Efficient Photodynamic Therapy on Human Retinoblastoma Cell Lines

International audiencePhotodynamic therapy (PDT) has shown to be a promising technique to treat various forms of malignant neoplasia. The photodynamic eradication of the tumor cells is achieved by applying a photosensitizer either locally or systemically and following local activation through irradiation of the tumor mass with light of a specific wavelength after a certain time of incubation. Due to preferential accumulation of the photosensitizer in tumor cells, this procedure allows a selective inactivation of the malignant tumor while sparing the surrounding tissue to the greatest extent. These features and requirements make the PDT an attractive therapeutic option for the treatment of retinoblastoma, especially when surgical enucleation is a curative option. This extreme solution is still in use in case of tumours that are resistant to conventional chemotherapy or handled too late due to poor access to medical care in less advanced country. In this study we initially conducted in-vitro investigations of the new cationic water-soluble photo sensitizer tetrahydroporphyrin-tetratosylat (THPTS) regarding its photodynamic effect on human Rb-1 and Y79 retinoblastoma cells. We were able to show, that neither the incubation with THPTS without following illumination, nor the sole illumination showed a considerable effect on the proliferation of the retinoblastoma cells, whereas the incubation with THPTS combined with following illumination led to a maximal cytotoxic effect on the tumor cells. Moreover the phototoxicity was lower in normal primary cells from retinal pigmented epithelium demonstrating a higher phototoxic effect of THPTS in cancer cells than in this normal retinal cell type. The results at hand form an encouraging foundation for further in-vivo studies on the therapeutic potential of this promising photosensitizer for the eyeball and vision preserving as well as potentially curative therapy of retinoblastoma

Dose and incubation time effects.

<p>The incubation of WERI Rb-1 with THPTS at concentrations of 25 µg/ml, 50 µg/ml, 100 µg/ml or 200 µg/ml for 1.5 h (red bars) lead to survival rates of 77.3%, 65.9%, 37.8% and 0.4%.respectively. An incubation time of 3 h (deep red bars) roughly doubled the cell death rates (at the lower concentrations) with survival as low as 41.5%, 32.2% 10.6% and 0.4% respectively, (*** p<0.001 comparison with control without THPTS; °°° p<0.001 for the comparison between 1.5h and 3h of THPTS incubation)</p

Rate of WERI-rb1 and primary RPE survival following THPTS treatment.

<p>As low as 50 µg/ml of THPTS induced an effect on retinoblastoma cell line as survival dropped to 62.9% after 90 min of incubation (light blue bars). In RPE, using the same condition the survival decreased slightly to 88.2% (light green bars). When increasing the THPTS concentration to 100 µg/ml the compound led to a reduced survival to 40.3% in retinoblastoma cell lines (deep blue bars) and only affect slightly the normal primary RPE cells over the same incubation period (84.5%, deep green bars). Increasing the incubation to 3 hours induced a more drastic effect of THPTS even with low concentration. 50 µg/ml of THPTS induced a survival of only 29.2% of the We-Rb1, a rate that dropped to 13.5% when using 100 µg/ml. Meanwhile the primary RPE cells survival is 83.7 and 42.4% with the same conditions. A survival significantly higher than for Rb1. After 6h the survival is not further affected in RPE cells as it was found to be at 78.3 and 42.1 using 50 and 100 µg/ml of THPTS. In Rb cells also the survival was only slightly further affected compared to 3 h of incubation (34.3% and 9.2% respectively). Asterisks indicate significant differences between Rb1 and RPE cells in comparable situations of concentration and incubation length.</p

THPTS-PDT on retinoblastoma cell lines.

<p>In vitro cell survival analysis of WERI Rb-1 (A) and Y79 (B) cells irradiated with laser after incubation with THPTS (200 µg/ml) for 0.5 h, 1.5 h, 3 h, 6 h, 12 h or 24 h (red bars) showed a minimal survival rate of 0% –5% after an incubation time of 1.5 hours (WERI Rb-1) or 3 hours (Y79). When incubated with THPTS (200µg/ml) without irradiation (dark toxicity) for 0.5 h, 1.5 h, 3 h, 6 h, 12 h or 24 h (brown bars), the survival rate relative to control varied from 85.2% (3 h) to 107.7% (24 h) in WERI Rb-1 and from 100.0% to 141.1% in Y79. Laser irradiation alone (light orange bars) lead to no significant changes of the survival rates relative to controls.</p

Quantitative gene expression.

