Research Article Effect of Purified Mushroom Tyrosinase on ...

Research ArticleEffect of Purified Mushroom Tyrosinase on Melanin Contentand Melanogenic Protein Expression

Kamal Uddin Zaidi1 Sharique A Ali2 and Ayesha S Ali2

1Biotechnology Pharmacology Laboratory Centre for Scientific Research amp Development Peoplersquos University Bhopal 462037 India2Department of Zoology amp Biotechnology Saifia College of Science Bhopal 462001 India

Correspondence should be addressed to Sharique A Ali drshariquealiyahoocoin

Received 8 April 2016 Revised 2 July 2016 Accepted 17 July 2016

Academic Editor Maxim Golovkin

Copyright copy 2016 Kamal Uddin Zaidi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In mammalian melanocytes melanosome is a highly specialized organelle where melanin is synthesized Melanin synthesis iscontrolled by tyrosinase the vital enzyme in melanogenic pathway The present investigation is based on an effect of purifiedmushroom tyrosinase ofAgaricus bisporus on B16F10melanocytes for themelanin production via blocking pigment cell machineryUsing B16F10 melanocytes showed that the stimulation of melanogenesis by purified tyrosinase is due to increased tyrosinaseabsorption Cellular tyrosinase activity and melanin content in B16F10 melanocytes were increased by purified tyrosinase in adose-dependent manner Western blot analysis revealed that cellular tyrosinase levels were enhanced after treatment with purifiedtyrosinase for 48 hours Furthermore tyrosinase induced phosphorylation of cyclic adenosine monophosphate (cAMP) responseelement-binding protein (CREB) in a dose-dependent manner The purified tyrosinase-mediated increase of tyrosinase activitywas significantly attenuated by H89 LY294002 Ro-32-0432 and PD98059 cAMP-dependent protein kinase inhibitors The resultsindicate that purified tyrosinase can be used as contestant for the treatment of vitiligous skin conditions

1 Introduction

Cutaneous pigmentation is a human phenotype determiningbody complexion andproviding protection against ultravioletray damage [1 2] Melanocyte the specialized skin cellis involved in regulating skin color by producing melaninpigment Loss of melanin in the epidermis can increasethe risk of acquiring skin cancers and hypopigmentationlike vitiligo [3 4] Defect in melanocytes or their functionsresults in pigmentary disorder leading to enhanced reducedor complete loss of skin pigmentation Individuals suffer-ing from any of hypopigmentarydepigmentary disordersparticularly disfiguring vitiligo are susceptible to the envi-ronmental assaults and cosmetic psychological stress Thusupregulating melanocytes activity in terms of growth andpigment synthesis in such condition is important In theera of modern medicine which is undergoing rapid changewhere genomic information is being accumulated the dataon vitiligo has not been appropriately archived or systemizedfor the disease analysis [5 6] In melanocytes and melanoma

cells melanogenesis is controlled via a cascade of enzymaticreactions synchronized at the intensity of tyrosinase Theenzyme synthesizes dopaquinone from tyrosine which is therate limiting step of melanogenesis [7 8]

Mushroom tyrosinase has been extensively studied inEastern Asia like China Korea and Japan While tropicalcountries such as India especially in Central India (Mad-hya Pradesh and Chhattisgarh) are less explored about53 edible mushrooms belonging to four orders 11 familiesand 18 genera of basidiomycetes are reported from MPAs stated it became evident that extracts from Agaricusbisporus have been used traditionally as well as medicinallyin various ailments such as antitumor immunomodulatoryhypocholesterolaemic anti-inflammatory antimicrobial andantiviral activities [9] Despite this to the best of our knowl-edge there are no studies indicating extract of mushroomas melanogenic agent except for the work of Zehtab etal [10] who reported that mushroom tyrosinase preventedexperimental autoimmune vitiligo Suppression of clinicaland histological disease was observed when animal received

Hindawi Publishing CorporationBiotechnology Research InternationalVolume 2016 Article ID 9706214 8 pageshttpdxdoiorg10115520169706214

2 Biotechnology Research International

mushroom tyrosinase but exact mechanism is still unknownso an attempt is made to explore the detailed mechanism ofmushroom tyrosinase on B16F10 melanocytes Furthermorethere are no studies at all on the effect of mushroomtyrosinase on cultured melanocytes to see the efficacy of themushroom tyrosinase as melanogenic agent

Thus the current investigation would provide a bench-mark in exploring the diversity of mushroom and the effectofmushroom tyrosinase on culturedmelanocytes for findingout melanogenic agents The present study was undertakenkeeping in view the above lacunae in literature and for the firsttime B16F10 melanocytes model has been studied in detailto block the signaling pathway and mechanism of inducedmelanogenesis by lyophilized purified tyrosinase of Agaricusbisporus The findings of the study have provided vitalinformation on these aspects and differentiating results havebeen obtained It may be mentioned here that this is the firstreport of its kind where purified mushroom tyrosinase hasbeen found to cause skin darkening viamelanin displacementwithin the B16F10 melanocytes The present study on B16F10melanocytes is the first study on the effect of mushroomtyrosinase especially from A bisporus that can serve as amelanogenic potent to vitiligo

