biology

Article Noninvasive and Safe Cell for Breast Cancer MCF-7 Cells Using Natural Food Pigment

Kyohei Yamashita 1,*, Ryoma Tagawa 2, Yoshikazu Higami 2 and Eiji Tokunaga 1

1 Department of Physics, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan; [email protected] 2 Laboratory of Molecular and Metabolic Disease, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan; [email protected] (R.T.); [email protected] (Y.H.) * Correspondence: [email protected]; Tel.: +81-3-5228-8214

 Received: 9 July 2020; Accepted: 10 August 2020; Published: 14 August 2020 

Abstract: A dye exclusion test (DET) was performed to determine the viability of human breast cancer cells MCF-7, using natural food pigments as compared with trypan blue (TB), a typical synthetic dye for DET known to exhibit teratogenicity and cytotoxicity. We demonstrated that Monascus pigment (MP) is noninvasive to living cells and can effectively stain only dead cells. This study is the first verification of the applicability of MP to cancer cells. The appropriate MP concentration was 0.4% (0.02% as the concentration of pure MP) and all the dead cells were stained within 10 min. We found that the cell proliferation or the reduced nicotinamide adenine dinucleotide (NADH) activity of living cells was maintained over 48 h. Although 0.1% TB did not show an increase in dead cells, a marked decrease in NADH activity was confirmed. In addition, even when MP coexisted with cisplatin, of dead cells was maintained for 47 h, indicating stability to drugs (reagents). The cost of MP is estimated to be about 1/10 of TB. The fact that MP can be used as a cell viability determination reagent for Euglena and Paramecium, as shown in preceding papers, and also for MCF-7, as shown in this paper, indicates the possibility of application in more cells of different species.

Keywords: viability assay; dye exclusion test; natural food pigment; Monascus; human breast cancer cell; MCF-7; noninvasive; trypan blue

1. Introduction In vitro tests are faster, easier to perform and to quantify, usually cheaper, and allow studies of isolated steps [1]. In vitro, chemical substances such as drugs and pesticides have various cytotoxic mechanisms such as destruction of cell membranes and prevention of protein synthesis [2]. To identify cell death caused by these toxicities, the fields of toxicology and pharmacology require inexpensive, reliable, and reproducible short-term cytotoxicity and cell viability assays. In particular, in the case of a harmless method for assessing the viability of a target cell, more detailed information can be obtained by long-term monitoring of the viability. Furthermore, since it is not necessary to prepare the same number of samples for viability assay, the cost and labor can be reduced, and also the problem of non-uniformity between samples does not occur. Therefore, there should be a potential demand for a long-term, noninvasive, safe viability assay of the same sample. The following conventional methods to distinguish between live and dead cells in culture have been established:

Dye exclusion test (DET) Cells stained with a synthetic dye such as trypan blue (TB) are judged as dead cells [3].

Biology 2020, 9, 227; doi:10.3390/biology9080227 www.mdpi.com/journal/biology Biology 2020, 9, 227 2 of 14

Colony formation assay The number of live cells is evaluated by the number of colonies formed on an agar culture after an inoculation of diluted cell suspension and following a definite time of culture [4]. Enzyme activity assay Enzymatic reaction in living cells or enzymes leaking out of dead cells are used for viability assay [5]. analysis Dead cells are labeled with a fluorescent dye [6] and detected by fluorescence flow cytometry [7,8]. Optical method The dead or living state of cells is diagnosed by deflection change of the probe light beam [9]. Electro-orientation-based assay The positions of alternative electro-orientation of living or dead cylindrical yeast cells are dependent on the applied frequency [10]. However, these methods have difficulties such as requiring specialized techniques and equipment, damaging cells, inability to perform in situ measurement in the cultivation process, or only applicable for cells of a specific shape (non-spherical) [9–11]. Therefore, in order to solve these problems, we propose a DET using natural edible pigments that are safe for humans and minimally invasive to cells, as an alternative to a DET using conventional synthetic dyes that are toxic to humans and invasive to cells. Trypan blue (TB), a vital dye, has been conventionally used for the DET. TB is a widely used diazo dye for selectively coloring dead cells or tissues. The mechanism for TB to stain dead cells is based on its negative charge which prevents the incorporation of it into the living cells with the membrane negatively charged. Therefore, living cells are not stained, but dead cells with the compromised cell membrane are stained by TB [12–14]. However, TB is known to cause cell health and environmental problems due to its potential teratogenic effects [15,16]. It has also been pointed out that pore formation in cell membranes is possibly induced to increase membrane permeability [13]. As other dyes for the DET, methylene blue [17,18], eosin [19], nile blue [20], and amethyst violet [21] have been used but it is known that the selective permeability of the plasma membrane is destroyed or severely impaired [3], indicating that these dyes are toxic for cells. In order to avoid these problems, six natural food pigments (Monascus, purple sweet potato, yellow gardenia, green gardenia, red beet, and Spirulina) and a traditional synthetic dye (TB) for viability assay are tested in this study, among which Monascus pigment (MP) is of particular interest. MP is extracted from the Monascus sp. which is a kind of filamentous fungus. MP exhibits a red color due to a molecule of the pigment whose main component is monascorubrin [22]. It does not have a significant pH dependence of the color (although it tends to precipitate in acidic solution), has excellent stainability to proteins, and is relatively stable to heat [22]. MP is not easily affected by metal ions other than copper [23], whereas it is unstable against light irradiation, especially in an acidic condition. MP has been used for more than 1000 years as a food pigment and a folk medicine in China because an efficient production method was established using fermentation of rice. MP is a cost-effective and reproducible substrate, has variation in colors, is highly safe, and shows good solubility in water and ethanol [24,25]. Moreover, it possesses biological activities such as anticancer properties, anti-mutagenic activity, antibacterial activity, and potential anti-obesity activity [26]. Regarding the quality assurance of MP, it is based on the component standards in the Japan’s Specifications and Standards for Food Additives (JSFA) [27]. The details are described in the article of the example applied to Paramecium [28]. Animal cells for various pathological experiments do not have motility or characteristic pigments, making it difficult to judge using only visual microscopic observation. The breast cancer cell line, MCF-7 cells, which is widely available as an in vitro model for cancer research, has been used [29]. Cisplatin (cis-diamminedichloroplatinum (II)) is a widely used chemotherapeutic drug for the treatment of various types of cancer, including breast cancer [30,31]. In particular, MCF-7 cells are widely used in studies of estrogen receptor (ER, positive breast cancer cells [32,33]. To verify that this method is applicable to a wide variety of cells, first, we examined whether natural pigments are not toxic to human breast cancer cells MCF-7 cells in the presence or absence Biology 2020, 9, 227 3 of 14 of cisplatin and if they can be replaced with conventional synthetic dyes for viability assay. Then, the effect of natural pigments on cells with different properties was investigated, and the results were compared between the unicellular green alga Euglena [34] and the protozoan Paramecium [28], which differ in cell structure.

