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LL-37, also called cathelicidin, is an important part of the human immune system, which can resist various pathogens. A plethora of experiments have demonstrated that it has the multifunctional effects of immune regulation, in addition to antimicrobial activity. Recently, there have been increasing interest in its immune function. It was found that LL-37 can have two distinct functions in different tissues and different microenvironments. Thus, it is necessary to investigate LL-37 immune functions from the two sides of the same coin. On the one side, LL-37 promotes inflammation and immune response and exerts its anti-infective and antitumor effects; on the other side, it has the ability to inhibit inflammation and promote carcinogenesis. This review presents a brief summary of its expression, structure, and immunomodulatory effects as well as brief discussions on the role of this small peptide as a key factor in the development and treatment of various inflammation-related diseases and cancers.

Salivary microRNAs (miRNAs) could potentially serve as biomarkers for the diagnosis and prognosis of various types of oral cancer but the significance of it is yet to be fully elucidated. This article reports our study results on the role of salivary miRNAs and their potential use as biomarkers in head and neck cancer (HNC) diagnosis with an emphasis on oral cancers (OC). Supernatant saliva samples from 24 subjects, including 12 OC patients and 12 healthy individuals as a control group, were initially profiled using next generation sequencing (NGS). A novel miR-7703 was further validated in 160 samples collected from OC patients (n = 80) and controls (n = 80) by quantitative real-time polymerase chain reaction (qRT-PCR) assays. Data analysis revealed that 373 miRNA expressions were significantly decreased and 265 miRNA expressions were significantly increased in patients with OC (p ≤ 0.05). Of the 638 miRNA, twenty-seven were non-redundant miRNAs associated with OC (p ≤ 0.0001). Validation of the novel miR-7703 showed a significant increase in the OC group when compared to the control group. Most importantly, this pattern of increased expression was also positively correlated with tumour stage, lymph node metastasis status, and clinical stage. This study identified twenty-seven non-redundant, differentially expressed miRNAs associated with OC. These signatures include a number of novel miRNAs as well as those that have been previously reported in either oral or other cancers. However, miR-7703 is a previously uncharacterised miRNA with the potential to be a significant biomarker for the diagnosis of OC.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by suppressing the target mRNA and inhibiting translation in order to regulate multiple biological processes. miRNAs play important roles as oncogenes or tumor suppressors in the development of various types of human cancer. The regulation of mammalian target of rapamycin (mTOR) by miRNAs has been studied in several types of cancer, including colorectal cancer (CRC). However, to the best of our knowledge, only limited information regarding the function of miRNAs in human CRC is available. In the present study, the expression of 22 miRNAs in CRC cell lines were investigated in regard to key genes in the mTOR pathway. Initially, it was revealed that mTOR, regulatory-associated protein of mTOR complex I and rapamycin-intensive companion of mTOR were overexpressed in CRC cell lines when compared with a normal colorectal cell line. Subsequently, putative miRNA-mRNA associations were identified via multiple miRNA target prediction programs. The expression levels for the candidate miRNAs were validated using quantitative real-time polymerase chain reaction. Expression analysis revealed that, among 20 miRNAs, five miRNAs (miR-496, miR-1185, miR-654, miR-3183 and miR-495) exhibited significant downregulation in association with the mTOR signaling pathway. Taken together, the results from the present study suggest that several miRNAs that are associated with CRC, with possible roles in mTOR signaling, may have potential therapeutic or diagnostic benefits in CRC treatment.

Background. AnaerobicClostridialspores (CG) cause significant oncolysis in hypoxic tumour microenvironment and result in tumour regression in both animal models and clinical trials. The immune mediated response plays a critical role in the antitumour effect by the anaerobic spore treatment.Method. Human papillomavirus 16 E6/E7 transformed TC-1 tumour bearing mice were intravenously administered with low (1 × 108CFU/kg) or high dosage (3 × 108CFU/kg) of DerivativeClostridialspore (DCG).Results. Intravenous administration of the derivative ofClostridial ghonii(DCG) spores leads to both tumour and systemic inflammatory responses characterized by increased IFNγ/IL-9 secreting T cells in the spleen and the tumour. Low numbers of antigen specific T cells (6spleen cells) in the spleen of the tumour bearing mice are also detected after intravenous DCG delivery. Interestingly, our results showed that a mixed IL-9/IFNγsecreting T cell response was induced when the tumour bearing mice received a low dose of DCG spore (1 × 108CFU/kg), while a strong IFNγresponse was elicited with a high dosage of DCG spore (3 × 108CFU/kg).Conclusion. The dosage of DCG spore will determine the types of the DCG induced immune responses.

Epidermal growth factor receptor (EGFR) supports colorectal cancer progression via oncogenic signaling. Anti-EGFR therapy is being investigated as a clinical option for colorectal cancer, and an observed interaction between EGFR and Prion protein has been detected in neuronal cells. We hypothesized that PrPC expression levels may regulate EGFR signaling and that detailed understanding of this signaling pathway may enable identification of resistance mechanisms and new actionable targets in colorectal cancer. We performed molecular pathway analysis following knockdown of PrPC or inhibition of EGFR signaling via gefitinib to identify changes in expression of key signaling proteins that determine cellular sensitivity or resistance to cisplatin. Expression of these proteins was examined in matched primary and metastatic patient samples and was correlated for resistance to therapy and progression of disease. Utilizing three colorectal cancer cell lines, we observed a correlation between high expression of PrPC and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrPC contributed to signaling via colocalization with EGFR, which could be overcome by targeting p38 mitogen-activated protein kinases (p38 MAPK). We revealed that the level of Kruppel-like factor 5 (KLF5), a target downstream of p38 MAPK, was predictive for cell line and patient response to platinum agents. Further, high KLF5 expression was observed in BRAF-mutant colorectal cancer. Our study indicates that the EGFR to KLF5 pathway is predictive of patient progression on platinum-based therapy.

