Antimalarial Drugs Enhance the Cytotoxicity of 5-Aminolevulinic Acid-Based Photodynamic Therapy Against the Mammary Tumor Cells of Mice in Vitro

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Article Antimalarial Drugs Enhance the Cytotoxicity of 5-Aminolevulinic Acid-Based Photodynamic Therapy against the Mammary Tumor Cells of Mice In Vitro

Tomohiro Osaki 1,* , Kiwamu Takahashi 2, Masahiro Ishizuka 2, Tohru Tanaka 2 and Yoshiharu Okamoto 1 1 Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan; [email protected] 2 SBI Pharmaceuticals Co., Ltd., Tokyo 106-6020, Japan; [email protected] (K.T.); [email protected] (M.I.); [email protected] (T.T.) * Correspondence: [email protected]; Tel.: +81-857-31-5434

Received: 30 September 2019; Accepted: 28 October 2019; Published: 29 October 2019

Abstract: Artemisinin and its derivatives, including artesunate (ART) and artemether (ARM), exert anticancer effects in the micromolar range in drug and radiation-resistant cell lines. Artemisinin has been reported to sensitize cervical cancer cells to radiotherapy. In the present study, we determined whether ART and ARM could enhance the cytotoxicity of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) against the mammary tumor cells of mice. The corrected PpIX fluorescence intensities in the control, 5-ALA, 5-ALA + ART, and 5-ALA + ARM groups were 3.385 3.730, 165.7 33.45, 139.0 52.77, and 165.4 51.10 a.u., respectively. At light doses of 3 ± ± ± ± and 5 J/cm2, the viability of 5-ALA-PDT-treated cells significantly decreased with ART (p < 0.01 and p < 0.01) and ARM treatment (p < 0.01 and p < 0.01). Besides, the number of annexin V-FITC and ethidium homodimer III-positive cells was greater in the 5-ALA-PDT with ARM group than that in the other groups. N-acetylcysteine could not significantly inhibit the percentages of apoptotic cells or inviable cells induced by 5-ALA-PDT with ARM. These reactive oxygen species-independent mechanisms might enhance cytotoxicity in 5-ALA-PDT with ARM-treated tumor cells, suggesting that the use of 5-ALA-PDT with ARM could be a new strategy to enhance PDT cytotoxicity against tumor cells. However, as these results are only based on in vitro studies, further in vivo investigations are required.

Keywords: 5-aminolevulinic acid; artemisinin; artesunate; antimalarial; artemether; cytotoxicity; photodynamic therapy; reactive oxygen species

1. Introduction Breast cancer is the most frequently diagnosed cancer and one of the most fatal diseases affecting women [1]. Surgery, radiation therapy, chemotherapy, and hormone therapy serve as the conventional treatment options for breast cancer. However, photodynamic therapy (PDT), a relatively new approach, has been recently adopted as a treatment strategy. PDT uses the synergistic effects of light and a photosensitizer to kill tumor cells. Photosensitizers, such as porphyrins, chlorins, and phthalocyanines are generally used in clinical PDT [2]. Another example of such a photosensitizer is protoporphyrin IX (PpIX), which is induced by the pro-drug, 5-aminolevulinic acid (5-ALA). Many studies have demonstrated that 5-ALA-induced PpIX can produce reactive oxygen species (ROS) when exposed to light, suggesting that 5-ALA-PDT may be a potential therapeutic modality for the treatment of malignant tumors [3,4]. Although 5-ALA-PDT is effective for malignant tumors, it may be insufficient to cure cancer [5]. Therefore, improving the efficacy of 5-ALA-PDT is needed.

