Accepted Manuscript Lycopene induces apoptosis in Candida albicans through reactive oxygen species production and mitochondrial dysfunction Hyemin Choi, Dong Gun Lee PII:

S0300-9084(15)00143-1

DOI:

10.1016/j.biochi.2015.05.009

Reference:

BIOCHI 4719

To appear in:

Biochimie

Received Date: 10 May 2015 Accepted Date: 12 May 2015

Please cite this article as: H. Choi, D.G. Lee, Lycopene induces apoptosis in Candida albicans through reactive oxygen species production and mitochondrial dysfunction, Biochimie (2015), doi: 10.1016/ j.biochi.2015.05.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Lycopene induces apoptosis in Candida albicans through reactive oxygen species production and mitochondrial

RI PT

dysfunction

SC

Hyemin Choi and Dong Gun Lee*

M AN U

School of Life Sciences, BK 21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu 702-701, Republic

AC C

EP

TE D

of Korea

* Corresponding author: Tel.: +82 53 950 5373; Fax: +82 53 955 5522; E-mail address: [email protected] (D.G. Lee)

1

ACCEPTED MANUSCRIPT ABSTRACT Lycopene, a well-known carotenoid pigment found in tomatoes, has shown various biological functions. In our previous report, we showed that lycopene induces two apoptotic

RI PT

hallmarks, plasma membrane depolarization and G2/M cell cycle arrest, in Candida albicans. In this study, we investigated the ability of lycopene to induce apoptosis, and the mechanism by which it regulates apoptosis. FITC-Annexin V staining, terminal deoxynucleotidyl

SC

transferase dUTP nick end labeling (TUNEL) analysis, and 4',6-diamidino-2-phenylindole (DAPI) assay showed that lycopene exerted its antifungal activity during the early and late

M AN U

stages of apoptosis in C. albicans. During apoptosis, intracellular reactive oxygen species (ROS) were increased, and specifically the hydroxyl radicals contributed to the fungal cell death. Furthermore, lycopene treatment caused intracellular Ca2+ overload and mitochondrial dysfunction, such as mitochondrial depolarization and cytochrome c release from the

TE D

mitochondria to the cytoplasm. At last caspase activation was triggered. In summary, lycopene exerted its antifungal effects against C. albicans by inducing apoptosis via ROS

AC C

EP

production and mitochondrial dysfunction.

2

ACCEPTED MANUSCRIPT Keywords: Lycopene; Apoptosis; Candida albicans; ROS production.

Abbreviations: 4'-6-diamino-2-phenylindole, DAPI; nitric oxide, NO; dihydrorhodamine-123,

RI PT

DHR-123; 3'-(p-hydroxylphenyl) fluorescein, HPF; 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylimidacarbocyanine iodide, JC-1; minimum inhibitory concentration, MIC; hydroxyl radical, ·

OH; reactive oxygen species, ROS; cytosolic free Ca2+ levels, [Ca2+]c; mitochondrial free

AC C

EP

TE D

M AN U

SC

Ca2+ levels, [Ca2+]m; mitochondrial membrane potential, ∆Ψm

3

ACCEPTED MANUSCRIPT 1. Introduction Lycopene is a red carotenoid pigment found in fruits and vegetables, including tomatoes, watermelons, pinkgrapefruits, and pinkguavas [1]. Carotenoids were observed to be highly

RI PT

involved in photosynthesis and phtoprotection in plants [2]. They were also found in animal tissues where they may act as antioxidants or as antimutagenic, immunomodulating and tumor-preventing agents. Due to its biological properties, carotenoids are used as medicine,

SC

cosmetics and food additives [3]. Recent studies have suggested that the carotenoids have antimicrobial activity. Carotenoids from the peel of Shatian pummelo (Citrus grandis Osbeck)

M AN U

exhibited antifungal activity against Rhizopus oryzae and Saccharomyces scerevisiae [4]. In addition, carotenoid pigment extracted from Sporobolomyces sp. isolated from natural source exhibited antibacterial activity against Enterococcus sp., Streptococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli [5].

TE D

Although it is not an essential nutrient in humans, lycopene has many health benefits. Several studies have reported that lycopene has anticancer [6-8], antioxidant [9], antiinflammatory [10, 11], and antimicrobial activities [12]. Lycopene may prevent certain

EP

cancers, including breast, colon, and prostate cancers, and lymphoma, by inducing apoptosis [6-8]. Interestingly, lycopene protects against H2O2- and7-ketocholesterol-induced apoptosis

AC C

in normal human cells, including endothelial cells andmacrophages, via the downregulation of p53 and caspase-3 expression [13, 14]. Further, lycopene inhibits inflammation by downregulating the production of pro-inflammatory mediators, includingnitric oxide (NO), interleukins, tumor necrosis factor (TNF) α, cyclooxygenase (COX)-2, and the transcription nuclear factor-κB (NF-κB) in macrophages. Our previous study showed that lycopene exerts antimicrobial activities against bacteria, such as Staphylococcus aureus and Escherichia coli O-157, and fungi, such as Candida albicans, Trichosporon beigelii, and Malassezia furfur 4

ACCEPTED MANUSCRIPT [12]. In C. albicans, lycopene treatment caused membrane damage [12]. However, the precise mechanism of lycopene antifungal action has not been reported. C. albicans is the most common pathogenic fungi in humans, causing cutaneous and life-

RI PT

threatening systemic infections [15]. There are four stages of C. albicans infection. In stage 1, C. albicans colonizes the epithelial surface, leading to superficial infections (stage 2). If the host has a weakened immune system, C. albicans triggers deep-seated infections (stage 3) by

SC

invasion into the epithelial tissue. Finally, C. albicans causes potentially fatal disseminated infections (stage 4), that allows the fungus to colonize and infect other host tissues [16]. C.

M AN U

albicans is a polymorphic fungus that can exist in a budded or yeast-like form, a pseudohyphal form, or a true hyphal filamentous form [17]. The ability of C. albicans to rapidly and reversibly shift between yeast and filamentous morphologies is important for its pathogenicity, because the hyphal form is more invasive than the yeast form [18, 19]. In

TE D

contrast, the yeast form is primarily involved in dissemination [20]. Candida infection is the leading cause of life-threatening nosocomial fungal infections, and antimycotic-resistant C. albicans strains are rapidly emerging [21, 22]. To overcome these problems, the development

EP

of antifungal agents possessing novel antifungal mechanisms is necessary. In this study, we assessed the antifungal mechanism of lycopene against C. albicans.