2. Materials and Methods

2.1. Sample Preparation Human breast cancer MCF-7 cells were cultured stationarily in Eagle’s Minimal Essential Medium (E-MEM) (Wako, Osaka, Japan) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin streptomycin (P/S) (Merck KGaA, Darmstadt, Germany), 10 µg/mL insulin (Wako), 1% MEM nonessential amino acids solution, and 1 mM sodium pyruvate solution at 37 ◦C and 5% CO2 on a 6-well plate (92006, TPP), 12-well plate (92012, TPP), and 96-well plate (92096, TPP). Cisplatin was dissolved in 90% dimethyl sulfoxide in phosphate buffered saline (90% DMSO in PBS) before use. Incubation period before and after addition of pigment for each experiment is shown in Table1.

2.2. Bright-Field Observation of Cells The following were used for bright-field observation in each test (listed in Table2):

A fluorescence microscope, BZ-9000; • A 4 objective lens, NA 0.20, CFI Plan Apochromat Lambda 4X, Nikon; • × A 20 objective lens, NA 0.75, CFI Plan Apochromat Lambda 20X, Nikon; • × Exposure time, 1/900 sec (4 objective lens, Figure1); • × Exposure time, 1/130 sec (20 objective lens, Figure2); • × Exposure time, 1/80 sec (20 objective lens, Figure3); • × Exposure time, 1/130 sec (20 objective lens, Figures4 and5). • × Only the experiments in Table3. “Pigment usable /unusable for viability assay” were performed under the following conditions (the objective lens is the same as above):

4 objective lens, 1/800 sec or 1/900 sec. • × 2.3. Preparation of Dead Cells of MCF-7 Cells (Common for All Experiments) Dead cells were obtained by the following two treatments:

Microwave (MW) treatment Cells were treated with microwave at 2.45 GHz until it was boiled. Cisplatin treatment Cisplatin (Wako) was added to the culture and adjusted to a final concentration of 20 µM. According to the report, the survival rate of MCF-7 cells at 20 µM of cisplatin was estimated to be about 80% [35]. In this report, the relationship between survival rate and cisplatin concentration was 85.67% (12 µM) and 76.03% (25 µM) (Figure 1B in reference [35], not in this paper). Biology 2020, 9, 227 4 of 14

Table 1. Incubation period before and after addition of pigment.

Table/Figure Incubation Period Incubation Period Experimental Item Number (Before Pigment Addition) (After Pigment Addition) Confirmation of staining by Table S3, Supplementary 2 day 12 h natural pigment Material Magnification of observation and staining Figure1 2 day 20 min of dead cells Confirmation of staining of dead cells Figure2 2 day 20 min Immersion time and staining of dead cells Figure3 2 day 1 min, 5 min, 10 min Staining of cells in a mixed solution of MP Figure4 2 day 14 h and cisplatin Confirmation of noninvasiveness Figure5 2 day 47 h of pigment Cytotoxicity assay (WST-8 *), Tukey test Figure6 2 day 24 h, 48 h * 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium.

2.4. Pigment for Viability Assay The following two types of pigments were used in this study for viability assay (the pigment concentrations in each experiment are shown in Table2):

Natural food pigment Food pigment Red (Monascus, Watashinodaidokoro Co., Ltd. http:// watashinodaidokoro.net/); Synthetic dye Trypan blue (Merck KGaA).

Here, the component of “Food pigment Red” contains 95% w/w dextrin by weight, and the remaining 5% is pure MP. However, in the preceding papers that used Euglena and Paramecium as samples [28,34], the concentration of “Food pigment Red” was described as the concentration of “MP”. (Cf. The concentration of MP shown in Table2 is the concentration of “Food pigment Red” and the concentration of “pure” MP is 5% of that value.).

