Photochemical internalization as a treatment option for radiation-resistant cancers

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Photochemical internalization as a treatment option for radiation-resistant

cancers

Photochemical internalization of Bleomycin in radiation resistant cells

Espen Wibe

Thesis for the Master’s degree at School of Pharmacy

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

August 2017

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Photochemical internalization as a treatment option for radiation-resistant

cancers

Author:

Espen Wibe

Main supervisor:

Kristian Berg

Department of Radiation Biology

Institute for Cancer Research Norwegian Radium Hospital

Oslo University Hospital

Department of Pharmacy School of Pharmacy

The Faculty of Mathematics and Natural Sciences

University of Oslo

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Photochemical internalization as a treatment option for radiation-resistant cancers Espen Wibe

http://www.duo.uio.no/

Trykk: Reprosentralen, Universitetet i Oslo

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Abstract

Photochemical internalization (PCI) is a light-triggered drug and gene therapy delivery technology. The technology utilizes light-triggered compounds known as photosensitizers to allow macromolecules to escape endo-/lysosomes and exert their effect in targeted cells. PCI has been proven effective in circumventing resistance to cytostatic agents, but little is known of the efficacy of PCI on radiation resistant cells. The two cell lines M059J and M059K, the first of which lacks expression of DNA-PKcs, were treated with PCI of the radiomimetic bleomycin and the protein toxin rGel. Several characteristics of the two cell lines were investigated using methods such as flow cytometry, epifluorescence microscopy and live cell imaging. The discovered characteristics commonly conflicted with earlier reports, leading to further investiagations. PCI was found to synergistically enhance the effects of rGel on both cell lines, but the effects of PCI of bleomycin was less clear. This work was thus not sufficent to accomplish its goal of fully elucidating the effects of PCI on radiation resistant cells.

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Acknowledgements

This master thesis was conducted at the Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, in the period August 2015 to August 2017. The thesis was written under the supervision of Prof. Kristian Berg, and is a part of the master's degree programme in pharmacy at the School of Pharmacy, University of Oslo.

I would like to thank my supervisor Kristian Berg for his guidance, supervision and patience throughout this period. A special thanks to Maria Berstad and Ellen Skarpen for help with microscopy, Idun Dale Rein for guiding me in the use of flow cytometry, and to Ane Sofie Viset Fremstedal for general instruction and supervision in the lab. I would also like to thank the entire PCI group and my fellow students for including me in the group and always being open to questions.

Dedicated to Morten Wibe

Oslo, August 2017

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Abbreviations

ATCC - American Type Culture Collection

BLM - Bleomycin

DAMP - Damage-associated molecular pattern

DMEM - Dulbecco’s Modified Eagle’s Medium

DMSO - Dimethyl sulfoxide

DNA - Deoxyribonucleic acid

DNA-PK - DNA-dependent protein kinase

DNA-PKcs - Catalytic subunit of DNA-PK

DSB - Double strand break

EDTA - Ethylenediaminetetraacetic acid

FBS - Fetal bovine serum

Gy - Gray (unit of IR)

HR - Homologous recombination

ISC - Intersystem crossover

IU - International unit

LDL - Low-density lipoprotein

NHEJ - Non-homologous end joining

nM - Nano-molar

P/S - Penicillin/Streptomycin solution

PBS - Phosphate Buffered Saline

PCC - Pearson correlation coefficient

MOC - Mander's overlap coefficient

PCI - Photochemical internalization

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PDT - Photodynamic therapy

PE - Plating efficiency

PS - Photosensitizer

rGel - Recombinant gelonin

RIP - Ribosome-inactivating protein

ROS - Reactive oxygen species

rpm - rotations per minute

RPMI - Rosswell Park Memorial Institute medium

SF2 - Surviving fraction after 2 Gy

SSA - Single strand annealing

TPCS2a - Disulfonated tetraphenyl chlorine

TPPS2a - disulfonated meso-tetraphenylporphine

UV - Ultraviolet

γ-H2AX - Phosphorylated histone H2A

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1 Introduction

1.1 Background

Radiation therapy and cytostatic agents are two of the main pillars of modern day cancer treatment. While effective, they both have their share of side-effects and problems reducing their efficacy. Radiotherapy resistance and lack of specificity of many cytostatic agents are among the greatest obstacles to achieving complete eradication cancer. Photochemical internalization (PCI) is a drug delivery method utilizing photosensitizers to allow

macromolecular drugs to escape endo/lysosomes and exert their effects in the cytosol of cells.

PCI has been proven to be effective against cytostatica-resistant cancer cells, but less is known of the effects of PCI on radiation-resistant cells

The aim of this study was to investigate the correlation between radiotherapy resistance and the efficacy of PCI. To this end, the radiomimetic drug bleomycin and the protein toxin recombinant gelonin was delivered by PCI to the cell lines M059J and M059K. Due to the lack of functional DNA-PK, a vital enzyme in the repair of double strand DNA breaks, M059J has been found to be 30 times more sensitive to ionizing radiation than M059K. The two cell lines should therefore provide a valuable model for establishing any relationship between radiosensitivity and efficacy of both bleomycin and rGel given by PCI.

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1.2 Photodynamic therapy

Photodynamic treatment (PDT) is a minimally invasive treatment modality with uses in the management of both malignant and nonmalignant disease (1). The treatment can with benefit be combined with ionizing radiation, surgery or chemotherapy when used to combat cancer, and can be utilized for skin cancers as well as internal cancers (1). The effects of PDT is dependent upon three components: a photosensitizer (PS), visible or near-infrared light and oxygen (2). None of these components are individually toxic, but result in selective

destruction of tissue when appropriately combined (3). This destruction is mediated through the generation of reactive oxygen species (ROS), amongst which singlet oxygen (¹O₂) plays a significant role (2). The resulting antineoplastic effects are attributed to 3 inter-related

mechanisms: direct inactivation of malignant cells by ROS, damage to tumor-associated vasculature, and activation of an immune response against tumor cells. (2).

1.2.1 Background

The use of light in treatment of diseases has a history stretching back thousands of years.

Ancient civilizations of Egypt, India and China all used sunlight to treat conditions such as psoriasis, rickets and cancer, even combining it with topical application of plants containing psoralens, a photoactive compound that binds to DNA and induces apoptosis when exposed to UV-radiation (4, 5).

Sunlight was again used to treat diseases such as tuberculosis, rickets and rheumatism in France during the 18^th and 19^th centuries (5). The use of light of more specific wavelengths however, could be said to have started in the late 19^th century when the Danish physician Niels Finsen used red light and UV-radiation in the treatment of smallpox and cutaneous tuberculosis, respectively (4).. The utilization of both light and chemicals to treat disease was first properly recorded at the start of the 20^th century when the medicine student Oscar Raab found that a combination of light and acridine had a more lethal effect than either alone (5).

This revelation occurred when Raab was doing research on a malaria-causing protozoa during a thunderstorm (5). Raab’s supervisor, von Tappeiner, later coined the term “photodynamic activity”, and proved the importance of oxygen in photosensitization reactions a few years later (5).