Glycolipids from Mycobacterium tuberculosis have a profound impact on the innate immune response of the host. Macrophage-inducible C-type lectin (Mincle) is a pattern-recognition receptor that has been shown to bind trehalose dimycolate (TDM) from the mycobacterium and instigate intracellular signalling in the immune cell. There are structural similarities between the structures of TDM and phosphatidyl inositol mannoside (PIM). We thus hypothesized that these latter structures might also modulate an immune response in a similar manner. To test this, we synthesized a series of new mannose derivatives modified with fatty esters at the 6-position and assessed the release of inflammatory cytokines in human U937 macrophages under the induction of lipopolysaccharides (LPS) after glycolipid treatment. The results showed that the amount of two major cytokines-tumour necrosis factor (TNF)-α and interleukin (IL)-6-released from LPS-stimulated U937 cells decreased significantly when compared to a control upon treatment with the prepared glycolipids, thus indicating a reduction in cytokine production by the macrophages.

Caerin is a family of peptides isolated from the glandular secretion of Australian tree frogs, the genusLitoria, and has been previously shown to have anticancer activity against several cancer cells. In this work, we used two host-defence peptides, caerin 1.1 and caerin 1.9, to investigate their ability to inhibit a murine derived TC-1 cell transformed with human papillomavirus 16 E6 and E7 growthin vitro. Caerin 1.9 inhibits TC-1 cell proliferation, although inhibition is more pronounced when applied in conjunction with caerin 1.1. To gain further insights into the antiproliferative mechanisms of caerin 1.9 and its additive effect with caerin 1.1, we used a proteomics strategy to quantitatively examine (i) the changes in the protein profiles of TC-1 cells and (ii) the excretory-secretory products of TC-1 cells following caerin peptides treatment. Caerin 1.9 treatment significantly altered the abundance of several immune-related proteins and related pathways, such as the Tec kinase and ILK signalling pathways, as well as the levels of proinflammatory cytokines and chemokines. In conclusion, caerin peptides inhibit TC-1 cell proliferation, associated with modification in signalling pathways that would change the tumour microenvironment which is normally immune suppressive.

Backgrounds MicroRNAs (miRNA) are a class of non-protein-coding RNAs that have significant biological and pathological functions. The importance of miRNAs as potential cancer diagnostic biomarkers is gaining attention due to their influence in the regulation of cellular processes such as cell differentiation, proliferation and apoptosis. The aim of this study was to identify significant miRNAs from saliva as potential diagnostic biomarkers in the early diagnosis and prognosis of head and neck squamous cell carcinoma (HNSCC). Materials and methods Five differentially expressed miRNAs (miR-7703, miR- let-7a-5p, miR- 345-5p, miR- 3928 and miR- 1470) were selected from Next Generation Sequencing (NGS) miRNA data generated from our previous study using saliva of 12 HNSCC patients and 12 healthy controls. Their differential expressed miRNAs were subsequently validated by RT-qPCR using saliva samples from healthy controls (n = 80) and HNSCC patients (n = 150). Total RNA was isolated from 150 saliva samples of HNSCC patients and was transcripted into cDNA by TaqMan MicroRNA Reverse Transcription Kit. Using quantitative RT-PCR analysis, salivary miRNAs were identified in HNSCC patients (n = 150) and healthy controlled cases (n = 80). T-tests were used to compare the differences among the various clinical variants. Results On average 160 ng/μl was isolated from 500 μl of saliva. Overall, a good correlation observed between the HNSCC and some of miRNAs expression levels. Salivary miR-let-7a-5p (P

Discovering the underlying signalling pathways that control cancer cells is crucial for understanding their biology and to develop therapeutic regimens. Thus, the aim of the present study was to determine the effect of Cripto-1 on pathways controlling glioblastoma (GBM) cell function. To this end, changes in protein phosphorylation in cells overexpressing Cripto-1 were analysed using the Kyoto Encyclopedia of Genes and Genomes pathway analysis tool, as well as the Uniprot resource to identify the functions of Cripto-1-dependent phosphorylated proteins. This revealed that proteins affected by Cripto-1 overexpression are involved in multiple signalling pathways. The mitogen-activated protein kinase (MAPK), focal adhesion (FA) and ErbB pathways were found to be enriched by Cripto-1 overexpression with 35, 27 and 24% of pathway proteins phosphorylated, respectively. These pathways control important cellular processes in cancer cells that correlate with the observed functional changes described in earlier studies. More specifically, Cripto-1 may regulate MAPK cellular proliferation and survival pathways by activating epithelial growth factor receptor (EGFR; Ser1070) or fibroblast GFR1 (Tyr654). Its effect on cellular proliferation and survival could be mediated through Src (Tyr418), FA kinase (FAK; Tyr396), p130CAS (Tyr410), c-Jun (Ser63), Paxillin (PXN; Tyr118) and BCL2 (Thr69) of the FA pathway. Cripto-1 may also control cellular motility and invasion by activating Src (Tyr418), FAK (Tyr396) and PXN (Tyr118) of the FA pathway. However, Cripto-1 regulation of cellular invasion and migration might be not limited to the FA pathway, it may also control these cellular mechanisms through signalling via EGFR (Ser1070)/Her2 (Tyr877) to mediate the Src (Tyr418) and FAK (Tyr396) cascade activation of the ErbB signalling pathway. Angiogenesis could be mediated by Cripto-1 by activating c-Jun (Ser63) through EGFR (Ser1070)/Her2 (Tyr877) of the ErbB pathway. To conclude, the present study has augmented and enriched our current knowledge on the crucial roles that Cripto-1 may play in controlling different cellular mechanisms in GBM cells.