AC C

Further, we confirmed that lycopene inhibits C. albicans via apoptosis.

5

ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Preparation of lycopene Lycopene derived from tomatoes was purchased from Sigma Chemical Co.. A stock solution

RI PT

of lycopene was prepared in tetrahydrofuran and stored at -80 °C.

2.2. Phosphatidylserine externalization assay

SC

C. albicans protoplasts were stained with propidium iodide (PI) and FITC-labeled annexin V using the FITC-Annexin V apoptosis detection kit [23]. To obtain viable

M AN U

protoplasts, C. albicans cells were lyzed with 0.1 M potassium phosphate buffer (pH 6.0) including 20 mg/mL lysing enzyme (Sigma-Aldrich) and 1 M sorbitol for 1 h at 30 °C with gentle agitation. After the protoplasts were harvested by filtration and centrifugation at 1500 rpm for 10 min, they (1 × 105 cells/mL) were suspended in 0.1 M potassium phosphate buffer (pH 6.0) including 1 M sorbitol. Afterwards the protoplasts were incubated with either 5

TE D

µg/mL lycopene or 10 mM H2O2 for 4 h at 28 °C and suspended in annexin binding buffer including 5 µL/mL FITC-Annexin-V and 5 µL/mL PI for 20 min. The stained cells were

EP

examined with a FACSCalibur flow cytometer (Becton Dickinson). Statistical significance

AC C

was calculated by Student’s t -test.

2.3. DNA and nuclear fragmentation assays DNA fragmentation was examined using the TUNEL assay [24]. The cells (1 × 105 cells/mL) were treated with 5 µg/mL lycopene or 10 mM H2O2 for 5 h at 28 °C. The samples were then washed in PBS (pH 7.4), suspended in permeabilization solution consisting of 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. After the cells were washed again in PBS, they were stained by an in situ cell death detection kit for 1 h at 37 °C, and observed 6

ACCEPTED MANUSCRIPT with a fluorescence microscope (Nikon Eclipse Ti-S). To ensure reproducibility, the experiment was repeated three times. Nuclear fragmentation and condensation were examined with 4'-6-diamino-2-

RI PT

phenylindole (DAPI) staining [23]. The cells (1 × 105 cells/mL) were treated with 5 µg/mL lycopene or 10 mM H2O2 for 5 h at 28 °C. After the cells were washed in PBS, they were stained by 1 µg/mL DAPI for 20 min, and examined under a fluorescence microscope (Nikon

SC

Eclipse Ti-S). To ensure reproducibility, the experiment was repeated three times.

M AN U

2.4. Reactive oxygen species (ROS) and hydroxyl radical (·OH) assessment Intracellular ROS and · OH accumulation were assessed using dihydrorhodamine-123 (DHR-123) [24] and 3'-(p-hydroxylphenyl) fluorescein (HPF) [25], respectively. For ROS detection, C. albicans cells (1 × 105 cells/mL) were treated with 5 µg/mL lycopene or 10 mM H2O2 for 4 h at 28°C. After the cells were washed in PBS, they were stained by 5 µg/mL

TE D

DHR-123. For · OH detection, C. albicans cells (1 × 105 cells/mL) were incubated with 5 µg/mL lycopene or 10 mM H2O2 for 4 h at 28°C, and then incubated in PBS containing 10

EP

µM HPF. The cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson).

AC C

Statistical significance was calculated by Student’s t -test.

2.5. Thiourea assay

To quench the hydroxyl radical, 150 mM thiourea (Sigma-Aldrich) was treated to the C. albicans culture with lycopene or H2O2. Importantly, we used a concentration of thiourea that minimized growth inhibition. The number of colony-forming units (CFU/mL) was monitored after exposing the C. albicans to 5 µg/mL lycopene or 10 mM H2O2 for 4 h. The culture (100 µL) was collected, washed with PBS, and serially diluted with PBS. Each dilution was 7

ACCEPTED MANUSCRIPT spreaded on YPD agar, and the plates were incubated overnight at 28 °C. Only dilutions which produced between 20-100 colonies were counted. The CFU/mL was represented as a percentage of survival using the following formula: [(CFU of sample treated with

Student’s t -test.

2.6. Analysis of cytosolic and mitochondrial Ca2+ levels

RI PT

agent)/(CFU of non-treated sample) × 100]. Statistical significance was calculated by

SC

To analyze cytosolic and mitochondrial Ca2+ levels, two fluorescent dyes such as Fura-

M AN U

2AM (Molecular Probes) and Rhod-2AM (Molecular Probes) were used, respectively. C. albicans cells (1 × 105 cells/mL) were incubated with 5 µg/mL lycopene or 10 mM H2O2 for 4 h at 28 °C. Subsequently, they were washed twice in Krebs buffer (pH 7.4) and treated with 0.01% Pluronic acid F-127 (Molecular Probes) and 1% BSA. Then, the cells were stained

TE D

with 5 µM Fura-2AM or 10 µM Rhod-2AM and incubated at 37 °C for 40 min. The cells were washed two times in calcium-free Krebs buffer and maintained in calcium and dye freemedium at 37 °C for 30 min, thereby allowing the cells to hydrolyze the acetoxymethyl ester

EP

completely. The fluorescence intensities of Fura-2AM (Ex=335 nm, Em=505 nm) and Rhod2AM (Ex=550 nm, Em=580 nm) were detected with a spectrofluorophotometer (Shimadzu

AC C

RF-5301PC, Shimadzu) [26]. Statistical significance was calculated by Student’s t -test.

2.7. Analysis of mitochondrial membrane potential To examine the changes in mitochondrial membrane potential (∆Ψm), 5,5',6,6'tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) (Molecular Probes) was used [27]. C. albicans cells (1 × 105 cells/mL) were incubated with 5 µg/mL lycopene or 10 mM H2O2 for 4 h at 28 °C. The cells were stained by 2.5 µg/mL JC-1 and incubated at 35 °C for 8

ACCEPTED MANUSCRIPT 15 min. The mean of the fluorescence intensities at FL1 (green fluorescence, 525 nm) and FL2 (red fluorescence, 595 nm) were analyzed with a FACSCaliburflow cytometer. The ratio of aggregated JC-1 (FL2) to monomer (FL1) intensity was calculated. A decrease in the ratio

RI PT

means mitochondrial depolarization. Statistical significance was calculated by Student’s t test.