2 Material and Methods

For the present study the compound mushroom tyrosi-nase (lyophilized powder ge 1000 unitsmg solid) and pro-tein kinase inhibitors protein kinase A (PKA) inhibitor(H89) protein kinase B (PKB) inhibitor (LY294002) proteinkinase C (PKC) inhibitor (Ro-32-0432) and MEK1 inhibitor(PD98059) were purchased from Sigma-Aldrich St LouisMissouri United States Goat anti-murine tyrosinase IgGantibody and Alexa Fluor 594 donkey anti-goat IgG (H+L)(2mgmL) were purchased from Life Technologies NorthAmerica United States Dulbeccorsquos Modified Eagle Medium(AT006A-5L) fetal bovine serum (RM10432-100mL) Anti-biotic Antimycotic Solution 100x (A002-20mL) Trypsin-EDTA Solution 1x (TCL042-5 times 100mL)MTT [3-(45-dim-ethylthiazol-2-yl)]-25-diphenyltetrazoliumbromide (TC191-500MG) 410158406-diamidino-2-phenylindole (DAPI) (TC229-5MG) phosphate buffered saline (RM7385-1PK) and Trypanblue Certified (RM263-5G) were purchased from HiMediaLaboratories Pvt Ltd Mumbai

21 Preparation of Tyrosinase In the previous study tyrosi-nase from Agaricus bisporus was purified by ammoniumsulphate precipitation dialysis followed by gel filtrationchromatography on Sephadex G-100 and DEAE-celluloseion exchange chromatography [11]

22 Preparation of Melanocyte Culture The melanocytecell line B16F10 was procured from National Center forCell Science Pune and was cultured in Dulbeccorsquos Modi-fied Eaglersquos Medium (DMEM) containing 10 heat inacti-vated fetal bovine serum (FBS) 15 gL NaHCO

3 2mM L-

glutamine 10000 units of penicillin 10 120583gmL streptomycinand 25 120583gmL amphotericin B and incubated at 37∘C with5 CO

2in a humidified atmosphere To inhibit the bacterial

contamination 2 benzalkonium chloride was kept in incu-bator The cells were subcultured in a ratio of 1 3 on everythird day For cell expansion and experiments with isolatedcells the B16F10 cells were detached with 1x Trypsin-EDTA(025 Trypsin and 01 EDTA in Hankrsquos balanced saltsolution) After 3-4 passages the cells were discarded andwhen necessary the cells preserved in liquid nitrogen wereused as fresh culture

23 Cell Viability Assay When 70 confluency of B16F10melanocytes was attained trypsinization and seeding weredone in 96-well microtitre plates at a density of 104 cellswellin DMEM supplemented with 10 FBS and 10000 unitsof penicillin 10 120583gmL streptomycin and 25 120583gmL ampho-tericin B antibiotic solutions After overnight incubationmedia of each well were replaced and the cells were treatedwith desired stimulants to performMTT assay [12] to exam-ine any cytotoxic effect of extracted tyrosinase of Agaricusbisporus along with standard control tyrosinase (Sigma) inB16F10 cells over the concentration range of 1 to 64120583gmL atdifferent incubation periods of 24 48 and 72 hr respectivelyAt the completion of incubation stimulant-containing mediawere discarded and fresh DMEM containing 1mgmL ofMTT was added to each well and incubated at 37∘C for4 h The solution was replaced with 004N HCl-isopropylalcohol solution and further incubated at room temperaturefor 30min Harvested solution was centrifuged at 11337timesg for5min and absorbance of supernatantwasmeasured at 570 nmusing microplate ELISA reader

24 Tyrosinase Assay of B16F10 Melanocytes Tyrosinaseactivity was performed using L-DOPA as the substratewith minor modifications of Sung and Cho [13] Prior toexperiment the B16F10 cells were cultured and 1 times 105cellswell were seeded in 24-well tissue culture plates Thecells were treated with different concentration range of 1 to64 120583gmL of extracted tyrosinase of Agaricus bisporus alongwith standard control tyrosinase (Sigma) for 24 48 and 72 hrAfter treatment the cells were washed twice with ice-cold PBSand extracted by sonication and then centrifuged at 11337timesgfor 10min at 4∘C For tyrosinase activity 100 120583L of extractfrom each well was added in 96-well microtitre plates and theenzymatic assay was commenced by adding 100 120583L L-DOPAsolution incubated at 37∘C for 1 h After the incubationabsorbance was measured at 475 nm using microplate inELISA reader and protein content was estimated [14]

25 Melanin Assay of B16F10 Melanocytes Melanin contentwas determined by the method of Tsuboi et al [15] withminor modifications 1 times 105 cellswell were seeded in 24-welltissue culture plates After overnight incubation the purifiedtyrosinase of Agaricus bisporus along with standard controltyrosinase (Sigma) was added in different concentration of 1to 64 120583gmL for 24 48 and 72 hours After trypsinization analiquot was divided into two parts Five hundred microlitreswas used for cell counting and the rest was centrifuged at1677timesg for 5min and lysed with 200120583L of 1N NaOH stirringat 80∘C for 1 h Melanin content was measured at 405 nmusing ELISA reader