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Artemisinin,tumors, it may a be chemical insufficient compound to cure cancer derived [5]. Therefore, from Artemisia improving annua the efficacy, has of been 5-ALA established-PDT is as a successfulneeded treatment. for malaria and is known to exert antineoplastic effects. In both cases, the effects of artemisininArtemisinin, are a chemical attributable compound to the derived endoperoxide from Artemisia component annua, has present been inestablished its structure. as a In the presencesuccessful of free iron,treatment iron-activated for malaria and artemisinin is known to induces exert antineoplastic damage by effects. the releasing In both cases of, highly the effects alkylating of artemisinin are attributable to the endoperoxide component present in its structure. In the presence carbon-centered radicals and ROS, such as the superoxide anion and hydroxyl radicals [6–8]. Cancer of free iron, iron-activated artemisinin induces damage by the releasing of highly alkylating carbon- cells arecentered characterized radicals and by ROS increased, such as expression the superoxide of transferrinanion and hydroxyl receptors, radicals which [6–8]. are Cancer responsible cells are for the iron uptakecharacterized and regulation by increased of intracellular expression of concentration. transferrin receptors Therefore,, which iron are responsible and heme metabolismfor the iron might have a relevantuptake and role regulation in the selectiveof intracellular antitumor concentration. activity Therefore, of artemisinin iron and [9]. heme metabolism might Varioushave a relevant derivatives role in the of selective artemisinin, antitumor includingactivity of artemisinin artesunate [9]. (ART), artemether (ARM), dihydroartemisininVarious (DHA),derivatives and of arteether, artemisinin, have including been identified artesunate [10, 11 (ART),]. First-generation artemether (ARM), semisynthetic dihydroartemisinin (DHA), and arteether, have been identified [10,11]. First-generation artemisinins include the lipophilic artemisinins, arteether, and ARM. ART, however, is a water-soluble semisynthetic artemisinins include the lipophilic artemisinins, arteether, and ARM. ART, however, derivativeis a [ water9]. Artemisinin-soluble derivative and its [9]. derivatives, Artemisinin which and its are derivatives, commonly which used are in commonly malaria used therapy, in have been reportedmalaria totherapy, exhibit have anticancer been reported effects to in exhibit the micromolar anticancer effects range in against the micromolar drug and range radiation-resistant against cell linesdrug [12 and–14 radiation]. In addition,-resistant they cell lines exhibit [12–14 anti-cancer]. In addition, activity they exhibit against anti- acancer variety activity of cancer against cellsa and have beenvariety reported of cancer to rapidly cells and convert have been to ROS reported inside to rapidly cells, ultimately convert to disrupting ROS inside cells,cellular ultimately functions [15]. ART is well-tolerateddisrupting cellular by functions patients, [15 with]. ART little is well toxicity-tolerated and by alack patients, of evident with little side toxicity effects and [16 a ].lack Artemisinin of evident side effects [16]. Artemisinin has been reported to sensitize cervical cancer cells to has beenradiotherapy, reported towhile sensitize DHA enhances cervical the cancer effects cells of 5-ALA to radiotherapy,-PDT in esophageal while cancer DHA cells enhances [17,18]. ART the effects of 5-ALA-PDThas been in reported esophageal to kill cancer breast cells cancer [17 cells,18]. via ART caspase has been-dependent reported apoptosis to kill mediated breast cancer by cells via caspase-dependentintracellular ROS accumulation apoptosis mediated promoted byby intracellulariron [19]. Previously, ROS accumulation we reported that promoted ART displays by iron [19]. Previously,great wepotential reported in enhancing that ART the displays efficacy of great 5-ALA potential-based sonodynamic in enhancing therapy the for effi thecacy treatment of 5-ALA-based of sonodynamiccancer [20 therapy]. for the treatment of cancer [20]. In the present study, we aimed to determine whether ART and ARM could enhance the In the present study, we aimed to determine whether ART and ARM could enhance the cytotoxicity cytotoxicity of 5-ALA-PDT against an in vitro human cancer model, the mammary tumor cells of of 5-ALA-PDTmice. against an in vitro human cancer model, the mammary tumor cells of mice. 2. Results 2. Results 2.1. Protoporphyrin IX Fluorescence in EMT-6 Cells 2.1. Protoporphyrin IX Fluorescence in EMT-6 Cells Microscopically, differences in fluorescence intensity and cell form were not observed among the Microscopically, differences in fluorescence intensity and cell form were not observed among groupsthe (Figure groups1). (Figure 1).

Figure 1. Protoporphyrin IX ﬂuorescence in EMT-6. Cells were incubated with 1 mM 5-aminolevulinic acid (5-ALA) (A,D,G), and 1 mM 5-ALA plus concentrations of 7.8 µM artesunate (B,E,H) or artemether (C,F,I) for 4 h. Transmission, ﬂuorescence, and merged images of EMT-6. Upper panel (A–C): Transmitted light images. Middle panel (D–F): Fluorescence images. Lower panel (G–I): Merged images. Scale bar = 20 µm. Molecules 2019, 24, x FOR PEER REVIEW 3 of 10 Molecules 2019, 24, x FOR PEER REVIEW 3 of 10