2.8. Assessment of cytochrome c release

SC

C. albicans cells (1 × 105 cells/mL) were incubated with 5 µg/mL lycopene or 10 mM

M AN U

H2O2 for 4 h at 28 °C. The cells were homogenized in buffer A (50 mM Tris, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, pH 7.5). To eliminate the impurities, 2% glucose was added and the mixture was centrifuged at 2,000 g for 10 min. The supernatants were gathered to detect released cytoplasmic cytochrome c. To get pure mitochondria, the pellet was

TE D

washed in buffer B (50 mM Tris, 2 mM EDTA, pH 5.0) by centrifugation at 5,000 g for 30 s. The mitochondria were then suspended in 2 mg/mL Tris-EDTA buffer. After treatment with 500 mg/mL ascorbic acid for 5 min, the cytochrome c contents in the cytoplasmic or

EP

mitochondrial samples were checked at 550 nm with a spectrophotometer (DU530, Beckman) [28]. The protein content was quantified using BSA as a standard [29]. Statistical significance

AC C

was calculated by Student’s t -test.

2.9. Caspase activation assay Using the CaspACE FITC-VAD-FMK in Situ Marker (Promega), caspase acitvation in C. albicans was investigated. C. albicans cells (1 × 105 cells/mL) were treated with 5 µg/mL lycopene or 10 mM H2O2 for 4 h at 28 °C. They were washed in PBS and stained by 2.5 µM CaspACE FITC-VAD-FMK. The cells were examined using a FACSCalibur flow cytometer 9

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

(Becton Dickinson) [30]. Statistical significance was calculated by Student’s t -test.

10

ACCEPTED MANUSCRIPT 3. Results 3.1. Lycopene induces early and late stages of apoptosis We hypothesized that lycopene induced apoptosis in C. albicans through plasma

RI PT

membrane depolarization and G2/M phase arrest from our previous study. In the following experiments, C. albicans were treated with the minimum inhibitory concentration (MIC) of

lycopene (5 µg/mL). At the concentration, lycopene showed no hemolytic activity against

SC

human erythrocytes [12]. To investigate the antifungal mechanism of lycopene, we assessed the externalization of phosphatidylserine from the inner to the outer layer of the fungal

M AN U

plasma membrane, an early marker of apoptosis. This staining technique allows for the detection of apoptotic cells by FITC-Annexin V and PI co-staining. Cells undergoing early apoptosis are Annexin V-FITC+/PI-, whereas cells undergoing late apoptosis or necrotic cell death are Annexin V-FITC+/PI+. In this study, 10 mM of H2O2 was used as a positive control because it can induce fungal apoptosis via oxidative stress [31]. Our results indicated that,

TE D

when treated with lycopene or H2O2, C. albicans cells showed an Annexin V-FITC+/PIphenotype (lycopene, 20.83%; H2O2, 30.74%), suggesting that the cells are undergoing early

EP

apoptosis. Few of the cells showed Annexin V-FITC+/PI+ phenotype (lycopene, 5.96%; H2O2, 1.42%), suggesting late apoptosis (Fig. 1A).

AC C

DNA fragmentation and nuclear condensation and fragmentation are hallmarks of late apoptosis [23]. To confirm that lycopene induces late stage of apoptosis, morphological changes in DNA were observed. The TUNEL assay is used to monitor apoptotic DNA breakage by labeling the free 3'-OH terminus with fluorescent dUTP catalyzed by terminal deoxynucleotidyl transferase [23, 24]. Fluorescence microscopy revealed that C. albicans cells exposed to lycopeneor H2O2 displayed green fluorescent spots, indicating DNA fragmentation (Fig. 1B). Furthermore, a DAPI assay was conducted to monitor 11

ACCEPTED MANUSCRIPT morphological changes in the nucleus. DAPI enters intact cell membranes and binds to the minor groove of A-T rich regions in DNA [32]. DAPI is used as a nuclear morphology indicator, based on the size and roundness of the nucleus. Fluorescence microscopy analysis

RI PT

revealed that cells treated with lycopene or H2O2 displayed more concentrated or cleaved fluorescence compared to untreated cells (Fig. 1B), indicating nuclear condensation and fragmentation.

SC

3.2. Lycopene induces accumulation of ROS, specifically ·OH, which contributes to cell death

M AN U

ROS are considered key factor in yeast apoptosis, because they induce and regulate apoptosis [31]. Thus, we used DHR-123 to assess the effect of lycopene on ROS production in C. albicans. DHR-123 is a ROS-sensitive dye that is oxidized by intracellular ROS to positively charged rhodamine-123 derivatives, resulting in increased fluorescence intensity

TE D

[33]. When the cells treated with lycopene or H2O2, 30.13% or 56.07% of fluorescence increased, respectively, compared to untreated cells (Fig. 2). These results suggested that lycopene causes ROS accumulation in C. albicans.

EP

We next assessed whether lycopene could induce the formation of · OH, a highly active ROS. HPF was used to selectively detect · OH. Upon entering cells, HPF is oxidized to the

AC C

highly fluorescent molecule fluorescein by · OH [34]. Indeed, fluorescent intensity of fluorescein was increased in C. albicans cells treated with lycopene (10.01%) and H2O2 (41.75%) (Fig. 3A). These results indicated that · OH is one of the ROS produced by lycopene treatment.

Excessive ROS accumulation induced by environmental stress causes progressive oxidative damage, and ultimately, cell death [35]. To investigate the effect of · OH production on fungal cell death, changes in fungal viability were observed following the addition of the 12

ACCEPTED MANUSCRIPT ·

OH scavenger thiourea. Thiourea (150 mM) alone did not exhibit a fungicidal activity on C.

albicans. However, thiourea treatment increased the viability from 56.1% to 73.3% or from

3.3 Lycopene elevates cytosolic and mitochondrial Ca2+ levels

RI PT

2.5% to 45.5%, in cells treated with lycopene or H2O2, respectively (Fig. 3B).

Ca2+ also plays an important role in initiating and executing apoptosis [36]. To examine changes in cytosolic and mitochondrial Ca2+ levels, Fura-2AM and Rhod-2AM were used.

SC

Cell permeable Fura-2AM is the ester form of Fura-2 and is selective for free cytosolic Ca2+.

