molecules

Article and : Antiproliferative, Necrotic and Apoptotic Activity in Tumor Cell Lines

Jesús J. Alvarado-Sansininea 1, Luis Sánchez-Sánchez 2, Hugo López-Muñoz 2, María L. Escobar 3 , Fernando Flores-Guzmán 2, Rosario Tavera-Hernández 1 and Manuel Jiménez-Estrada 1,*

1 Laboratorio 2-10, Departamento de Productos Naturales, Instituto de Química, Universidad Nacional Autónoma de México, 04510 Ciudad de México, Mexico; [email protected] (J.J.A.-S.); [email protected] (R.T.-H.) 2 Laboratorio 6, 2do piso, UMIEZ, Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, 09230 Ciudad de México, Mexico; [email protected] (L.S.-S.); [email protected] (H.L.-M.); [email protected] (F.F.-G.) 3 Laboratorio de Microscopía Electrónica, Departamento de Biología Celular, Facultad de Ciencias, Universidad Nacional Autónoma de México, 04510 Ciudad de México, Mexico; [email protected] * Correspondence: [email protected]; Tel.: +52-(55)-56-22-44-30

 Received: 22 August 2018; Accepted: 4 October 2018; Published: 9 October 2018 

Abstract: Quercetagetin and patuletin were extracted by the same method from two different Tagetes species that have multiple uses in folk medicine in Mexico and around the globe, one of which is as an anticancer agent. Their biological activity (IC50 and necrotic, apoptotic and selective activities of these flavonols) was evaluated and compared to that of , examining specifically the effects of C6 substitution among quercetin, quercetagetin and patuletin. We find that the presence of a methoxyl group in C6 enhances their potency.

Keywords: necrotic; apoptosis; quercetin; quercetagetin; patuletin

1. Introduction Cancer is one of the most severe public health problems worldwide. The urgent need to obtain new drugs to treat it arises from the fact that conventional treatments cause many side effects that negatively impact patients’ quality of life. This has fostered the use of plant extracts and their metabolites as sources of active compounds [1]. Plants are still widely-used for medicinal purposes by the indigenous peoples in Mexico, and the biological activity of many plant derivatives has been evaluated and shown to have medicinal importance [2]. The genus Tagetes belongs to the family Asteraceae. It is highly-esteemed as an ornamental plant, and in some parts of the world is has ritual-cultural importance [3]. Several species of Tagetes are found in Mexico, some known commonly as Cempasuchil or Cempoalxochitl [4]. Two representative species are Tagetes erecta and Tagetes patula [5], both of which have important therapeutic potential due to their antiparasitic, antimalarial, anti-inflammatory, antitumorogenic and antiviral effects [6]. Tagetes flowers contain different classes of natural products [7], including terpenes, carotenoids and flavonoids, which are known to have pharmacological effects [8]. Differences in their molecular structures may be responsible for their different pharmacological properties [9]. Specifically, the genus Tagetes has many kinds of flavonoids [10]. Of these, quercetagetin and patuletin present distinct inhibitory activity [11]. In the present study, we implemented a method to obtain quercetagetin and patuletin from Tagetes erecta and Tagetes patula, respectively [12,13], and performed a structure-activity

Molecules 2018, 23, 2579; doi:10.3390/molecules23102579 www.mdpi.com/journal/molecules Molecules 2018, 23, 2579 2 of 18

Molecules 2017, 22, x FOR PEER REVIEW 2 of 18 relationship (SAR) study [14], focused on their anticancer properties by comparing them to Molecules 2017, 22, x FOR PEER REVIEW 2 of 18 quercetinFlavonoids [15]. are polyphenolic compounds with a C6-C3-C6 phenylbenzopyrone structure (Figure 1a) and they exhibit great structural diversity due to their various hydroxylation, methoxylation, FlavonoidsFlavonoids are are polyphenolic polyphenolic compounds compounds with with a C6 a-C C3-C6-C6 phenylbenzopyrone3-C6 phenylbenzopyrone structure structure (Figure (Figureglycosylation1a) and1a) they and exhibitand they acylation exhibit great great structural patterns structural [16]. diversity diversityThe basic du duee structureto totheir their various of various the flavonoid hydroxylation, hydroxylation, nucleus methoxylation,methoxylation, has different glycosylationsubstitutionglycosylation patterns andand acylationacylation in the A, patternspatterns B and [[16].C16 rings]. TheThe that basicbasic result structurestructure in various ofof thethe subgroups, flavonoidflavonoidnucleus nucleusone of which hashas differentdifferent are the substitutionflavonols,substitution which patterns possess in the thea 3-hydroxyflavone A, A, B B and and C C rings rings structure that that result result (Figure in invarious various1b). Examples subgroups, subgroups, of thisone one type of which of of which flavonoid are arethe theareflavonols, flavonols,the compounds which which possess quercetin, possess a 3-hydroxyflavone amyricetin 3-hydroxyflavone and structurekaempfer structure (Figureol [17]. (Figure 1b).Flavonoids Examples1b). Examplesdisplay of this diverse type of this of biologicalflavonoid type of flavonoidactivities,are the compounds areincluding the compounds quercetin, chemopreventive quercetin, myricetinandand kaempferchemot andherapeuticol [17]. Flavonoids effects [17]. Flavonoidson display cancer. diverse displayThe followingbiological diverse biologicalmechanismsactivities, activities,including of action including chemopreventivehave been chemopreventive reported: and carcinogen chemot and chemotherapeutic herapeuticinactivation, effects antiproliferation, effects on oncancer. cancer. cell-cycleThe The followingfollowing arrest, mechanismsinductionmechanisms of ofapoptosis,of actionaction havehave inhibition beenbeen reported:reported: of angiogenic carcinogen processes, inactivation, modulation antiproliferation, of multi-drug cell-cycle resistance, arrest, and inductionantioxidativeinduction of apoptosis, apoptosis,activity [18,19]. inhibition inhibition of of angiogenic angiogenic pr processes,ocesses, modulation modulation of multi-drug of multi-drug resistance, resistance, and andantioxidative antioxidative activity activity [18,19]. [18, 19].

Figure 1. (a) Base structure of flavonoids; (b) Structure of . FigureFigure 1.1. ((aa)) BaseBase structurestructure ofof flavonoids;flavonoids; ((bb)) StructureStructure ofof flavonols.flavonols. Some structure-activity relationship studies have been conducted, and they indicate that the presenceSomeSome of structure-activitystructure-activity the C2=C3 double relationshiprelationship bond and studiesstudiesthe hydroxylation havehave beenbeen conducted,patternconducted, of flavonoids andand theythey indicateindicateinfluence thatthat tumor thethe presenceproliferationpresence ofof the themodulation C2=C3C2=C3 doubledouble [20]. bondbondIn this andand regard, thethe hydroxylationhydroxylation research has patternpatternshown ofofthat flavonoidsflavonoids hydroxylated influenceinfluence flavonoids tumortumor proliferationpossessproliferation stronger modulation modulation inhibitory [20 [20]. ].activity In thisIn this regard,on cancerregard, research cells research than has shown permethoxylatedhas shown that hydroxylated that hydroxylatedflavonoids, flavonoids and flavonoids that possess C3- strongerhydroxylationpossess inhibitorystronger is inhibitoryimportant activity on activityfor cancer this cells onactivity. cancer than permethoxylated Apigenin,cells than forpermethoxylated example, flavonoids, which and flavonoids, thatlacks C3-hydroxylation a 3-OHand that group, C3- ispresentshydroxylation important lower for antiproliferativeis thisimportant activity. for Apigenin, activitythis activity. than for kaempf example,Apigenin,erol which [21].for example,The lacks catechol a 3-OHwhich group group, lacks in ring presentsa 3-OH B enhances group, lower antiproliferativeanticancerpresents lower activity antiproliferative activity [22]. There than are kaempferol activity few studies than [21 kaempf ].of Thesubserol catecholtituents [21]. atThe group C6 catechol in in relation ring group B to enhances anticancer in ring B anticancer enhances activity, activitybutanticancer flavonols [22 ].activity There like are[22].quercetin, few There studies quareercetagetin offew substituents studies and of subs atpatuletin C6tituents in relation all at have C6 to in anticancer the relation aforementioned activity,to anticancer but structural flavonols activity, likecharacteristicsbut quercetin,flavonols quercetagetinlikethat quercetin,seem to andbe qu important patuletinercetagetin allfor and haveantiproliferative patuletin the aforementioned all activity.have the structural The aforementioned only characteristics difference structural among that seemthesecharacteristics tocompounds be important that is seem the for C6 antiproliferativeto group be important substituent, activity.for antiproliferativeas querce The onlytin has difference activity.a C6-H, amongquercetagetinThe only these difference compounds has a C6-OH, among is theandthese C6 patuletin compounds group substituent,has ais C6-OMethe C6 as group quercetin(Figure substituent, 2). has Given a C6-H, asthis querce quercetagetinbackground,tin has a theC6-H, has present a C6-OH,quercetagetin study and was patuletin has designed a C6-OH, has to a C6-OMecontributeand patuletin (Figure to the has2 ).study a Given C6-OMe of this the background,(Figurerole of 2).C6 Givensubstitution the present this background, in study ring was A in designedthe the present cytotoxic to contributestudy activity was to ofdesigned theflavonols study to oflikecontribute the quercetin, role of to C6 the quercetagetin substitution study of the in and ringrole patu Aof inC6letin. the substitution cytotoxicThe fact that activity in somering of A cancer flavonols in the cells cytotoxic like show quercetin, activityresistance quercetagetin of toflavonols certain andkindslike patuletin.quercetin, of cell death Thequercetagetin fact can that pose some and a problem cancerpatuletin. cells for The show treatment fact resistance that somesuccess, to cancer certain so identifying cells kinds show of cell resistanceand death characterizing can to certain pose a problemmoleculeskinds of forcell that treatment death induce can success, apoptosis pose a so problem identifyingor necrosis for andtreatment[23] characterizing will allow success, us moleculestoso addidentifying new that strategies induce and characterizing apoptosis to existing or necrosistherapeuticmolecules [23 that] approaches. will induce allow usapoptosis to add new or necrosis strategies [23] to existingwill allow therapeutic us to add approaches. new strategies to existing therapeutic approaches.

