Wayne F. Beyer

The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles

The 2.2 A resolution crystal structure of recombinant human manganese superoxide dismutase, a homotetrameric enzyme that protects mitochondria against oxygen-mediated free radical damage, has been determined. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical 4-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form novel tetrameric interfaces that stabilize the active sites. Structurally altered polymorphic variants with reduced activity, such as tetrameric interface mutant Ile-58 to Thr, may produce not only an early selective advantage, through enhanced cytotoxicity of tumor necrosis factor for virus-infected cells, but also detrimental effects from increased mitochondrial oxidative damage, contributing to degenerative conditions, including diabetes, aging, and Parkinsons and Alzheimers diseases.

Characterization of the interaction between alpha2-macroglobulin and fibroblast growth factor-2: the role of hydrophobic interactions.

Basic fibroblast growth factor (FGF-2) is important in development, wound healing and angiogenesis. The human plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) binds to and regulates the biological activity of various growth factors, including FGF-2. FGF-2 binds specifically and saturably to native alpha2M and conformationally modified alpha2M (alpha2M*); however, the KD for FGF-2 binding to alpha2M* is 10-fold lower. This study investigates the biochemical nature of the interaction between FGF-2 and alpha2M* and localizes a possible FGF-2 binding site in the alpha2M subunit. FGF-2 binding to alpha2M* was not affected by shifts in pH between 6.5 and 10; however, increasing temperature decreased the KD for this interaction. The binding affinity of FGF-2 for alpha2M* also increased with increasing ionic strength. These results are consistent with the hypothesis that hydrophobic interactions predominate in promoting FGF-2 association with alpha2M*. Consistent with this hypothesis, FGF-2 bound to a glutathione S-transferase fusion protein containing amino acids 591-774 of the alpha2M subunit (FP3) and to a hydrophobic 16-amino-acid peptide (amino acids 718-733) within FP3. Specific binding of FGF-2 to the 16-amino-acid peptide was inhibited by excess transforming growth factor-beta1. When the 16-amino-acid peptide was chemically modified to neutralize the only two charged amino acids, FGF-2-binding activity was unaffected, supporting the predominant role of hydrophobic interactions. FGF-2 presentation to signalling receptors is influenced by growth factor binding to heparan sulphate proteoglycans (HSPGs), which is electrostatic in nature. Our results demonstrate that the interactions of FGF-2 with alpha2M* and HSPGs are biochemically distinct, suggesting that different FGF-2 sequences are involved.

Magnetic Fluid Hyperthermia for Bladder Cancer: A Preclinical Dosimetry Study

Abstract Purpose: This paper describes a preclinical investigation of the feasibility of thermotherapy treatment of bladder cancer with magnetic fluid hyperthermia (MFH), performed by analysing the thermal dosimetry of nanoparticle heating in a rat bladder model. Materials and methods: The bladders of 25 female rats were instilled with magnetite-based nanoparticles, and hyperthermia was induced using a novel small animal magnetic field applicator (Actium Biosystems, Boulder, CO). We aimed to increase the bladder lumen temperature to 42 °C in <10 min and maintain that temperature for 60 min. Temperatures were measured within the bladder lumen and throughout the rat with seven fibre-optic probes (OpSens Technologies, Quebec, Canada). An MRI analysis was used to confirm the effectiveness of the catheterisation method to deliver and maintain various nanoparticle volumes within the bladder. Thermal dosimetry measurements recorded the temperature rise of rat tissues for a variety of nanoparticle exposure conditions. Results: Thermal dosimetry data demonstrated our ability to raise and control the temperature of rat bladder lumen ≥1 °C/min to a steady state of 42 °C with minimal heating of surrounding normal tissues. MRI scans confirmed the homogenous nanoparticle distribution throughout the bladder. Conclusion: These data demonstrate that our MFH system with magnetite-based nanoparticles provides well-localised heating of rat bladder lumen with effective control of temperature in the bladder and minimal heating of surrounding tissues.

[23] Superoxide dismutase mimic prepared from desferrioxamine and manganese dioxide

Publisher Summary This chapter discusses superoxide dismutase (SOD) mimic prepared from desferrioxamine and manganese dioxide. It describes the preparation and properties of a soluble green complex prepared from desferrioxamine and manganese dioxide (MnO2)4, which can catalyze the dismutation of O2– in vitro and protect green algae against the toxic effects of paraquat or of sulfite. In addition, the preparation of a more active and more stable pink variant of this complex is described in the chapter. Results show that both the green and the pink complexes of manganese with Desferal contain one atom of Mn(III) per molecule of Desferal. As the green complex can be prepared by reacting MnO2 with Desferal, it follows that some moiety of the Desferal must have been oxidized in providing the electron that reduced Mn(IV) to Mn(III). The pink complex is obtained when ascorbate is present during the reaction of MnO2 with Desferal, and this suggests that the ascorbate then provided the electron. In this view, the difference between the green and the pink complexes is that the former contains oxidized Desferal while the latter contains intact Desferal. The greater stability and catalytic activity of the pink complex suggest that it may be a more useful mimic of SOD activity than the green complex.

Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85ErbB2) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85ErbB2. Here we show that PUVA reduced p85ErbB2 phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.

X-Ray Psoralen Activated Cancer Therapy (X-PACT).

This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of solid cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with current application to proliferative disease and extracorporeal photopheresis (ECP) of cutaneous T Cell Lymphoma. An immunogenic role for light-activated psoralen has been reported, contributing to long-term clinical responses. Psoralen therapies have to-date been limited to superficial or extracorporeal scenarios due to the requirement for psoralen activation by UVA light, which has limited penetration in tissue. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and re-radiate (phosphoresce) at UV wavelengths. The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry in combination with complimentary assays, and the in-vivo mouse study. In an in-vitro study, we show that X-PACT induces significant tumor cell apoptosis and cytotoxicity, unlike psoralen or phosphor alone (p<0.0001). We also show that apoptosis increases as doses of phosphor, psoralen, or radiation increase. Finally, in an in-vivo pilot study of BALBc mice with syngeneic 4T1 tumors, we show that the rate of tumor growth is slower with X-PACT than with saline or AMT + X-ray (p<0.0001). Overall these studies demonstrate a potential therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. In summary, X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation are delivered to a specific tumor site to generate UVA light which in-turn unleashes both short- and potentially long-term antitumor activity of photo-active therapeutics like psoralen.

WE-FG-BRA-01: Cancer Treatment Utilizing Photo-Activation of Psoralen with KV X-Rays

PURPOSE This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with immunogenic anti-cancer potential. Psoralen therapies have been limited due to the requirement for psoralen activation by UVA light. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and reradiate (phosphoresce) at UV wavelengths. METHODS The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry to investigate treatment induced apoptosis. Methylene blue staining, and WST assays were also used. X-PACT was then evaluated in an in-vivo pilot study on BALBc mice with syngeneic 4T1 tumors, including control arms for X-PACT components. Analysis focused on tumor growth delay. RESULTS A multivariable regression analysis of 36 independent in-vitro irradiation experiments demonstrated that X-PACT induces significant tumor cell apoptosis and cytotoxicity on all three tumor cell lines in-vitro (p<0.0001). Neither psoralen nor phosphor alone had a strongly significant effect. The in-vivo studies show a pronounced tumor growth delay when compared to controls (42% reduction at 25 days, p=0.0002). CONCLUSIONS These studies demonstrate for the first time a therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation generate UVA light in-situ (including deep seated lesions) which in-turn photo-activates powerful anticancer therapeutics which may lead to short and long term therapeutic effect. This work was supported by Immunolight Llc.

SU-E-T-93: Activation of Psoralen at Depth Using Kilovoltage X-Rays: Physics Considerations in Implementing a New Teletherapy Paradigm

Purpose: Psoralen is a UV-light activated anti-cancer biotherapeutic used for treating skin lesions (PUVA) and advanced cutaneous T-cell lymphoma (ECP). To date psoralen has not been used to treat deep seated tumors due to difficulty in generating UV-light at depth. We recently demonstrated psoralen activation at depth by introducing energy converting particles that absorb kV x-ray radiation and re-emit UV-light. Our in-vitro work found that 0.2–1Gy using 40–100kVp x-rays combined with psoralen and particles can induce a substantial apoptotic response beyond that expected from the sum of individual components. In preparation for a phase I clinical trial of canine companion animals, we address the physics and dosimetry considerations for applying this new teletherapy paradigm to an in-vivo setting. Methods: The kV on-board imaging (OBI) system mounted on a medical linear accelerator (Varian) was commissioned to deliver the prescribed dose (0.6Gy) using 80 and 100kVp. Dosimetric measurements included kVp, HVL, depth dose, backscatter factors, collimator and phantom scatter factors, field size factors, and blade leakage. Absolute dosimetry was performed following AAPM TG61 recommendations and verified with an independent kV dose meter. We also investigated collimated rotational delivery to minimize skin dose using simple dose calculations on homogeneous cylindrical phantoms. Results: Single beam delivery is feasible for shallow targets (<5cm) without exceeding skin tolerance, while a rotational delivery may be utilized for deeper targets; skin dose is ∼75% of target dose for 80kVp collimated rotational delivery to a 3cm target within a 20cm phantom. Heat loading was tolerable; 0.6Gy to 5cm can be delivered before the anode reaches 75% capacity. Conclusion: KV teletherapy for Psoralen activation in deep seated tissue was successfully commissioned for a Varian OBI machine for use in a phase I clinical trial in canines. Future work will use Monte Carlo dosimetry to investigate dose in presence of bone. Research funded by Immunolight LLC. H. Walder, Z. Fathi, & W. Beyer are employees of Immunolight LLC which holds a patent on the technology. Drs. Adamson and Oldham are consultants to Immunolight LLC.