M AN U

After crossing the membrane, Fura-2AM is quickly metabolized by cytoplasmic esterases to the membrane impermeable dye, Fura-2 [37]. When C. albicans cells were exposed to lycopene and H2O2, the fluorescence intensity of Fura-2 was increased compared with the control cells (Fig. 4A). Rhod-2AM is converted to the free mitochondrial Ca2+-sensitive

TE D

probe, Rhod-2, via intracellular esterases [38]. The fluorescence intensity of Rhod-2AM also increased in the cells treated with lycopene and H2O2 (Fig. 4B). These results indicate that lycopene treatment causes increase in free cytosolic Ca2+ level ([Ca2+]c) and free

EP

mitochondrial Ca2+ level ([Ca2+]m).

AC C

3.4. Lycopene induces mitochondrial depolarization and cytochrome c release to the cytoplasm

Mitochondria have a pivotal role in yeast apoptosis because they are the major site of ROS production and contain many pro-apoptotic factors [39]. Particularly, the loss of ∆Ψm and enhancement of mitochondrial permeability are hallmarks in the early stage of apoptosis [40]. Thus, we assessed mitochondrial involvement in lycopene-induced apoptosis by JC-1 staining. In the mitochondrial matrix of normal cells, the lipophilic cationic JC-1 dye exists as 13

ACCEPTED MANUSCRIPT red aggregates. In contrast, in apoptotic cells with depolarized mitochondria, JC-1 remains in the cytoplasm and appears green [40]. As shown in Fig. 5A, the ratio of FL2/FL1 was decreased in both lycopene-treated cells (5.47) and H2O2-treated cells (0.61), compared to the

RI PT

control cells (6.58) (Fig. 5A). These results mean that mitochondrial depolarization is involved in lycopene-induced apoptosis.

Cytochrome c release from the mitochondria to the cytoplasm is a crucial step in

SC

apoptosis. Hence, the translocation of cytochrome c from mitochondria to cytoplasm was examined. Our results indicate that treatment with lycopene decreased the cytochrome c level

M AN U

in the mitochondria, while increasing the level of cytochrome c in the cytoplasm, as compared to the control cells. H2O2 caused a more potent release of cytochrome c than lycopene (Fig. 5B).

TE D

3.5. Lycopene induces caspase activation

ROS production and mitochondrial dysfunction are associated with caspase activation, which plays a crucial role in the apoptotic signaling network [41]. Therefore, using FITC-

EP

VAD-FMK, we assessed the activation of caspases during lycopene-induced apoptosis. The FITC-labeled caspase inhibitor VAD-FMK irreversibly binds to activated caspases in

AC C

apoptotic cells [30]. Flow cytometric analysis revealed that caspase activation was shown in the cells incubated with lycopene (11.01%) or H2O2 (40.71%) (Fig. 6).

14

ACCEPTED MANUSCRIPT 4. Discussion Apoptosis is a form of programmed cell death that plays an important role in metazoan homeostasis and maintenance by eliminating unwanted, mutated, damaged, and superfluous

RI PT

cells [42]. Recent studies have reported that yeast cells, like metazoans, undergo apoptosis and display representative apoptotic phenotypes, such as phosphatidylserine externalization, DNA fragmentation, chromatin condensation and fragmentation, and ROS production [23,

SC

43]. Thus, antifungal-induced yeast apoptosis is considered a novel mechanism to inhibit yeast proliferation [43].

M AN U

According to our previous study, lycopene induced plasma membrane depolarization and G2/M cell cycle arrest, suggesting that it induced apoptosis [12]. Plasma membrane depolarization has been reported to occur in response to different apoptotic stimuli, including stress-induced, receptor-induced, and drug-induced apoptosis [44]. For example, plasma

TE D

membrane depolarization is observed during Styraxjaponoside C-induced apoptosis in C. albicans [45]. In addition, G2/M cell cycle arrest commonly occurs during yeast apoptosis induced by antifungal agents, such as amphotericin B [24], plagiochin E [46], and benzyl

yeast.

EP

benzoate [47]. Thus, we hypothesized that lycopene has the potential to induce apoptosis in

AC C

To confirm that the antifungal effects of lycopene occur via apoptosis, we assessed several markers of apoptosis in C. albicans. Similar to mammalian cells, yeasts have an asymmetric distribution of phospholipids within the cytoplasmic membrane, with 90% of phosphatidylserines

oriented

toward

the

cytoplasm

[48].

During

apoptosis,

phosphatidylserine is externalized from the inner to the outer layer [49]. FITC-labeled Annexin V binds to phosphatidylserine in the presence of Ca2+ and fluoresces [23]. PI is a membrane-impermeable fluorescent dye that binds between the bases of DNA [50]. The 15

ACCEPTED MANUSCRIPT FITC-Annexin V and PI double staining assay revealed that phosphatidylserine was localized to the outer layer of the plasma membrane without change in membrane permeability following lycopene treatment, indicating that lycopene induced early apoptosis.

RI PT

Phosphatidylserine externalization generally precedes DNA cleavage in the late apoptosis. TUNEL and DAPI assays showed that lycopene induced DNA fragmentation, as well as nuclear fragmentation and condensation, indicating that early apoptosis progressed to the late

SC

stage apoptosis. Taken together, these results suggest that lycopene can affect all stages of apoptosis in C. albicans.

M AN U

It has been reported that lycopene exerts its antifungal activity through fungicidal effects in C. albicans [12]. Fungicidal drugs promote ROS-dependent cellular death, mediated by a common signaling and metabolic cascade [51]. Further, ROS act as an essential intracellular messenger during yeast apoptosis [52]. Thus, we assessed whether ROS production is altered

TE D

during lycopene-induced apoptosis. The DHR-123 assay revealed that lycopene induced ROS accumulation. Excessive ROS generation during apoptosis causes protein and lipid peroxidation, reduced mitochondrial enzyme activity, and direct DNA damage [53].