Table 2. Concentration of added reagent and observation magnification in each examination.

Pigment/Dye Concentration (%) Experimental Item Table/Figure Cisplatin (µM) Objective Lens Number Control MP TB Confirmation of staining by Table3-1 ※ - 0 4 /20 natural pigment × × Magnification of observation and staining of Figure1 0 1, 5 - 0 4 /20 × × dead cells Confirmation of staining of Figure2 0 0.4 0.1 0 20 dead cells × Immersion time and Figure3 0 0.4 0.1 0 20 staining of dead cells × Staining of cells in a mixed Figure4 0 1, 5 - 20 20 solution of MP and cisplatin × Confirmation of Figure5 0 0.4 0.1 0 20 noninvasiveness of pigment × Cytotoxicity assay (WST-8) Figure6 0 0.4 0.1 0 - ※ The pure MP concentration is 5% of the value in the table (cf. 0.4% MP in the table means 0.02% “pure MP” concentration).

2.5. Confirmation of Staining by Natural Pigment The MCF-7 cells were plated at a density of 5.0 105 cells/well in 6-well plates (10 mm2/well). × After 2 days, the MCF-7 cells were treated with six natural food pigments (Monascus, 0.5% W/W (purple sweet potato) or 1% W/W (Monascus, yellow gardenia, green gardenia, red beet, or Spirulina) in the presence or absence of 20 µM cisplatin, for 12 h. Then, cells mixed with Monascus or green gardenia were washed with PBS. Samples mixed with other pigments were not washed with PBS. Cells were Biology 2020, 9, 227 5 of 14 observed with a bright field microscope (4 objective lens). The pigment that enabled easy visual × discrimination of stained cells was defined as usable as a reagent for viability assay. The results are shown in Table3.

2.6. Magnification of Observation and Staining of Dead Cells The MCF-7 cells were plated at a density of 3.8 105 cells/well in 6-well plates. After 2 days, × the MCF-7 cells were microwaved until the medium boiled. After discarding the medium, cells were treated with 1 or 5% MP for 20 min, and then washed three times with PBS at room temperature. Cells treated with no pigment were used as the “control”. Cells on the bottom of the dish were observed with a bright-field microscope (4 or 20 objective lens). The results are shown in Figure1. × × 2.7. Confirmation of Staining of Dead Cells The MCF-7 cells were plated at a density of 1.5 105 cells/well in 12-well plates (4 mm2/well). × After 2 days, the MCF-7 cells were microwaved until the medium boiled. After discarding the medium, cells were treated with 0.4% MP or 0.1% TB for 20 min, and then washed three times with PBS at room temperature. Cells treated with no pigment were used as the “control”. Cells on the bottom of the dish were observed with a bright-field microscope (20 objective lens). The results are shown in Figure2. × 2.8. Immersion Time and Staining of Dead Cells The MCF-7 cells were plated at a density of 1.5 105 cells/well in 12-well plates (4 mm2/well). × After 2 days, the MCF-7 cells were microwaved until the medium was boiled. After discarding the medium, cells were treated with 0.4% MP or 0.1% TB for 20 min, and then washed three times with PBS at room temperature. Cells treated with no pigment were used as the “control”. Cells on the bottom of the dish were observed with a bright-field microscope (20 objective lens). The results are shown in × Figure3.

2.9. Staining of Cells in A Mixed Solution of MP and Cisplatin The MCF-7 cells were plated at a density of 3.8 105 cells/well in 6-well plates (10 mm2/well). × After 2 days, the MCF-7 cells were treated with 1 or 5% MP in the presence or absence of 20 µM cisplatin for 14 h. The medium was collected and the cells on the bottom of 6-well plates were washed three times with PBS (“bottom” sample). The medium was centrifuged at 200 g for 5 min, at 4 C. × ◦ After discarding the supernatant, the pellet was washed by PBS and centrifuged again. The pellet was suspended by PBS and moved to new 12-well plates as floating cell sample (“floating” sample). Cells treated with no pigment were used as the “control”. Cells were observed with a bright-field microscope (20 objective lens). The results are shown in Figure4. × 2.10. Confirmation of Noninvasiveness of Pigment The MCF-7 cells were plated at a density of 1.5 105 cells/well in 12-well plates (4 mm2/well). × After 2 days, the MCF-7 cells were treated with 0.4% MP or 0.1% TB for 47 h. The medium was collected and the cells on the bottom of the 6-well plates were washed three times by PBS (bottom sample). The medium was centrifuged at 200 g for 5 min, at 4 C. After discarding the supernatant, the × ◦ pellet was washed by PBS and centrifuged again. The pellet was suspended by PBS and moving new 12-well plates as floating cell sample (floating sample). Cells treated with no pigment were used as the “control”. Cells were observed with a bright-field microscope (20 objective lens). The results are × shown in Figure5.

