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PIN1, the peptidyl-prolyl isomerase (PPIase), is an enzyme that changes the conformation of phosphoproteins. The conformational change induced by PIN1 alters the function and stability of the target proteins. PIN1 is overexpressed in many different types of malignancies, including breast, lung, cervical, brain and colorectal tumors. PIN1 overexpression has been associated with activation of multiple oncogenic signaling pathways during tumor development. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor activated in hypoxia, plays a role in erythropoiesis, glycolysis, tissue invasion, metastasis and angiogenesis. In this study, we found the direct interaction between HIF-2α and PIN1 in colorectal cancer HCT116 cells. Notably, serine 16 and lysine 63 residues of PIN1 were critical for its interaction with HIF-2α. When PIN1 protein was silenced by transient transfection of PIN1 short interfering RNA, the expression of HIF-2α was attenuated under a hypoxic condition. Moreover, genetic and pharmacologic inhibition of PIN1 abrogated the expression of vascular endothelial growth factor and angiogenesis. The cycloheximide chase experiment revealed the stabilization of HIF-2α by PIN1. Both WW and PPIase domains of PIN1 appear to be critical for its interaction with HIF-2α.

Histone modifications, including histone lysine methylation, regulate gene expression in the vasculature, and targeting tumor blood vessels through histone modification decreases tumor growth. SETD8, a methyltransferase that catalyzes the mono-methylation of histone H4 lysine 20 is known to promote tumorigenesis in various cancers and its high levels of expression are related to poor prognosis. However, the detailed mechanisms by which SETD8 stimulates tumor progression and angiogenesis are still not well understood. Recent studies have demonstrated that, in vitro, BVT-948 efficiently and selectively suppresses SETD8 activity and histone methylation levels. In this study, we showed that BVT-948-mediated SETD8 inhibition in HUVECs results in an inhibition of angiogenesis. Inhibition of SETD8 not only inhibited angiogenesis but also disrupted actin stress fiber formation and induced cell cycle arrest at S phase. These effects were accompanied by increased HES-1 expression levels, decreased osteopontin levels, and a decreased differentiation of human induced pluripotent stem cells into endothelial cells. Interestingly, BVT-948 treatment reduced pathological angiogenesis in mouse OIR model. These data illustrate the mechanisms by which SETD8 regulates angiogenesis and may enable the use of a SETD8 inhibitor to treat various pathological conditions that are known to be associated with excessive angiogenesis, including and tumor growth.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with an overall 5-year survival rate of only 30%. EOC is associated with drug resistance, frequent recurrence, and poor prognosis. A major contributor toward drug resistance might be cancer stem cells (CSCs), which may remain after chemotherapy. Here, we aimed to find therapeutic agents that target ovarian CSCs. We performed a high-throughput screening using the Clinical Compound Library with a sphere culture of A2780 EOCs. Poziotinib, a pan-human epidermal growth factor receptor (HER) inhibitor, decreased sphere formation, viability, and proliferation, and induced G1 cell cycle arrest and apoptosis in ovarian CSCs. In addition, poziotinib suppressed stemness and disrupted downstream signaling of Wnt/β-catenin, Notch, and Hedgehog pathways, which contribute to many characteristics of CSCs. Interestingly, HER4 was overexpressed in ovarian CSCs and Poziotinib reduced the phosphorylation of STAT5, AKT, and ERK, which are regulated by HER4. Our results suggest that HER4 may be a promising therapeutic target for ovarian CSCs, and that poziotinib may be an effective therapeutic option for the prevention of ovarian cancer recurrence.

Anterior gradient protein 2 homolog (AGR2), an endoplasmic reticulum protein, is secreted in the tumor microenvironment. AGR2 is a member of the disulfide isomerase family, is highly expressed in multiple cancers, and promotes cancer metastasis. In this study, we found that etravirine, which is a non-nucleoside reverse transcriptase inhibitor, could induce AGR2 degradation via autophagy. Moreover, etravirine diminished proliferation, migration, and invasion in vitro. Moreover, in an orthotopic xenograft mouse model, the combination of etravirine and paclitaxel significantly suppressed cancer progression and metastasis. This drug may be a promising therapeutic agent for the treatment of ovarian cancer.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) has been frequently overexpressed in many types of malignancy, suggesting its oncogenic function. It recognizes phosphorylated serine or threonine (pSer/Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. The oncogenic transcription factor, Nrf2 harbors the pSer/Thr-Pro motif. This prompted us to investigate whether Pin1 could bind to Nrf2 and influence its stability and function in the context of implications for breast cancer development and progression. The correlation between Pin1 and Nrf2 in the triple-negative breast cancer cells was validated by RNASeq analysis as well as immunofluorescence staining. Interaction between Pin1 and Nrf2 was assessed by co-immunoprecipitation and an in situ proximity ligation assay. We found that mRNA and protein levels of Pin1 were highly increased in the tumor tissues of triple-negative breast cancer patients and the human breast cancer cell line. Genetic or pharmacologic inhibition of Pin1 enhanced the ubiquitination and degradation of Nrf2. In contrast, the overexpression of Pin1 resulted in the accumulation of Nrf2 in the nucleus, without affecting its transcription. Notably, the phosphorylation of Nrf2 at serine 215, 408, and 577 is essential for its interaction with Pin1. We also identified phosphorylated Ser104 and Thr277 residues in Keap1, a negative regulator of Nrf2, for Pin1 binding. Pin1 plays a role in breast cancer progression through stabilization and constitutive activation of Nrf2 by competing with Keap1 for Nrf2 binding.

