br JOURNAL br This intensity relates the ability
This intensity relates the ability of each tested cell line to produce the ROS responsible for the oxidation of the boron-carbon bond leading to the release of coumarin. Since oxidation and subsequent self-immolation of precursor 3 is rather slow and dependent of the ROS concentration, we chose to: (i) incubate 3 in the presence of a large number of Dihydrorhodamine 123 in order to produce as much ROS as possible using six-well plates; and (ii) maintain contact for 6 hours to obtain a significant amount of liberated coumarin. The A549 and U87 cell lines demonstrated a low production of ROS as only a weak fluorescence intensity was observed. On the other hand, Hep G2, MiaPaCa-2, MCF-7 and MCF-7 MDR revealed a higher production of ROS as the fluorescence intensity was much higher compared to the other tested cell lines. Further, Hep G2 and MiaPaCa-2 were the most active cell lines for conversion of precursor 3 into coumarin 2 and these two cell lines seem to be the most suitable for further investigation of ROS-activatable prodrugs. The comparison of the ROS production by these cancer cells versus the cell line producing the least ROS (U87) was unequivocal and confirmed using a Kruskall-Wallis test with p < 0.0001.
In vitro cytotoxicity
As opposed to the probe 3, the prodrugs are built of an aryl self-immolative spacer bonded to DOX through a carbamate linker to enhance the kinetic of disassembly as it is beneficial for the biological activity to release high level of drug rapidly (data not shown). The IC50 of all the designed prodrugs (8a-c) and controls (9 and 12) were evaluated on the panel of cell line and the results are depicted in Table 1. No activity was observed on the multidrug resistant cell line MCF-7 MDR. DOX showed an important cytotoxic activity on all the other tested cell lines with nanomolar IC50 ranging from 10 to 600 nM. The cell lines U87, MCF-7, Hep G2 and MiaPaCa-2 were sensitive to DOX withIC50 around 100 nM. A549 cell line on the other hand demonstrated a lower activity with an IC50 of 600 nM. The control compounds 9 and 12 had no activity on the studied cell lines even at our highest tested concentration of 50 µM. These results are coherent as these control molecules have no trigger unit (12) or deliver a non-toxic motif (9).
Table 1. In vitro cytotoxicity of 8a-c, 9 and 12 on the different cell lines. Percentage of restored activity is displayed in bracket. Nd: Not determined.
All the ROS-activatable doxorubicin prodrugs (8a-c) demonstrated activities on the DOX-sensitive cell line and variable micromolar activities were obtained ranging from 0.1 to 10 µM underlining moderate activities of the tested prodrugs. The activity of the different prodrugs on a particular cell line was comparable except in the case of MiaPaca-2 where 8a was more cytotoxic than 8b and 8c. Interestingly, 8a showed activity on the MiPaCa-2 cell line with an IC50 close to the one obtained with the free DOX, 0.3 µM for 8a versus 0.2 µM for DOX. Although the conversion of 8a is not complete: ~ 67 %, as deducted from the activity obtained with the same concentration of free doxorubicin, we observe a greater conversion than those obtained for related structures (~ 40 %).8a was however not as active against the Hep G2 cells (IC50 = 4 µM, ~ 4 % estimated conversion), a cell line able to produce similar level of ROS as the MiPaCa-2 cell line. As described by Doskey et al., the H2O2 removal rate constants differ greatly between cell lines. In exponential growth phase, Hep G2 cells were shown to remove H2O2 four times faster than the MiaPaCa-2 cells. In this study, the cytotoxic activities were
determined after 72 hours of incubation when cells have reached their exponential growth phase and this can therefore rationalize the strongest activity of 8a against MiaPaCa-2 in comparison with Hep G2 cell lines.
Chick Chorioallantoic Membrane (CAM) Assay and HPLC Detection of DOX
The HET-CAM assay was developed to investigate the efficacy of our most active arylboronate doxorubicin prodrug 8a using the MiaPaCa-2 pancreatic cancer cell line for tumor production on the CAM as it showed the most promising cytotoxicity and an elevated release of ROS. Different protocol of intratumoral injection of 8a or DOX were performed. In our case and according to the literature, we first determined the highest dose of DOX that was not lethal for the embryo. Our maximum dose of 184 nmol (100 µg) of DOX in a single injection was well tolerated in a statistically significant sample size of chick embryos. For the evaluation of the tumor regression, we chose to enhance the number of injections rather than testing a high concentration as a single dose taking into account the poor solubility of 8a. Optimized protocol was found with the administration of DOX or 8a (20 nmol) intratumorally twice a day and two days in a row followed by an observation of the tumor size after three days. This protocol led us to observe a significant decrease of the tumor in DOX group (~ 50 % regression) and a comparable result was obtained for 8a (~ 50 % regression) (Figure 3). Statistically relevant images of MiaPaCa tumor implanted on the CAM are shown in Figure 3. Necrosis seen in Figure 3B was noticed in several experiments as the result of DOX/8a action. In this vascularized tumor model, the tested drug has also some antivascular effect often leading to partial necrosis of the tumor.