Iain D. Nicholl

The many futures for cell-penetrating peptides: how soon is now?

Studies of CPPs (cell-penetrating peptides), sequences that are also commonly designated as protein transduction domains, now extend to a second decade of exciting and far-reaching discoveries. CPPs are proven vehicles for the intracellular delivery of macromolecules that include oligonucleotides, peptides and proteins, low-molecular-mass drugs, nanoparticles and liposomes. The biochemical properties of different classes of CPP, including various sequences derived from the HIV-1 Tat (transactivator of transcription) [e.g. Tat-(48-60), GRKKRRQRRRPPQ], and the homeodomain of the Drosophila homeoprotein Antennapaedia (residues 43-58, commonly named penetratin, RQIKIWFQNRRMKWKK), also provide novel insights into the fundamental mechanisms of translocation across biological membranes. Thus the efficacy of CPP-mediated cargo delivery continues to provide valuable tools for biomedical research and, as witnessed in 2007, candidate and emerging therapeutics. Thus it is anticipated that the further refinement of CPP technologies will provide drug-delivery vectors, cellular imaging tools, nanoparticulate devices and molecular therapeutics that will have a positive impact on the healthcare arena. The intention of this article is to provide both a succinct overview of current developments and applications of CPP technologies, and to illustrate key developments that the concerted efforts of the many researchers contributing to the Biochemical Societys Focused Meeting in Telford predict for the future. The accompanying papers in this issue of Biochemical Society Transactions provide additional details and appropriate references. Hopefully, the important and eagerly anticipated biomedical and clinical developments within the CPP field will occur sooner rather than later.

Anionic Antimicrobial and Anticancer Peptides from Plants

Anionic antimicrobial peptides (AAMPs) have been identified in a wide variety of plant species with net charges that range between −1 and −7 and structures that include: extended conformations, α-helical architecture and cysteine stabilized scaffolds. These peptides commonly exist as multiple isoforms within a given plant and have a range of biological activities including the ability to kill cancer cells as well as phytopathogenic bacteria, fungi, pests, molluscs, and other predatory species. In general, the killing mechanisms underpinning these activities are poorly understood although they appear to involve attack on intracellular targets such as DNA along with compromise of cell envelope integrity through lysis of the cell wall via chitin-binding and/or permeabilisation of the plasma membrane via lipid interaction. It is now becoming clear that AAMPs participate in the innate immune response of plants and make a major contribution to the arsenal of defence toxins produced by these organisms to compensate for their lack of some defence mechanisms possessed by mammals, such as mobility and a somatic adaptive immune system. Based on their biological properties, a number of potential uses for plant AAMPs have been suggested, including therapeutically useful anticancer agents and novel antimicrobial compounds, which could be utilized in a variety of scenarios, ranging from the protection of crops to the disinfection of hospital environments.

A sychnological cell penetrating peptide mimic of p21(WAF1/CIP1) is pro-apoptogenic.

Targeting chemotherapeutic agents directly to sites of DNA replication and repair within cancerous cells is problematic. This study attempts to address the issue of nuclear delivery of biologically active peptides with the potential to disrupt cancer cell growth. Herein, the protein transduction domain of the HIV-1 transactivator of transcription, Tat (Tat(48-60)), is used to deliver a cytotoxic peptide mimic of the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1) into the nucleus. This construct, which we designate as Tat(48-60)-P10, contains the PCNA interacting protein (PIP) box. We demonstrate the utility of Tat(48-60) for peptide delivery to the nucleus and show that Tat(48-60)-P10 induces apoptosis specific to the inclusion of the wild type PIP box containing sequence. Colocalization of Tat(48-60)-P10 with nuclear PCNA was observed by immunofluorescence analysis, supporting the hypothesis that cytotoxicity is potentially related to disruption of nuclear PCNA function. The U251 and U373 glioma cell lines exhibited particular sensitivity to the construct.