Figure 1. Protoporphyrin IX fluorescence in EMT-6. Cells were incubated with 1 mM 5-aminolevulinic Figure 1. Protoporphyrin IX fluorescence in EMT-6. Cells were incubated with 1 mM 5-aminolevulinic acid (5-ALA) (A,D,G), and 1 mM 5-ALA plus concentrations of 7.8 μM artesunate (B,E,H) or acid (5-ALA) (A,D,G), and 1 mM 5-ALA plus concentrations of 7.8 μM artesunate (B,E,H) or artemether (C,F,I) for 4 h. Transmission, fluorescence, and merged images of EMT-6. Upper panel (A– artemether (C,F,I) for 4 h. Transmission, fluorescence, and merged images of EMT-6. Upper panel (A– C): Transmitted light images. Middle panel (D–F): Fluorescence images. Lower panel (G–I): Merged MoleculesC): 2019Transmitted, 24, 3891 light images. Middle panel (D–F): Fluorescence images. Lower panel (G–I): Merged3 of 10 images. Scale bar = 20 μm. images. Scale bar = 20 μm. The level of 5-ALA-induced PpIX in EMT-6 is shown in Figure 2. After a 4-h incubation with 1 The level of of 5 5-ALA-induced-ALA-induced PpIX PpIX in in EMT EMT-6-6 is is shown shown in in Figure Figure 2.2. After After a a 4 4-h-h incubation incubation with with 1 mM 5-ALA, fluorescence intensity was approximately 55-fold higher than that of the control. The 1mM mM 5- 5-ALA,ALA, fluorescence ﬂuorescence intensity intensity was was approximately approximately 55 55-fold-fold higher higher than than that that of of the control. The corrected PpIX fluorescence intensities of the control, 5-ALA, 5-ALA + ART, and 5-ALA + ARM corrected PpIX PpIX ﬂuorescence fluorescence intensities intensities of theof the control, control, 5-ALA, 5-ALA, 5-ALA 5-AL+ ART,A + ART, and 5-ALA and 5+-ALAARM + groups ARM weregroups 3.385 were 3.730,3.385 ± 165.7 3.730, 165.733.45, ± 139.0 33.45, 139.052.77, ± and52.77 165.4, and 165.451.10 ± a.u., 51.10 respectively. a.u., respectively. groups were± 3.385 ± 3.730±, 165.7 ± 33.45,± 139.0 ± 52.77, and± 165.4 ± 51.10 a.u., respectively.

Figure 2. Corrected protoporphyrin IX fluorescence intensity in EMT-6 cells. Cells were incubated Figure 2. Corrected protoporphyrinprotoporphyrin IX IX fluorescence fluorescence intensity intensity in EMT-6in EMT cells.-6 cells. Cells Cells were were incubated incubated with with 1 mM 5-aminolevulinic acid (5-ALA), 1 mM 5-ALA, and concentrations of 7.8 μM artesunate with1 mM 1 5-aminolevulinicmM 5-aminolevulinic acid (5-ALA), acid (5-ALA), 1 mM 5-ALA,1 mM 5 and-ALA concentrations, and concentrations of 7.8 µM ofartesunate 7.8 μM artesunate (ART) or (ART) or artemether (ARM) for 4 h. Results are presented as means ± standard deviations (n = 5). artemether (ARM) for 4 h. Results are presented as means standard deviations (n = 5). (ART) or artemether (ARM) for 4 h. Results are presented as± means ± standard deviations (n = 5). 2.2. Cytotoxicity Analysis of the ART or ARM Dose 2.2. Cytotoxicity Analysis of the ART or ARM Dose Figure3 3 shows shows the the viability viability of of 5-ALA-PDT-treated 5-ALA-PDT-treate cellsd cells in thein the presence presence of ART of ART or ARM. or ARM. At 0 AtµM 0 Figure 3 shows the viability of 5-ALA-PDT-treated cells in the presence of ART or ARM. At 0 ofμM ART of ART or ARM, or ARM, viability viability of 5-ALA-PDT-treated of 5-ALA-PDT-treated cells was cells dependent was dependent on the lighton the dose. light In dose. the absence In the μM of ART or ARM, viability of 5-ALA-PDT-treated cells was dependent on the light dose. In the ofabsence irradiation of irradiation (0 J/cm2), (0 increasing J/cm2), increasing the concentration the concentration of ART or of ARM ART didor ARM not significantly did not significantly affect cell absence of irradiation (0 J/cm2), increasing the concentration of ART or ARM did not significantly survival.affect cell surviv However,al. However, at light dosesat light of doses 3 and of 5 3 J /andcm2 5, J/cm viability2, viability of the of 5-ALA-PDT-treated the 5-ALA-PDT-treated cells cells was affect cell survival. However, at light doses of 3 and 5 J/cm2, viability of the 5-ALA-PDT-treated cells significantlywas significantly decreased decreased in the in presence the presence of ART of (ARTp < 0.01 (p < and 0.01p and< 0.01) p < or0.01) ARM or ARM (p < 0.01 (p < and 0.01p and< 0.01), p < was significantly decreased in the presence of ART (p < 0.01 and p < 0.01) or ARM (p < 0.01 and p < respectively.0.01), respectively. At a light At dosea light of dose 3 J/cm of2, 3 cell J/cm viability2, cell viability in 5-ALA-PDT in 5-ALA with-PDT 7.8 µ withM ARM 7.8 groupμM ARM was group lower 0.01), respectively. At a light dose of 3 J/cm2, cell viability in 5-ALA-PDT with 7.8 μM ARM group thanwas lower that in than 5-ALA-PDT that in 5 with-ALA 7.8-PDTµM ARTwith group7.8 μM (p ART= 0.0165). group At (pa = light 0.0165). dose At of a 5 Jlight/cm2 ,dose cell viabilityof 5 J/cm in2, was lower than that in 5-ALA-PDT with 7.8 μM ART group (p = 0.0165). At a light dose of 5 J/cm2, 5-ALA-PDTcell viability forin 5 2.0-ALA and-PDT 7.8 µ forM ARM2.0 and groups 7.8 μM was ARM lower group thans was that lower in 5-ALA-PDT than that forin 5 2.0-ALA and-PDT 7.8 µ forM cell viability in 5-ALA-PDT for 2.0 and 7.8 μM ARM groups was lower than that in 5-ALA-PDT for ART2.0 and groups 7.8 μM (p