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3 The studies performed by Lipson and Scwartz at the Mayo Clinic in 1960 is commonly said to have ushered in the current era of PDT (6) Using injections containing hematoporphyrin (Hp), they observed fluorescence of neoplastic lesions during surgery. Further work by T.J.

Dougherty and others during the 70’s eventually led to the development of PDT as a modern therapeutic modality (3)

1.2.2 Photosensitizers (PS)

Photosensitizers are chemical or synthetic compounds that can absorb light and transfer it to nearby molecules, inducing chemical and physical alterations (7). This effect is achieved when the PS absorbs a photon and is transformed from its ground state (S₀) into an

electronically excited triplet state (T₁) (4). This happens though a short-lived intermediary excited singlet state (S1) (8). See section 1.2.3 for more information.

There are a number of properties an ideal PS should possess in order to be utilized reliably during PDT. Firstly, a high quantum yield of singlet oxygen is considered important, as type II reactions generally are responsible for the majority of the effects of PDT (8). Secondly, a high absorption coefficient at wavelengths capable of penetrating deep into tissue (700-850 nm) is necessary to be able to treat larger tumors or tumors located deeper into the body. This also contributes to prevent skin photosensitivity, as the power in sunlight generally cuts of at λ > 600 nm (8, 9) Further, a swift and high preferential uptake in target tissue and rapid clearance from normal tissues is desirable in order to prevent toxicity to non-neoplastic tissue (8). Likewise, both dark toxicity and administrative toxicity should ideally be minimal for the same reason (9). For production and formulation purposes, the PS should be relatively easy to synthesize, with starting materials sufficiently available for large-scale production. The end product should be stable and pure, with low batch-to-batch variation (9).

The first PS widely studied and attempted used was hematoporphyrin derivative (Photofrin®), a complex mixture of porphyrins derived from hematoporphyrin (10). As one might expect, this PS turned out to fulfill few of the above criteria. Significant problems with determining and isolating the various compounds in the mixture, along with other deficiencies such as high batch-to-batch variation as well as long-lasting skin photosensitivity, resulted in multiple efforts to develop novel PS with improved qualities (9).

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The majority of PS in development and use today are based on the tetrapyrrole macrocycle structure porphyrin, found in many naturally occurring pigments, including heme and chlorophyll (9). Porphyrins and related tetrapyrrolic substances exhibit many of the qualities desired in photosensitizers, including low dark toxicity and suitability for therapeutic

administration (11). Consisting of four pyrrole units joined via methylene subunits, the porphyrin structure is aromatic and extensively conjugated. This makes the molecule fluorescent, and able to be excited by visible light to produce triplet states (8). Multiple methods of altering the properties of porphyrins through chemical modifications have been explored. Reduction of one or both of the two cross-conjugated double bonds in the structure results in chlorins and bacteriochlorins, respectively (see figure 1). For every reduced bond the Q-bands in the absorption spectrum of the molecule is bathochromically shifted, allowing for deeper tissue penetration (8).

Figure 1 - Chemical structures of groups of photosensitizers used in PDT. Numbers indicate an estimate of wavelengths commonly used to activate the groups (nm) (9). Chlorins and bacteriochlorins are porphyrin molecules with one and two double bonds reduced, respectively. Phthalocyanine and Naphthalocyanine are porphyrin molecules with extended ring structures. Adapted from Berg (2009) (7).

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5 Another way of causing a bathochromic shift in the absorption spectrum of a photosensitizer is by expanding the conjugation in their structure. Two relatively commonly used

photosensitizers made taking advantage of this are phthalocyanine and naphthalocyanine (7).

In their case, the tetrapyrrolic structure has been modified with extended ring systems, thus causing increased conjugation and a red shift of their absorption spectrum (7).

Much work has been put into investigating the tumor localizing ability of photosensitizers.

While charting the exact correlation between chemical structure and localization has proven difficult, several theories have been put forward (12). Theories on the preferential

accumulation of the photosensitizers often include the increased vascular permeability

commonly found in neoplastic tissue, combined with the tendency of photosensitizers to bind to serum proteins. Albumin-bound-photosensitizers have been found to be of the perfect size to pass through the endothelium in tumor microvasculature, and consequentially accumulate in the interstitial space of the tumor tissue (12). It has further been a general observation that the tumor selectivity of a photosensitizer increases to some extent with its lipophilicity and degree of binding to low density lipoproteins (LDL) (3). This is believed to contribute to preferential retention due to tumor cells often expressing an abnormally high level of LDL- receptors, and thus taking up LDL-bound photosensitizers through receptor-mediated endocytosis (12). Other factors believed to contribute to the preferential retention of

photosensitizers in tumor tissue is the reduced lymphatic drainage and low pH often found in these tissues. While reduced lymphatic drainage likely results in the entrapment of

macromolecule-bound photosensitizers in general, low pH is believed to increase entrapment of anionic photosensitizers through their protonation and increased lipophilicity (12).

1.2.3 Mechanism

Photosensitizers are activated upon administration of light of a wavelength within the absorption range of the specific photosensitizer (1). Most molecules exist naturally in an energetically favorable ground state (S₀) where their electrons are paired such that the sum of their quantum spin numbers are zero (8). Absorption of a photon by the photosensitizer will result in the elevation of one of the electrons of the photosensitizer molecule from its ground state to a higher molecular orbit (2). This elevation causes the molecule to enter an unstable excited singlet state (S₁) with a half-life of 10^-6 to 10^-9 seconds (3). Excitation to even higher states (S₂ or higher) are possible, but relaxation through internal conversion (IC) to the lowest

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excited state (S₁) generally occur so rapidly that these states are of little relevance in

photosensitizer photophysics (8). From the S₁ state the molecule can relax back to S₀ by non- radioactive decay/vibrational relaxation, or through radioactive decay in the form of

fluorescence (13). Alternatively, it may enter a more stable triplet state (T₁) through intersystem crossover (ISC), meaning that the excited electron reverses its quantum spin, resulting in an overall spin of 1 (14). Owing to spin multiplicity, this T₁ state is generally far more long-lived than excited singlet states, and is thus more likely to induce the biomolecular reactions required for the effects of PDT (8, 13). From the T1 state, the photosensitizer

molecule can, similarly to S1, return to the S0 state through vibrational relaxation, or through radioactive decay in the form of phosphorescence (14).

Type I & type II reactions

The T1 photosensitizer molecule can further initiate two different photodynamic reactions.

Energy can be transferred to ground-state molecular oxygen ³O2 through a collision (type II reaction), resulting in the generation of highly reactive singlet oxygen, or an electron or proton can be transferred from the photosensitizer molecule to nearby molecules (type I reaction), resulting in the formation of reactive oxygen species (ROS) (15). Both type I and type II reactions occur in parallel, in ratios dependent upon the photosensitizer being used, its affinity for the substrates, and the concentration of oxygen. The type II reaction is however widely considered to be the dominant process in most situations (13).

