In vivo and in vitro characterisation of a protoporphyrin IX-cyclic RGD peptide conjugate for use in photodynamic therapy.

Increasing treatment specificity is one of the major aims of cancer research. Photodynamic therapy is a clinically proven treatment for some cancers and certain other diseases. Photosensitisers generally have little intrinsic selectivity for tumours and any accumulation is dependent upon the type of tumour involved. Increasing tumour selective accumulation could improve the efficacy of PDT and reduce any risk of side effects caused by photosensitiser accumulation in non-target tissue. In order to target photosensitisers to tumours, a cyclic peptide, cRGDfK (arginine-glycine-aspartic acid-phenylalanine-lysine) has been synthesised using solid phase peptide chemistry and conjugated to the porphyrin photosensitiser, protoporphyrin IX. The arginine-glycine-aspartic acid (RGD) motif has been shown to specifically bind alphavbeta3 integrins, heterodimeric glycoproteins upregulated on the surface of proliferating endothelial cells such as those in tumour neovasculature. This study reports the synthesis, in vitro and in vivo characterisation of this novel compound and compares its properties to the free photosensitiser. The individual components in our system, protoporphyrin IX and cRGDfK retain their respective photodynamic and integrin binding activity following the coupling step and produce a conjugate of high purity. The PpIX:cRGDfK conjugate is shown to be a good photosensitiser in vitro in the integrin positive human SiHa cell line and in vivo in a mouse CaNT tumour model. Moreover, pharmacokinetic analysis of PpIX:cRGDfK treated mice shows significant retention and accumulation of photosensitiser in tumour tissue with higher tumour : normal tissue ratios than the free photosensitiser. However, although the conjugate shows this higher accumulation and improved tumour : non-target tissue ratios, the overall in vivo PDT effect, between dose-light intervals of 0 and 6 h, is not significantly better than for free protoporphyrin IX This is possibly due to differences in the target environment or in the subcellular localisation of the compounds.     

Low‐dose angiostatic tyrosine kinase inhibitors improve photodynamic therapy for cancer: lack of vascular normalization

Photodynamic therapy (PDT) is an effective clinical treatment for a number of different cancers. PDT can induce hypoxia and inflammation, pro‐angiogenic side effects, which may counteract its angio‐occlusive mechanism. The combination of PDT with anti‐angiogenic drugs offers a possibility for improved anti‐tumour outcome. We used two tumour models to test the effects of the clinically approved angiostatic tyrosine kinase inhibitors sunitinib, sorafenib and axitinib in combination with PDT, and compared these results with the effects of bevacizumab, the anti‐VEGF antibody, for the improvement of PDT. Best results were obtained from the combination of PDT and low‐dose axitinib or sorafenib. Molecular analysis by PCR revealed that PDT in combination with axitinib suppressed VEGFR‐2 expression in tumour vasculature. Treatment with bevacizumab, although effective as monotherapy, did not improve PDT outcome. In order to test for tumour vessel normalization effects, axitinib was also applied prior to PDT. The absence of improved PDT outcome in these experiments, as well as the lack of increased oxygenation in axitinib‐treated tumours, suggests that vascular normalization did not occur. The current data imply that there is a future for certain anti‐angiogenic agents to further improve the efficacy of photodynamic anti‐cancer therapy.     

Angiogenesis inhibition for the improvement of photodynamic therapy: The revival of a promising idea

Photodynamic therapy (PDT) is a minimally invasive form of treatment, which is clinically approved for the treatment of angiogenic disorders, including certain forms of cancer and neovascular eye diseases. Although the concept of PDT has existed for a long time now, it has never made a solid entrance into the clinical management of cancer. This is likely due to secondary tissue reactions, such as inflammation and neoangiogenesis. The recent development of clinically effective angiogenesis inhibitors has lead to the initiation of research on the combination of PDT with such angiostatic targeted therapies. Preclinical studies in this research field have shown promising results, causing a revival in the field of PDT. This review reports on the current research efforts on PDT and vascular targeted combination therapies. Different combination strategies with angiogenesis inhibition and vascular targeting approaches are discussed. In addition, the concept of increasing PDT selectivity by targeted delivery of photosensitizers is presented. Furthermore, the current insights on sequencing the therapy arms of such combinations will be discussed in light of vascular normalization induced by angiogenesis inhibition.     