Generation of proper models for studying human genetic diseases has been hindered until recently by the scarcity of primary cell samples from genetic disease patients and inefficient genetic modification tools. However, recent advances in clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and human induced pluripotent stem cells (hiPSCs) have provided an opportunity to explore the function of pathogenic variants and obtain gene-corrected cells for autologous cell therapy. In this chapter, we address recent applications of CRISPR/Cas9 to hiPSCs in genetic diseases, including neurodegenerative, cardiovascular, and rare diseases.

The presence of brown adipocytes within white adipose tissue is associated with phenotypes that exhibit improved metabolism and proper body weight maintenance. Therefore, a variety of dietary agents that facilitate the browning of white adipocytes have been investigated. In this study, we screened a natural product library comprising 133 compounds with the potential to promote the browning of white adipocytes, and found that D-mannitol induces the browning of 3T3-L1 adipocytes by enhancing the expression of brown fat-specific genes and proteins, and upregulating lipid metabolism markers. D-mannitol also increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 (ACC), suggesting a possible role in lipolysis and fat oxidation. Moreover, an increase in the expression of genes associated with D-mannitol-induced browning was strongly correlated with the activation of the β3-adrenergic receptor as well as AMPK, protein kinase A (PKA), and PPARγ coactivator 1α (PGC1α). D-mannitol effectively reduced the body weight of mice fed a high-fat diet, and increased the expression of β1-oxidation and energy expenditure markers, such as Cidea, carnitine palmityl transferase 1 (CPT1), uncoupling protein 1 (UCP1), PGC1α, and acyl-coenzyme A oxidase (ACOX1) in the inguinal white adipose tissue. Our findings suggest that D-mannitol plays a dual regulatory role by inducing the generation of a brown fat-like phenotype and enhancing lipid metabolism. These results indicate that D-mannitol can function as an anti-obesity supplement.

Generation of proper models for studying human genetic diseases has been hindered until recently by the scarcity of primary cell samples from genetic disease patients and inefficient genetic modification tools. However, recent advances in clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and human induced pluripotent stem cells (hiPSCs) have provided an opportunity to explore the function of pathogenic variants and obtain gene-corrected cells for autologous cell therapy. In this chapter, we address recent applications of CRISPR/Cas9 to hiPSCs in genetic diseases, including neurodegenerative, cardiovascular, and rare diseases.

Osteoclasts are large, multinucleated cells responsible for bone resorption and are induced in response to the regulatory activity of receptor activator of nuclear factor-kappa B ligand (RANKL). Excessive osteoclast activity causes pathological bone loss and destruction. Many studies have investigated molecules that specifically inhibit osteoclast activity by blocking RANKL signaling or bone resorption. In recent years, we screened compounds from commercial libraries to identify molecules capable of inhibiting RANKL-induced osteoclast differentiation. Consequently, we reported some compounds that are effective at attenuating osteoclast activity. In this study, we found that N-[2-(4-acetyl-1-piperazinyl)phenyl]-2-(3-methylphenoxy)acetamide (NAPMA) significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells from bone marrow-derived macrophages in a dose-dependent manner, without cytotoxic effects. NAPMA downregulated the expression of osteoclast-specific markers, such as c-Fos, NFATc1, DC-STAMP, cathepsin K, and MMP-9, at the transcript and protein levels. Accordingly, bone resorption and actin ring formation were decreased in response to NAPMA treatment. Furthermore, we demonstrated the protective effect of NAPMA against ovariectomy-induced bone loss using micro-CT and histological analysis. Collectively, the results showed that NAPMA inhibited osteoclast differentiation and attenuated bone resorption. It is thus a potential drug candidate for the treatment of osteoporosis and other bone diseases associated with excessive bone resorption.

