Figure 4. Decreased dipole potential enhances cellular uptake and endo-lysosomal escape of penetratin. Cells (SKBR-3, MDA-MB-231, displayed on the left) were treated with a mixture of AFDye532-penetratin and NF-penetratin at 37°C and their fluorescence intensity was measured by flow cytometry. The membrane dipole potential of cells was decreased and increased by pre-treatment with phloretin and 6-ketocholestanol, respectively. Time-dependent intensities and their ratio were determined after gating out debris and dead cells. The error bars represent the standard error of the mean calculated from 12-14 samples from five biological replicates. Asterisks indicate significant difference of the phloretin-treated sample compared to the control at 20 min (p<0.05).
Statins increase the endo-lysosomal release of penetratin due to decreased dipole potential
Increasing the uptake of cell penetrating peptides has great potential medical benefit. Since the treatment used for decreasing the dipole potential in the previous section cannot be applied in humans, we sought an alternative approach to enhance the uptake of penetratin by modulating the dipole potential. The dipole potential correlates with membrane cholesterol content (Kovács, Batta, Zákány, Szöllősi & Nagy, 2017; Sarkar, Chakraborty & Chattopadhyay, 2017), and statins are known to decrease the dipole potential (Sarkar, Chakraborty & Chattopadhyay, 2017). We opted for atorvastatin since it is one of the most effective statins and it is the active substance not requiring enzymatic activation (Corsini, Maggi & Catapano, 1995; Jones, Kafonek, Laurora & Hunninghake, 1998; Schaefer et al., 2004). Atorvastatin, used at a concentration of 1-10 nM corresponding to the serum concentration in human patients (Bjorkhem-Bergman, Lindh & Bergman, 2011), significantly decreased the total cholesterol content of MDA-MB-231 cells by 40-50%. Albeit to a lesser extent, atorvastatin also decreased the cholesterol content of SKBR-3 cells (Suppl. Fig. 3). In perfect agreement with these results, atorvastatin decreased the dipole potential in both cell lines, but SKBR-3 displayed lower sensitivity (Fig. 3A). Treatment of MDA-MB-231 cells with the same concentration range of atorvastatin significantly enhanced the endo-lysosomal release of penetratin with only a marginal effect on total cellular uptake (Fig. 5). At the same time, the effect of atorvastatin on SKBR-3 cells was smaller and it did not reach statistical significance. Since the extent of decrease in the cholesterol content of this cell line turned out to be smaller compared to MDA-MB-231, we also tested the effect of higher atorvastatin concentrations. 100 nM and 10 µM of atorvastatin decreased the total cellular uptake of penetratin in SKBR-3 cells. Although cells with increased membrane permeability were discarded from the analysis, we attribute this finding to compromised cell viability. However, the amount of penetratin leaving the endo-lysosomal compartment was significantly higher in cells treated with these high atorvastatin concentrations even though the total cellular uptake was lower (Fig. 5). This finding is evidenced by the almost two-times higher NF/AFDye532 intensity ratio characterizing the fraction of penetratin escaping from endosomes. In conclusion, we have convincingly shown that release of penetratin from the endo-lysosomal compartment is the step that is the most significantly increased by atorvastatin treatment.