Figure 2 - Jablonski's energy level diagram for PDT. S0 = PS ground state. S1 and Sn =PS excited singlet states. T1 = PS excited tripler state. IC = Internal conversion. vr = Vibrational relaxation. ³O₂= Oxygen triplet state. ¹O₂ = Oxygen singlet state. Adapted from Sibata et al. (2001) (16) and Agnostini et al. (2011) (2).

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7 The type II reaction is based on the transfer of energy, not electrons, to molecular oxygen (13). Unlike other molecules, molecular oxygen is in a triplet state when not activated. The activated singlet state of molecular oxygen (¹O₂) has a solvent-dependent lifetime, found to be 3-4 µs in pure water (8, 17). The actual lifetime of ¹O₂ in cells, however, has been found to be far shorter in most nanoenvironments (10-40 ns) due to the presence of reacting molecules (17). Taking into consideration the diffusion constant of O₂, this gives singlet oxygen a radius of action of 10-20 nm, meaning that the photodynamic damage caused occurs close to the location of light absorption (17). It follows that the mutagenicity of PDT is low, provided the photosensitizers utilized do not enter the nucleus of the cell (17). Being an electrophile, singlet oxygen commonly reacts with double bonds and ring systems. Major biomolecule substrates thus include cholesterol, unsaturated lipids, proteins and nucleic acid bases (guanine in particular) (8). Oxidation of these substrates results in numerous products, often inducing radical chain reactions commonly involving various forms of peroxides or similar compounds (18). Examples of the consequences of these reactions include membrane permeabilization and/or lysis through formation of lipid hydroperoxides, and induction of apoptosis through damage to anti-apoptotic proteins such as Bcl-2 or mTOR (18). The precise mechanisms of PDT-induced cytotoxicity have yet to be fully charted, but are highly

dependent upon the localization of the photosensitizer, and numerous pathways of apoptosis, necrosis or autophagy can be induced depending on the choice of photosensitizers and time of light administration (16, 18).

The type I pathway involves electron or proton transfers between the triplet photosensitizers and a substrate, resulting in radical forms of the substrate or the photosensitizer itself. These radicals go on to react with other molecules, and commonly results in the propagation of radical chain reactions (18)The reactions involved result in a wider range of products than the type II pathway, some of which result in the destruction or inactivation of the photosensitizer.

This is a common concern regarding electron transfer reaction, and one of the reasons why photosensitizers generally are made to favor type II reactions (18). The initiated radical chain reaction can continue long after the irradiation is ended, and terminate only by antioxidant- action or depletion of reactants. Unlike type II reactions, the reaction can occur in part without oxygen, though the rate at which the radicals formed react with molecular oxygen to form reactive oxygen species (ROS) means that the effect would be highly reduced in hypoxic environments (3, 14).

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In the case of type II reactions, the photosensitizer returns to its ground state after transferring energy to the oxygen molecule, and can again be activated. In a type II reaction the

photosensitizer will however undergo a redox reaction, and will require an electron or a proton in order to return to its original state (18).

Figure 3 - Type I and type II reactions in PDT. Type I: PS in excited triplet state reacts directly with a substrate and forms radicals. These radicals interact with molecular oxygen to produce oxygenated products. Type II: PS in excited triplet state transfers its energy directly to molecular oxygen, forming highly reactive singlet oxygen molecules (¹O₂). These oxygen molecules oxidize substrates, again producing oxygenated products. Adapted from Dolmans et al. (2003) (4).

1.2.4 Light

The light sources used for PDT in modern times have progressed greatly from the usage of the broad-spectrum radiation of the sun in ancient and not so ancient times. While the basic concept of PDT has been known for well over a century, much of the progress made in recent years can be ascribed to the development of photosensitizers with high photo-physical

efficiency, and the increased availability of lasers and other high-brightness light sources (19).

Owing to the necessity of localized light delivery, PDT can only be used for tumors where light can be applied directly or indirectly through an optical fiber. Therefore, both light source and light delivery are aspects of great importance in PDT, and the optimal protocols for each can vary significantly depending on the photosensitizer being used, the localization of the

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9 tumor, and the light dose to be delivered (20). When it comes to light penetration through tissue, there are a number of processes one has to take into consideration, including refraction, reflection, absorption and scattering, all of which vary depending on the composition of the tissue being penetrated (13). While these parameters won’t be discussed further here, it should be mentioned that the chromophores typically present in tissue limit the wavelength range suitable for PDT to about 650-1200 nm, with the penetration of light increasing with longer wavelengths (13). It should also be noted that photosensitizers absorbing light above 850 nm typically won’t be able to form triplet states with high enough energy to excite ¹O2, meaning that absorption in the range of 650-850 nm is desirable for photosensitizers utilized in PDT (17).

Figure 4 - Absorbance spectrum for a porphyrin type photosensitzer. Characteristic Soret and Q-bands are shown, with comparative peaks often used with common classes of photosensitizers indicated (Not to scale). Borrowed with permission from Berg (2009) (7).

Tetrapyrrole photosensitizers are all characterized by a large absorption band between 400- 430 nm (Soret band), and smaller absorption bands above 550 nm (Q-bands) (20). A weak absorption peak at 630 nm is typically used in PDT treatments with the earlier mentioned photosensitizer Photofrin®. This results in the light only being able to penetrate 5-10 mm through the tissue, and illustrates why significant work has been put into developing novel photosensitizers with absorption peaks at longer wavelengths (16). Considering this, it

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follows that most light sources used for PDT have been developed to optimize their output in the ideal wavelength range mentioned above.

Light sources for PDT can generally be divided into two groups: lasers and lamps. The range of lasers utilized include argon lasers, metal vapor lasers, solid state lasers and diode lasers, and commonly used lamps include xenon arc lamps and metal halide lamps (20). Lasers have the advantage of providing a monochromatic and powerful source of light capable of

delivering the desired light dose over a limited area in a short amount of time. A laser is also ideal for delivering light via optic fibers to sites where direct illumination is impossible (19).

On the downside, the use of lasers has traditionally involved high levels of maintenance and expense, as well as being limited to using photosensitizers with absorption wavelengths matching the one output of the laser (3, 20). Although these problems are less pronounced in newer types and models of lasers, lamps are now being used in several settings. Lamps are generally far cheaper and easier to maintain than lasers, but has a far wider spectral output, often requiring filters to be used (20). While this enables multiple photosensitizers to be excited by the same lamp, it can also complicate identifying the exact wavelengths being applied, cause unforeseen excitation of photoproducts of the photosensitizer, and potentially increase the necessary treatment time (20).

PDT has traditionally been performed using external light sources with light penetrating the skin. In recent years however, there has been performed multiple successful studies involving light given internally through endoscopes or fiber optic catheters inserted intracranially or intraabdominally. This allows for treatment of tumors in areas where use of transcutaneous light is impractical or impossible, and in which surgery would require extensive resection (1)..