2.11. Cytotoxicity Assay (WST-8) Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.) is a cell proliferation/ cytotoxicity measurement kit that uses the water-soluble tetrazolium salt WST-8 (2-(2-methoxy- Biology 2020, 9, 227 6 of 14

4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) as a coloring reagent. When WST-8 is reduced, WST-8 produces orange, water-soluble formazan with a maximum absorption around 460 nm. WST-8 is reduced by reduced nicotinamide adenine dinucleotide (NADH) produced by dehydrogenase in the cell via 1-methoxy PMS [36]. Since the absorbance is proportional to the number of cells and the enzymatic activity of the cells, it serves as an indicator of the health of the cells. The MCF-7 cells were plated at a density of 1.3 104 cells/well in 96-well plates (0.35 mm2/well). × After 2 days, the MCF-7 cells were treated with 100 µL of 0.4% MP or 0.1% TB for 24 or 48 h. Then, 10 µL of WST-8 was added. Samples were incubated for 3 h. Cells treated with no pigment were used as the “control”. Samples were measured for absorbance (TECAN Nano Quant Infinite M200 Pro, Tecan, Zurich, Switzerland). From the sample absorbance (450 nm), the absorbance of a solution in which an equivalent amount of the pigment (Figure6) and WST-8 were added to the fresh growth medium as a background was subtracted. Samples were measured in six independent wells. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey test using BellCurve for Excel software (Social Survey Research Information Co., Ltd., Tokyo, Japan). The average value of “the absorbance standard error (n = 6)” is shown in Figure6 and the results of the Tukey test are shown ± in Figure6.A p-value <0.05 was considered to be significant.

3. Results

3.1. Confirmation of Staining by Natural Pigment Six natural food pigments (Monascus, purple sweet potato, yellow gardenia, green gardenia, red beet, and Spirulina) were added to the cell suspension (1%). For each sample with pigment added, a sample with cisplatin added (20 µM) and a control sample with no pigment added were prepared. The pigments that could be easily visually discriminated by bright field microscope observation after 12 h, and in which more cells were stained in the cisplatin-added sample than in the control sample, were made usable as a reagent for viability assay. Trypan blue was determined to be usable because we confirmed that it was a conventional reagent for viability assay and that it could be usable by other tests (Figures2,3 and5). The data are shown in Supplementary Material, and the results are summarized in Table3.

Table 3. Pigment usable/unusable for viability assay.

Natural Food Pigment Synthetic Dye Purple Yellow Green Monascus Red beet Spirulina Trypan blue sweet potato gardenia gardenia

× × × × × # , usable as a reagent for viability assay; , unusable as a reagent for viability assay. # × # As a result of DET with six types of natural pigments, it was found that only MP could be used as a reagent for viability assay. A characteristic of MP that is different from other natural pigments is strong staining of intracellular proteins. The mechanism of staining with TB also results from binding to intracellular proteins [14]. In the preceding papers on DET of Euglena and Paramecium, long-term viability could be determined with anthocyanin pigments (purple sweet potato and red cabbage dye) in addition to MP [28,34]. This was thought to be due to differences in cell structure. Therefore, it was suggested that high affinity for the intracellular protein is a more suitable dye condition for DET.

3.2. Magnification of Observation and Staining of Dead Cells Cell death of MCF-7 cells was induced by microwave exposure. After discarding the medium, dead cells were treated with MP (1 or 5%) for 20 min, and then washed with PBS. Cells treated with no pigment were used as the “control”. Cells on the bottom of the dish were observed with a bright-field microscope (4 or 20 objective lens). × × Biology 2020, 9, 227 7 of 14

From Figure1, one can visually confirm the staining of the MW treated dead cell when observing the 1% MP with a 4 objective lens. In the observation of 5% MP dead cells with a 4 objective lens, × × even deeper staining was obtained, and it was found that in this concentration range, dead cells were deeply stained in proportion to the MP concentration. In addition, observation with a 20 objective lens × enabled us to distinguish individual stained cells with higher visibility. However, with respect to living cells, at a concentration of 0.6%, although the morphology of the cells did not change in 24 h, the results showed that the cells deformed into spheres in 48 h (data not shown). By contrast, no morphological Biology 2020, 9, x 7 of 14 change was observed at a concentration of 0.4%. Therefore, in the case where MP is used instead of TB in conventionalused instead DET of without TB in conventional performing DET without long-term performing monitoring long-term of monitoring viability assay,of viability it is assay, advantageous it is advantageous to use high concentration for the purpose of enhancing visibility. Nevertheless, to use highBiology concentration 2020, 9, x for the purpose of enhancing visibility. Nevertheless, when7 of 14 performing long-termwhen monitoring performing of viabilitylong-term assay,monitoring it is necessaryof viability toassay, adjust it is the necessary MP concentration to adjust the to MP 0.4%. concentrationused instead of to TB 0.4%. in conventional DET without performing long-term monitoring of viability assay, it is advantageous to use high concentration for the purpose of enhancing visibility. Nevertheless, when performing long-term monitoring of viability assay, it is necessary to adjust the MP concentration to 0.4%.

Figure 1. MagnificationFigure 1. Magnification of observation of observation and and staining staining of of dead dead cellscells (microwave (microwave treatment). treatment). All scale All scale bars bars designate 100 μm. designate 100 µm.