Biotechnology Research International 3

26 Determining theMechanistic Action of Purified Tyrosinasevia Using Melanin Content and Western Blot Assay Effectsof purified tyrosinase on the different pathways involvedin melanogenesis in B16F10 melanocytes were exposedby using the protein kinase inhibitors protein kinase A(PKA) inhibitor (H89) protein kinase B (PKB) inhibitor(LY294002) protein kinase C (PKC) inhibitor (Ro-32-0432)and MEK1 inhibitor (PD98059) B16F10 melanocytes werepretreated with 3 120583M of protein kinase inhibitors at 37∘C for1 hr and then treated with 64 120583gmL of purified tyrosinaseAfter treatment the cells were collected and analyzed byusing a melanin content assay For western blot the cellswere homogenized using ice-cold lyses buffer (01MTris-HClbuffer 1 Nonidet P-40 001 SDS 100 120583M phenyl methylsulfonyl fluoride and 1 120583gmL aprotinin) The homogenatescontaining 15 120583g of protein were separated by SDS-PAGEAfter electrophoresis a gel was transferred to polyvinylidenedifluoride (PVDF) membranes (HiMedia) in western blotapparatus (Axygen Scientific Pvt Ltd India) at 15 volts for25 to 30min The membranes were washed with 1x TBST pH72 (137M Sodium Chloride 20mM Tris and 01 Tween20) and blocked with blocking buffer (5 skim milk 137MSodiumChloride 20mMTris and 01 Tween 20) overnightat 4∘C Following blocking the membranes were washedthrice in 1x TBST for 5min each at room temperature Firstlythe membranes were washed and then treated with primaryantibody buffer of goat anti-murine tyrosinase IgG (1 100dilutions) with gentle agitation at 4∘C for 1 h After incubationwith primary antibody themembranes were washed thrice in1x TBST for 5min each at room temperaturewith gentle shak-ing after extensive washing with secondary buffer of donkeyanti-goat IgG conjugated with horseradish peroxides (1 2000dilution) and washed with stripping buffer (10 SDS 05MTris 08 120573-mercaptoethanol and ultrapure water) Theproteins were visualized using Gel documentation analyzer(Bio-Rad Laboratories Pvt Ltd India) and densitometry wasperformed using Quantity One software [16]

27 Statistical Analysis The data is presented as mean plusmnSEM (standard error of the mean) where 119899 represents thenumber of dose concentrations (treated) used for a partic-ular experiment Comparisons were made between treatedand control groups by using Studentrsquos 119905-test All data wereanalyzed using GraphPad Prism 5 software (UK) 119901 lt 0005indicates statistically significant difference

3 Results and Discussion

31 Purified Tyrosinase Stimulates Dendrite Formation inB16F10 Melanocytes To assess the functional significanceof purified tyrosinase in B16F10 melanocytes we deter-mined the effects of purified tyrosinase stimulation onB16F10 melanocytes morphology Prior to treatment B16F10melanocytes exhibited no dendrite formation (Figure 1(a))The minimum concentration of 1ndash4 120583gmL purified tyrosi-nase of A bisporus caused the morphological change inthe B16F10 melanocytes it was found that the B16F10melanocytes initially showed dendritic network processesin which the pigment granules appeared on treatment

(Figure 1(b)) Increasing the concentrations of purifiedtyrosinase of A bisporus from 8 to 32 120583gmL it was observedthat there were more pronounced morphological changeswhere the melanocytes dendritic process increased furtheras compared to the untreated B16F10 melanocytes Thusthere was distinct increase in the dendritic process morpho-logically which is exhibited (Figures 1(c) and 1(d)) at thehighest concentration of 64120583gmLof purified tyrosinase ofAbisporus under the same culture conditions It was found thatafter the incubation period the B16F10 melanocytes becamemultipolar with highly branched dendritic network and alsoshowed dense pigmented granules in the cytoplasm of thetreated cells and clusters of growing cell assembly were alsovisible (Figure 1(e)) Tyrosinase (Sigma) also increased themelanocytes dendricity and proliferation at concentration of64 120583gmL (Figure 1(f)) The present data are in fairly goodagreement with the suggestion of Mallick et al [17] whoreported the effect of placental total lipid fraction in B16F10melanoma cells It was found that B16F10 melanoma cellsgrew with large dendrites and formed a confluent monolayerand multipolar highly branched dendritic network and alsodense pigmented granules appeared in the cytoplasm of thetreated cells

32 Purified Mushroom Tyrosinase Induced both CellularMelanin Content and Tyrosinase Activity without Affectingthe Cell Viability To examine the cell viability due to thetreatment of purifiedmushroom tyrosinaseMTTcytotoxicityassays were performed in B16F10 cells A wide concentrationrange of purified mushroom tyrosinase (1ndash64120583mL) waspreferred It was observed that cell viability significantlyincreases in B16F10 culture system at a higher concentrationAfter 24 h of treatment all the concentration of purifiedmushroom tyrosinase (1ndash64120583gmL) showed significant cellviability as 103 to 121 respectively as compared to controlas 100 (0382 plusmn 05mg1 times 105 cells) Maximum cell viabilitywas observed at concentration of 64 120583gmL 1212 (0462plusmn 05mg1 times 105 cells) (119901 lt 0005) whereas tyrosinase(Sigma) possessed maximum proliferation of 1263 (0482plusmn 05mg1 times 105 cells) (119901 lt 0005) with respect to control as100Moreover extending the period of treatment from24 to48 h and 72 h and increasing the dose of purified mushroomtyrosinase above 4120583gmL no further enhancement in cellularproliferation was observed rather growth stimulation startedto decline gradually above this dose reaching 1289 (0385plusmn 05mg1 times 105 cells) (119901 lt 0005) as compared tocontrol as 100 (0371 plusmn 05mg1 times 105 cells) value at theconsiderable higher concentration of 64 120583gmL (Figure 2(a))These findings are in agreement with those of Mallick et al[17] who reported the similar effects of placental total lipidfractions on B16F10 melanocytes viability It had been alsoreported by Lee et al 2005 [7] that the serial concentration ofglycyrrhizin significantly increased the cellrsquos viability in B16melanoma cells but significantly decreased when at higherconcentration The present findings are in full agreementwith those of Yoon et al [18] who reported the effects ofisopanduratin A on cell viability percentages of viablemelan-cells