EP

Considering that lycopene caused ROS accumulation in C. albicans, it seems reasonable to hypothesize that ROS accumulation contributed to the DNA and nucleus damage, observed in

AC C

the TUNEL and DAPI assays. Cells in the G2 phase synthesize additional mRNAs and proteins in preparation for cell division or mitosis (the M phase), where the cell divides into two daughter cells [54]. Cells with incompletely replicated or damaged DNA are prevented from entering mitosis at the G2/M cell cycle checkpoint [55]. Thus, lycopene-induced DNA damage could prevent the G2/M transition, contributing to the G2/M cell cycle arrest in C. albicans [12]. ·

OH participates in various biological processes and mediates apoptosis inmany different 16

ACCEPTED MANUSCRIPT cell types [56]. For example, · OH induces apoptosis in HeLa, MW451, and HL-60 cells [57]. The ROS accumulated by lycopene may be converted into · OH via the Fenton or Haber– Weiss reaction. Therefore, we conducted an HPF assay to detect · OH, and investigated the

RI PT

viability of C. albicans in the presence of thiourea to understand effect of · OH in fungal cell death. Our results revealed that lycopene induced · OH generation, and that thiourea increased the viability of cells treated with lycopene. Thus, we confirmed that ROS accumulation triggered subsequent · OH generation, eventually leading to fungal cell death.

SC

Recently, several studies reported that the Ca2+ signaling pathway is linked to yeast

M AN U

apoptosis. For example, a rise in cytoplasmic Ca2+ has been observed in pheromone- and amiodarone-induced Saccharomycescerevisiae apoptosis and H2O2-induced C. albicans apoptosis [58, 59]. To examine cytosolic and mitochondrial Ca2+ handling in lycopeneinduced apoptosis, Fura-2AM and Rhod-2AM assays were done. The results showed that

TE D

lycopene induced Ca2+ overload in both the cytosol and the mitochondria. During apoptosis, lycopene caused depolarization of the plasma membrane [12], leading to an imbalance in Ca2+ influx and Ca2+ export at the plasma membrane and a sustained increase in [Ca2+]c. This

EP

further led to a progressive increase in mitochondrial Ca2+ uptake. The [Ca2+]c and [Ca2+]m can also be regulated by the release of Ca2+ from intracellular stores, such as the endoplasmic

AC C

reticulum (ER). In mammalian cells, prolonged [Ca2+]m elevation facilitates cytochrome c release from the mitochondria to the cytoplasm [60]. The released cytochrome c binds to the inositol-3-phosphate receptor, which is a calcium channel in the outer membrane of the ER, resulting in release of Ca2+ from the ER to the cytoplasm, and thereby promoting of apoptosis [61]. Like mammalian cells, yeast cells store Ca2+ in organelles such as Golgi apparatus, ER, and vacuoles [62]. Therefore, lycopene could induce an elevation in [Ca2+]c and [Ca2+]m by damaging Ca2+ influx and Ca2+ export across the plasma membrane or by releasing Ca2+ from 17

ACCEPTED MANUSCRIPT intracellular stores in the apoptotic pathway. During apoptosis in yeast, ROS production triggers mitochondrial dysfunction [63]. This mitochondrial dysfunction leads to depolarization of mitochondrial membrane and

RI PT

translocation of pro-apoptotic factors from the mitochondria to the cytoplasm (cytochrome c) or to the nucleus (Aif1p and Nuc1p) [64]. We therefore investigated mitochondrial signs of apoptosis in C. albicans following lycopene treatment. The JC-1 and cytochrome c assays

SC

indicated that lycopene induced mitochondrial depolarization and cytochrome c release from the mitochondria to the cytoplasm. These results suggested that lycopene causes

M AN U

mitochondrial dysfunction via ROS production, thereby inducing apoptotic cell death. Thus, the mitochondria play a key role in lycopene-induced apoptosis in C. albicans. Similar to caspase in mammalian cells, structural homologues of the caspase family (metacaspases) have been identified in yeast cells and have been shown to regulate apoptosis

TE D

[30, 65]. For example, the Saccharomyces cerevisiae metacaspase Yeast Caspase 1 (Yca1p) has enzymatic peptidase activity analogous to mammalian caspase activity [30]. In C. albicans, CaMCA1, a homologue of the Saccharomyces cerevisiae metacaspase Yca1, was

EP

identified and shown to be involved in oxidative stress-induced apoptosis [66]. In addition, some unidentified caspase(s) or caspase-like proteases in C. albicans have been recently

AC C

reported [67]. Released cytochrome c into the cytoplasm can cause caspase activation [68]. The loss of mitochondrial membrane potential also coincides with caspase activation [40]. Thus lycopene-induced cytochrome c release and mitochondrial depolarization may regulate activation of caspase in C. albicans. The caspase activity analysis indicated that lycopene activated caspases. Taken together, lycopene induced an accumulation of ROS, including ·

OH. The accumulated ROS triggered DNA damage, thereby contributing to G2/M cell cycle

arrest and apoptosis in C. albicans. Lycopene-induced apoptosis involved Ca2+- and 18

ACCEPTED MANUSCRIPT mitochondria-mediated pathway. During apoptotic cell death, intracellular Ca2+ was increased, and mitochondrial dysfunction, such as mitochondrial depolarization and cytochrome c release, was triggered. At last the released cytochrome c led to caspase

RI PT

activation. In conclusion, lycopene exerted its antifungal effects against C. albicans by inducing apoptosis via ROS production and mitochondria dysfunction. The present study provides an

SC

understanding of the mechanisms of lycopene-induced apoptosis in C. albicans, and suggests

AC C

EP

TE D

M AN U

that lycopene has potential as an antifungal compounds with apoptotic activity.

19

ACCEPTED MANUSCRIPT Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant

AC C

EP

TE D

M AN U

SC

RI PT

funded by the Korea government (MSIP) (No. 2008-0062618).

20

ACCEPTED MANUSCRIPT References [1]

W. Stahl, H. Sies, Lycopene: a biologically important carotenoid for humans? Arch. Biochem. Biophys. 336 (1996) 1-9. N.G. Tao, Z.Y. Hu, Q. Liu, J. Xu, Y.J. Cheng, L.L. Guo, W.W. Guo, X.X. Deng,

RI PT

[2]

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck), Plant Cell Rep. 26 (2007) 837-843.

S. Machmudah, Y. Kawahito, M. Sasaki, M. Goto, Process optimization and extraction

SC

[3]

rate analysis of carotenoids extraction from rosehip fruit using supercritical CO2. J.

[4]

M AN U

Supercrit. Fluids 44 (2008) 308-314.