Figure 2. Structures of (11)) quercetin,quercetin, ((22)) quercetagetinquercetagetin andand ((33)) patuletin.patuletin. 2. Results Figure 2. Structures of (1) quercetin, (2) quercetagetin and (3) patuletin. 2. Results In the present study, quercetagetin (2) and patuletin (3) were isolated by the same method from 2. ResultsIn the present study, quercetagetin (2) and patuletin (3) were isolated by the same method from different species of Tagetes plants. Upon comparing the structure of isolated compound 2 and quercetin, differentIn the species present of study, Tagetes quercetagetin plants. Upon (2) andcomparing patuletin the (3 )structure were isolated of isolated by the samecompound method 2 fromand quercetin,different species we found of thatTagetes they plants. share aUpon basic flavonolcomparing structure the structure type and of that isolated the only compound difference 2 is andthe quercetin, we found that they share a basic flavonol structure type and that the only difference is the

Molecules 2018, 23, 2579 3 of 18 we found that they share a basic flavonol structure type and that the only difference is the substituent groupMolecules at the 2017 ring-A, 22, x FOR C6 PEER position REVIEW (Figure 2). These structures were confirmed by 1D and 2D3 of NMR18 experimentssubstituent that group assigned at the the ring-A quercetagetin C6 position hydrogen (Figure 2). and These carbon structures positions. were confirmed by 1D and 2D NMR experiments that assigned the quercetagetin hydrogen and carbon positions. 2.1. Identification of Compounds 2 and 3 Compound2.1. Identification2 was of Compounds isolated as 2 aand yellow 3 powder. The structure was elucidated and the compound 1 identifiedCompound as quercetagetin 2 was isolated by 700 as MHz a yellow 1D powder. and 2D The NMR stru experimentscture was elucidated in DMSO- and thed6. compound The H-NMR 1 spectrumidentified displayed as quercetagetin six hydroxyl by 700 proton MHz signals 1D and and 2D fourNMRaromatic experiments proton in DMSO- signalsd6. atTheδH H-NMR7.66 (1H, d, 0 0 0 J = 2.2spectrum Hz, H-2 displayed), 7.53 (1H, six hydroxyl dd, J = 8.5,proton 2.2 sign Hz,als H-6 and), four 6.88 aromatic (1H, d, protonJ = 8.5 signals Hz, H-5 at δH) 7.66 and (1H, 6.49 d, (1H, J s, H-8).= This 2.2 Hz, assignment H-2′), 7.53 disagrees (1H, dd, J with= 8.5, a2.2 previously Hz, H-6′), 6.88 reported (1H, d, one J = 8.5 [13 Hz,] because H-5′) and the 6.49 HSQC (1H, experiments, H-8). This assignment disagrees with a previously reported one [13] because the HSQC experiment showed showed correlations between the protons mentioned above with the aromatic carbons at δc 115.05 correlations between the protons mentioned above with the aromatic carbons at δc 115.05 (C-2′), (C-20), 119.91 (C-60), 115.03 (C-50) and 93.22 (C-8). Additionally, the 13C-NMR spectrum showed 15 119.91 (C-6′), 115.03 (C-5′) and 93.22 (C-8). Additionally, the 13C-NMR spectrum showed 15 signals signals corresponding to the base structure of flavonols and a signal at δc 175.84 corresponding to corresponding to the base structure of flavonols and a signal at δc 175.84 corresponding to a carbonyl a carbonylgroup group(C-4). The (C-4). other The carbon other atom carbon assignments atom assignments were made with were the made support with of HMBC the support experiments of HMBC experiments(see Supplementary (see Supplementary Material) and Material) the corresponding and the corresponding correlations are correlations shown in Figure are shown 3. Complete in Figure 3. Completeassignments assignments are listed are in listed Table in 1. Table1.

H H O H H O O H H O H H O O OH O H FigureFigure 3. 3.HMBC HMBC correlations correlations of quercetagetin. quercetagetin.

1 13 TableTable 1. H 1. and1H andC-NMR 13C-NMR data data of of quercetagetinquercetagetin (2 (2) )and and patuletin patuletin (3) ((ppm).3) (ppm).

3 3 2 a 2 a Measured b Literature c Measured b Literature c δc (J in δc (J in Position δ δH (J in Hz) Position δ δH (J in Hz) δc (J in Hz)δ δH (J in Hz) Position c Hz) δ (J in Hz) Position Hz)c δ (J in Hz) c δ (J in Hz) (J in Hz) H (J in Hz) H (J in Hz) H 4 175.84 4 177.58 177.86 45 175.84153.58 4 9 158.46 177.58 d 158.76 177.86 d d d 57 153.58148.81 9 5 153.61158.46 d 153.89158.76 d d d 74′ 148.81147.57 5 7 153.00153.61 d 153.28153.89 d 0 d d 4 2 147.57146.64 74′ 153.00148.82 149.10153.28 2 146.64 40 148.82 149.10 6 145.88 2 148.28 148.56 6 145.88 2 148.28 148.56 3′ 145.04 3′ 146.22 146.51 30 145.04 30 146.22 146.51 33 135.32135.32 3 3 136.96 136.96 137.25 137.25 99 128.53128.53 6 6 132.17 132.17 132.47 132.47 10 1′ 122.21122.21 1 10′ 124.14124.14 124.43 124.43 60 119.91 7.537.53 (dd, (dd,J = 8.5, J = 2.2)8.5, 60 121.72 7.647.64 dddd ((JJ= = 8.5, 2.2) 122.027.65 7.65 dd dd (J ( J= =8.4, 8.4, 2.2) 6′ 119.91 6′ 121.72 122.02 50 115.59 6.88 (d, 2.2)J =8.5) 20 116.228.5, 7.74 2.2) d (J = 2.2) 116.52 7.762.2) (J = 2.2) 0 0 2 5′ 115.03115.59 7.666.88 (d, (d,J = J 2.2)= 8.5) 5 2′ 116.22116.02 7.74 6.89 d (Jd = (2.2)J = 8.5) 116.52 116.31 7.76 6.92 (J = d 2.2) (J = 8.5) 102′ 103.31115.03 7.66 (d, J = 2.2) 10 5′ 116.02 104.95 6.89 d (J = 8.5) 116.31 105.25 6.92 d (J = 8.5) 810 93.22103.31 6.49 (s) 10 8 104.95 94.70 6.50 s 105.25 95.00 6.52 s OCH 60.97 3.89 s 61.27 3.92 s 8 93.22 6.49 (s) 8 3 94.70 6.50 s 95.00 6.52 s C5-OH 12.25 (s) OCH3 60.97 3.89 s 61.27 3.92 s C6-OH 10.48 (s) C30-OHC5-OH 9.55 12.25 (s) (s) C40-OHC6-OH 9.28 10.48 (s) (s) C3-OHC3’-OH 9.19 9.55 (s) (s) C7-OHC4’-OH 8.65 9.28 (s) (s) C3-OHa b 9.19 (s) c d 700 MHz, DMSO-d6; 700 MHz, CD3OD; 400 MHz, CD3OD; these values may be interchangeable. C7-OH 8.65 (s)