1.2.5 Oxygen

The amount of ¹O₂ produced in the tumor tissue strongly affects the efficacy of PDT. The production of ¹O₂ is dependent on the concentration of oxygen in the tissue, in addition to the quantum yield of singlet oxygen specific to the photosensitizer, i.e. the fraction of excited photosensitizer molecules that produces singlet oxygen (2). PDT has been shown to have a reduced effect on hypoxic cells, and the mechanism itself can reduce oxygen levels

sufficiently to inhibit further PDT effects (21). The loss of oxygenation in treated tissue can be the result of direct photochemical consumption of O₂, as well as a result of reduced blow flow ensuing the antivascular effects of PDT (22). Treatments at low fluence rates or using

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11 fractionated light doses could potentially increase the efficacy of PDT (22). Initial studies also seem to indicate that the use of hyperbaric oxygenation combined with PDT is a feasible method of similarly enhancing PDT efficacy (3).

1.2.6 Effects on tumors

Three distinct but likely interrelated mechanisms are believed to be responsible for the effects of PDT on tumor tissue. The direct killing of tumor cells by PDT-generated ROS, vascular collapse as a result of antivascular effects, and the activation of an immune response against tumor cells (4). The importance of each of these mechanisms are not yet defined, and are modulated by factors such as structure, localization and dose of the photosensitizer, fluence, light fluence rate, time between administration of photosensitizer and irradiation, as well as the conditions in the tumor itself (23). It does however appear clear that a combination of all three are required for long term-tumor control (4).

Direct cytotoxic effects

Some experiments have predicted that total tumor eradication should be feasible through the direct cytotoxic effects of PDT alone by using certain photosensitizers in combination with high light doses (12). In practice this has rarely proven true, in part due to oxygen shortage and non-homologous localization of the photosensitizers (4). Though direct cellular damage certainly is a major component of the antitumor effects achieved with PDT, the cell killing observed achieved by this mechanism alone has so far been insufficient for complete tumor eradication (12). PDT treatments generally results in the evocation of all three main cell death pathways, apoptosis, necrosis, and autophagy, to degrees varying with the type of cell being treated and the treatment protocol being used (2).

Apoptosis, or so called “programmed cell death” , is used to describe a distinct form of cell death commonly occurring during tissue homeostasis (24). No universal mechanism has been found for PDT-induced apoptosis (25). Mechanisms suspected involved to lesser or greater degrees include oxidative damage to lipids or proteins of the plasma membranes, release of lysosomal enzymes from compromised lysosomes, or damage to organelles such as

mitochondria or endoplasmic reticulum (2). All of these have the potential to result in the release, production or destruction of various receptors or intracellular signal transductors, including, but certainly not limited to, Ca⁺, arachidonic acid and cytochrome c (3).

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Imbalances in the cytoplasmic levels of such cellular signal transductors can result in cascades inhibiting or promoting pro- or antiapoptotic pathways. Some of the primary

“gatekeepers” involved in these pathways are Bcl-2 and caspase-8 or -9 (3).

Necrosis is another death pathway commonly induced by PDT. In contrast to apoptosis, necrosis is a passive and degenerative process, most often initiated by overexposure to cytotoxic agents (2). In the case of PDT, necrosis is suspected to be instigated through severe mitochondria damage or loss of integrity of the plasma membrane (2, 23)

Autophagy is a process in which the cytoplasmic components and organelles of a cell are sequestered within double-membrane structures called autophagosomes. Autophagy is thought to be an adaptive mechanism of recycling cytoplasmic components during nutrient- limiting conditions, as well as for the removal damages organelles, toxic metabolites and intracellular pathogens. Through excessive self-digestion and degradation of essential cellular constituents, autophagy is also capable of promoting cell death, however (23). Whether this promotion serves a purpose or is a failed effort of survival is not yet fully elucidated (23). It is however suspected that the autophagy seen during PDT is initiated either in order to remove faulty or cytotoxic products of oxidation, or through some signaling cascade induced through photochemical damage (23). It is clear that further information on the causes and processes of these mechanisms would be of great value in order to optimize and evaluate the therapeutic outcomes of PDT (23).

Antivascular effects

An additional mechanism contributing to long-term tumor control is the effects of PDT on vasculature (12). As the viability of a tumor is greatly dependent upon the nutrients supplied by nearby blood vessels, any potential damage caused to tumor vasculature may result in hypoxia, nutritional deprivation and eventual cell death in tumor tissue (4). The means through which the antivascular effects occur seems to differ with different photosensitizers.

PDT with Photofrin® appears to be mediated primarily through vascular constriction and subsequent platelet activation and thrombus formation, whilst PDT with certain

phthalocyanines seems to predominantly cause vascular leakage (26, 27). In general, it seems that hydrophilic photosensitizers are more likely to inflict vascular damage compared to

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13 hydrophobic ones (25). The underlying workings of the antivascular effects are still under investigation, but appear to be linked in part with the release of vasoactive compounds including thromboxane and other eicosanoids (22).

The vascular effects of PDT has been shown to depend on the drug-light interval, as an earlier administration of light will result in more of the photosensitizer still being located in the blood vessel walls (12). Fractioned photosensitizer dosing protocols, in which the

photosensitizer is administered at two separate times to allow for localization both in vascular and tumor cell compartments, have been explored with promising results in order to optimize the separate mechanisms of PDT (28)

Effects on immune system

The effects of PDT on the immune system were first demonstrated through experiments in which immunocompetent and immunodeficient mice were both exposed to identical treatments of PDT. Initial ablation of the implanted tumor were seen in all mice, but long- term cures were only obtained in the immunocompetent mice, indicating that a functioning immune system is required for full response to PDT (12). The immune response to PDT are believed to be both immediate and non-specific through a rapid recruitment of inflammatory cells to the directly affected tissue, and more lasting through generation of a specific, long- term anti-tumor response (12). As is the case with most other responses to PDT, the degree inflammation and immune response is highly dependent on the concentration of

photosensitizer, fluence and fluence rate (29). Treatments inducing higher rates of necrosis have in some cases been shown to result in a greater immune response due to the release of antigenic cellular components from the interior of tumor cells (30, 31). In other studies, the immune responses provoked primarily by phagocytosis of apoptotic blebs by macrophages during apoptosis appear to surpass the immune response induced by necrosis (31)

The non-specific parts of the immune system are assumed to get involved as the PDT-induced oxidative damage causes changes in the plasma membrane and organelle membranes of tumor cells. This leads to the activation of membraneous phospholipases, causing an accelerated phospholipid degradation and a subsequent release of inflammatory mediators, including cytokines, leukocyte chemoattractants and growth factors (12, 32). The massive release of immunostimulants result in an infiltration of neutrophils, macrophages, mast cells and natural killer cells (32). Interleukin 1β appears to be the most important among the cytokines

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involved, and studies have indicated that performing PDT combined with local administration of recombinant cytokines could cause increased antitumor response (33, 34).