N. Tao, Y. Gao, Y. Liu, F. Ge, Carotenoids from the Peel of Shatian Pummelo (Citrus grandis Osbeck) and its antimicrobial activity, American-Eurasian J. Agric. & Environ. Sci. 7 (2010) 110-115.

M.R.A. Manimala, R. Murugesan, In vitro antioxidant and antimicrobial activity of

TE D

[5]

carotenoid pigment extracted from Sporobolomyces sp. isolated from natural source, J. Appl. Nat. Sci. 6 (2014) 649-653.

H. Salman, M. Bergman, M. Djaldetti, H. Bessler, Lycopene affects proliferation and

EP

[6]

apoptosis of four malignant cell lines, Biomed. Pharmacother. 61 (2007) 366-369. A.J. Teodoro, F.L. Oliveira, N.B. Martins, A. Maia Gde, R.B. Martucci, R. Borojevic,

AC C

[7]

Effect of lycopene on cell viability and cell cycle progression in human cancer cell lines, Cancer Cell Int. 12 (2012) 36. [8]

C. Soares Nda, A.J. Teodoro, F.L. Oliveira, C.A. Santos, C.M. Takiya, O.S. Junior, M. Bianco, A.P. Junior, L.E. Nasciutti, L.B. Ferreira, E.R. Gimba, R. Borojevic, Influence of lycopene on cell viability, cell cycle, and apoptosis of human prostate cancer and benign hyperplastic cells, Nutr. Cancer 65 (2013) 1076-1085. 21

ACCEPTED MANUSCRIPT [9]

P.D. Mascio, S. Kaiser, H. Sies, Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Arch. Biochem. Biophys. 274 (1989) 532-538.

[10] M.M. Rafi, P.N. Yadav, M. Reyes, Lycopene inhibits LPS-induced proinflammatory

RI PT

mediator inducible nitric oxide synthase in mouse macrophage cells, J. Food Sci. 72 (2007) S069-074.

[11] D. Feng, W.H. Ling, R.D. Duan, Lycopene suppresses LPS-induced NO and IL-6

macrophages, Inflamm. Res. 59 (2010) 115-121.

SC

production by inhibiting the activation of ERK, p38MAPK, and NF-kappaB in

M AN U

[12] W.S. Sung, I.S. Lee, D.G. Lee, Damage to the cytoplasmic membrane and cell death caused by lycopene in Candida albicans, J. Microbiol. Biotechnol. 17 (2007) 17971804.

[13] X. Tang, X. Yang, Y. Peng, J. Lin, Protective effects of lycopene against H2O2-induced

(2009) 439-448.

TE D

oxidative injury and apoptosis in human endothelial cells, Cardiovasc. Drugs Ther. 23

[14] P. Palozza, R. Simone, A. Catalano, A. Boninsegna, V. Böhm, K. Fröhlich, M.C. Mele,

EP

G. Monego, F.O. Ranelletti, Lycopene prevents 7-ketocholesterol-induced oxidative stress, cell cycle arrest and apoptosis in human macrophages, J. Nutr. Biochem. 21

AC C

(2010) 34-46.

[15] T. Meri, A.M. Blom, A. Hartmann, D. Lenk, S. Meri, P.F. Zipfel, The hyphal and yeast forms of Candida albicans bind the complement regulator C4b-binding protein, Infect. Immun. 72 (2004) 6633-6641. [16] J.R. Naglik, S.J. Challacombe, B. Hube, Candida albicans secreted aspartyl proteinases in virulence and pathogenesis, Microbiol. Mol. Biol. Rev. 67 (2003) 400-428.

22

ACCEPTED MANUSCRIPT [17] K.A. Toenjes, S.M. Munsee, A.S. Ibrahim, R. Jeffrey, J.E. Edwards, D.I. Jr, Johnson, Small-molecule inhibitors of the budded-to-hyphal-form transition in the pathogenic yeast Candida albicans, Antimicrob. Agents Chemother. 49 (2005) 963-972.

9 (2001) 327-335.

RI PT

[18] R.A. Calderone, W.A. Fonzi, Virulence factors of Candida albicans, Trends Microbiol.

[19] J. Berman, P.E. Sudbery, Candida Albicans: a molecular revolution built on lessons

SC

from budding yeast, Nat. Rev. Genet. 3 (2002) 918-930.

[20] S.P. Saville, A.L. Lazzell, C. Monteagudo, J.L. Lopez-Ribot, Engineered control of cell

M AN U

morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection, Eukaryot. Cell 2 (2003) 1053-1060. [21] D.R. Snydman, Shifting patterns in the epidemiology of nosocomial Candida infections, Chest 123 (2003) 500S-503S.

TE D

[22] M.A. Pfaller, L. Boyken, R.J. Hollis, S.A. Messer, S. Tendolkar, D.J. Diekema, In vitro activities of anidulafungin against more than 2,500 clinical isolates of Candida spp., including 315 isolates resistant to fluconazole, J. Clin. Microbiol. 43 (2005) 5425-5427.

EP

[23] F. Madeo, E. Fröhlich, K.U. Fröhlich, A yeast mutant showing diagnostic markers of early and late apoptosis, J. Cell Biol. 139 (1997) 729-734.

AC C

[24] A.J. Phillips, I. Sudbery, M. Ramsdale, Apoptosis induced by environmental stresses and amphotericin B in Candida albicans, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 14327-14332.

[25] S.J. Zunino, J.M. Ducore, D.H. Storms, Parthenolide induces significant apoptosis and production of reactive oxygen species in high-risk pre-B leukemia cells, Cancer Lett. 254 (2007) 119-127.

23

ACCEPTED MANUSCRIPT [26] D.M. Arduino, A.R. Esteves, A.F. Domingues, C.M. Pereira, S.M. Cardoso, C.R. Oliveira, ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells, BMB Rep. 42 (2009) 719-724.

RI PT

[27] A. Almeida, J. Almeida, J.P. Bolaños, S. Moncada, Different responses of astrocytes and neurons to nitric oxide: the role of glycolytically generated ATP in astrocyte protection, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 15294-15299.

SC

[28] E.M. Barbu, F. Shirazi, D.M. McGrath, N. Albert, R.L. Sidman, R. Pasqualini, W. Arap, D.P. Kontoyiannis, An antimicrobial peptidomimetic induces mucorales cell death

M AN U

through mitochondria-mediated apoptosis, PLoS One 8 (2013) e76981. [29] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265-275.