Subcellular localization of Photofrin determines the death phenotype of human epidermoid carcinoma A431 cells triggered by photodynamic therapy: when plasma membranes are the main targets

Photodynamic therapy (PDT) is a kind of photochemo-therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane-localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 micro g/ml, and at higher dose (28 micro g/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin-PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c-Jun N-terminal kinase, (iii) delayed activation of caspase-3 and cleavage of polyADP-ribose polymerase and p21-activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin-PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like.     

Intratumoural light distribution in an experimental mouse tumour irradiated by a diffuse-light irradiator compared with unilateral helium-neon light for photodynamic therapy

Investigations on dosimetry in photodynamic therapy (PDT) of experimental mouse tumours transplanted into the right hind leg revealed a significant variability in the fluence rate reaching tumour cells in different parts of the tumour when irradiated by a 50 mW collimated He-Ne laser. Based on intratumoural fibre-optical light measurements, a new irradiation source was constructed, in which variability in the radiant energy fluence rate between different parts of the solid tumour was reduced. The new diffuse-light irradiator was constructed basically from two concentric water chambers surrounded by three linear 3000 W xenon flash lamps. The outer chamber was an optical band filter, and the inner chamber contained a light-dispersing solution of lipid droplets which created an isotropic light field in which the tumours were submerged for PDT. Compared with unilateral He-Ne laser irradiation, an enhancement factor of 7.3 in radiance was obtained for the diffuse-light irradiator measured in the tumour. The new apparatus provides a nearly isotropic light field for in vivo experimental PDT.     

Inhibition of angiogenesis and the angiogenesis/invasion shift

Angiogenesis has become a major target in cancer therapy. However, current therapeutic strategies have their limitations and raise several problems. In most tumours, anti-angiogenesis treatment targeting VEGF (vascular endothelial growth factor) has only limited overall survival benefit compared with conventional chemotherapy alone, and reveals several specific forms of resistance to anti-VEGF treatment. There is growing evidence that anti-VEGF treatment may induce tumour cell invasion by selecting highly invasive tumour cells or hypoxia-resistant cells, or by up-regulating angiogenic alternative pathways such as FGFs (fibroblast growth factors) or genes triggering new invasive programmes. We have identified new genes up-regulated during glioma growth on the chick CAM (chorioallantoic membrane). Our results indicate that anti-angiogenesis treatment in the experimental glioma model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. We have identified a molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme. Furthermore, we are focusing our research on alternative inhibitors that act, in part, independently of VEGF. These are endogenous molecules that play a role in the control of tumour growth and may constitute a starting point for further development of novel therapeutic or diagnostic tools.     

Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency

Photodynamic therapy (PDT) is a novel treatment, used mainly for anticancer therapy, that depends on the retention of photosensitizers (PS) in tumour cells and irradiation of the tumour with appropriate wavelength light. Photosensitizers are molecules such as porphyrins and chlorins that, on photoactivation, effect strongly localized oxidative damage within target cells. The PS used for PDT localize in various cytoplasmic membranous structures, but are not found in the most vulnerable intracellular sites for reactive oxygen species, such as the cell nucleus. The experimental approaches discussed in the present paper indicate that it is possible to design highly efficient molecular constructs, PS carriers, with specific modules conferring cell-specific targeting, internalization, escape from intracellular vesicles and targeting to the most vulnerable intracellular compartments, such as the nucleus. Nuclear targeting of these PS-carrying constructs results in enhanced photodynamic activity, maximally about 2500-fold that of free PS. Future work is intended to optimize this approach to the point at which tumour cells can be killed rapidly and efficiently, while minimizing normal cell and tissue damage.     

Role of mitochondria in cell death induced by Photofrin-PDT and ursodeoxycholic acid by means of SLIM.

