In vitro antifungal activity of Shikonin against Candida albicans by inducing cellular apoptosis and necrosis

Our previous studies showed that Shikonin (SK) had a strong anti-Candida albican (C. albicans) activity, especially against some fluconazole-resistant strains, which is probably due to the oxidative damage of SK to C. albicans. In this study, we expanded the antifungal spectrum and evaluate the toxicity of SK. The results indicated that SK also exhibited potent invitro antifungal activities against other pathogenic fungi such as other Candida, Aspergillus, Cryptococcus, and Dermatophytes, but did not display apparent toxicity to the mammalian cells, suggesting that SK is safe to be a potential antifungal drug. Furtherly, we analyze the exact mechanism of SK against C. albicans. We found that SK could induce a series of apoptosis characteristics, including phosphatidylserine externalization, chromatin condensation and fragmentation, decreased cytochrome c oxidase activity as well as caspase activation. In summary, this study highlighted the antifungal activity and mechanism of SK against C. albicans, providing a potential therapeutic strategy for C. albicans infection.


Introduction
Candida albican is an endophytic fungus, which usually lives in the human gastrointestinal tract and skin. However, as an opportunistic pathogen, C. albicans would cause severe mucosal infections and fatal invasive infections in people with lowered immune function due to AIDS or cancer radioor chemotherapy. Besides, C. albicans can provide suitable living and establishing conditions for other fungus, with such complicated infections leading to more severe infections and antimicrobial resistance [1,2]. Nowadays, with the mass application of broad-spectrum antifungal drugs, more and more multidrug-resistant strains are isolated from C. albicans-infected patients [3]. Therefore, it is necessary to explore new antifungal drugs with high efficiency and low drug resistance to deal with fatal oidiomycosis.
Many studies have shown that extracts from plants used in traditional medicine have broad antifungal activities, such as berberine, forsythiaside, baicalein, etc. [4][5][6]. Shikonin (SK) is the major constituent of the fat-soluble naphthoquinone pigment extracted from the rhizome of the traditional Chinese herbal medicine named Zicao [7]. It has been reported that SK can induce apoptosis of many kinds of tumor cells, thereby achieving antitumor effects [8]. Our previous study found that SK possessed potent activities against almost all C. albicans isolates, with MIC 80 value ranging from 2 to 8 µg/mL. It should be noted that, for some FCZ-resistant C. albicans strains, the action of SK (MIC 80 value 4 µg/ mL) was shown to be over 16 times higher than that of FCZ (MIC 80 > 64 µg/mL) [9]. Furtherly, in this research, SK also exhibited potent invitro antifungal activities against other pathogenic fungi such as other Candida, Aspergillus, Cryptococcus, and Dermatophytes, but did not display apparent toxicity to the normal mouse hepatocytes and human skin fibroblast (HSF) cells. Nevertheless, the detailed mechanisms of SK against C. albicans remain unclear. Based on our original works, this study will further explore the mechanism of SK-induced apoptosis of C. albicans, in order to provide an important experimental basis for its antifungal infection.

Strains, media and compounds
Candida albican SC5314 and Cryptococcus neoformans ATCC32609 are the standard strains used in many fungal studies. Other strains mentioned above are clinical isolates, which were obtained from Changhai Hospital in Shanghai and identified by morphology and biochemistry. For all vitro experiments, 6.4 mg/mL SK (purity > 99%, Beijing Suolaibao Technology Co. Ltd, Beijing, China, dissolved in DMSO (Sigma)) was used as the stock solution stored at − 20 °C and added to the culture medium to obtain the required concentration. Media used in this study included Sabouraud dextrose agar plate (SDA, 1% peptone, 4% glucose, and 1.8% agar), yeast extract peptone dextrose (YPD, 1% w/v yeast extract, 2% w/v peptone, 2% w/v dextrose), phosphate buffered saline (PBS) [10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride (pH 7.4)] and RPMI 1640 (Gibco, Bethesda, MD, USA) supplemented with L-glutamine and buffered with morpholinepropanesulfonic acid (MOPS). C. albicans strains were grown in SDA plates medium, and cultivated in a liquid complete medium YPD medium at 30 °C with constant shaking (200 rpm).