Figure 3. Cytotoxicity analysis of the artesunate (ART) or artemether (ARM) dose. Cells were incubated Figure 3. Cytotoxicity analysis of the artesunate (ART) or artemether (ARM) dose. Cells were Figurefor 4 h with 3. Cytotoxicity 1 mM 5-aminolevulinic analysis of acidthe artesunate (5-ALA) and (ART) various or concentrations artemether (ARM) (0, 2, dose.7.8, 31.3, Cells 125, were and incubated for 4 h with 1 mM 5-aminolevulinic acid (5-ALA) and various concentrations (0, 2, 7.8, 31.3, incubated500 µM) of for (A )4 ARTh with or 1 (B mM) ARM. 5-aminolevulinic After washing acid with (5 fresh-ALA) medium, and various cells wereconcentrations irradiated with(0, 2, a7.8, 630-nm 31.3, light (20 mW/cm2, 10 J/cm2) emitted by LED lights. At ﬂuorescence intensities of 3 and 5 J/cm2, the

viability of the 5-ALA-photodynamic therapy (PDT)-treated cells was signiﬁcantly decreased in the presence of ART (p < 0.01 and p < 0.01) or ARM (p < 0.01 and p < 0.01). Results are presented as means standard deviations (n = 5). ± Molecules 2019, 24, x FOR PEER REVIEW 4 of 10

125, and 500 μM) of (A) ART or (B) ARM. After washing with fresh medium, cells were irradiated with a 630-nm light (20 mW/cm2, 10 J/cm2) emitted by LED lights. At fluorescence intensities of 3 and 5 J/cm2, the viability of the 5-ALA-photodynamic therapy (PDT)-treated cells was significantly Moleculesdecreased2019, 24 in, 3891 the presence of ART (p < 0.01 and p < 0.01) or ARM (p < 0.01 and p < 0.01). Results are 4 of 10 presented as means ± standard deviations (n = 5).

2.3.2.3. Morphological Morphological Changes Changes in in EMT EMT-6-6 C Cellsells FigureFigure 44AA–D–D shows the results of Hoechst 33342 staining of EMT-6EMT-6 cells incubated with 1 mM 2 2 55-ALA-ALA for for 4 4 h, h, irradiated irradiated (20 (20 mW/cm mW/cm2,, 3 3 J/cm J/cm2),), and and stained stained with with Hoechst Hoechst 33342 33342 for for 6 6 h h after after PDT. PDT. No No signssigns of of apoptosis apoptosis were were observed observed in in the the control control group. group. Furthermore, Furthermore, cells cells treated treated with with 5 5-ALA-PDT-ALA-PDT with/withoutwith/without A ARTRT displayed displayed slight slight nuclear nuclear condensation, condensation, while while cells cells treated treated with with 5 5-ALA-PDT-ALA-PDT and and ARMARM displayed displayed more more nuclear nuclear condensation. condensation.

FigureFigure 4. 4. MorphologicalMorphological changes changes in in EMT EMT-6-6 cells cells 6 6 h h after after PDT PDT with with artesunate artesunate (ART) (ART) or or artemether artemether (ARM).(ARM). EMT EMT-6-6 cells cells were were incubated incubated w withith 1 1 mM mM 5 5-aminolevulinic-aminolevulinic acid acid (5 (5-ALA)-ALA) for for 4 4 h h prior prior to to washing washing andand exposure exposure to to a a 635 635-nm-nm LED LED light. light. Subsequently, Subsequently, cells cells were were incubated incubated for for an an additional additional 6 6 h h before before HoechstHoechst 33342 33342 staining staining.. The The 5 5-ALA-PDT-ALA-PDT with with ARM ARM group group had had a a marked marked increase increase in in condensation condensation o off nuclearnuclear chromatin chromatin relative relative to the other groups. Images represent the (AA, Eand) control, E) control, (B,F) (5-ALA-PDT,B and F) 5- ALA(C,G-)PDT, 5-ALA-PDT (C and andG) 5 ART,-ALA and-PDT (D ,Hand) 5-ALA-PDT ART, and and(D and ARM H groups.) 5-ALA Upper-PDT and panel ARM (A–D groups.): Fluorescence Upper panelimages. (A– LowerD): Fluorescence panel (E–H images.): Transmitted Lower lightpanel images. (E–H): ScaleTransmitted bar = 50 lightµm. images. Scale bar = 50 μm.