The function of natural killer (NK) cell-derived interferon-&gamma, (IFN-&gamma, ) expands to remove pathogens by increasing the ability of innate immune cells. Here, we identified the critical role of thioredoxin-interacting protein (TXNIP) in the production of IFN-&gamma, in NK cells during bacterial infection. TXNIP inhibited the production of IFN-&gamma, and the activation of transforming growth factor &beta, activated kinase 1 (TAK1) activity in primary mouse and human NK cells. TXNIP directly interacted with TAK1 and inhibited TAK1 activity by interfering with the complex formation between TAK1 and TAK1 binding protein 1 (TAB1). Txnip&minus, /&minus, (KO) NK cells enhanced the activation of macrophages by inducing IFN-&gamma, production during Pam3CSK4 stimulation or Staphylococcus aureus (S. aureus) infection and contributed to expedite the bacterial clearance. Our findings suggest that NK cell-derived IFN-&gamma, is critical for host defense and that TXNIP plays an important role as an inhibitor of NK cell-mediated macrophage activation by inhibiting the production of IFN-&gamma, during bacterial infection.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women worldwide, with an overall 5 year survival rate below 30%. The low survival rate is associated with the persistence of cancer stem cells (CSCs) after chemotherapy. Therefore, CSC-targeting strategies are required for successful EOC treatment. Pan-human epidermal growth factor receptor 4 (HER4) and L-type calcium channels are highly expressed in ovarian CSCs, and treatment with the pan-HER inhibitor poziotinib or calcium channel blockers (CCBs) selectively inhibits the growth of ovarian CSCs via distinct molecular mechanisms. In this study, we tested the hypothesis that combination treatment with poziotinib and CCBs can synergistically inhibit the growth of ovarian CSCs. Combined treatment with poziotinib and manidipine (an L-type CCB) synergistically suppressed ovarian CSC sphere formation and viability compared with either drug alone. Moreover, combination treatment synergistically reduced the expression of stemness markers, including CD133, KLF4, and NANOG, and stemness-related signaling molecules, such as phospho-STAT5, phospho-AKT, phospho-ERK, and Wnt/&beta, catenin. Moreover, poziotinib with manidipine dramatically induced apoptosis in ovarian CSCs. Our results suggest that the combinatorial use of poziotinib with a CCB can effectively inhibit ovarian CSC survival and function.

Non-alcoholic fatty liver disease (NAFLD) is a leading form of chronic liver disease, with few biomarkers and treatment options currently available. Non-alcoholic steatohepatitis (NASH), a progressive disease of NAFLD, may lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Epigenetic modification can contribute to the progression of NAFLD causing non-alcoholic steatohepatitis (NASH), in which the exact role of epigenetics remains poorly understood. To identify potential therapeutics for NASH, we tested small-molecule inhibitors of the epigenetic target histone methyltransferase EZH2, Tazemetostat (EPZ-6438), and UNC1999 in STAM NASH mice. The results demonstrate that treatment with EZH2 inhibitors decreased serum TNF-alpha in NASH. In this study, we investigated that inhibition of EZH2 reduced mRNA expression of inflammatory cytokines and fibrosis markers in NASH mice. In conclusion, these results suggest that EZH2 may present a promising therapeutic target in the treatment of NASH.

Drug resistance in epithelial ovarian cancer (EOC) is reportedly attributed to the existence of cancer stem cells (CSC), because in most cancers, CSCs still remain after chemotherapy. To overcome this limitation, novel therapeutic strategies are required to prevent cancer recurrence and chemotherapy-resistant cancers by targeting cancer stem cells (CSCs). We screened an FDA-approved compound library and found four voltage-gated calcium channel blockers (manidipine, lacidipine, benidipine, and lomerizine) that target ovarian CSCs. Four calcium channel blockers (CCBs) decreased sphere formation, viability, and proliferation, and induced apoptosis in ovarian CSCs. CCBs destroyed stemness and inhibited the AKT and ERK signaling pathway in ovarian CSCs. Among calcium channel subunit genes, three L- and T-type calcium channel genes were overexpressed in ovarian CSCs, and downregulation of calcium channel genes reduced the stem-cell-like properties of ovarian CSCs. Expressions of these three genes are negatively correlated with the survival rate of patient groups. In combination therapy with cisplatin, synergistic effect was shown in inhibiting the viability and proliferation of ovarian CSCs. Moreover, combinatorial usage of manidipine and paclitaxel showed enhanced effect in ovarian CSCs xenograft mouse models. Our results suggested that four CCBs may be potential therapeutic drugs for preventing ovarian cancer recurrence.

Peptidyl-prolyl isomerase (PIN1) specifically binds and isomerizes the phosphorylated serine/threonine–proline (pSer/Thr–Pro) motif, which results in the alteration of protein structure, function, and stability. The altered structure and function of these phosphorylated proteins regulated by PIN1 are closely related to cancer development. PIN1 is highly expressed in human cancers and promotes cancer as well as cancer stem cells by breaking the balance of oncogenes and tumor suppressors. In this review, we discuss the roles of PIN1 in cancer and PIN1-targeted small-molecule compounds.

Alcoholism is a serious addiction that can lead to various health complications such as liver fibrosis, steatosis, and cirrhosis. Carvacrol is present in many plant-based essential oils and used as a preservative in the food industry. In this study, we have investigated the hepatoprotective role of carvacrol against ethanol-induced liver toxicity in mice. To determine the effect of carvacrol on liver injury parameters, 5 doses of 50% ethanol (10 mL/kg body weight) were orally administered every 12 h for inducing the hepatotoxicity in experimental mice. Interestingly, carvacrol pre-treatment (50 and 100 mg/kg) reversed the ethanol-induced effects on liver function, antioxidant markers, matrix metalloproteinases activities, and histological changes. Moreover, carvacrol binds to the active pocket of cytochrome P450 (Cyt P450) and inhibits its expression. Thus, our finding suggests carvacrol can be used as an adjuvant for the amelioration of alcohol-induced hepatotoxicity.

