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It is known that Photodynamic Therapy (PDT) induces changes in the cytoskeleton, the cell shape, and the adhesion properties of tumour cells. In addition, these targets have also been demonstrated to be involved in the development of PDT resistance. The reversal of PDT resistance by manipulating the cell adhesion process to substrata has been out of reach. Even though the existence of cell adhesion-mediated PDT resistance has not been reported so far, it cannot be ruled out. In addition to its impact on the apoptotic response to photodamage, the cytoskeleton alterations are thought to be associated with the processes of metastasis and invasion after PDT. In this review, we will address the impact of photodamage on the microfilament and microtubule cytoskeleton components and its regulators on PDT-treated cells as well as on cell adhesion. We will also summarise the impact of PDT on the surviving and resistant cells and their metastatic potential. Possible strategies aimed at taking advantage of the changes induced by PDT on actin, tubulin and cell adhesion proteins by targeting these molecules will also be discussed.

Photodynamic therapy (PDT) is a novel cancer treatment utilising a photosensitiser, visible light and oxygen. PDT often leaves a significant number of surviving tumour cells. In a previous work, we isolated and studied two PDT resistant clones derived from the mammary adenocarcinoma LM3 line (Int. J. Oncol. 29 (2006) 397-405). The isolated Clon 4 and Clon 8 exhibited a more fibroblastic, dendritic pattern and were larger than the parentals. In the present work we studied the metastatic potential of the two clones in comparison with LM3. We found that 100% of LM3 invaded Matrigel, whereas only 19+/-6% and 24+/-7% of Clon 4 and Clon 8 cells invaded. In addition, 100% of LM3 cells migrated towards a chemotactic stimulus whereas 38+/-8% and 73+/-10% of Clones 4 and 8, respectively, were able to migrate. In vivo, 100% of the LM3 injected mice developed spontaneous lung metastasis, whereas none of the Clon 8 did, and only one of the mice injected with Clon 4 did. No differences were found in the proteolytic enzyme profiles among the cells. Anchorage-dependent adhesion was also impaired in vivo in the resistant clones, evidenced by the lower tumour take, latency time and growth rates, although both clones showed in vitro higher binding to collagen I without overexpression of beta1 integrin. This is the first work where the metastatic potential of cells surviving to PDT has been studied. PDT strongly affects the invasive phenotype of these cells, probably related to a higher binding to collagen. These findings may be crucial for the outcome of ALA-PDT of metastatic tumours, although further studies are needed to extrapolate the results to the clinic employing another photosensitisers and cell types.

We isolated and characterized cell lines resistant to aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) derived from a murine adenocarcinoma and studied cross resistance with other injuries. The most resistant clones were numbers 4 and 8, which exhibited 6.7- and 4.2-fold increase in resistance respectively. Several characteristics were altered in these clones. A 2-fold increase in cell volume, higher cell spreading, and a more fibroblastic, dendritic pattern, were the morphology features that led us to think they could have different adhesive, invasive or metastatic phenotypes. The amount of porphyrins synthesized per cell in the resistant clones was similar to the parental line but, when it was expressed per mg protein, there was a 2-fold decrease, with a higher proportion of hydrophilic porphyrins. These cells were not cross-resistant to photosensitization with Benzoporphyrin derivative and Merocyanine 540, but exhibited a slight resistance to exogenous protoporphyrin IX treatment. Both clones displayed higher protein content and increased number of mitochondria, together with a higher oxygen consumption. The distinctive features found in the resistant lines led as to think how to exploit the changes induced by PDT treatment to target surviving cells. Those hypoxic cells can be also a preferential target of bioreductive drugs and hypoxia-directed gene therapy, and would be sensitive to treatment with other photosensitizers.

Photodynamic therapy (PDT) interactions with nitric oxide (NO) are not well understood. In this work, we attempted to elucidate whether NO cytotoxicity and PDT from aminolevulinic acid (ALA) have independent cell damage mechanisms. We employed the murine mammary adenocarcinoma cell line LM3 and its NO-resistant variant LM3-SNP obtained after successive exposures to sodium nitroprusside (SNP). No cross-resistance was found between NO cytotoxicity and ALA-PDT; LM3-SNP cells were not more resistant to ALA-PDT than the parental line, instead they were more sensitive. We also induced resistance to ALA-PDT in LM3-SNP cells after multiple cycles of photodynamic treatment. We isolated two clones, identified as Clon 1 and Clon 3, which were 9.2 and 12.5 times more resistant to ALA-PDT than the parental lines, showing that resistance to NO did not interfere in the development of PDT resistance. In addition, the sensitivity to NO decreased in Clon 1 and increased in Clon 3, but they did not show any modifications in NO production. All the cell lines have similar GSH content and GSH transferases activities. However, GSSG content is markedly lower in LM3-SNP, Clon 1, and Clon 3 compared to parental LM3 line and consequently GSH/GSSG ratios are also higher. Our results suggest that different degrees of NO resistance of tumours would not correlate with resistance to PDT.