FigureFigure 44EE–H–H shows shows the the results results of of the the morphological morphological evaluation evaluation performed performed with with EMT-6. EMT-6. No No morphologicalmorphological changes changes were were observed observed in in the the control control group. group. However, However, cells cells treated treated with with 5 5-ALA-PDT-ALA-PDT with/withoutwith/without ART ART displaye displayedd minimal minimal shrinkage shrinkage and and plasma plasma membrane membrane blebbing blebbing,, while while those those treated treated withwith 5 5-ALA-PDT-ALA-PDT and and ARM ARM displayed displayed more more shrinkage and plasma membrane blebbing. FigureFigure 55 showsshows imagesimages of of EMT-6 EMT-6 cells cells stained stained with with annexin-V-FITC annexin-V-FITC and and ethidium ethidium homodimer homodimer III IIIand and analyzed analyzed 6 h 6 after h after 5-ALA-PDT. 5-ALA-PDT. Cells Cells treated treated with 5-ALA-PDTwith 5-ALA- withPDT/without with/without ART were ART only were slightly only slightlystained with stained annexin with V-FITCannexin and V- ethidiumFITC and homodimer ethidium III.homodimer In addition, III. the In number addition, of positive the number cells for of positiveannexin cells V-FITC for andannexin ethidium V-FITC homodimer and ethidium III staining homodimer in the III 5-ALA-PDT staining in with the 5 ARM-ALA group-PDT wit (Figureh ARM5D) groupwas found (Figure to 5 beD) greaterwas found than to thosebe greater of the than 5-ALA-PDT those of the with 5-ALA/without-PDT ARTwith/without groups (Figure ART groups5B,C). (EMT-6Figure cells5B,C). treated EMT-6 with cells 5-ALA-PDT treated with plus 5-ALA ARM-PDT were plus positively ARM were stained positively with annexin stained Vwith or ethidiumannexin Vhomodimer or ethidium III, homodimer indicating lateIII, indicating apoptosis late or necrosis. apoptosis or necrosis.

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FigureFigure 5. Fluorescent 5. Fluorescent staining staining to to detectdetect cell death death in in EMT EMT-6-6 cells. cells. EMT EMT-6-6 cells cellswere wereincubated incubated with 1 with 1 mMmM 5-aminolevulinic 5-aminolevulinic acid acid (5-ALA) (5-ALA) forfor 4 h prior prior to to washing washing and and exposure exposure to a to 635 a- 635-nmnm LED LEDlight. light. Cells Cells were further incubated for 6 h before staining with annexin V (green) and ethidium homodimer III were further incubated for 6 h before staining with annexin V (green) and ethidium homodimer III (red). (red). Images represent the (A) control, (B) 5-ALA-PDT, (C) 5-ALA-PDT and artesunate, and (D) 5- Images represent the (A) control, (B) 5-ALA-PDT, (C) 5-ALA-PDT and artesunate, and (D) 5-ALA-PDT ALA-PDT and artemether groups. Transmitted light images. Scale bar = 100 μm. and artemether groups. Transmitted light images. Scale bar = 100 µm. 2.4. The Effect of an Antioxidant on PDT-Induced Reactive Oxygen Species (ROS) and Apoptosis 2.4. The Effect of an Antioxidant on PDT-Induced Reactive Oxygen Species (ROS) and Apoptosis To further investigate whether 5-ALA-PDT only or 5-ALA-PDT with ART or ARM induced ROS andTo further apoptosis, investigate EMT-6 cells whether were treated 5-ALA-PDT with 5-ALA only-PDT or 5-ALA-PDT only or 5-ALA with-PDT ART with or ART ARM or inducedARM in ROS and apoptosis,the presence EMT-6 (10 mM) cells or wereabsence treated of N-acetylcysteine with 5-ALA-PDT (NAC). only As ora result, 5-ALA-PDT NAC could with significantly ART or ARM in the presenceinhibit ROS (10 induction mM) or by absence 5-ALA of-PDT N-acetylcysteine only (p = 0.0023) (NAC). or via 5 As-ALA a result,-PDT with NAC ARM could (p < significantly0.0001) inhibit(Figure ROS 6A). induction In addition, by 5-ALA-PDT it could significantly only (p =inhibit0.0023) apoptosis or via’s 5-ALA-PDTinduction caused with by ARM 5-ALA (p-PDT< 0.0001) (Figureonly6A). (p < In 0.0001) addition, (Figure it could 6B). The significantly cytotoxicity inhibit due to 5 apoptosis’s-ALA-PDT only induction (p < 0.0001) caused or 5 by-ALA 5-ALA-PDT-PDT with only (p < 0.0001)ART (p (Figure= 0.0003)6 B).could The be cytotoxicity significantly due inhibited to 5-ALA-PDT by NAC (Figure only ( p6C).< 0.0001) NAC could or 5-ALA-PDT not significantly with ART (p = 0.0003)inhibit the could percentage be significantly of apoptotic inhibited cells, nor by inviable NAC cell (Figures induced6C). NACby 5-ALA could-PDT not with significantly ARM. inhibit theMolecules percentage 2019, 24 of, x FOR apoptotic PEER REVIEW cells, nor inviable cells induced by 5-ALA-PDT with ARM. 5 of 10

Figure 6. Cont.