The phosphatase of regenerating liver (PRL) family, including PRL-1, PRL-2, and PRL-3, comprises protein tyrosine phosphatases whose deregulation is associated with the tumorigenesis and metastasis of many types of cancer. However, the underlying mechanism is poorly understood. In this study, aiming to increase understanding of the molecular mechanisms underlying the functions of PRL-1 and PRL-3, a yeast two-hybrid system was employed to screen for their interacting proteins. Alignment with the NCBI BLAST database revealed 12 interactive proteins: Synaptic nuclear envelope protein 2, emerin, mannose 6-phosphate receptor-binding protein 1, low-density lipoprotein receptor-related protein 10, Rab acceptor 1, tumor protein D52-like 2, selectin P ligand (SELPLG), guanylate binding protein 1, transmembrane and ubiquitin-like domain-containing 2, NADH:ubiquinone oxidoreductase subunit B8, syndecan 4 and FK506-binding protein 8 (FKBP8). These proteins are associated with cell proliferation, apoptosis, immune response, cell fate specification and metabolic process in biological process categories, and involved in various signaling pathways, including Alzheimer's disease, Parkinson's disease, Huntington's disease, hypertrophic cardiomyopathy and cell adhesion molecules. Interactions of PRL-1 with the prey proteins SELPLG and FKBP8 were confirmed by immunoprecipitation or immunostaining. Furthermore, SELPLG and FKBP8 suppressed PRL-1- or PRL-3-mediated p53 activity. Identification of the proteins interacting with PRL family proteins may provide valuable information to better understand the mechanism of PRL-mediated signal transduction in cancer and other diverse diseases.

MDM2, a critical negative regulator of p53, is often overexpressed in leukemia, but few p53 mutations are found, suggesting that p53-independent MDM2 expression occurs due to alterations in MDM2 upstream regulators. In this study, a high MDM2 transcription level was observed (41.17%) regardless of p53 expression in patient with acute myeloid leukemia (AML). Therefore, we performed genome-scale functional screening of the human genes modulating MDM2 expression in a p53-independent manner. We searched co-expression profiles of genes showing a positive or negative pattern with MDM2 expression in a DNA microarray database, selected1089 links, and composed a screening library of 368 genes. Using MDM2 P1 and P2 promoter-reporter systems, we screened clones regulating MDM2 transcriptions in a p53-independent manner by overexpression. Nine clones from the screening library showed enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells. Among them, six clones, including NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, and YWHAB mediated MAPK signaling for expressing MDM2. These results indicate that p53-independent upregulation of MDM2 by increasing selected clones may lead to oncogenesis in AML and that MDM2-modulating genes are novel potential targets for AML treatment.

XBP1 (X-box-binding protein 1) is activated in cancer and has a pivotal role in tumorigenesis and progression of human cancer. In particular, the XBP1 transcriptional regulatory network is well known to drive cancer development, but little is known about whether the stability of XBP1 is regulated and, if so, what controls the stability of XBP1. In the present study we show that PIN1 prolyl isomerase interacts with the active form of XBP1 (XBP1s) in a phosphorylation-dependent manner and promotes XBP1s-induced cell proliferation and transformation through the regulation of XBP1 stability. By contrast, depletion of Pin1 in cancer cells reduced XBP1s expression, which subsequently inhibits cell proliferation and transformation. Interestingly, XBP1s activates multiple oncogenic pathways including NF-κB (nuclear factor κB), AP1 (activator protein 1) and Myc, and down-regulates PIN1 transcription via a negative-feedback mechanism through p53 induction. Ultimately, reciprocal regulation of Pin1 and XBP1s is associated with the activation of oncogenic pathways, and the relationship of PIN1 and XBP1 may be an attractive target for novel therapy in cancers.

Protein phosphorylation and post-phosphorylation events regulate many cellular signaling pathways. Peptidyl-prolyl isomerase (Pin1) is the only peptidyl-prolyl cis/trans isomerase that interacts with numerous oncogenic or tumor suppressive phosphorylated proteins, causes conformational changes in target proteins, and eventually regulates the activities of such proteins. These alterations in activity play a pivotal role in tumorigenesis. Since Pin1 is overexpressed and/or activated in various types of cancers, and the dysregulation of proline-directed phosphorylation contributes to tumorigenesis, Pin1 represents an attractive target for cancer therapy. This review will describe the role of Pin1 in cancer and the current status of Pin1 inhibitor development.

