Argania Spinosa Fruit Shell Extract-Induced Melanogenesis Via Camp Signaling Pathway Activation

International Journal of Molecular Sciences

Article Argania Spinosa Fruit Shell Extract-Induced Melanogenesis via cAMP Signaling Pathway Activation

1,2, 1,3, 1,2, 2 Rachida Makbal †, Myra O. Villareal †, Chemseddoha Gadhi *, Abdellatif Haﬁdi and Hiroko Isoda 1,3,*

1 Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Tennodai 1-1-1, Tsukuba City, Ibaraki 305-8572, Japan; [email protected] (R.M.); [email protected] (M.O.V.) 2 Faculty of Sciences Semlalia, Cadi Ayyad University, Avenue Prince Moulay Abdellah, BP 2390, Marrakesh 40000, Morocco; a.haﬁ[email protected] 3 Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba City, Ibaraki 305-8572, Japan * Correspondence: [email protected] (H.I.); [email protected] (C.G.); Tel.: +8129-853-5775 (H.I.); +212-524-434-649 (C.G.) These authors contributed equally to this work. † Received: 3 March 2020; Accepted: 3 April 2020; Published: 6 April 2020

Abstract: We have previously reported that argan oil and argan press-cake from the kernels of Argania spinosa have an anti-melanogenesis effect. Here, the effect of argan fruit shell ethanol extract (AFSEE) on melanogenesis in B16F10 cells was determined, and the mechanism underlying its effect was elucidated. The proliferation of AFSEE-treated B16F10 cells was evaluated using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, while the melanin content was quantified using a spectrophotometric method. The expression of melanogenesis-related proteins was determined by Western blot and real-time PCR, while global gene expression was determined using a DNA microarray. In vitro analysis results showed that the melanin content of B16F10 cells was significantly increased by AFSEE, without cytotoxicity, by increasing the melanogenic enzyme tyrosinase (TRY), tyrosinase related-protein 1 (TRP1), and dopachrome tautomerase (DCT) protein and mRNA expression, as well as upregulating microphthalmia-associated transcription factor (MITF) expression through mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38, and the cyclic adenosine monophosphate (cAMP) signaling pathway, as indicated by the microarray analysis results. AFSEE’s melanogenesis promotion effect is primarily attributed to its polyphenolic components. In conclusion, AFSEE promotes melanogenesis in B16F10 cells by upregulating the expression of the melanogenic enzymes through the cAMP–MITF signaling pathway.AFSEE may be used as a cosmetics product component to promote melanogenesis, or as a therapeutic against hypopigmentation disorders.

Keywords: B16F10 cells; pigmentation; MITF; MAPKs; argan fruit shell

1. Introduction Cutaneous pigmentation is the primary defense of the human skin against harmful UV radiation, and it is imparted by the pigment melanin, a polymer resulting from melanogenesis in melanocytes [1]. Defects in melanocyte functions can lead to the loss of melanin pigment, bringing about hypopigmentation disorders, such as progressive macular hypomelanosis. At present, the treatment for hypopigmentation involves the use of topical corticosteroids, laser treatment, or skin

Int. J. Mol. Sci. 2020, 21, 2539; doi:10.3390/ijms21072539 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 17 treatmentInt. J. Mol. for Sci. hypopigmentation2020, 21, 2539 involves the use of topical corticosteroids, laser treatment, or skin2 of 17 surgery, which often have unwanted side effects. Thus, the discovery of natural compounds that can stimulate melanogenesis would be an interesting alternative to popular but carcinogenic means of surgery, which often have unwanted side eﬀects. Thus, the discovery of natural compounds that can promoting pigmentation. stimulate melanogenesis would be an interesting alternative to popular but carcinogenic means of Melanogenesis is a multi-step biosynthetic process catalyzed by tyrosinase (TYR), promoting pigmentation. tyrosinase-related protein-1 (TRP1), and dopachrome tautomerase (DCT). TYR catalyzes the Melanogenesis is a multi-step biosynthetic process catalyzed by tyrosinase (TYR), hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA), and the oxidation of tyrosinase-related protein-1 (TRP1), and dopachrome tautomerase (DCT). TYR catalyzes the L-DOPA to DOPA-quinone. DCT catalyzes the rearrangement of DOPA-chrome to hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA), and the oxidation of L-DOPA to dihydroxyindole-2-carboxylic acid (DHICA), while TRP-1 oxidizes DHICA to a DOPA-quinone. DCT catalyzes the rearrangement of DOPA-chrome to dihydroxyindole-2-carboxylic carboxylatedindole-quinone that polymerizes into melanin[2]. These enzymes are under the acid (DHICA), while TRP-1 oxidizes DHICA to a carboxylatedindole-quinone that polymerizes into transcriptional regulation of the microphthalmia-associated transcription factor (MITF),which in melanin [2]. These enzymes are under the transcriptional regulation of the microphthalmia-associated turn is regulated by a number of signaling pathways that include the cyclic adenosine transcription factor (MITF),which in turn is regulated by a number of signaling pathways that include monophosphate (cAMP) and Wnt signaling pathways[3]. cAMP is one of the key factors involved in the cyclic adenosine monophosphate (cAMP) and Wnt signaling pathways [3]. cAMP is one of the the signal transduction pathways that regulate melanogenesis. The principal intracellular target of key factors involved in the signal transduction pathways that regulate melanogenesis. The principal cAMP in mammalian cells is protein kinase A (PKA), which phosphorylates serine and threonine intracellular target of cAMP in mammalian cells is protein kinase A (PKA), which phosphorylates serine residues on target proteins, such as the cAMP responsive element binding protein (CREB) and and threonine residues on target proteins, such as the cAMP responsive element binding protein (CREB) CREB-binding protein (CBP). Phosphorylated CREB interacts with CBP to activate MITF[4]. and CREB-binding protein (CBP). Phosphorylated CREB interacts with CBP to activate MITF [4]. Argania spinosa oil and argan press-cake have been reported to regulate melanogenesis [5,6]. Argania spinosa oil and argan press-cake have been reported to regulate melanogenesis [5,6]. However, there are no studies that have examined the potential of argan fruit shell in regulating However, there are no studies that have examined the potential of argan fruit shell in regulating melanogenesis. Argan fruit shell contains procyanidin, phloridzin, epicatechin, rutin, and melanogenesis. Argan fruit shell contains procyanidin, phloridzin, epicatechin, rutin, and isoquercitrin, isoquercitrin, among others [7], some of which have been reported to regulate melanogenesis [8]. among others [7], some of which have been reported to regulate melanogenesis [8]. Thus, in the current Thus, in the current study, we determine the regulatory effect of argan fruit shell ethanol extract study, we determine the regulatory eﬀect of argan fruit shell ethanol extract (AFSEE) on melanogenesis (AFSEE) on melanogenesis using B16F10 melanoma cells. using B16F10 melanoma cells.