Interleukin 21 inhibits cancer-mediated FOXP3 induction in naïve human CD4 T cells

IL-21 is known to promote anti-tumour immunity due to its ability to promote T cell responses and counteract Treg-mediated suppression. It has also been shown to limit Treg frequencies during tumour-antigen stimulations. However, whether this represents inhibition of FOXP3 induction in naïve CD4 T cells or curtailed expansion of natural Treg remains unclear. Moreover, whether this effect is maintained in an environment of tumour-derived immunosuppressive factors is not known. Here, we show that in the context of a number of cancers, naïve CD45RA+ CD4 T cells are induced to express high levels of FOXP3, and that FOXP3 expression correlates with inhibition of T cell proliferation. FOXP3 expression was most potently induced by tumours secreting higher levels of total and active TGFβ1 and this induction could be potently counteracted with IL-21, restoring T cell proliferation. We conclude that Treg induction in naïve T cells is a common phenomenon amongst a number of different cancers and that the ability of IL-21 to counteract this effect is further evidence of its promise in cancer therapy.

α-Catenin Structure and Nanoscale Dynamics in Solution and in Complex with F-Actin

As a core component of the adherens junction, α-catenin stabilizes the cadherin/catenin complexes to the actin cytoskeleton for the mechanical coupling of cell-cell adhesion. α-catenin also modulates actin dynamics, cell polarity, and cell-migration functions that are independent of the adherens junction. We have determined the solution structures of the α-catenin monomer and dimer using in-line size-exclusion chromatography small-angle X-ray scattering, as well as the structure of α-catenin dimer in complex to F-actin filament using selective deuteration and contrast-matching small angle neutron scattering. We further present the first observation, to our knowledge, of the nanoscale dynamics of α-catenin by neutron spin-echo spectroscopy, which explicitly reveals the mobile regions of α-catenin that are crucial for binding to F-actin. In solution, the α-catenin monomer is more expanded than either protomer shown in the crystal structure dimer, with the vinculin-binding M fragment and the actin-binding domain being able to adopt different configurations. The α-catenin dimer in solution is also significantly more expanded than the dimer crystal structure, with fewer interdomain and intersubunit contacts than the crystal structure. When in complex to F-actin, the α-catenin dimer has an even more open and extended conformation than in solution, with the actin-binding domain further separated from the main body of the dimer. The α-catenin-assembled F-actin bundle develops into an ordered filament packing arrangement at increasing α-catenin/F-actin molar ratios. Together, the structural and dynamic studies reveal that α-catenin possesses dynamic molecular conformations that prime this protein to function as a mechanosensor protein.

Abstract 4589: Perturbation of epidermal growth factor (EGF) receptor internalization by novel aspirin analogues in SW480 cells

BACKGROUND Colorectal cancer (CRC) is a global phenomenon with over 135 million new cases and over 600,000 deaths worldwide annually. The EGFR regulates normal growth and differentiation of cells, however its dysregulation is the cause of a wide range of cancers, including CRC. Activation of the EGFR requires phosphorylation at specific sites, which then leads to the recruitment of proteins and ultimately its internalization. The use of low-dose aspirin as a preventative agent is a controversial topic, particularly as the mechanism of action is hotly disputed. We have previously determined that novel aspirin analogues dramatically reduce EGFR phosphorylation in SW480 CRC cells; these cells express neither COX-1 nor COX-2, the perceived target for aspirin-like compounds. We have seen these aspirin analogues to also perturb the internalization of this receptor. METHODS SW 480 CRC cells were cultured in Leibovitz9s L-15 medium on glass slides and starved for 48 hours. The cells were then treated with 0.5 mM aspirin analogues (ortho-aspirin, meta-aspirin, para-aspirin, ortho-thioaspirin, meta-thioaspirin and para-thioaspirin) for 30 minutes and then treated with EGF-tagged with AlexaFluor 555 for an hour. The reaction was the quenched with cold PBS, fixed and mounted on microscope slides with VectaShield + DAPI. Confocal microscopy was used to study the effect of these aspirin analogues on EGFR internalization. RESULTS EGF increased phosphorylation at all sites tested. We have peviously shown that these effects were prevented by preincubation with thioaspirins more potently than with aspirin and its positional isomers. These novel thioaspirins are also more potent in inhibiting proliferation of SW480 CRC cells. We now show these analogues prevent the internalization of EGFR. Instead they permit the formation of clusters. Aspirin and its analogues can perturb EGFR internalization and signalling in SW 480 cells. CONCLUSION The EGFR signalling pathway is a target for aspirin and aspirin analogues. The inhibition of receptor phosphorylation is accompanied by the loss of receptor internalization. Citation Format: Asma’U I.J. Bashir, Sarah Jones, Christopher J. Perry, Stephen T. Safrany, Iain D. Nicholl. Perturbation of epidermal growth factor (EGF) receptor internalization by novel aspirin analogues in SW480 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4589.