Osteoclasts are large multinucleated cells which are induced by the regulation of the receptor activator of nuclear factor kappa-&Beta, ligand (RANKL), which is important in bone resorption. Excessive osteoclast differentiation can cause pathologic bone loss and destruction. Numerous studies have targeted molecules inhibiting RANKL signaling or bone resorption activity. In this study, 11 compounds from commercial libraries were examined for their effect on RANKL-induced osteoclast differentiation. Of these compounds, only 2-(3-(2-fluoro-4-methoxyphenyl)-6-oxo-1(6H)-pyridazinyl)-N-1H-indol-5-ylacetamide (2N1HIA) caused a significant decrease in multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cell formation in a dose-dependent manner, without inducing cytotoxicity. The 2N1HIA compound neither affected the expression of osteoclast-specific gene markers such as TRAF6, NFATc1, RANK, OC-STAMP, and DC-STAMP, nor the RANKL signaling pathways, including p38, ERK, JNK, and NF-&kappa, B. However, 2N1HIA exhibited a significant impact on the expression levels of CD47 and cathepsin K, the early fusion marker and critical protease for bone resorption, respectively. The activity of matrix metalloprotease-9 (MMP-9) decreased due to 2N1HIA treatment. Accordingly, bone resorption activity and actin ring formation decreased in the presence of 2N1HIA. Taken together, 2N1HIA acts as an inhibitor of osteoclast differentiation by attenuating bone resorption activity and may serve as a potential candidate in preventing and/or treating osteoporosis, or other bone diseases associated with excessive bone resorption.

Osteoporosis is caused by an imbalance of osteoclast and osteoblast activities and it is characterized by enhanced osteoclast formation and function. Peptidyl-prolyl cis-trans isomerase never in mitosis A (NIMA)-interacting 1 (Pin1) is a key mediator of osteoclast cell-cell fusion via suppression of the dendritic cell-specific transmembrane protein (DC-STAMP). We found that N,N&prime, 1,4-butanediylbis[3-(2-chlorophenyl)acrylamide] (BCPA) inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in a dose-dependent manner without cytotoxicity. In addition, BCPA attenuated the reduction of Pin1 protein during osteoclast differentiation without changing Pin1 mRNA levels. BCPA repressed the expression of osteoclast-related genes, such as DC-STAMP and osteoclast-associated receptor (OSCAR), without altering the mRNA expression of nuclear factor of activated T cells (NFATc1) and cellular oncogene fos (c-Fos). Furthermore, Tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells were significantly decreased by BCPA treatment compared to treatment with the Pin1 inhibitor juglone. These data suggest that BCPA can inhibit osteoclastogenesis by regulating the expression of the DC-STAMP osteoclast fusion protein by attenuating Pin1 reduction. Therefore, BCPA may be used to treat osteoporosis.

Activation of adiponectin receptors (AdipoRs) by its natural ligand, adiponectin has been known to be involved in modulating critical metabolic processes such as glucose metabolism and fatty acid oxidation as demonstrated by a number of in vitro and in vivo studies over last two decades. These findings suggest that AdipoRs' agonists could be developed into a potential therapeutic agent for metabolic diseases, such as diabetes mellitus, especially for type II diabetes, a long-term metabolic disorder characterized by high blood sugar, insulin resistance, and relative lack of insulin. Because of limitations in production of biologically active adiponectin, adiponectin-mimetic AdipoRs' agonists have been suggested as alternative ways to expand the opportunity to develop anti-diabetic agents. Based on crystal structure of AdipoR1, we designed AdipoR1's peptide agonists using protein-peptide docking simulation and screened their receptor binding abilities and biological functions via surface plasmon resonance (SPR) and biological analysis. Three candidate peptides, BHD1028, BHD43, and BHD44 were selected and confirmed to activate AdipoR1-mediated signal pathways. In order to enhance the stability and solubility of peptide agonists, candidate peptides were PEGylated. PEGylated BHD1028 exhibited its biological activity at nano-molar concentration and could be a potential therapeutic agent for the treatment of diabetes. Also, SPR and virtual screening techniques utilized in this study may potentially be applied to other peptide-drug screening processes against membrane receptor proteins.

Phosphorylation, the addition of a phosphate group to a molecule, is an effective way of regulating the biological properties of that molecule. Protein phosphorylation is a post-translational modification of proteins and affects cellular signaling transduction. Protein kinases induce phosphorylation by catalyzing the transfer of phosphate groups to serine, threonine, and tyrosine residues on protein substrates. Consistent with their roles in cancer, protein kinases have emerged as one of the most clinically useful target molecules in pharmacological cancer therapy. Intrinsic or acquired resistance of cancers against anti-cancer therapeutics, such as ionizing radiation, is a major obstacle for the effective treatment of many cancers. In this review, we describe key aspects of various kinases acting on proteins. We also discuss the roles of protein kinases in the pathophysiology and treatment of cancer. Because protein kinases correlate with radiation resistance in various types of cancer, we focus on several kinases responsible for radiation resistance and/or sensitivity and their therapeutic implications. Finally, we suggest some ongoing radiation-sensitization strategies using genetic loss and/or kinase inhibitors that can counteract radiation resistance-related protein kinases.