a 700 MHz, DMSO-d6; b 700 MHz, CD3OD; c 400 MHz, CD3OD; d these values may be interchangeable. + The molecular formula of 2 was verified by HR-DART-MS as C15H11O8 by an [M + H] ion + peak at m/Thez 319.04553, molecular formula indicating of 2 thewas molecular verified by formulaHR-DART-MS was C as15 HC1510HO118O.8 Compoundby an [M + H]3 wasion peak isolated as a yellowat m/z 319.04553, powder and indicating identified the molecular as patuletin. formula The was HR-DART-MS C15H10O8. Compound of 3 showed 3 was a ionisolated peak as at a m/z + 333.6114 [M + H] (calcd. for C16H13O8: 333.06104) indicating the molecular formula was C16H12O8. MoleculesMolecules2018 2017, 23,, 22 2579, x FOR PEER REVIEW 4 of4 18 of 18

yellow powder and identified as patuletin. The HR-DART-MS of 3 showed a ion peak at m/z 333.6114 + The[M structure + H] (calcd. was for elucidated C16H13O8: 333.06104) by 1D and indicating 2D NMR the experiments molecular formula carried was out C at16H 70012O MHz8. The instructure CD3OD, andwas compared elucidated with by 1D the and structure 2D NMR reported experiments in [11 carried] the displacements out at 700 MHz were in CD very3OD, similar, and compared however, with the structure reported in [11] the displacements were very similar, however, the values of the the values of the quaternary carbons at δc 158.46, 153.61 and 153.00 could be interchangeable due to quaternary carbons at δc 158.46, 153.61 and 153.00 could be interchangeable due to1 their proximity their proximity and the last two only correlate with the proton H-8 (δH 6.50). The H-NMR spectrum and the last two only correlate with the proton H-8 (δH 6.50). The 1H-NMR0 spectrum displayed four displayed four aromatic protons signals at δH 7.74 (1H, d, J = 2.2 Hz, H-5 ), 7.64 (1H, dd, J = 8.5, 2.2 Hz, aromatic protons signals at δH 7.74 (1H, d, J = 2.2 Hz, H-5′), 7.64 (1H, dd, J = 8.5, 2.2 Hz, H-6′), 6.89 H-60), 6.89 (1H, d, J = 8.5 Hz, H-20) and 6.50 (1H, s, H-8), methyl connected to an oxygen protons (1H, d, J = 8.5 Hz, H-2′) and 6.50 (1H, s, H-8), methyl connected to an oxygen protons were displayed were displayed at δH 3.88 (OCH3, s, 3H). The assignments of the carbons and protons of patuletin in at δH 3.88 (OCH3, s, 3H). The assignments of the carbons and protons of patuletin in comparison with comparison with NMR literature data are given in Table1. NMR literature data are given in Table 1. 2.2. Antiproliferative Activity 2.2. Antiproliferative Activity To determine the concentration of flavonols required to inhibit the proliferation of CaSki, To determine the concentration of flavonols required to inhibit the proliferation of CaSki, MDA- MDA-MB-231 and SK-Lu-1 by half (IC50), 7500 cells were cultured for 24 h with 6, 12, 25, 50 and MB-231 and SK-Lu-1 by half (IC50), 7500 cells were cultured for 24 h with 6, 12, 25, 50 and 100 μg/mL µ 100of g/mLquercetin, of quercetin,quercetagetin quercetagetin or patuletin. or After patuletin. 24 h, the After number 24 h, of the cells number was evaluated of cells wasusing evaluated crystal usingviolet crystal staining violet (Figure staining 4, Table (Figure 2). 4, Table2).

FigureFigure 4. 4.Dose-response Dose-response curves curves of of thethe antiproliferativeantiproliferative effecteffect of quercetin, quercetagetin quercetagetin and and patuletin. patuletin.

Table 2. Antiproliferative activity of the quercetin, quercetagetin and patuletin compounds in tumor Tablecell 2.linesAntiproliferative 1. activity of the quercetin, quercetagetin and patuletin compounds in tumor cell lines 1. Compound/Cell line CaSki MDA-MB-231 SK-LU-1 QuercetinCompound/Cell line CaSki39 (129) MDA-MB-231 55 (129) SK-LU-1 39 (181) QuercetagetinQuercetin 3988 (129) (276) 55 (129) 60 (188) 39 (181) 19 (59) PatuletinQuercetagetin 8837 (276) (111) 60 (188) 86 (258) 19 (59) 18 (54.17) Patuletin 137 IC (111)50 μg/mL (μM). 86 (258) 18 (54.17) 1 IC50 µg/mL (µM).

Molecules 2018, 23, 2579 5 of 18

Results showed a dose-dependent proliferation inhibition (Table2). The IC 50 values calculated were between 37 and 88 µg/mL (Table2). It is interesting to note that the three cell lines showed Molecules 2017, 22, x FOR PEER REVIEW 5 of 18 different susceptibilities. The IC50 values of the three compounds for the SK-Lu-1 cell line suggest that they could beResults more showed effective a dose-dependent for this particular proliferation type of inhibition cancer. (Table 2). The IC50 values calculated were between 37 and 88 μg/mL (Table 2). It is interesting to note that the three cell lines showed 2.3. Necroticdifferent Activity susceptibilities. in Tumor The Cells IC50 values of the three compounds for the SK-Lu-1 cell line suggest that they could be more effective for this particular type of cancer. The necrotic activity of quercetin, quercetagetin and patuletin was evaluated to determine whether2.3. they Necrotic induce Activity necrosis in Tumor (Figure Cells 5). CaSki, MDA-MB-231 and SK-Lu-1 cultures were exposed to the compoundsThe necrotic at theactivity aforementioned of quercetin, querceta IC50 values,getin and and patuletin the amount was evaluated of LDH to released determine into the culturewhether supernatant they induce after treatmentnecrosis (Figure was 5). used CaSki, as MDA-MB-231 a measure of and the SK-Lu-1 loss of cultures plasma-membrane were exposed integrity.to In addition,the compounds the three at cancer the aforementioned cell lines were IC50 values, treated and with the Tritonamount X-100 of LDH in released independent into the experiments,culture and thesupernatant LDH released after treatment was adjusted was used to 100% as a measur to servee of asthe a loss positive of plasma-membrane control. Our resultsintegrity. show In that quercetinaddition, and patuletin the three cancer do not cell manifest lines were necrotic treated cellwith deathTriton activity,X-100 in independent as evidenced experiments, by the fact and that the the LDH released was adjusted to 100% to serve as a positive control. Our results show that quercetin cellular decrease observed in the treated cultures was not a consequence of a necrotic process. This and patuletin do not manifest necrotic cell death activity, as evidenced by the fact that the cellular findingdecrease suggests observed that some in the other treated type cultures of cell was death, not a consequence distinct from of a necrosis, necrotic process. was responsible This finding for the decreasesuggests in the that number some other of cells. type Inof cell contrast, death, dist theinct amount from necrosis, of LDH was released responsible in the for cellsthe decrease treated with quercetagetinin the number did indicate of cells.significant In contrast, the necrotic amount activity of LDH (15–30%), released in suggestingthe cells treated that with part quercetagetin of the decrease in the numberdid indicate of cells significant using this necrotic compound activity was (15–30%), due to su aggesting necrotic that process. part of the As decrease a result, in we the would number suggest that theof structure cells using of this quercetagetin, compound was which due to hasa necrotic a hydroxyl process. group As a result, in C6, we could would besuggest important that the for this structure of quercetagetin, which has a hydroxyl group in C6, could be important for this compound’s compound’s necrotic activity. It is important to mention that there are no existing reports of necrotic necrotic activity. It is important to mention that there are no existing reports of necrotic activity in activityrelation in relation to any to of any these of three these molecules. three molecules.

Figure 5. LDH activity induced by quercetin, quercetagetin and patuletin on CaSki, MDA-MB-231 Figure 5. LDH activity induced by quercetin, quercetagetin and patuletin on CaSki, MDA-MB-231 and SK-Lu-1 cultures. In each case, 7500 cells/well were seeded in 96-well tissue culture plates. and SK-Lu-1 cultures. In each case, 7500 cells/well were seeded in 96-well tissue culture plates. After After 24 h, the medium was removed, and the cells were stimulated with the IC values for quercetin, 24 h, the medium was removed, and the cells were stimulated with the IC50 values50 for quercetin, quercetagetinquercetagetin and patuletin, and patuletin, or EtOH or EtOH (10 µ L/mL).(10 μL/mL). The amountThe amount of lactate of lactate dehydrogenase dehydrogenase (LDH) (LDH) released into the culture supernatant was measured after 24 h of treatment. Experimental data are presented as

mean ± S.D. for three independent experiments with three repetitions. * p < 0.05 versus EtOH (ANOVA followed by a Tukey’s test). Molecules 2017, 22, x FOR PEER REVIEW 6 of 18

released into the culture supernatant was measured after 24 h of treatment. Experimental data are Moleculespresented2018, 23, as 2579 mean ± S.D. for three independent experiments with three repetitions. * p < 0.05 versus6 of 18 EtOH (ANOVA followed by a Tukey’s test).