2. 2.Results Results

2.1.2.1. AFSE AFSEEE Has Has No No Cytotoxic Cytotoxic Effect Effect on onB16F10 B16F10 Cells Cells TheThe cytotoxicity cytotoxicity of ofa drug a drug is isof ofchief chief importance importance when when it is it isused used,, either either as asa medicine a medicine or oras asa a cosmeticcosmetic agent agent [9]. [9 ].The The results results of ofthe the evaluation evaluation of of different different doses doses of of AFSEE AFSEE on on cell cell proliferation proliferation (24 (24 h, h, 48 h, and 72 h h treatment) treatment) showed showed that that the the proliferation proliferation of of B16F10 B16F10 cells cells was was not not affected affected by by AFSEE AFSEE in ina dose a dose-- and and time time-dependent-dependent manner manner (Figure (Figure 1). 1AFSEE). AFSEE at 6 atand 6 and30 μg 30/mLµg wer/mLe wereconsidered considered to be to optimumbe optimum concentrations concentrations for use for in use the in theinvestigation investigation of AFSEE’s of AFSEE’s effecton effecton melanogenesis melanogenesis and and in in elucidatingelucidating the the molecular molecular mechanism mechanism underlying underlying its its effect. effect.

FigureFigure 1. Effect 1. Effect of ofargan argan fruit fruit shell shell ethanol ethanol extract extract (AFSEE) (AFSEE) on onB16F10 B16F10 cell cell prolif proliferation,eration, determined determined usingusing the the 3-(4,5 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide bromide (MTT (MTT)) assay. assay. B16F10 B16F10 cells cells µ werewere treated treated with with AFSEE AFSEE (0– (0–6060 µg/mL)g/mL) for for 24 24h, h,48 48h, h,or or72 72h. h.Each Each bar bar represents represents the the percentage percentage of of viable cells relative to the control, expressed as mean standard deviation (SD) of four independent viable cells relative to the control, expressed as mean ± standard± deviation (SD) of four independent experiments, each one performed in triplicate. Data was subjected to ANOVA (n = 3). All comparisons were made between treatments. Different letters indicate treatment differences at the p 0.05 level. ≤ Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 17

Int. J. Mol.experiments, Sci. 2020, 21 each, 2539 one performed in triplicate. Data was subjected to ANOVA (n=3). All comparisons3 of 17 were made between treatments. Different letters indicate treatment differences at the p ≤ 0.05 level.

2.2.2.2. AFSEE AFSEE Enhances Enhances Melanogenesis Melanogenesis in in B16F10 B16F10 Cells Cells TheThe melanin melanin content content of of B16F10 B16F10 cells cells was was evaluated evaluated using usingα α-MSH-MSH as as a a positive positive control, control, and and the the resultsresults showed showed that that AFSEE AFSEE promoted promoted melanogenesis melanogenesis in in a a dose-dependent dose-dependent manner. manner. Compared Compared to to αα-MSH,-MSH, surprisingly, surprisingly AFSEE-treated, AFSEE-treated cells cells had had a a higher higher melanin melanin content content (185%) (185%) (Figure (Figure2 ).2). Treatment Treatment withwith AFSEE AFSEE for for 48 48 h h signiﬁcantly significantly increased increased the the melanin melanin content content of B16F10of B16F10 cells cells to 349%,to 349% compared, compared to theto the control control without without cytotoxicity, cytotoxicity while, whileα-MSH α-MSH increased increased the melaninthe melan contentin content to 185%. to 185%. Increasing Increasing the treatmentthe treatment time to time 72 h to also 72 showed h also anshowed increase an in increase the melanin in the content melanin (159% content and 219% (159% in α and-MSH- 219% and in AFSEE-treatedα-MSH- and AFSEE cells, respectively),-treated cells, respectively) but not after, only but not 48 h.after The only highest 48 h. concentration The highest concentration of AFSEE that of promotedAFSEE that melanin promoted synthesis melanin without synthesis cytotoxicity without was cytotoxicity 30 µg/mL. was 30 µg/mL.

Figure 2. Effect of argan fruit shell ethanol extract (AFSEE) on the melanin content (bar graph) and Figure 2.Effect of argan fruit shell ethanol extract (AFSEE) on the melanin content (bar graph) and cell viability (line graph) of B16F10 cells cultured in a 100 mm dish at a density of 5 105 cells/dish, cell viability (line graph) of B16F10 cells cultured in a 100 mm dish at a density of× 5×105 cells/dish, and treated without (control) or with α-MSH (200 mM) or AFSEE (6, 10, and 30 µg/mL) for 48 or 72 h. and treated without (control) or with α-MSH (200 mM) or AFSEE (6, 10, and 30 µg/mL) for 48 or 72 h. Each bar represents the percentage of viable cells versus control, expressed as mean SD of four Each bar represents the percentage of viable cells versus control, expressed as mean± ± SD of four independent experiments, each one performed in triplicate. Data were subjected to ANOVA (n = 3). independent experiments, each one performed in triplicate. Data were subjected to ANOVA (n=3). All comparisons were made between treatments. Different letters indicate treatment differences at All comparisons were made between treatments. Different letters indicate treatment differences at p p 0.05 level. ≤≤ 0.05 level. 2.3. Melanogenic Enzymes Expression Level in B16 Melanoma Cells 2.3. Melanogenic Enzymes Expression Level in B16 Melanoma Cells In order to clarify the mechanism underlying the observed AFSEE-induced melanogenesis, the expressionIn order to of clarify melanogenic the mech enzymesanism underlying TYR, TRP1, the DCT, observed and their AFSEE respective-induced intensities melanogenesis, (Figure 3the) wereexpression determined. of melanogenic AFSEE (30 enzymesµg/mL) significantly TYR, TRP1, increased DCT, and the their expression respective level intensities of TYR, TRP1, (Figure and 3)DCT were bydetermined 194%, 175%,. AFSEE and 384%,(30 µg respectively,/mL) significantly compared increased to the the control. expression The significantlevel of TYR, eff TRP1,ect on and enzyme DCT expressionby 194%, 175%, was observed and 384%, after respectively, 48 h of treatment; comparedα-MSH, to the ascontrol. expected, The alsosignificant enhanced effect melanogenic on enzyme enzymeexpression expression, was observed but not after as much 48 h asof AFSEEtreatment (Figure; α-MSH,3). A as time-dependent expected, also eenhancedffect (12 h,melanoge 24 h, 48nic h, andenzyme 72 h) expression on the melanogenesis, but not as promotionmuch as AFSEE of AFSEE (Figure was 3). also A time observed-dependent (Supplementary effect (12 h, Figure 24 h, S1).48 h, and 72 h) on the melanogenesis promotion of AFSEE was also observed (Supplementary Figure 1).