<p>The expression of genes related to apoptosis showed rapid and significant increases after treatment with THPTS-PDT compared to the controls (depicted as the red line reflecting the expression baseline). Y axis corresponds to the relative expression vs control while x axis corresponds to the different time points (0.5, 1.5, 3, 4.5, 6, 12 and 24h). The pink bars reflect the Y79 and the violet bars the WERI Rb-1 cells. ATF3 showed a significant increase of up to 67.25 fold (p<0.01) in Y79 cells and a 19.87 fold (p<0.001) increase in WERI Rb-1 cells within 12 hours after treatment. Maximal increase for hJUN expression was obtained with values up to 99.30 fold (p<0.001) in Y79 cells and up to 100.60 fold (p<0.05) in WERI-Rb-1, 12 and 3 hours after PDT respectively. hHSP70B was increased significantly after 1.5 and 0.5 hours for Y79(6.09 fold, p<0.01) and WERI Rb-1 (43.84 fold, p<0.001) respectively. With maximal increase values up to 584.24 fold (Y79, p<0.001) and 979.00 fold (WERI Rb-1, p<0.001). hHSP105 levels increased significantly after 3 h and 0,5h in Y79 (1.43 fold, P<0.001) and in WERI Rb-1 cells (1.87 fold, p<0.05) respectively; reaching a maximum of 8.55 fold (Y79, p<0.01) to 19.20 fold (WERI Rb-1, p<0.001) within 24 h. The DNA damage related mRNA levels of hGADD45G (Y79: max. 16.81 fold increase after 12 h, p<0.001; WERI Rb-1: 6.88 fold increase after 3h, p<0.001) as well as hGADD153 (Y79: max. 21.57 fold increase after 12 h, p<0.01; WERI Rb-1: 11.90 fold increase after 1.5h, p<0.001) were significantly increased within the first 4.5 h after PDT for both cell lines. The levels of hp27 were increased significantly increased in both cell lines. The maximum reached a 3.34 fold increase after 12 h (p<0.001) for the Y79 cells and a 3.75 (p<0.05) fold increase for the WERI Rb-1 cells 4.5 h after the treatment. Note that for hHSP70B, y axis is in log scale.</p

Immunocytochemistry.

<p>Y79 cells were cultured in 10% lipumin and incubated with low dose of THPTS (25 µg/ml) for 90 min. After formaldehyde fixation, cells were stained with DAPI nuclear marker and phalloidin coupled to Alexa 488. In control without laser illumination Rb cells showed a strong development of filopodia as seen by actin cytoskeleton staining (arrow in A and C). Nuclei were found homogenous in diameter and for DAPI staining intensity (B and C). Rarely, fragmented nucleus can be identified (arrowhead in B and C). THPTS-PDT treatment induced a reduction of filopodia (D, F) and many Rb cells exhibited heterogeneous nucleus aspect as seen with DAPI staining (E, F). Many fragmented nucleus (arrowheads in E and F) can be observed. Condensed genomic material was detected by the increased intensity for the DAPI signal (arrows in E and F). Many nuclei were found reduced in diameter compared to the control situation. Scale bar 30 µm.</p

THPTS subcellular localization.

<p>MitoTracker green (M7514) original scan identifying mitochondria in Y79 cells (A) and resembling to subcellular THPTS distribution (B). (C) Control scan reveals no staining of THPTS at 488 nm excitation and detection with 650 nm long pass filter. (D-G) FRET pair identification in Y79 mitochondria as excited at 488 nm and observed at 505–530 nm (D) and above 650 nm (E). (F) Merged images revealed colocalization of M7514 and THPTS by double staining in numerous, though not all, mitochondria. Exclusive THPTS fluorescence distribution (arrows) account for a complete energy transfer from M7514 at those localizations while signal seen at 505–530 nm account for an excess of donor (D, F). M7514 and THPTS establish a FRET pair of fluorescence dyes as the emission maximum of MT7514 is 516 nm, which is the excitation maximum of THPTS; blue colored area highlights M7514 excitation curve; pale green color indicates M7514 emission spectrum and dark green area corresponds to the overlap with THPTS excitation spectrum (G). (H-I) Evidence for mitochondrial ROS production by THPTS excitation. Basic mitotracker M7511 fluorescence (H) was clearly enhanced following THPTS excitation at 514nm (I), indicating an oxidative process specifically due to THPTS-PDT (DAPI filter set, 5 min). Insets in indicate M7511 fluorescence intensity histogram (H-I). Scale bar in (A) applies to all images except for (B-C) where scale bar is shown in (C).</p