2.4.2.3. ApoptoticApoptotic BodiesBodies underunder DAPIDAPI StainingStaining ApoptosisApoptosis isis characterizedcharacterized byby morphologicalmorphological changeschanges andand chromatinchromatin condensationcondensation thatthat produceproduce smaller,smaller, moremore compactcompact nucleinuclei and/orand/or thethe formationformation ofof apoptoticapoptotic bodies.bodies. OurOur CaSki,CaSki, MDA-MB-231MDA-MB-231 andand SK-Lu-1SK-Lu-1 cellcell culturescultures werewere stimulatedstimulated withwith quercetin,quercetin, quercetagetinquercetagetin andand patuletinpatuletin toto evaluateevaluate morphologicalmorphological changes, changes, chromatin chromatin condensation condensation and and the the formation formation of apoptotic of apoptotic bodies. bodies. The latter The werelatter identifiedwere identified by staining by withstaining fluorochrome with fluorochrome 4,6-diamidino-2-phenylindole 4,6-diamidino-2-phenylindole (DAPI) and epifluorescence (DAPI) and microscopyepifluorescence observation microscopy (Figures observation6–8). (Figures 6–8).

FigureFigure 6.6.Morphology Morphology and and DAPI DAPI staining. staining. CaSki cellCaSki cultures cell werecultures exposed were to quercetin,exposed quercetagetinto quercetin,

andquercetagetin patuletin atand the patuletin aforementioned at the aforementioned IC50 values. CamptothecinIC50 values. Camptothecin was used as was a positive used as control a positive for apoptosis.control for Theapoptosis. phase-contrast The phase-contrast images evidenced images cellular evidenced shapes cellular under shapes the different under experimentalthe different conditions.experimental The conditions. control and The EtOH control cells and had extendedEtOH cells cytoplasm had extended with chromatin cytoplasm distributed with chromatin in the nucleardistributed space, in asthe shown nuclear by space, the fluorescent as shown distribution by the fluorescent of DAPI. distribution After treatment, of DAPI. both After the cytoplasmtreatment, andboth nucleus the cytoplasm were contracted, and nucleus and severalwere contracted, apoptotic bodiesand several were visibleapoptotic (arrows), bodies Scale were bars visible 100 (arrows), microns. Scale bars 100 microns.

Molecules 2018, 23, 2579 7 of 18 Molecules 2017, 22, x FOR PEER REVIEW 7 of 18

FigureFigure 7. Morphology7. Morphology and and DAPI DAPI staining. staining. MDA-MB-231 MDA-MB-231 cell cell cultures cultures were were exposed exposed to to quercetin, quercetin,

quercetagetinquercetagetin and and patuletin patuletin at theat the aforementioned aforementioned IC50 ICvalues.50 values. Camptothecin Camptothecin was was used used as aas positive a positive controlcontrol for for apoptosis. apoptosis. The The phase-contrast phase-contrast images images evidenced evidenced cellular cellular shapes shapes under under the the different different experimentalexperimental conditions. conditions. The The control control and and EtOH EtOH cells cells had had extended extended cytoplasm cytoplasm with with chromatin chromatin distributeddistributed in thein the nuclear nuclear space, space, as shownas shown by by the the fluorescent fluorescent distribution distribution of DAPI.of DAPI. After After treatment, treatment, bothboth the the cytoplasm cytoplasm and and nucleus nucleus were were contracted, contracted, and and several several apoptotic apoptotic bodies bodies were were visible visible (arrows), (arrows), ScaleScale bars bars 100 100 microns. microns.

Observations under phase-contrast illumination revealed that the morphology of the control and vehicle cells was conserved, with a polyhedral shape and extended cytoplasm. In these conditions, chromatin is distributed throughout the nucleoplasm (see DAPI staining). After treatment with quercetin, quercetagetin or patuletin, the morphology of the cells was clearly altered, as they showed shrinkage, evidenced by the loss of the polyhedral form. Compact nuclei and apoptotic bodies were also visible, suggesting the presence of the apoptotic process in the CaSki (Figure6), MDA-MB-231 (Figure7), and SK-Lu-1 (Figure8), cells. Despite these results, it is still necessary to detect the activation of effector caspases such as active caspase-3, to establish that a caspase-dependent apoptotic process has been activated. Thus, more conclusive experiments were performed, as described below.

Molecules 2018, 23, 2579 8 of 18 Molecules 2017, 22, x FOR PEER REVIEW 8 of 18

Figure 8. Morphology and DAPI staining. view of SK-Lu-1 cultures, were exposed to quercetin, Figure 8. Morphology and DAPI staining. view of SK-Lu-1 cultures, were exposed to quercetin, quercetagetin and patuletin at the aforementioned IC50 values. Camptothecin was used as a positive quercetagetin and patuletin at the aforementioned IC50 values. Camptothecin was used as a positive controlcontrol forfor apoptosis.apoptosis. The The phase-contrast phase-contrast images images evidence cellular shapes under thethe differentdifferent experimentalexperimental conditions.conditions. The The control control and and EtOH cells had extended cytoplasmcytoplasm withwith chromatinchromatin distributeddistributed in the nuclear space, as shown by the fluorescentfluorescent distribution of DAPI. After treatment, bothboth thethe cytoplasmcytoplasm and nucleus were contracted, and several apoptotic bodies were visible (arrows), ScaleScale barsbars 100100 microns.microns.

DetectionObservations of Active under Caspase-3, phase-contrast Active Caspase-8, illumination Active revealed Caspase-9 that the and morphology PARP of the control and vehicleThe cells caspases was conserved, are a family with of cysteinea polyhedral proteases shape that and function extended as cytoplasm. crucial mediators In these of conditions, apoptosis. Polychromatin (ADP-ribose) is distributed polymerases throughout (PARPs) the are nucleopl a familyasm of related(see DAPI enzymes staining). that playAfter an treatment important with role inquercetin, DNA repair. quercetagetin PARP is or a cleavagepatuletin, that the morphology occurs during of increasedthe cells was levels clearly of active altered, caspase-3, as they showed which isshrinkage, likely the evidenced most well-understood by the loss of ofthe the polyhedral mammalian form. caspases Compact in termsnuclei of and its apoptotic specificity bodies and role were in apoptosis.also visible, Caspase-3 suggesting is the also presence required of for the some apoptotic typical process hallmarks in the of CaSki apoptosis (Figure and 6), is indispensableMDA-MB-231 for(Figure apoptotic 7), and chromatin SK-Lu-1 condensation (Figure 8), cells. and DNADespite fragmentation. these results, Likewise, it is still itnecessary is essential to fordetect certain the processesactivation associated of effector with caspases the formation such as of active apoptotic caspase-3, bodies. Theto establish apoptosis that process a caspase-dependent can be conducted inapoptotic two classic process signaling has pathways:been activated. one extrinsic, Thus, more the other conclusive intrinsic. experiments were performed, as describedThe extrinsic below. pathway is mediated by caspase-8, while caspase-9 is determinant in the intrinsic pathway. In this study, we identified active caspase-3 and PARP by means of western blots to Detection of Active Caspase-3, Active Caspase-8, Active Caspase-9 and PARP determine the presence of the apoptotic process. In addition, we immunodetected active caspase-3, activeThe caspase-8 caspases and are active a family caspase-9 of cysteine to evaluate proteases the presencethat function of these as crucial enzymes mediators in the three of apoptosis. cell lines Poly (ADP-ribose) polymerases (PARPs) are a family of related enzymes that play an important role in DNA repair. PARP is a cleavage that occurs during increased levels of active caspase-3, which is