Polyethyleneimine (PEI) has been widely used in gene delivery systems because of its outstanding ability to promote DNA condensation and endosome escape. However, its use for the introduction of foreign DNA into host cells is compromised by high cytotoxicity and serum reactivity. In this study we examined the possibility of improving the PEI-mediated gene delivery using the serum-stable and biodegradable polymer, chitosan. Among chitosans of different molecular weights, the 45 kD form exhibited significantly higher gene delivery efficiency than the 15 and 100 kD forms. Using the 45 kDa form of chitosan, we formulated composite PEI/DNA/chitosan (PDC) complexes consisting of the core part where DNA chains were tightly condensed by PEI and the outer layer lined with chitosan, and found that an optimal PDC complex exhibits a significantly higher transfection efficiency in a high serum condition and a lower toxicity than monolithic PEI/DNA (PD) complex. Moreover, addition of chitosan to PEI/DNA complex reduced nonspecific interactions with erythrocytes, which may eventually contribute to improved transfection efficiency in high serum concentrations. These results demonstrate that chitosan coating of PD complex might increase the efficiency of PEI-mediated gene delivery in vivo as well as in vitro.

Ageing is a natural process in living organisms throughout their lifetime, and most elderly people suffer from ageing-associated diseases. One suggested way to tackle such diseases is to rejuvenate stem cells, which also undergo ageing. Here we report that the thioredoxin-interacting protein (TXNIP)-p38 mitogen-activated protein kinase (p38) axis regulates the ageing of haematopoietic stem cells (HSCs), by causing a higher frequency of long-term HSCs, lineage skewing, a decrease in engraftment, an increase in reactive oxygen species and loss of Cdc42 polarity. TXNIP inhibits p38 activity via direct interaction in HSCs. Furthermore, cell-penetrating peptide (CPP)-conjugated peptide derived from the TXNIP-p38 interaction motif inhibits p38 activity via this docking interaction. This peptide dramatically rejuvenates aged HSCs in vitro and in vivo. Our findings suggest that the TXNIP-p38 axis acts as a regulatory mechanism in HSC ageing and indicate the potent therapeutic potential of using CPP-conjugated peptide to rejuvenate aged HSCs., The processes regulating the ageing of stem cells are not clearly defined. Here, the authors report that in haematopoietic stem cells (HSC) thioredoxin-interacting protein, known to regulate the cell cycle, binds to p38 mitogen-activated protein kinase and regulates HSC ageing and rejuvenation.

Salmonella have been experimentally used as anti-cancer agents, because they show selective growth in tumours. In this study, we genetically modified attenuated Salmonella typhimurium to express and secrete interferon-gamma (IFN-γ) as a tumouricidal agent to enhance the therapeutic efficacy of Salmonella. IFN-γ was fused to the N-terminal region (residues 1-160) of SipB (SipB160) for secretion from bacterial cells. Attenuated S. typhimurium expressing recombinant IFN-γ (S. typhimurium (IFN-γ)) invaded the melanoma cells and induced cytotoxicity. Subcutaneous administration of S. typhimurium (IFN-γ) also efficiently inhibited tumour growth and prolonged the survival of C57BL/6 mice bearing B16F10 melanoma compared with administration of phosphate-buffered saline (PBS), unmodified S. typhimurium or S. typhimurium expressing empty vector (S. typhimurium [Vec]) in a natural killer (NK) cell-dependent manner. Moreover, genetically modified Salmonella, including S. typhimurium (IFN-γ), showed little toxicity to normal tissues with no observable adverse effects. However, S. typhimurium (IFN-γ)-mediated tumour suppression was attributed to direct killing of tumour cells rather than to stable anti-tumour immunity. Collectively, these results suggest that tumour-targeted therapy using S. typhimurium (IFN-γ) has potential for melanoma treatment.

Due to their intracellular permeability, protein transduction domains (PTDs) have been widely used to deliver proteins and peptides to mammalian cells. However, their performance in gene delivery has been relatively poor. To improve the efficiency of PTD-mediated gene delivery, we synthesized a new peptide, KALA-Antp (K-Antp), which contains the sequences for PTD of the third alpha-helix of Antennapedia (Antp) homeodomain and the fusogenic peptide KALA. In this configuration, Antp is designed to provide the cell permeation capacity and nuclear localization signal, while the KALA moiety to promote cellular entry of the peptide-DNA complex. An optimal K-Antp/DNA formula was nearly 400-600 fold more efficient than Antp or poly-lysine-Antp (L-Antp) in gene delivery, and comparable or superior to a commercial liposome. The K-Antp-mediated plasmid DNA transfection not only exhibited temperature sensitivity, reflecting the involvement of an endocytosis-mediated gene transfer mechanism similar to other known PTDs, but also temperature insensitivity, suggesting the role of an energy-independent mechanism. Incorporation of an endosomolytic polymer polyethylenimine (PEI) into the system or treatment with chloroquine further increased the efficiency of K-Antp-mediated gene delivery. These results demonstrate the potential of the combinatorial use of KALA, Antp and PEI in the development of efficient PTD-derived gene carriers.