Recent evidences show that activation of serotonin 2A receptors (5-HT2A R) by agonists is significant in improving therapeutic activity of disease conditions, such as obsessive-compulsive disorder (OCD). Though the exact molecular mechanism is still not well understood, it is thought to involve agonist-driven, enhanced expression of 5-HT2A R in certain areas of brain, such as the pre-frontal cortex (PFC). Several other reports have also demonstrated association of OCD with lower dopamine receptor (D2 R) availability, primarily in the striatum of the brain along with dysfunction of 5-HT2A R-D2 R heteromer regulation. We thus hypothesized that compound(s) interacting with this molecular mechanism could be developed as drugs for long-term beneficial effects against OCD. In the present study, we have obtained experimental evidence in cultured neuronal cells (CLU213) that aqueous extract (AE, 50 μg/mL, P < 0.05) of the Australian cane toad skin significantly increased the levels of 5-HT2A R and D2 R protein and mRNA expression. AE was also found to enhance the interaction between 5-HT2A R and D2 R and formation of expression of 5-HT2A R-D2 R heteromer using co-immunoprecipitation and Western blot. Further investigation showed the involvement of classical signaling pathway (Gq/11 -PLCβ) along with c-FOS transcription factor preferentially in 5-HT2A -mediated agonist activation. These results obtained demonstrated that AE upregulates 5-HT2A R by a mechanism that appears to involve Gq/11 -PLCβ signaling pathway and c-FOS transcription factor activation. We indicate this enhanced 5-HT2A R and D2 R expression and their interaction to induce increased 5-HT2A R-D2 R heteromer formation by exposure to AE might provide a molecular mechanism to develop potential novel drug candidates to ameliorate OCD symptoms. J. Cell. Biochem. 118: 979-993, 2017. © 2016 Wiley Periodicals, Inc.

Indolealkylamines (IAAs) are biogenic amines and derivatives of 5-hydroxytryptamine, acting primarily on serotonin receptors. IAAs are often considered the most thoroughly investigated group of aromatic amines in the amphibian skin. On the contrary, at present the detailed knowledge of these compounds in lower organisms is still limited and the biogenic amine receptors, mediating hormonal and modulatory functions, are largely unknown in primitive invertebrates. However, some active research is currently underway investigating this class of biogenic amines. Notably, during the last three decades several investigations have demonstrated the biological activity of endogenous biogenic amines in cnidarians, which are known to be the lowest beings equipped with an effective, even though rudimentary, nervous system. Toads, especially those from the Bufonidae family, constitute a significant part of the amphibian family and are an identified source of IAAs. To date fourteen IAAs have been identified in the skins of toad species. All are 5-substituted IAA derivatives acting mainly on the central nervous system (CNS), with most exhibiting some degrees of 5-HT2A receptor selectivity. This selective ability presents potential for their use in the development of treatments for various disorders such as schizophrenia, depression, anxiety, obsessive-compulsive disorders and chronic pain conditions. There are indications that some IAAs may also show subclass selectivity through binding to multiple 5-HT receptor subtypes. Thus, there exists an additional promising platform for the development of therapeutics targeting multiple 5-HT receptors. In this review, IAAs occurring naturally in various species of toad skins, which have been identified and isolated since 1944 are summarized and comparisons are made with similar biogenic amines recognized in cnidarians to date. Such comparisons highlight the potential to utilize existing knowledge gathered from vertebrates, such as toads in order to improve the understanding of the activities of such compounds in lower invertebrates.

Toad skin extracts, such as aqueous extracts (AE) of Chinese toad skins, have demonstrated therapeutic benefits for a range of diseases including pain, inflammation, swelling, heart failure, and various types of cancers. In this study, we investigated the anti-inflammatory potential of an AE (0.1-10 μg/mL) and a 60% ethanol extract (EE; 0.1-10 μg/mL) from Australian cane toad (Bufo marinus) skins and the known bioactive compound, bufotenine (BT; 0.1-10 nM). The assay employed a model of the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) for the release of tumor necrosis factor (TNF)-α and interleukin (IL)-6. We demonstrated that AE, EE, and BT significantly inhibited the release and expression of TNF-α and IL-6 in a dose-dependent manner when the cells were pre-treated at non-cytotoxic concentrations. Further investigation revealed that the inhibition of TNF-α and IL-6 release and expression was associated with the suppression of nuclear factor (NF)-kappa (κ)B activation. These results indicate that AE, EE, and BT are strong inflammation inhibitors, thus have the potential for further development as anti-inflammatory therapeutic agents from a natural source regarded as a feral pest in Australia. J. Cell. Biochem. 117: 2769-2780, 2016. © 2016 Wiley Periodicals, Inc.

Recent culture-independent studies have enabled detailed mapping of human microbiome that has not been hitherto achievable by culture-based methods. DNA extraction is a key element of bacterial culture-independent studies that critically impacts on the outcome of the detected microbial profile. Despite the variations in DNA extraction methods described in the literature, no standardized technique is available for the purpose of microbiome profiling. Hence, standardization of DNA extraction methods is urgently needed to yield comparable data from different studies. We examined the effect of eight different cell lysis protocols on the yield and quality of the extracted DNA from oral rinse samples. These samples were exposed to cell lysis techniques based on enzymatic, mechanical, and a combination of enzymatic-mechanical methods. The outcome measures evaluated were total bacterial population, Firmicutes levels and human DNA contamination (in terms of surrogate GAPDH levels). We noted that all three parameters were significantly affected by the method of cell lysis employed. Although the highest yield of gDNA was obtained using lysozyme-achromopeptidase method, the lysozyme-zirconium beads method yielded the peak quantity of total bacterial DNA and Firmicutes with a lower degree of GAPDH contamination compared with the other methods. Taken together our data clearly points to an urgent need for a consensus, standardized DNA extraction technique to evaluate the oral microbiome using oral rinse samples. Further, if Firmicutes levels are the focus of investigation in oral rinse microbiome analyses then the lysozyme-zirconium bead method would be the method of choice in preference to others.

Blocking cytokine interleukin 10 (IL-10) at the time of immunisation enhances vaccine induced T cell responses and improves control of tumour cell growth in vivo. However, the effect of an IL-10 blockade on the biological function of macrophages has not been explored. In the current paper, a macrophage precursor cell line, U937 cells, was selected to investigate the differential expression of proteins and relevant cell signalling pathway changes, when stimulated with lipopolysaccharide (LPS) in the presence of antibodies to IL-10 or IL-10 receptor. We used a quantitative proteomic strategy to investigate variations in protein profiles of U937 cells following the treatments with LPS, LPS plus human anti-IL10 antibody and anti-IL10R antibody in 24hrs, respectively. The LPS treatment significantly activated actin-related cell matrix formation and immune response pathways. The addition of anti-IL10 and anti-IL10R antibody further promoted the immune response and potentially effect macrophage survival through PI3K/AKT signalling; however, the latter appeared to also upregulated oncogene XRCC5 and Cajal body associated processes.