The initial and possibly most important step in the recruitment of immune cells to the site of treatment is the rapid invasion of first-responder neutrophils (12). Neutrophils further

coordinate inflammatory action by recruiting and activating and/or stimulating differentiation of monocytes, dendritic cells and T-cells through secretion of chemotactic signals and

cytokines (29). Combined with the recognition of damage-associated molecular patterns (DAMPs) and cell-death-associated molecular patterns (CDAMPs) released by injured or dead PDT-treated cells, the eventual result is an inflammatory response involving

phagocytosis, lysis and apoptosis signaling, and subsequent activation of the adaptive immune system (32).

While the PDT-induced tumor specific reaction of the adaptive immune system may not be of great relevance to the initial tumor ablation, it is likely instrumental in attaining long-term tumor control (12). Activated dendritic cells will upon recognition of DAMPs migrate to secondary lymphoid tissue, and proceed to present T cells with tumor-associated antigens (32). A similar presentation is performed with macrophages presenting antigens in the context of major histocompatibility complex class II molecules to CD4⁺-helpers cells, which in turn sensitize CD8⁺-cells. (12). Upon activation and maturation, CD8⁺T cells migrate back to the tumor and participate in the elimination of tumor cells (12, 32). While the direct cytotoxic effects of PDT are limited to the site originally treated, the tumor-sensitized cells of the adaptive immune system have been found to operate on distant or metastasized lesions of the same cancer (12)

1.2.7 Clinical applications in oncology

Despite promising results in multiple clinical trials, PDT is as of yet not widely used in the treatment of cancer. Emerging as an useful treatment option for localized cancers, PDT offers multiple potential advantages when compared to the traditional treatment modalities in clinical settings, namely surgery, chemotherapy and radiation therapy (16). Some of these advantages include precise targeting through both the selectivity of the utilized

photosensitizer and the local administration of light, improved cosmetic outcomes and less invasiveness than surgery (16). Other advantages that should be mentioned are short treatment times, few long term side effects, the possibility of multiple applications if necessary, and

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15 comparatively low costs. However, in all but a few cases, the efficacy of PDT on various forms of tumors has not yet been sufficiently explored through adequately powered

randomized controlled clinical studies (2). Problems limiting current use include the necessity of light being able to reach the areas to be treated, the lack of efficiency on metastasized cancers, and the photosensitivity side effects of early photosensitizers (16)

The first approved usage of PDT was that of prophylactic treatment of bladder cancer using Photofrin® in Canada in 1993 (16). Other photosensitizers clinically available for the treatment of cancer today include Forscan®, Metvix® and Levulan®.

1.3 Photochemical internalization

Photochemical internalization (PCI) is a light-triggered drug and gene delivery technology developed at the Norwegian Radium Hospital. The technology is based on the same principles as PDT, and is used to allow therapeutic molecules to escape from endocytic vesicles and exert their effect inside the cells (35). In use against cancer PCI therefore allows for the exploitation of the effects of PDT as well as the effects of the therapeutic molecule released within the cell.

1.3.1 Background

Macromolecular therapeutics, such as antibodies, immunotoxins and nucleic acids, are of particular interest in cancer treatment due to their potential advantage of exerting a higher therapeutic specificity than is possible with most smaller chemotherapeutics (36). Many macromolecular therapeutics have intracellular targets, but lack an efficient method of gaining entry through the plasma membrane. As a result, macromolecules are often taken up into cells by endocytosis. Endocytosis involves internalization of extracellular substances through invagination of the cell membrane (37). This invagination results in the formation of an endosome enclosing the substance on the inside of the cell. The endosome will eventually fuse with a lysosome, an acidic endocytic vesicle containing numerous hydrolytic enzymes, and the substance will be degraded and inactivated unless it can somehow escape from the endosome (35). PCI was developed as a means of selectively allowing these macromolecules to escape from endosomes in cancer cells in a functionally intact form (36). The method can

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also be used for smaller molecules that doesn’t necessarily qualify as macromolecules, but nevertheless accumulate in endocytic vesicles (36).

1.3.2 Principles of PCI

PCI utilizes the same main mechanisms as those described for PDT in section 1.2.3. The technology is dependent on the use of amphiphilic photosensitizers that localize specifically to the membrane of endocytic vesicles, as well as the administration of a drug that localizes within the same endocytic vesicles (38). Administration of light results in ROS generation similar to that seen in PDT treatment, and endocytic vesicles will be partially ruptured through the oxidation of membrane components (38). Through proper timing and

administration of drug and photosensitizer, this rupture will result in the subsequent release of the drug into the cytosol, where it can interact with its therapeutic target (36). PCI does involve is a potential for photochemical degradation of the drug being delivered. The damage caused by the ROS is however largely contained to the membrane of the vesicles due to the short range of action. The molecules delivered through PCI are on the other hand generally located in the lumen of the endosomal vesicles, provided they are sufficiently hydrophilic (35). In contrast to PDT, the contained photochemical effects of PCI also mean that the initial photochemical damage induced rarely is expected to kill the cell by itself. This is an important consideration when it comes to the utilization of PCI in other fields than oncology (38).

It should be noted that though standard protocols of PCI often involve co-incubation of drug and photosensitizers prior to the administration of light (“light after”), it is also possible to incubate the cells with the photosensitizer and perform the illumination up to 6-8 hours prior to the incubation of the drug (35, 37). This procedure, known as the “light first” strategy, has been shown to increase the efficacy of the treatment in certain cases (35). A suggested mechanism for how this works is the merging of newly endocytosed vesicles containing the drug with vesicles that were damaged during the prior illumination (35).

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Figure 5 - Mechanism of PCI. Both macromolecules and photosensitizer gains entry to the cell via endocytosis.

Photosensitizer molecules localized to the membrane of endocytic vesicles are activated by light, and causes the vesicles to rupture, preventing hydrolytic breakdown of the macromolecules in lysosomes, and instead releases the macromolecules into the cytoplasma. Adapted from Norum et al. (2009) (37)

1.3.3 Photosensitizers

Photosensitizers used in PCI should ideally fulfill the requirements for photosensitizers used in PDT (section 1.2.2). They are additionally required to eventually localize to the membrane of endocytic vesicles. This necessitates an amphiphilic structure, with both hydrophilic and hydrophobic regions. An appropriately amphiphilic photosensitizer is prevented from complete penetration through the plasma membrane of the cell, but allowed a partial intercalation sufficient for the production of ROS within the membrane (2). The

photosensitizer is thus expected to be lodged in the outer leaflet of the plasma membrane upon reaching the cell, with the hydrophobic region associating with the membrane, and hydrophilic regions facing the extracellular space. Subsequent endocytosis results in the photosensitizers being located in the inner leaflet of endocytic vesicles (37).