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Figure 3. 3.E ffEffectect of of argan argan fruit fruit shell shell ethanol ethanol extract extract (AFSEE) (AF onSEE) the expression on the expression level of the level melanogenic of the Figure 3. Effect of argan fruit shell ethanol extract (AFSEE) on the expression level of the enzymesmelanogenic tyrosinase enzymes (TYR), tyrosinase tyrosinase-related (TYR), tyrosinase protein-related 1 (TRP1), protein and dopachrome 1 (TRP1), tautomeraseand dopachrome (DCT). melanogenic enzymes tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome (tautomeraseA) The expression (DCT). level(A) The of TYR, expression TRP1, andlevel DCT of TYR, was TRP1, determined and DCT by Westernwas determined blotting. by B16F10 Western cells tautomerase (DCT). (A) The expression level of TYR, 5TRP1, and DCT was determined by Western wereblotting. cultured B16F10 in acells 100 were mm dishcultured at a density in a 100 of mm 3 10dishcells at a/dish, density and of treated 3×105 withoutcells/dish (control), and treated or with blotting. B16F10 cells were cultured in a 100 mm× dish at a density of 3×105 cells/dish, and treated αwithout-MSH (200 (control) mM) or or with AFSEE α-MSH (6 µg (200/mL mM)and 30 or µ AFSEEg/mL) for(6 µg 48/mL h. (andB) The 30 proteinµg/mL) bandfor 48 intensities h. (B) The of without (control) or with α-MSH (200 mM) or AFSEE (6 µg/mL and 30 µg/mL) for 48 h. (B) The TYR,protein TRP1, band and intensities DCT were of TYR, obtained TRP1, using andLi-COR DCT were Software. obtained Data using were Li subjected-COR Software. to ANOVA Data (weren = 3) . protein band intensities of TYR, TRP1, and DCT were obtained using Li-COR Software. Data were Allsubjected comparisons to ANOVA weremade (n=3) between. All comparisons treatments. were Different made letters between indicate treatments. treatment Different differences letters at the subjected to ANOVA (n=3). All comparisons were made between treatments. Different letters pindicate0.05 level.treatment differences at the p ≤ 0.05 level. indicate≤ treatment differences at the p ≤ 0.05 level. 2.4. AFSEE Inhibited MITF Phosphorylation in B16F10 Cells 2.4. AFSEE InhibitedInhibited MITFMITF Phosphorylation Phosphorylation in in B16F10 B16F10 Cells Cells To understand the mechanism underlying the effect of AFSEE on melanogenesis, we determined To understand the the mechanism mechanism underlying underlying the the effect effect of of AFSEE AFSEE on on m elanogenesis, melanogenesis, we we the effect AFSEE on MITF phosphorylation. AFSEE (30 µg/mL) significantly decreased MITF determined thethe effecteffect AFSEEAFSEE on on MITF MITF phosphorylation. phosphorylation. AFSEE AFSEE (30 (30 μg μg/mL)/mL) significantly significantly decreased decreased phosphorylation (Figure4). AFSEE-decreased MITF phosphorylation was observed after 12 h and 24 h MITF phosphorylation (Figure(Figure 4). 4). AFSEE AFSEE-decreased-decreased MITF MITF phosphorylation phosphorylation was was observed observed after after 12 12h h (54% and 84%, respectively (p 0.05)). and 24 h (54% andand 84%,84%, respectively respectively≤ ( p(p ≤ ≤ 0.05) 0.05)).) .

Figure 4. Effect of argan fruit shell ethanol extract (AFSEE) on the expression level of phosphorylated Figure 4. EffectEffect ofof arganargan fruit fruit shell shell ethanol ethanol extract extract (AFSEE) (AFSEE) on on the the expression expression level level of phosphorylatedof phosphorylated microphthalmia-associated transcription factor (pMITF) and total MITF. (A) The expression level of microphthalmia--associatedassociated transcription transcription factor factor (pMITF) (pMITF) and and total total MITF. MITF. (A ()A The) The expression expression level level of of pMITFpMITF and MITF MITF were were determined determined by by Western Western blotting. blotting. B16F10 B16F10 cells cells were were cultured cultured in a in100 a mm 100 dish mm dish pMITF and MITF were5 determined by Western blotting. B16F10 cells were cultured in a 100 mm dish at a density of 3 510 cells/dish and treated without (control) or with α-MSH (200 mM) or AFSEE at a density ofof 3×103×10× 5 cells/dishcells/dish and and treated treated without without (control) (control) or or with with α -αMSH-MSH (200 (200 mM) mM) or orAFSEE AFSEE (6 (6 (6µgµ/mLg/mL and and 30 30µgµ/mL)g/mL) forfor 12 12h and h and 24 24h. ( h.B) (TheB) The protein protein band band intensities intensities of pMITF of pMITF and MITF and MITF were were µg/mL and 30 µg/mL) for 12 h and 24 h. (B) The protein band intensities of pMITF and MITF were obtained using Li Li-COR-COR Software. Software. Data Data were were subjected subjected to toANOVA ANOVA (n=3 (n)=. All3). comparisons All comparisons werewere made made obtained using Li-COR Software. Data were subjected to ANOVA (n=3). All comparisons were made between treatments. Different Different letters letters indicate indicate treatment treatment differences differences at the atthe p ≤ p0.050.05 level. level. between treatments. Different letters indicate treatment differences at the p ≤≤ 0.05 level. 2.5.2.5. InfluenceInfluence of of AFSEE AFSEE on on ERK ERK and and p38 p38 Mitogen Mitogen-Activated-Activated Protein Protein Kinases Kinases in B16F10 in B16F10 Cells Cells 2.5. Influence of AFSEE on ERK and p38 Mitogen-Activated Protein Kinases in B16F10 Cells To confirmconfirm the the effect effect of of AFSEE AFSEE on mitogen on mitogen-activated-activated protein protein kinase kinase (MAPK (MAPK)) signaling, signaling, the To confirm the effect of AFSEE on mitogen-activated protein kinase (MAPK) signaling, the theexpression expression level level of MAPKs of MAPKs p38, p38, extracellular extracellular signal signal-regulated-regulated kinase kinase 1/2 (ERK1/2 1/2 (ERK1) was/2) wasinvestigated investigated expression level of MAPKs p38, extracellular signal-regulated kinase 1/2 (ERK1/2) was investigated usingusing Western blot (Figure (Figure 5).5). Incubation Incubation of of B16F10 B16F10 cells cells with with AFSEE AFSEE (6 or (6 30 or µg 30/mL)µg/mL) for 15 for min 15 minand and using Western blot (Figure 5). Incubation of B16F10 cells with AFSEE (6 or 30 µg/mL) for 15 min and 3030 minmin increasedincreased thethe phosphorylationphosphorylation of p38 to 126% and 113%, respectively. A decrease in ERK1 and ERK230 min phosphorylation increased the phosphorylation by about 35% andof p38 27%, to respectively,126% and 113%, was respectively. observed only A decrease at a higher in ERK1 AFSEE

Int.Int. J. Mol. Sci. Sci. 20202020, 2211, x 2539 FOR PEER REVIEW 55 ofof 1717 and ERK2 phosphorylation by about 35% and 27%, respectively, was observed only at a higher concentrationAFSEE concentration (30 µg/mL), (30 µg and/mL) at 15, and min at of 15 AFSEE min of treatment. AFSEE treatment. Extending Extending the treatment the treatment time (30 min)time further(30 min) decreased further decreased the level ofthe phosphorylated level of phosphorylated ERK1 (~58%) ERK1 and (~58%) ERK2 and (~81%) ERK2 (Figure (~81%)5). (Figure 5).