Abstract 335: Aspirin, di-aspirin and their toxicity to colorectal cancer

Introduction The lifetime risk for developing colorectal cancer (CRC) is approximately 7%. In the USA over 140,000 people a year develop CRC, with approximately 51,000 deaths/year. Epidemiological studies indicate that long-term aspirin usage reduces the incidence of CRC, and may be protective against other non-CRC related adenocarcinoma, including esophageal cancer. We have investigated the mechanism by which aspirin exhibits toxicity to CRC by synthesising novel analogues of aspirin in an effort to identify functionally significant moieties. We previously suggested that “di-aspirin” (bis-carboxyphenol succinate, BCS) and analogues thereof retain relatively specific toxicity to colorectal cancer cells cultured in vitro. The cellular functions of the Inhibitors of Apoptosis (IAPs) correlate with suppression of apoptotic cell death, tumor progression and metastases. Aspirin triggered apoptosis correlates with its capacity to decrease expression of Survivin, a member of the IAP family. Survivin exhibits dual functions; as an inhibitor of apoptosis and regulator of mitosis. We hypothesised that studying the alterations in the expression of IAPs would give an insight into the cytotoxic nature of the compounds. Experimental procedure A cell viability assay (MTT) was employed to investigate the cytotoxicity of the analogues in various CRC (SW480, HCT116 and LoVo) and non-CRC cell lines (A549, MCF7 and EA.hy926). Expression levels of IAPs (c-IAP1, cIAP2, XIAP, Livin and Survivin) were examined in the SW480 cell line by immunoblotting. Survivin expression was further examined by immunofluorescence analysis. Changes in Survivin expression in the CRC and non-CRC cell lines was also determined by immunoblotting. Summary of data Herein, we show that the “di-aspirin” analogues are relatively more toxic to colorectal cancer cells compared to non-colorectal cancer cell lines. In addition we suggest that these analogues can potently reduce the expression of Survivin in comparison to other IAPs. Conclusion We conclude that the analogues of aspirin used in the study promote cell death in CRC and non-CRC cell lines. We propose that the compounds induce apoptosis by decreasing the expression of Survivin. We also propose that the anti-proliferative activity of “di-aspirins” and their analogues have potential benefit in tackling human cancer. Citation Format: Chandra S. Kankipati, Christopher J. Perry, Iain D. Nicholl. Aspirin, di-aspirin and their toxicity to colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 335. doi:10.1158/1538-7445.AM2014-335

