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.