells were sensitized by this heptapeptide. The above data strongly show that the heptapeptide of the PKCδ-V5 region sensitized human cancer cells through its interaction with HSP27, thereby sequestering HSP27. The heptapeptide may provide a novel strategy for selective neutralization of HSP27. [Cancer Res 2007;67(13):6333–9]

Supplementary Methods and Figure Legends 1-6 from Heat Shock Factor 1–Mediated Aneuploidy Requires a Defective Function of p53

Previously, heat shock factor 1 (HSF1) had been reported to induce genomic instability and aneuploidy by interaction with Cdc20. Here, we have further examined the functions of HSF1 in the regulation of mitosis. A null mutant or knockdown of HSF1 caused defective mitotic progression. By monitoring chromosomes in living cells, we determined that HSF1 was localized to the centrosome in mitosis and especially to the spindle poles in metaphase. HSF1 was phosphorylated by Plk1 at Ser216 of the DSGXXS motif during the timing of mitosis and a phospho-defective mutant form of HSF1 inhibited mitotic progression. Phosphorylated HSF1 during spindle pole localization underwent ubiquitin degradation through the SCFβ-TrCP pathway. However, binding of HSF1 with Cdc20 stabilized the phosphorylation of HSF1. Moreover, SCFβ-TrCP–mediated degradation only occurred when phosphorylated HSF1 was released from Cdc20. HSF1 phosphorylation at Ser216 occurred in the early mitotic period with simultaneous binding of Cdc20. The interaction of HSF1 with SCFβ-TrCP was followed and then the interaction of APC/Cdc20 was subsequently observed. From these findings, it was shown that Plk1 phosphorylates HSF1 in early mitosis and that the binding of phosphorylated HSF1 with Cdc20 and ubiquitin degradation by SCFβ-TrCP regulates mitotic progression. [Cancer Res 2008;68(18):7550–60]

Supplementary Figure Legends 1-6 from HSF1 as a Mitotic Regulator: Phosphorylation of HSF1 by Plk1 Is Essential for Mitotic Progression

Supplementary Figures 1-7 from Inhibition of Heat Shock Protein 27–Mediated Resistance to DNA Damaging Agents by a Novel PKCδ-V5 Heptapeptide

Background: The coexistence of magnetite within protein aggregates in the brain is a typical pathologic feature of Alzheimer’s disease (AD), and the formation of amyloid-β (Aβ) plaques induces critical impairment of cognitive function. Objective: This study aimed to investigate the therapeutic effect of proton stimulation (PS) targeting plaque magnetite in the transgenic AD mouse brain. Methods: A proton transmission beam was applied to the whole mouse brain at a single entrance dose of 2 or 4 Gy to test the effect of disruption of magnetite-containing Aβ plaques by electron emission from magnetite. The reduction in Aβ plaque burden and the cognitive function of the PS-treated mouse group were assayed by histochemical analysis and memory tests, respectively. Aβ-magnetite and Aβ fibrils were treated with PS to investigate the breakdown of the amyloid protein matrix. Results: Single PS induced a 48–87%reduction in both the amyloid plaque burden and ferrous-containing magnetite level in the early-onset AD mouse brain while saving normal tissue. The overall Aβ plaque burden (68–82%) and (94–97%) hippocampal magnetite levels were reduced in late onset AD mice that showed improvements in cognitive function after PS compared with untreated AD mice (p