Figure 5. Effect of argan fruit shell ethanol extract (AFSEE) on the expression level of mitogen-activated Figure 5. Effect of argan fruit shell ethanol extract (AFSEE) on the expression level of protein kinases (MAPKs). (A) The expression level of phosphorylated MAPK p38 (phospho-p38), total mitogen-activated protein kinases (MAPKs). (A) The expression level of phosphorylated MAPK p38 p38 MAPK (p38), phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), and ERK1/2 were(phospho determined-p38), total by Western p38 MAPK blotting. (p38), B16F10 phosphorylated cells were extracellular cultured in 100 signal mm-regulated dish at a densitykinase 1/2 of 3(pERK1/2),105 cells and/dish ERK1/2and treated were determined without (control) by Western or with blotting.α-MSH B16F10 (200 mM) cells were or AFSEE cultured (6 µ ing/ mL100 andmm × 5 30dishµg at/mL) a density for 15 minof 3×10 and cells/dish 30 min. (B and, C) Thetreated protein without band (control) intensities or ofwith p38 α and-MSH ERK1 (200/2 mM) were or obtained AFSEE using(6 µg/mL Li-COR and Software. 30 µg/mL) Data for were 15 min subjected and 30 to min.ANOVA (B, C (n) = The3). All protein comparisons band intensities were made of betweenp38 and treatments.ERK1/2 were Di ff obtainederent letters using indicate Li-COR treatment Software. differences Data atwere the p subjected0.05 level. to ANOVA (n=3). All comparisons were made between treatments. Different letters indicate≤ treatment differences at the p 2.6. AFSEE≤ 0.05 level. Upregulated Tyr, Trp1, and Dct Gene Expression Real-time PCR results showed that AFSEE increased the mRNA expression level of Tyr, Trp1, 2.6. AFSEE Upregulated Tyr, Trp1, and Dct Gene Expression and Dct by 192%, 268%, and 407%, respectively, compared to the control, 24 h after treatment with 30 µgReal/mL- AFSEE.time PCRTyr resultsexpression, showed however, that AFSEE was observed increased to decrease the mRNA when expression the incubation level was of Tyr extended, Trp1, byand another Dct by 24 192%, h, although 268%, and the mRNA407%, respectively, levels of AFSEE-treated compared to cells the remained control, 24 higher h after than treatment the control with at 179%,30 μg/mL 176%, AFSEE. and 174% Tyr for expressionTyr, Trp1,, andhowever,Dct, respectively was observed (Figure to decrease6). when the incubation was extended by another 24 h, although the mRNA levels of AFSEE-treated cells remained higher than the control at 179%, 176%, and 174% for Tyr, Trp1, and Dct, respectively (Figure 6).

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Figure 6. Effect of argan fruit shell ethanol extract (AFSEE) on tyrosinase (Tyr), tyrosinase-related protein Figure 6. Effect of argan fruit shell ethanol extract (AFSEE) on tyrosinase (Tyr), tyrosinase-related 1 (Trp1), dopachrome tautomerase (Dct), and microphthalmia-associated transcription factor (Mitf ) protein 1 (Trp1), dopachrome tautomerase (Dct), and microphthalmia-associated transcription factor mRNA expression. B16F10 cells were cultured in a 100 mm dish at a density of 3 105 cells/dish and (Mitf) mRNA expression. B16F10 cells were cultured in a 100 mm dish at a density× of 3 × 105 cells/dish treated without (control) or with α-MSH (200 mM) or AFSEE (6 µg/mL and 30 µg/mL). The expression leveland treatedof Tyr, Trp1, withoutand Dct (control)mRNA or expression with α-MSH were (200 quantified mM) orusing AFSEE TaqMan (6 µg real-time/mL and PCR 30 ( µgA)/mL) following. The treatmentexpression without level of (control) Tyr, Trp1, or and with Dctα-MSH mRNA or AFSEEexpression for 24were h and quantified (B) following using treatmentTaqMan real without-time orPCR with (Aα) following-MSH or AFSEE treatment for 48without h. (C) (control) The effect or of with AFSEE α-MSH on Mitf or AFSEEexpression for 24 was h and quantified (B) following using TaqMantreatment real-time withoutPCR or with following α-MSH treatment or AFSEE without for 48 h. or (C with) Theα effect-MSH of or AFSEE AFSEE on for Mitf 8 h expression and 24 h. Data was wasquantified subjected using to ANOVATaqMan real (n =-time3). All PCR comparisons following treatment were made without between or treatments.with α-MSH Di orff erentAFSEE letters for 8 indicateh and 24 treatment h. Data was diff erencessubjected at to the ANOVAp 0.05 ( level.n = 3). All comparisons were made between treatments. Different letters indicate treatment differences≤ at the p ≤ 0.05 level. 2.7. AFSEE Upregulated Mitf Expression 2.7. AFSEE Upregulated Mitf Expression AFSEE enhanced Tyr, Trp1 and Dct gene expression, suggesting that this could be due to an upregulationAFSEE enhanced of MITF. Tyr Quantification, Trp1 and Dct ofgene the expressionMitf mRNA, suggesting levels using that real-time this could PCR be showeddue to an a dose-dependentupregulation of increase MITF. Quantificationin Mitf levels after of the 8 h ofMitf AFSEE mRNA treatment, levels compared using real to-time the control, PCR showed but was a observeddose-dependent to decrease increase after in 24 Mitf h of treatmentlevels after (Figure 8 h of6 ).AFSEE treatment, compared to the control, but was observed to decrease after 24 h of treatment (Figure 6). 2.8. Transcriptome Changes Induced by AFSEE in B16F10 Cells 2.8. TranscriptomeAnalysis of the Changes global Induced gene expression by AFSEE in in B16F10 B16F10 Cells cells showed that AFSEE caused significant changesAnalysis in the of B16F10 the global gene gene expression expression profile in (FigureB16F107 ).cells Treatment showed withthat 30AFSEEµg/mL caused AFSEE significant elicited higherchanges gene in the expression B16F10 gene fold changeexpression compared profile to(Figure 6 µg/mL 7). (TableTreatment1). Out with of a30 total µg/m ofL 1700 AFSEE genes elicited that were significantly altered, 1200 genes were up-regulated ( 1.5-fold change) while 500 genes were higher gene expression fold change compared to 6 µg/mL (Table≥ 1). Out of a total of 1700 genes that down-regulated( 1.5-fold change). Hierarchical clustering analysis of the top 100 genes revealed the were significantly≤ −altered, 1200 genes were up-regulated (≥ 1.5-fold change) while 500 genes were concentration-dependentdown-regulated(≤ −1.5-fold eff ectchange). of AFSEE Hierarchical (Figure7). clustering analysis of the top 100 genes revealed the concentrationGene ontology-dependent (GO) annotations effect of revealed AFSEE that(Figure the upregulated7). genes belong to multiple functional biologicalGene pathways ontology that (GO) are annotations significant for revealed the positive that regulationthe upregulated of transcription, genes belong the regulation to multiple of proteinfunctional kinase biological activity pathways signal transduction, that are significant and cell adhesion,for the positive while theregulation downregulated of transcription, genes were the involvedregulation in of pathways protein kinase regulating activity the signal protein transduction, kinase cascade and and cell cellular adhesion di,ff whileerentiation the downregulated (Table1).

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 17 Int. J. Mol. Sci. 2020, 21, 2539 7 of 17 genes were involved in pathways regulating the protein kinase cascade and cellular differentiation (Table 1). GenesGenes associated associated with with pigmentation pigmentation were were upregulated upregulated by by AFSEE. AFSEE. The The cAMP cAMP signaling signaling pathwaypathway-associated-associated genes genes Akap13 Akap13 (A-kinase (A-kinase anchor anchor protein protein 13) and 13) Cxcl10 and (C–X–C Cxcl10 motif (C–X chemokine–C motif chemokineligand 10) expressionligand 10) wereexpression increased were 2.1- increased and 2-fold, 2.1 respectively,- and 2-fold, followingrespectively, treatment following with treatment 30 µg/mL withof AFSEE 30 μg (Table/mL of1 ).AFSEE (Table 1).