3.3. Confirmation of Staining of Dead Cells 3.3. ConfirmationFigure of 1. Staining Magnification of Dead of observation Cells and staining of dead cells (microwave treatment). All scale Cellbars designatedeath of 100MCF-7 μm. cells was induced by microwave exposure. After discarding the medium, Cell deathdead cells of were MCF-7 treated cells with was 0.4% induced MP or 0.1% by TB microwave for 20 min, and exposure. then washed After with PBS. discarding Cells treated the medium, dead cellswith3.3. were Confirmation no treatedpigment of withwere Staining used 0.4% of as Dead MP the orCells“control”. 0.1% TB Cells for on 20 the min, bottom and of thenthe dish washed were observed with PBS. with Cells a treated with no pigmentbright-fieldCell death were microscope of used MCF-7 as(20× cells the objective was “control”. induced lens). byBecause Cells microwave on of the exposure. light bottom red Afterbackground, of the discarding dish it werelooks the medium, as observed if the with a cellsdead arecells not were clearly treated stained, with 0.4% however, MP or staining 0.1% TB offor dead 20 min, cells and was then sufficiently washed with confirmed PBS. Cells by treated visual bright-field microscope (20 objective lens). Because of the light red background, it looks as if the cells observationwith no pigment with awere microscope.× used as the “control”. Cells on the bottom of the dish were observed with a are not clearlybright-fieldFigure stained, 2microscope shows however, the result(20× staining objective of confirming oflens). dead theBecause cellsstaining of was theof su MWlightffi cientlytreated red background, dead confirmed cells byit looks0.4% by visual MP,as if and the observation with a microscope.stainingcells are wasnot clearlyclearly stained,confirmed however, against staining the “control” of dead containing cells was nosufficiently pigment. confirmed Clear staining by visual was Figureconfirmedobservation2 shows also with the with a resultmicroscope. 0.1% TB. of confirming the staining of MW treated dead cells by 0.4% MP, Figure 2 shows the result of confirming the staining of MW treated dead cells by 0.4% MP, and and staining was clearly confirmed against the “control” containing no pigment. Clear staining was staining was clearly confirmed against the “control” containing no pigment. Clear staining was confirmedconfirmed also with also 0.1% with TB.0.1% TB.

Figure 2. Confirmation of staining of dead cells (microwave treatment). All scale bars designate 100 μm.

3.4. Immersion Time and Staining of Dead Cells Figure 2. FigureConfirmation 2. Confirmation of staining of staining of of deaddead cells cells (microwave (microwave treatment) treatment).. All scale bars All designate scale bars 100 designate μm. 100 µm.

3.4. Immersion Time and Staining of Dead Cells

Biology 2020, 9, 227 8 of 14

3.4. Immersion Time and Staining of Dead Cells Cell death of MCF-7 cells was induced by microwave exposure. After discarding the medium, dead cells were treated with MP (1 or 5%) for the indicated times, and then washed with PBS. Biology 2020, 9, x 8 of 14 Cells treated with no pigment were used as the “control”. Cells on the bottom of the dish were observed with a bright-fieldCell death microscope of MCF-7 (20cells wasobjective induced lens).by microwave Because exposure. of the After light discarding red background, the medium, it looks as if × the cells aredead not cells clearly were treated stained, with however, MP (1 or 5%) staining for the indicated of dead times, cells and was then su ffiwashedciently with confirmed PBS. Cells by visual observationtreated with with a microscope. no pigment were used as the “control”. Cells on the bottom of the dish were observed with a bright-field microscope (20× objective lens). Because of the light red background, it looks as if Figurethe3 cellsshows are not the clearly state stained, of staining however, of staining MW treatedof dead cells dead was cells sufficiently at each confirmed elapsed by visual time from the addition ofobservation the pigment with a to microscope. the cell suspension. On the one hand, in the 0.4% MP, almost all cells were stained in 5 min,Figure and 3 shows staining the ofstate all of cells staining was of confirmed MW treated in dead 10min. cells at The each time elapsed until time this from dyeing the is within a practicaladdition range. of On the the pigment other to hand, the cell almost suspension. all cellsOn the of one the hand, TB(0.1%) in the 0.4% were MP, stained almost all in cells 1 min. were There is no stained in 5 min, and staining of all cells was confirmed in 10 min. The time until this dyeing is within rigorous comparisona practical range. of visibility,On the other but hand, TB almost is at aall higher cells of the concentration, TB (0.1%) were consideringstained in 1 min. that There the is “pure MP” concentrationno rigorous is 5% comparison of the amount of visibility, of MP but added TB is at to a thehigher medium concentration, (i.e., pure considering MP concentration that the “pure is 0.02%). Therefore,MP” it is concentration reasonable is to 5% judge of the thatamount the of higher MP adde pigmentd to the medium concentration (i.e., pure MP in TBconcentration resulted is in a shorter staining time.0.02%). Therefore, it is reasonable to judge that the higher pigment concentration in TB resulted in a shorter staining time.