Molecules 2017, 22, x FOR PEER REVIEW 9 of 18 likely the most well-understood of the mammalian caspases in terms of its specificity and role in apoptosis. Caspase-3 is also required for some typical hallmarks of apoptosis and is indispensable for apoptoticMolecules chromatin 2017, 22, x FOR condensation PEER REVIEW and DNA fragmentation. Likewise, it is essential for9 ofcertain 18 processeslikely associated the most well-understoodwith the formation of the of mammalianapoptotic bodies. caspases The in apoptosisterms of its process specificity can andbe conducted role in in twoapoptosis. classic signaling Caspase-3 pathways: is also required one extrinsic, for some typical the other hallmarks intrinsic. of apoptosis and is indispensable for Theapoptotic extrinsic chromatin pathway condensation is mediated and by caspase-8,DNA fragment whileation. caspase-9 Likewise, is determinant it is essential in for the certain intrinsic pathway.processes In this associated study, withwe identifiedthe formation active of apoptoti caspase-3c bodies. and The PARP apoptosis by means process of can western be conducted blots to determinein two the classic presence signaling of the pathways: apoptotic one process.extrinsic, Inthe addition, other intrinsic. we immunodetected active caspase-3, active caspase-8The extrinsic and active pathway caspase-9 is mediated to evaluate by caspase-8, the presence while caspase-9 of these isenzymes determinant in the in threethe intrinsic cell lines treatedpathway. with quercetin, In this study, quercetagetin we identified and active patuletin. caspase-3 Finally, and allPARP of theseby means caspases of western were blotsevaluated to Molecules 2018, 23, 2579 9 of 18 using determineflow cytometry the presence to obtain of the quantitative apoptotic process. results. In addition, we immunodetected active caspase-3, Resultsactive caspase-8 show that and the active expression caspase-9 of toactive evaluate caspas the e-3presence and PARP of these proteins enzymes occurred in the three in all cell three lines cell treated with quercetin, quercetagetin and patuletin. Finally, all of these caspases were evaluated lines treatedtreated with with quercetin, querceti quercetagetinn, quercetagetin and patuletin. and patuletin Finally, (Figure all of these 9). caspasesThis confirms were evaluated the participation using using flow cytometry to obtain quantitative results. of theflow apoptotic cytometry process to obtain during quantitative elimination results. of cells treated with the compounds used in this study. ResultsResults show show that that the the expression expression of of active active caspase-3 caspase-3 and and PARP PARP proteins proteins occurred occurred in in all all three three cell cell The quantitative evaluation of active caspase-3 by cytometry (Figure 10) showed that the number of lineslines treated treated with with quercetin, quercetin, quercetagetin quercetagetin and and patuletin patuletin (Figure (Figure9). 9). This This confirms confirms the the participation participation cells positive to active caspase-3 had different percentages: SK-Lu-1 cells presented greater ofof the the apoptotic apoptotic process process during during elimination elimination of of cells cells treated treatedwith withthe the compoundscompounds usedused inin thisthis study.study. susceptibilityTheThe quantitative quantitative to quercetin evaluation evaluation and ofquercetagetin,of activeactive caspase-3caspase-3 sinc bye cytometry these presented (Figure (Figure a 10)10 higher) showed showed percentage that that the the number numberof positive of cells ofcomparedcells cells positive positive to theto to otheractive active twocaspase-3 caspase-3 cell types had (Table differentdifferen 3).t percentages:percentages:Conversely, SK-Lu-1SK-Lu-1MDA-MB-231 cellscells presentedpresented proved to greatergreater be more sensitivesusceptibilitysusceptibility to patuletin to to quercetin quercetin(Table 3). and and Our quercetagetin, quercetagetin, morphological since sinc ande these these biochemical presented presented a aresults higher higher percentageevidence percentage the of of positivepresence positive of apoptosiscellscells compared duringcompared elimination to to the the other other of two twothe cell cancercell types types cells (Table (Table treated3). 3). Conversely, Conversely, with quercetin, MDA-MB-231 MDA-MB-231 quercetagetin proved proved toand to be bepatuletin. more more sensitivesensitive to to patuletin patuletin (Table (Table3). 3). Our Our morphological morphological and and biochemical biochemical results results evidence evidence the the presence presence of of apoptosisapoptosis during during elimination elimination of of the the cancer cancer cells cells treated treated with with quercetin, quercetin, quercetagetin quercetagetin and and patuletin. patuletin.

Figure 9. Expression of active caspase-3 and PARP in CaSki, MDA-MB-231 and SK-Lu-1 cell cultures FigureFigure 9. 9.Expression Expression of of active active caspase-3 caspase-3 and and PARP PARP in in CaSki, CaSki, MDA-MB-231 MDA-MB-231 and and SK-Lu-1 SK-Lu-1 cell cell cultures cultures exposed to quercetin, quercetagetin and patuletin at the aforementioned IC50 values. exposedexposed to to quercetin, quercetin, quercetagetin quercetagetin and and patuletin patuletin at at the the aforementioned aforementioned IC IC5050values. values.

Figure 10. Detection of active caspase-3 in CaSki, MDA-MB-231 and SK-Lu-1 cell cultures exposed to FigureFigurequercetin, 10. Detection 10. Detection quercetagetin of active of active and caspase-3 caspase-3 patuletin in in CaSki,at CaSki, the aforementioned MDA-MB-231 MDA-MB-231 and andIC50 SK-Lu-1 values.SK-Lu-1 cellCells cell cultures werecultures analyzed exposed exposed toby to quercetin,FACS from quercetagetin Cell Quest; and numbers patuletin indicate at the the aforementioned percentages of IC caspase-350 values. activity. Cells were analyzed by FACS quercetin, quercetagetin and patuletin at the aforementioned IC50 values. Cells were analyzed by from Cell Quest; numbers indicate the percentages of caspase-3 activity. FACS from Cell Quest; numbers indicate the percentages of caspase-3 activity.

Molecules 2018, 23, 2579 10 of 18

Molecules 2017, 22, x FOR PEER REVIEW 10 of 18 Table 3. Percentage of positive cells to active caspase-3 in cultures of CaSki, MDA-MB-231 and SK-Lu-1 Table 3. Percentage of positive cells to active caspase-3 in cultures of CaSki, MDA-MB-231 and SK- cells treated with IC50 values of quercetin, quercetagetin and patuletin. Lu-1 cells treated with IC50 values of quercetin, quercetagetin and patuletin. CaSki MDA-MB-231 SK-LU-1 CaSki MDA-MB-231 SK-LU-1 QuercetinQuercetin Control 4 3 4 Control 4 3 4 EtOH 5 3 5 EtOH 5 3 5 Quercetin 14 37 45 QuercetinCamptothecin 14 30 35 37 60 45 Camptothecin 30 35 60 Quercetagetin Quercetagetin Control 4 4 4 Control EtOH4 6 5 4 5 4 EtOH Quercetagetin6 31 20 5 45 5 QuercetagetinCamptothecin 31 50 25 20 50 45 Camptothecin 50 25 50 Patuletin Control 3 5Patuletin 3 Control EtOH3 4 5 5 5 3 EtOH Patuletin4 40 43 5 36 5 PatuletinCamptothecin 40 65 32 43 43 36 Camptothecin 65 32 43

Active caspase-9 and active caspase-8 were also present in the treated cancer cells, though the Active caspase-9 and active caspase-8 were also present in the treated cancer cells, though the numbers of cells positive to caspase-9 detection were significantly higher than those that were positive numbers of cells positive to caspase-9 detection were significantly higher than those that were to caspase-8 (Figure 11). This finding suggests that quercetin, quercetagetin and patuletin induce the positive to caspase-8 (Figure 11). This finding suggests that quercetin, quercetagetin and patuletin apoptotic process through the intrinsic apoptotic pathway (see Supplementary Material). induce the apoptotic process through the intrinsic apoptotic pathway (see Supplementary Material).

Figure 11.11. PercentagePercentage of of positive positive cells cells to to active ca caspase-8spase-8 and caspase-9 treated with quercetin, quercetagetin, andand patuletin.patuletin.

