Novel Rotational Combination Regimen of Skin Topicals Improves Facial Photoaging: Efficacy Demonstrated in Double-Blinded Clinical Trials and Laboratory Validation

From Frontiers via Jisc Publications RouterHistory: collection 2021, received 2021-06-12, accepted 2021-08-05, epub 2021-09-17Publication status: PublishedTopical antiaging products are often a first-line intervention to counter visible signs of facial photoaging, aiming for sustained cosmetic improvement. However, prolonged application of a single active topical compound was observed clinically to lead to a plateau effect in improving facial photoaging. In view of this, we set out to reduce this effect systematically using a multi-tiered approach with laboratory evidence and clinical trials. The objective of the study was to evaluate the effects of active topical ingredients applied either alone, in combination, or in a rotational manner on modulation of facial photoaging. The study methodology included in vitro, organotypic, and ex vivo skin explants; in vivo biopsy study; as well as clinical trials. We demonstrate for the first time that a pair of known antiaging ingredients applied rotationally, on human dermal fibroblasts, maximized pro-collagen I production. Indeed, rotational treatment with retinol and phytol/glycolic acid (PGA) resulted in better efficacy than application of each active ingredient alone as shown by explants and in vivo biopsy study, with penetration of active ingredients confirmed by Raman spectroscopy. Furthermore, two split-face, randomized, double-blinded clinical trials were conducted, one for 12 months to compare treated vs. untreated and the other for 6 months followed by a 2-month regression to compare treated vs. commercially marketed products. In both studies, rotational regimen showed superior results to its matching comparison as assessed by clinical grading and image analysis of crow's feet wrinkles. In conclusion, rotational regimen using retinol and PGA is effective in treating facial photoaging signs with long-lasting benefits

Skn1 and Ipt1 negatively regulate autophagy in Saccharomyces cerevisiae

We demonstrated that a yeast deletion mutant in IPT1 and SKN1 , encoding proteins involved in the biosynthesis of mannosyldiinositolphosphoryl ceramides, is characterized by increased autophagy and DNA fragmentation upon nitrogen (N) starvation as compared with the single deletion mutants or wild type (WT). Apoptotic features were not significantly different between single and double deletion mutants upon N starvation, pointing to increased autophagy in the double Δ ipt1 Δ skn1 deletion mutant independent of apoptosis. We observed increased basal levels of phytosphingosine in membranes of the double Δ ipt1 Δ skn1 deletion mutant as compared with the single deletion mutants or WT. These data point to a negative regulation of autophagy by both Ipt1 and Skn1 in yeast, with a putative involvement of phytosphingosine in this process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78623/1/j.1574-6968.2009.01869.x.pd

Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

<p>Abstract</p> <p>Background</p> <p><it>N</it>-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model.</p> <p>Methods</p> <p>CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling.</p> <p>Results</p> <p>No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells.</p> <p>Conclusions</p> <p>In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.</p

Sustained activation of protein kinase C induces delayed phosphorylation and regulates the fate of epidermal growth factor receptor.

It is well established that acute activation of members of the protein kinase C (PKC) family induced by activation of cellular receptors can transduce extracellular stimuli to intracellular signaling. However, the functions of sustained activation of PKC are not well studied. We have previously shown that sustained activation of classical PKC isoforms over 15-60 min induced the formation of the pericentrion, a subset of recycling endosomes that are sequestered perinuclearly in a PKC- and phospholipase D (PLD)-dependent manner. In this study, we investigated the role of this process in the phosphorylation of EGFR on threonine 654 (Thr-654) and in the regulation of intracellular trafficking and fate of epidermal growth factor receptor (EGFR). Sustained stimulation of the angiotensin II receptor induced translocation of the EGFR to the pericentrion, which in turn prevents full access of EGF to the EGFR. These effects required PKC and PLD activities, and direct stimulation of PKC with phorbol esters was sufficient to reproduce these effects. Furthermore, activation of PKC induced delayed phosphorylation of EGFR on Thr-654 that coincided with the formation of the pericentrion and which was dependent on PLD and endocytosis of EGFR. Thus, Thr-654 phosphorylation required the formation of the pericentrion. On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF. Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR

Growth differentiation factor 11 (GDF11) has pronounced effects on skin biology.