Antifungal susceptibility test
Assays were tested according to the methods of the CLSI (formerly NCCLS) (M27-A3), with a few modifications [10]. Briefly, Candida or Cryptococcus was activated in YPD medium for overnight. Aspergillus or Dermatophytes was cultured on SDA plates and resuspended in RPMI 1640 for counting. Next, the initial concentration of the fungal suspension in RPMI 1640 medium was 1 × 10 3 colonyforming units (CFU)/mL, Then, the fungal suspension and drugs were added to a 96-well plate, and the final drug concentrations were 0.125-64 µg/mL for SK. The 96-well plates were incubated at 30 °C or 35 °C for indicated time. Candida was cultured for 24 h. Aspergillus and Cryptococcus were cultured for 72 h. Dermatophytes were cultured for 7 days. Optical density was measured at 630 nm and background optical densities were subtracted from that of each well. Each isolate was tested in triplicate. The MIC 80 values were determined as the lowest concentration of the drugs that inhibited growth by 80% compared with that of drug-free wells.

Cytotoxicity studies
Cytotoxicity of SK was assessed by cell counting kit-8 (CCK-8) as described [11]. Both AML12 (alpha-mouse-liver-12) mouse hepatocytes and human skin fibroblast (HSF) cells were used to evaluate the toxicity of SK, these two cells were seeds into 96-well plates at 5 × 10 4 cells/mL and grown at 37 °C with 5% CO 2 over-night. The media was removed and replaced with fresh media containing different concentrations of SK followed by incubation for further 24 h. After inoculation, 10 µL of CCK-8 solution was added to each well incubated for another 2 h. Cell viability was assessed by detecting absorbance at 450 nm. Cells incubated with DMSO treatment were calculated as the standard for 100% viability. Three independent experiments were conducted.

Protoplast preparation
To study the effect of test SK on apoptotic markers, protoplasts were prepared as described previously [12], with modifications. Briefly, mid-exponential phase cells were harvested and exposed to various concentrations of SK shaken at 30 °C for 2 h at 200 rpm. The cells were then washed twice with PBS. Next, the cells was resuspended at a concentration of 3 × 10 7 CFU/mL and digested with lyticase (100 U/mL) at 30 °C for 30 min. The digested cells were centrifuged at 1500 rpm for 5 min again and the supernatant was discarded. Finally, the obtained protoplasts were washed twice with PBS and resuspended for further use.

Annexin V-FITC/PI-staining
This assay was used to identify necrosis (cellular integrity) and apoptosis (externalization of phosphatidylserine). Annexin-V-FLUOS kit (Roche Applied Science, Germany) was used following the manufacturer's instructions [13]. Briefly, protoplasts of C. albicans cells were treated with different concentrations of SK, as mentioned in above section, and 10 mM H 2 O 2 was used as a positive control. Then these preparation protoplasts were resuspended and incubated at 25 °C for 15 min by 5 uL propidium iodide (PI) and FITC-labeled annexin-V. After incubation, 100 µL binding buffer was added to resuspend each sample, then which was analyzed using the flow cytometry (FACSCalibur, USA). The detection conditions are as follows: Annexin V-FITC: excitation wavelength 488 nm, emission wavelength 518 nm; PI: excitation wavelength 488-540 nm, emission wavelength 617 nm.

Transmission electron microscope (TEM) [14]
The C. albicans SC5314 (3 × 10 7 CFU/mL) were firstly administrated by 4 µg/mL SK for 2 h at 37 °C, and then harvested by centrifugation (3000×g, 5 min). The collected cells were promptly placed in 3% glutaraldehyde at 4 °C overnight, fixed with 1% (w/v) osmium acid for 1 h. After gradient dehydration with ethanol and acetone, it was immersed, embedded, polymerized, and sliced. Finally, thin sections were prepared and stained by uranyl acetate for 2 h, being observed under Hitachi H-800 transmission electron microscope.

Cytochrome c oxidase (COX) activity
Firstly, C. albicans mitochondria of the cells were separated by Cell Mitochondria Isolation Kit (Beyotime). Then, COX detection kit (GENMED) was used to detect mitochondrial COX activity [15]. All experiments were performed according to the manufacturer's instructions. Briefly, protoplasts of C. albicans cells were treated with 1, 2, 4 μg/mL SK as mentioned in above section, and 10 mM H 2 O 2 was used as a positive control. Then these preparation protoplasts were homogenized in order to release the mitochondria. After centrifugation at a speed of 10,000×g for 10 min at 4 °C, the supernatant was discarded, and the precipitation particles (mitochondrial precipitate) were resuspended in 200 μL of storage solution. Protein concentration was determined by the TCA Lowry method [16]. After that, the mitochondrial COX activity need be determined. Firstly, these prepared mitochondrial samples were melted in ice. Secondly, set the spectrophotometer parameters: temperature 25 °C, wavelength 550 nm, reading at 0 s and 60 s each time. Thirdly, 850 μL buffer solution and 50 μL reaction working solution were added into the colorimetric cup, then which was detected for the background reading (0-60 s reading) by spectrophotometer. Fourthly, added another 100 μL sample (complete mitochondria containing 2 μg mitochondrial protein) into the foregoing colorimetric cup for detecting the sample reading (0-60 s reading). Finally, calculation of sample activity: (sample reading − background reading)/[0.1 (sample volume, mL) × 21.84 (molar absorptivity)] = Unit/mL or μmol/ min/mL.