Although cancer is a leading cause of death, significant breakthroughs have been made in its treatment in recent years. In particular, increasingly effective cancer vaccines are being developed, including some for colorectal cancer. There are also currently a variety of compounds that can act as adjuvants, such as signalling molecules called cytokines. Other adjuvants target and inhibit the specific mechanisms by which cancers evade the immune system. One of them is a galectin inhibitor, which targets galectins—proteins produced by cancer cells that can cause the death of immune cells. Likewise, immune checkpoint inhibitors affect immune checkpoints—natural host proteins that usually control inflammation but can be exploited by cancers to weaken the body’s defences. Equally, regulatory T cells may contribute to the progression of cancer by inhibiting the functions of other T cells. The main advantages of cancer vaccines include their low toxicity and their ability to strengthen the immune system. Nevertheless, significant limitations include their slow effects and their inability to treat cancer at times due to immunosuppression. Ultimately, ongoing trials provide hope for the development of more effective methods of immunotherapeutic inoculation that can target a greater variety of cancers.

Background Anaerobic Clostridial spores (CG) cause significant oncolysis in hypoxic tumour microenvironment and result in tumour regression in both animal models and clinical trials. The immune mediated response plays a critical role in the antitumour effect by the anaerobic spore treatment. Method Human papillomavirus 16 E6/E7 transformed TC-1 tumour bearing mice were intravenously administered with low (1 × 108 CFU/kg) or high dosage (3 × 108 CFU/kg) of Derivative Clostridial spore (DCG). Results Intravenous administration of the derivative of Clostridial ghonii (DCG) spores leads to both tumour and systemic inflammatory responses characterized by increased IFNγ/IL-9 secreting T cells in the spleen and the tumour. Low numbers of antigen specific T cells (

Cancer cells have been known to overexpress the epidermal growth factor receptor (EGFR) and hence relevant multiple‑targeted therapies have been developed, with a recent clinical application of the antibody‑mediated inhibition of the EGFR. However, this strategy is not useful in cancer cells with mutations in KRAS; a GTPase downstream of EGFR which constitutively activates the pathway without EGF stimulation. Furthermore, mutations in EGFR also reduce the binding of monoclonal antibodies and thereby render them ineffective. In the present study, we designed a chimeric EGF protein fused to the truncated N‑terminal domain fragment of Pseudomonas aeruginosa exotoxin A (EGF‑ETA), which has ADP‑ribosylation activity and induces apoptosis. The EGF‑ETA protein was expressed in E. coli as a His‑tagged fusion. Our results showed that EGF‑ETA significantly inhibited the proliferation of EGFR‑positive A431 epidermoid carcinoma (IC50 27 ng/ml) and HN5 head and neck squamous cell carcinoma (IC50 36 ng/ml) cells. However, its effect on cancer cells with little or no EGFR expression was limited (A549‑IC50 1,000 ng/ml; MCF‑7‑IC50 >10,000 ng/ml). Compared to cetuximab, EGF‑ETA was highly potent in its killing capacity of HN5 cancer cells at 1,000 ng/ml, while cetuximab had little effect at 1,000 ng/ml. Furthermore, EGF‑ETA was just as potent in HCT116 (KRAS G13D) and SW480 (KRAS G12V) colon cancer cell lines harbouring KRAS hyperactivating mutations when compared to KRAS wild‑type HT29 colon cancer cells. Finally, co‑incubation of EGF‑ETA with an anti‑EGF antibody abrogated its effect on the EGFR‑positive A431 cells. Our results show that the chimeric EGF‑ETA toxin is extremely effective against EGFR‑positive cancers and raises the potential to further develop this chimera for use in targeting EGFR‑positive tumours resistant to monoclonal antibodies.

Toxins from toads have long been known to contain rich chemicals with great pharmaceutical potential. Recent studies have shown more than 100 such chemical components, including peptides, steroids, indole alkaloids, bufogargarizanines, organic acids, and others, in the parotoid and skins gland secretions from different species of toads. In traditional Chinese medicine (TCM), processed toad toxins have been used for treating various diseases for hundreds of years. Modern studies, including both experimental and clinical trials, have also revealed the molecular mechanisms that support the development of these components into medicines for the treatment of inflammatory diseases and cancers. More recently, there have been studies that demonstrated the therapeutic potential of toxins from other species of toads, such as Australian cane toads. Previous reviews mostly focused on the pharmaceutical effects of the whole extracts from parotoid glands or skins of toads. However, to fully understand the molecular basis of toad toxins in their use for therapy, a comprehensive understanding of the individual compound contained in toad toxins is necessary; thus, this paper seeks to review the recent studies of some typical compounds frequently identified in toad secretions.

Cancer as a genetic disorder is one of the leading causes of death worldwide. Conventional anticancer options such as chemo- and/or radio-therapy have their own drawbacks and could not provide a cure in most cases at present. More effective therapeutic strategies with less side effects are urgently needed. Aptamers, also known as chemical antibodies, are single strand DNA or RNA molecules that can bind to their target molecules with high affinity and specificity. Such site-specific binding ability of aptamers facilitates the delivery and interaction of exogenous nucleic acids with diseased genes. Thus, aptamer-guided gene therapy has emerged as a promising anticancer strategy in addition to the classic treatment regimen. Aptamers can directly deliver anti-cancer nucleic acids, e.g. small interfering RNA, micro RNA, antimicroRNA and small hairpin RNA, to cancer cells or function as a targeting ligand to guide nanoparticles containing therapeutic nucleic acids. This review focuses on recent progress in aptamer-mediated gene therapy for the treatment of hepatocellular carcinoma and other types of cancers, shedding light on the potential of this novel approach of targeted cancer gene therapy.