SVCT2, Sodium-dependent Vitamin C Transporter 2, uniquely transports ascorbic acid (also known as vitamin C and ascorbate) into all types of cells. Vitamin C is an essential nutrient that must be obtained through the diet and plasma levels are tightly regulated by transporter activity. Vitamin C plays an important role in antioxidant defenses and is a cofactor for many enzymes that enable hormone synthesis, oxygen sensing, collagen synthesis and epigenetic pathways. Although SVCT2 has various functions, regulation of its expression/activity remains poorly understood. We found a p53-binding site, within the SVCT2 promoter, using a transcription factor binding-site prediction tool. In this study, we show that p53 can directly repress SVCT2 transcription by binding a proximal- (~−185 to −171 bp) and a distal- (~−1800 to −1787 bp) p53-responsive element (PRE), Chromatin immunoprecipitation assays showed that PRE-bound p53 interacts with the corepressor-histone deacetylase 3 (HDAC3), resulting in deacetylation of histones Ac-H4, at the proximal promoter, resulting in transcriptional silencing of SVCT2. Overall, our data suggests that p53 is a potent transcriptional repressor of SVCT2, a critical transporter of diet-derived ascorbic acid, across the plasma membranes of numerous essential tissue cell types.

Glioblastoma multiforme (GBM) or glioblastoma is the most deadly malignant brain tumor in adults. GBM is difficult to treat mainly due to the presence of glioblastoma stem cells (GSCs). Epidermal growth factor receptor variant III (EGFRvIII) has been linked to stemness and malignancy of GSCs; however, the regulatory mechanism of EGFRvIII is largely unknown. Here, we demonstrated that Anoctamin-1 (ANO1), a Ca2+-activated Cl– channel, interacts with EGFRvIII, increases its protein stability, and supports the maintenance of stemness and tumor progression in GSCs. Specifically, shRNA-mediated knockdown and pharmacological inhibition of ANO1 suppressed the self-renewal, invasion activities, and expression of EGFRvIII and related stem cell factors, including NOTCH1, nestin, and SOX2 in GSCs. Conversely, ANO1 overexpression enhanced the above phenomena. Mechanistically, ANO1 protected EGFRvIII from proteasomal degradation by directly binding to it. ANO1 knockdown significantly increased survival in mice and strongly suppressed local invasion of GSCs in an in vivo intracranial mouse model. Collectively, these results suggest that ANO1 plays a crucial role in the maintenance of stemness and invasiveness of GSCs by regulating the expression of EGFRvIII and related signaling molecules, and can be considered a promising therapeutic target for GBM treatment.

Social media is a powerful tool for brands to entice and retain customers. However, research on how para-social interaction (PSI) through social media leads to brand loyalty is lacking. To bridge this gap, this study examines the sequential mediating roles of PSI and consumer engagement as well as the moderating effect of self-construal in building brand loyalty on Facebook brand fan pages. Using 497 responses from survey participants, we find that consumers with high interdependent self-construal, regardless of their levels of perceived interactivity and openness, have high PSI, consumer engagement, and brand loyalty. Moreover, the positive effects of interdependent self-construal on brand loyalty are mediated sequentially by PSI and consumer engagement. However, for those with high independent self-construal, both high perceived interactivity and high openness have positive effects on para-social interaction, consumer engagement, and brand loyalty. Moreover, the positive effects of perceived interactivity and openness on brand loyalty confirm the sequential mediating roles of PSI and consumer engagement. These findings would help brands streamline their operations to optimize brand loyalty.

Dramatic cellular reorganization in mitosis critically depends on the timely and temporal phosphorylation of a broad range of proteins, which is mediated by the activation of the mitotic kinases and repression of counteracting phosphatases. The mitosis-to-interphase transition, which is termed mitotic exit, involves the removal of mitotic phosphorylation by protein phosphatases. Although protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) drive this reversal in animal cells, the phosphatase network associated with ordered bulk dephosphorylation in mitotic exit is not fully understood. Here, we describe a new mitotic phosphatase relay in which Wip1/PPM1D phosphatase activity is essential for chromosomal passenger complex (CPC) translocation to the anaphase central spindle after release from the chromosome via PP1-mediated dephosphorylation of histone H3T3. Depletion of endogenous Wip1 and overexpression of the phosphatase-dead mutant disturbed CPC translocation to the central spindle, leading to failure of cytokinesis. While Wip1 was degraded in early mitosis, its levels recovered in anaphase and the protein functioned as a Cdk1-counteracting phosphatase at the anaphase central spindle and midbody. Mechanistically, Wip1 dephosphorylated Thr-59 in inner centromere protein (INCENP), which, subsequently bound to MKLP2 and recruited other components to the central spindle. Furthermore, Wip1 overexpression is associated with the overall survival rate of patients with breast cancer, suggesting that Wip1 not only functions as a weak oncogene in the DNA damage network but also as a tumor suppressor in mitotic exit. Altogether, our findings reveal that sequential dephosphorylation of mitotic phosphatases provides spatiotemporal regulation of mitotic exit to prevent tumor initiation and progression.