Figure 3. ImmersionFigure 3. Immersion time andtime stainingand staining of of dead dead cellscells (m (microwaveicrowave treatment). treatment). All scale All bars scale designate bars designate 100 μm. 100 µm. 3.5. Staining of Cells in a Mixed Solution of MP and Cisplatin 3.5. Staining of Cells in a Mixed Solution of MP and Cisplatin The MCF-7 cells were treated with MP (1 or 5%) in the presence or absence of cisplatin for 14 h. The MCF-7(a) Cells on cells the bottom were treatedof the dish with and (b) MP floating (1 or cells 5%) in the in medium the presence were washed or absence with PBS. of Cells cisplatin for 14 h. (a) Cellstreated on with the no bottom pigment of thewere dish usedand as the (b) “con floatingtrol”. Cells cells were in the observed medium with were a bright-field washed with PBS. microscope (20× objective lens). Cells treated withMP stained no pigment dead cells werewhen 20 used μM ascisplatin the “control”. was present Cellsin the live were cell observedsuspension, withas well a as bright-field microscope (20 objective lens). when it× was not (Figure 4). Living MCF-7 attached to the bottom of the dish, whereas dead cells MP stainedtended to dead float away cells from when it. The 20 detachmentµM cisplatin of dead was cells present in MCF-7 in could the live be due cell to suspension,the weakening as well as when it wasof cell not adhesion (Figure 4due). Living to cleavage MCF-7 of β attached-catenin because to the bottomβ-cateninof forms the dish,a complex whereas with deadcadherin cells tended involved in cell adhesion [37]. Regardless of the presence or absence of cisplatin, the “control” to float awaycontaining from no it. pigment The detachment and the sample of dead with 1% cells MP in showed MCF-7 very could few floating be due cells, to the and weakening 5% MP of cell β β adhesion due to cleavage of -catenin because -catenin forms a complex with cadherin involved in cell adhesion [37]. Regardless of the presence or absence of cisplatin, the “control” containing no pigment and the sample with 1% MP showed very few floating cells, and 5% MP showed many cells. Regarding cell staining, at 24 h and 48 h, 1% MP stained only floating cells, and 5% MP stained both Biology 2020, 9, 227 9 of 14 Biology 2020, 9, x 9 of 14 cells onshowed the dish many and cells. floating Regarding cells, cell regardless staining, ofat the24 h presenceand 48 h, 1% or MP absence stained of only cisplatin. floating Therefore, cells, and it was confirmed5% MP that stained MP was both not cells a ffonected the dish by theand presencefloating cells, of cisplatinregardlessfor of the 48 hpresence and stably or absence functions of as a reagentcisplatin. of viability Therefore, assay. it Thiswas confirmed is thought that to MP be was due not to theaffected fact by that the MPpresence is less of acisplatinffected for by 48 metal h ions and stably functions as a reagent of viability assay. This is thought to be due to the fact that MP is other than copper [23], and the platinum complex cisplatin did not affect staining. High concentrations less affected by metal ions other than copper [23], and the platinum complex cisplatin did not affect of MP werestaining. toxic High to MCF-7,concentrations as almost of MP all were cells toxic were to stainedMCF-7, as with almost 5% all MP, cells in were thepresence stained with or absence5% of cisplatin.MP, From in the the presence previous or absence report [of38 cisplatin.], the cell From proliferation the previous of MCF-7report [38], at MP the concentrationscell proliferation ofof 0.001%, 0.01%, andMCF-7 0.1% at MP were concentrations 100%, 140%, of 0.001%, and 0%, 0.01%, respectively. and 0.1% were The 100%, cell proliferation 140%, and 0%, valuesrespectively. shown The here are read fromcell the proliferation graphs in values the report. shown Whenhere are 1% read and from 5% the of graphs the MP in concentration the report. When in 1% Figure and 45% are of convertedthe MP concentration in Figure 4 are converted into “pure MP concentration”, they are 0.05% and 0.25%, into “pure MP concentration”, they are 0.05% and 0.25%, respectively. Therefore, the tendency about respectively. Therefore, the tendency about the MP concentration and the health condition of the cell the MPcoincide. concentration and the health condition of the cell coincide.

Figure 4.FigureStaining 4. Staining of cells of cells in a in mixed a mixed solution solution ofof MPMP and and cisplatin. cisplatin. (a) (Cellsa) Cells on the on bottom the bottom of dishes; of dishes; (b) Floating cells. All scale bars designate 100 μm. (b) Floating cells. All scale bars designate 100 µm.

3.6. Confirmation3.6. Confirmation of Noninvasiveness of Noninvasiveness of Pigmentof Pigment

The MCF-7 cells were treated with 0.4% MP or 0.1% TB for 47 h. (a) Cells on the bottom of the dish and (b) floating cells from the medium were washed with PBS. Cells treated with no pigment were used as the “control”. Cells were observed with a bright-field microscope (20 objective lens). × Due to the light red background, it looks as if the cells are not clearly stained, however, staining of dead cells was sufficiently confirmed by visual observation with a microscope. Biology 2020, 9, x 10 of 14

Biology 2020, 9,The 227 MCF-7 cells were treated with 0.4% MP or 0.1% TB for 47 h. (a) Cells on the bottom of the 10 of 14 dish and (b) floating cells from the medium were washed with PBS. Cells treated with no pigment were used as the “control”. Cells were observed with a bright-field microscope (20× objective lens). FigureDue 5to shows the light a bright-fieldred background, microscopic it looks as image if the cells of the are living not clearly cells afterstained, 47 however, h at the MP staining concentration of (0.4%) atdead which cells thewas viabilitysufficiently assay confirmed can beby visual performed. observation Almost with alla microscope. cells attached to the bottom of the Figure 5 shows a bright-field microscopic image of the living cells after 47 h at the MP dish were not stained by 0.4% MP. This was the same for 0.1% TB. The number of cells at 0.4% MP was concentration (0.4%) at which the viability assay can be performed. Almost all cells attached to the increasedbottom against of the the dish “control” were not containingstained by 0.4% no MP. pigment, This was while the same that for at 0.1% 0.1% TB. TB The was number comparable of cells to that of the “control”.at 0.4% MP Thewas numberincreased ofagainst floating the “control” cells was containing almost theno pigment, same as while that that of the at 0.1% “control” TB was for both pigments,comparable and the to number that of the was “control”. small. InThe addition, number of the floating staining cells ofwas floating almost cellsthe same is not as that so clear of the for both pigments“control” as compared for both pigments, with the and cells the attached number was to the small. bottom In addition, of the the dish. staining In particular, of floating cells the 0.4%is MP floatingnot cells so didclear not for appearboth pigments to be stained, as compared but thiswith was the cells due attached to the defocus, to the bottom and it of was the confirmeddish. In that particular, the 0.4% MP floating cells did not appear to be stained, but this was due to the defocus, they couldand beit was visually confirmed discriminated. that they could be visually discriminated.