Aims The phosphatase of regenerating liver (PRL) family is related to tumorigenesis and metastasis in various cancer types. Its overexpression increases cell motility and proliferation via the downregulation of p21 expression. In a previous study, we reported that PRL-1 downregulates p53 and is a target gene of p53. In this study, we investigated whether a member of the PRL family, PRL-3, could regulate p53 like PRL-1 in cancer cells. Main methods To elucidate the role of PRL-3 in regulating p53 in cancer cells, we used a cell culture system to measure protein level, transcriptional level, apoptosis or localization. Key findings We determined that PRL-3 overexpression reduced the activity of the p21 and p53 reporters. Additionally, the levels of endogenous and exogenous p53 protein were reduced in cells transiently expressing PRL-3, whereas the ablation of PRL-3 by siRNA increased levels of the p53 protein. The downregulation of p53 by PRL-3 inhibited p53-mediated apoptosis. However, the phosphatase-dead mutant C104S, prenylated-site mutant C170S, and C104S/C170S PRL-3 evidenced minimal effects on the downregulation of p53 protein as compared with wild-type PRL-3. Further examinations revealed that PRL-3 expression reduced the stability of p53 by inducing the transcription of p53 induced protein with a RING-H2 domain (PIRH2) through early growth response (EGR) and by increasing the phosphorylation of mouse double minute 2 (MDM2), and then both negatively regulated p53. Significance These findings demonstrated that PRL-3, like PRL-1, can negatively regulate p53 via the activation of PIRH2 and MDM2 in cancer cells.

Transforming growth factor-beta1 (TGF-beta1) is a multi-functional cytokine involved in the regulation of cell proliferation, differentiation and extracellular matrix formation. In search for novel genes mediating the TGF-beta1 function at downstream signaling, we performed a cDNA microarray analysis and identified 60 genes whose expression is regulated by TGF-beta1 in the liver cancer cell line PLC/PRF/5. Among them, we report here lysyl oxidase like 4 (LOXL4) as a novel target of TGF-beta1 signaling, and provide experimental evidence for its expression regulation and function. LOXL4 was found to be the only member of LOX family whose expression is induced by TGF-beta1 in hepatoma cells. Deletion mapping of the LOXL4 promoter indicated that the TGF-beta1 regulation of LOXL4 expression is mediated through the binding of AP1 transcription factor to a conserved region of the promoter. This was confirmed by the chromatin immunoprecipitation assay that captured c-Fos-bound chromatin from TGF-beta1-treated cells. Forced expression of LOXL4 in PLC/PRF/5 cells resulted in inhibition of cell motility through Matrigel in the presence of TGF-beta1 treatment. In parallel, LOXL4 suppressed the expression of laminins and alpha3 integrin and the activity of MMP2. These results suggest that LOXL4 may function as a negative feedback regulator of TGF-beta1 in cell invasion by inhibiting the metabolism of extracellular matrix (ECM) components.

A novel non-viral gene delivery system comprised of a DNA/PEI/Alginate (DPA) polyplex was prepared and assessedin vitro andin vivo. Coating the positively charged DNA/PEI (DP) complex with a polyanion resulted in a high level ofin vitro reporter gene transfection in the presence of 50 vol% serum due to the minimized cytotoxicity of PEI and the reduced nonspecific interactions with serum components. Among the tested anionic polymers, which included sodium alginate, poly(methacrylic acid) and poly(acrylic acid), the sodium alginate showed the highest gene transfection efficiency. The DPA polyplex also showed a reduced level of erythrocyte aggregation in target cells when compared with the DP complex. According toin vivo studies in which reporter genes encoding green fluorescent protein (GFP) and luciferase were used, injection of the DPA polyplex into tumor cells in six week old female C57/BL6 mice resulted in a much higher level of GFP expression and approximately 7 fold higher luciferase expression than treatment with the DP complex. Taken together, these results demonstrated that the anionic alginate coating of the DP complex contributed to efficient gene deliveryin vitro andin vivo.

Alzheimer's disease (AD) is a chronic neurodegenerative disease and the risk of developing it increases with advancing age. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the main bioactive components produced by the traditional Chinese herb, radix bupleuri, the root of Bupleurum falcatum, against AD in various neuronal models. Interestingly, we found that SSc has dual effects on AD by targeting amyloid beta (Aβ) and tau, two key proteins in AD. SSc significantly suppressed the release of both Aβ peptides 1-40 and 1-42 into cell culture supernatants, though it does not affect BACE1 activity and expression. SSc also inhibited abnormal tau phosphorylation at multiple AD-related residues. Moreover, SSc seems to have beneficial effects on cellular tau function; it accelerated nerve growth factor-mediated neurite outgrowth and increased the assembly of microtubules. In addition, SSc increased synaptic marker proteins such as synaptophysin and PSD-95. Considering its various biological activities, our results suggest that SSc might be a novel therapeutic tool for treating human AD and other neurodegenerative diseases. Tau and amyloid beta are two key features in Alzheimer's disease. Saikosaponin C, an active component of Bupleuri Radix, inhibits abnormal tau phosphorylation and amyloid beta production, thereby promoting synaptic integrity. Saikosaponin C also prevents amyloid beta-induced apoptosis in brain vascular endothelial cells. Therefore, Saikosaponin C may provide a new therapeutic strategy for treatment of neurodegenerative diseases, including Alzheimer's disease.