FigureFigure 7. 7. HeatHeat map map representing representing the the effect eﬀect of of argan argan fruit fruit shell shell ethanol ethanol extract extract ( (AFSEE)AFSEE) on on global global gene gene expressionexpression in B16F10 cells and thethe hierarchicalhierarchical clusteringclustering ofof genes genes that that were were di differentiallyﬀerentially expressed expressed in inB16F10 B16F10 cells cells treated treated without without (control) (control) or or with with (a) (a) AFSEE AFSEE (6 (6µ gµg/mL)/mL) or or (b) (b) AFSEE AFSEE (30 (30µ gµg/mL)/mL) for for 4 4 h. The 25 downregulated genes subjected to hierarchical clustering had a fold-change value of –1.5 h. The 25 downregulated genes subjected to hierarchical clustering had a fold-change value of ≤ –1.5 (vs.control), while for the upregulated genes, genes with 1.5-fold change values were chosen. The (vs.control), while for the upregulated genes, genes with ≥ 1.5-fold change values were chosen. The mmapap is splitsplit into into two two (A (,AB )and for clarity.B) for The clarity. Euclidian The Euclidian distance method distance was method used for was the used comparison, for the comparisonand the resulting, and red the and resulting green colors red and represent green gene colors up- represent and down-regulation, gene up- and respectively. down-regulation, respectively.Table 1. List of up- and down-regulated genes in B16F10 melanoma cells treated with AFSEE (6 µg/mL and 30 µg/mL), as determined by DNA microarray. Table 1. List of up- and down-regulated genes in B16F10 melanoma cells treated with AFSEE (6 µgGene/mL Symbol and 30 µg/mL) Gene, as Title determined by DNA Biological microarray. Process AFSEE 6 µg/mL AFSEE 30 µg/mL PRP4 pre-mRNA mRNA processing, protein GenePrpf4b processing factor 2.5AFSEE 6 5.8AFSEE 30 Gene Title phosphorylation, RNABiological splicing. Process Symbol 4 homolog B (yeast) µg/mL µg/mL Ankyrin repeat Regulation of canonical Wnt Ankrd10 PRP4 pre-mRNA processing mRNA processing, protein phosphorylation,1.9 4.0 Prpf4b domain 10 signaling pathway. 2.5 5.8 factor 4 homolog B (yeast)Cell cycle, cell division, chromosomeRNA splicing. Centromere segregation,Regulation establishment of ofcanonical protein Wnt signaling Ankrd10Cenpe Ankyrin repeat domain 10 1.71.9 3.6 4.0 protein E localization, positive regulationpathway of protein. kinaseCell cycle, activity. cell division, chromosome Cenpe Centromere protein E segregation, establishment of protein 1.7 3.6 localization, positive regulation of protein

Int. J. Mol. Sci. 2020, 21, 2539 8 of 17

Table 1. Cont.

Gene Symbol Gene Title Biological Process AFSEE 6 µg/mL AFSEE 30 µg/mL Alpha Cellular response to DNA damage thalassemia/mental stimulus, signal transduction by p53 DNA Atrx retardation 1.9 3.4 repair, DNA replication-independent syndrome X-linked nucleosome assembly. homolog (human) Nuclear export, regulation of cardiac muscle hypertrophy, regulation of A kinase (PRKA) Akap13 glucocorticoid mediated signaling 2.1 2.1 anchorprotein 13 pathway, regulation of protein kinase activity. Cellular response to interferon-alpha, Translocated Tpr MAPK import into nucleus, response to 1.2 2.1 promoter region epidermal growth factor. Positive regulation of cAMP metabolic Chemokine process, positive regulation of Cxcl10 (C–X–C motif) 1.2 2.0 cAMP-mediated signaling, positive ligand 10 regulation of cell proliferation. Chromatin silencing, DNA methylation, Helicase, Hells mitotic nuclear division, multicellular 1.1 2.0 lymphoid specific organismal development. Angiogenesis, keratinocyte differentiation, lactation-positive regulation of keratinocyte differentiation, ERK1 and ERK2 cascade, cellular Mediator complex Med1 response to epidermal growth factor 1.3 1.5 subunit 1 stimulus, positive regulation of receptor activity, protein ubiquitination, positive regulation of protein import into nucleus, translocation. EDAR (ectodysplasin-A Edaradd Cell differentiation 1.1 1.5 receptor)-associated death domain Regulation of transcription, Jumonji domain Jmjd6 DNA-template, cell surface receptor 1.1 1.5 containing 6 signaling pathway. Embryonic Positive regulation of histone H3-K27 Eed ectoderm methylation, covalent 1.2 1.5 development chromatin modification. Guanine nucleotide Regulation of melanocyte differentiation, binding protein, Gnaq regulation of protein kinase activity, 1.1 1.5 alpha q signal transduction. polypeptide Golgi vesicle transport, protein transport, Arcn1 Archain 1 1.0 1.5 vesicle-mediated transport. Solute carrier Amino acid transmembrane transport, Slc7a11 1.3 1.8 family 7 member 11 response to toxic substance. − − G protein-coupled Regulation of melanosome transport, Gpr143 1.1 2.1 receptor 143 regulation of melanosome organization. − − Oculo-cutaneous Melanocyte differentiation, cell Oca2 1.3 2.6 albinism II proliferation, transmembrane transport. − −

2.9. Validation of DNA Microarray Results by Real-Time PCR DNA microarray analysis showed that AFSEE modulated the expression of the genes associated with the transcription and signaling regulation of Mitf and Mitf -related signaling. Results of the validation of the DNA microarray using RT-PCR made it clear that the expression of Mitf transcription regulators Crebbp, Pax3, Lef1, and Sox10 was indeed changed by AFSEE in a time-dependent manner (Figure8). Crebbp was increased 2.2-fold, while Pax3 expression was increased 1.5-fold. Treatment with AFSEE for 4 h did not aﬀect the expression of Lef1 (1-fold), but it downregulated Sox10 expression by 0.8-fold. Int. J. Mol. Sci. 2020, 21, 2539 9 of 17 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 17