The present study was undertaken to find new ways to improve efficacy of photodynamic therapy (PDT). We investigated the combinatory effect of the photosensitizer Photofrin and ursodeoxycholic acid (UDCA). UDCA is a relatively non-toxic bile acid which is used inter alia as a treatment for cholestatic disorders and was reported to enhance PDT efficiency of two other photosensitizers. Since besides necrosis and autophagic processes apoptosis has been found to be a prominent form of cell death in response to PDT for many cells in culture, several appropriate tests, such as cytochrome c release, caspase activation and DNA fragmentation were performed. Furthermore spectral resolved fluorescence lifetime imaging (SLIM) was used to analyse the cellular composition of Photofrin and the status of the enzymes of the respiratory chain. Our experiments with two human hepatoblastoma cell lines revealed that the combination of Photofrin with UDCA significantly enhanced efficacy of PDT for both cell lines even though the underlying molecular mechanism for the mode of action of Photofrin seems to be different to some extent. In HepG2 cells cell death was clearly the consequence of mitochondrial disturbance as shown by cytochrome c release and DNA fragmentation, whereas in Huh7 cells these features were not observed. Other mechanisms seem to be more important in this case. One reason for the enhanced PDT effect when UDCA is also applied could be that UDCA destabilizes the mitochondrial membrane. This could be concluded from the fluorescence lifetime of the respiratory chain enzymes which turned out to be longer in the presence of UDCA in HepG2 cells, suggesting a perturbation of the mitochondrial membrane. The threshold at which PDT damages the mitochondrial membrane was therefore lower and correlated with the enhanced cytochrome c release observed post PDT. Thus enforced photodamage leads to a higher loss of cell viability.     

Photodynamic therapy combined with a cysteine proteinase inhibitor synergistically decrease VEGF production and promote tumour necrosis in a rat mammary carcinoma

OBJECTIVES: Photodynamic therapy (PDT) and inhibition of cathepsin B proteases by cystatin (cysteine proteinase inhibitor, CPI) are potential new tumour treatment modalities. We have investigated the efficacy of PDT and CPI alone and in combination on a solid mammary carcinoma transplanted into Wistar rats. MATERIALS AND METHODS: Intraperitoneally injected single doses of chlorine e6 or HpD as photosensitizers were excited at 630 nm (90 J/cm(2)). CPI (500 micro g per animal) was injected around the tumour daily during the 8-day treatment. Inoculation of tumour was either on day 1 of the protocol, or 8 days before. On day 8, tumour size was measured, tumour necrosis and vascularization were determined based on haematoxylin and eosin (H&E)-stained sections and serum vascular endothelial growth factor (VEGF) levels measured using an enzyme-linked immunosorbent assay kit. RESULTS: No differences (two-way anova) were found for treatments started with various time lags. At doses where CPI or PDT alone had no or negligible effect, their combination caused a marked (P < 0.001) decrease in serum VEGF, paralleled by a significant decrease in tumour size and number of capillary vessels, and a significant increase in necrosis (up to 80% of the tumour tissue). CONCLUSIONS: The combination of PDT and CPI could be a useful approach in tumour therapy as the two agents appear to be synergistic and probably decrease VEGF production by the tumour tissue.     

Targeting the NG2/CSPG4 proteoglycan retards tumour growth and angiogenesis in preclinical models of GBM and melanoma

Aberrant expression of the progenitor marker Neuron-glia 2 (NG2/CSPG4) or melanoma proteoglycan on cancer cells and angiogenic vasculature is associated with an aggressive disease course in several malignancies including glioblastoma multiforme (GBM) and melanoma. Thus, we investigated the mechanism of NG2 mediated malignant progression and its potential as a therapeutic target in clinically relevant GBM and melanoma animal models. Xenografting NG2 overexpressing GBM cell lines resulted in increased growth rate, angiogenesis and vascular permeability compared to control, NG2 negative tumours. The effect of abrogating NG2 function was investigated after intracerebral delivery of lentivirally encoded shRNAs targeting NG2 in patient GBM xenografts as well as in established subcutaneous A375 melanoma tumours. NG2 knockdown reduced melanoma proliferation and increased apoptosis and necrosis. Targeting NG2 in two heterogeneous GBM xenografts significantly reduced tumour growth and oedema levels, angiogenesis and normalised vascular function. Vascular normalisation resulted in increased tumour invasion and decreased apoptosis and necrosis. We conclude that NG2 promotes tumour progression by multiple mechanisms and represents an amenable target for cancer molecular therapy.     

Targeting the Tumour Vasculature: Exploitation of Low Oxygenation and Sensitivity to NOS Inhibition by Treatment with a Hypoxic Cytotoxin

Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels.     