Photosensitizers most commonly used in PCI include AlPcS2a (aluminum phthalocyanine disulfonate), TPPS_2a (disulfonated tetraphenyl porphine) and TPCS_2a (disulfonated

tetraphenyl chlorin) (39). These three are all based on the porphyrin structure, with TPPS_2a

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being classified as a porphyrin, TPCS_2a as a chlorin and AlPcS_2a as a phthalocyanine. At a physiological pH the two sulfonate groups included in each of the structures will be ionized, whilst the tetrapyrrole nucleus remains uncharged (40). This provides these photosensitizers with the amphiphilic nature required to be of use in PCI. TPPS_2a, being a porphyrin, has an absorption spectrum that makes it less than ideal for absorption of light through tissue. The phthalocyanine AlPcS_2a, capable of strong absorption at 670 nm, has thus traditionally been the photosensitizer of choice in in vivo studies (39). A significant drawback with AlPcS2a

however, is that is consists of a large number of isomers that potentially varies from batch to batch. Worries that this would result in clinical responses varying with the batch of the photosensitizers lead to the development of a novel photosensitizer for use in clinical trials.

TPCS2a, the photosensitizer used in this study, was therefore developed by di-imide reduction of TPPS2a, and contains 3 isomers with low inter-batch variations (39). TPCS2a absorbs light at 652 nm, well within the optical window optimal for penetration of light through tissue, and localizes in the membrane of endocytic vesicles (39)

Figure 6 - Chemical structures of TPPS_2a, TPCS_2a, and AlPcS_2a. Adapted from Berg et al. (2004) (41) and Berg et al.

(2011) (39).

1.3.4 PCI of bleomycin

Bleomycin is a class of water-soluble glycopeptide antibiotics with a molecular weight of approximately 1.5 kDa. Bleomycin used clinically primarily consist of the two molecules bleomycin A2 and bleomycin B2 (see figure 7 below)(42) . Commonly used as a

chemotherapeutic, bleomycin exerts its cytotoxic effects by inducing single. and double- strand breaks in DNA (43). The size and hydrophilicity of bleomycin prevents it from

efficiently diffusing through the plasma membrane of cells, resulting in the necessity of a high

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19 dosage in traditional treatment regimens used on cancer patients (44). The dosage required for a sufficient intracellular concentration of bleomycin often results in serious side effects, chief among them lung fibrosis, which affects up to 46 % of the patients treated (44). Bleomycin has however been found to accumulate in endocytic vesicles, and has therefore long been considered a good subject for enhanced intracellular delivery via PCI (36).

Figure 7 - Chemical structure of Bleomycin A2. Bleomycin B2 contains a methylguanidine group in place of the terminal sulfonium group in Bleomycin A2 (i.e. to the far right in this figure).

It should be noted that the dosage of BLM is referred to by different terms, milligram- potency, international units (IU) and United States Pharmacopoeia units (USP units) (45).

Traditionally, 1 mg by weight of BLM was equal to 1 mg-potency unit, which again was equal to 1 USP unit, or 1000 IU. Later improvements in the purification of BLM means that 1 mg-weight unit of BLM now is equal to approximately 1.5 mg-potency units, or 1500 IU and 1.5-2 USP units, as stated in the European and United States Pharmacopoeia, respectively (45). In this thesis, only international units are used when referring to BLM.

1.3.5 Clinical applications

PCI has been shown to increase the biological activity of a number of molecules that normally have no efficient method of passing through the plasma membrane, including immunotoxins, type I ribosome inactivating proteins (RIPs), plasmids, adenoviruses, oligonucleotides, and unconjugated chemotherapeutics such as bleomycin (2). A total of four clinical studies

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exploring the safety and efficacy of PCI are listed at clinicaltrials.gov. A phase I clinical study was carried out in the UK from 2009 to 2014 in which TPCS_2a-induced PCI of bleomycin was used in patients with cutaneous or subcutaneous malignancies (46). The conclusion at the end of the study was that TPCS_2a-mediated PCI was safe and tolerated, and 0.25 mg/kg was found to be the recommended treatment dose of TPCS_2a (46).

1.4 Ionizing radiation

Ionizing radiation (IR) is defined as radiation with enough energy to ionize atoms. Whereas the excitation caused by lower energy radiation can result in alteration of the energy state of an electron (ie. elevation to a higher orbit), ionization involves supplying enough energy to completely remove an electron from the orbits of the atom (47). The lower energy threshold required for radiation to be considered ionizing is somewhat debated, but usually set at either 33 eV (the energy required to ionize water) or at 124 eV (47). This means that the upper portions of the electromagnetic spectrum (gamma-, x-ray-, and potentially high energy UV- radiation) as well as particulate radiation (neutrons, alpha- and beta radiation) are considered ionizing radiation (47).

Ionization and deionization of biological molecules cause formation of highly reactive free radicals as well as disruption of hydrogen bonds, leading to disruption of molecular structures such as DNA. These and other qualities have led to IR having multiple applications in the industry, medical imaging and cancer therapies of today (47).

1.4.1 Background

Ionizing radiation was first discovered in 1895 when the German scientist William Röntgen discovered X-rays. While researching the fluorescence generated when applying voltage to evacuated glass tubes (Crookes tubes), Röntgen found that something was passing through a cardboard layer completely covering the glass tube and causing fluorescence on a barium- platinum covered screen nearby (48, 49). Further experimentation, including being able to see the bones in his hand when holding it in front of the screen, caused him to conclude that some new kind of invisible and penetrating radiation was being emitted from the fluorescing

portion of the glass tube (48).

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21 This discovery prompted further research into potential radiation emitted from other

fluorescing materials. Hearing of Röntgen's results, Henri Becquerel began experimenting with uranium a couple of months after the discovery (50). He eventually concluded that similar invisible radiation was emitted from uranium salts, regardless of whether they were fluorescent/phosphorescent or not. Further research conducted by Pierre and Marie Curie solidified the concept of radioactivity, and led to Becquerel and the Curies receiving a shared Nobel Prize in physics in 1903 (50).

The nature of these new kinds of radiation was explored further by Ernest Rutherford during the same time period. Building on the results of both Röntgen and Becquerel he discovered two kinds of rays that differed from X-rays in their penetrating power (51). Rutherford named these alpha and beta rays, and later named a third kind of radiation gamma rays, though the discovery of this ray was made by French chemist Paul Villard (51, 52)

It would however be several years until the danger of exposure to these new kinds of radiation was fully understood and appreciated. During those early years, this lack of knowledge led to a somewhat frivolous application of these marvelous new rays, including use in cosmetics, children's toys and toothpaste (53)

1.4.2 Types of ionizing radiation

Electromagnetic radiation:

Gamma- and X-rays are the two primary forms of electromagnetic radiation considered ionizing. There is no true difference in the nature or properties of these two sub-designations of radiation, though X-rays typically are used at longer wavelengths and lower energy levels than gamma radiation for medical purposes (54). The difference therefore, lies in how they are produced. Simply put, gamma radiation is produced within the nucleus of an atom, and X- rays are produced outside the nucleus. Gamma radiation is produced spontaneously within the nucleus of a radioactive isotope, while X-rays tend to be produced purposely in an X-ray- machine by making an accelerated electron collide with a dense material such as tungsten (47). Gamma rays, and to a slightly lesser degree X-rays, are generally highly penetrating and capable of passing through a human body. X-ray radiation is commonly used for diagnostic purposes in contraptions such as CT-machines, and both gamma and X-rays are used in radiotherapy of cancers through so called external-beam radiation therapy (55).