(a) (b)

(c) (d)

(e) (f)

Figure 1 Morphological appearance of B16F10 melanocytes Melanocytes exhibited no dendrite formation (a) Dendritic network processesin which the pigment granules appeared (b) Increased dendritic process (c d) Multipolar highly branched dendritic network with densepigmented granules (e) Increased melanocytes dentricity and proliferation (f) All photographs are under phase contrast microscope equalmagnification of 200x

B16F10 melanocytes were exposed to purified mush-room tyrosinase at various concentrations ranging from 1to 64 120583gmL for 24 48 and 72 hours Treatment of B16F10melanocytes with PMT caused increase in the melanincontent in time and concentration-dependentmanner At theconcentration of 64120583gmLmelanin synthesis was stimulatedmaximally to 159 (119901 lt 0005) with respect to untreatedcontrol (100) while tyrosinase (Sigma) was found to havemore potent melanogenic activity in B16F10 cells at 171(119901 lt 0005) with respect to control Meanwhile extendingthe treatment period of purified mushroom tyrosinase from24 to 48 h at the concentration range of 1ndash64 120583gmL results inthe further enhancement of melanin production wheremax-imum melanin production was observed at a concentration

of 64 120583gmL 169 (119901 lt 0005) and standard control 176(119901 lt 0005) with respect to untreated control (100)After the treatment of 72 h B16F10 cells showed decreasedmelanin production at all the concentration of purifiedmushroom tyrosinase 1ndash64120583gmL (Figure 2(b)) The presentfindings suggest that purified tyrosinase induced melaninproduction in the B16F10 melanocytes significantly these arewell supported by earlier classic work of Raman et al [19]who reported that aqueous extract of Angelica sinensis rootcommonly used in the treatment of vitiligo was very activeon mouse melanocytes Similar data have been reportedby Smit et al [20] where tomato extract was used on thegrowth and pigmentation of melanocytes Moreira et al[21] reported melanogenic activity of hydroalcoholic extracts


24 h48 h72 h

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64

Concentration (120583gmL)

0

20

40

60

80

100

120

140C

ell v

iabi

lity

()

(a)

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

50

100

150

200

Mela

nin

cont

ent (

)

24 h48 h72 h

(b)

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

20

40

60

80

100

120

140

160

Tyro

sinas

e act

ivity

()

24 h48 h72 h

(c)

Figure 2 Purifiedmushroom tyrosinase induced both pigmentation and the cellular content of tyrosinase without affecting cell viability Cellviability (a) Melanin synthesis in B16F10 melanocytes (b) Tyrosinase activity (c) Data represented as mean plusmn SE from triplicate experiments(119901 lt 0005)

from the leaves and flowers of P venusta on murine B16F10melanoma cells Both extracts increased the melanin contentin a concentration-dependent manner on melanoma cells

To gain insight about the pigment inducing ability ofA bisporus tyrosinaseas observed in B16F10 melanocyteswe assessed their effect on the activity of key melanogenicenzyme tyrosinase in the same cellular systems It wasobserved that tyrosinase activity of purified mushroomtyrosinase of A bisporus significantly increased at differentconcentration (1ndash64120583gmL) after 24 h of treatment All theconcentration showed increase in cellular tyrosinase rangingfrom 112 (0438 plusmn 05mg1 times 105 cells) to 148 (0581 plusmn05mg1 times 105 cells) respectively as compared to controlat 100 (0438 plusmn 05mg1 times 105 cells) Maximum enhance-ment in tyrosinase activity was observed at concentrationof 64 120583gmL that is 148 (0581 plusmn 05mg1 times 105 cells)(119901 lt 0005) With extension of treatment period from 24to 48 h the tyrosinase activity increased from 124 (0486plusmn 05mg1 times 105 cells) (1120583gmL) to maximum tyrosinaseactivity of 153 (0601 plusmn 05mg1 times 105 cells) at 64 120583gmL(119901 lt 0005) compared to control After the treatment of 72 h

no further enhancement of cellular tyrosinase was observed(Figure 2(c)) The present finding confirmed that purifiedtyrosinase increases tyrosinase activity significantly in B16F10melanocytes is in corroboration with those of Jeon et al [22]who studied the essential oil from lotus flower extract and itseffects onmelanogenesis in humanmelanocytes It was foundthat the effective compound induced significant amount oftyrosinase Similarly Park et al [23] had reported that theeffect of harmaline and harmalol on B16F10 cells tyrosinaseactivity occurred in a time-dependent manner

33 Mechanistic Action of Purified Tyrosinase InducingMelanogenesis via Using Melanin Content and Western BlotAssay It is well recognized that cAMP accumulation inducesactivation of PKA resulting in melanin synthesis Becauseactivation of CREB is required for cAMP responsiveness ofthe MITF promoter [24] we investigated the involvementof the cAMPPKA pathway in purified tyrosinase-regulatedmelanogenesis 3120583M of PKA (H-89) PKB (LY294002)PKC (Ro-32-0432) and MEK1 (PD98059) was used toinhibit signaling pathways involved in melanogenesis in


Fold

of s

timul

atio

n

10

9

8

7

6

5

4

3

2

1

0

Con

trol

Purifi

ed

tyro

sinas

e

PKA

(H-8

9)

PKB

(LY2

9400

2)

PKC

(Ro-

32-0

432)

MEK

1 (P

D98

059)

Purified tyrosinase (64 120583gmL)

(a)

Control Purified tyrosinase

PKA(H-89)

PKB (LY294002)

PKC (Ro-32-0432)

MEK1 (PD98059)