Real time RT-PCR
RNA isolation and real-time RT-PCR were performed as described previously [9]. Total RNA was extracted according to the instructions of C. albicans RNA extraction kit (TIANZ, Beijing, China). Isolated RNA was resuspended in diethyl pyrocarbonate-treated water. The OD260 and OD280 were measured. First-strand cDNA was obtained using the cDNA synthesis kit (TaKaRa Biotechnology, Dalian, China) for RT-PCR according to the manufacturer's instructions. Real-time PCR was performed using the 7500 Applied Biosystems. SYBR green I (TaKaRa Biotechnology, Dalian, China) is used for real-time monitoring of amplification products. CaMCA1 was amplified with the forward primer 5-TAT AAT AGA CCT TCT GGA C-3 and the reverse primer 5-TTG GTG GAC GAG AAT AAT G-3.
The PCR protocol consisted of denaturation program (95 °C for 10 s); 40 cycles of amplification and quantification program: 95 °C for 10 s (denaturation), 60 °C for 20 s (annealing), 72 °C for 30 s (extension); melting curve program: 60-95 °C with a heating rate of 0.1 °C per second; finally, a cooling step to 40 °C. The changes in SYBR Green I fluorescence in every cycle were monitored by the Light Cycler system software, and the threshold cycle (CT) above background for each reaction was calculated. The CT value of 18S ribosomal RNA (amplified with the forward primer 5-TCT TTC TTG ATT TTG TGG GTGG-3 and the reverse primer 5-TCG ATA GTC CCT CTA AGA AGTG-3) was subtracted from that of the tested gene to obtain a ΔCT value. The ΔCT value of an arbitrary calibrator (e.g., an untreated group) was subtracted from the ΔCT value for each sample to obtain a ΔΔCT value. The gene expression level relative to the calibrator was expressed as 2 −ΔΔCT . Triplicate experiments were conducted to generate a mean value.

Assessment of caspase activity
Caspase activity was detected by the CaspSCREEN Flow Cytometric Apoptosis Detection Kit (BioVision, USA) [17]. The kit used dye D 2 R (rhodamine 110 containing two aspartic acid residues). The experimental procedures are as follows: protoplasts of C. albicans cells were treated with 1, 2, 4 μg/ mL SK as mentioned in above section, then were resuspended in D 2 R staining solution (296 μL incubation buffer, 3 μL 1 M DTT and 1μL D 2 R reagent) at 30 °C for 45 min before viewing and counting under a fluorescence microscope.

Statistical analysis
Data were analyzed using SPSS 19.0 (IBM Corp.) and presented as the mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. P < 0.05 was considered to indicate a statistically significant difference.

SK exhibited fungistatic activity with low toxicity to mammalian cells
In this study, we expanded the antifungal spectrum, and the results showed that MIC 80 of SK against other common pathogenic Candida was ranged from 2 to 8 µg/mL, which contains 3 strains of Candida glabratas, 3 strains of Candida tropicalis, 3 strains of Candida parapsilosis, 3 strains of Candida krusei (Table 1). To further ensure that antifungal effect of SK is not strain-specific, we also tested Aspergillus, Cryptococcus, and Dermatophytes which contain 1 strain of Aspergillus fumigatus, 1 strain of Cryptococcus neoformans, 3 strains of Trichophyton mentagrophytes and 3 strains of Microsporum gypseum. As expected, SK also exhibited similar antifungal effect on all the strains, and the MIC 80 is listed in Table 2. To investigate the selectivity of SK, we assessed its toxicity to both AML12 mouse hepatocytes and HSF cells using cell counting kit-8 (CCK-8). As shown in Fig. 1, no significant influence on the viability of AML12 or HSF was observed when the concentration of SK was below 8 µg/mL. Collectively, these results confirmed low cytotoxicity to mammalian cells of SK, suggesting that SK is safe to be a potential antifungal drug.