2.5.2.4. Effect of Quercetin, Quercetagetin and Patuletin on Non-Tumor CellsCells The mainmain compounds compounds currently currently used used in chemotherapy in chemotherapy present present problems problems in terms in of terms their selectiveof their activityselective on activity malignant on malignant cells and becausecells and they because produce they undesirable produce undesirable side effects. side We effects. thus considered We thus itconsidered crucial to it determine crucial to thedetermine selectivity the ofselectivity the compounds of the compounds tested in the tested present in the work, present and work, to evaluate and to theirevaluate antiproliferative their antiproliferative activity inactivity non-tumor in non-tu cellsmor in cells order in toorder reach to conclusionsreach conclusions concerning concerning their potentialtheir potential anticancer anticancer activity. activity. Our research Our research has demonstrated has demonstrated the anti-proliferative the anti-proliferative activity of activity quercetin, of quercetagetinquercetin, quercetagetin and patuletin and in patuletin CaSki, MDA-MB-231, in CaSki, MDA-MB-231, and SK-Lu-1 and tumor SK-Lu-1 cells; tumor however, cells; their however, effect their effect on non-tumor cells has not yet been determined. To test the selectivity of the compounds evaluated, we used lymphocytic cells. A sample of an enriched lymphocyte population (ELP) from peripheral blood was treated with the three compounds to assess the proliferation of this cellular

Molecules 2018, 23, 2579 11 of 18 on non-tumor cells has not yet been determined. To test the selectivity of the compounds evaluated, we used lymphocytic cells. A sample of an enriched lymphocyte population (ELP) from peripheral bloodMolecules was 2017 treated, 22, x FOR with PEER the REVIEW three compounds to assess the proliferation of this cellular fraction.11 ELPs of 18 from a normal blood donor were induced for three cycles of proliferation by phyto-hemagglutinin and labeledfraction. with ELPs 5(6)-carboxyfluorescein from a normal blood donor diacetate wereN-succinimidyl induced for three ester cycles (CFSE). of Theseproliferation cells were by treatedphyto- withhemagglutinin quercetin, quercetagetinand labeled with and patuletin5(6)-carboxyfluorescein after 48 h of culture diacetate (second N proliferation-succinimidyl cycle) ester and (CFSE). then culturedThese cells for were another treated 72 h. with The cellsquercetin, were harvested, quercetagetin and theirand patuletin proliferative after potential 48 h of wasculture analyzed (second by flowproliferation cytometry. cycle) and then cultured for another 72 h. The cells were harvested, and their proliferativeTo evaluate potential the anti-proliferativewas analyzed by flow potential cytometry. of the flavonoids on lymphocytes, cells were culturedTo evaluate at the IC the50 anti-proliferativeconcentration values potential for 24of the h and flavonoids then labeled on lymphocytes, with 5(6)-carboxyfluorescein cells were cultured diacetateat the IC50N concentration-succinimidyl values ester (CSFE).for 24 h Fluorescenceand then labeled was with measured 5(6)-carboxyfluorescein using a cytometer. diacetate A culture N- withoutsuccinimidyl flavonoids ester was(CSFE). used Fluorescence as a positive was control, measured while eluentsusing a werecytometer. used as A negative culture controls.without Normalflavonoids lymphocytes was used as were a positive induced control, for twowhile cycles eluents of were proliferation used as negative by phyto-hemagglutinin controls. Normal (Figurelymphocytes 12). Quercetin,were induced quercetagetin for two cycles and patuletinof proliferation all produced by phyto-hemagglutinin significant proliferation (Figure at 12). the concentrationQuercetin, quercetagetin employed. and patuletin all produced significant proliferation at the concentration employed.

Figure 12.12. Evaluation of the anti-proliferative effect of quercetin, quercetagetin and patuletin in lymphocytes.lymphocytes. The compoundscompounds showedshowed nono evidenceevidence ofof anyany anti-proliferativeanti-proliferative effecteffect onon normalnormal cells.cells.

Cells treatedtreated only only with with the the vehicle vehicle only only were were used used as an as experimental an experimental control control (EtOH). (EtOH). They showed They 100%showed proliferation 100% proliferation (Figure 12 );(Figure however, 12); when howeve the lymphocytesr, when the werelymphocytes treated at were the aforementioned treated at the ICaforementioned50 values of the IC compounds50 values of their the behaviorcompounds differed, their asbehavior the proliferative differed, potentialas the proliferative of lymphocytes potential was negatively-affectedof lymphocytes was by negatively-affected treatment with 86 µbyg/mL treatment of patuletin. with 86 The μg/mL treatment of patuletin. with quercetin The treatment affected with all thequercetin cells evaluated affected inall a proportionthe cells evaluated of approximately in a proportion 50%. In contrast, of approximately quercetagetin 50%. did notIn affectcontrast, the proliferationquercetagetin activity did not of affect the lymphocytes. the proliferation activity of the lymphocytes.

2.6.2.5. Necrotic Effect of Quercetin, QuercetagetinQuercetagetin andand PatuletinPatuletin inin Non-TumorNon-Tumor CellsCells To determinedetermine thethe necroticnecrotic effecteffect ofof differentdifferent flavonoidsflavonoids onon normalnormal cells,cells, humanhuman lymphocyteslymphocytes werewere inducedinduced toto proliferateproliferate byby phytohemagglutininphytohemagglutinin for 72 h, and thethe amountamount ofof LDHLDH releasedreleased intointo thethe mediamedia waswas measured.measured. The The percentage percentage of of LDH released after treating thethe lymphocyteslymphocytes withwith quercetin,quercetin, quercetagetinquercetagetin andand patuletinpatuletin waswas belowbelow 8%.8%. Upon comparing this percentage to the amount releasedreleased byby thethe tumortumor cells,cells, itit waswas determineddetermined thatthat the necrotic effecteffect insideinside non-tumornon-tumor cellscells waswas notnot significant. Our results show that treatment with the compounds evaluated—quercetin, quercetagetin and patuletin—did not cause any necrotic effect in the lymphocytes. This result is suggestive of a selective effect of quercetin, quercetagetin and patuletin in non-tumor cells (Figure 13).

Molecules 2018, 23, 2579 12 of 18 significant. Our results show that treatment with the compounds evaluated—quercetin, quercetagetin and patuletin—did not cause any necrotic effect in the lymphocytes. This result is suggestive of a selectiveMolecules effect 2017, 22 of, x quercetin, FOR PEER REVIEW quercetagetin and patuletin in non-tumor cells (Figure 13). 12 of 18

Figure 13. Evaluation of necrosis by quercetin, quercetagetin and patuletin on normal cell lymphocytes. Figure 13. Evaluation of necrosis by quercetin, quercetagetin and patuletin on normal cell The medium was removed, and the cells were stimulated to the level of IC50 values for quercetin, quercetagetinlymphocytes. and The patuletin, medium aswas well removed, as with and EtOH the (10cellsµ wereL/mL). stimulated After 24 to h, the the level amount of IC of50 values lactate for dehydrogenasequercetin, quercetagetin (LDH) released and patule from thetin, culture as well supernatant as with EtOH was (10 evaluated. μL/mL). After Experimental 24 h, the dataamount are of presentedlactate dehydrogenase as mean ± S.D. for(LDH) three released independent from experimentsthe culture supernatant with three repetitions. was evaluated. Experimental data are presented as mean ± S.D. for three independent experiments with three repetitions. 3. Discussion 3. Discussion Cancer is still considered the most detrimental disease for humans. Extensive research suggests that quercetinCancer mayis still be considered an antiproliferative the most agentdetrimenta [24] andl disease that it for interacts humans. with Exte DNAnsive [25 research], causes suggests tumor regressionthat quercetin [26] and may apoptosis be an antiproliferative [27]. For this agent reason, [24] itand is that important it interacts to broaden with DNA our [25], knowledge causes tumor of otherregression compounds [26] and derived apoptosis from [27]. natural For products, this reason, since it is it important is well-known to broade thatn changes our knowledge in the general of other structurecompounds of flavonoids derived functionfrom natural to enhance products, many since biological it is activitieswell-known [28]. that In terms changes of their in structure,the general however,structure the of flavonoids flavonoids function quercetin, to enhance quercetagetin many biol andogical patuletin activities differ [28]. only In terms in their of their substitution structure, patternshowever, in C6, the as flavonoids quercetin hasquercetin, a hydrogen quercetagetin atom, quercetagetin and patuletin has a diff hydroxyler only group, in their and substitution patuletin haspatterns a methoxy in C6, group, as quercetin at this has position. a hydrogen Additionally, atom, quercetagetin all three compounds has a hydrox haveyl agroup, catechol, and C2–C3patuletin unsaturationhas a methoxy and 3-hydroxylgroup, at this groups, position. which Addition have beenally, reportedall three tocompounds participate have in various a catechol, biological C2–C3 activitiesunsaturation [29]. Biological and 3-hydroxyl activity hasgroups been, which attributed have to been phytochemicals reported to obtained participate from in species various of biologicalTagetes worldwideactivities [[29].30], includingBiological cytotoxicactivity has and been antiproliferative attributed to action phytochemicals [31]. However, obtained before from the presentspecies of study,Tagetes no worldwide one knew if [30], quercetagetin including cytotoxic and patuletin and antiproliferative molecules were capableaction [31]. of inducing However, apoptosis before the and/orpresent necrosis, study, orno if one they knew would if alsoquercetagetin affect normal and cells patuletin at the samemolecules time andwere in capable the same of modelsinducing whenapoptosis compared and/or to quercetinnecrosis, or [32 if– 35they]. Ourwould study also presents affect normal interesting cells at evidence the same which time demonstratesand in the same thatmodels quercetin, when quercetagetin compared to and quercetin patuletin [32–35]. do indeed Our havestudy anti-proliferative presents interesting activity. evidence Moreover, which it appearsdemonstrates likely that thequercetin, change quer in C6cetagetin of the flavonoid and patuletin skeleton do ofindeed quercetagetin have anti-proliferative and patuletin couldactivity. beMoreover, a potentiator it appears of anti-proliferative likely that the activity change in SK-Lu-1in C6 of cells,the flavonoid as evidenced skeleton by comparing of quercetagetin the IC50 ’sand usedpatuletin in the treatmentscould be described.a potentiator of anti-proliferative activity in SK-Lu-1 cells, as evidenced by comparingOur results the also IC50 show’s used that in quercetinthe treatments and patuletin described. do not produce a necrotic effect. Quercetagetin was presentOur results only in also tumor show cell lines.that quercetin These findings and suggestpatuletin that do cell not elimination produce a is notnecrotic achieved effect. throughQuercetagetin necrotic was cell present death; rather,only in treatingtumor cell these lines. cancer These cell findings lines withsuggest flavonoids that cell elimination evidenced theis not presenceachieved of apoptosis,through necrotic distinguished cell death; by rather, the classic treating morphological these cancer changes cell lines of cytoplasmicwith flavonoids and evidenced nuclear compaction.the presence The of presence apoptosis, of activedistinguished caspase-3, bycaspase-8, the classic caspase-9 morphological and cleaved changes PARP of cytoplasmic in the treated and cellsnuclear lends compaction. additional support The presence to this statement.of active caspase-3, We could caspase-8, speculate caspase-9 that the substitution and cleaved of PARP the –OH in the grouptreated for thecells methoxyl lends additional group in support C6 between to this quercetagetin statement. andWe could patuletin speculate [21] accounts that the for substitution this. What of hasthe been –OH ascertained group for isthe that methoxyl quercetin group is capable in C6 betwee of inducingn quercetagetin apoptosis and [36 ].patuletin Our work [21] shows accounts that for quercetin,this. What quercetagetin has been ascertained and patuletin is that all causequercetin significant is capable nuclear of inducing fragmentation apoptosis and [36]. have Our a great work capacityshows tothat induce quercetin, caspase-3 quercetagetin activation, and sometimes patuletin even all cause greater significant than camptothecin, nuclear fragmentation a well-known and inducerhave a of great apoptosis capacity [37]. to induce caspase-3 activation, sometimes even greater than camptothecin, a well-known inducer of apoptosis [37]. Our results clearly show that quercetin, quercetagetin and patuletin all have good cytotoxic activity against different cancer cell lines, and that they achieve cell elimination through the ordered cell-death process called apoptosis. In addition, the results indicate that these compounds induce the intrinsic apoptotic pathway. Also, quercetin and quercetagetin evidenced a selective pro-apoptotic activity against the SK-Lu-1 lung cancer cell line. In this regard, it is important to emphasize that the