Tyrosinase expression ()

(b)

Figure 3 Effects of protein kinase inhibitors on purified tyrosinase induced melanogenesis Melanin content (a) Tyrosinase expression oftreated cells (b) Data represented as mean plusmn SE from triplicate experiments (119901 lt 0005)

B16F10 melanocytes PKA PKC PKB and MEK1 inhibitorsdecreased the stimulatory effects of tyrosinase from 836-fold value to 792- 486- 3347- and 531-fold value respec-tively They reduced the percentage of purified tyrosinase-stimulated melanin content from 100 in nonpretreated cellsto 947 5813 4003 and 6351 in PKA PKC PKB andMEK1 inhibitor pretreated cells respectively (Figures 3(a)and 3(b)) These findings suggest that protein kinase was themajor signaling pathway mechanism of purified tyrosinaseinduced melanogenesis in B16F10 melanocytes Howeverthe results also show that purified tyrosinase may partiallyinducemelanin synthesis via PKC PKB andMAPK signalingpathways

Protein kinase inhibitors of PKB (LY294002) and MEK1(PD98069) also slightly decreased the purified tyrosinaseincreasedmelanin content and the cellular tyrosinase expres-sions (Figures 3(a) and 3(b)) These findings were similarto a previous study that demonstrated that cAMP not onlyinitiates PKA activation but also activates other signalingpathwaysThese pathways include PI3KAKT and PKBGSK-3120573 pathways [25]

PKC Ro-32-0432 has been shown to decrease purifiedtyrosinase-stimulated melanogenesis and expressions of cel-lular tyrosinase (Figures 3(a) and 3(b)) These results suggestthat purified tyrosinase may also enhance melanin synthesisthrough PKC signaling pathwayThePKC- (protein kinase C-) dependent pathway has emerged as a second intracellularsignaling pathway regulating melanogenesis It was first

observed that addition of diacylglycerol the endogenousactivator of PKC to cultured human melanocytes causeda rapid 3-4-fold increase in total melanin content [26]Some extracellular signals such as endothelin-1 (ET-1) havebeen shown to increase melanogenesis by activating PKCActive PKC activates tyrosinase by directly phosphorylatingtyrosinase into melanosome [27]

Since tyrosinase is the key in regulation of melaninproduction the study conducted on the effect of purifiedtyrosinase absorption and its effect has shown mushroomtyrosinase as an effective agent for melanogenesis The studyconducted by Zehtab et al [10] indicated that the tyrosi-nase of A bisporus is a key enzyme in melanin syntheticpathway and is expected to prevent autoimmune vitiligoby oral administration The present study supports thatthe tyrosinase of A bisporus in B16F10 melanocytes caninduce melanin production directly as it was observed bythe cells This might be due to their characteristic similarityto the mammalian tyrosinase A bisporus has been alreadyreported to be structurally similar to mammalian tyrosinase[28ndash30] The physiological role of tyrosinase is related tomelanin biosynthesis and has been extracted from differentsources such as fungi fruits and mammalian melanomatumors [29 31] In addition tyrosinase is associated withwound healing with the immune response in plants [32 33]In humans tyrosinase is involved in the pigmentation inmelanocytes [34 35] as a marker in melanoma patients [36]and as a target for the activation of prodrugs [37]


Based on the pharmacological reports presented abovewe hereby postulate that purified tyrosinase could havepotential of a pigment cell acting drug In order to confirmthe finding in the present study an attempt has been madeto explore the possibility of initiation of the pigment cellmachinery by purified tyrosinase an enzyme from Agari-cus bisporus to induce melanin displacement causing skindarkening It may be mentioned here that this is the firstreport of its kind where purified tyrosinase has been foundto cause skin darkening via melanin displacement within themelanocytes The mushroom extracts containing the activecompound tyrosinase can be used as novel contestant for thetreatment of vitiligous skin conditions

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to Peoplersquos University Bhopal forproviding laboratory facilities and for granting financialassistance to carry out this work and Principal and SecretarySaifia College of Science Bhopal for encouragement

References

[1] M Brenner and V J Hearing ldquoThe protective role of melaninagainst UV damage in human skinrdquo Photochemistry and Photo-biology vol 84 no 3 pp 539ndash549 2008

[2] R Narayanaswamy and I S Ismail ldquoCosmetic potential ofSoutheast Asian herbs an overviewrdquo Phytochemistry Reviewsvol 14 no 3 pp 419ndash428 2015

[3] J Lee Y S Kim and D Park ldquoRosmarinic acid inducesmelanogenesis through protein kinase A activation signalingrdquoBiochemical Pharmacology vol 74 no 7 pp 960ndash968 2007

[4] P Jahan S Tippisetty and P L Komaravalli ldquoFOXP3 is apromising and potential candidate gene in generalised vitiligosusceptibilityrdquo Frontiers in Genetics vol 6 article 249 2015

[5] A Alikhan L M Felsten M Daly and R V Petronic ldquoVitiligoA comprehensive overview Part I Introduction epidemiologyquality of life diagnosis differential diagnosis associationshistopathology etiology and work-uprdquo Journal of the AmericanAcademy of Dermatology vol 65 no 3 pp 473ndash491 2011

[6] R A Spritz ldquoRecent progress in the genetics of generalizedvitiligordquo Journal of Genetics andGenomics vol 38 no 7 pp 271ndash278 2011

[7] J Lee E Jung J Park et al ldquoGlycyrrhizin induces melanogene-sis by elevating a cAMP level in B16 melanoma cellsrdquo Journal ofInvestigative Dermatology vol 124 no 2 pp 405ndash411 2005