Figure 5.FigureConfirmation 5. Confirmation of noninvasiveness of noninvasiveness ofof pigmentpigment (47 (47 h hpigment pigment immersion). immersion). (a) Cells (a )on Cells the on the bottom of dishes; (b) Floating cells. All scale bars designate 100 μm. bottom of dishes; (b) Floating cells. All scale bars designate 100 µm.

3.7. Immersion3.7. Immersion Time andTime Stainingand Staining of Deadof Dead Cells Cells The MCF-7 cells were treated with 0.4% MP or 0.1% TB for (a) 24 h or (b) 48 h. Then, the WST-8 Theassay MCF-7 was cellsperformed. were treatedCells treated with with 0.4% no MP pigm orent 0.1% were TB used for (a)as 24theh “control”. or (b) 48 Samples h. Then, were the WST-8 assay wasmeasured performed. for absorbance Cells (450 treated nm) and with the noaverage pigment value wereof “the usedabsorbance as the ± standard “control”. error Samples(n = 6)” were measuredwas for calculated. absorbance Differences (450 nm) between and thevalues average were analyzed value of using “the a absorbance Tukey test (* p standard< 0.05 and error** p < (n = 6)” ± was calculated.0.01). The Di resultsfferences of the between Tukey test values are shown were in analyzed Figure 6. using Although a Tukey no significant test (* p < decrease0.05 and in **cellp < 0.01). The resultsnumber of thewas Tukeyobserved test at 0.1% are shownTB, the absorbance in Figure6 shows. Although significantly no significant lower values decrease than the “control” in cell number and 0.4% MP. was observed at 0.1% TB, the absorbance shows significantly lower values than the “control” and Since 0.4% MP was the upper limit concentration without cell morphological change, the toxicity 0.4% MP.test was conducted only at this concentration. The WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4- Sincenitrophenyl)-5-(2,4- 0.4% MP wasdisulfophenyl)-2H-tetrazolium) the upper limit concentration assay was performed without cellat 24 morphologicalh and 48 h after change, the toxicitypigment test addition was to examine conducted the health only of atlive thiscells in concentration. the dye solution (Figure The 6). WST-8At both times, (2-(2-methoxy- the 4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium)absorbance of 0.1% TB was significantly lower than the other samples, suggesting assay a wasdecrease performed in cell at 24 h and 48number h after or pigmenta decrease in addition enzyme activity to examine (Figure the 6). Microscopic health of live observation cells in showed the dye that solution the number (Figure 6). of cells in 0.1% TB was comparable to that of the other samples (control, 0.4% MP), and that almost At bothno times cells, thewere absorbance stained, as shown of 0.1% in TBFigure was 5. significantly Therefore, on lowerthe one than hand, the the other enzymatic samples, activity suggesting is a decreaseconsidered in cell number to be remarkably or a decrease suppressed in enzyme in the livi activityng cells (Figure of 0.1%6 ).TB. Microscopic On the other observationhand, 0.4% MP showed that theis number noninvasive of cells to cells in due 0.1% to TBits higher was comparableabsorbance than to the that “control”. of the other samples (control, 0.4% MP), and that almost no cells were stained, as shown in Figure5. Therefore, on the one hand, the enzymatic activity is considered to be remarkably suppressed in the living cells of 0.1% TB. On the other hand, 0.4% MP is noninvasive to cells due to its higher absorbance than the “control”. Biology 2020, 9, 227 11 of 14 Biology 2020, 9, x 11 of 14

FigureFigure 6. 6.Cytotoxicity Cytotoxicity assayassay (WST-8). (a ()a 24) 24 h; h;(b) ( b48) 48h h.