[30] F. Madeo, E. Herker, C. Maldener, S. Wissing, S. Lächelt, M. Herlan, M. Fehr, K.

TE D

Lauber, S.J. Sigrist, S. Wesselborg, K.U. Frolich, A caspase-related protease regulates apoptosis in yeast, Mol. Cell 9 (2002) 911-917. [31] F. Madeo, E. Fröhlich, M. Ligr, M. Grey, S.J. Sigrist, D.H. Wolf, K.U. Fröhlich,

EP

Oxygen stress: a regulator of apoptosis in yeast, J. Cell Biol.145 (1999) 757-767. [32] A.J. Munoz, K. Wanichthanarak, E. Meza, D. Petranovic, Systems biology of yeast cell

AC C

death, FEMS Yeast Res. 12 (2012) 249-265. [33] Y. Jiang, S. Zhou, G.E. Sandusky, M.R. Kelley, M.L. Fishel, Reduced expression of DNA repair and redox signaling protein APE1/Ref-1 impairs human pancreatic cancer cell survival, proliferation, and cell cycle progression, Cancer Invest. 28 (2010) 885895.

24

ACCEPTED MANUSCRIPT [34] K. Setsukinai, Y. Urano, K. Kakinuma, H.J. Majima, T. Nagano, Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species, J. Biol. Chem. 278 (2003) 3170-3175.

RI PT

[35] A. Alboresi, L. Dall'osto, A. Aprile, P. Carillo, E. Roncaglia, L. Cattivelli, R. Bassi, Reactive oxygen species and transcript analysis upon excess light treatment in wildtype Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein,

SC

BMC Plant Biol. 11 (2011) 62.

[36] M.P. Mattson, Apoptosis in neurodegenerative disorders, Nat. Rev. Mol. Cell Biol. 1

M AN U

(2000) 120-129.

[37] J. Vallipuram, J. Grenville, D.A. Crawford, The E646D-ATP13A4 mutation associated with autism reveals a defect in calcium regulation, Cell. Mol. Neurobiol. 30 (2010) 233-246.

TE D

[38] G.R. Monteith, Seeing is believing: recent trends in the measurement of Ca2+ in subcellular domains and intracellular organelles, Immunol. Cell Biol. 78 (2000) 403407.

EP

[39] C. Pereira, R.D. Silva, L. Saraiva, B. Johansson, M.J. Sousa, M. Côrte-Real, Mitochondria-dependent apoptosis in yeast, Biochim. Biophys. Acta 1783 (2008) 1286-

AC C

1302.

[40] G. Yao, L. Yang, Y. Hu, J. Liang, J. Liang, Y. Hou, Nonylphenol-induced thymocyte apoptosis involved caspase-3 activation and mitochondrial depolarization, Mol. Immunol. 43 (2006) 915-926. [41] F. Shirazi, D.P. Kontoyiannis, Mitochondrial respiratory pathways inhibition in Rhizopusoryzae potentiates activity of posaconazole and itraconazole via apoptosis, PLoS One 8 (2013) e63393. 25

ACCEPTED MANUSCRIPT [42] P. Rockenfeller, F. Madeo, Apoptotic death of ageing yeast, Exp. Gerontol. 43 (2008) 876-881. [43] B. Almeida, A. Silva, A. Mesquita, B. Sampaio-Marques, F. Rodrigues, P. Ludovico,

RI PT

Drug-induced apoptosis in yeast, Biochim. Biophys. Acta 1783 (2008) 1436-1448. [44] R. Franco, C.D. Bortner, J.A. Cidlowski, Potential roles of electrogenic ion transport and plasma membrane depolarization in apoptosis, J. Membr. Biol. 209 (2006) 43-58.

SC

[45] C. Park, E.R. Woo, D.G. Lee, Antifungal effect with apoptotic mechanism(s) of Styraxjaponoside C, Biochem. Biophys. Res. Commun. 390 (2009) 1255-1259.

M AN U

[46] W.Z. Wu, W.Q. Chang, A.X. Cheng, L.M. Sun, H.X. Lou, Plagiochin E, an antifungal active macrocyclicbis (bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway, Biochim. Biophys. Acta 1800 (2010) 439447.

TE D

[47] G.B. Zore, A.D. Thakre, S. Jadhav, S.M. Karuppayil, Terpenoids inhibit Candida albicans growth by affecting membrane integrity and arrest of cell cycle, Phytomedicine 18 (2011) 1181-1190.

EP

[48] J. Cerbón, V. Calderón, Changes of the compositional asymmetry of phospholipids associated to the increment in the membrane surface potential, Biochim. Biophys. Acta

AC C

1067 (1991) 139-144.

[49] J. Reiter, E. Herker, F. Madeo, M.J. Schmitt, Viral killer toxins induce caspasemediated apoptosis in yeast, J. Cell Biol. 168 (2005) 353-358. [50] D.V. Krysko, T. VandenBerghe, K. D'Herde, P. Vandenabeele, Apoptosis and necrosis: detection, discrimination and phagocytosis, Methods 44 (2008) 205-221. [51] P. Belenky, D. Camacho, J.J. Collins, Fungicidal drugs induce a common oxidativedamage cellular death pathway, Cell Rep. 3 (2013) 350-358. 26

ACCEPTED MANUSCRIPT [52] C. Mazzoni, E. Herker, V. Palermo, H. Jungwirth, T. Eisenberg, F. Madeo, C. Falcone, Yeast caspase 1 links messenger RNA stability to apoptosis in yeast, EMBO Rep. 6 (2005) 1076-1081.

RI PT

[53] V. Costa, P. Moradas-Ferreira, Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases, Mol. Aspects Med. 22 (2001) 217–246.

SC

[54] C. Hutchison, D.M. Glover, Cell Cycle Control, Oxford University Press, New York, 1995.

M AN U

[55] J.M. Li, G. Brooks, Cell cycle regulatory molecules (cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors) and the cardiovascular system; potential targets for therapy? Eur. Heart J. 20 (1999) 406–420.

[56] K. Setsukinai, Y. Urano, K. Kakinuma, H.J. Majima, T. Nagano, Development of novel

TE D

fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species, J. Biol. Chem. 278 (2003) 3170-3175. [57] J.G. Ren, H.L. Xia, T. Just, Y.R. Dai, Hydroxyl radical-induced apoptosis in human

EP

tumor cells is associated with telomere shortening but not telomerase inhibition and caspase activation, FEBS Lett. 488 (2001) 123-132.