SK concentration-dependently induces C. albicans cells death
C. albicans SC5314 cells treated with SK at different concentrations were analyzed by double staining with annexin V-FITC and propidium iodide (PI). Annexin V-FITC was used to detect the apoptotic cells with externalized PS, whereas PI staining was used to detect necrotic cells.
As shown in Fig. 2, after being treated with 2 μg/mL SK, the percentage of death cells was 30.51% (17.42% as apoptosis and 13.09% as necrosis). When the concentration of SK increased to 4 μg/mL, the proportion of death cells was about 60.82% (34.12% as apoptosis and 26.70% as necrosis). When the concentration of SK reached 8 μg/mL, the proportion of death cells further increased (87.77% were necrotic). The negative control (unstimulated cells) resulted in apoptosis (5.50%) and necrosis (4.73%), whereas positive control (10 mM H 2 O 2 ) showed apoptosis (51.43%) and necrosis (19.75%). The results also show that cell death was dose-dependent with a significant increase in the percentage of apoptosis and necrosis compared to unstimulated control.

Effect of SK on the ultrastructure changes of C. albicans
The effect of SK on the ultrastructure changes of C. albicans SC5314 cells was observed by transmission electron microscopy (TEM). As shown in Fig. 3, compared with the untreated control, the cells exposed to 4 µg/mL SK showed extensive chromatin condensation and fragmentation along the nuclear envelope, a leading mark of cellular apoptosis. Cells treated at high fungicidal concentration of SK (8 µg/mL) showed extensive granular cytoplasm with little evidence of an organized internal structure, and nuclear bodies were only rarely observed, a leading mark of cellular necrosis.

SK concentration-dependently reduced C. albicans mitochondrial COX activity
Mitochondrial COX activity is an important indicator for the evaluation of cell function. In this study, the mitochondrial COX activity of C. albicans cells was determined by the transformation of reduced cytochrome c into oxidized cytochrome c. The results showed that the enzyme activity was significantly decreased in a concentration-dependent manner after exposure of C. albicans SC5314 cells to various concentrations of SK compared to the unstimulated control. As shown in Fig. 4, the enzyme activity was reduced to 74.54%, 50.48%, and 24.91% at concentration of 1, 2 and 4 μg/mL SK, respectively. As a positive control, the COX enzyme activity of the 10 mM H 2 O 2 treatment group was 11.09% of the unstimulated control.

SK concentration-dependently improved caspase enzyme activity and CaMCA1 gene transcription level in C. albicans
Since C. albicans caspase and its encoding gene CaMCA1 has been found to play an important role in apoptosis, here we investigated the caspase activity by staining the cells with D 2 R and the staining rate can reflect the activation of caspase in cells. As shown in Fig. 5A, after exposure of C. albicans SC5314 strains to various concentrations of SK for 2 h, an increase in caspase activity was observed in a dose dependent manner. Briefly, the cells staining rates in the 1 μg/mL, 2 μg/mL and 4 μg/mL SK treatment groups were 11.3%, 19.57% and 33.80% respectively, significantly higher than the unstimulated control (5.2%). As the positive control, the cell staining rates in the 10 mM H 2 O 2 treatment group were 37.50%.
In addition, the transcription of caspase activity-related gene CaMCA1 in C. albicans was studied by real time RT-PCR. As shown in Fig. 5B, the expression levels of CaMCA1 in different concentrations of SK-treated groups were increased in a concentration-dependent manner.