The unfolded protein response (UPR) regulates endoplasmic reticulum (ER) homeostasis and protects cells from ER stress. IRE1α is a central regulator of the UPR that activates the transcription factor XBP1s through an unconventional splicing mechanism using its endoribonuclease activity. IRE1α also cleaves certain mRNAs containing XBP1-like secondary structures to promote the degradation of these mRNAs, a process known as regulated IRE1α-dependent decay (RIDD). We show here that the mRNA of CReP/Ppp1r15b, a regulatory subunit of eukaryotic translation initiation factor 2α (eIF2α) phosphatase, is a RIDD substrate. eIF2α plays a central role in the integrated stress response by mediating the translational attenuation to decrease the stress level in the cell. CReP expression was markedly suppressed in XBP1-deficient mice livers due to hyperactivated IRE1α. Decreased CReP expression caused the induction of eIF2α phosphorylation and the attenuation of protein synthesis in XBP1-deficient livers. ER stress also suppressed CReP expression in an IRE1α-dependent manner, which increased eIF2α phosphorylation and consequently attenuated protein synthesis. Taken together, the results of our study reveal a novel function of IRE1α in the regulation of eIF2α phosphorylation and the translational control.

Polyethylene glycol (PEG) has been coupled to many cationic polymers such as polyethylenimine (PEI) to improve the stability and transfection efficiency. We prepared PEG-grafted PEI with different lengths and amounts of PEG and used these graft copolymers as nonviral gene vectors. We measured the complex size and zeta-potential of polymer-DNA complexes in the presence of salt to estimate the stability of polymer-DNA complexes. We also investigated the cytotoxicity and transfection efficiency in C3 cells. In the case of graft copolymers, the stability of polymer-DNA complexes and transfection efficiency were affected by the graft length and amount of PEG side chain. PEG side chains stabilize the polymer-DNA complexes in the presence of salt, but the longer PEG side chains also interrupt the gene delivery in the cells due to the more efficient steric hindrance by longer PEG side chains, and therefore the transfection efficiency is decreased. Short PEG side chains with molecular weight of 350 kDa stabilized the polymer-DNA complexes without decreased transfection efficiency.

Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

Pin1 contains two functional domains, an N-terminal WW domain and a C-terminal peptidyl-prolyl cis/trans isomerase (PPIase) domain. The WW domain is a phosphorylation specific protein interaction m...

An all CMOS variable gain amplifier (VGA) which features wide dB-linear gain range per stage (45dB), low power consumption (1.32mW), small chip size (0.13mm2), and low supply voltage (1.2V) is described. The dB-linear range is extended by reducing the supply voltage of the conventional V-to-I converter. The two-stage VGA implemented in 0.18μm CMOS offers 90dB of gain variation, 3dB bandwidth of greater than 21MHz, and max/min input IP3 and P1 dB, respectively, of -5/-42 and -12/-50 dBm.

There is growing interest in identifying regulators of autophagy. The molecular mechanism underlying transforming growth factor-β activated kinase 1 (TAK1)-induced autophagy is poorly understood. We found that TAK1 inhibits p70 S6 kinase1 (S6K1) phosphorylation by interfering interaction of raptor with S6K1, thus inducing autophagy. The factors that determine whether autophagy is cytoprotective or cytotoxic have not been fully elucidated. In Drosophila, TAK1 overexpression leads to an impaired eye phenotype despite inhibition of apoptosis, indicating that the phenotype was mainly due to autophagy. Also, TAK1 overexpression increases lactate dehydrogenase (LDH) level in mammalian cells. When treated with autophagy inhibitors, the level of TAK1-induced cytotoxicity or cell death was significantly attenuated, indicating that TAK1 induces cytotoxic autophagic cell death. This study provides the first in vitro and in vivo evidence of TAK1-induced autophagy and we believe that our findings significantly contribute to the understanding of the mechanisms underlying the induction of autophagy.

Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1 alpha in human colon cancer (HCT116) cells. PIN1 binding to HIF-1 alpha occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1 alpha at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1 alpha protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1 alpha resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1 alpha. These results suggest that PIN1 interacting with HIF-1 alpha is a potential cancer chemopreventive and therapeutic target.

Fbw7 is the substrate recognition component of the SCF (Skp1-Cullin-F-box)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers, however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, over-expressing Pin1 reduces Fbw7 abundance and suppresses Fbw7’s ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis and Pin1 may be a promising drug target for anti-cancer therapy.