Jean-Louis Merlin

MTT assays allow quick and reliable measurement of the response of human tumour cells to photodynamic therapy.

MCF-7 and HT-29 cell lines were selected as a reliable model to examine the possible parameters affecting the sensitivity of tumour cells to photodynamic therapy (PDT) using a dye-laser at 630 nm. The chemical composition of haematoporphyrin derivative (HPD) was determined by high-performance liquid chromatography (HPLC) analysis and was in agreement with reported values. MTT assays were performed to assess the time-dependency of PDT and the influence of the output power and light fluence. The results showed a maximal cytotoxicity 48 h after photoirradiation. The output power (1 or 2 W) did not significantly affect the cytotoxicity when the fluence was constant (20 J/cm2). However, an increase in fluence (10-40 J/cm2) led to a significant enhancement of cytotoxicity until maximal values were reached (30-40 J/cm2). A further increase in fluence (50 J/cm2) proved to induce a fall-off in cytotoxicity related to the intense photobleaching of HPD.

Spheroids in radiobiology and photodynamic therapy

Spheroids are tridimensional aggregates of tumor cells coming from one or several cell clones. This model, which mimics the micro-tumors structure and some of their properties, shows oxygen, pH and nutrient gradients inducing a necrotic area in the center of the spheroid. Analysis of spheroids, cultured under static or stirred conditions, can be performed on whole spheroids or dissociated spheroids. The spheroids sensitivity to ionizing radiation and photodynamic therapy can be altered by oxygen status, damage repair, intercellular commmunications and apoptosis induction, as in experimental tumor models. In radiobiology, the similarity of radiation response between spheroids and tumor xenograft bearing mice makes the spheroids to be a good alternative model to in vivo irradiation studies. In photodynamic therapy, spheroids lead to a better understanding of the own tumor response without interactions with vascular system. Finally, despite the quality of spheroid model, only the use of new technology for analysis of spheroid populations will help to increase their experimental use, particularly in preclinical oncology.

Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status.

p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC50values ranging from 0.2–22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r= 0.880,P= 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity.

Effect of meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy on sensitive and multidrug-resistant human breast cancer cells

Meta-tetra(hydroxyphenyl)chlorin (mTHPC) is in clinical trials for the photodynamic therapy (PDT) of localized-stage cancer. The PDT susceptibility of cells expressing multidrug resistance (MDR) phenotype is an attractive possibility to overcome the resistance to cytotoxic drugs observed during cancer chemotherapy. The accumulation, photocytotoxicity and intracellular localization of mTHPC were examined using the doxorubicin selected MCF-7/DXR human breast cancer cells, expressing P-glycoprotein (P-gp), and the wild-type parental cell line, MCF-7. No significant difference in mTHPC accumulation was observed between the two cell lines up to 3 h contact. The photodynamic activity of mTHPC, measured 24 h after irradiation with red laser light (lambda=650 nm), was significantly greater in MCF-7/DXR as compared to MCF-7 cells. A light dose of 2.5 J cm(-2) inducing 50% of cytotoxicity in MCF-7, resulted in 85% cytotoxicity in MCF-7/DXR. The presence of P-gp inhibitors SDZ-PSC-833 and cyclosporin A did not modify the mTHPC-induced cytotoxicity. The difference in intracellular mTHPC distribution pattern between two cell lines may contribute to different photocytotoxicity. Our results indicate that mTHPC mediated PDT could be useful in killing cells expressing MDR phenotype.

Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant MCF-7 cells using fluorescence microscopy image analysis