Discussion
In recent decades, the incidence of invasive fungal infections, mainly including Candida, Aspergillus, Cryptococcus, and Dermatophytes infection, has increased dramatically. Fig. 4 Effect of various concentrations of SK on mitochondria COX activity in C. albicans SC5314. Untreated cells and cells exposed to H 2 O 2 (10 mM) were used as negative and positive controls respectively. The sample of untreated control was regarded as 100%. These data were mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 compared with the control Fig. 5 Effect of various concentrations of SK on caspase activity in C. albicans SC5314. Untreated cells and cells exposed to H 2 O 2 (10 mM) were used as negative and positive controls respectively. A Percentage of cells with activated caspases staining with D 2 R. B Transcription levels of CaMCA1 were determined by real-time RT-PCR. The mRNA levels were normalized on the basis of their 18S rRNA levels. Gene expression was calculated as the fold increase relative to the control group. These data were mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 compared with the control Meanwhile, due to the wide application of various antifungal drugs, the cross-resistance of fungi has been widely reported [1]. Therefore, it is urgent to research highly effective and low-toxic antifungal compounds with new mechanisms. In our previous study, we identified SK exhibited obvious antifungal activities against almost all C. albicans isolates. However, the systematic evaluation of antifungal activities and related mechanism studies of SK have not been totally clarified. In this study, our research demonstrated SK possessed broad-spectrum antifungal effects against many pathogenic fungal species including Candida, Aspergillus, Cryptococcus, and Dermatophytes with the MIC 80 ranging from 1 to 8 µg/mL. In vitro cytotoxicity tests by two mammalian cells indicated that SK was less toxic. These results suggested that SK may specifically target fungal proteins, which are less homologous to mammalian cells.
Apoptosis refers to one of main pathways of programmed cell death under certain physical and pathological conditions, following the activation of extrinsic or intrinsic death signals regulated by genes. It is a common process in eukaryotes [18]. Inducing or accelerating tumor cell apoptosis has become an important strategy in anti-tumor therapy [19]. In recent years, it has been reported that some natural products of traditional Chinese medicine, such as resveratrol, baicalein and pterostilbene, can induce the apoptosis of C. albicans at a suitable concentration, suggesting that inducing apoptosis of pathogenic fungi is expected to become a potential mean in clinical therapy of fungal infection [20].
In this study, the effect of SK on C. albicans apoptosis was investigated. The results suggested that C. albicans SC5314 strains after being exposed to different concentrations of SK exhibited concentration-dependent effect on the classical traits of yeast apoptosis such as PS externalization, chromatin condensation, decrease in cytochrome c oxidase activity as well as increase in caspase activity and mRNA expression of the gene encoding C. albicans caspase, CaMCA1.
We first analyzed the proportion of apoptotic cells, necrotic cells, and living cells by Annexin V-FITC/PI double staining, which is the most direct method to detect apoptosis. The results showed that apoptosis occurred in C. albicans cells treated with a low concentration of SK, and the proportion of apoptotic and necrotic cells gradually increased with the increase of SK concentration. In addition, compared to the unstimulated control, the chromatin of C. albicans cells with 4 μg/mL SK treatment showed obvious condensation and fragmentation along the nuclear envelope, further suggesting that SK could be an inducer of apoptosis in C. albicans. However, at higher concentration of SK (8 μg/mL), most cells were in necrosis. It suggested high concentration of SK exert direct cytotoxic effect leading to necrotic rather than apoptotic cell death, which could be the reason of rapid and irreversible antifungal activity of SK and can be correlated to the ROS accumulation-induced internal structural and functional damage of many organelles [13,21].
It is well known that apoptosis can be activated by two main signaling pathways: the death receptor-mediated extrinsic apoptosis pathway and the mitochondrial-mediated (cytochrome c, caspase) intrinsic apoptosis pathway [22]. Because SK is a lipophilic quinone natural compound [23]. it is very important to pay attention to the intrinsic pathway rather than extrinsic pathway, which is the classical pathway of apoptosis. Previously, our study found that SK could significantly reduce mitochondrial membrane potential and ATP level, as well as increase production of ROS in C. albicans [9]. In this study, mitochondrial COX and caspase activity were further measured to understand the effect of SK on the mitochondrial pathway. Mitochondria has been reported an important cell organelle and play a key role in the physiological of cell death [24]. When apoptotic signaling molecules act on mitochondria, mitochondrial permeability changes, membrane potential being decreased, resulting in a large amount of ROS, then cytochrome c being released into the cytoplasm [25,26]. The release of cytochrome c from mitochondria to cytoplasm is directly related to the decrease of cytochrome c oxidase activity [27]. After cytochrome c being released from the mitochondria to cytoplasm, yeast metacaspase Yca1p (it is the only caspase that plays an important role in yeast apoptosis, and it is also an ortholog of mammalian caspases.) is activated, thereby in turn resulting in the activation of caspase cascade and inducing apoptosis [28]. Our results demonstrated significant decrease in COX activity in C. albicans after treatment with different concentrations of SK. In addition, the caspase activity and the expression of related gene CaMCA1 increased. Previous studies have found that CaMCA1 is a homologous of metacaspase YCA1 in Saccharomyces cerevisiae. As the only gene responsible for caspase activity in C. albicans, CaMCA1 is involved in H 2 O 2 -induced apoptosis [29].
In addition to the mitochondrial pathway, the Ras-cAMP-PKA signaling pathway can also mediate the yeast apoptosis [30]. Our study found that SK could affect C. albicans biofilm formation by inhibiting the Ras-cAMP-PKA pathway (data not shown). Therefore, whether the apoptosis of C. albicans induced by SK is also related to the Ras-cAMP-PKA pathway remains to be further studied.
In summary, this study reported for the first time that SK could induce apoptosis of C. albicans, showing some typical apoptosis characteristics including of PS externalization and chromatin condensation. It is related to the accumulation of intracellular ROS, the decrease of mitochondrial membrane potential, the release of cytochrome c, and the increase of caspase activity caused by mitochondrial injury. This study provides an effective laboratory basis for SK in the clinical treatment of C. albicans infection and further enriches the theory of Chinese traditional medicine antifungals.