Abstract 2527: Aspirin and aspirin analogs on esophageal cancer

Background: Esophageal cancer is the eighth most common cancer seen worldwide and is the sixth most common cause of death from cancer. The UK alone has over 8,000 new cases of esophageal cancer every year. Epidemiological studies have shown that low-dose daily intake of aspirin can decrease the incidence of esophageal cancer. However, its use as an anti-cancer drug has been restrained because of its side effects exerted through inhibition of cyclooxygenase (COX) enzymes. In our study, we have investigated the effects of a number of novel aspirin analogs on esophageal cancer cell lines. Methods: The effects of aspirin and its analogs on the viability of esophageal cancer cell lines were tested using the MTT assay. ApoSense and flow cytometric analysis were performed to examine whether aspirin analog-mediated tumor cell death is due to apoptosis or necrosis. Colorimetric assays measuring peroxidase component of cyclooxygenases were employed to screen aspirin analogs for COX inhibition. Results: Our data suggests that the anti-proliferative property of certain aspirin analogs is greater than that of aspirin itself. Benzoylsalicylates and fumaroyl diaspirin were more effective than aspirin against the oe21 squamous cell carcinoma cells and oe33 esophageal adenocarcinoma cells. Flo-1 esophageal adenocarcinoma cells showed resistance to aspirin and most of the aspirin analogs other than the benzoylsalicylates. Both diaspirin and benzoylsalicylates inhibited metabolic activity in all these esophageal cells. However, apoptosis was induced in only a small proportion. We have also shown that these aspirin analogs do not appear to inhibit COX enzymes. Conclusion: We have synthesized and characterized a number of novel aspirin analogs that are more effective against esophageal cancer cell lines than aspirin. These compounds do not exert their anti-proliferative effect through induction of apoptosis. Moreover, these analogs inability to inhibit COX enzymes suggests that they may cause fewer or no side effects compared to aspirin.

Non-Steroidal Anti-Inflammatory Drugs, DNA Repair and Cancer

Colorectal cancer is the third most common cancer in women and fourth in men, with respect to incidence, and 529,000 deaths occurred worldwide in 2002 (Parkin et al., 2005). Approximately 2 ‐ 5% of cases of colorectal cancer are due to a genetic predisposition of which the most common is hereditary non‐polyposis colorectal cancer (HNPCC). HNPCC is an autosomal dominant disorder with high penetrance and exhibits allelic and locus heterogeneity (Aarnio et al., 1995; de la Chapelle, 2004; Dunlop et al., 1997). In HNPCC there are heterozygous germline mutations in the DNA mismatch repair (MMR) genes MutS homologue 2 (MSH2), MutL homologue 1 (MLH1), MutS homologue 6 (MSH6), post‐meiotic segregation increased 2 (PMS2) and post‐meiotic segregation increased 1 (PMS1) (Bocker et al., 1999; Buermeyer et al., 1999; Jiricny, 1998; Jiricny & Marra, 2003; Lucci-Cordisco et al., 2003; Mitchell et al., 2002; Narayan & Roy, 2003; Nicolaides et al., 1998; Plaschke et al., 2004; Zabkiewicz & Clarke, 2004). Germline mutations in hMLH1 and hMSH2 are the most common with abnormalities in these genes found in more than 90% of HNPCC mutation carriers (Abdel-Rahman et al., 2006; de la Chapelle, 2004; Hampel et al., 2005; Lagerstedt Robinson et al., 2007). The phenomenon of transmission of an epimutation in hMLH1 has also been reported (Hitchins et al., 2007). The DNA MMR system plays an essential role in identifying and correcting any replication errors and any additional errors which arise through physical or chemical damage. These errors may be base‐base mismatches, short insertions/deletions and heteroduplexes, which can occur during DNA replication and recombination (Jiricny, 1998; Jiricny & Marra, 2003). The DNA MMR system therefore maintains genomic integrity and stability and in essence provides a tumour suppressor function. Deficiencies in DNA MMR lead to the accumulation of mutations in repetitive nucleotide regions, a phenomenon termed microsatellite instability (MSI) (Parsons et al., 1993; Parsons et al., 1995; Thibodeau et al., 1993; Thibodeau et al., 1998). Microsatellites are classically defined as simple tandem nucleotide sequence repeats of 1 – 6 base pairs in the genome (Hancock, 1999). Changes in the number of the repeat units due to defective DNA MMR are potentially cancer causing (Riccio et al., 1999; Yamamoto et al., 1998). The MSI phenotype or replication error positive (RER+) phenotype can be considered as an almost canonical feature of DNA MMR deficiency (Kinzler & Vogelstein, 1996; Parsons et al., 1993). This MSI phenotype is observed in approximately 15% of all human colorectal cancer, gastric and endometrial carcinomas (Lothe et al., 1993; Seruca et al., 1995; Shibata, 1999; Umar et al., 1994). Somatic inactivation of DNA MMR largely