The mammalian target of rapamycin (mTOR), a downstream effector of the PI3K/Akt signalling pathway, is a critical regulator of cell metabolism, growth and survival in response to oncogenic factors. Activation of mTOR frequently occurs in human tumours making it a crucial and validated target in the treatment of cancer. mTOR inhibitors such as rapamycin and its analogues decrease cancer progression in experimental models including colorectal cancer (CRC). Recently, the second generation ATP‑competitive mTOR kinase (such as PP242) and dual mTOR/PI3K (such as NVP‑BEZ235) inhibitors have entered clinical trials as anticancer agents. However, in CRC, the efficacy of these novel drugs needs to be fully investigated. In the present study, we examined five human CRC cell lines, HT29, HCT116, SW480, SW620 and CSC480 to evaluate their sensitivity to three mTOR inhibitors, RAD001, PP242 and NVP‑BEZ235. We observed that compared to RAD001 and PP242, NVP‑BEZ235 markedly reduced cell proliferation of CRC cells. Furthermore, we found that the reduced cell proliferation caused by NVP‑BEZ235 was not achieved through the disruption of mitochondrial potential. Using an mTOR‑specific signalling pathway phospho array we revealed that NVP‑BEZ235 significantly decreased phosphorylation of 4E‑BP1 (Thr70), the downstream target of mTORC1. In addition, NVP‑BEZ235 decreased phosphorylation of AKT (Ser473), the downstream target of mTORC2. Immunoblotting analysis revealed that NVP‑BEZ235 effectively inhibited 4E‑BP1 phosphorylation, while PP242 had a weak inhibitory effect. However, PP242 and NVP‑BEZ235 decreased AKT levels in all cell lines. RAD001 demonstrated no effect on 4E‑BP1. Based on the above‑mentioned results, the dual PI3K/mTOR and ATP‑competitive mTOR inhibitors have demonstrated high potential for targeting the mTOR pathway in CRC.

Photodynamic therapy (PDT) using photosensitized reaction to produce cytotoxicity was used for cancer therapy in recent years. To study the effectiveness of PDT mediated by a novel photosensitizer (PS), DTPP 5-(4'-(2″-dicarboxymethylamino)acetamidophenyl)-10, 15, 20-triphenylporphyrin, on lung cancer A549 cell lines in vitro, DTPP was employed in different concentrations (2, 4, 6, 8, 10, 12, 15, 20, 25, and 30 μg/ml) and combined with 650 nm laser of different power densities (0.6, 1.2, 2.4, 4.8, 7.2, and 9.6 J/cm(2)) that resulted in obvious inhibition of cell proliferation and apoptosis. Results showed that cell survival rates have a dependent relationship with time and PS concentrations and no significant cytotoxicity was induced by DTPP itself. Apoptosis and cell cycle S arrest were observed; cytoskeleton morphologic observation revealed collapse, sparkling, and shrunken shapes. Apoptosis-related protein caspase-3 overexpression was detected while caspase-9, bcl-2, and cytoskeleton protein beta-catenin were in low levels of expression than the control. Cleavage of beta-catenin by caspase-3 or other proteases from the lysosome might be the main reason for the cytoskeleton collapse as beta-tubulin and actin were at a stable level 12 h after PDT. This paper gives a better understanding of the effectiveness of DTPP-mediated PDT in lung cancer A549 cells both with regard to dosimetry and apoptosis changes.

Layered Double Hydroxides (LDHs)-based drug delivery systems have, for many years, shown great promises for the delivery of chemical therapeutics and bioactive molecules to mammalian cells in vitro and in vivo. This system offers high efficiency and drug loading density, as well as excellent protection of loaded molecules from undesired degradation. Toxicological studies have also found LDHs to be biocompatible compared with other widely used nanoparticles, such as iron oxide, silica, and single-walled carbon nanotubes. A plethora of bio-molecules have been reported to either attach to the surface of or intercalate into LDH materials through co-precipitation or anion-exchange reaction, including amino acid and peptides, ATPs, vitamins, and even polysaccharides. Recently, LDHs have been used for gene delivery of small molecular nucleic acids, such as antisense, oligonucleotides, PCR fragments, siRNA molecules or sheared genomic DNA. These nano-medicines have been applied to target cells or organs in gene therapeutic approaches. This review summarizes current progress of the development of LDHs nanoparticle drug carriers for nucleotides, anti-inflammatory, anti-cancer drugs and recent LDH application in medical research. Ground breaking studies will be highlighted and an outlook of the possible future progress proposed. It is hoped that the layered inorganic material will open up new frontier of research, leading to new nano-drugs in clinical applications.

The intracellular delivery and functionalization of genetic molecules play critical roles in gene-based theranostics. In particular, the delivery of plasmid DNA (pDNA) with safe nonviral vectors for efficient intracellular gene expression has received increasing attention; however, it still has some limitations. A facile one-pot method is employed to encapsulate pDNA into zeolitic imidazole framework-8 (ZIF-8) and ZIF-8-polymer vectors via biomimetic mineralization and coprecipitation. The pDNA molecules are found to be well distributed inside both nanostructures and benefit from their protection against enzymatic degradation. Moreover, through the use of a polyethyleneimine (PEI) 25 kD capping agent, the nanostructures exhibit enhanced loading capacity, better pH responsive release, and stronger binding affinity to pDNA. From in vitro experiments, the cellular uptake and endosomal escape of the protected pDNA are greatly improved with the superior ZIF-8-PEI 25 kD vector, leading to successful gene expression with high transfection efficacy, comparable to expensive commercial agents. New cost-effective avenues to develop metal-organic-framework-based nonviral vectors for efficient gene delivery and expression are provided.

Aptamers, and the selection process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) used to generate them, were first described more than twenty years ago. Since then, there have been numerous modifications to the selection procedures. This review discusses the use of modified bases as a means of enhancing serum stability and producing effective therapeutic tools, as well as functionalising these nucleic acids to be used as potential diagnostic agents.