Figure 8. Effect of argan fruit shell ethanol extract (AFSEE) on mRNA expression of CREB binding Figureprotein 8. ( CrebbpEffect ),of pairedargan fruit box geneshell ethanol 3 (Pax3 ),extract lymphoid (AFSEE) enhancer on mRNA binding expression factor 1of ( Lef1CREB), andbinding SRY proteinbox-containing (Crebbp), gene paired 10 box(Sox gene 10). 3 B16F10 (Pax3), cells lymphoid were cultured enhancer in binding a 100 mm factor dish 1 (Lef1 at a), density and SRY of 5 box3 -10containing5 cells/dish geneand 10 treated (Sox 10 without). B16F10 (control) cells were or cultured with α-MSH in a 100 (200 mm mM) dish or AFSEEat a density (6 µ gof/mL 3 × and10 × cells/dish30 µg/mL) and for 1treated h and 4without h (A) The (control) effect of or AFSEE with α on-MSHCrebbp (200,(B mM)) Pax3 or,( AFSEEC) Lef1, (6 and µg (/mLD) Sox10 and 30expression µg/mL) forwas 1 quantifiedh and 4 h using(A) The TaqMan effect real-timeof AFSEE PCR. on Crebbp Data were, (B) Pax3 subjected, (C) Lef1 to ANOVA, and (D ()n Sox10= 4). Allexpression comparisons was quantifiedwere made using between TaqMan treatments. real-time Diff PCR.erent lettersData were indicate subjected treatment to ANOVA differences (n = at 4 the). Allp comparisons0.05 level. were made between treatments. Different letters indicate treatment differences at the p ≤≤ 0.05 level. 2.10. Phytochemical Characterization of AFSEE 2.10. Phytochemical Characterization of AFSEE The qualitative phytochemical screening of AFSEE revealed the presence of polyphenols (flavonoids,The qualitative tannins and phytochemical coumarins), the screening presence of of AFSEE saponins, revealed and the the absence presence of alkaloids of polyphenols (Table2). (flavonoids,The spectrophotometric tannins and quantificationcoumarins), the of presence the two mainof saponins, families and of secondarythe absence metabolites of alkaloids detected (Table 2).in AFSEE The sp showedectrophotometric that the AFSEE quantification polyphenol of and the saponintwo main content families was of 22.1 secondary0.87 mg metabolites gallic acid ± detectedequivalent in/ gAFSEE dry weight showed and that 16.23 the AFSEE0.13 mg polyphenol oleanolic acidand saponin equivalent content/g dry was weight, 22.1 respectively.± 0.87 mg gallic ± acid equivalent/g dry weight and 16.23 ± 0.13 mg oleanolic acid equivalent/g dry weight, respectivelyTable 2.. Qualitative and quantitative phytochemical characterization of AFSEE, determined using colorimetric and spectrophotometric methods. Table 2. Qualitative and quantitative phytochemical characterization of AFSEE, determined using Parameters Visual Detection colorimetric and spectrophotometric methods. Alkaloids - Qualitative characterization FlavonoidsParameters Visual+++ Detection SaponinsAlkaloids ++- Qualitative characterization CoumarinsFlavonoids ++++ ParametersSaponins Content in++ mg /g DW Coumarins + Total polyphenols (mg gallic acid eq/g DW) 22.11 0.87 Quantitative characterization Parameters Content in± mg/g DW Total flavonoids (mg catechin eq/g DW) 9.9 0.2 ± CondensedTotal polyphenols tannins (mg (mg gallic catechin acid eqeq/g/g DW) 1.6022.11 ±0.08 0.87 ± Quantitative characterization SaponinsTotal flavonoids (mg oleanolic (mg catechin acid eq eq/g/g DW)DW) 16.239.9 ± 0.20.13 Condensed tannins (mg catechin eq/g DW) 1.60 ± 0.08 (+++) strong presence; (++) moderate presence; (+): weak presence; (-): absence; (eq): equivalent; DW: dry weight. Saponins (mg oleanolic acid eq/g DW) 16.23 ± 0.13 (+++) strong presence; (++) moderate presence; (+): weak presence; (-): absence; (eq): equivalent; DW: dry weight The chemical characterization of AFSEE polyphenols was performed by HPLC, by comparison of The chemical characterization of AFSEE polyphenols was performed by HPLC, by comparison the retention times and the UV spectrum of AFSEE peaks with those of reference standards. The results of the retention times and the UV spectrum of AFSEE peaks with those of reference standards. The

Int. J. Mol. Sci. 2020, 21, 2539 10 of 17 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 17 demonstrateresults demonstrate that quercetin that isquercetin the major is phenolic the major compound phenolic in compound AFSEE (Figure in 9 AFSEE, Supplementary (Figure 9, FigureSupplementary S2). Figure S2).

Figure 9. High-performance liquid chromatography (HPLC) fingerprinting of AFSEE: chromatograms Figure 9. High-performance liquid chromatography (HPLC) fingerprinting of AFSEE: of AFSEE and quercetin, AFSEE’s major polyphenol compound, were acquired at 280 nm. HPLC chromatograms of AFSEE and quercetin, AFSEE’s major polyphenol compound, were acquired at chromatograms of standards used in the analysis are included as supplementary figures. 280 nm. HPLC chromatograms of standards used in the analysis are included as supplementary 3. Discussionfigures. Melanin is a photoprotective skin pigment that plays a critical role in protecting human skin from 3. Discussion the harmful effects of UV radiation, and pathologies characterized by hypo- or hyperpigmentation are common.Melanin Melanin is a photoprotective biosynthesis in the skin skin pigment is initiated that uponplays exposure a critical torole UV in radiation, protecting promoting human skin the expressionfrom the of harmful the proopiomelanocortin effects of UV (POMC) radiation, protein and in the pathologies keratinocytes characterized [10], which causes by α hypo-MSH- toor cleavehyperpigmentation from its precursor are POMCcommon. protein Melanin [11] andbiosynthesis bind to the in receptorthe skin MC1R, is initiated activating upon adenylylexposure cyclase to UV thatradiation, will generate promoting cAMP the [12 ]. expression The accumulation of the of proopiomelanocortin cAMP activates PKA, (POMC) causing phosphorylation protein in the ofkeratinocytes the cAMP responsive [10], which binding causes α element-MSH to (CREB), cleave from which its inprecursor turn promotes POMC theprotein transcription [11] and bind of the to microphthalmiathe receptor MC1R, transcription activating factor adenylyl (MITF). cyclase MITF that acts will as generate the master cAMP regulator [12]. The of the accumulation expression ofof multiplecAMP activates enzymes PKA, that catalyzecausing melaninphosphorylation biosynthesis, of the including cAMP responsive tyrosinase (TYR),binding tyrosinase-related element (CREB), proteinwhich in 1 (TRP1),turn promotes and dopachrome the transcription tautomerase of the (DCT) microphthalmia [13]. Previous transcription studies have factor indicated (MITF). that MITF the activationacts as the of MAP maste kinaser regulator family of members the expression ERK, c-Jun of N-terminal multiple enzymes kinase (JNK), that and catalyze p38 plays melanin an importantbiosynthesis role, including in MITF regulation tyrosinase [ 14 (TYR),–16]. MITFtyrosinase contains-related a basic protein helix–loop–helix–leucine 1 (TRP1), and dopachrome zipper domaintautomerase in its structure(DCT) [13]. [13 ],Previous and specifically studies binds have toindicated the M-box that and the E-box activation motifs inof the MAP promoter kinase regions family ofmembersTyr, Trp1 ERK,, and Dctc-Junto regulateN-terminal their kinase expression (JNK), [17 and]. Within p38 plays this context,an important natural role compounds in MITF regulation that affect the[14– expression16]. MITF or contains activity a of basic these helix enzymes–loop or–helix MITF–leucine may be zipper considered domain as potential in its structure therapeutics [13], and for thespecifically regulation binds of pigmentation to the M-box or and treatment E-box ofmotifs pigmentary in the p disordersromoter regions [18]. In thisof Tyr study,, Trp1 we, and evaluated Dct to theregulate effect their of argan expression fruit shell [17]. ethanol Within extractthis context, (AFSEE) natural on melanogenesis compounds that in affect B16F10 the cells. expression In order or toactivity avoid of interference these enzymes in melanogenesis or MITF may causedbe considered by the reductionas potential of therapeutics cell numbers, for due the to regulation the testing of extract,pigmentation only non-toxic or treatment concentrations of pigmentary of AFSEE disorders (6 µg/ mL[18]. to In 30 thisµg/ mL)study, were we used evaluated in the experiments.the effect of argan fruit shell ethanol extract (AFSEE) on melanogenesis in B16F10 cells. In order to avoid