Figure 6. The percentage of ROS-positive cells in the presence or absence of N-acetylcysteine (NAC). EMT-6 cells were treated with 5-aminolevulinic acid (5-ALA)-PDT only or 5-ALA-PDT with artesunate (ART) or artemether (ARM) in the presence (10 mM) or absence of NAC. ROS’ induction by 5-ALA-PDT only (p = 0.0023) or 5-ALA-PDT with ARM (p < 0.0001) was significantly inhibited by NAC (A). Apoptosis’s induction by 5-ALA-PDT only (p < 0.0001) was significantly inhibited by NAC

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FigureFigure 6. 6.The The percentage percentage ofof ROS-positiveROS-positive cells cells in in the the presence presence or or absence absence of ofN- N-acetylcysteineacetylcysteine (NAC). (NAC). EMT-6EMT- cells6 cells were were treated treated with with 5-aminolevulinic 5-aminolevulinic acid (5-ALA)-PDT acid (5-ALA)- onlyPDT or only 5-ALA-PDT or 5-ALA with-PDT artesunate with (ART)artesunate or artemether (ART) or (ARM) artemether in the (ARM) presence in the (10 presence mM) or absence (10 mM) of or NAC. absence ROS’ of inductionNAC. ROS by’ induction 5-ALA-PDT by 5-ALA-PDT only (p = 0.0023) or 5-ALA-PDT with ARM (p < 0.0001) was significantly inhibited by only (p = 0.0023) or 5-ALA-PDT with ARM (p < 0.0001) was signiﬁcantly inhibited by NAC (A). NAC (A). Apoptosis’s induction by 5-ALA-PDT only (p < 0.0001) was significantly inhibited by NAC Apoptosis’s induction by 5-ALA-PDT only (p < 0.0001) was signiﬁcantly inhibited by NAC (B).

Cytotoxicity, exhibited by 5-ALA-PDT only (p < 0.0001) or 5-ALA-PDT with ART (p = 0.0003), was significantly inhibited by NAC (C). Data were analyzed using Sidak’s multiple comparison test (** p < 0.01; NAC ( ) vs. NAC (+)). Results are presented as means standard deviations. − ± 3. Discussion ART has been reported to kill breast cancer cells via caspase-dependent apoptosis mediated by intracellular ROS accumulation promoted by iron [19]. Herein, we hypothesized that ART and ARM could be promising candidates in the search for low-toxicity compounds that could increase the efficacy of 5-ALA-based PDT. In the present study, with a light dose of 3 and 5 J/cm2, the viability of 5-ALA-PDT-treated cells significantly decreased following ART (p < 0.01 and p < 0.01) and ARM (p < 0.01 and p < 0.01) treatment. There were no differences in corrected fluorescence intensities and percentages of ROS-positive cells among the 5-ALA-PDT, 5-ALA-PDT with ART, and 5-ALA-PDT with ARM groups. However, the cytotoxicity of 5-ALA-PDT was enhanced when combined with ART or ARM. Besides, the cytotoxicity of 5-ALA with/without ART and ARM-based PDT did not depend on intracellular PpIX. Previously, ART and DHA, as monotherapies, were reported to exert higher anticancer effects against human Molecules 2019, 24, 3891 7 of 10 hepatoma cells than ARM [21]. However, in this study, viability was lower in cells treated with 5-ALA-PDT with ARM than in those treated with 5-ALA-PDT with ART. Hence, the mechanism of ART or ARM cytotoxicity might be complicated when combined with other therapies, such as PDT. The active fraction of the artemisinin analog is an endoperoxide bridge that generates carbon-centered free radicals or ROS and oxidative stress upon cleavage [22]. ROS plays a significant role in the killing of specific tumor cells via the induction of apoptosis and oxidative DNA damage [23]. The intracellular bioactivation of the endoperoxide to carbon-centered radicals was found to be dependent on cellular heme with the addition of PpIX, a precursor of heme that could increase heme synthesis to levels approximately 2.5-fold greater than normal, thereby significantly increasing the sensitivity to ART-induced cytotoxicity [24]. ART treatment alone for 24 h resulted in an IC50 of approximately 2 µM for human colorectal cancer cells, while the addition of 5-ALA lowered the IC50 by approximately 10-fold to around 200 nM [25]. As addition of 5-ALA increased intracellular heme level and enhanced ART activation in cancer cells, ART/5-ALA combination therapy could be more effective than ART monotherapy [25]. In this study, 5-ALA-PDT with ART or ARM was more effective than 5-ALA-PDT alone at 3 and 5 J/cm2. The cytotoxicity of 5-ALA-PDT with ART or ARM might be endoperoxide-dependent. However, further studies in mouse models of EMT6 mammary tumors are required to evaluate the in vivo photodynamic effects of 5-ALA-PDT-ARM. Although NAC significantly inhibited the generation of ROS induced by 5-ALA-PDT with/without ARM, NAC could not significantly inhibit the percentage of apoptotic cells and inviable cells induced by 5-ALA-PDT with ARM. Hence, we speculate that mechanisms other than the generation of ROS are involved in cell death induced by 5-ALA-PDT with ARM. A recent report revealed that the major metabolites of artemisinin and its derivative, DHA, induced the binding of the nuclear protein, murine double minutes 2, and downregulated the levels of the cell surface transferrin receptor to inhibit p53 ubiquitination [9,21,23,26]. Hence, such ROS-independent mechanisms might enhance the efficacy of 5-ALA-PDT with ARM.