In this work, we studied the in vitro interactions between aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) and nitric oxide (NO), as well as the interactions between ALA, porphyrins and some NO donors and precursors. We employed three murine adenocarcinoma cell lines: LM2, which does not produce NO; LM3, which produces NO, and LM3-SNP, a variant of LM3 resistant to NO producing the same amount of NO as the parental. We did not find cross-resistance between NO-induced cytotoxicity and ALA-PDT. In spite of the lower porphyrin synthesis, LM2 cells show the highest sensitivity to ALA-PDT. However, we hypothesised that this is not related to the lack of endogenous NO production, because modulation of NO levels did not modify the response to PDT in any of the cell lines. Two unexpected results were found: the enhancement of NO production from the donor sodium nitroprusside (SNP) induced by ALA in both cells and medium, and the inhibition by ALA of NO production from arginine. We also found that SNP strongly protected the cells from ALA-PDT by impairing porphyrin biosynthesis as a consequence of an inhibition of the enzyme ALA dehydratase. We were not able to evaluate the action of NO derived from SNP because of the unexpected porphyrin impairment. On the other hand, impairment of NO from Arginine driven by ALA, although not modulating in vitro the ALA-PDT response, by increasing in vivo blood flow, may be contributing to the mechanism of tumour cures.

The aim of this work was to test in vitro and in vivo the efficacy of the derivatives of 5-aminolevulinic acid (ALA): hexyl-ALA (He-ALA), undecanoyl-ALA and R,S-2-(hydroximethyl)tetrahydropyranyl-ALA (THP-ALA) as pro-photosensitising agents. The compounds were assayed in a cell line derived from a murine mammary tumour, in tumour explants and after injection of the cells into mice. In vitro, undecanoyl-ALA and THP-ALA did not improve ALA efficacy in terms of porphyrin synthesis. On the other hand, half of the amount of ALA is required to obtain the same plateau amount of photosensitiser from He-ALA. However, this plateau value cannot be surpassed in spite of the four-times higher accumulation of ALA/He-ALA from the ALA derivative. This shows that He-ALA conversion to porphyrins but not He-ALA entry to the cells is limiting. Employing ionic exchange chromatography, we found that 80% of total uptake was He-ALA whereas only 20% was ALA. This suggests that the esterases, probably themselves regulated by the heme pathway, are limiting the conversion of ALA derivatives into porphyrins. A similar situation occurs with THP-ALA. Tumour explant porphyrin results correlate well with cell line data. However, i.p. injection of ALA derivatives to mice resulted in a lower porphyrin concentration in the tumour when compared to the administration of equimolar amounts of ALA, indicating that there should be retention of ALA derivatives either within the blood vessels in the initial phase of distribution and/or within the capillaries of the tumour.

In spite of the wide range of tumours successfully treated with 5-aminolevulinic acid mediated photodynamic therapy, the fact that 5-aminolevulinic acid has low lipid solubility, limits its clinical application. More lipophilic 5-aminolevulinic acid prodrugs and the use of liposomal carriers are two approaches aimed at improving 5-aminolevulinic acid transmembrane access. In this study we used both 5-aminolevulinic acid and its hexyl ester in their free and encapsulated formulations to compare their corresponding endogenous synthesis of porphyrins. Employing murine tumour cultures, we found that neither the use of hexyl ester nor the entrapment of either 5-aminolevulinic acid or hexyl ester into liposomes increase the rate of tumour porphyrin synthesis. By light and electronic microscopy it was demonstrated that exposure of tumour explants to either free or liposomal 5-aminolevulinic acid and subsequent illumination induces the same type of subcellular damage. Mitochondria, endoplasmic reticulum and plasma membrane are the structures mostly injured in the early steps of photodynamic treatment. In a later stage, cytoplasmic and nuclear disintegration are observed. By electronic microscopy the involvement of the endocytic pathway in the incorporation of liposomal 5-aminolevulinic acid into the cells was shown., (Copyright 2002 Cancer Research UK)

