Figure 5. Atorvastatin enhances endo-lysosomal escape of penetratin. Two
different cell types were pretreated with the indicated concentrations
of atorvastatin for 72 h and the uptake of AFDye-532-penetratin and
NF-penetratin were followed by flow cytometry. 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 13-15 samples from five biological replicates. Asterisks
indicate significant difference of the sample treated with the highest
concentration of atorvastatin in the figure compared to the control at
20 min (p<0.05).
Characterization of the
effects of atorvastatin and the dipole potential modifying compounds on
membrane biophysical properties
The previous experiments revealed that treatments reducing the membrane
dipole potential enhance the total cellular uptake and the release of
penetratin from endosomes, while increasing the dipole potential results
in an opposite, weak effect. The validity of this conclusion was checked
by calculating the rank-correlation between the dipole potential and
penetratin uptake (Suppl. Fig. 4). A significant, negative correlation
was revealed between the dipole potential and the total uptake (AFDye532
fluorescence), the concentration of penetratin in non-acidic
compartments (NF fluorescence) and the fractional release of penetratin
from the endo-lysosomal compartment (ratio of NF and AFDye532
intensities). Since the experimental data corresponding to the effect of
phloretin on total cellular uptake of penetratin seemed to deviate from
the trend established by the other conditions, the correlation was also
tested by removing these outlier data points. The significant and
biologically relevant negative correlation between the endo-lysosomal
release of penetratin and the dipole potential was retained. However,
removing the data points corresponding to phloretin treatment eliminated
most of the dipole potential-dependent changes in total cellular uptake
(Suppl. Fig. 4). This observation indicates that although both
atorvastatin and phloretin decrease the dipole potential, their cellular
effects and their influence on penetratin uptake are different. Since
our measurement approach based on the pH-dependent fluorescence of NF
assumes that the pH of the endo-lysosomal compartment is equally acidic
in all compared samples, a possible explanation for the different
results obtained with atorvastatin and phloretin is that they alter the
pH of lysosomes. Therefore, we performed fluorescence ratiometric
measurements of lysosomal pH, which revealed no significant effect of
any of the treatments on lysosomal pH (Suppl. Fig. 5). These results
imply that the ratio of the fluorescence of AFDye532-penetratin and
NF-penetratin correctly reports the release of penetratin from acidic
compartments. The issue of the different effect of phloretin and
atorvastatin on penetratin release will be further considered in the
Discussion.
Since treatments altering the dipole potential could potentially also
modify other biophysical properties of the membrane, the correlations
between penetratin uptake and two other fluorescent indicators were also
tested. Membrane fluidity, inversely proportional to microviscosity and
the fluorescence anisotropy of TMA-DPH (Kuhry, Fonteneau, Duportail,
Maechling & Laustriat, 1983), was not changed by any of the treatments
in the SKBR-3 cell line, and phloretin and 6-ketocholestanol were also
without effect in MDA-MB-231 cells (Fig. 3B). In contrast, an unexpected
decrease in the membrane fluidity of this cell line was induced by
atorvastatin, an effect most likely attributable to the compensatory
increase in the synthesis of certain lipid species in order to
compensate for inhibited cholesterol synthesis (Dawaliby et al., 2016).
Membrane fluidity and total cellular uptake of penetratin did not
correlate with each other, but there was a strong correlation between
membrane fluidity and the fractional escape of penetratin from acidic
compartments (Suppl. Fig. 4). However, the power of membrane fluidity to
predict endo-lysosomal escape of penetratin is limited by two
observations: (i) the comparable effects of phloretin and atorvastatin
on penetratin uptake are not correlated with their distinct effects on
membrane fluidity in MDA-MB-231 cells; (ii) none of the treatments
induced any significant change in the membrane fluidity of SKBR-3 cells,
while phloretin increased endosomal release of penetratin.
The effect of the treatments on the generalized polarization of Laurdan,
known to be proportional to membrane compactness and inversely related
to membrane hydration (Parasassi, De Stasio, Ravagnan, Rusch & Gratton,
1991), was also tested. None of the conditions led to any significant
change in membrane compactness in the two cell lines (Fig. 3C). The
generalized polarization of Laurdan did not show appreciable correlation
with total penetratin uptake or its concentration in non-acidic
compartments (Suppl. Fig. 4). Given the fold-changes induced and the
statistical significance of the correlations we can conclude that the
dipole potential has the largest power for predicting penetratin uptake,
and that release of the cell-penetrating peptide from endosomes and its
concentration in the cytoplasm are both strongly correlated with the
dipole potential.