4. Materials and Methods

4.1. Cell Line and Culture Conditions Mouse mammary tumor EMT6 cells were supplied by Professor Yoshihiro Uto of Tokushima University (Tokushima, Japan). The cells were maintained as an adherent monolayer culture, and incubated in 5% CO2 at 37 ◦C. RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Nichirei Biosciences Inc., Tokyo, Japan) and PSN (5 mg/mL penicillin, 5 mg/mL streptomycin, and 10 mg/mL neomycin; Invitrogen) was employed as the culture medium. For the experiments, cells were harvested from near-conﬂuent cultures via brief exposure to a solution containing 0.25% trypsin and 1 mmol/L EDTA-4Na with phenol red (Invitrogen). Trypsinization was terminated using RPMI 1640 medium containing 10% fetal bovine serum. Thereafter, cells were centrifuged and re-suspended in RPMI 1640 medium. Trypan blue staining was used to assess cell viability.

4.2. Chemicals 5-ALA was donated by SBI Pharma (Tokyo, Japan). A stock solution of 100 mM 5-ALA in phosphate-buﬀered saline (PBS), stored at 4 ◦C, was used for the in vitro experiments. Stock solutions of ART and ARM (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) at 1 mM were prepared with PBS and 1% (v/v) DMSO and diluted to the ﬁnal concentrations indicated in the cell cultures.

4.3. Protoporphyrin IX Fluorescence in EMT-6 Cells To investigate the formation of PpIX from 5-ALA, PpIX ﬂuorescence was examined using an Olympus Fluoview FV1000 (Olympus Co., Tokyo, Japan) and read with a ﬂuorescence spectrometer SH-9000Lab (Hitachi High-Technologies Co., Tokyo, Japan). Molecules 2019, 24, 3891 8 of 10

First, 1 105 EMT-6 cells were seeded in 35-mm Petri dishes containing 2 mL of cultivation × medium. After 24 h of incubation, the dishes were divided into the following four groups: (1) Control group (no treatment), (2) 5-ALA group (treated with 1 mM 5-ALA), (3) 5-ALA + ART group (treated with 1 mM 5-ALA and 7.8 µM ART), and (4) 5-ALA + ARM group (treated with 1 mM 5-ALA and 7.8 µM ARM). Cells were incubated with 1 mM 5-ALA and concentrations of 7.8 µM ART or ARM for 4 h. After washing with PBS, PpIX fluorescence was examined using an Olympus Fluoview FV1000. To obtain the cell extracts, cells were harvested by trypsinization, and centrifuged at 300 g for 5 min × at room temperature. Whole-cell extracts were prepared with 50 µL Triton buffer (1% Triton X-100 in PBS). The fluorescence of cell lysates was read at Ex = 405 nm and Em = 635 nm with a fluorescence spectrometer SH-9000Lab.

4.4. Cytotoxicity Analysis of the ART or ARM Dose A total of 4–5 104 of primary cells was seeded into each well of 96-well plates (Corning Inc., × Corning, NY, USA) prior to overnight incubation. Cells were then incubated with 1 mM 5-ALA and various concentrations of ART or ARM (0, 2.0, 7.8, 31.3, 125, and 500 µM) for 4 h. After washing with fresh medium, cells were irradiated with a 630-nm light (20 mW/cm2, 10 J/cm2) emitted by LED lights (Pleiades Technologies LLC, Fukuoka, Japan). Subsequently, the cells were re-incubated for 24 h in the dark. Cell viability was examined by using Cell Counting Kit-8 in accordance with the manufacturer’s instructions.

