Discussion
In this study, correlative measurement of the biophysical properties of
the plasma membrane and the uptake of penetratin allowed us to reach the
following major conclusions: (i) The physiological, positive membrane
dipole potential inhibits the total cellular uptake of penetratin and
its release from acidic endo-lysosomes. These conclusions are based on
temporal measurement of the cellular intensity of AFDye532-labeled and
NF-labeled penetratin, respectively. The effect of the dipole potential
on both steps of penetratin uptake is most likely mediated by an
alteration in the membrane insertion of the cell-penetrating peptide,
since incorporation and penetration of peptides and small, hydrophobic
molecules into the membrane are known to depend on the dipole potential
(Asawakarn, Cladera & O’Shea, 2001; Cladera & O’Shea, 1998). (ii)
Treatment with phloretin and atorvastatin reduced the membrane dipole
potential and led to a significantly enhanced cytoplasmic concentration
of penetratin. Since the applied, nanomolar concentration of
atorvastatin is identical to that used in the clinical setting, the
finding of statin-boosted uptake of penetratin is of potential medical
significance. (iii) By analyzing the correlation between several
membrane biophysical properties and penetratin uptake the dipole
potential turned out to be the only characteristic of sufficient value
for predicting penetratin uptake.
While both phloretin and atorvastatin decreased the dipole potential and
increased penetratin uptake, their differential effects raise question
about which step in the uptake process is the most sensitive to the
dipole potential. In particular, although atorvastatin decreased the
dipole potential more substantially than phloretin, only the latter
induced an increase in the total cellular uptake of penetratin, while
atorvastatin only increased the escape from acidic endo-lysosomes. These
apparent contradictions can be resolved by the following three points:
(i) The fact that phloretin does not increase the fraction of penetratin
released from the endo-lysosomal compartment is explained by the fact
that this compound is unlikely to reach sufficient concentrations in
intracellular membranes during the brief, 10-minute incubation and
therefore the dipole potential of these compartments remains largely
unaffected. On the other hand, the three-day treatment with atorvastatin
is sufficiently long so that substantial decrease in the cholesterol
content and consequent reduction in the dipole potential of
endo-lysosomal membranes can take place. (ii) The minuscule,
phloretin-induced decrease of the dipole potential and the significantly
elevated uptake of penetratin after phloretin treatment also require
clarification. We assume that the physiological level of the dipole
potential already limits penetratin uptake as much as potentially
achievable by the dipole potential. This conclusion is supported by the
fact that the significantly enhanced dipole potential after
6-ketocholestanol treatment had hardly any effect on the characteristics
of penetratin uptake. On the other hand, even the relatively minor
decrease in the dipole potential, achieved by phloretin, may be
sufficient to facilitate penetratin uptake. However, the magnitude of
this change in the dipole potential is not sufficiently large to reach
statistical significance given the measurement errors. (iii) Although
the reduction in the dipole potential achieved by atorvastatin is
significantly larger than the phloretin-induced change, atorvastatin had
no effect on the total cellular concentration of penetratin. The sudden
rise in AFDye532-penetratin intensity and the protracted increase in the
intensity of NF-penetratin indicate that initial uptake is mediated by
endocytosis, a characteristic left unaltered by any of the treatments.
Therefore, the lack of statin-induced increase in the total uptake of
penetratin implies that endocytosis must be hindered in
atorvastatin-treated samples. Indeed, it has been shown that statins
inhibit endocytosis by interfering with the prenylation-dependent
function of certain G proteins (Sidaway et al., 2004; Yilmaz et al.,
2006). Alternatively, the compensatory increase in membrane viscosity
after atorvastatin treatment may also impede endocytosis.
Despite the aforementioned uncertainties, two of the treatments,
phloretin and atorvastatin, significantly enhanced the concentration of
penetratin in the cytosol, the compartment most relevant from a
therapeutical point of view. Analysis of the correlation between
penetratin uptake and different biophysical properties of the membrane
revealed that the dipole potential is the strongest predictor of
penetratin uptake. Although the dipole potential, membrane compactness
and viscosity characterize different membrane properties, all of them
are related to membrane order. Therefore, it is not surprising that
besides the dipole potential, other membrane characteristics also
correlate with penetratin uptake. Membrane viscosity is correlated with
endo-lysosomal release of penetratin. However, the predictive value of
this correlation is undermined by the following two points: (i) While
phloretin exerts no effect on membrane fluidity, it significantly
enhances penetratin uptake in both SKBR-3 and MDA-MB-231 cells. (ii)
Although penetratin uptake was modulated by the treatments in SKBR-3,
none of them altered membrane viscosity in this cell line. Membrane
compactness or hydration, characterized by the generalized polarization
of Laurdan, was correlated weakly and in a statistically non-significant
way with penetratin uptake. The predictive value of this correlation is
further deteriorated by the fact that the generalized polarization of
Laurdan was non-significantly modified by any of the treatments in
MDA-MB-231 cells, while large and significant effects in penetratin
uptake were observed.