The exogenously stimulated formation of intracellularly generated protoporphyrin IX, a precursor of haem, is becoming one of the fastest developing areas in the field of photodynamic therapy (PDT). We tested the action of several free radical scavengers, amino acids, antioxidants and sulphur-containing compounds as protectors from photodamage induced by 5-aminolaevulinic acid (ALA)-mediated PDT, employing the LM2 cell line, derived from a mammary murine adenocarcinoma. We exposed the cells to different concentrations of the compounds, 24 h before PDT, during PDT, and 19 h after treatment. We defined the protection grade (PG) as the ratio between cell survival after ALA-PDT treatment in the presence of the protector and cell survival of ALA-PDT treatment alone. We found that l -tryptophan (PG=9.2 at 2 m m ), reduced glutathione (GSH) (PG=5.8 at 0.8 m m ), N-acetyl- l -cysteine (PG=4.86 at 30 m m ), melatonin (PG=4.5 at 8 m m ) and l -methionine (PG=4.0 at 0.8 m m ) are the best protectors from PDT damage, followed by l -cysteine (PG=2.8 at 0.8 m m ), mannitol (PG=2.6 at 20 m m ) and glycine (PG=2.4 at 40 m m ) whereas oxidised glutathione and S-adenosyl- l -methionine do not exert any protection. We did not found any photoactive action of the protectors in absence of ALA. These results can be considered to modulate the photodamage induced by ALA-PDT.

In this work we have studied the effects of ALA-mediated photodynamic therapy (PDT) on resting and mitogen-activated murine splenic lymphocytes, evaluating its impact on cell viability. We have also characterised the stress response, measuring the levels of antioxidant enzymes. A 2h exposure to ALA produced 50% lethality upon irradiation of activated cells with 2.1J/cm2. The decrease in cell survival with increasing time exposure to ALA, correlated well with the higher porphyrin accumulation. In resting lymphocytes, in spite of the low amount of porphyrins formed during 2h incubation with ALA, 40% of the cells died after irradiation, this response was not further increased when higher amounts of porphyrins were synthesised. Superoxide dismutase was impaired by light treatment independently of ALA exposure in activated lymphocytes and, to a lesser extent, in resting lymphocytes. PDT induced an antioxidant adaptive response in activated cells 19 h after irradiation, reflected as a net increase in GSHPx activity and a slight reversion of the catalase (CAT) activity already impaired by light treatment. PDT treatment of activated cells also produced a diminution in the GSH/GSSG ratio. Only activated cells are capable of developing an antioxidant adaptive response to PDT treatment.

Delta-aminolevulinic acid (ALA) is the precursor in the biosynthesis of porphyrins. The knowledge of both the regulation of ALA entrance and efflux from the cells and the control of porphyrin biosynthesis is essential to improve ALA-mediated photodynamic therapy. In this work, we studied the regulation of ALA uptake and efflux by endogenously accumulated ALA and/or porphyrins in murine mammary adenocarcinoma cells. Under our set of conditions, the haem synthesis inhibitor succinyl acetone completely prevented porphobilinogen and porphyrin synthesis from ALA, and led to an increase in the intracellular ALA pool. However, neither intracellular ALA nor porphyrin pools regulate ALA uptake or efflux during the first 15 min of the process. Based on temperature dependence data, ALA but not gamma-aminobutyric acid (GABA) efflux is mediated by a diffusion mechanism. Moreover, the addition of extracellular GABA not only did not influence the rate of ALA efflux but on the contrary it affected ALA uptake, showing the contribution of a saturable mechanism for the uptake, but not for the efflux of ALA from the cells. [ABSTRACT FROM AUTHOR]

Although 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has proven to be clinically beneficial for the treatment of certain cancers, including a variety of skin cancers, optimal tissue localisation still remains a problem. An approach to improve the bioavailability of protoporphyrin IX (PpIX) is the use of ALA derivatives instead of ALA. In this work, we employed a subcutaneous murine mammary adenocarcinoma to study the tissue distribution pattern of the ALA hexyl ester (He-ALA) in comparison with ALA after their topical application in different vehicles. He-ALA induced porphyrin synthesis in the skin overlying the tumour (SOT), but it did not reach the tumour tissue as efficiently. Only 5 h after He-ALA lotion application, tumour porphyrin levels surpassed control values. He-ALA delivered in cream induced a substantially lower porphyrin synthesis in SOT, reinforcing the importance of the vehicle in the use of topical PDT. Porphyrin levels in internal organs remained almost within control values when He-ALA was employed. The addition of DMSO to ALA formulation slightly increased tumour and SOT porphyrin biosynthesis, but it did not when added to He-ALA lotion. [ABSTRACT FROM AUTHOR]