Photodynamic therapy: illuminating the road from cell death towards anti-tumour immunity

Photodynamic therapy (PDT) utilizes the destructive power of reactive oxygen species generated via visible light irradiation of a photosensitive dye accumulated in the cancerous tissue/cells, to bring about their obliteration. PDT activates multiple signalling pathways in cancer cells, which could give rise to all three cell death modalities (at least in vitro). Simultaneously, PDT is capable of eliciting various effects in the tumour microenvironment thereby affecting the tumour-associated/-infiltrating immune cells and by extension, leading to infiltration of various immune cells (e.g. neutrophils) into the treated site. PDT is also associated to the activation of different immune phenomena, e.g. acute-phase response, complement cascade and production of cytokines/chemokines. It has also come to light that, PDT is capable of activating ‘anti-tumour adaptive immunity’ in both pre-clinical as well as clinical settings. Although the ability of PDT to induce ‘anti-cancer vaccine effect’ is still debatable, yet it has been shown to be capable of inducing exposure/release of certain damage-associated molecular patterns (DAMPs) like HSP70. Therefore, it seems that PDT is unique among other approved therapeutic procedures in generating a microenvironment suitable for development of systemic anti-tumour immunity. Apart from this, recent times have seen the emergence of certain promising modalities based on PDT like-photoimmunotherapy and PDT-based cancer vaccines. This review mainly discusses the effects exerted by PDT on cancer cells, immune cells as well as tumour microenvironment in terms of anti-tumour immunity. The ability of PDT to expose/release DAMPs and the future perspectives of this paradigm have also been discussed.     

VEGF-mediated tumour angiogenesis: a new target for cancer therapy

Considerable evidence is gathering for the involvement of vascular endothelial growth factor (VEGF) in the vascularization and growth of primary tumours as well as in the formation of metastases. The expression of VEGF depends on activated oncogenes and inactivated tumour suppressor genes as well as several other factors (e.g. growth factors, tumour promoters and hypoxia). Substantial expression of the receptors for VEGF is restricted mainly to the tumour blood vessels. The causal involvement of this angiogenic factor in the progression of disease has been successfully evaluated by means of monoclonal antibodies against VEGF, dominant-negative receptor mutants and the use of antisense oligonucleotides against the VEGF mRNA. Thus, the VEGF signalling system seems to be an appropriate target to inhibit tumour angiogenesis and metastases formation.     

Properties and applications of photodynamic therapy

Photodynamic therapy (PDT) is the treatment of malignant lesions with visible light following the systemic administration of a tumor-localizing photosensitizer. Pharmacological and photochemical properties of the photosensitizer are combined with precise delivery of laser-generated light to produce a treatment which can offer selective tumoricidal action. Hematoporphyrin derivative (HD) and a purified component called Photofrin II are currently being used in clinical PDT. Initial patient results have been encouraging, and considerable interest has developed in the synthesis and evaluation of new photosensitizers with improved photochemical and pharmacological characteristics. In addition, there has been a gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT. This report provides an overview of the properties and applications of PDT. Information and data related to drug development, photochemistry, subcellular targets, in vivo responses, and clinical trials of PDT are presented.     

Characterization of photodynamic therapy responses elicited in A431 cells containing intracellular organelle‐localized photofrin

Photodynamic therapy (PDT), a photochemotherapeutic regimen used to treat several diseases, including cancer, exerts its effects mainly through induction of cell death. Using human epidermoid carcinoma A431 cells as a model, we previously showed that distinct cell death types could be triggered by protocols that selectively delivered Photofrin (a clinically approved photosensitizer) to different subcellular sites (Hsieh et al. [2003] J Cell Physiol 194: 363–375]. Here, the responses elicited by PDT in A431 cells containing intracellular organelle‐localized Photofrin were further characterized. Two prominent cell phenotypes were observed under these conditions: one characterized by perinuclear vacuole (PV) formation 2–8 h after PDT followed by cell recovery or shrinkage within 48 h, and a second characterized by typical apoptotic features appearing within 4 h after PDT. DCFDA‐sensitive reactive oxygen species formed proximal to PVs during the response to PDT, covering areas in which both endoplasmic reticulum (ER) and the Golgi complex were located. Biochemical analyses showed that Photofrin‐PDT also induced JNK activation and altered the protein secretion profile. A more detailed examination of PV formation revealed that PVs were derived from the ER. The alteration of ER structure induced by PDT was similar to that triggered by thapsigargin, an ER Ca²⁺‐ATPase inhibitor that perturbs Ca²⁺ homeostasis, suggesting a role for Ca²⁺ in the formation of PVs. Microtubule dynamics did not significantly affect PV formation. This study demonstrates that cells in which intracellular organelles are selectively loaded with Photofrin mount a novel response to ER stress induced by PDT. J. Cell. Biochem. 111: 821–833, 2010. © 2010 Wiley‐Liss, Inc.     