In the present manuscript, not only did we identify the positive,
intramembrane dipole potential inhibiting the uptake and endo-lysosomal
release of penetratin, but we could also enhance the cytoplasmic
concentration of the cell-penetrating peptide in a medically relevant
way by statin treatment. In order for a treatment modality to be of
potential medical importance, it must be well-tolerated and
therapeutically effective. In contrast to many in vitro studies, in
which statins are often overdosed, the nanomolar concentration range of
atorvastatin used in the presented experiments is identical to the serum
concentration at therapeutic doses (Bjorkhem-Bergman, Lindh & Bergman,
2011). By applying atorvastatin not requiring metabolic activation, we
also circumvented another pitfall of in vitro cellular studies, the
application of statins in a prodrug form, which are unlikely to be
converted to the active metabolite in cell cultures. Although certain
side-effects are associated with statin treatment, extensive experience
indicates that they are safe, well-tolerated even in long-term
applications in combinations with other drugs (Fievet & Staels, 2009;
Luo, Wang, Zhu, Du, Wang & Ding, 2016). These circumstances further
support the potential medical relevance of our findings.
Although reduction of the dipole potential in both MDA-MD-231 and SKBR-3
cells resulted in enhanced penetratin accumulation in the cytosol after
phloretin treatment, the latter cell line exhibited lower sensitivity to
atorvastatin. Sensitivity to statins correlates inversely with HMG-CoA
reductase activity (Göbel, Breining, Rauner, Hofbauer & Rachner, 2019;
Kimbung, Lettiero, Feldt, Bosch & Borgquist, 2016). While the
expression of this enzyme is higher by ∼20-30% in SKBR-3 cells
according to a publication (Kimbung, Lettiero, Feldt, Bosch &
Borgquist, 2016) and the Expression Atlas of the European Bioinformatics
Institute (https://www.ebi.ac.uk/gxa/home), the confidence
intervals of the expression of HMG-CoA reductase in the two cell lines
completely overlap according to the Genevestigator platform comparing
transcriptomic data from several public repositories, with the level of
expression corresponding to the MDA-MB-231 cell line spanning three
orders of magnitude. In addition, atorvastatin, albeit at a micromolar
concentration, resulted in 4-fold induction of HMG-CoA reductase
expression in SKBR-3 cells, while no change in enzyme expression was
observed in MDA-MD-231 cells (Kimbung, Lettiero, Feldt, Bosch &
Borgquist, 2016). Cellular uptake of statins is believed to be mediated
by transporters to a large extent. Organic anion transporter
polypeptides (OATP) have been implicated in transmembrane import of
statins (Dobson & Kell, 2008; Kalliokoski & Niemi, 2009; Wu, Whitfield
& Stewart, 2000). However, none of the OATP transporters is expressed
significantly differently according to the previous databases.
Therefore, the most likely cause for the different atorvastatin
sensitivity of the two cell lines in terms of penetratin uptake and
reduction in cellular cholesterol content seems to be the difference in
the baseline and statin-induced expression of HMG-CoA reductase
expression, but solid conclusions cannot be drawn due to inconsistencies
in the literature.
In conclusion, we have shown that a decreased, positive membrane dipole
potential significantly increases both the total cellular uptake and
endocytic escape of penetratin depending on what kind of treatment is
used for modifying the dipole potential. As a result, both medically
relevant (atorvastatin) and irrelevant (phloretin) treatments decreasing
the dipole potential enhance the concentration of penetratin in the
cytoplasm, the compartment most relevant for its therapeutic action.
This discovery could permit the delivery of drugs and drug candidates
exhibiting low or no transmembrane permeability into cells in animal
experiments, human trials or in the clinical setting after further
studies clarify the cell type dependence and the in vivo potential of
this treatment.