Molecules 2018, 23, 2579 13 of 18

Our results clearly show that quercetin, quercetagetin and patuletin all have good cytotoxic activity against different cancer cell lines, and that they achieve cell elimination through the ordered cell-death process called apoptosis. In addition, the results indicate that these compounds induce the intrinsic apoptotic pathway. Also, quercetin and quercetagetin evidenced a selective pro-apoptotic activity against the SK-Lu-1 lung cancer cell line. In this regard, it is important to emphasize that the occurrence of side effects during current chemotherapy treatments are related to the necrotic activity caused by the compound(s) applied. In addition to demonstrating the pro-apoptotic action exerted by quercetin, quercetagetin and patuletin inside tumor cells, we were also able to determine that these compounds show a selective effect on non-tumor cells, since when normal lymphocytes were treated with the doses applied to the cancer cell lines, the necrotic effect measured was not statistically-significant. Finally, and contrary to their effect on tumor cells, the compounds evaluated herein exhibited a non-antiproliferative effect inside non-tumor cells, relevant data that could be used in other investigations [38] with flavonoids [39]. All these results allow us to propose these flavonoids and modifications in substitution patterns in C6 as options that have selective activity against cancer cells but do not damage normal cells, indicating that they may have future utilization in cancer treatment or other biological activities.

4. Materials and Methods

4.1. Instrumentation Melting points were determined using a Fisher Scientific (Pittsburg, PA, USA) apparatus. NMR spectra were recorded on an Advance III 700 MHz spectrometer (Bruker, Billerica, MA, USA) with the residual solvent peak as reference. HR-MS-DART data were obtained by an AccuTOF JMS-T100LC mass spectrometry system (Jeol Ltd., Tokyo, Japan). HPLC-UV analysis were run by an Agilent 1200 Series Binary SL system (Agilent, Carpinteria, CA, USA) equipped with a 2996 UV-Vis diode array detector (Waters Corporation, Milford, MA, USA). The samples were dissolved in methanol and analyzed on a Luna 3 µm C18 (100 × 2.0 mm, 100 Å) column (Phenomenex, Torrance, CA, USA); the mobile phase was composed by water (A) and methanol (B). The elution was performed in gradient conditions, starting from 20 to 100 of B in 30 min; the rate flow was 0.2 mL/min and the UV detection was fixed at 254 nm. The separation of quercetagetin and patuletin were carry out by reverse-phase thin layer chromatography plates (20 × 10 cm, 0.25 mm) (Macherel-Nagel, Düren, Germany). The extract was obtained concentrating in under vacuum using a rotary evaporator (R-100, Büchi, Meierseggstrasse, Switzerland). Quercetin was purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA).

4.2. Plant Materials Flowers of Tagetes erecta and Tagetes patula were collected from October–November in Puebla and the State of Mexico; Mexico. Two prepared herbarium specimens were authenticated and deposited in the National Herbarium of Mexico (MEXU), under folio numbers 1409057 and 1409058 respectively.

4.3. Extraction and Isolation Quercetagetin and patuletin were isolated from Tagetes erecta and Tagetes patula flowers respectively. Once the flowers were cut, we separated the ligules from the receptacles, then, 500 g of each flower were subjected to a static maceration at room temperature in ethanol (1L) for 24 h, the solution was filtered, and ethanol was concentrated in vacuum using a rotary evaporator to give a residue (10 g). The separation of extracts (100 mg) was performed utilizing reverse-phase thin layer chromatography plates (20 × 10 cm, 0.25 mm) eluting with ethyl acetate/methanol/water 7.8:1.2:1.0. This separation conditions were utilized for each extract to afford quercetagetin (70 mg) and patuletin (30 mg). Molecules 2018, 23, 2579 14 of 18

4.4. Compound Characterization

◦ 1 13 Quercetagetin (2). Yellow solid; m.p. 255–260 C; H-NMR (700 MHz, DMSO-d6) and C-NMR (176 MHz, DMSO) see Table1. HR-DART-MS (positive ion mode) m/z 319.04553 [M + H]+ (Calcd. for C15H11O8: 319.04539); HPLC-UV area peak = 95.17%, retention time = 16.454 min; UV (CH3OH) λmax 258.0, 361.7 nm.

◦ 1 13 Patuletin (3). Yellow solid, 260–263 C. H-NMR (700 MHz, Methanol-d4) and C-NMR (175 MHz, + Methanol-d4) see Table1. HR-DART-MS (positive ion mode) m/z 333.06114 [M + H] (Calcd. for C16H13O8: 333.06104); HPLC-UV area peak = 89.47%, retention time = 19.865 min; UV (CH3OH) λmax 256.8, 367.1 nm.

4.5. Cell Culture The following cell lines were purchased from the American Type Culture Collection (ATCC Rockville, MD, USA): CaSki, (cervical cancer), MDA-MB-231 (breast cancer), and SK-Lu-1 (lung cancer). They were cultured in RPMI-1640 medium (GIBCO, Invitrogen Corp., Grand Island, NY, USA) containing 5% Newborn Calf Serum (NCS, GIBCO, Invitrogen Corp., Grand Island, NY, USA) with red phenol supplemented by benzylpenicillin. All cultures were stored in a humidified atmosphere with ◦ 5% CO2 at 37 C. All cell-based assays were performed using cells in the exponential growth phase.

4.6. Cell Proliferation Assay Assays were performed by seeding 7500 cells/well in 96-well tissue cultured plates in a volume of 100 µL of RPMI-1640 medium supplemented with 5% NCS per well. Cells were allowed to grow for 24 h in the culture medium prior to exposure to the compounds. Also, 1% of vehicle (EtOH) was added to the control cells. Antiproliferative activity (IC50) was determined after 24 h by crystal violet staining [40]. Cellular density was determined by measuring absorbance at 590 nm on an Enzyme-linked ImmunoSorbent Assay (ELISA) plate reader (Tecan, Seestrasse, MÄ, Switzerland).