BACKGROUND Anthracycline resistance is known to be mediated by P-glycoprotein (P-gp) or multidrug-resistance related protein (MRP) as well as intracellular sequestration of drugs. METHODS The resistance phenotype of doxorubicin-selected MCF-7(DXR) human breast adenocarcinoma cell line was characterized by cellular and nuclear daunorubicin efflux, P-gp and MRP expression and apoptosis induction. Daunorubicin sequestration was investigated through organelle markers (lysosomes, endoplasmic reticulum and Golgi apparatus) and daunorubicin co-localization by dual-color image analysis fluorescence microscopy using high numerical aperture objective lenses to achieve the smallest field depth and the best lateral resolution. Signal to noise and specificity ratios were optimized for daunorubicin and organelle fluorescent probes labeling. RESULTS An original image analysis procedure was developed to investigate daunorubicin and organelles co-localization. The reliability of the image analysis was controlled through chromatic shift and intensity linearity measurement using calibrated microbeads. The main contribution (65%) of Golgi vesicles in daunorubicin sequestration was demonstrated. Although no rational relationship could be established between daunorubicin sequestration and apoptosis induction, no apoptosis was observed in MCF-7(DXR) cells. CONCLUSIONS In addition to P-glycoprotein mediated drug efflux and without MRP overexpression, MCF-7(DXR) daunorubicin resistance phenotype involves drug sequestration within intracellular vesicles identified as Golgi vesicles and resistance to apoptosis induction.

Long-term alterations of cytokines and growth factors expression in irradiated tissues and relation with histological severity scoring.

Purpose Beside its efficacy in cancer treatment, radiotherapy induces degeneration of healthy tissues within the irradiated area. The aim of this study was to analyze the variations of proinflammatory (IL-1α, IL-2, IL-6, TNF-α, IFN-γ), profibrotic (TGF-β1), proangiogneic (VEGF) and stem cell mobilizing (GM-CSF) cytokines and growth factors in an animal model of radiation-induced tissue degeneration. Materials and Methods 24 rats were irradiated unilaterally on the hindlimb at a monodose of 30 Gy. Six weeks (n = 8), 6 months (n = 8) and 1 year (n = 8) after irradiation the mediators expression in skin and muscle were analyzed using Western blot and the Bio-Plex® protein array (BPA) technology. Additional histological severity for fibrosis, inflammation, vascularity and cellularity alterations scoring was defined from histology and immnunohistochemistry analyses. Results A significant increase of histological severity scoring was found in irradiated tissue. Skin tissues were more radio-sensitive than muscle. A high level of TGF-β1 expression was found throughout the study and a significant relation was evidenced between TGF-β1 expression and fibrosis scoring. Irradiated tissue showed a chronic inflammation (IL-2 and TNF-α significantly increased). Moreover a persistent expression of GM-CSF and VEGF was found in all irradiated tissues. The vascular score was related to TGF-β1 expression and the cellular alterations score was significantly related with the level of IL-2, VEGF and GM-CSF. Conclusion The results achieved in the present study underline the complexity and multiplicity of radio-induced alterations of cytokine network. It offers many perspectives of development, for the comprehension of the mechanisms of late injuries or for the histological and molecular evaluation of the mode of action and the efficacy of rehabilitation techniques.

Foscan® (mTHPC) photosensitized macrophage activation : enhancement of phagocytosis, nitric oxide release and tumour necrosis factor-α-mediated cytolytic activity

SummaryPhotodynamic activation of macrophage-like cells contributes to an effective outcome of photodynamic therapy (PDT) treatment. The possibility for an enhancement of macrophage activity by photosensitization with meta-tetra(hydroxyphenyl)chlorin (mTHPC) (1 μg ml–1) was studied in U937, monocyte cell line differentiated into macrophages (U937Φ cells). Phagocytic activity of U937Φ cells was evaluated by flow-cytometry monitoring of ingestion of fluorescein-labelled Escherichia coli particles. Increase in irradiation fluence up to 3.45 mJ cm–2 (corresponding irradiation time 15 s) resulted in significant increase in fluorescence signal (145%), while at higher light fluences the signal lowered down to the untreated control values. A light energy-dependent production of tumour necrosis factor-alpha (TNF-α) by photosensitized macrophages was further demonstrated using the L929 assay. The maximum TNF-α mediated cytolysis was observed at 28 mJ cm–2 and was 1.7-fold greater than that in control. In addition, we demonstrated a fluence-dependent increase in nitric oxide (NO) production by mTHPC-photosensitized macrophages. NO release increased gradually and reached a plateau after irradiation fluence of 6.9 mJ cm–2. Cytotoxicity measurements indicated that the observed manifestations of mTHPC-photosensitized macrophage activation took place under the sublethal light doses. The relevance of the present findings to clinical mTHPC-PDT is discussed.