[8] S A Ali and I Naaz ldquoUnderstanding the ultrastructuralaspects of berberine-induced skin-darkening activity in thetoad Bufo melanostictus melanophoresrdquo Journal of Microscopyand Ultrastructure vol 3 pp 210ndash219 2015

[9] M E Valverde T Hernandez-Perez and O Paredes-LopezldquoEdible mushrooms improving human health and promotingquality liferdquo International Journal of Microbiology vol 2015Article ID 376387 14 pages 2015

[10] T Zehtab R Yazdanparast and S Rafieii ldquoPrevention ofexperimental autoimmune vitiligo by oral administration ofmushroom tyrosinaserdquo Iranian Biomedical Journal vol 4 no1 pp 31ndash36 2000

[11] K U Zaidi A S Ali and S A Ali ldquoPurification and char-acterization of melanogenic enzyme tyrosinase from buttonmushroomrdquo Enzyme Research vol 2014 Article ID 120739 6pages 2014

[12] D-S Kim S-H Park S-B Kwon et al ldquoTemperature regu-lates melanin synthesis in melanocytesrdquo Archives of PharmacalResearch vol 26 no 10 pp 840ndash845 2003

[13] C K Sung and S H Cho ldquoThe purification and characteristicsof tyrosinase from gingerrdquo Journal of Biochemistry and Molecu-lar Biology vol 25 pp 564ndash572 1992

[14] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[15] T Tsuboi H Kondoh J Hiratsuka and Y Mishima ldquoEnhancedmelanogenesis induced by tyrosinase gene-transfer increasesboron-uptake and killing effect of boron neutron capturetherapy for amelanotic melanomardquo Pigment Cell Research vol11 no 5 pp 275ndash282 1998

[16] N Lv J-H Koo H-Y Yoon et al ldquoEffect of Angelica gigasextract on melanogenesis in B16 melanoma cellsrdquo InternationalJournal of Molecular Medicine vol 20 no 5 pp 763ndash767 2007

[17] S Mallick S KMandal and R Bhadra ldquoHuman placental lipidinduces mitogenesis and melanogenesis in B16F10 melanomacellsrdquo Journal of Biosciences vol 27 no 3 pp 243ndash249 2002

[18] J-H Yoon J-S Shim Y Cho et al ldquoDepigmentation ofmelanocytes by isopanduratin A and 4-hydroxypanduratin Aisolated from Kaempferia pandurata ROXBrdquo Biological andPharmaceutical Bulletin vol 30 no 11 pp 2141ndash2145 2007

[19] A Raman Z X Lin E Sviderskaya and D Kowalska ldquoInves-tigation of the effect of Angelica sinensis root extract on theproliferation of melanocytes in culturerdquo Journal of Ethnophar-macology vol 54 no 2-3 pp 165ndash170 1996

[20] N Smit J Vicanova P Cramers H Vrolijk and S PavelldquoThe combined effects of extracts containing carotenoids andvitamins E and C on growth and pigmentation of culturedhumanmelanocytesrdquo Skin Pharmacology and Physiology vol 17no 5 pp 238ndash245 2004

[21] C G Moreira C D S Horinouchi C S Souza-Filho et alldquoHyperpigmentant activity of leaves and flowers extracts ofPyrostegia venusta on murine B16F10 melanomardquo Journal ofEthnopharmacology vol 141 no 3 pp 1005ndash1011 2012

[22] S Jeon N-H Kim B-S Koo J-Y Kim and A-Y Lee ldquoLotus(Nelumbo nuficera) flower essential oil increasedmelanogenesisin normal human melanocytesrdquo Experimental and MolecularMedicine vol 41 no 7 pp 517ndash524 2009

[23] K-C Park S Y Huh H R Choi and D-S Kim ldquoBiologyof melanogenesis and the search for hypopigmenting agentsrdquoDermatologica Sinica vol 28 no 2 pp 53ndash58 2010

[24] R Busca and R Ballotti ldquoCyclic AMP a key messenger in theregulation of skin pigmentationrdquo Pigment Cell Research vol 13no 2 pp 60ndash69 2000

[25] A Slominski D J Tobin S Shibahara and J WortsmanldquoMelanin pigmentation in mammalian skin and its hormonalregulationrdquo Physiological Reviews vol 84 no 4 pp 1155ndash12282004

[26] P R Gordon and B A Gilchrest ldquoHuman melanogenesis isstimulated by diacylglycerolrdquo Journal of Investigative Dermatol-ogy vol 93 no 5 pp 700ndash702 1989


[27] G Imokawa Y Yada and M Kimura ldquoSignalling mecha-nisms of endothelin-induced mitogenesis and melanogenesisin human melanocytesrdquo Biochemical Journal vol 314 no 1 pp305ndash312 1996

[28] S-Y Seo V K Sharma and N Sharma ldquoMushroom tyrosinaserecent prospectsrdquo Journal of Agricultural and Food Chemistryvol 51 no 10 pp 2837ndash2853 2003

[29] K U Zaidi A S Ali and S A Ali ldquoPurification and char-acterization of high potential tyrosinase from macrofungi andits appliance in food engineeringrdquo Journal of MicrobiologyBiotechnology and Food Sciences vol 5 no 3 pp 203ndash206 2015

[30] K U Zaidi and A S Ali ldquoComparative evaluation of purifiedand characterized tyrosinases from two edible mushroomsAgaricus bisporus and Pleurotus ostreatus and their clinicalpotentialrdquo Bioscience Biotechnology Research Communicationsvol 8 no 2 pp 161ndash170 2015