4. Discussion4. Discussion As aAs result a result of DET of DET using using six six types types of naturalof natural pigments, pigments, only only MP MP stained stained deaddead cellscells ofof MCF-7MCF-7 with highwith visibility. high visibility. A unique A property unique property of MP is of that MP itis has that a it high has aaffi highnity affinity for intracellular for intracellular proteins. proteins. Since MP can beSince applied MP can to be DET applied even to in DETEuglena even andin EuglenaParamecium and Paramecium, the abovementioned, the abovementioned properties properties of MP of were MP were expected to be versatile in that they could target more cells of different species. The MP expected to be versatile in that they could target more cells of different species. The MP concentrations concentrations shown below are that of the commercial products and the concentration of pure MP shownis 5% below of that. are By that adjusting of the the commercial MP concentration products to 0.4% and (pure the concentration MP concentration of pure0.02%), MP all isthe 5% MW of that. By adjustingtreated dead the MPcells concentrationwere stained within to 0.4% 10 (pure min af MPter concentrationthe addition of0.02%), the pigment, all the and MW long-term treated dead cellsviability were stained assay withinover 48 10h was min realized. after the At addition this concentration of the pigment, there is andno morphological long-term viability change of assay the over 48 h wascells realized.and no inhibition At this concentrationof cell growth or there reduction is no morphologicalof NADH, so it is change noninvasive. of the Dead cells andcells nobecame inhibition of cellmore growth intense or and reduction the visibility of NADH, increased so itin isproportion noninvasive. to the Deadconcentration cells became of MP. moreIn fact, intense almost andall the visibilitycells increasedwere stained in at proportion 1% (Figures to 1 the and concentration 4). In this case, of long-term MP. In fact, noninvasiv almoste all DET cells could were be staineddifficult at 1% (Figuresbecause1 and the4). Inmorphological this case, long-term change occurred noninvasive on the DET second could day be diat ffi0.6%,cult but because it would the morphologicalhave the advantages of higher safety and less environmental impact than conventional DET with TB. change occurred on the second day at 0.6%, but it would have the advantages of higher safety and less Furthermore, when the MP concentration is 0.4% and the TB concentration is 0.1%, the cost of MP is environmental impact than conventional DET with TB. Furthermore, when the MP concentration is estimated to be about 1/10 of TB, which is economical. As a similar method, a technique with 0.4%erythrosine and the TB B concentration(EB, also known is 0.1%,as Red the No. cost 3) used of MP as isa estimatedfood additive to bewas about developed 1/10 of [39]. TB, This which is economical.synthetic Ascolorant a similar is an method,edible tar a dye technique that does with noterythrosine pass through B biological (EB, also membranes known as Redand is No. 3) usedcompatible as a food additivewith automatic was developed cell counters. [39 Because]. This syntheticEB was suspected colorant of is being an edible carcinogenic, tar dye the that FDA does not pass(Food through and biological Drug Administration) membranes banned and is the compatible use in food with (1990) automatic [40,41]. Although cell counters. the ban Because on EB was EB was suspectedremoved of beingafterwards, carcinogenic, most of the the EB FDA in the (Food USA ha ands been Drug replaced Administration) with Allura bannedRed (also the known use inas food (1990)Red [40 40).,41 ].However, Although Allura the Red ban is on banned EB was in many removed European afterwards, countries most because of the it is EB an in azo the dye USA [42,43]. has been replacedTherefore, with Allurait is difficult Red (alsoto monitor known the as noninvasive Red 40). However, viability assay Allura of a Red synthetic is banned food colorant in many over European a long period of time because of the concern regarding the influence on the human body. At a countries because it is an azo dye [42,43]. Therefore, it is difficult to monitor the noninvasive viability concentration of TB of 0.1%, no increase in dead cells was observed, but NADH activity was assay of a synthetic food colorant over a long period of time because of the concern regarding the significantly suppressed, indicating that a heavy load was imposed on the cells. Therefore, MP was influenceconfirmed on the to human be less body.invasive At aand concentration less costly th ofan TB TB. of In 0.1%, addition, no increase even when in dead a metal cells wascomplex observed, but NADHanticancer activity agent was such significantly as cisplatin coexists, suppressed, stable indicatingstaining of thatdead a cells heavy is maintained. load was imposed For example, on the a cells. Therefore,further MPpractical was confirmeduse of this technique to be less is invasiveexpected in and cooperation less costly with than a technique TB. In addition, for distinguishing even when a metalnormal complex cells anticancer from cancer agent cells such based as on cisplatin the difference coexists, in stablestaining staining properties of deadof edible cells natural is maintained. or For example,synthetic dyes a further under practical a multipho useton of laser this microscope technique [44]. is expected in cooperation with a technique for distinguishing normal cells from cancer cells based on the difference in staining properties of edible 5. Conclusions natural or synthetic dyes under a multiphoton laser microscope [44]. As a result of DET using six kinds of natural pigments, only MP with high affinity for proteins 5. Conclusionsstably stained dead cells of MCF-7 with cisplatin. This property of MP is expected to be versatile in that they can target more cells of various species. Dead cells stained more strongly in proportion to As a result of DET using six kinds of natural pigments, only MP with high affinity for proteins stably stained dead cells of MCF-7 with cisplatin. This property of MP is expected to be versatile in that they can target more cells of various species. Dead cells stained more strongly in proportion Biology 2020, 9, 227 12 of 14 to the concentration of MP, and visibility was increased, but morphological changes of cells were observed. In this case, long-term non-invasive DET may be difficult, but it has the advantage of being more safely, less environmentally impactive, and more economical than traditional DET using TB. Therefore, if synthetic dyes for DET are replaced with a natural food pigment, the burden on the cells in the viability assay in basic research such as drug discovery will be reduced, and it will be used as an additive to the culture solution. These advantages are not seen with synthetic dyes.

6. Patents Title, “Method and Kit for Cell Viability Assay”; patent application number, 2018-241789; date, 25 December 2018.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-7737/9/8/227/s1, Figure S1: Confirmation of staining of dead cells, Figure S2: Confirmation of staining of dead cells. Author Contributions: K.Y. and R.T. conceived the experiments; R.T. conducted the experiments; R.T. and K.Y. analyzed the results; K.Y., E.T., R.T., and Y.H. wrote the manuscript and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: In this section you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments). Conflicts of Interest: The authors declare no conflict of interest.

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