Growth differentiation factor 11 (GDF11) belongs to the TGF-β superfamily of proteins and is closely related to myostatin. Recent findings show that GDF11 has rejuvenating properties with pronounced effects on the cardiovascular system, brain, skeletal muscle, and skeleton in mice. Several human studies were also conducted, some implicating decreasing levels of circulating GDF11 with age. To date, however, there have not been any reports on its role in human skin. This study examined the impact of GDF11 on human skin, specifically related to skin aging. The effect of recombinant GDF11 on the function of various skin cells was examined in human epidermal keratinocytes, dermal fibroblasts, melanocytes, dermal microvascular endothelial cells and 3D skin equivalents, as well as in ex vivo human skin explants. GDF11 had significant effects on the production of dermal matrix components in multiple skin models in vitro and ex vivo. In addition, it had a pronounced effect on expression of multiple skin related genes in full thickness 3D skin equivalents. This work, for the first time, demonstrates an important role for GDF11 in skin biology and a potential impact on skin health and aging

Scheme illustrating sustained activation of PKC induces PLD- and endocytosis- dependent phosphorylation of Thr-654 on EGFR and sequestration of EGFR to a cPKC- dependent subset of recycling compartment (pericentrion).

<p>Prolonged treatment with ATII or PMA could induce translocation of EGFR to pericentrion and phosphorylation of EGFR on Thr-654 on a cPKC- and PLD- dependent manner. Sequestration of EGFR to pericentrion protects EGFR accessed by EGF. </p

Effects of PMA on phosphorylation of EGFR and the role of the pericentrion.

<p>A, HEK293 cells were serum starved for 5 hours followed by 100nM PMA for 2 min, 5 min, 10 min, 30 min or 60 min. Phospho-Thr654 and total EGFR were determined as described above. B. HEK293 cells were starved for 5 hours and then pretreated with vehicle, Gö6976, 1-butanol, depleted of potassium (K-), or preincubated 400mM sucrose followed by 1-hour 100nM PMA treatment. The procedure for potassium-depletion is as described previously (30). Levels of P-Thr654 and total EGFR were determined as described. C, HEK293 cells were transfected with dominate negative constructs of PLD1 or PLD2. After 24 hours post-transfection, cells were starved for 5 hours and then treated with 100nM PMA for 1 hour. Levels of P-Thr654 and total EGFR were determined as shown before. D, HEK293 cells were starved for 5 hours and then pretreated with 100nM PMA for the indicated time followed by 5min 5ng/ml EGF treatment. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) and total EGFR were determined as described. E, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol, FIPI, or vehicle followed with 100nM PMA or vehicle for 1 hour and then treated with 5ng/ml EGF or vehicle for 5 min. P-Tyr1045 and EGFR were determined by western blotting. F, HEK293 cells were starved for 5 hours and then pretreated with vehicle or 100nM for 1 hour followed by treatment with 10ng/ml EGF for 2 min. Phospho-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.</p

Effects of ATII on localization and fate of EGFR.

<p>A, HEK293 cells stably transfected with AT_1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, location of AT_1AR (green) and endogenous Rab11 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells stably transfected with AT_1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (red) was determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). C, HEK293 cells stably transfected with AT_1AR were serum starved for 5 hours and treated with 2ng/ml of EGF for 3 hour with or without 1 hour pretreated with 100nM ATII. Protein level of EGFR was determined by Western Blotting. Blots were stripped and reprobed for Na⁺K⁺ATPase to normalize for loading. These results are representative of three independent experiments. Pictures are representative of at least three experiments.</p

ATII-induced sequestration and protection of EGFR loss require the pericentrion.

<p>A, HEK293 cells were transfected with WT-EGFR-GFP. After 24 hours, cells were starved for 5 hours and then treated with 100nM PMA for 5 or 60 min. After fixation and permeabilization, location of EGFR (green) and endogenous EEA1 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (Leica TSC SP8). B, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol or vehicle followed by 100nM PMA or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (red) was determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). C, HEK293 cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (green) and Lamp1 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (Leica TSC SP8). D, HEK293 cells were transfected with Rab11-GFP. After 24 hours, cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation and permeabilization, location of Rab11 (green) and endogenous EGFR (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (Leica TSC SP8). E, HEK293 cells starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour following with 5ng/ml EGF or vehicle for 3 hours. Protein levels of EGFR and actin were determined by western blotting. F, HEK293 cells starved for 5 hours and then were pretreated with Gö6976, Bis, 1-butanol or vehicle followed with 100nM PMA for 1 hour and then treated with 5ng/ml EGF or vehicle for 3 hours. Protein levels of EGFR and actin were determined by western blotting. G, HEK293 cells or HEK293 cells stably overexpressing AT_1AR were starved for 5 hours and then treated with vehicle, 100nM PMA or 100nM ATII for 1 hour. After treatments, cells were collected immediately and the mRNA level of EGFR were determined by real-time PCR. Pictures are representative of at least three experiments. E: **** <i>P</i><0.0001 compared to control, two-way ANOVA.</p