Immunotoxins are engineered chimeric proteins that consist of a fragment of a toxin fused to a modified antibody or growth factor capable of targeting specific cells. Furthermore, these proteins can be targeted to receptors that are commonly overexpressed on cancer cells. The majority of immunotoxins function by binding to cells, translocating into the cytosol and inhibiting protein synthesis. In this study, the expression of claudin‑4 (CLDN4) in various cancer cells was analysed as a potential target for immunotoxins. To target CLDN4-expressing cancer cells, the c-terminal CLDN4‑binding domain of Clostridium perfringens enterotoxin (CPE) was fused to the Pseudomonas aeruginosa exotoxin A (ETA) domain to create an immunotoxin (CPE‑ETA'). Subsequently, the capacity of such an immunotoxin in suppressing the proliferation of CLDN4-positive cancer cells was investigated. We report that head and neck squamous carcinoma cells (HN5) have an elevated CLDN4 expression compared to the other cell lines tested. Our findings further demonstrate that CPE‑ETA' is highly potent against MCF-7 breast [50% inhibitory concentration (IC50) 9.8 ng/ml] and HN5 head/neck (IC50 8.8 ng/ml) cancer cell lines, while it has no cytotoxic effects on HeLa cells (CLDN4‑negative). The immunotoxin was subsequently expressed in the tumour colonising oncolytic strain, Clostridium ghonii. Most importantly, the strictly anaerobic Clostridium ghonii was able to overexpress and secrete a functional CPE‑ETA' fusion protein. Our findings open the possibility of the targeted delivery of the immunotoxin locally to tumour sites at a high concentration using strictly anaerobic Clostridium ghonii for the treatment of CLDN4-positive cancer cells.

Graft-versus-host disease (GVHD) is a common and often fatal complication of bone marrow transplantation. Antigen-presenting cells from donor and recipient play a critical role in the initiation and maintenance of GVHD. CD83, which is expressed in activated lymphocytes and dendritic cells, is regarded as a marker of mature dendritic cells. Targeting CD83 using soluble CD83 molecules or antibodies has been demonstrated to have therapeutic effects against GVHD in preclinical models. Understanding the biological function of CD83 and the underlying mechanisms through which targeting CD83 attenuates GVHD is likely to greatly improve current treatments and provide new methods for the treatment of GVHD.

The mammalian target of rapamycin (mTOR) is a critical regulator of many fundamental features in response to upstream cellular signals, such as growth factors, energy, stress and nutrients, controlling cell growth, proliferation and metabolism through two complexes, mTORC1 and mTORC2. Dysregulation of mTOR signalling often occurs in a variety of human malignant diseases making it a crucial and validated target in the treatment of cancer. Tumour cells have shown high susceptibility to mTOR inhibitors. Rapamycin and its derivatives (rapalogs) have been tested in clinical trials in several tumour types and found to be effective as anticancer agents in patients with advanced cancers. To block mTOR function, they form a complex with FKBP12 and then bind the FRB domain of mTOR. Furthermore, a new generation of mTOR inhibitors targeting ATP-binding in the catalytic site of mTOR showed potent and more selective inhibition. More recently, microRNAs (miRNA) have emerged as modulators of biological pathways that are essential in cancer initiation, development and progression. Evidence collected to date shows that miRNAs may function as tumour suppressors or oncogenes in several human neoplasms. The mTOR pathway is a promising target by miRNAs for anticancer therapy. Extensive studies have indicated that regulation of the mTOR pathway by miRNAs plays a major role in cancer progression, indicating a novel way to investigate the tumorigenesis and therapy of cancer. Here, we summarize current findings of the role of mTOR inhibitors and miRNAs in carcinogenesis through targeting mTOR signalling pathways and determine their potential as novel anti-cancer therapeutics.

Background Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing b-cells of the pancreas. The present study aimed to reverse T1D by gene therapy. Methods We used a novel surgical technique, which involves isolating the liver from the circulation before the delivery of a lentiviral vector carrying furin-cleavable human insulin (INS-FUR) or empty vector to the livers of diabetic non-obese diabetic mice (NOD). This was compared with the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests were performed. Expression of insulin and pancreatic transcription factors was determined by the reverse transcriptase-polymerase chain reaction and immunohistochemistry and immunoelectron microscopy was used to localise insulin. Results Using the novel surgical technique, we achieved long-term transduction (42% efficiency) of hepatocytes, restored normoglycaemia for 150 days (experimental endpoint) and re-established normal glucose tolerance. We showed the expression of b-cell transcription factors, murine insulin, glucagon and somatostatin, and hepatic storage of insulin in granules. The expression of hepatic markers, C/EBP-b, G6PC, AAT and GLUI was down-regulated in INS-FUR-treated livers. Liver function tests remained normal, with no evidence of intrahepatic inflammation or autoimmune destruction of the insulin-secreting liver tissue. By comparison, direct injection of INS-FUR reduced blood glucose levels, and no pancreatic transdifferentiation or normal glucose tolerance was observed. Conclusions This gene therapy protocol has, for the first time, permanently reversed T1D with normal glucose tolerance in NOD mice and, as such, represents a novel therapeutic strategy for the treatment of T1D. Copyright © 2013 John Wiley & Sons, Ltd.

Chansu is one of the most widely used traditional Chinese medicines in China, Japan, and other Southeast Asian countries primarily for antipain, anti-inflammation, and recently anticancer. Over 10 recipes and remedies contained Chansu, which are easily available in pharmacies and hospitals, but the mechanisms of action were not clearly articulated. In the present study, Cinobufagin (CBF), the major compound of Chansu, was employed as a surrogate marker to determine its ability in inducing cancer cell death. As expected, CBF has significant cancer-killing capacity for a range of cancers, but such ability differs markedly. Colon and prostate cancers are more sensitive than skin and lung cancers. Interestingly, cancer cells die through apoptotic pathway either being biphasic caspase-3-dependent (HCT116) or independent (HT29). Multipathway analysis reveals that CBF-induced apoptosis is likely modulated by the hypoxia-inducing factor-1 alpha subunit (HIF-1α) as its inhibition was evidentin vitroandin vivo. Taken together, these results demonstrate that CBF is a potent apoptotic inducer with potential for further development as a novel and effective anticancer agent for a range of cancers, especially colon cancer.

Recent evidences show that activation of serotonin 2A receptors (5-HT

Blockade of IL-10 signalling clears chronic viral and bacterial infections. Immunization together with blockade of IL-10 signalling or relatively low level of IL-10 further enhances viral and bacterial clearance. IL-10 functions through binding to interleukin 10 receptor (IL-10R). Here we showed that peptides P1 and P2 with the hydrophobic and hydrophilic pattern of the IL10R-binding helix in IL-10 could bind with either IL-10R1 or IL-10, and inhibit inflammatory signals with long duration and negligible cytotoxicity in vitro. Furthermore, P2 can enhance antigen specific CD8+ T cell responses in mice induced by the vaccine based on a long peptide of protein E7 in a human papillomavirus type 16.