To investigate whether carbon-ion beam alone, or in combination with lapatinib, has a beneficial effect in targeting HER2-positive breast cancer stem-like cells (CSCs) compared to that of X-rays, human breast CSCs derived from BT474 and SKBR3 cell lines were treated with a carbon-ion beam or X-rays irradiation alone or in combination with lapatinib, and then cell viability, spheroid formation assays, apoptotic analyses, gene expression analysis of related genes, and immunofluorescent gamma-H2AX foci assays were performed. Spheroid formation assays confirmed that ESA+/CD24- cells have CSC properties compared to ESA-/CD24+ cells. CSCs were more highly enriched after X-ray irradiation combined with lapatinib, whereas carbon-ion beam combined with lapatinib significantly decreased the proportion of CSCs. Carbon-ion beam combined with lapatinib significantly suppressed spheroid formation compared to X-rays combined with lapatinib or carbon ion beam alone. Cell cycle analysis showed that carbon ion beam combined with lapatinib predominantly enhanced sub-G1 and G2/M arrested population compared to that of carbon-ion beam, X-ray treatments alone. Carbon-ion beam combined with lapatinib significantly enhanced apoptosis and carbon-ion beam alone dose-dependently increased autophagy-related expression of Beclin1 and in combination with lapatinib greatly enhanced ATG7 expression at protein levels. In addition, a large sized gamma H2AX foci in CSCs were induced by carbon ion beam combined with lapatinib treatment in CSCs compared to cells receiving X-rays or carbon-ion beam alone. Altogether, combination of carbon-ion beam irradiation and lapatinib has a high potential to kill HER2-positive breast CSCs, causing severe irreparable DNA damage, enhanced autophagy, and apoptosis.

This study was conducted to construct basic data for the selection of elite cows by analyzing the estimated breeding value (EBV) and accuracy using the pedigree of Hanwoo cows in Gyeongnam. The phenotype trait used in the analysis are the carcass weight (CWT), eye muscle area (EMA), backfat thickness (BFT) and marbling score (MS). The pedigree of the test group and reference group was collected to build a pedigree structure and a numeric relationship matrix (NRM). The EBV, genetic parameters and accuracy were estimated by applying NRM to the best linear unbiased prediction (BLUP) multiple-trait animal model of the BLUPF90 program. Looking at the pedigree structure of the test group, there were a total of 2,371 cows born between 2003 to 2009, of these 603 cows had basic registration (25%), 562 cows had pedigree registration (24%) and 1,206 cows had advanced registration (51%). The proportion of pedigree registered cows was relatively low but it gradually increased and reached a point of 20,847 cows (68%) between 2010 to 2017. Looking at the change in the EBV, the CWT improved from 4.992 kg to 9.885 kg, the EMA from 0.970 cm2 to 2.466 cm2, the BFT from −0.186 mm to −0.357 mm, and the MS from 0.328 to 0.559 points. As a result of genetic parameter estimation, the heritability of CWT, EMA, BFT, and MS were 0.587, 0.416, 0.476, and 0.571, respectively, and the accuracy of those were estimated to be 0.559, 0.551, 0.554, and 0.558, respectively. Selection of superior genetic breed and efficient improvement could be possible if cow ability verification is implemented by using the accurate pedigree of each individual in the farms.