4.7. Determination of LDH Necrotic activity was determined by means of the LDH Cytotoxicity Assay Kit (BioVision, Milpitas, CA, USA). following the manufacturer’s instructions. LDH oxidizes lactate to pyruvate, which then reacts with the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium (INT) to produce formazan. The increase in the amount of formazan generated in the culture supernatant directly correlates to the increase in the number of lysed cells. Formazan dye is water-soluble and can be detected with a spectrophotometer at 500 nm [41].

4.8. Observation of Morphological Characteristics of Apoptotic Cells by Fluorescence Microscopy

Cells were cultured in glass coverslips and treated with the IC50 of quercetin, quercetagetin and patuletin for 24 h. They were then fixed in 2% paraformaldehyde in PBS at pH 7.2 for 20 min. After rinsing in the same buffer, they were stained with 4,6-diamidino-2-phenylindole (DAPI) and evaluated under an Eclipse E600 microscope (Nikon, Melville, NY, USA). Images were recorded with a DXM1200F digital camera (Nikon, Melville, NY, USA).

4.9. CFSE-Labeling Assay Heparinized blood samples were obtained from healthy human volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated using standard Hypaque (Sigma-Aldrich, St. Louis, MO, USA) gradient density centrifugation. The PBMCs were washed twice with RPMI 1640 medium (GIBCO) containing 10% NCS, penicillin (100 U/mL), and streptomycin (100 U/mL). The lymphocyte population was further enriched (ELP) by eliminating adherent cells (i.e., the cells were incubated ◦ at 37 C, 5% CO2 for 1 h, and non-adherent cells were harvested). The ELPs were re-suspended in RPMI-1640 medium at a concentration of 1 × 106 cells/mL and 5(6)-carboxyfluorescein diacetate Molecules 2018, 23, 2579 15 of 18

N-succinimidyl ester (CFSE) (Sigma-Aldrich) was added to the cell suspension to a final concentration of 12 µM, and then incubated for 15 min at room temperature in the dark. Labeling was completed by adding the same volume of NCS during 5 min at room temperature to quench the free CFSE. The labeled cells were washed five times with sterile PBS containing 10% NCS, counted, and then re-suspended in RPMI-1640 medium at 1 × 106 cells/mL [42]. Unstimulated, phytohaemagglutinin (PHA)-stimulated, or treated, cells were plated at 2 × 105 cells/mL in 96-well, flat-bottomed cell culture plates, and five replicate samples for each treated amount were prepared. Cells were incubated ◦ in a 5% CO2 incubator at 37 C for 72 h. Cultured cells were harvested, washed twice with PBS, fixed with 1% formaldehyde, and then analyzed by flow cytometry to acquire a minimum of 20,000 events from each sample. Data analysis was performed using CellQuest software (Becton–Dickinson, Franklin Lakes, NJ, USA).

4.10. Immunolocalization of Active Caspase-3, Active Caspase 8 and Active Caspase-9 by Flow Cytometry The CaSki, MDA-MB-231 and SK-Lu-1 cells were seeded at 105 cells/mL in 60-mm tissue culture plates and allowed to grow for 24 h in culture medium before being treated with their respective IC50. Cells were harvested with versene solution. For active caspase-3, caspase-8 and caspase-9 immunodetection, the cells were fixed and permeabilized in 50% methanol in PBS, washed in PBS, incubated with primary antibody anti-active-caspase-3 (Novous Biologicals, Littleton, CO, USA), anti-active-caspase-8 (Novous Biologicals, Littleton, CO, USA) or anti-active-caspase-9 (Novous Biologicals, Littleton, CO, USA), and then diluted in PBS (1:500) for 18 h. Next, the cells were washed and incubated with the secondary goat anti-rabbit antibody with FITC (SIGMA, St. Louis, MO, USA), and then diluted to 1:200 in PBS for 2 h. The samples were analyzed by flow cytometry, acquiring a minimum of 20,000 events from each sample. Data analysis was performed using CellQuest software (Becton–Dickinson, Franklin Lakes, NJ, USA) [43].

4.11. Western Blot Analysis Protein expression levels were evaluated using western blot assays. The CaSki, MDA-MB-231 and SK-Lu-1 cells were seeded at 105 cells/mL in 60-mm tissue culture plates and allowed to grow for 24 h in culture medium before being treated with their respective IC50. Cells were harvested with versene solution. Next, the cells were incubated for 15 min in lysis buffer (50 mMTris–Cl, pH 7.5; 150 mM NaCl, 0.1% SDS, 1 mM PMSF, 0.5% sodium deoxycholate, and 1% Nonidet P-40), supplemented with the complete protease inhibitor cocktail (Roche, Mannheim, Germany). Total proteins were measured by the Qubit system using the Qubit 3 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), as follows: 100 µg of total proteins were loaded onto a 12% SDS-PAGE gel before being transferred to polyvinylidene fluoride (PVDF) membranes, which were blotted using the SNAP i.d.® 2.0 Protein Detection System. Briefly: membranes were incubated in blocking buffer, then incubated with anti-active caspase-3 and anti-PARP antibodies at a dilution of 1:5000. After that, the proteins were tagged by incubation with peroxidase-conjugated secondary antibody (Jackson, Newmarket, UK) at 1:10,000 in blocking buffer. Using Horse Radish Peroxidase (HRP) as the substrate (Immobilon Western, Millipore Co., Darmstadt, Germany.), specific labeling was detected by chemiluminescence. Amersham Biosciences Hyperfilm (Amersham Biosciences, Corston, UK) was exposed to the membranes to detect chemiluminescence.

4.12. Statistical Analysis Means and standard deviations (SD) were calculated using Excel (Microsoft Office, 2016 Version, Redmond, WA, USA). Statistical analysis of differences was carried out by analysis of variance (ANOVA) using SPSS 10.0 for Windows (Microsoft, Redmond, WA, USA). A p value < 0.05 (Student’s t test) was considered significant. In all cases, the data represent three independent experiments performed in triplicate. Molecules 2018, 23, 2579 16 of 18

5. Conclusions Our conclusion, therefore, is that quercetin quercetagetin and patuletin all demonstrate antiproliferative activity in a dose-dependent manner, with only quercetagetin showing significant necrotic activity. All three compounds induced the intrinsic apoptotic route, but none of them affected the proliferation of normal cells. These results suggest that the presence of a methoxyl group in C6 enhances the potency of flavonols. In future studies, structural modifications will be made as we continue to conduct research on this type of biological activity in an effort to improve our understanding of these compounds and how they could be incorporated into applications to combat different types of cancer in the future.

Supplementary Materials: The following are available online. Figure S1: 1H-NMR spectrum of quercetagetin (2), Figure S2: 13C-NMR spectrum of quercetagetin (2), Figure S3: HSQC spectrum of quercetagetin (2), Figure S4: HMBC spectrum of quercetagetin (2), Figure S5: HR-DART-MS positive ion mode spectrum of patuletin (3), Figure S6: HR-DART-MS positive ion mode spectrum of quercetagetin (2), Figure S7: Detection of active caspase-8 in CaSki, MDA-MB-231 and SK-Lu-1 cell cultures exposed to quercetin, quercetagetin and patuletin at the aforementioned IC50 values, Figure S8: Detection of active caspase-9 in CaSki, MDA-MB-231 and SK-Lu-1 cell cultures exposed to quercetin, quercetagetin and patuletin at the aforementioned IC50 values. Author Contributions: J.J.A.-S. designed the study and performed the research. M.J.-E. and L.S.-S. were involved in the study design, organization, and resourcing and wrote the paper together with all other author. M.L.E. designed and performed microscopy assays. R.T.-H. designed and performed the chemical analysis. H.L.-M. and F.F.-G. designed and performed Flow Cytometry and LDH assays. All authors read and approved the final manuscript. Funding: This research was funded by SEP-CONACYT, Investigacion Cientifica Basica: 253979 and 255881; DGAPA, PAPIIT IG 200418, PAPIIT IN216718, IN220916 and IN225117; Fernando Flores Guzman acknowledge a postdoctoral fellowship from DGAPA/UNAM and Jesus Javier Alvarado Sansininea received fellowship from CONACYT, 440947. Acknowledgments: Jesus Javier Alvarado Sansininea is a doctoral student from Posgrado en Ciencias Biologicas. The authors are grateful to, Instituto de Química, UNAM; Beatriz Quiroz Garcia, Isabel Chavez Uribe, Luis Velazco Ibarra and Francisco Javier Perez Lopez for technical assistance; Paul C. Kersey Johnson for reviewing the English word usage and grammar and Evaristo Javier, the Tagetes plantation owner. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds quercetin, quercetagetin and patuletin are available from the authors.

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