Aurélie François

Relationship between subcellular localisation of Foscan and caspase activation in photosensitised MCF-7 cells.

The present study investigates the relationship between the subcellular localisation of Foscan® and intrinsic apoptotic pathway post Foscan®-based photodynamic therapy (PDT). With this purpose, mammary carcinoma MCF-7 cells were incubated with Foscan® for 3 or 24 h and then subjected to equitoxic light doses. Fluorescence microscopy revealed very good Foscan® co-localization to endoplasmic reticulum (ER) and Golgi apparatus after 3 h incubation with MCF-7 cells. Progressive increase in incubation time shows leakage of Foscan® from Golgi apparatus. Twenty-four hours incubation yielded a fluence-dependent enhanced induction of the ER-resident glucose-regulated protein 78 (Bip/GRP78), along with a weak mitochondrial damage, thus underscoring the ER as the main site of photodamage after prolonged incubation. Analysis of events implicated in apoptotic pathway after 24 h incubation demonstrated photodamage to Bcl-2 protein in total cellular extract, but not in the mitochondrial fraction. We further determined an increase in caspases-7 and -6 activation, which was strongly related to the expression of GRP78. The above findings demonstrate that Foscan® localisation in ER improves the photoactivation of the caspase-7 apoptotic pathway, which is poorly related to mitochondrial damage.

Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo

A major challenge in the application of a nanoparticle-based drug delivery system for anticancer agents is the knowledge of the critical properties that influence their in vivo behavior and the therapeutic performance of the drug. The effect of a liposomal formulation, as an example of a widely-used delivery system, on all aspects of the drug delivery process, including the drug’s behavior in blood and in the tumor, has to be considered when optimizing treatment with liposomal drugs, but that is rarely done. This article presents a comparison of conventional (Foslip®) and polyethylene glycosylated (Fospeg®) liposomal formulations of temoporfin (meta-tetra[hydroxyphenyl]chlorin) in tumor-grafted mice, with a set of comparison parameters not reported before in one model. Foslip® and Fospeg® pharmacokinetics, drug release, liposome stability, tumor uptake, and intratumoral distribution are evaluated, and their influence on the efficacy of the photodynamic treatment at different light–drug intervals is discussed. The use of whole-tumor multiphoton fluorescence macroscopy imaging is reported for visualization of the in vivo intratumoral distribution of the photosensitizer. The combination of enhanced permeability and retention-based tumor accumulation, stability in the circulation, and release properties leads to a higher efficacy of the treatment with Fospeg® compared to Foslip®. A significant advantage of Fospeg® lies in a major decrease in the light–drug interval, while preserving treatment efficacy.

mTHPC-based photodynamic therapy induction of autophagy and apoptosis in cultured cells in relation to mitochondria and endoplasmic reticulum stress

Photodynamic therapy (PDT), an approved anticancer treatment, is reported as a potent inducer of programmed cell death (PCD) by both apoptosis and autophagy. The present study investigated the kinetics of both autophagy and caspase activation in MCF-7 cells submitted to mTHPC-PDT upon condition of treatment promoting ER accumulation of mTHPC. Fluence-dependent immediate cytochrome c (cyt C) release followed by caspase-9 and -7 activation at 1 h post-PDT evidenced a mitochondrial oxidative stress triggered by high light doses leading to >90% of cell death. ER oxidative stress was monitored by the induction of the glucose-related protein chaperone GRP78. From 6 h post-PDT, GRP78 induction was accompanied by the conversion of LC3-I into LC3-II, the hallmark of autophagosome formation. The formation of acid vesicles evidenced by fluorescence microscopy was obvious from 22 h post-PDT. Twenty-four hours post-PDT, cyt C release decreased and caspase-9 cleavage disappeared, while the expression of cleaved caspase-7 remained significant. At the same time, the profiles of GRP78, cleaved caspase-7 and LC3-II expression were similar irrespective of light doses. In contrast to an inhibitor of caspase activation Z-VAD-FMK, the use of autophagy inhibitor, Wortmannin, impaired cytotoxicity along with an increase in caspase-7 activation. These results demonstrate a valuable contribution of autophagy to cell death in mTHPC-photosensitized MCF-7 cells.

Photodynamic therapy targeting neuropilin-1: Interest of pseudopeptides with improved stability properties.

The general strategy developed aims to favor the vascular effect of photodynamic therapy by targeting tumor vasculature. Since angiogenic endothelial cells represent an interesting target to potentiate this vascular effect, we previously described the conjugation of a photosensitizer to a peptide targeting neuropilins (NRPs) over-expressed specially in tumor angiogenic vessels and we recently characterized the mechanism of photosensitization-induced thrombogenic events. Nevertheless, in glioma-bearing nude mice, we demonstrated that the peptide moiety was degraded to various rates according to time after intravenous administration. In this study, new peptidases-resistant pseudopeptides were tested, demonstrating a molecular affinity for NRP-1 and NRP-2 recombinant chimeric proteins and devoid of affinity for VEGF receptor type 1 (Flt-1). To argue the involvement of NRP-1, MDA-MB-231 breast cancer cells were used, strongly over-expressing NRP-1 receptor. We evidenced a statistically significant decrease of the different peptides-conjugated photosensitizers uptake after RNA interference-mediated silencing of NRP-1. Peptides-conjugated photosensitizers allowed a selective accumulation into cells. In mice, no degradation was observed in plasma in vivo 4h after intravenous injection by MALDI-TOF mass spectrometry. This study draws attention to this potential problem with peptides, especially in the case of targeting strategies, and provides useful information for the future design of more stable molecules.