δ-aminolevulinic acid, the precursor of porphyrin biosynthesis has been used to induce the endogenous synthesis of the photosensitiser protoporphyrin IX for photodynamic therapy in the treatment of various turnouts. The aim of this work was to characterise the δ-aminolevulinic acid transport system in the murine mammary adenocarcinoma cell line LM3 using [sup 14]C-δaminolevulinic acid, to finally improve δ-aminolevulinic acid incorporation in mammalian cells. Our results showed that δaminotevulinic acid is incorporated into these cells by two different mechanisms, passive diffusion which s important at the beginning of the incubation, and active transport. Specificity assays suggested that the transporter involved in δ-aminolevulinic acid incorporation is a BETA transporter, probably GAT-2. [ABSTRACT FROM AUTHOR]

Exogenous administration of 5-aminolevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of Protoporphyrin IX (PpIX) in photodynamic therapy. We analysed porphyrin formation in chemically induced squamous papillomas, after topical application of ALA and ALA hexyl ester (He-ALA) administered in different formulations, as well as the pattern of distribution in the internal organs, and the synthesis of porphyrins in distant tumoural and normal skins. A lotion formulation containing DMSO and ethanol was the best vehicle for topical ALA delivery to papillomas, whereas cream was the most efficient formulation for He-ALA application. Similar porphyrin concentration can be accumulated in the skin tumours employing either ALA or He-ALA delivered in their optimal formulations. The use of cream as a vehicle of both ALA and He-ALA, induces highest porphyrin tumour/normal skin ratios. The main advantage of using He-ALA is that porphyrins synthesized from the ester are more confined to the site of application, thus inducing low porphyrin levels in normal skin, liver, blood and spleen, as well as in papillomas distant from the point of application, independently on the vehicle employed, so reducing potential side effects of photodynamic therapy. [ABSTRACT FROM AUTHOR]

Background: Extracorporeal photochemotherapy (EPC) has recently been proposed for the treatment of adults with either acute or chronic GVHD. However, data on children given this therapy are scarce. A Phase I-II study was carried out on EPC in children experiencing GVHD after allogeneic transplantation of HPCs.Study Design and Methods: Nine patients with steroid-resistant, grade II-IV acute GVHD and 14 with chronic GVHD, all of whom had been refractory to at least one line of treatment, were enrolled in this study and analyzed. The median age was 10.3 years (range, 5.4-18.1), and the median body weight was 35 kg (range, 17-89).Results: Seven of the nine patients with acute GVHD showed a response to EPC, whereas the disease progressed in the remaining two children (both with skin, gastrointestinal, and liver GVHD), and they died of grade IV acute GVHD. Among the seven children who responded to EPC, it was possible to completely discontinue immunosuppressive treatment in three. In the 14 children with chronic GVHD, 4 and 5 patients experienced complete and partial response to EPC, respectively, whereas the remaining 5 patients, all with extensive chronic GVHD, had stable disease or disease that progressed during EPC. Among these latter 5 patients, 3 died. In 6 of the 9 patients with chronic GVHD responding to EPC, immunosuppressive therapy was discontinued.Conclusion: EPC is safe, feasible, and effective in children with either acute or chronic GVHD occurring after an allograft. [ABSTRACT FROM AUTHOR]

Recently, MacoPharma released a new UV-A cell irradiator device (Macogenic G2) for extracorporeal photopheresis ( ECP), smaller and lighter than the Macogenic G1 but with no integrated cooling system. We compared the two devices at different working temperatures (G1 at standard irradiation temperature - 21°C - and G2 set by purpose at 34°C) in patients affected with chronic graft-versus-host disease and chronic lung allograft dysfunction treated by ECP. We demonstrate that both G1 and G2 devices are efficient in inducing the inhibition of lymphocytic proliferation and mononuclear cells apoptosis after 48 h even when G2 is set at higher-than-standard temperature. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the treatment of graft versus host disease using first-line extracorporeal photochemotherapy after bone marrow transplantation (BMT) in the November 11, 2013 issue.