Immunotherapy: a way to improve the therapeutic outcome of photodynamic therapy?

Photodynamic therapy (PDT) is a treatment for cancer and non-cancerous lesions involving light and a sensitizing drug, a so-called photosensitizer. Photosensitizers for PDT usually accumulate in tumour tissues with some selectivity. Thus, malignant and abnormal cells can be destroyed by PDT which acts by producing singlet oxygen and possible other reactive oxygen species. However, the efficiency of PDT is often limited by shallow light penetration into tissue. In some cases one treatment modality cannot cure a patient because of treatment limitations and/or side effects. In recent years, many preclinical studies have indicated that the therapeutic outcome of PDT can be improved, doses and side effects lowered by combination with immunotherapy. Most experiments have been done with animals and cell lines. This review summarizes the current knowledge about different immunotherapeutic approaches which can be used to improve effectiveness and extend the applications of PDT in clinics.     

PV1 down‐regulation via shRNA inhibits the growth of pancreatic adenocarcinoma xenografts

PV1 is an endothelial‐specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour‐bearing mice by single‐dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down‐regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC‐1 and BxPC‐3). The effect observed is because of down‐regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down‐regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis.     

Gene therapy strategies for intracranial tumours: glioma and pituitary adenomas

Intracranial tumours such as brain gliomas and pituitary adenomas pose a challenging area of research for the development of gene therapy strategies, both from the point of view of the severity of the diseases, to the physiological implication of gene delivery into the central nervous system and pituitary gland. On the one hand, brain gliomas are very malignant tumours, with a life expectancy of six months to a year at the most after the time of diagnosis, in spite of advances in treatment modalities which involve chemotherapy, surgery and radiotherapy. Gene therapy for these tumours is therefore a very attractive therapeutic modality which due to the severity of the disease is already in clinical trials. On the other hand, pituitary tumours are usually benign, and in most cases, treatment is successful. Nevertheless, there are some instances, especially with the macroadenomas and some invasive tumours in which treatment fails. Gene therapy strategies for these adenomas therefore needs to progress substantially in terms of safety, adverse side effects and physiological impact on the normal pituitary gland before clinical implementation. In this paper, we will review gene delivery systems both viral and non-viral and several therapeutic strategies which could be implemented for the treatment of these diseases. These include cytotoxic approaches both conditional and direct, immune-stimulatory strategies, anti-angiogenic strategies and approaches which harness pro-apoptotic and tumour suppressor gene targets. We will also review the models which are currently available in which these gene therapy strategies can be tested experimentally. This new therapeutic modality holds enormous promise, but we still need substantial improvements both from the delivery, efficacy and safety stand points before it can become a clinical reality.     

Use of bacteria in anti-cancer therapies

While a number of valid molecular targets have been discovered for tumours over the past decade, finding an effective way of delivering therapeutic genes specifically to tumours has proved more problematic. A variety of viral and non-viral delivery vehicles have been developed and applied in anti-cancer gene therapies. However, these suffer from either inefficient and/or short-lived gene transfer to target cells, instability in the bloodstream and inadequate tumour targeting. Recently, various types of non-pathogenic obligate anaerobic and facultative anaerobic bacteria have been shown to infiltrate and selectively replicate within solid tumours when delivered systemically. This has prompted the development of cancer gene therapy protocols that use such bacteria as gene delivery vehicles. Here, we review the evidence for the success of these in pre-clinical models and clinical trials, as single modality treatments and in combination with conventional cancer therapies.