[31] K U Zaidi A Mani S A Ali and A S Ali ldquoEvaluationof Tyrosinase producing endophytic fungi from CalotropisgiganteaAzadirachta indicaOcimum tenuiflorum and Lantanacamarardquo Annual Review amp Research in Biology vol 3 no 4 pp389ndash396 2013

[32] C W G Van Gelder W H Flurkey and H J WichersldquoSequence and structural features of plant and fungal tyrosi-nasesrdquo Phytochemistry vol 45 no 7 pp 1309ndash1323 1997

[33] L Cerenius andK Soderhall ldquoTheprophenoloxidase-activatingsystem in invertebratesrdquo Immunological Reviews vol 198 pp116ndash126 2004

[34] Y Jin S A Birlea P R Fain et al ldquoVariant of TYR andautoimmunity susceptibility loci in generalized vitiligordquo TheNew England Journal ofMedicine vol 362 no 18 pp 1686ndash16972010

[35] K U Schallreuter MM A E L Salem S Hasse andH RokosldquoThe redoxmdashbiochemistry of human hair pigmentationrdquo Pig-ment Cell andMelanoma Research vol 24 no 1 pp 51ndash62 2011

[36] A Gradilone E Cigna AM Agliano and L Frati ldquoTyrosinaseexpression as a molecular marker for investigating the presenceof circulating tumor cells in melanoma patientsrdquo CurrentCancer Drug Targets vol 10 no 5 pp 529ndash538 2010

[37] S Jawaid T H Khan H M I Osborn and N A O WilliamsldquoTyrosinase activatedmelanoma prodrugsrdquoAnti-Cancer Agentsin Medicinal Chemistry vol 9 no 7 pp 717ndash727 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology


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Signal TransductionJournal of


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Genetics Research International


Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


mushroom tyrosinase but exact mechanism is still unknownso an attempt is made to explore the detailed mechanism ofmushroom tyrosinase on B16F10 melanocytes Furthermorethere are no studies at all on the effect of mushroomtyrosinase on cultured melanocytes to see the efficacy of themushroom tyrosinase as melanogenic agent

Thus the current investigation would provide a bench-mark in exploring the diversity of mushroom and the effectofmushroom tyrosinase on culturedmelanocytes for findingout melanogenic agents The present study was undertakenkeeping in view the above lacunae in literature and for the firsttime B16F10 melanocytes model has been studied in detailto block the signaling pathway and mechanism of inducedmelanogenesis by lyophilized purified tyrosinase of Agaricusbisporus The findings of the study have provided vitalinformation on these aspects and differentiating results havebeen obtained It may be mentioned here that this is the firstreport of its kind where purified mushroom tyrosinase hasbeen found to cause skin darkening viamelanin displacementwithin the B16F10 melanocytes The present study on B16F10melanocytes is the first study on the effect of mushroomtyrosinase especially from A bisporus that can serve as amelanogenic potent to vitiligo

2 Material and Methods

For the present study the compound mushroom tyrosi-nase (lyophilized powder ge 1000 unitsmg solid) and pro-tein kinase inhibitors protein kinase A (PKA) inhibitor(H89) protein kinase B (PKB) inhibitor (LY294002) proteinkinase C (PKC) inhibitor (Ro-32-0432) and MEK1 inhibitor(PD98059) were purchased from Sigma-Aldrich St LouisMissouri United States Goat anti-murine tyrosinase IgGantibody and Alexa Fluor 594 donkey anti-goat IgG (H+L)(2mgmL) were purchased from Life Technologies NorthAmerica United States Dulbeccorsquos Modified Eagle Medium(AT006A-5L) fetal bovine serum (RM10432-100mL) Anti-biotic Antimycotic Solution 100x (A002-20mL) Trypsin-EDTA Solution 1x (TCL042-5 times 100mL)MTT [3-(45-dim-ethylthiazol-2-yl)]-25-diphenyltetrazoliumbromide (TC191-500MG) 410158406-diamidino-2-phenylindole (DAPI) (TC229-5MG) phosphate buffered saline (RM7385-1PK) and Trypanblue Certified (RM263-5G) were purchased from HiMediaLaboratories Pvt Ltd Mumbai

21 Preparation of Tyrosinase In the previous study tyrosi-nase from Agaricus bisporus was purified by ammoniumsulphate precipitation dialysis followed by gel filtrationchromatography on Sephadex G-100 and DEAE-celluloseion exchange chromatography [11]

22 Preparation of Melanocyte Culture The melanocytecell line B16F10 was procured from National Center forCell Science Pune and was cultured in Dulbeccorsquos Modi-fied Eaglersquos Medium (DMEM) containing 10 heat inacti-vated fetal bovine serum (FBS) 15 gL NaHCO

3 2mM L-

glutamine 10000 units of penicillin 10 120583gmL streptomycinand 25 120583gmL amphotericin B and incubated at 37∘C with5 CO

2in a humidified atmosphere To inhibit the bacterial

contamination 2 benzalkonium chloride was kept in incu-bator The cells were subcultured in a ratio of 1 3 on everythird day For cell expansion and experiments with isolatedcells the B16F10 cells were detached with 1x Trypsin-EDTA(025 Trypsin and 01 EDTA in Hankrsquos balanced saltsolution) After 3-4 passages the cells were discarded andwhen necessary the cells preserved in liquid nitrogen wereused as fresh culture