Fluorescence diagnosis of bladder cancer: a novel in vivo approach using 5-aminolevulinic acid (ALA) dendrimers.

Whats known on the subject? and What does the study add?

Photodynamic diagnosis with methyl-5-aminolevulinate in squamous intraepithelial lesions of the vulva: Experimental research

The incidence of the High-grade Squamous Intraepithelial Lesion of the vulva, formerly vulvar intra-epithelial neoplasia is progressively increasing. Today, an early detection and a precise localization of vulvar lesions are still problematic issues, due to the lack of accuracy of the available diagnostic tool. A new approach is the photodynamic diagnosis based on the fluorescence detection of protoporphyrin IX (PpIX) in cancer cells after topical application of a cream of methyl amino-levulinic acid. This study aimed to evaluate the effectiveness of photodiagnosis in order to discriminate the intensity of PpIX fluorescence between vulvar tumor and healthy skin. After topical application of the cream, the fluorescence on xenografted A431 tumor and adjacent skin was non-invasively measured with optical fiber. The tumor to skin fluorescence ratios were 1.38 and 1.41 at respectively 3h and 6h after application, which were significantly higher compared to those observed before application. PpIX accumulation at different depths of the tumor was investigated by spectrofluorimetry after PpIX chemical extraction from tumor sections at 3h and 6h post-application. It was noticed at both application times that the concentration of PpIX within the tumor progressively decreased. However PpIX fluorescence was always detectable up to 2.5 mm, a depth equivalent to more than three quarters of the tumor. The tumor to exposed skin ratios of PpIX fluorescence showed a good selectivity up to1mm depth at 3h post-application and up to 1.5mm at 6h post-m-ALA. Thus, the photodynamic diagnosis using in vivo topical methyl amino-levulinic acid appears to be a promising way to detect the intraepithelial lesions of the vulva. Our results open the possibility for implementation of topical methyl amino-levulinic acid in clinical settings for recognition of vulvar high-grade squamous intraepithelial lesions.

Abstract 498: ALA dendrimers to improve diagnostic specificity of fluorescence guided diagnosis of bladder cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Fluorescence guided cystoscopy with 5-amino levulinic acid (ALA) or its hexyl derivative (h-ALA, Hexvix®) has been shown to increase bladder cancer diagnosis and offer the possibility to perform a more complete resection, thereby reducing the recurrence and progression rate. However, aspects of this procedure could be further improved. The fast photobleaching of the fluorophore protoporphyrin IX (PpIX) limits the visualization during cystoscopy and the specificity is low. We thus investigated the possibility to use dendrimers ALA in order to overcome these drawbacks. We have previously synthesized a dendrimer containing 18 ALA molecules (18-ALA) that provoked a sustained synthesis of PpIX following IP administration. We investigated the in vitro hydrolysis of ALA, the PpIX synthesis rate as well as the photobleaching rate of equimolar concentrations of ALA, h-ALA and 18-ALA. We further instilled 18-ALA intravesically in rats, bearing orthotopic bladder tumors. Frozen sections were analyzed with fluorescence microscopy. We observed a very slow mono-exponential hydrolysis of the dendrimers (k 6.87 h−1), as opposed to the rather fast and bi-exponential liberation of free ALA from Hexvix® (k1 32.06 h−1, k2 3.88 h−1). This can be attributed to steric hindrance and limited access to esterases. Only ALA esters induce PpIX synthesis after withdrawal of the produg. Due to the slow hydrolysis of dendrimers, a minimal concentration is needed to obtain a sustained release. PpIX synthesis is continuous for the first 24 H for both h-ALA and 18-ALA but rapidly decreases thereafter for h-ALA, whereas PPIX is still synthesized after several days for dendrimers. This is compatible with the very slow but prolonged hydrolysis and production of free ALA. This continuous synthesis of PpIX in itself would thus ensure a more prolonged presence of fluorescence during cystoscopy since the tumor cells having endocytosed the dendrimer keep producing the fluorophore. Furthermore, when comparing in vitro photobleaching of h-ALA and 18-ALA, we observe a slower photobleaching of dendrimer induced PpIX. We futher investigated PpIX synthesis following intravesical instillation of 18-ALA. One hour instillation of dendrimers is sufficient to enable endocytosis of the prodrug but a minimum of 3 H resting time is required to observe fluorescence. Unlike ALA or other derivatives administered to rats, dendrimers induce a tumor vs. normal urothelium fluorescence ratio of 3, with a tumor to muscle ratio superior to 7. Intravesical administration of ALA dendrimers in rats results in a PpIX synthesis which is much more selective towards the tumor as opposed to ALA or h-ALA. In vitro experiments show that there is a prolonged and sustained PpIX synthesis with a reduced photobleaching as compared to h-ALA induced PpIX. These are primary indicators that ALA dendrimers could possibly overcome drawbacks of ALA / h-ALA fluorescence guided cystoscopy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 498.