4.5. Morphological Changes in EMT-6 Cells A total of 1 105 EMT-6 cells were seeded in 35-mm Petri dishes containing 2 mL of cultivation × medium. After 24 h of incubation, the dishes were divided into the following four groups: (1) Control group (no treatment), (2) 5-ALA-based PDT group (treatment with 1 mM 5-ALA and irradiated with a light dose of 3 J/cm2), (3) 5-ALA + ART-based PDT group (treatment with 1 mM 5-ALA and 7.8 µM ART, and irradiated with a light dose of 3 J/cm2), and (4) 5-ALA + ARM-based PDT group (treatment with 1 mM 5-ALA and 7.8 µM ARM, and irradiated with a light dose of 3 J/cm2). Cells were incubated with 1 mM 5-ALA and 7.8 µM ART or ARM for 4 h. After washing with fresh media, cells were irradiated with a 630-nm laser light (20 mW/cm2, 3 J/cm2) emitted by LED lights. After 6 h of PDT, cells were stained using the Promokine Apoptotic/Necrotic/Healthy cell detection kit, according to the manufacturer’s instructions. Subsequently, cells were stained with Hoechst 33342. Nuclear morphology was examined using an Olympus BX51 (Olympus Co., Tokyo, Japan). To assess apoptosis and necrosis, cells were stained with annexin V-ﬂuorescein isothiocyanate (FITC) and ethidium homodimer III at 6 h after laser irradiation. Annexin V was used to detect phosphatidylserine on the external membrane of apoptotic cells. Cells were analyzed by Olympus Fluoview FV1000 using the FITC and Texas Red ﬁlter settings.

4.6. Analysis of Reactive Oxygen Species (ROS) and Apoptosis Induced by PDT EMT-6 cells were seeded at 1 104 cells in 35-mm petri dishes containing 2 mL of culture medium. × Following 24 h of incubation, cells were divided into the following four groups: (1) Control group (no treatment), (2) 5-ALA-based PDT group (treatment with 1 mM 5-ALA and irradiated with a light dose of 3 J/cm2), (3) 5-ALA + ART-based PDT group (treatment with 1 mM 5-ALA and 7.8 µM ART, and irradiated with a light dose of 3 J/cm2), and (4) 5-ALA + ARM-based PDT group (treatment with 1 mM 5-ALA and 7.8 µM ARM, and irradiated with a light dose of 3 J/cm2). Cells were incubated with 1 mM 5-ALA and 7.8 µM ART or ARM for 4 h in the presence (10 mM) or absence of the antioxidant, N-acetylcysteine (NAC). After washing with fresh media, cells were irradiated with a 630-nm laser light (20 mW/cm2, 3 J/cm2) emitted by LED lights. Molecules 2019, 24, 3891 9 of 10

To determine the percentage of cells that were negative (healthy cells) and positive for ROS (cells containing ROS), ROS generation was assessed 6 h after laser irradiation using the Muse Oxidative Stress kit (EMD Millipore Co., Billerica, MA, USA) according to the manufacturer’s protocols. The total apoptotic cells of early and late stages and cell viability were assessed 6 h after LED irradiation using the Muse Annexin V (EMD Millipore Co.) and Dead Cell Assay kit (EMD Millipore Co.), according to the manufacturers’ protocols. Cells were harvested for the experiments and single-cell suspensions were loaded onto the Muse Cell Analyzer (EMD Millipore Co.).

4.7. Statistical Analysis Data were analyzed by Dunnett and Sidak’s multiple comparisons test. p < 0.05 was considered to indicate a statistical signiﬁcance. Statistical analyses were performed using GraphPad Prism software (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA).

5. Conclusions Herein, we identified that ARM could enhance 5-ALA-PDT-induced EMT-6 cell death. To our knowledge, this is the first study to evaluate 5-ALA-PDT with ARM as a new therapeutic strategy to enhance the cytotoxicity of PDT against tumor cells. However, because the results are only based on in vitro studies, in vivo studies should be performed. In addition, further studies are needed to investigate the unknown mechanisms of action in vitro and the difference in antitumor efficacy against different tumor cell lines.

Author Contributions: Conceptualization, T.O.; methodology, T.O.; investigation, T.O.; resources, K.T., M.I., and T.T.; data curation, T.O.; writing—original draft preparation, T.O.; writing—reviewing and editing, K.T., M.I., T.T., and Y.O.; visualization, T.O.; supervision, T.T.; project administration, T.T. Funding: This study was partly funded by SBI Pharmaceuticals Co., Ltd. (Tokyo, Japan). Acknowledgments: We would like to thank Kei Sakanoue for providing support with the LED light source. Conﬂicts of Interest: Kiwamu Takahashi, Masahiro Ishizuka, and Tohru Tanaka are employees of SBI Pharmaceuticals Co., Ltd.

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