Thymidylate synthase expression and activity: relation to S-phase parameters and 5-fluorouracil sensitivity

Six human cancer cell lines exhibiting a large range of sensitivity to 5-fluorouracil (5-FU) were evaluated for thymidylate synthase (TS) and p53 gene expression, TS and dihydropyrimidine dehydrogenase (DPD) activity, as well as cell cycle parameters, S-phase fraction (SPF), bromodeoxyuridine labelling index (LI) and S-phase duration (SPD). All these parameters were investigated for 7 days in asynchronously growing cell populations and compared with the cell sensitivity to 5-FU. No significant correlation was found between S-phase parameters and TS gene expression and/or activity. TS activity was higher in proliferating cells; however, it was not significantly higher in rapidly growing cell lines with short SPD. Neither TS gene expression nor activity was found to correlate with 5-FU sensitivity. On the another hand, a statistically significant correlation (P < 0.0001) was observed between LI and SPD and 5-FU sensitivity. The present results suggest that cell cycle parameters such as SPD and/or LI could be better parameters for 5-FU sensitivity prediction than TS gene expression and/or activity. This could be especially informative in cases of concomitant radio-chemotherapy as S-phase parameters are already proposed for hyperfractionated radiotherapy planning.

Involvement of neutrophilic granulocytes in the uptake of biodegradable non-stealth and stealth nanoparticles in guinea pig.

The in vivo behavior of monomethoxypoly(ethylene oxide)-poly(lactic acid) (MPEO20-PLA45/PLA (75/25)) nanoparticles in comparison with PLA ones was studied in guinea pig. Indeed, the aim of this study was to bring to the fore the in vivo stealth character of these copolymer nanoparticles and to identify the phagocytic circulating cells involved in their uptake. After the intravascular administration of fluorescent nanoparticles (rubrene), their phagocytosis by granulocytes and monocytes was assayed by flow cytometry. At the same time, the evolution of the number of these phagocytic cells was realized in order to identify their function in the nanoparticle uptake. Finally, a histological study of the spleen (30 h after the nanoparticle administration) was investigated to highlight the splenic trapping of these stealth nanoparticles. This study has shown that the phagocytic circulating cells involved in the nanoparticle uptake were mainly neutrophilic granulocytes and some of them were found in the spleen.

In vivo growth inhibitory effect of iterative wild-type p53 gene transfer in human head and neck carcinoma xenografts using glucosylated polyethylenimine nonviral vector.

Polyethylenimine (PEI) derivatives are polycationic nonviral vectors for gene transfer. Previous results achieved in vitro in head and neck cancer cells demonstrated that glucosylated PEI yields higher gene transfer efficiency and longer transgene expression than unsubstituted PEI. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of P53 protein expression and induction of spontaneous apoptosis. The present study reports in vivo data achieved in human head and neck squamous cell carcinoma xenografted mice. Using biotinylated PEI and histochemistry analysis, the vector was found to diffuse in the proliferating cells of the tumor tissue, sparing necrotic areas. No diffusion was observed inside keratinized area composed of nonproliferating, mature differentiated cells. Using green fluorescent protein (GFP) transfection and fluorescence microscopy, the transgene expression was mainly observed at the periphery of the tumor containing proliferating cells. GFP expression appeared lower inside the tumor depth. Quantitative transgene expression kinetics was then determined using luciferase as reporter gene. The maximal transgene expression was achieved 48 hours after intratumoral injection of glucosylated PEI/DNA complexes. The highest gene transfer efficacy was achieved 48 hours after two intratumoral injection. After transfection of wild-type p53, tumor growth inhibition was observed in tumor-bearing mice receiving intratumoral injection of glucosylated PEI/DNA complexes repeated twice weekly. Tumor growth inhibition was maintained under continuous treatment using the same schedule. In all experiments, no noticeable toxicity was observed. The present results demonstrate the feasibility and the tumor growth inhibition potency of nonviral gene transfer using glucosylated polyethylenimine.