23 Cell Viability Assay When 70 confluency of B16F10melanocytes was attained trypsinization and seeding weredone in 96-well microtitre plates at a density of 104 cellswellin DMEM supplemented with 10 FBS and 10000 unitsof penicillin 10 120583gmL streptomycin and 25 120583gmL ampho-tericin B antibiotic solutions After overnight incubationmedia of each well were replaced and the cells were treatedwith desired stimulants to performMTT assay [12] to exam-ine any cytotoxic effect of extracted tyrosinase of Agaricusbisporus along with standard control tyrosinase (Sigma) inB16F10 cells over the concentration range of 1 to 64120583gmL atdifferent incubation periods of 24 48 and 72 hr respectivelyAt the completion of incubation stimulant-containing mediawere discarded and fresh DMEM containing 1mgmL ofMTT was added to each well and incubated at 37∘C for4 h The solution was replaced with 004N HCl-isopropylalcohol solution and further incubated at room temperaturefor 30min Harvested solution was centrifuged at 11337timesg for5min and absorbance of supernatantwasmeasured at 570 nmusing microplate ELISA reader

24 Tyrosinase Assay of B16F10 Melanocytes Tyrosinaseactivity was performed using L-DOPA as the substratewith minor modifications of Sung and Cho [13] Prior toexperiment the B16F10 cells were cultured and 1 times 105cellswell were seeded in 24-well tissue culture plates Thecells were treated with different concentration range of 1 to64 120583gmL of extracted tyrosinase of Agaricus bisporus alongwith standard control tyrosinase (Sigma) for 24 48 and 72 hrAfter treatment the cells were washed twice with ice-cold PBSand extracted by sonication and then centrifuged at 11337timesgfor 10min at 4∘C For tyrosinase activity 100 120583L of extractfrom each well was added in 96-well microtitre plates and theenzymatic assay was commenced by adding 100 120583L L-DOPAsolution incubated at 37∘C for 1 h After the incubationabsorbance was measured at 475 nm using microplate inELISA reader and protein content was estimated [14]

25 Melanin Assay of B16F10 Melanocytes Melanin contentwas determined by the method of Tsuboi et al [15] withminor modifications 1 times 105 cellswell were seeded in 24-welltissue culture plates After overnight incubation the purifiedtyrosinase of Agaricus bisporus along with standard controltyrosinase (Sigma) was added in different concentration of 1to 64 120583gmL for 24 48 and 72 hours After trypsinization analiquot was divided into two parts Five hundred microlitreswas used for cell counting and the rest was centrifuged at1677timesg for 5min and lysed with 200120583L of 1N NaOH stirringat 80∘C for 1 h Melanin content was measured at 405 nmusing ELISA reader









(a) (b)

(c) (d)

(e) (f)





24 h48 h72 h

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

20

40

60

80

100

120

140C

ell v

iabi

lity

()

(a)

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

50

100

150

200

Mela

nin

cont

ent (

)

24 h48 h72 h

(b)

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

20

40

60

80

100

120

140

160

Tyro

sinas

e act

ivity

()

24 h48 h72 h

(c)







Fold

of s

timul

atio

n

10

9

8

7

6

5

4

3

2

1

0

Con

trol

Purifi

ed

tyro

sinas

e

PKA

(H-8

9)

PKB

(LY2

9400

2)

PKC

(Ro-

32-0

432)

MEK

1 (P

D98

059)


(a)


PKA(H-89)

PKB (LY294002)

PKC (Ro-32-0432)

MEK1 (PD98059)


(b)









Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









(a) (b)

(c) (d)

(e) (f)





24 h48 h72 h

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

20

40

60

80

100

120

140C

ell v

iabi

lity

()

(a)

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

50

100

150

200

Mela

nin

cont

ent (

)

24 h48 h72 h

(b)

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

20

40

60

80

100

120

140

160

Tyro

sinas

e act

ivity

()

24 h48 h72 h

(c)







Fold

of s

timul

atio

n

10

9

8

7

6

5

4

3

2

1

0

Con

trol

Purifi

ed

tyro

sinas

e

PKA

(H-8

9)

PKB

(LY2

9400

2)

PKC

(Ro-

32-0

432)

MEK

1 (P

D98

059)


(a)


PKA(H-89)

PKB (LY294002)

PKC (Ro-32-0432)

MEK1 (PD98059)


(b)









Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


24 h48 h72 h

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

20

40

60

80

100

120

140C

ell v

iabi

lity

()

(a)

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

50

100

150

200

Mela

nin

cont

ent (

)

24 h48 h72 h

(b)

Con

trol

Tyro

sinas

e(S

igm

a)

1 2 4 8

16

32

64


0

20

40

60

80

100

120

140

160

Tyro

sinas

e act

ivity

()

24 h48 h72 h

(c)







Fold

of s

timul

atio

n

10

9

8

7

6

5

4

3

2

1

0

Con

trol

Purifi

ed

tyro

sinas

e

PKA

(H-8

9)

PKB

(LY2

9400

2)

PKC

(Ro-

32-0

432)

MEK

1 (P

D98

059)


(a)


PKA(H-89)

PKB (LY294002)

PKC (Ro-32-0432)

MEK1 (PD98059)


(b)









Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Fold

of s

timul

atio

n

10

9

8

7

6

5

4

3

2

1

0

Con

trol

Purifi

ed

tyro

sinas

e

PKA

(H-8

9)

PKB

(LY2

9400

2)

PKC

(Ro-

32-0

432)

MEK

1 (P

D98

059)


(a)


PKA(H-89)

PKB (LY294002)

PKC (Ro-32-0432)

MEK1 (PD98059)


(b)









Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Competing Interests


Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Effect of Purified Mushroom Tyrosinase on ...

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