AC C

[58] A.I. Pozniakovsky, D.A. Knorre, O.V. Markova, A.A. Hyman, V.P. Skulachev, F.F. Severin, Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast, J. Cell Biol. 168 (2005) 257-269. [59] H. Lu, Z. Zhu, L. Dong, X. Jia, X. Sun, L. Yan, Y. Chai, Y. Jiang, Y. Cao, Lack of trehalose accelerates H2O2-induced Candida albicans apoptosis through regulating Ca2+ signaling pathway and caspase activity, PLoS One 6 (2011) e15808. [60] M. Narita, S. Shimizu, T. Ito, T. Chittenden, R.J. Lutz, H. Matsuda, Y. Tsujimoto, Bax 27

ACCEPTED MANUSCRIPT interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 14681-14686.

RI PT

[61] X. Liu, C.N. Kim, J. Yang, R. Jemmerson, X. Wang, Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c, Cell 86 (1996) 147-157. [62] A. Miseta, L. Fu, R. Kellermayer, J. Buckley, D.M. Bedwell, The Golgi apparatus plays

SC

a significant role in the maintenance of Ca2+ homeostasis in the vps33Delta vacuolar biogenesis mutant of Saccharomyces cerevisiae, J. Biol. Chem. 274 (1999) 5939-5947.

M AN U

[63] C. Xu, J. Wang, Y. Gao, H. Lin, L. Du, S. Yang, S. Long, Z. She, X, Cai, S. Zhou, Y. Lu, The anthracenedione compound bostrycin induces mitochondria-mediated apoptosis in the yeast Saccharomyces cerevisiae, FEMS Yeast Res. 10 (2010) 297-308. [64] P. Ludovico, F. Rodrigues, A. Almeida, M.T. Silva, A. Barrientos, M. Côrte-Real,

TE D

Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae, Mol. Biol. Cell 13 (2002) 2598-2606. [65] A.G. Uren, K. O’Rourke, L.A. Aravind, M.T. Pisabarro, S. Seshagiri, Identification of

EP

paracaspases and metacaspases: two ancient families of caspase like proteins, one of which plays a key role in MALT lymphoma, Mol. Cell 6 (2000) 961–967.

AC C

[66] Y. Cao, S. Huang, B. Dai, Z. Zhu, H. Lu, L. Dong, Y. Cao, Y. Wang, P. Gao, Y. Chai, Y. Jiang, Candida albicans cells lacking CaMCA1-encoded metacaspase show resistance to oxidative stress-induced death and change in energy metabolism, Fungal Genet. Biol. 46 (2009) 183-189.

[67] A.M. Aerts, D. Carmona-Gutierrez, S. Lefevre, G. Govaert, I.E. François, F. Madeo, R. Santos, B.P. Cammue, K. Thevissen, The antifungal plant defensin RsAFP2 from radish

28

ACCEPTED MANUSCRIPT induces apoptosis in a metacaspase independent way in Candida albicans, FEBS Lett. 583 (2009) 2513-2516. [68] C. Pereira, N. Camougrand, S. Manon, M.J. Sousa, M. Côrte-Real, ADP/ATP carrier is

AC C

EP

TE D

M AN U

SC

in yeast apoptosis, Mol. Biol. 66 (2007) 571-582.

RI PT

required for mitochondrial outer membrane permeabilization and cytochrome c release

29

ACCEPTED MANUSCRIPT Figure captions Fig. 1. The early and late stages of apoptosis in C. albicans. (A) Phosphatidylserine externalization during early apoptosis was assessed by FITC-Annexin V and PI double

RI PT

staining. (a) Phosphatidylserine externalization was analyzed by flow cytometry. (b) The data display the mean ± SD from three independent experiments. *P < 0.05 (Student's t test). (B) DNA and nuclear damage during late stage apoptosis was visualized by fluorescence

SC

microscopy. (a) DNA fragmentation was observed by TUNEL assay. (b) Concentrated or split fluorescence of DAPI indicates nuclear condensation and fragmentation.

M AN U

Fig. 2. ROS generation induced by lycopene treatment. (a) Changes in intracellular ROS were assessed by flow cytometry using DHR-123. (b) The data display the mean ± SD from three independent experiments. **P < 0.01 (Student's t test).

Fig. 3. Hydroxyl radical accumulation contributing to the fungal cell death. (A) Hydroxyl

TE D

radical generation was assessed by HPF staining. (a) Hydroxyl radical generation was monitored by flow cytometry. (b) The data display the mean ± SD from three independent experiments. **P < 0.01 (Student's t test). (B) Percentage of survival was evaluated by

EP

counting CFUs after treatment with the agents for 4 h at 28 °C. The data display the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 (Student's t test).

AC C

Fig. 4. Increase in the cytosolic and mitochondrial Ca2+ levels was detected by Fura-2AM (A) and Rhod-2AM (B) assays. The data display the mean ± SD from three independent experiments. *P < 0.05 (Student's t test). Fig. 5. (A) Mitochondrial depolarization was detected using JC-1. (a) Mitochondrial depolarization was analyzed by flow cytometry. (b) Mitochondrial membrane potential was shown as the ratio of FL2/FL1. The FL2/FL1 ratio was measured using the mean value of FL1 or FL2 from 10000 cells. A decrease in the ratio was considered as depolarization of 30

ACCEPTED MANUSCRIPT mitochondrial membrane. The data display the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 (Student's t test). (B) The release of cytochrome c from the mitochondria (a) to the cytoplasm (b) in C. albicans was analyzed by measuring the

experiments using the mean ± SD. *P < 0.05 (Student's t test).

RI PT

absorbance at 550 nm with a spectrophotometer. Data are presented from three independent

Fig. 6. Caspase activity in C. albicans was evaluated by FITC-VAD-FMK assay. (A)

SC

Caspase activation was analyzed by flow cytometry. (B) The data display the mean ± SD

AC C

EP

TE D

M AN U

from three independent experiments. **P < 0.01 (Student's t test).

31

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT ㆍWe have focused on the antifungal mechanism of lycopene. ㆍLyopene exerted its antifungal effect against C. albicans by inducing apoptosis.

AC C

EP

TE D

M AN U

SC

RI PT

ㆍDuring apoptosis, lycopene induces ROS production and mitochondrial dysfunction.