Int. J. Mol. Sci. 2020, 21, 2539 11 of 17

AFSEE significantly increased the melanin content in B16F10 cells in a dose- and time-dependent manner (Figure2), and this e ffect may be attributed to the flavonoid content of argan fruit shells; since quercetin which was identified as the major polyphenol compound in AFSEE (Figure9), it has been reported to be an enhancer of melanin in human melanoma cells [19]. Argan fruit shell also contains bidesmosidic saponins with oleanane-type aglycone, namely misaponin A, arganin M, and arganin N[20,21]. Oleanane-type saponins have been reported as inhibitors of melanogenesis [21]. It could therefore be assumed that the melanogenesis promotion effect of AFSEE is a result of the synergistic effects of these compounds. Treatment with AFSEE for 48 h increased the expression level of TYR, TRP1, and DCT in a dose-dependent manner, demonstrating that the AFSEE-induced melanogenesis was primarily due to the elevated TYR, TRP-1, and DCT expression (Figure3). It appears that 48 h of treatment is the most effective treatment time, since incubation with AFSEE at 12 h, 24 h, and 72 h was not as effective as at 48 h (Supplementary Figure S1). This is what was observed as the effect of AFSEE on the melanogenic enzymes, with regards to treatment time. The increased melanogenic enzyme expression was caused by the inhibition of MITF phosphorylation (Figure4). MITF phosphorylation leads to ubiquitination and proteasome-mediated degradation of MITF [22], suggesting that the inhibition of MITF phosphorylation plays an important role in AFSEE-induced melanogenesis promotion, and the time when the melanogenic enzymes expression is affected will depend on when the MITF is activated. Other natural products or plant extracts were observed to have different effects on MITF, depending on treatment time. In one of our previous reports, transcription factor Mitf RNA expression may be observed after 4 h [23]. In another, the significant effect of hirsein A from T. hirsuta on MITF (protein expression) was observed after 24 h and 48 h [5]. The MAPK family includes extracellular responsive kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK [24]. p38 MAPK is a major intracellular signaling molecule critical to pigmentation, as its activation is associated with increased melanin synthesis, while activation of ERK 1/2 and JNK is associated with decreased melanogenesis [14]; p38 MAPK signaling has been reported to activate MITF [25], while ERK activation leads to MITF degradation [26]. Argan oil has been demonstrated to promote MITF degradation, as we have previously reported [5], and which the work of Caporarello et al. [27] on uveal melanocytes has corroborated. The MITF phosphorylation was inhibited by AFSEE (Figure4). Moreover, AFSEE significantly decreased ERK1 and ERK2 phosphorylation (Figure5). MITF appears to be regulated through the MAPK pathway, but also by the cAMP pathway [3]. The cAMP pathway plays a key role in the regulation of melanogenesis at the transcription and translation levels [28]. The cAMP pathway activates CREB, which binds to the MITF promoter; at the same time, cAMP activates MAPK, which will also activate MITF. At the transcriptional level, AFSEE enhanced the expression of Mitf, and as a result, the melanogenic enzymes (Figure6). DNA microarray analysis of AFSEE-treated B16F10 cells showed significant changes in the expression of genes associated with the cAMP signaling pathway and in the modulation of MITF transcriptional regulators Pax3 and Crebbp (Figure8). Crebbp encodes for the protein CREBBP and binds to phosphorylated CREB to enhance its activity [4]. Activation of p38 has been shown to contribute positively to melanogenesis by activating CREB. In addition, the activation of CREB-binding protein is required for cAMP responsiveness of the MITF promoter [29]. Interestingly, based on the DNA microarray results, among all the genes modulated by AFSEE (Table1), cAMP signaling pathway genes Akap13 and Cxcl10 were upregulated, and could have contributed to the overall effect of AFSEE. Akap13 plays a critical role in coordinating cAMP signaling in adrenocortical cells [30], while Cxcl10 increases the level of activated CREB [31]. Therefore, the AFSEE-enhanced Crebbp expression activated MITF and led to the promotion of melanin synthesis in B16F10 cells. Int. J. Mol. Sci. 2020, 21, 2539 12 of 17

4. Materials and Methods

4.1. Reagents For cell culture, RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), fetal bovine serum (FBS;Bio West, Miami, FL, USA), alpha-melanocyte-stimulating hormone (α-MSH; Sigma Aldrich, St. Louis, MO, USA), phosphate-buffered saline (PBS; SigmaChemical Co., Deisenhofen, Germany), and trypsin/EDTA (0.25% trypsin/0.02% EDTA in PBS; Sigma Chemical Co., Deisenhofen, Germany) were used. The MTT assay used 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT; Dojindo, Japan), sodium diodecyl sulfate (SDS; WAKO, Osaka, Japan), triton-x 100 (Sigma Aldrich, St. Louis, MO, USA), trichloroacetic acid (TCA; Wako, Japan), and 8N sodium hydroxide solution (NaOH; Wako, Japan). The Western blot analysis used resolving gel buffer 0.5 and 1.5M Tris-HCL (pH=8.8, BIO-RAD, Hercules, CA, USA), acrylamide/Bis mixed solution (BIO-RAD, Hercules, CA, USA), TEMED (GE Healthcare, Buckinghamshire, UK), mercapto-ethanol (Sigma Aldrich, St. Louis, MO, USA), ammonium persulfate (GE Healthcare, Uppsala, Sweden), tween 20 (Sigma, Germany), and Odyssey Blocking buffer (Li-Cor Biosciences, Lincoln, Nebraska, USA). The real-time PCR included an ISOGEN kit (Nippon Gene, Tokyo, Japan), chloroform (Wako, Japan), 2-propanol (Molecular Biology, Wako, Japan), ethanol 99.5% (Wako, Japan), tris-EDTA buffer solution (Fluka, Japan), and TaqMan Gene Expression Master Mix (Applied Biosystems, United States). Lastly, for HPLC, acetonitrile, quercetin, rutin, catechin, epicatechin, caffeic acid, 3–4 dihydroxybenzoïc acid, chlorogenic acid, p-coumaric acid, gallic acid, and rosmarinic acid standards, were all from Sigma (Germany).

4.2. Preparation of Argan Fruit Shell Extract Powdered argan fruit shells (10 g) were extracted using 100 mL ethanol (70%) in the dark at room temperature for two weeks. The extraction yield was 3.5%. Argan fruit shell ethanol extract (AFSEE) was ﬁlter sterilized using 0.22 µm ﬁlter (Millex GV, Millipore, Bedford, USA) and stored at 80 C − ◦ until use. Argan (Argania spinosa) drupe-like fruits, containing the shell, were collected in June 2016 from Mirght (Sidi Ifni region), in the southwestern part of Morocco, and were authenticated by Prof. Ahmed Ouhammou from Cadi Ayyad University, Faculty of Sciences Semlalia, Department of Biology, Marrakech, Morocco. A voucher specimen of plant material (MARK 10888) was deposited in the Herbarium of the same institution

4.3. AFSEE Chemical Characterization AFSEE was screened for the presence of alkaloids, flavonoids, coumarins, tannins, and saponins. The qualitative determination of these phytochemicals was conducted using previously reported methods [32,33]. The AFSEE total phenol content, flavonoid content, condensed tannins, and saponin content were determined according to methods previously reported [34–37]. They were expressed as mg of gallic acid equivalents per g of dry weight, mg of catechin equivalent per g of dry weight, mg of catechin equivalent per g of dry weight, and mg of oleanolic acid equivalent per g of dry weight, respectively. The chemical characterization of AFSEE was performed using high-performance liquid chromatography (HPLC; Knauer; Germany), equipped with a smart line pump (K-1001) and a photometric diode array (PDA) detector 28000 (200-700 UV-Vis) operating at 280 nm. The device was equipped with a Eurospher II 100-5 C 18 (250 4.6 mm) column, and the temperature was maintained × at 25 ◦C. A mixture of (A) acidified water (pH 2.6) (A) and acetonitrile (B) was used as a mobile phase, for a total running time of 60 min.The flow rate was 1 mL/min with gradient program (0–54 min, 5–100% B). The sample volume injected was 10 µL. The tentative identification of phenolic compounds Int. J. Mol. Sci. 2020, 21, 2539 13 of 17 in AFSEE was performed by a comparison of retention times and UV-visible spectra of unknown peaks with the standards (quercetin, rutin, catechin, epicatechin, caffeic acid, 3-4 dihydroxibenzoïc acid, chlorogenic acid, p-coumaric acid, gallic acid, and rosmarinic acid).