Toad skins and venom glandular secretions have been widely used for centuries in traditional Chinese and Japanese medicine for the treatment of various ailments such as cancer, sores, toothache, local inflammation and pain. The active chemical constituents from traditional oriental medicines have demonstrated potential in the development of effective therapeutic pharmaceuticals. Our primary focus in this research was to identify and characterise ‘active’ compounds or groups of compounds for their potential as neuropsychiatric disorder therapeutics. For this aim, we utilised a variety of solvents, i.e., the aqueous, 60% ethanol (aqueous) and acetic acid (aq) (at two different pHs) for extractions of Australian cane toad skins to identify chemical constituents. The identification of compounds was carried out using HPLC-ESI-Q-TOF-MS/MS based on the accurate mass measurement for molecular ions and MS/MS analysis, whereby accurate mass pseudo-molecular ions and characteristic fragment ions were compared to published reference data, including mass bank and NIST. As a result, we have to date identified 42 major constituents including alkaloids, amino acids, bufadienolides, fatty acids, nucleobases, nucleosides and vitamins mostly from the aqueous and 60% ethanol extracts. Of the 42 constituents identified, 29 were found in the aqueous extract, 35 were found in the ethanol (aq) extract and only 10 in the pH 1.78 acetic acid extract and 11 in the pH 2.17 acetic acid extract of the cane toad skins. Therefore, the aqueous and 60% ethanolic extracts present the greatest potential for ongoing development in our assays. There have been no previous reports on the identification of many of the constituents we have here identified in Australian cane toad skins. These findings, while somewhat consistent with findings in toad skins in other countries, identifies the presence of potential bioactive constituents. Our results showed that HPLC-ESI-Q-TOF-MS/MS is an effective method to characterise and identify components in Australian cane toad skin extracts. Chemical profiling is an essential initial step in the identification and therapeutic exploitation of bioactive agents present in Australian cane toad skin extracts.

With a view to improving the solubility and delivery characteristics of poorly water-soluble drugs, we prepared β-cyclodextrin-curcumin (βCD-C) inclusion complexes (hydrophilic curcumin) and entrapped both native curcumin (hydrophobic) and the complexes separately into liposomes; these were then assessed for in vitro cytotoxicity in lung and colon cancer cell lines. Optimization of curcumin entrapment within βCD was achieved, with the resultant βCD-C complexes prepared by methanol reflux. Inclusion complexes were confirmed using UV spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction. The water solubility of βCD-C complexes improved markedly (c.f. native curcumin) and successful entrapment of complexes into liposomes, prepared using a thin-film hydration approach, was also achieved. All the liposomal formulations were characterized for curcumin and βCD-C complex entrapment efficiency, particle size, polydispersity and stability at 2-8°C. Curcumin, βCD-C complex and their optimized liposomal formulations were evaluated for anticancer activity in lung (A-459) and colon (SW-620) cancer cell lines. All curcumin-containing formulations tested were effective in inhibiting cell proliferation, as determined via an MTT assay. The median effective dose (EC(50)) for all curcumin formulations was found to be in the low µM range for both lung and colon cancer cell lines tested. Our results confirm that βCD inclusion complexes of poorly water soluble drugs, such as curcumin can be entrapped within biocompatible vesicles such as liposomes, and this does not preclude their anticancer activity.

Summary - Carcinogenesis arises from the malfunction of genes that control cell growth and division. Therefore, the most effective method of hindering tumourigenesis is to induce the death of immortalized cancer cells. - Apoptosis or programmed cell death has shown the most promises in impairing cancer growth. A variety of proteins is involved in the regulation of apoptosis and the malfunction of any these regulators may cause cell proliferation. - The microRNAs have been shown to play a central role in the regulation of the cell cycle, including apoptosis. The microRNAs are involved in post‐transcriptional gene suppression and have been implicated in the regulation of cell differentiation and development. - Aberrations in the microRNA regulation of apoptosis lead to tumourigenesis. - The present review assesses the current knowledge of apoptotic regulation in cancer and the effect of microRNA aberrations in tumourigenesis.

Aim: To isolate and characterize lactic acid bacteria (LAB) and determine whether they could potentially be used as heavy metal (cadmium and lead) absorbing probiotics. Methods and Results: The study used 53 environmental (mud and sludge) samples to isolate cadmium- and lead-resistant LAB, by following spared plate technique. A total of 255 cadmium- and lead-resistant LAB were isolated from these samples. The survival of 26 of the LAB was found after passing through sequential probiotic characterizations. These 26 probiotic LAB exhibited remarkable variations in their metal-resistant and metal-removal abilities. Of 26, seven (Cd54-2, Cd61-7, Cd69-12, Cd70-13, Pb82-8, Pb96-19 and Cd109-16) and four (Pb71-1, Pb73-2, Pb85-9 and Pb96-19) strains displayed relatively elevated cadmium- and lead-removal efficiencies from water, respectively, compare with that of the remaining strains. Strains Cd70-13 and Pb71-1 showed the highest cadmium (25%) and lead (59%) removal capacity from MRS (De Man, Rogosa and Sharpe) culture medium, respectively, amongst the selected strains and showed a good adhesive ability on fish mucus. A phylogenetic analysis of their 16S rDNA sequences revealed that the strains Cd70-13 and Pb71-1 belong to Lactobacillus reuteri. Conclusion: Excellent probiotic, metal sorption and adhesive characteristics of newly identified Lact. reuteri strains Cd70-13 and Pb71-1 were isolated, which indicated their high potential abilities to survive in the intestinal milieu and to uptake the tested metals from the environment. Significance and Impact of the Study: To our knowledge, this is the first study that has aimed to isolate, characterize and identify metal-resistant LAB strains that have potential to be a probiotic candidate for food and in vivo challenge studies in the intestinal milieu of fish for the uptake and control of heavy metal bioaccumulation.

Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMP-7) are key regulators of angiogenesis and osteogenesis during bone regeneration. The aim of this study was to investigate the possibility of realizing sequential release of the two growth factors using a novel composite scaffold. Poly(lactic-co-glycolic acid) (PLGA)-Akermanite (AK) microspheres were used to make the composite scaffold, which was then loaded with BMP-7, followed by embedding in a gelatin hydrogel matrix loaded with VEGF. The release profiles of the growth factors were studied and selected osteogenic related markers of bone marrow stromal cells (BMSCs) were analysed. It was shown that the composite scaffolds exhibited a fast initial burst release of VEGF within the first 3 days and a sustained slow release of BMP-7 over the full period of 20 days. The in vitro proliferation and differentiation of the BMSCs cultured in the osteogenic medium were enhanced by 1 to 2 times, resulting from the additionally and sequentially release of growth factors from the PLGA-AK/gelatin composite scaffolds.

The lack of a specific targeting strategy against cancer stem cells in current cancer treatment regimens is at least partly responsible for life-threatening cytotoxicity for patients undergoing traditional chemotherapy. An effective cancer stem cell targeting system is urgently required for the next generation of cancer medicine. Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and it has recently been identified as a cancer stem cell marker. In this study, we isolated a 40-base RNA aptamer that binds to EpCAM from a random oligonucleotide library using systematic evolution of ligands by exponential enrichment. The aptamer was further truncated to 19 bases. This 19-nt RNA aptamer interacts specifically with a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM, but not with those not expressing EpCAM, as analyzed using flow cytometry and confocal microscopy. The binding affinity of the EpCAM RNA aptamer to human cancer cells is approximately 55 nM. Importantly, this EpCAM RNA aptamer is efficiently internalized after binding to cell surface EpCAM. To our knowledge, this is the first RNA aptamer against a cancer stem cell surface marker being developed. Such cancer stem cell aptamers will greatly facilitate the development of novel targeted nanomedicine and molecular imaging agents for cancer theranostics.

In the quest to developing novel cancer therapies, oncolytic Clostridia are re-emerging as promising candidates due to their ability to specifically target and lyse tumours with great efficacy. Clostridial spores have the innate abilities that exploit the unique tumour microenvironment by directly penetrating, colonising and killing tumour cells. These unique features have prompted many studies to investigate their oncolytic potency. In addition, Clostridia possess a number of characteristics that enable them to be developed as oncolytic gene delivery vectors, such as their unlimited capacity, antibiotic sensitivity, and extracellular existence. Similarly, numerous strategies are being devised and tested to take advantage of these features with modern molecular technologies. In this review, we detail the traits that distinguish Clostridia from other agents, and describe the potential therapeutic effects that Clostridial spores demonstrate, but are not achievable with other treatment modalities. Furthermore, we will also summarize the recent advances in the use of Clostridial spores as viable therapeutic candidates, incorporating the latest progresses in genetic engineering tools, such as ClosTron. Finally, we will highlight some avenues deserving further studies in order to realise the ultimate goal of utilizing oncolytic Clostridia clinically in patients.

1. To date, cancer persists as one of the most devastating diseases worldwide. Problems such as metastasis and tumour resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of existing clinical treatments. 2. To address these problems, cancer gene therapy has been developing over the past two decades, specifically designed to deliver therapeutic genes to treat cancers using vector systems. So far, a number of genes and delivery vehicles have been evaluated and significant progress has been made with several gene therapy modalities in clinical trials. However, the lack of an ideal gene delivery system remains a major obstacle for the successful translation of regimen to the clinic. 3. Recent understanding of hypoxic and necrotic regions within solid tumours and rapid development of recombinant DNA technology have reignited the idea of using anaerobic bacteria as novel gene delivery systems. These bacterial vectors have unique advantages over other delivery systems and are likely to become the vector of choice for cancer gene therapy in the near future. 4. Meanwhile, complicated tumour pathophysiology and associated metastasis make it hard to rely on a single therapeutic modality for complete tumour eradication. Therefore, the combination of cancer gene therapy with other conventional treatments has become paramount. 5. The present review introduces important cancer gene therapy strategies and major vector systems that have been studied so far with an emphasis on bacteria-mediated cancer gene therapy. In addition, exemplary combined therapies are briefly reviewed.

Part I of this article published in the previous issue of Current Drug Metabolism discussed the substrate specificity, inhibitor selectivity and structure-activity relationship (SAR) of human CYP2C9. The features of CYP2C9 pharmacophore and SAR models have been elaborated. Part II of this article will address the homology models of CYP2C9, data from site-directed mutagenesis studies, and crystal structural features of CYP2C9. The heteroactivation of CYP2C9 and its interactions with other CYPs will also be discussed. A number of ligand-based and homology models of CYP2C9 have been reported and this has provided insights into the binding of ligands to the active site of CYP2C9. Site-directed mutagenesis studies have revealed that a number of residues (e.g. R97, F110, F114, R132, R144, D293, F476 and A477) play an important role in ligand binding and determination of substrate specificity. The resolved crystal structures of CYP2C9 have confirmed the importance of these residues in substrate recognition and ligand orientation. Currently, there are three X-ray structures of the human CYP2C9 in Protein Database (PDB): one ligand-free protein (1OG2), and two in complex with S-warfarin (1OG5) or flurbiprofen (1R9O). The published structures of 1OG2 and 1OG5 differ in comparison with 1R9O in residues 30-53 of N-termini, residues 97-121 of B/C-loops, and residues 196-233 of helix F and F/G-loops. CYP2C9 is a two-domain protein with typical fold characteristics of the CYPs. The B-C loop forms part of the active site and contributes to substrate specificity. In the structures of CYP2C9 without ligand bound or with bound S-warfarin, residues 101-106 in the B-C loop form helix B'. In addition, residues 212-222 in the F-G loop form helices F' and G', which was not observed in rabbit CYP2C5 and bacterial CYPs. In the 1OG2 and 1OG5 structures, the heme is stabilized by hydrogen bonds between the propionates and the side chains of W120, R124, H368 and R433. In addition, R97 forms hydrogen bonds to the propionates as well as the carbonyl oxygen atoms of V113 and P367. CYP2C9 is activated by dapsone and its analogues and R-lansoprazole in a stereo-specific and substrate-dependent manner, probably through binding to the active site and inducing positive cooperativity. Further studies are needed to investigate the molecular determinants for ligand-CYP2C9 interactions.