Sophie Marchal

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.

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.

Primary Photodamage Sites and Mitochondrial Events after Foscan® Photosensitization of MCF-7 Human Breast Cancer Cells¶

To determine the initial photodamage sites of Foscan®‐mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF‐7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5′‐diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan®‐mediated PDT in MCF‐7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan® photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan®‐based PDT were very different from that of overall cell death. By 24 h post‐PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan®‐mediated MCF‐7 cell death by late and indirect mechanism.

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.

Damaged DNA binding protein 2 plays a role in breast cancer cell growth.

The Damaged DNA binding protein 2 (DDB2), is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER)-positive (MCF-7 and T47D), but not in ER-negative breast cancer (MDA-MB231 and SKBR3) or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold) higher in ER-positive than in ER-negative tumor samples (P = 0.0208) from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase). These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.

Antiproliferative activity of thermosensitive liposome-encapsulated doxorubicin combined with 43°C hyperthermia in sensitive and multidrug-resistant MCF-7 cells

Thermosensitive liposome-encapsulated doxorubicin (TLED) was compared to free doxorubicin, at 37 degrees C or combined with 43 degrees C hyperthermia, in sensitive and multidrug-resistant MCF-7 human tumour cells using clonogenic assays. In the resistant subline, TLED was found to partly circumvent multidrug resistance (MDR). The reversal was comparable to that obtained when verapamil was added to free doxorubicin. When hyperthermic treatment was applied, no difference in thermosensitivity was found between sensitive and resistant cells. The combination of hyperthermia with free doxorubicin did not reverse MDR. Hyperthermia and TLED yielded additive effects in the resistant cells while potentiation was observed in the sensitive cells. These results confirmed the usefulness of the liposome encapsulation of doxorubicin in reversing MDR. The possibility of obtaining additive cytotoxicity using TLED combined with hyperthermia may represent an alternative way of intensification of doxorubicin cytotoxicity concomitant with the circumvention of MDR without using MDR reversing agents, which often generate limiting toxic side-effects.

Radiosensitivity of multicellular tumour spheroids obtained from human ovarian cancers

The radioresponsiveness of immunologically characterised (KL1, antivimentin and OC125) human ovarian carcinoma cells, obtained from effusions or solid tumours, was assayed in vitro using the multicellular tumour spheroids (MTS) three-dimensional model. Great interspecimen variabilities were observed in MTS doubling time (1.0-8.5 days), as well as in the doses inducing a 50% decrease in the MTS individual volume (ID50) (0.56-9.15 Gy), or in the overall population MTS number (SCD50) (1.9-15.7 Gy) and the residual/initial MTS individual volume ratio after 2 Gy irradiation (RSV2) (10-88%). The doubling time, DNA-ploidy and S-phase fraction did not correlate with the ID50. Significant correlations were found between the new parameters defined (RSV2 and ID50) and the SCD50, a well-accepted local control parameter. These parameters demonstrated their usefulness for studying the radiosensitivity of MTS prepared from human ovarian tumour biopsies.

Influence of SDZ‐PSC833 on daunorubicin intracellular accumulation in bone marrow specimens from patients with acute myeloid leukaemia

The multidrug resistance (MDR) modulating activity of SDZ‐PSC833 (PSC), a non‐immunosuppressive cyclosporine analogue, was investigated and compared with cyclosporin A (CSA) in bone marrow clinical specimens from 45 patients with acute myeloid leukaemia (AML) taken at diagnosis, using double‐labelling flow cytometry with simultaneous determination of P‐glycoprotein (PGP) expression and intracellular daunorubicin fluorescence (IDF). On the basis of pre‐clinical results in multidrug‐resistant K562 leukaemic cells, concentrations leading to iso‐effective complete restoration of IDF were used: 5 and 10 μmol/l, respectively for PSC and CSA.  In the clinical specimens, PGP expression was correlated with a significant decrease in IDF. PSC was found to be significantly more potent than CSA since it was found to induce a significant increase in IDF in a higher number of cases and to a higher extent than CSA. PGP‐unrelated activity of PSC was also observed in specimens expressing no PGP but exhibiting low IDF, thus probably expressing alternative resistance mechanisms. The results confirm the potency of PSC as MDR‐modulating agent in clinical AML specimens whose resistance pattern differed from that of highly resistant cell models and suggest that the activity of PSC is not limited to P‐glycoprotein inhibition.