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Figure 8 – Electromagnetic spectrum diagram Adapted from NASA w/Chambers, L (56)

Particulate radiation

The three main types of particulate radiation are alpha-, beta- and neutron-radiation. Alpha particles are made up of two protons and two neutrons, similar to a helium nucleus (57).

Amongst the common types of ionizing radiation, alpha radiation possesses the shortest penetration ability (up to 0.1 mm in tissue), and is in most cases incapable of passing through paper or skin (58). It is however far more ionizing than beta or gamma radiation, and causes extensive damage in a limited area if a source is introduced into the body. Beta particles are high-speed electrons or positrons with a penetration and ionization potential in between alpha and gamma radiation (57). In contrast to electromagnetic radiation, both alpha and beta particles possess a charge, causing them to continually lose energy as they interact with surrounding particles (59). A final type of ionizing particulate radiation is neutron radiation.

Consisting of a single neutral particle, neutron radiation shares some qualities with the above- mentioned kinds of particulate radiation, but has far more potential for penetration due to its lack of a charge. Neutron radiation is in limited use for medical imaging and therapy (60, 61).

1.4.3 Mechanism of biological damage

The biological effects of radiation are widely thought to be caused primarily through damage to DNA (62). Depending on the type of radiation used, the damage to DNA is caused either by photons or ionizing particles. This damage can occur through direct interaction of the ionizing particle or photon with a DNA strand (direct action), or via free radical production as a result of interaction with other molecules or atoms in the cell (indirect action) (47).

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23 Direct action is the dominant process for particulate radiation, where the charged particles directly ionize a DNA strand through a direct energy transfer independent of oxygen. This process is also induced to some degree by x-rays and gamma rays, but for these kinds of radiation the majority of the effect is mediated through indirect action (47) The indirect action of IR is believed to be primarily mediated through the ionization of cellular water, though ionization can occur in any molecule in the cell upon irradiation (47, 54). The effects of IR on water results in a rapid generation of ROS such as hydroxyl radicals (·OH) and ionized water (H2O⁺), as well as the reducing agent hydrogen radical (·H) (54). Hydroxyl radicals are capable of diffusing over distances to interact with DNA, but the production of these reactive species also results in chemical cascades involving the production of additional cell-damaging molecules, including superoxide (O2-

), hydrogen peroxide (H2O2), and reactive nitrogen species (RNS) (54). Both the direct and the indirect action of IR results in the induction of several kinds of DNA damage, chiefly damage to nucleotide bases, single strand breaks (SSBs), double strand breaks (DSBs), and DNA-protein cross-links (DPC) (47). Depending on the extent of damage and the capabilities of the affected cells, these lesions may result in cell-cycle arrest and a possible subsequent DNA repair, or it may result in the induction of apoptosis or necrosis (47).

1.4.4 Ionizing radiation in oncology

The use of IR in treatment of cancer, radiotherapy, can be divided into two categories, based on how the radiation is delivered. External beam therapy, also known as teletherapy, is used to describe treatment of cancer in which the source of radiation is distant from the target.

Internal radiation therapy, brachytherapy, describes treatment where the source of radiation is placed in or near the tumor (63). Altogether, radiotherapy is used to treat up to 50 % of cancer patients (64)

Teletherapy typically involve the use of a machine to deliver x-rays, beta or gamma radiation, though teletherapy using protons, neutrons or ions are under experimental use and

development (65-67). Improvements in technologies relating to both pre-therapeutic imaging and radiation delivery have led to teletherapy being the most common form of radiotherapy, and it is used for a number of different cancers, including head and neck, breast, lung and prostate cancer (55, 68, 69).

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Brachytherapy has been in use since the early 20^th century, when radium needles were inserted directly into tumors (70). The crude methods used in these early attempts inevitably led to poor accuracy and consequent side effects, and modern methods of brachytherapy commonly rely on the use of metabolic pathways or ligand-receptors to guide radionuclides given intravenously or orally to their desired targets. Examples involve the use of radioactive iodine-131 for thyroid cancer, and CD20 receptor-targeted antibodies conjugated to yttrium- 90 in use against non-Hodgkin lymphoma (71). Radionuclides introduced to the body typically emit beta particles, and some emit alpha particles, x-rays and gamma radiation (71, 72). As the radiation from these radionuclides only travel short distances (~0,4 mm for beta particles), relatively low levels of damage to nearby tissues are possible (58, 71).

1.4.5 Resistance to ionizing radiation

A major issue with radiotherapy in cancer is the tendency of cancerous tissue to possess (intrinsic) or develop (acquired) a resistance to the effects of radiation (73). Resistance to radiotherapy can be mediated at multiple levels, from entire organisms, to tumor regions, to single cell populations (73) The mechanisms behind radioresistance are far from fully elucidated and can vary significantly from patient to patient. Some of the major factors believed to contribute this resistance will be briefly discussed below.

Hypoxia:

Hypoxia has long been known to be of importance in radiotherapy resistance, and is a key regulatory factor in tumor growth (74). Hypoxic microenvironments are commonly found within solid tumors, whose rapid growth has outgrown its blood supply. Hypoxia appears to affect tumor radiotherapy resistance through a chemical oxygen effect, as well as a biological oxygen effect (75). Molecular oxygen, O2, is a potent chemical radiosensitizer that contributes to inducing DNA damage after absorption of energy from ionizing radiation, and depletion of O2 reduces the production of radiation induced reactive and cytotoxic species (76). In addition to this direct chemical effect, hypoxia has also been known to induce tumor radioresistance through the stimulation of a number of cell signaling networks, including the activation of HIF-1 (Hypoxia-inducible factor 1) (75). One of the components of HIF-1, HIF-1α, is swiftly degraded under normoxic condition, but is more stable under hypoxia. This allows it to interact with HIF-1β, forming the heterodimer that is HIF-1 (75). HIF-1 exerts its effect

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25 through binding with an enhancer sequence, and induces gene expression leading to increased adaption of the cellular metabolism to hypoxia. These adaptations include the switch from oxidative to anoxic respiration, increased metastasis, and induction of angiogenesis (75). Cells that are anoxic during irradiation have been found to be three times more resistant to radiation than well-oxygenated cells (76).

Cancer stem cells:

Another likely reason for radioresistance is the presence of cancer stem cells (CSCs) (77).

CSCs are cancer cells that have retain stem cell properties of pluripotency and self-renewal, and are intrinsically more resistant to multiple clinical therapies (77). This resistance is believed to be related to improved DNA-repair capabilities and ROS defenses (77). As such, the survival of a single CSC in tumor could results in repopulation and tumor recurrence (78).

