Somatostatin receptor mediated targeting of acute myeloid leukemia by photodynamic metal complexes for light induced apoptosis

Abstract

Acute myeloid leukemia (AML) is characterized by relapse and treatment resistance in a major fraction of patients, underlining the need of innovative AML targeting therapies. Here we analysed the therapeutic potential of an innovative biohybrid consisting of the tumor-associated peptide somatostatin and the photosensitizer ruthenium in AML cell lines and primary AML patient samples. Selective toxicity was analyzed by using CD34 enriched cord blood cells as control. Treatment of OCI AML3, HL60 and THP1 resulted in a 92, and 99 and 97% decrease in clonogenic growth compared to the controls. Primary AML cells demonstrated a major response with a 74 to 99% reduction in clonogenicity in 5 of 6 patient samples. In contrast, treatment of CD34⁺ CB cells resulted in substantially less reduction in colony numbers. Subcellular localization assays of RU-SST in OCI-AML3 cells confirmed strong co-localization of RU-SST in the lysosomes compared to the other cellular organelles. Our data demonstrate that conjugation of a Ruthenium complex with somatostatin is efficiently eradicating LSC candidates of patients with AML. This indicates that receptor mediated lysosomal accumulation of photodynamic metal complexes is a highly attractive approach for targeting AML cells.

Figure 1

Uptake and IC50 studies of RU complex: confocal images of OCI-AML3 cells incubated with RU-SST (50 μm) (A) and RU-Alkyne control (RU-Alk) (100μm) (B) for 4 hrs. The laser intensity was measured for alexaflour (430 nm). Uptake characteristics with values of the integrated intensity (a.u.) per cell (C). Bar graphs indicate the average integrated intensity per cell in both experimental arms. Cells analysed for RU-SST (n = 45) and RU-Alk (n = 44). Data are represented as mean ± SEM. (D) IC50 was calculated in HL60 AML cell lines. Percent cell survival was measured by trypan blue exclusion after 4 h incubation with RU-Alk (control), RU-SST (irradiated) compared to RU-SST in the absence of light (dark RU). Experiments performed for dark RU-SST (n = 6); RU-Alk (n = 3), for RU-SST (n = 3). Data are represented as mean ± SEM.

Figure 2

Determination of clonogenic potential of AML cell lines with RU-complexes: (A) Bar graph shows the number of colonies after day 14 of plating cells on methylcellulose in OCI-AML3 AML cells. Significance was calculated using the Mann-Whitney test (** < 0.01). (B) Percent reduction in the number of colonies compared to dark control in OCI-AML3, HL60 and THP-1 AML cells. The bars represent mean ± SEM (n = 3).

Figure 3

Effect of RU-complexes on clonogenicity of normal CD34⁺ cells: cells from four CD34⁺ enriched cord blood (CB) samples (n = 6) were tested for the effect of both RUSST and RU-Alkyne compounds. The bar graphs represent the average colony numbers of each experimental arm performed in duplicates. Percent reduction was determined based on the colony numbers generated from CB cells treated with RU-SST and exposed to light compared to dark RU-SST. C) Bar graphs show the percent decrease in CFC growth of AML samples compared to normal CB CD34⁺ cells. Significance calculated by Mann-Whitney test ** < 0.01.

Figure 4

Evaluation of localization of the RU complexes: (A–D): (A) Confocal microscopy images of OCI-AML3 cells incubated with RU-SST (Panel 1) and RU-Alkyne (Panel 2) and treated with organelle trackers for (A) lysosomes, (B) mitochondria, (C) membrane and (D) nuclei. The panels of each figure show (a) RU-SST emission, (b) corresponding bright field images, (c) emission from the organelle trackers, and (d) overlay of all three images.

Figure 5

Determination of reactive oxygen species (ROS) levels (A,C) Confocal microscopy images of OCI-AML3 cells incubated with RU-SST (A) and RU-Alkyne (C) and treated with ROS reporter and kept in the dark (Panel 1) or exposed to light (Panel 2). (a) ROS reporter, (b) corresponding bright field image, (c) overlay. (B) Bar graph indicates the fluorescence intensity corresponding to A) for untreated OCI-AML3 cells, after treatment with RU-SST in the dark as well as after exposure to light (irradiatíon). Significance calculated by Mann-Whitney test *** < 0.001 and * < 0.05. (D) Bar gaph indicates the fold CTCF (The corrected total cell fluorescence) of ROS reporter before and after irradiation compared to untreated cells. (E) Bar graph indicated the Mean fluorescence intensity (MFI) of ROS measurement in primary AML (n = 3) and nCB-CD34⁺ (n = 3) cells. (F) Bar graph indicates the fold increase of MFI after irradiation with RU-SST compared to Dark control in primary AML samples and were compared to the fold reduction in nCB-CD34⁺ cells (n = 3). Significance was calculated by 2way ANOVA with *p < 0.05, **p < 0.01.