4.4. Cell Culture Murine melanoma cell line B16F10 (RCB2630) was purchased from Riken Cell Bank in Tsukuba, Japan. Melanoma cells were maintained as a monolayer culture in RPMI 1640 medium, supplemented with 10% FBS and incubated at 37 ◦C in a humidiﬁed atmosphere of 5% CO2.

4.5. Cell Proliferation The eﬀect on B16F10 cells proliferation was evaluated using the MTT assay, as we have previously reported [38]. The B16F10 cells were treated with various concentrations of AFSEE (0, 0.03, 0.06, 0.3, 0.6, 3, 6, 30, 60 µg/mL) for 24 h, 48 h, or 72 h.

4.6. Melanin Quantiﬁcation The melanin content of the B16F10 cells treated with or without AFSEE was quantiﬁed spectrophotometrically, as previously reported [6]. B16F10 cells (5 104 cells/mL) were treated × without (control), or with α-MSH (200 mM) (positive control) or AFSEE (6, 10 and 30 µg/mL) for 48 h and 72 h. Cells lysates were centrifuged at 3000 rpm for 5 min after 2 h (room temperature) heating in NaOH. All samples were assayed in triplicates.

4.7. Western Blot The total protein (10 µg) extracted from the B16F10 cells treated without (control) or with α- MSH (200 mM) or AFSEE (6 or 30 µg/mL) at diﬀerent time points was separated, following the manufacturer’s instructions for BIO RAD Mini-Protean II for 10% gel preparation using 30% acrylamide gel and blotted onto PVDF membrane (Millipore, Germany). The blots were probed with primary antibodies for MITF, phosphorylated MITF, TYR, TRP1, DCT, MAPKs (phosphorylated-p38, p38, phosphorylated ERK1/2, ERK1/2), and GAPDH. The proteins were visualized using a LiCor Odyssey Infrared Imaging System (LI-COR Biosciences, United States) after reaction with goat anti-mouse IRDye 680LT or goat anti-rabbit IRDye 800CW (LI-COR). Antibodies were diluted as recommended by the manufacturer, and in case there were none, the antibodies were diluted 1/500 v/v for primary antibodies and 1/1000 v/v for the secondary antibodies.

4.8. Real-Time PCR RNA from samples was extracted using ISOGEN (Wako, Japan) and used as templates for the reverse transcription polymerase chain reaction (RT-PCR), using SuperScript III reverse transcriptase kit (Invitrogen, Carlsbad, CA, United States), following the manufacturer’s instructions. Real–time PCR analysis (RT-PCR) was performed in 7500 Fast Real-time PCR (Applied Biosystems, United States). Real-time Primers for Mitf (Mm0043495-m1), Tyr (Mm00495817-m1), Trp1 (Mm00453201-m1), Dct (Mm01225584-m1), Crebbp (Mm01342452-m1), Pax3 (Mm00435491-m1), Lef1 (Mm00550265-m1), and Sox10 (Mm00569909-m1) were used (Applied Biosystems, Foster City, CA, United States). Gapdh (Mm99999915-g1) was used as an endogenous control. The thermal cycling protocol was as follows: 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min.

4.9. DNA Microarray Analysis The global gene expression changes in B16F10 cells were analyzed following the protocol for the Affymetrix MG-430 PM Array Strip (Affymetrix, Santa Clara, CA, USA). Partek Express Software (Affymetrix) was used to analyze the data, by running comparisons of gene expression in treated and control cells based on mathematical algorithms. The generated data (significant fold change in gene Int. J. Mol. Sci. 2020, 21, 2539 14 of 17 expression) was then analyzed using the Pathway Studio Explore 1.1 software (Affymetrix). The DNA microarray data comply with MIAME guidelines, and have been deposited in the ArrayExpress database at EMBI-EBI (www.ebi.ac.uk/arrayexpress), under the reference number E-MTAB-8836.

4.10. Statistical Analysis The results are expressed as mean standard deviation (SD) of at least three independent ± experiments. The data were statistically analyzed using one-way analysis of variance, with a Fisher’s least significant difference (LSD) post-hoc test, using CoStat 6.451 software, and two-way ANOVA, followed by a Tukey post-hoc test, using IBM SPSS statistics software. From these, p-values less than 0.05 were considered to be statistically significant.

5. Conclusions The differentiation (or melanin biosynthesis) and associated signaling mechanism of melanoma cells (i.e., B16 cell line) and melanocytes (pigment cells) have been discussed by Bennett [39]. Both melanoma cells and melanocytes require the activation of tyrosinase (TYR) to produce melanin, and α-MSH and other inducers may be used to activate TYR. In this study, AFSEE has been demonstrated to promote melanogenesis in B16F10 cells by its effect on the melanogenic enzymes, through the cAMP–MITF signaling pathway. A follow-up study using human epidermal melanocytes and animal models may be necessary to demonstrate its effect on human skin cells, and as a preliminary step before evaluation as an active component of a cosmetic or therapeutics designed to promote melanogenesis.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/7/2539/ s1. Author Contributions: Conceptualization, H.I., C.G., and A.H.; methodology, H.I., M.O.V., and C.G.; investigation, R.M. and M.O.V.; writing—original draft preparation, R.M.; writing—review and editing, M.O.V. and C.G.; supervision, C.G. and H.I.; funding acquisition, H.I., A.H., and C.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Japan International Cooperation Agency (JICA)-Japan Science and Technology Agency (JST)’s Science and Techonology Research Partnership for Sustainable Development (SATREPS) project entitled, “Valorization of Bioresources Based on Scientific Evidence in Semi- and Arid Land for Creation of New Industry”, and by the Ministry of Higher Education, Scientific Research and Executive Training (MHESRET) of the Kingdom of Morocco. Acknowledgments: We thank the Centre d’Analyse et de Caractérisation (CAC) of Cadi Ayyad University of Marrakech for the HPLC analysis. R.M received a grant from the Japan Student Services Organization (JASSO), as well as and a grant from MHESRET of the Kingdom of Morocco. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

AFSEE Argan fruit shell ethanol extract Akap13 A-kinase anchor protein 13 cAMP Cyclic adenosine monophosphate CREB cAMP-responsive element binding protein Crebbp CREB binding protein Cxcl10 C–X–C motif chemokine ligand 10 DCT Dopachrome tautomerase ERK Extracellular signal-regulated kinase GAPDH Glyceraldehyde 3-phosphate dehydrogenase HPLC High-performance liquid chromatography Lef1 Lymphoid enhancer binding factor 1 MAPK Mitogen-activated protein kinase MITF Microphthalmia-associated transcription factor MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide Int. J. Mol. Sci. 2020, 21, 2539 15 of 17

Pax3 Paired box gene 3 PBS Phosphate-buﬀered saline PKA Protein kinase A RT-PCR Real-time polymerase chain reaction Sox 10 SRY-box containing gene 10 TRP1 Tyrosinase-related protein 1 TYR Tyrosinase α-MSH alpha-melanocyte-stimulating hormone α-MSH alpha-melanocyte-stimulating hormone

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