Cellular pathways:

A number of cellular pathways are involved in the response to radiotherapy. The first step in DNA damage response is the sensing of the DNA damage by ATM and ATR, whose

downstream signaling influences the tumor suppressor protein p53 (79). Based on the degree of damage inflicted, p53 has a significant role in determining whether to activate further pathways for initiating cell death, or cell cycle arrest and attempts at DNA repair (80). This pivotal role has led to p53 being termed "the guardian of the genome" (79). The two chief pathways for repair of DNA damage caused by IR is NHEJ and HR. More can be read on these pathways in section 1.5.1. p53 also contributes to the initiation of the pathways of both intrinsic mitochondria-mediated and extrinsic death-receptor-mediated apoptosis (79). All these pathways are dependent upon genes, modulators and proteins too numerous to mention here, most of which has to potential to contribute to radiotherapy resistance in some manner if altered.

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1.5 DNA damage and repair

The DNA molecule plays an essential role in the functioning, development, reproduction, and growth of every known living organism. Maintaining the correct sequence of nucleotides in the DNA is therefore a task of paramount importance (81). Mutations, chromosomal

abberations and other alterations to the DNA molecules in a cell can lead to disease and death, both for the individual cell and for the organism containing said cell. A short overview of types of DNA damage and the main pathways of repair of double strand breaks will be given below as both ionizing radiation and bleomycin are believed to exert their primary cytotoxic effects through the induction of double strand breaks (82, 83)

Causes of DNA damage

The causes of DNA damage can be divided into two main categories. The first includes all forms of endogenous damage. This includes damage caused by ROS-formation as a result of normal metabolic processes, and replication errors occurring during mitosis. The second category includes damage caused by exogenous factors. These include viruses, toxins, ionizing radiation and radiomimetic agents such as bleomycin (84).

Double strand breaks (DSBs)

The DNA double strand break is considered the most toxic out of the various kinds of DNA lesions (85). A DSB occurs when both strands of the DNA helix are severed, leading to a potential for genome rearrangements. A single DSB can result in cell death if unrepaired, or chromosomal aberrations if misrepaired, underscoring the importance of a functioning pathway for reparation lesions of this kind.(85). In mammalian cells, two primary pathways have been found to mediate the repair of DSBs - homologous recombination (HR) and non- homologous end-joining (NHEJ).

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1.5.1 DNA repair pathways:

Figure 9- Repair of DSB's via A: Homologous recombination (HR), B: Single-strand annealing (SSA) and C: Non- homologous end joining (NHEJ). Note that the exact mechanism likely varies, and different versions can be found in different works. Adapted from Sancar et al. (2004) (86) and van der Bosch et al. (2002).

Homologous recombination (HR)

Homologous recombination is a process in which DNA damage is repaired using homologous stretches of chromosomal or sister chromatid DNA as a template (87). As such, HR is

primarily active during the G2/M phases of the cell cycle (88). The exact process through which HR occurs can vary, but the main steps involved are strand invasion, branch migration and Holliday junction formation (86). Strand invasion and branch migration is reported to be initiated by Rad51 in eukaryotes. Additional proteins believed to be involved include Rad52, Rad54, Rad55, Rad57, BRCA1 and BRCA2 (86). Out of the three main methods of DSB- repair, HR is the only process which results in essentially error-free repair (81).

The single-strand annealing (SSA) pathway is often considered a sub-pathway of HR. SSA is initiated when a DSB occurs between two repeating sequences oriented in the same direction (86). Unlike the other pathways of HR, no separate molecule of DNA is required, with the two repeating sequences instead being used as the template. During SSA, the ends of the DNA duplex is cleaved by an endonuclease at the repeating sequence, forming two single

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strand overhangs which are stabilized by a protein (89). The two overhangs are then annealed, and the 3'-flaps are cleaved. Finally, gaps are filled in by a polymerase and the two threads joined by a ligase (89). The exact enzymes involved in SSA in mammals aren't well charted, but likely include the MRN complex, Rad52 and RPA (86). The DNA sequence between the two repeating sequences, as well as one repeating sequence, are always lost during SSA, resulting in the deletion of genetic material (86).

Non-homologous end joining (NHEJ)

Non-homologous end joining is a versatile pathway that mediates the ligation of a wide range of DSB, including those that are complex, have incompatible ends, and contain base damages (87). NHEJ appears to be the predominant repair pathway in mammalian cells, and is of particular importance in the repair of breaks induced by IR or radiomimetic agents (86, 87).

Unlike HR, NHEJ does not require a complementary DNA sequence, and can theoretically be active during any stage of the cell cycle (87).

NHEJ is initiated by the sensing and binding of the DSB by the Ku70/80 heterodimer within seconds of its creation (85). The Ku heterodimer has an extremely high affinity for dsDNA ends, and forms a ring-shaped protein that slides onto the ends of the broken DNA molecule in a sequence independent manner. The Ku heterodimer serves as a scaffold to recruit the canonical NHEJ factors; DNA-PKcs, XRCC4 and DNA ligase IV, as well as additional factors potentially required for processing of the break (85). The exact progression of end- processing prior to ligation is not entirely elucidated and depends on the type and complexity of the DSB to be repaired (84). WRN (Werner syndrome protein), MRN (Mre11-Rad50-Nbs1 complex), and Artemis are examples of factors believed to contribute to processing of DNA breaks through helicase, transferase or nuclease activity (84). The role of DNA-PKcs in NHEJ is believed to be mediated through its kinase activity when in complex with DNA and the Ku heterodimer. All the factors currently known to be required for NHEJ, including the

individual components of DNA-PK, are phosphorylated by DNA-PK in vitro, though the importance of each phosphorylation site is still under investigation (90). The repair of the DSB is finalized by ligation of the two duplex termini by the ligase IV-XRCC4 heterodimer (86).

Photochemical internalization as a treatment option for radiation-resistant cancers

Photochemical internalization as a treatment option for radiation-resistant

cancers

Photochemical internalization of Bleomycin in radiation resistant cells

Espen Wibe

Thesis for the Master’s degree at School of Pharmacy

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

Photochemical internalization as a treatment option for radiation-resistant

cancers

Author:

Espen Wibe

Main supervisor:

Kristian Berg

Department of Radiation Biology

Institute for Cancer Research Norwegian Radium Hospital

Oslo University Hospital

Department of Pharmacy School of Pharmacy

The Faculty of Mathematics and Natural Sciences

University of Oslo

Abstract

Acknowledgements

Table of Contents

Abbreviations

1 Introduction

1.1 Background

1.2 Photodynamic therapy

1.2.1 Background

1.2.2 Photosensitizers (PS)

1.2.3 Mechanism

1.2.4 Light

1.2.5 Oxygen

1.2.6 Effects on tumors

1.2.7 Clinical applications in oncology

1.3 Photochemical internalization

1.3.1 Background

1.3.2 Principles of PCI

1.3.3 Photosensitizers

1.3.4 PCI of bleomycin

1.3.5 Clinical applications

1.4 Ionizing radiation

1.4.1 Background

1.4.2 Types of ionizing radiation

1.4.3 Mechanism of biological damage

1.4.4 Ionizing radiation in oncology

1.4.5 Resistance to ionizing radiation

1.5 DNA damage and repair

1.5.1 DNA repair pathways: