s2fieldsofstudy: Biology Medicine

text:
Effects of Magnolol and Honokiol on Adhesion, Yeast-Hyphal Transition, and Formation of Biofilm by Candida albicans

Background The first step in infection by Candida albicans is adhesion to host cells or implanted medical devices and this followed by hyphal growth and biofilm formation. Yeast-to-hyphal transition has long been identified as a key factor in fungal virulence. Following biofilm formation, C. albicans is usually less sensitive or insensitive to antifungals. Therefore, development of new antifungals with inhibitory action on adhesion, yeast-hyphal transition and biofilm formation by C. albicans is very necessary. Methods The effects of magnolol and honokiol on hypha growth were investigated using different induction media. Their inhibitory effects were determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide assay, and biofilm thickness and viability were observed by a confocal scanning laser microscope. Mammalian cells were used in adhesion assays. Genes related to hyphae development and cell adhesions were analyzed by real-time reverse transcription-polymerase chain reaction. The exogenous cyclic adenosine monophosphate was used to determine the mechanisms of action of magnolol and honokiol. Caenorhabditis elegans was used as an in vivo model to estimate the antifungal activities of magnolol and honokiol. Results and conclusions Magnolol and honokiol inhibited adhesion, the transition from yeast to hypha, and biofilm formation by C. albicans through the Ras1-cAMP-Efg1 pathway. Moreover, magnolol and honokiol prolonged the survival of nematodes infected by C. albicans. Magnolol and honokiol have potential inhibitory effects against biofilm formation by C. albicans. General Significance This study provides useful information towards the development of new strategies to reduce the incidence of C. albicans biofilm-associated infection.

Introduction
Candida albicans is one of the most common pathogens, and is associated with the occurrence of mortality rates as high as 35-50% [1,2]. The estimated annual cost of treating Candida infection exceeds $1 billion [1,2]. Infection by C. albicans involves adhesion to host cells or implanted medical devices as the first step, and this is followed by hyphal growth and biofilm formation [3][4][5][6][7]. The pathogenicity of C. albicans is closely related to its ability to change between two morphological forms, yeast and hyphae [7,8]. This transition is highly regulated and involves multiple interconnected signaling pathways, including the cyclic AMP-dependent Protein (cAMP) Kinase A and the Cph1-mediated Mitogen-Activated Protein Kinase (MAPK) [7]. C. albicans biofilm formation is very complex and the developmental processes involves the adhesion of yeast cells to the substrate, with the subsequent cell proliferation, elevation of hyphal growth, and production of extracellular matrix materials [8][9][10]. . The matured cells finally disperse from the biofilm into the surrounding environment [8]. Biofilm formation is an important step in pathogenesis because there after, C. albicans is usually less sensitive or insensitive to antifungals [11]. Therefore, development of new antifungals with inhibit action on adhesion, yeast-hyphal transition and biofilm formation of C. albicans would be a crucial strategy.
Natural products play an important role in drug discovery and development [12][13][14]. In the area of infectious disease, over 75% of drugs used in therapy are of natural origin [15]. Magnolia officinalis is a major component of herbal formulations such as Banxia-Houpu decoction and Huoxiang Zhengqi liquid which are used as remedies for illnesses like depression, coughing, asthma, liver disease, shoulder pain, urinary problems and diarrhea. Magnolol and honokiol, two neolignan compounds, are primarily isolated from the dried stem, root, or branch bark of M. officinalis. Previous studies have demonstrated that magnolol and honokiol have various pharmacological activities including antifungal [16][17][18]. However, the role of magnolol and honokiol in the inhibition of adhesion, transition from yeast to hypha, and biofilm formation by C. albicans as well the underlying mechanisms involved are still largely unclear.
In the present study, we first evaluated the inhibitory action of magnolol and honokiol against adhesion, yeast-hyphal transition and biofilm formation by C. albicans in vitro. The mechanisms of inhibition of biofilm formation by magnolol and honokiol were associated with the Ras1-cAMP-Efg1 pathway. Finally, the activity of magnolol and honokiol were investigated in vivo, using a nematode infection model. We believe our findings will be helpful in the future development of magnolol and honokiol as drugs against the formation of biofilm by C. albicans.

Antifungal activities of compounds
The antifungal activities of all tested compounds were tested by the broth microdilution method according to CLSI standard M27-A3 [23] with a final inoculum of 0.5 × 10 3 to 2.5 × 10 3 cells/mL. The test was carried out in RPMI 1640 medium adjusted to pH 7.0 with 0.165 M morpholinepropanesulfonic acid [MOPS] buffer in 96-well flat-bottomed microtitration plates. After incubation at 37°C for 48 h, minimum inhibitory concentrations (MICs) were determined by measuring the optical density at 600 nm with the BioTek Synergy 4 microplate reader (BioTek Instruments Inc., USA). The background optical densities were subtracted from that of each well. The MICs were defined as the concentrations of drug that reduced growth by 80% compared to that of organisms grown in the absence of drug. The experiments were performed in triplicate.

Measurement of minimum fungicidal concentrations (MFCs)
MFCs were determined as described previously [24]. After the MIC was determined, a 5-μL aliquot was taken from each well and added to 200μL of compound-free fresh YPD medium. After 48 h of incubation, the MFC was determined as the lowest concentration of the test compound in which no recovery of microorganisms was observed.
Effect of magnolol and honokiol on C. albicans growth C. albicans SC5314 with different starting inoculum of 10 6 , 10 7 , 10 8 cells/mL in YPD medium were exposed at different concentrations of magnolol and honokiol. Samples were taken at 4, 8, 12, and 24 h and yeast counts were determined using 10-fold dilutions plated on YPD plates which were incubated at 37°C for 48 h. Using a 20-μL sampling volume, the lower limit of accurate and reproducible quantitation was 50 CFU/mL for each of the isolates. All experiments were performed in triplicate and repeated on two different days. A fungicidal effect was defined as a reduction in !3 log 10 CFU/mL compared to initial inoculum [25].
Filamentation assay-solid spider media for colony morphology assay Spider media agar plates with or without various concentrations of compounds were used in this study. SC5314 cells pregrown overnight in YPD media were diluted into spider liquid media to a final concentration of 1000 cells/mL. And then 50 μL of the solution were spread onto the plates. Plates were incubated at 37°C for 3 days. Images of colony edges were obtained using a microscope (Olympus IX71, Olympus Co., Tokyo, Japan). The experiments were performed in triplicate.
Filamentation assay-RPMI 1640, GlcNAc, or 10% FBS YPD liquid medium C. albicans SC5314 were grown overnight in YPD medium. And then, 1 × 10 6 cells/mL with or without compounds were incubated in RPMI 1640 medium, GlcNAc (0.5% GlcNAc, 0.5% peptone, 0.3% KH 2 PO 4 ) or 10% FBS YPD medium at 37°C for 4 h. Inhibition quantification of the yeast-to-hyphal-form transition was accomplished by counting the number of individual budded cells versus the number of hyphae in the population as previously described [26]. More than 100 cells were counted for each well in duplicate, and all assays were repeated for five times. Images of cells were obtained using a microscope. The experiments were performed in triplicate.

Biofilm formation-before the biofilm adhesion phase
Various concentrations of compounds ranging from 8 to 64 μg/mL were prepared in RPMI 1640 medium in 96 well plates (Costar, Corning Inc., USA). Wells without test compounds served as controls. Amphotericin B (8 to 64 μg/mL) was used as a standard antifungal agent. Cell suspensions of 1 × 10 6 cells/mL were prepared in RPMI 1640 medium. And then 100 μL of solution was inoculated into 96-well polystyrene plates. After incubation at 37°C for 48 h, nonadhered cells were removed by sterile PBS, and biofilm growth was analyzed with XTT assay, as described [20]. The absorbance of wells was measured with the BioTek Synergy 4 microplate reader at 490 nm. The experiments were performed in triplicate.
to allow attachment of cells to the solid surface. Nonadhered cells were removed by sterile PBS. And then 100 μL of various concentrations of compounds in RPMI 1640 medium were added to each well. The plates were incubated at 37°C for 48 h to allow for biofilm formation. Biofilm growth was analyzed with XTT assay as described above. The experiments were performed in triplicate.
Analysis of C. albicans biofilm formation using confocal scanning laser microscope (CLSM) The C. albicans strain SC5314 (1 × 10 5 cells/mL) was cultured in RPMI 1640 medium at 37°C for 48 h with or without tested compounds. After incubation, the supernatant was aspirated, and nonadherent cells were removed by washing with sterile PBS. The biofilm were co-stained with 10 μg/mL of fluorescein diacetate (FDA) and 5 μg/mL of propidium iodide (PI) for 30 min. The images were taken by confocal laser-scanning microscope (Olympus Fluoview FV1000). Laser beams with 488 and 555-nm excitation wavelengths were used for FDA and PI imaging, respectively. A detailed three-dimensional image of biofilm was performed using Z-stacks (depending on the height of the biofilm). Cell viability was also able to be assessed since the healthy cells would hydrolyze FDA so as to accumulate the green fluorescence, whereas the dead cells were stained as red due to the PI labeling. Red or green fluorescence intensity was calculated using histogram in ImageJ.

In vitro biofilm growth on titanium sheet
Titanium is widely employed for implant manufacturing due to its good biocompatibility and mechanical properties, but infection remains a cause of failure leading to removal. The titanium surface is not antimicrobial by itself, so it could be used as support for a Candida biofilm in this investigation [27]. The effect of honokiol or magnolol on biofilm growth on titanium sheet was examined as described previous [28]. After autoclaved, the squares (1.0 × 1.0 cm) were transferred to 35 mm glass bottom dishes (NEST Biotech, China) and incubated with 1 × 10 5 cells/mL SC5314 in RPMI 1640 medium at 37°C for 2 h. The squares were washes with PBS and transferred to new dishes with or without tested compounds. After incubation at 37°C for 48 h, the dishes were photographed, and images of titanium sheet edges were visualized under a microscope. The bound biofilm was measured after drying of titanium sheet in a chemical hood. The experiments were performed in triplicate.

Cell adhesion assay
An immortalized rat hepatic stellate cell line HSC-T6 was used in this study [29]. HSC-T6 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere of 5% CO 2 . HSC-T6 cells were grown to confluence on 48 well plates. Media were decanted, and the plates were carefully washed three times with PBS to remove unbound cells. And then 500 μL of 1 × 10 7 cells/mL C. albicans CaSA1 cells mixed with various concentrations of compounds were plated into each well. After incubation at 37°C for 2 h, the media were decanted, and the monolayers were washed carefully three times with PBS. The number of adherent C. albicans cells was determined by scraping the bottom of the 48well plate with a sterile scraper. Five-fold serial dilutions were plated on YPD agar to determine viability. Also, both DIC and fluorescence images were acquired using a fluorescence microscope (Olympus IX71, Olympus Co., Tokyo, Japan). The experiments were performed in triplicate.

Quantification analysis by real-time reverse transcription-polymerase chain reaction (RT-PCR)
The total RNAs were isolated using the hot phenol method as previously described [30]. C. albicnas was grown overnight in YPD medium and diluted to a cell density of 1.0 × 10 6 . Yeasts were incubated at 37°C for 12 h followed by centrifugation at 4°C. Approximately 1 μg of total RNA was used to synthesize cDNA using random primers and AMV reverse transcriptase (Promega, WI, USA). Gene expression levels of GSP1, RAS1, EFG1, TEC1, CDC35, ALS3, HWP1 and ECE1 were analyzed by the real-time RT-PCR. Primer sequences used for amplification of specific genes are shown in S1 Table. Real-time RT-PCR was performed in 8-tube strips in triplicate. Each reaction containing 1 × GoTaq qPCR Master mix (Promega, Madison, WI), 0.2 μM forward primer and reverse primer, and 1 μL template cDNA in a final volume of 20 μL. GSP1, which was not transcriptionally regulated in the morphogenesis switch, served as the internal control. Cycling profile included 35 cycles of 95°C for 60 sec, 60°C for 60 sec and 72°C for 45 sec. Data acquisition and the analysis of the RT-PCR assay were performed using an ABI 7500 Real-Time PCR System (Applied Biosystems). The transcript level of detected genes was calculated using the formula 2 -ΔΔCT .

cAMP Rescue Experiments
Grown overnight C. albicans SC5314 cells were diluted to 2×10 5 cells/mL with RPMI1640 medium. Dibutyryl-cAMP (Sigma) was added to the cultures with a final concentration of 5 mM immediately following the addition of 8 μg/mL magnolol or honokiol. The free-drug treatment cells with or without dibutyryl-cAMP (dbc-AMP) served as control. After 4 h incubation, cells were visualized by an Olympus microscope.

Toxicity assessment of compounds in HSC-T6 cells
The HSC-T6 cells were seeds into 96-well plates at 1 × 10 4 cells/mL and grown at 37°C overnight. The media was removed and replaced with fresh media containing compounds followed by incubation for further 24 h. The CCK-8 assay was used to evaluate cell viability. The cells were washed with D-Hank's buffer, and 200 μl of WST-8 solution (Dojindo Molecular Technologies, Inc., USA) was introduced to each well to incubate for an additional 3 h at 37°C. The optical density (OD) of each well at 450 nm was examined with the BioTek Synergy 4 microplate reader (BioTek Instruments Inc., USA). The experiments were performed in triplicate.
Antifungal activity and evaluation of fugal burden in a nematode model of infection C. elegans survival analysis was performed as previously described [31]. Age synchronous populations of L4-larvae were prepared, and exposed to C. albicans SC5314. For the pathogen assay, 75 mg/mL of fluoro-29-deoxyuridine (FUdR) was added to the assay plates to prevent the growth of progeny. C. albicans was seeded on killing plates containing modified NGM (0.35% instead of 0.25% peptone). C. albicans was incubated first for 24 h at 37°C and then for 24 h at 25°C. Young adult animals were exposed to the C. albicans lawns for 2 h and washed three times with sterile M9 buffer, then moved to NGM in the presence or absence of antifungal agents. The infection was started by adding 60 animals to each plate at 25°C, and scored for dead or live every 12 h. Animals were scored as dead if no response was detected after prodding with a platinum wire. Three replicates were analyzed for each experiment.
The number of C. albicans colony-forming Units (CFU) in C. elegans was quantified based on the protocol described previously [32]. C. albicans lawns were prepared on BHI as described above [32]. Young adult animals were exposed to the lawns for 2 h and then moved to liquid media (80% M9 buffer, 20% BHI, and 90 μg/mL kanamycin) and incubated at 20°C in the presence or absence of antifungal agents. After 24 h incubation, three groups of twenty worms each were washed three times with 1 mL of M9 medium to remove surface Candida. Each group of twenty worms was then disrupted using a homogenizer and plated on YPD agar containing kanamycin (45 μg/mL), ampicillin (100 μg/mL), and streptomycin (100 μg/mL). The plates were incubated for 48 h at 37°C and colonies were counted to determine CFU per nematode. Three replicates were analyzed for each experiment.

Statistical analysis
All data were presented as means ± standard error of the mean (S.E.M.). Graphs were generated using Microsoft Excel (Microsoft Corp., Redmond, WA). Statistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicage, USA). Differences between groups were determined using analysis of variance (ANOVA). A P-value<0.05 was defined as statistically significant. The lifespan data were statistically analyzed using a 2-tailed 2 sample t-test (Minitab Ltd., Coventry, UK).

Results
Inhibitory activity of magnolol and honokiol against planktonic growth of C. albicans Magnolol and honokiol exhibited similar and potent inhibitory activitiy against clinically isolated C. albicans strains (SC5314, CA1, CA2, CA3, CA4, CA10, CA127, CA129, CA132, CA135, and CA137), with MICs in the range of 16-32 μg/mL (S2 Table). In all cases the MIC was equivalent to the MFC. After 48 h of exposure to treatments, samples were re-inoculated in YPD medium. After an additional 48 h incubation, no growth was detected in cultures exposed to the MIC values of magnolol and honokiol with the MIC values.
The effect of magnolol and honokiol on growth of C. albicans was further investigated. It was observed that the fungicidal activities of these compounds depended greatly on the initial inoculum (Fig. 2). Time-growth curves indicated that, compared with the control group, 4, 8, or 16 μg/mL of compounds could not affect the growth of C. albicans after 24 h of incubation. But 32 μg/mL of magnolol and honokiol slightly slowed down the growth of C. albicans when incubated with initial inoculum of 1.0×10 6 or 1.0×10 7 cells/mL ( Fig. 2A and 2B). A fungicidal effect was observed when 64 μg/mL of magnolol or honokiol was used with initial inoculum of 1.0×10 6 or 1.0×10 7 cells/mL. When the initial inoculum of 1.0×10 8 cells /mL was used, only 64μg/mL of magnolol or honokiol had a reduction in the growth of C. albicans, and no fungicidal activity was observed (Fig. 2C). Although the chemical structures of magnolol and honokiol are different, particularly in the position of the hydroxyl group on the aromatic ring, this difference appears not to influence their biological activity on C. albicans.
Inhibitory activity of magnolol and honokiol against the transition of C. albicans from yeast to hyphae The transition from yeast to hyphae is an important virulence-mediating attribute of C. albicans. Formation of hyphae helps C. albicans in penetrating the host tissues with subsequent invasive growth that leads to the establishment of systemic infection. Conversion of yeast cells to filamentous forms is critical for biofilm biogenesis, which provides strength and support to the developing heterogeneous biofilm structure. In the present study, we observed that magnolol and honokiol at a low concentration of 4 μg/mL effectively inhibit transition from yeast to hyphae on the solid spider agar plates (Fig. 3A-3G) and at 16 μg/mL both compounds completely blocked this transition ( Fig. 3D and 3G).
Hyphal morphogenesis can be induced by multiple stimuli [7]. In addition to the nutrientpoor spider medium, liquid RPMI 1640, GlcNAc and mammalian serum can also induce the formation of hyphae [7]. We further discovered that both magnolol and honokiol at a low concentration of 4μg/mL significantly inhibited the hyphae formation in RPMI 1640 or GlcNAc media (Fig. 3H-3I, and 3K). In contrast, 4 μg/mL of magnolol or honokiol did not obviously influence the hyphae formation in 10% FBS YPD medium (Fig. 3J-3K). Magnolol and honokiol at different concentrations had similar effects on hyphae growth in different hyphae-inducing media (Fig. 3H-3K). Therefore, our data suggest that both magnolol and honokiol have the potential to inhibit the transition of C. albicans from yeast to hyphae. Effects of Magnolol and Honokiol on Candida albicans Biofilm Inhibitory effects of magnolol and honokiol on biofilm growth at different stages in C. albicans The development of C. albicans biofilm involves several specific stages including the early phase, the developmental phase, and the biofilm maturation stage. It was found that magnolol and honokiol inhibited biofilm formation in a dose-dependent manner (Fig. 4). During the early phase (that is immediately after adhesion), biofilm formation in the 16 μg/mL magnolol or honokiol group was less than 50% as compared with the control group (Fig. 4A). At the developmental phase, treatment with 16 μg/mL of magnolol and honokiol inhibited more than 80% biofilm formation by C. albicans (Fig. 4B). Furthermore, the inhibitory effects of 16-64 μg/mL magnolol or honokiol on biofilm formation were comparable to those of amphotericin B, a "gold standard" drug used in the treatment of Candida infections (Fig. 4B). At the biofilm maturation stage, treatment with 64 μg/mL of magnolol and honokiol decreased more than 50% biofilm formation, which was similar to the function of amphotericin B (Fig. 4C).

The effects of magnolol and honokiol on biofilm thickness and viability
The anti-biofilm effects of magnolol and honokiol were further confirmed by CLSM analysis. To analyze the effects of magnolol and honokiol on biofilm thickness, three dimensions (3D) views were taken for each sample and Z-stacks were prepared to compare the thickness. In the Effects of Magnolol and Honokiol on Candida albicans Biofilm control experiment, C. albicans cells formed a thick biofilm with an average Z-axis of 280 μm (Fig. 5A). In contrast, treatment with magnolol at concentrations of 16 and 32 μg/mL reduced the thickness of the biofilm to 58.6 μm and 49.6 μm, respectively (Fig. 5A). Similarly, treatment with honokiol at concentrations of 16 and 32 μg/mL decreased the thickness of biofilm to 67.6 μm and 45.1 μm, respectively (Fig. 5A).
Furthermore, both magnolol and honokiol at 16 μg/mL significantly decreased the viability of the biofilm as indicated by FDA labeling and increased the dead C. albicans cells as indicated by PI labeling (Fig. 5B-5C).

Inhibition of biofilm formation on titanium sheet by magnolol and honokiol
Fungal biofilm formation on implanted medical devices is a serious medical problem, which can lead to life-threatening systemic infections [6,28]. We used the titanium sheet assay [27] to simulate the formation of biofilm by C. albicans on medical devices. We confirmed that C. albicans SC5314 cells efficiently formed biofilms on titanium sheet (Fig. 6). In contrast, magnolol and honokiol at concentrations of 8-32 μg/mL significantly suppressed biofilm formation ( Fig. 6A and 6B). The edges of the titanium sheets treated with 16 μg/mL of magnolol or honokiol were clearer with less C. albicans growth than that of the vehicle control (Fig. 6B).

Inhibition of C. albicans adhesion to the surface of HSC-T6 cells by magnolol and honokiol
In addition to the experiments on inert surfaces, we further investigated the effects of magnolol and honokiol treatment on C. albicans adherence to the surface of mammalian cells. Magnolol and honokiol at concentrations of 4-32 μg/mL significantly inhibited the adhesion of C. albicans cells to the surface of HSC-T6 cells (Fig. 7). Moreover, the fluorescent images also confirmed that treatment with 16 μg/mL of magnolol or honokiol significantly inhibited adhesion to HSC-T6 cells (S1 Fig.).

Ras1-cAMP-Efg1 pathway is involved in the mechanisms of action of magnolol and honokiol
To understand the molecular basis for magnolol and honokiol inhibiting hyphae growth and cell adhesion, we investigated the expression of genes related to hyphae growth and cell adhesion in C. albicans treated with both compounds. Four genes (RAS1, EFG1, TEC1, and CDC35) associated with the Ras1-cAMP-Efg1 pathway were examined. After treatment with 16 μg/mL of magnolol or honokiol, the expression levels of RAS1, EFG1, TEC1, and CDC35 genes were significantly decreased (Fig. 8A). Ras1p is a GTPase that plays a role in inducing hyphal formation by activating both the Ras1-cAMP-Efg1 pathway and MAPK cascade pathway. EFG1 is a transcription factor in the Ras1-cAMP-Efg1 pathway which plays an important role in regulating the expression of some hypha-specific genes, including ECE1, HWP1 and ALS3. In our study, following treatment with 16 μg/mL of magnolol or honokiol, the expression levels of all three genes examined including ECE1, HWP1, and ALS3 were significantly decreased (Fig. 8A). Next, we investigated whether magnolol and honokiol can also down-regulate the expression levels of these genes in other C. albians strains. For this purpose, we used the strain YEM30, and the results in YEM30 were similar to those in SC5314 (S2 Fig.). Based on these results, we therefore speculated that the inhibition of the transition from yeast to hyphae by magnolol and honokiol may be related to down-regulation of the Ras1-cAMP-Efg1 pathway. To verify this hypothesis, we conducted the exogenous cAMP rescue experiments. The results revealed that exogenous cAMP restored the hyphal formation in the magnolol and honokiol treatment groups (Fig. 8B). Together, these data suggest that magnolol and honokiol may inhibit the filamentous growth through the Ras1-cAMP-Efg1 pathway.
Magnolol and honokiol exhibited antifungal activity in the in vivo C. elegans assay system In C. elegans, we found that infection with C. albicans SC5314 induced a significant (P < 0.0001) decrease in lifespan of the nematodes compared with control (Fig. 9A). Treatment with 16 μg/mL of magnolol or honokiol significantly (P < 0.0001) increased the lifespan of nematodes infected by C. albicans SC5314 (Fig. 9A). Moreover, we found that treatment with 16 μg/mL of magnolol or honokiol significantly reduced the CFU in the body of nematodes (Fig. 9B). Thus, magnolol and honokiol reduced the colonization of C. albicans in the body of nematodes and thereby increase the lifespan of infected nematodes.  Toxicity assessment of magnolol and honokiol in HSC-T6 cells Finally, we investigated the safety profile of magnolol and honokiol using mammalian HSC-T6 cells. We found that both magnolol and honokiol at concentrations of 4-32 μg/mL did not influence the viability of mammalian HSC-T6 cells (S3 Fig.) or affect the morphology, longevity, development, and reproduction of nematodes (data not shown). These data suggest that magnolol and honokiol at the concentrations tested may not have adverse effects on these organisms.

Discussion
In the present study we investigated the effects of magnolol and honokiol, two neolignan compounds isolated from M. officinalis, on adhesion, yeast-hyphal transition, and biofilm formation by C. albicans. It has been previously reported that the MICs of both magnolol and honokiol were 25 μg/mL against C. albicans KCTC 1940 and Epidermophyton floccosum KCTC 1246, while for other strains such as Trichophyton mentagrophytes KCTC 6077, Microsporium gypseum KCTC 1252, and Cryptococcus neoformans KCTC 7224, the antifungal effect of honokiol was evaluated to be higher than that of magnolol [16]. Although the chemical structures between magnolol and honokiol differ in the position of the hydroxyl group on aromatic ring, this may not influence their biological activity on C. albicans (Fig. 1). Our data demonstrate that both magnolol and honokiol have activity against the clinically isolated C. albicans strains in the concentration range of 16-32 μg/mL. According to the definition of fungistatic versus fungicidal effect [24,25], in our study magnolol and honokiol were considered to have a fungicidal effect, because cultures treated with magnolol or honokiol at MICs were unable to be recovered. It is reported that C. albicans at high cell density is tolerant to various antifungal drugs at high concentrations [33]. The results presented in this paper suggest the same behavior in the effect of magnolol and honokiol on C. albicans. We discovered that both magnolol and honokiol at a low concentration of 4μg/mL had the potential to inhibit the transition of C. albicans from yeast to hyphae on solid spider agar plates (Fig. 3). Then, we found that the inhibitory effects of magnolol and honokiol on transition of C. albicans from yeast to hyphae were dependent on different environmental stimuli. The nutrient-poor spider medium, liquid RPMI 1640, GlcNAc, and mammalian serum are all able to induce hyphae growth [7]. Our data demonstrated that 4 μg/mL of magnolol and honokiol inhibited the hyphae growth in nutrient-poor spider medium, liquid RPMI 1640, and GlcNAc medium (Fig. 3). In contrast, 4 μg/mL of magnolol or honokiol did not suppress the hyphae growth in 10% FBS YPD medium (Fig. 3). These results suggest that environmental stimuli may need to be considered in the potential use of magnolol or honokiol as inhibitors of hyphae growth of C. albicans.
Our results further suggest that magnolol and honokiol inhibit both the formation and development of biofilm by C. albicans. Notably, magnolol and honokiol could not only inhibit the formation of biofilm but also destroy the maintenance of mature biofilm. Magnolol and honokiol at the concentration of 32 μg/mL (1×MIC) inhibited more than 90% biofilm formation in the early phase and the developmental phase. In addition, 64 μg/mL (2×MIC) of magnolol and honokiol destroyed the maintenance of about 50% mature biofilm (Fig. 4). The effects of these two compounds were similar with amphotericin B. Moreover, both compounds at a concentration of 16 μg/mL effectively reduced the thickness and viability of biofilm (Fig. 5). Furthermore, our results demonstrated that both compounds at concentrations of 8-32 μg/mL inhibited C. albicans cells adhesion to HSC-T6 cells and biofilm formation on titanium sheets ( Fig. 6 and Fig. 7). In summary, our data indicate that magnolol and honokiol could inhibit biofilm formation through decreasing adhesion, morphological transition, and their fungicidal effect.
Treatment with magnolol and honokiol significantly decreased expression levels of RAS1, EFG1, TEC1, and CDC35, which are members of the Ras1-cAMP-Efg1 pathway (Fig. 8A). RAS1 encoding the Ras-family GTPase is involved in the induction of hyphal formation by activation of Ras1-cAMP-Efg1 and MAPK pathways [34]. EFG1 and TEC1 genes encoding transcription factors positively regulate the expression of hypha-specific genes such as ECE1 [6], [34]. In addition, our results indicate that 3 adhesion-related genes HWP1, ALS3, and ECE1 were significantly down-regulated following treatment with magnolol and honokiol (Fig. 8A). In C. albicans, HWP1 which encodes a cell-wall protein is a unique adhesion gene expressed on hyphal surface. The biofilm lacking HWP1 gene was prone to detachment from the abiotic substrate [35]. The ALS3 gene encoding a protein similar to alpha-agglutinin plays an essential role in the adhesion of C. albicans, and serves as an upstream regulator of both the Ras1-cAMP-Efg1 and MAPK pathways [36]. ECE1 is associated with both cell adhesion and hyphae formation by its regulation of the extent of cell elongation [37]. Interestingly, these genes: HWP1, ALS3, and ECE1 which were down regulated after magnolol or honokiol treatment, are regulated by Ras1-cAMP-Efg1 pathway [38]. Exogenous cAMP restored hyphal formation in the magnolol and honokiol treatment groups (Fig. 8B). These results indicate that magnolol and honokiol may inhibit adhesion, transition from yeast to hyphae, and biofilm formation by C. albicans by down-regulating the Ras1-cAMP-Efg1 pathway (Fig. 10).
Using the C. elegans infection model, we found that treatment with 16 μg/mL of magnolol or honokiol increased the lifespan and reduced the CFU content of nematodes infected by C. albicans SC5314 (Fig. 9). These data further demonstrate the potential of magnolol and honokiol to be used against C. albicans infection in vivo. Proposed model of mechanism underlying the magnolol and honokiol-induced inhibition of biofilm formation by C. albicans. Magnolol or honokiol significantly decreased the expression levels of RAS1, EFG1, TEC1, and CDC35 genes encoding the Ras1-cAMP-Efg1 pathway. In addition, exogenous cAMP restored hyphal formation in the magnolol and honokiol treatment groups. Collectively, these magnolol or honokiol-mediated effects


extfieldsofstudy: Medicine Biology

text:
Sexual selection and cancer biology

Darwin suggested that when males and females “differ in structure, colour or ornament” it is the result of sexual selection (“On the origin of species” by Charles Darwin). It has become increasingly apparent that in addition to overt sexual dimorphisms, there are substantial differences in underlying male and female biology that have relevance to human health and disease. It was recently reported that male astrocytes exhibit markedly greater tumorigenic responses to combined loss of neurofibromin and p53 function and activation of the EGF receptor pathway than do female astrocytes [1]. Moreover, differential RB regulation was shown to be essential for the manifestation of sex differences in tumorigenicity. It was suggested that cell intrinsic dissimilarities in tumor suppressor and oncogene effects provided novel insight into the basis for disparities that exist in glioblastoma rates and outcomes. It is likely however, that these findings are more broadly relevant to the overall greater rate of cancer diagnoses and cancer deaths in males. The experimental paradigm that was used for measuring sex-specific effects provides new opportunities and possibly, new requirements for basic and translational research. What makes this position so compelling is the ways in which sex differences in tumorigenicity can be related to fundamental sexual dimorphisms in growth, the vigorous evolutionary forces that created them, and the multiple developmental mechanisms that execute sex-specific growth strategies [2].

Sexual selection and cancer biology Joshua B. Rubin
Darwin suggested that when males and females "differ in structure, colour or ornament" it is the result of sexual selection ("On the origin of species" by Charles Darwin). It has become increasingly apparent that in addition to overt sexual dimorphisms, there are substantial differences in underlying male and female biology that have relevance to human health and disease. It was recently reported that male astrocytes exhibit markedly greater tumorigenic responses to combined loss of neurofibromin and p53 function and activation of the EGF receptor pathway than do female astrocytes [1]. Moreover, differential RB regulation was shown to be essential for the manifestation of sex differences in tumorigenicity. It was suggested that cell intrinsic dissimilarities in tumor suppressor and oncogene effects provided novel insight into the basis for disparities that exist in glioblastoma rates and outcomes. It is likely however, that these findings are more broadly relevant to the overall greater rate of cancer diagnoses and cancer deaths in males. The experimental paradigm that was used for measuring sexspecific effects provides new opportunities and possibly, new requirements for basic and translational research. What makes this position so compelling is the ways in which sex differences in tumorigenicity can be related to fundamental sexual dimorphisms in growth, the vigorous evolutionary forces that created them, and the multiple developmental mechanisms that execute sex-specific growth strategies [2].
As any parent knows normative growth for boys and girls is substantially different. Not only do males typically grow to a greater size than females, but they also grow for a longer period of time. Sex related size differences that result in larger males are pervasive throughout the animal kingdom with few exceptions. Greater male size is a manifestation of greater male growth rates from the pre-implantation stage of embryogenesis to adulthood, with the brief period of exception during early puberty. The important questions are: Why are there differences in growth between males and females? What are the mechanistic foundations of sexual dimorphism? What are the implications of sexual dimorphism for the risks and manifestations of disorders in growth regulation such as cancer? What are the opportunities and requirements for studying sex differences in lab-based and clinical investigations in oncology?
Darwin recognized that sex differences were evolutionarily determined and he promulgated a theory of sexual selection, a distinct evolutionary process for shaping phenotypic differences within a species through competition between individuals of the same sex for reproductive success. Markedly different reproductive pressures on males and females have produced colorfully distinct strategies for success. Male reproductive success is limited by access to mates and consequently their reproductive strategy involves body size and displays that enhance their attractiveness to females and their ability to vanquish other males. This has been referred to as the "live fast, die young" blueprint for success [3]. Resources, not mates, limit female reproductive success. Consequently large body size and ostentatious displays are counterproductive. Instead, balancing growth with viability, particularly from gestation through weaning stages of offspring development is paramount for female reproductive success. Thus, there are differing growth optima for males and females and genetic conflict in growth regulatory pathways [4].
It is appealing to think about the relationship between differences in body size, the numbers of cells required and cancer risk as a simple equation in which bigger size and more cells results in greater cancer risk. However, the normal distribution of size between males and females overlaps significantly and is not likely to be the primary determinant of differences in cancer risk. Instead, the importance of sex-specific growth to cancer biology is more likely to lie in the homeostatic mechanisms that are in place to defend and preserve the differing optima. Normal distribution in size is a balance between male-derived influences that favor being larger (e.g. paternal imprinting of the H19 locus) and femalederived influences that favor being smaller (e.g. maternal imprinting of the IGF2 locus). The critical question may be, what happens when perturbations in growth regulation occur that shift normal growth towards greater or smaller growth rates and size. If loss of growth control were to move growth closer to the male optima but further from the female optima would the counter-regulatory response be the same, or would this be "allowed" in the male cell but "disallowed" in the female cell? This may be the key to the relationship between differences in growth and cancer at the earliest stages of cancer initiation when loss of tumor suppressor function or gain of oncogene function first alters growth. This could be the basis for the differential effects of combined loss of neurofibromin and p53 function.
Developmental consequences of in utero stressors provide important insight into these issues. Multiple Editorial www.impactjournals.com/oncotarget studies of the impact that maternal nutrient deprivation and other in utero stressors have on fetal development reveal sex differences in viability and body size at birth [5]. Normal growth rates but increased fetal demise is more commonly observed for males while greater viability but decreased body size is more commonly observed for females. Underlying these differences are epigenetic mechanisms that preserve metabolism and growth stimulatory pathways in males and differing epigenetic mechanisms that slow growth, reduce nutrient requirements and increase immune activity in females [6]. A related set of observations suggests that epigenetic sex differences like these may be prevalent. The bed nucleus of the stria terminalis (BNST) is a highly sexually dimorphic region of the brain. An analysis of histone bivalency in male and female BNST revealed H3K4/K27 trimethylation at developmental and growth regulatory genes in males but in synaptic function genes in females [7]. These data may indicate that in response to perturbations in growth, males are poised to maintain their size, while females are poised to preserve their function.
With such strong evolutionarily designed and developmentally ensconced mechanisms, sexual dimorphism in growth, metabolism and immune function are bound to determine cancer risk and therapeutic responses. The importance of sex differences as a variable in research design should not be ignored. These findings should prompt investigators to examine oncogenic mechanisms separately in male and female experimental systems and to anticipate that therapeutics targeting growth regulatory pathways may have differing effects in male and female cancer patients.


extfieldsofstudy: Biology Medicine

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Co-regulation of mitochondrial respiration by proline dehydrogenase/oxidase and succinate

Proline dehydrogenase/oxidase (PRODH/POX) is a mitochondrial protein critical to multiple stress pathways. Because of the roles of PRODH/POX in signaling, and its shared localization to the mitochondrial inner membrane with the electron transport chain (ETC), we investigated whether there was a direct relationship between PRODH/POX and regulation of the ETC. We found that PRODH/POX binds directly to CoQ1 and that CoQ1-dependent PRODH/POX activity required functional Complex III and Complex IV. PRODH/POX supported respiration in living cells during nutrient stress; however, expression of PRODH/POX resulted in an overall decrease in respiratory fitness. Effects on respiratory fitness were inhibited by DHP and NAC, indicating that these effects were mediated by PRODH/POX-dependent reactive oxygen species (ROS) generation. PRODH/POX expression resulted in a dose-dependent down-regulation of Complexes I–IV of the ETC, and this effect was also mitigated by the addition of DHP and NAC. We found that succinate was an uncompetitive inhibitor of PRODH/POX activity, inhibited ROS generation by PRODH/POX, and alleviated PRODH/POX effects on respiratory fitness. The findings demonstrate novel cross-talk between proline and succinate respiration in vivo and provide mechanistic insights into observations from previous animal studies. Our results suggest a potential regulatory loop between PRODH/POX and succinate in regulation of mitochondrial respiration.

Introduction
Proline dehydrogenase (PRODH), a.k.a. proline oxidase (POX), is a mitochondrial inner membrane protein. Oxidation of proline to pyrroline-5-carboxylate (P5C) mediates the proline cycle that shuttles NADP(H)/NADP(+) redox equivalents between mitochondria and cytosol and forms a metabolic interlock with the pentose phosphate pathway (Hagedorn and Phang 1983;Phang 1985). Conversion of P5C to glutamine or ornithine also connects the proline cycle to the TCA and urea cycles, respectively.
PRODH/POX is a mediator of genotoxic, inflammatory, and metabolic stress signaling. Originally identified in a screen of p53-induced genes (Polyak et al. 1997;Campbell et al. 1997), induction of PRODH/POX by p53 was subsequently shown to result in mitochondrial-mediated apoptosis through generation of reactive oxygen species (ROS) in the form of mitochondrial superoxide (Donald et al. 2001;Hu et al. 2007;Liu et al. 2005). During hypoxia, tumor cells respond with either PRODH/POX-mediated ATP generation under conditions of low glucose, or pro-survival, ROS-mediated autophagy induction when glucose levels were normal (Liu et al. 2012). Activation of PRODH/POX by PPAR-γ results in PRODH/POX-dependent superoxide production that induces beclin-1 gene expression and activates protective autophagy versus the toxic effects of oxidized low-density lipoproteins (Zabirnyk et al. 2010). In adipocytes, PRODH/POX activation of PPAR-α through ROS-dependent and FOXO1-mediated induction prevents cell death and inflammation (Lettieri Barbato et al. 2014). In RKO colorectal cancer cells, induction of nutrient stress by either glucose withdrawal or treatment of cells with rapamycin results in increased AMP-activated protein kinase (AMPK)-dependent PRODH/POX catalytic activity, PRODH/POX-dependent cellular ATP generation, and activation of the pentose phosphate pathway (Pandhare et al. 2009). In C. elegans, AMPK-mediated upregulation of proline catabolism results in generation of ROS that promote an adaptive endogenous stress defense and increased lifespan (Zarse et al. 2012). Treatment of embryonic stem cells with proline induces their differentiation into either a primitive, ectoderm-type cell (Washington et al. 2010) or a novel, reversible epiblast stem cell-like state (Casalino et al. 2011). Thus, depending on cellular and environmental context, PRODH/POX can mediate programmed cell death, promote cell survival, or induce differentiation.
The mitochondrial respiratory electron transport chain (ETC) consists of a series of four protein complexes, known as Complexes I-IV, that pass electrons from the electron donors NADH and FADH through a series of redox reactions, creating an electrochemical proton gradient that is coupled with oxidative phosphorylation and the synthesis of ATP by ATP synthase (Chaban et al. 2014). Complex I, aka NADH dehydrogenase, passes electrons from NADH to reduce ubiquinone to ubiquinol. Complex II, aka succinate dehydrogenase (SDH), couples the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle with the reduction of ubiquinone to ubiquinol in the ETC (Hagerhall 1997). Electrons are transferred from this ubiquinol pool to Complex III, coenzyme Q-cytochrome C reductase, which passes the electrons to the hemeprotein cytochrome C. The final step is the transfer of electrons to Complex IV, cytochrome C oxidase, which passes the electrons to molecular oxygen to form water and drive the proton gradient that powers ATP synthase.
The SDH holoenzyme consists of four subunits, known as SDHA-D. SDHA contains the succinate binding site and flavin-adenine dinucleotide (FAD) redox center; SDHB contains three iron-sulfur centers required for electron transfer to ubiquinone, and SDHC and SDHD are two hydrophilic subunits responsible for anchoring the enzyme in the mitochondrial inner membrane. The ubiquinonebinding site of the holoenzyme is in a pocket formed by SDHB, SDHC, and SDHD at the surface of the matrix face of the mitochondrial inner membrane (Lancaster 2002;Horsefield et al. 2004;Sun et al. 2005). Mutations in SDHB, SDHC, and SDHD have been found in hereditary paraganglioma (Baysal et al. 2000;Niemann and Muller 2000;Astuti et al. 2001), gastrointestinal stromal tumors (GISTs) (Pasini et al. 2008), thyroid (Ricketts et al. 2010;Zantour et al. 2004), and renal tumors (Ricketts et al. 2008;Vanharanta et al. 2004). There is increasing evidence that mutations in SDH can lead to aberrant ROS generation through errors introduced in electron transport, and that this can result in tumorigenesis. ROS can be generated at the SDHA subunit during impaired electron transport (Yankovskaya et al. 2003). SDHC inactivation mutation in C. elegans and 3T3 mouse fibroblasts and electron leakage, oxidative stress, apoptosis and increased transformation and tumor growth (Adachi et al. 1998;Ishii et al. 2005).
Because of the pleiotropic role of PRODH/POX in cellular energetics and signaling, and its shared localization with the ETC on the inner membrane of the mitochondria, we sought to determine whether there was a direct relationship between PRODH/POX and regulation of the ETC. Using a PRODH/POX-expressing DLD colorectal cancer cell model and mouse mitochondria, we demonstrate that PRODH/POX passes electrons directly to Coenzyme Q1 (CoQ1), and that acute proline treatment in PRODH/ POX-expressing cells resulted in Complex I-and Complex II-independent oxidative respiration during nutrient stress conditions. In contrast, exposure of cells to PRODH/ POX and proline resulted in a significant time and dependent decrease in total oxidative respiration due to PRODH/ POX-dependent ROS production. PRODH/POX had dosedependent effect on the protein levels of individual subunits of Complexes I-IV of the ETC, which was reversed with the PRODH/POX inhibitor DHP and the antioxidant l-NAC. We show here that succinate inhibits PRODH/ POX through uncompetitive inhibition, and treatment of cells with succinate inhibits production of PRODH/POXdependent ROS, mitigates inhibition of respiration by PRODH/POX, and restores protein levels of ETC complexes in PRODH/POX-treated cells. These results suggest that PRODH/POX acts as a regulator of cellular respiration and that PRODH/POX activity is functionally linked to levels of succinate, potentially linking them as metabolic regulators.

Mouse liver mitochondria isolation
Mouse liver mitochondria were prepared in the method of Chappell and Hansford (Birnie 1972). In brief, freshly harvested livers were minced and washed 3× in ice-cold sucrose buffer (0.25 M sucrose, 3.4 mM tris-HCl, 1 mM EDTA, pH 7.4). Liver tissue was homogenized by 5 passes in a Dounce homogenizer. Homogenates were then centrifuged once at 478×g for 10 min, and the supernatant centrifuged at 10,000×g for 7 min. The pellet was washed with 25 ml ice-cold sucrose buffer and centrifuged 4 times at 10,000×g for 7 min, then resuspended in 3 ml ice-cold sucrose buffer. Protein concentration was determined using a BCA kit following the manufacturer's instructions (Life Technologies).

Measurement of PRODH/POX catalytic activity
PRODH/POX activity was measured as previously described, with minor modification (Pandhare et al. 2009). After treatment, cells were washed with cold PBS and harvested by trypsinization. Cells were resuspended in cold sucrose buffer [0.25 M sucrose, 3.5 mM Tris, and 1 mM EDTA (pH 7.4)] containing 1× protease inhibitor cocktail 1 and 2 (Sigma) and then sonicated for 20 s at a setting of 20 % (Branson Sonifier 450; Branson Ultrasonics Corp., Danbury, CT). Total protein was determined using the BCA protein assay (Pierce). A 1 ml reaction mixture containing 0.1 M KPO 4 , pH 7.2, 0.12 mg/ml o-aminobenzaldehyde (OAB), 0.012 mg/ml cytochrome C, 5 mM proline, and cell extract containing 50-100 μg protein was incubated for 20-60 min at 37 °C. The reaction was terminated by addition of 20 μl of OAB (10 mg/ml in 6 N HCl). The samples were centrifuged and the absorbance of the OAB-P5C complex was measured at 440 nm. A standard calibration curve was generated using purified P5C.

Oxygen consumption rate assays
XF24 cell culture plates were seeded with 5 × 10 5 cells and allowed to attach overnight. For PRODH/POX expression, cells were washed 3× with PBS and plated in DMEM containing the indicated amount of DOX and media treatment. One hour prior to the assay, cells were washed 2× with PBS and plated in Seahorse assay media containing 5 mM glucose and 2 mM glutamine, and adjusted to pH 7.4. Cells were incubated for 1 h at 37 °C in a no CO 2 incubator. Assay reagents (DMSO, oligomycin, FCCP, rotenone, and antimycin A) were loaded at a final concentration of 2.5 µM. Seahorse calibration, mitochondrial fitness tests, and oxygen consumption measurements were performed as per manufacturer's instructions (Seahorse Biotechnology, Massachusetts).

Measurement of reactive oxygen species
Cells were washed 3× with Dulbecco's PBS (DPBS), then placed in DPBS containing 5 µM of 5-(and-6)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA; Life Technologies). Cells were exposed to the dye for 20 min in a 37 °C incubator. The fluorescence intensity was determined on an adherent cell laser cytometer (ACAS; Meridian Instruments, Inc. Okemos, MI) using 488 nm excitation and 560 nm fluorescence detection. Quantitation was based on total protein measured per well using the BCA protein quantitation kit as per manufacturer's protocol (Life Technologies).

Measurement of electron transfer
Catalytic activity of SDH and PRODH/POX was measured using 2,6-dichlorophenolindophenol (DCIP) in the method of Hatefi and Stiggall with modification (Hatefi and Stiggall 1978). 50-100 µg of mouse mitochondria was suspended in 1 ml assay buffer containing 2 mM KCN, 20 mM KPO 4 , 50 µM DCIP, 100 µM CoQ1, and either 10 mM methylsuccinate or proline. Reactions were incubated at 37 °C for 10 min. Changes in absorbance at 600 nm were calculated versus negative control and blank.

Crosslinking and co-immunoprecipitation
Mitochondrial proteins were cross-linked using dithiobi s(succinimidylpropionate) (DSP) according to manufacturer's instructions (Thermo Fisher Scientific; Waltham, MA). Briefly, 200 mg of mouse mitochondrial fractions was washed 3× in ice-cold PBS. Mitochondria were suspended in cold PBS containing 2.5 mM of DSP and incubated on ice for 2 h. Reaction was quenched by addition of 50 mM Tris-HCl. Immunoprecipitation of Complex II was performed using the monoclonal Complex II immunocapture antibody (Abcam, Cambridge, MA) following manufacturer's protocol. Mitochondria were solubilized by the addition of 1 % lauryl maltoside (LM) and 1× protease inhibitor cocktail 1 and 2 (Sigma Aldrich) and incubated on ice for 30 min. Mitochondria were centrifuged at 14,000×g at 4 °C for 10 min to pellet insoluble material. Lysates were incubated with either 10 µg of Complex II antibody, PRODH monoclonal antibody (A-11; Santa Cruz Biotechnology), or mouse IgG control (Abcam) on ice for 1.5 h. 50 µl of a 50 % slurry of TrueBlot Anti-Mouse IP Beads (Rockland Antibodies, Limerick, PA) was added and samples placed on a rotator overnight at 4 °C. Beads were washed 3× with cold PBS containing 0.1 % LM and suspended in 100 µl of Laemelli SDS-page buffer.

Western blotting
Cell lysates were prepared and quantified according to established methods. To each well of a 4-12 % or 12 % SDS-polyacrylamide gel, 15-30 μg total protein was applied, electrophoresed, and transferred to nitrocellulose membrane using an iBlot semi-dry transfer apparatus (Life Technologies). Membranes were blocked using Tris-buffered saline with 5 % nonfat milk (pH 7.6; Sigma).

Statistical methods
For analysis of mechanisms of inhibition of POX by SDH inhibitors and succinate, data in this study were evaluated using linear and nonlinear regression analysis, Lineweaver-Burk double-reciprocal plot analysis, and analysis of covariance (ANCOVA). Precise estimates of V max and K m were estimated using modern nonlinear regression (Bates and Watts 1988) methods. Intercepts (1/v0) and slopes (K m /V max ), which were back-calculated from the V max and K m estimates obtained from nonlinear regression analyses, were found to be virtually identical to intercept and slope estimates obtained from linear regression analyses using the double-reciprocal Lineweaver-Burk strategy. ANCOVA was used to determine statistical equivalence of intercept and slope estimates in connection with inferences regarding competitive or uncompetitive inhibition in the CoQ1, SUCC, and TTFA analyses (N AWaC2014; Alvord 2014; Venables et al. 2002). Statistical analyses were performed with the R Statistical Language and Environment (Team RC 2014).

Coenzyme Q1 is electron acceptor for PRODH/POX
It was recently shown that recombinant PRODH/POX isolated from S. Cerevisae bound directly to CoQ1 and that this was the mechanism by which PRODH/POX fed electrons from proline into the electron transport chain (Wanduragala et al. 2010). In addition, we have previously demonstrated that PRODH/POX catalysis of proline can be used to support ATP generation under conditions of acute nutrient stress (Pandhare et al. 2009).
To assess PRODH/POX utilization of CoQ1 as an electron acceptor in our DLD-POX cell model, we added increasing amounts of CoQ1 to DLD-POX lysates and monitored PRODH/POX catalytic activity. As shown in supports respiration independent of Complex I and II activity. a POX activity assay showing the increase in POX activity in the presence of increasing CoQ1. DLD-POX cells were grown in 0.2 ng/ml DOX to allow POX expression. Homogenized cell lysate containing 100 μg protein was incubated in assay buffer with 0, 25, 50, 100, 250, and 500 μM of CoQ1 at 37 °C for 20 min. Absorbance at 440 nm was measured to assess the amount of OAB-P5C complex formed. A P5C standard curve was used to calculate the P5C concentration. Data shown represent mean ± SEM. Compared to CoQ1 = 0, all values are significant to p < 0.001. b Double-reciprocal analysis of CoQ1-dependent POX activity. DLD-POX lysate with 100 µg protein was incubated with 1, 2, 5, or 10 mM of proline and 25, 50, 75, or 100 µM of CoQ1 in assay buffer at 37 °C for 20 min. Absorbance at 440 nm was used to measure the amount of OAB-P5Ccomplex formed and a P5C standard curve used to calculate the P5C concentration. c PRODH/POX activity in DLD-POX lysates and mouse mitochondria show similar dependence on ETC function. PRODH/POX-containing lysates (200 µg protein) or mouse mitochondria (50 µg protein) were incubated in assay buffer at 37 °C for 30 min. Sensitivity of PRODH/POX activity to ETC function was measured by the addition of 50 µM ROT, 2 mM TTFA, 5 µM of AA5, 100 µM of AMA, and 1 mM KCN. Data shown represent mean ± SEM. *p < 0.01 compared to its respective control. d PRODH/POX expression is required for proline to support respira-tion during acute nutrient stress. DLD-VEC control cells (VEC) and DLD-POX cells (POX) were grown in 0.2 ng/ml DOX for 48 h. Cells were incubated in glucose-free media alone (CON) or with the addition of 5 mM proline (+PRO) for 1 h prior to Seahorse XF24 analysis. OCR was measured and a respiratory profile was established by the addition of 2.5 µM DMSO, OLIGO, FCCP, and ROT/AMA at the indicated time points. *p < 0.01 compared to vector control. e Seahorse XF24 oxygen consumption analysis of DLD-POX control cells. Cells were grown in 20 ng/ml DOX to suppress PRODH/POX expression. 1 h prior to analysis, cells were incubated in assay media containing 5 mM glucose and either 0 (CON), 1, or 5 mM proline (PRO). OCR was measured at the indicated timepoints. ROT and AA5 were added at 2.5 uM to inhibit Complex I and Complex II activity, respectively. AMA was added at 2.5 μM to inhibit Complex III. Compared to control, *p < 0.01; + p < 0.02. f Experimental details as described for (e), except in this case, DLD-POX cells were grown in 0.2 ng/ ml DOX for 48 h prior to assay. Compared to control, + p < 0.001; *p < 0.01. The following abbreviations apply: AA5 atpenin A5, AMA antimycin A, CoQ1 coenzyme Q1, DMSO dimethyl sulfoxide, DOX doxycycline, FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone, KCN potassium cyanide, OAB 2-aminobenzaldehyde, OCR oxygen consumption rate, OLIGO oligomycin, PRODH/POX proline oxidase, ROT rotenone, TTFA 2-thenoyltrifluoroacetone CoQ1 in a dose-dependent and saturable manner. These data indicate that PRODH/POX utilizes CoQ1 as an electron acceptor in the oxidation of proline to P5C.
To further characterize its interaction with CoQ1, we performed PRODH/POX activity assays using increasing concentrations of proline and CoQ1 to generate a Lineweaver-Burk plot (Fig. 1b). Under our experimental conditions, the V max and K m values were 9.72 ± 0.35 µM P5C min −1 and 0.42 ± 0.098 mM proline, respectively, using 25 µM of CoQ1. The V max and K m increased to 21.97 ± 0.47 µM P5C min −1 and 1.2 ± 0.09 mM proline, respectively, using 100 µM CoQ1. In a global analysis of covariance of the four data sets, there was no significant difference in the slopes at the α = 0.01 level of confidence, p = 0.011. This indicates that PRODH/POX binds directly to CoQ1 without the formation of a tertiary complex, consistent with recent reports (Wanduragala et al. 2010).
To determine whether PRODH/POX activity was dependent on ETC function, we compared PRODH/POX activity in DLD-POX cell lysates and mouse mitochondria in the presence of ETC inhibitors. We examined PRODH/ POX activity in the presence of CoQ1 alone or in combination with rotenone (ROT), an inhibitor of Complex I, 2-thenoyltrifluoroacetone (TTFA) or atpenin A5 (AA5), inhibitors of Complex II, antimycin A (AMA), an inhibitor of Complex III, or potassium cyanide (KCN), an inhibitor of Complex IV. In both isolated mitochondria and DLD-POX cell lysates, addition of ROT only modestly affected PRODH/POX activity, whereas addition of TTFA significantly reduced PRODH/POX activity (Fig. 1c). This was not the case with AA5, which had no effect on PRODH/ POX activity. When AMA or KCN was added to the reaction, PRODH/POX catalytic activity was dramatically reduced. Thus, in both DLD-POX cells and mouse mitochondria, the transfer of electrons from proline to CoQ1 by PRODH/POX was dependent on downstream electron transfer from CoQ1 to Complexes III and IV.
Acute expression of PRODH/POX during nutrient stress led to PRODH/POX-and proline-dependent ATP generation and cell survival (Pandhare et al. 2009). To determine whether PRODH/POX supported oxidative respiration during acute nutrient stress, we incubated either DLD-VEC cells, containing the vector construct, or DLD-POX cells, grown in 0.2 ng/ml doxycycline (DOX) to allow PRODH/ POX expression, for 48 h in proline-free media. We then placed the cells in glucose-free media with or without the addition of 5 mM proline for 1 h, and measured cellular oxygen consumption rate (OCR). Whereas the DLD-VEC cells did not respond to proline treatment, the DLD-POX cells were able to support a stable respiratory profile (Fig. 1d).
For additional evidence that PRODH/POX could support respiration solely through proline oxidation, we incubated induced and uninduced DLD-POX cells in proline-free media for 48 h, and then in media containing 0, 1, and 5 mM proline for 1 h, and measured cellular OCR. After measurement of basal respiration, ROT and AA5 were added to eliminate the contributions of Complex I and Complex II to respiration. As shown in Fig. 1e, f, addition of ROT and AA5 reduced respiration to approximately 30 % of normal basal respiration in cells in which PRODH/ POX expression was uninduced. The addition of 5 mM proline to uninduced control cells resulted in only a slight recovery of respiration (Fig. 1e). In contrast, addition of proline to PRODH/POX-expressing cells restored respiration at both 1 mM (approximately 50 % basal) and 5 mM (approximately 80 % basal; Fig. 1f). Addition of AMA inhibited respiration in all conditions shown in Fig. 1e, f, indicating that the observed oxygen consumption was dependent on Complex III. This data indicates that proline is a PRODH/POX-dependent respiratory substrate during acute nutrient stress and that it acts independently of Complex I and Complex II but requires functional Complex III.

Effect of PRODH/POX on respiratory fitness
Our data indicated that PRODH/POX and proline can support respiration during acute nutrient stress. We investigated the effects of PRODH/POX expression on overall respiratory fitness and capacity. We grew DLD-VEC control cells and DLD-POX cells for 3 days with 20, 0.2, 0.02 and 0 ng/ ml of DOX to allow for increasing levels of PRODH/POX expression. Manipulation of DOX concentration in DLD-VEC cells had no effect on respiration (Fig. 2a), but DLD-POX cells showed a decrease in both basal and maximal respiratory capacity that was directly proportional to DOX concentration (Fig. 2b).
We examined the effects on respiration resulting from increasing duration of PRODH/POX expression alone or with the addition of proline to the media. DLD-POX cells were grown in either 20 ng/ml DOX as a negative control or 0.2 ng/ml of DOX to allow PRODH/POX expression for 3 or 5 days in the presence or absence of 5 mM added proline. In control cells, proline alone had no effect on basal or maximal respiration, even after 5 days (Fig. 2c). In contrast, expression of PRODH/POX significantly decreased basal and maximal respiration at both 3 and 5 days, and proline addition exacerbated this effect (Fig. 2c). Thus, PRODH/POX-dependent inhibition of respiration can be modulated by the duration of PRODH/POX expression and the availability of proline.
PRODH/POX is a well-established superoxide generator, and the majority of effects of PRODH/POX on signaling are due to generation of ROS (Liu et al. 2006;D'Aniello et al. 2015;Pang and Curran 2014). We investigated whether effects of PRODH/POX on respiration were due to ROS production. DLD-POX cells were grown Oxygen consumption rate (OCR) was measured and a cellular respiratory profile was established by the addition of 2.5 µM DMSO, OLIGO, FCCP, and ROT/AMA at the indicated timepoints. b Increasing PRODH/ POX expression correlates with lower cellular respiration. DLD-POX cells were grown in the indicated amount of DOX and oxygen consumption rate was analyzed as in (a). Values with DOX = 0.02 and 0.0 were compared to DOX = 20; *p ≤ 0.01; + p < 0.02. c Prolonged PRODH/ POX expression and addition of proline correlates with greater decreases in respiration. DLD-POX cells were grown in 0.2 ng/ml DOX to allow PRODH/POX expression for 3 days (3D POX) or 5 days (5D POX) alone or in media supplemented with 5 mM proline (3D POX + PRO and 5D POX + PRO). Respiration by these cells was compared to DLD-POX cells that had been grown for 5 days in 20 ng/ml DOX to suppress PRODH/ POX expression, alone or in combination with 5 mM proline (5D CON and 5D CON + PRO, respectively). Oxygen consumption rate was measured and a respiratory profile established as described in (a). Values for 3D POX + PRO, 5D POX, and 5D POX + PRO were compared to 5D CON. + p ≤ 0.05; *p < 0.01. d Inhibition of PRODH/POX-mediated ROS decreases effects on respiration. DLD-POX cells were grown for 48 h in either 20 ng/ml DOX (CON), or 0.2 ng/ml DOX alone (POX) or in combination with 5 or 10 mM of N-acetyl-l-cysteine (NAC). Oxygen consumption rate was measured and a respiratory profile established as described in (a). Compared to control, *p < 0.001; Compared to POX, + p < 0.01. e PRODH/POX expression down-regulated ETC component proteins. DLD-POX cells were grown in the indicated ng/ml concentration of doxycycline (DOX) for 48 h, alone or in combination with 10 mM DHP or NAC. Whole cell lysates were harvested, and protein expression of subunits of Complex I (NDUFA10), Complex II (SDHA and SDHB), Complex III (CIII-R, Reiske Fe-S subunit), and Complex IV (COX IV) were analyzed by Western blotting. β-Actin was used as a protein loading control. The band intensities shown below each panel were quantified using Image Studio, normalized to β-actin control, and expressed as the level relative to untreated control (lane 1 20 ng/ml DOX). Values for DHP and NAC were compared to the mean ± STD of the ratios for 0.2 ng/mL DOX (lane 3 e and lane 2, Fig. 5a) combined with those for 0.02 ng/ml DOX (lane 4 e) Although the DOX concentration for DHP and NAC treatment was 0.02 ng/mL DOX, the values at 0.02 ng/ml DOX were consistently lower than those at 0.2 ng/ml DOX. Thus, the values used represent a higher distribution for the PRODH/POX-mediated effect on ETC proteins. DHP and NAC increased the values of the ETC proteins, i.e., decreased the effect of PRODH/POX. *Value greater than 2 standard deviations of aforementioned distribution, denoting 95 % confidence limits in 0.2 ng/ml DOX for 3 days with 5 mM proline alone or with the addition of 5 or 10 mM of the antioxidant N-acetyl-l-cysteine (NAC). Expression of PRODH/POX with proline showed a dramatic suppression of oxidative respiration, and NAC mitigated their inhibitory effects on and preserved both basal and maximal respiratory capacity (Fig. 2d). These results demonstrate that basal and maximal respiratory capacity are modulated by levels of PRODH/ POX induction and proline availability, and that this effect is mediated by ROS generation.
As shown in Fig. 2, PRODH/POX down-regulates cellular respiration through ROS production. We examined the effects of PRODH/POX-dependent ROS on ETC component proteins. To correlate changes in proteins to functional effects on respiration, we grew DLD-POX cells in 20, 2, 0.2, and 0.02 ng/ml of DOX and monitored ETC proteins at increasing levels of PRODH/POX expression (Fig. 2e,  lanes 1-4, respectively). In addition, cells were incubated in 0.02 ng/ml of DOX together with either the PRODH/ POX inhibitor dehydroproline (DHP) (Fig. 2e, lane 5) or NAC (Fig. 2e, lane 6). To assess statistical significance of DHP and NAC, we calculated mean ± standard deviation of the values for 0.2 and 0.02 ng/ml DOX from Figs. 2e and 5a. Increasing PRODH/POX expression decreased levels of the NDUFA10 subunit of Complex I, SDHA and SDHB, the Reiske subunit of cytochrome C reductase (Complex III), and subunit IV of cytochrome C oxidase (COX IV, Complex IV). Importantly, SDHA, SDHB and CIII-R were restored by either NAC or DHP or by both (Fig. 2e, lanes 5 and 6, respectively. See legend to Fig. 2e). Compared to the POX levels, the values for DHP and NAC indicated by an asterisk were outside of 2 standard deviations from the mean of the POX controls. Taken together, these results provide preliminary evidence that PRODH/POX modulates respiration through ROS-mediated down-regulation of ETC component proteins.

PRODH/POX is inhibited by Complex II inhibitors and succinate
We showed in Fig. 1c that TTFA inhibited PRODH/POX activity whereas AA5 did not. The crystal structure of SDH with bound TTFA or AA5 has been solved; both are competitive inhibitors of the ubiquitin-binding site, with AA5 binding at a site more deeply embedded in the enzyme's catalytic site (Sun et al. 2005;Miyadera et al. 2003;Horsefield et al. 2006). To compare the inhibitory potency of TTFA and AA5 for PRODH/POX versus SDH, we performed enzyme activity assays with either proline or succinate as the substrate. Carboxin, an additional competitive inhibitor of the ubiquitin-binding site of SDH (Ruprecht et al. 2009), was also tested. Both TTFA and carboxin inhibited SDH and PRODH/POX activity with similar efficacy, with TTFA inhibiting 60 % and 35 % and carboxin inhibiting 36 and 42 % of SDH and PRODH/POX activity, respectively. In contrast, AA5 was selective toward SDH, inhibiting its activity by 83 % whereas it only inhibited PRODH/POX activity by 9 % versus control (Fig. 3a).
To elucidate the mechanism by which TTFA inhibited PRODH/POX activity, we performed POX activity assays to construct a Lineweaver-Burk plot (Fig. 3b). V max was only modestly decreased, being 17.2 ± 0.4 µM P5C min −1 for control and 16.4 ± 0.3 µM P5C min −1 with the addition of 2 mM TTFA. In contrast, 2 mM TTFA increased K m from 0.65 ± 0.05 mM proline to 2.7 ± 0.35 mM proline. Statistical tests were performed for the equivalence of the y-intercepts for TTFA data pairs. All p values were greater than 0.05, indicating that the y-intercepts for TTFA data were statistically equivalent. Together, these results suggest that TTFA acts as a competitive inhibitor of PRODH/POX. We compared the mechanism by which succinate inhibits PRODH/POX activity. We again performed PRODH/ POX activity assays to construct a Lineweaver-Burk plot (Fig. 3c). Addition of 5 mM succinate decreased both V max and K m from 19.1 ± 0.4 to 8.8 ± 0.3 µM P5C min −1 and 1.1 ± 0.08 mM proline and 0.55 ± 0.09 mM proline, respectively, versus control. In a global analysis of covariance of the four experimental data sets, there was no significant difference in the slopes at the α = 0.05 level of confidence, p = 0.75. This indicates succinate inhibits PRODH/ POX activity through uncompetitive inhibition.
To examine whether PRODH/POX and SDH could be physically associated on the mitochondrial inner membrane, we performed cross-linking and co-immunoprecipitation assays. We incubated mouse mitochondria with the protein cross-linker DSP, dissolved the mitochondria with 1 % laurel maltoside and immunoprecipitated using an antibody versus either PRODH/POX or Complex II. We then immunoblotted for PRODH/POX and the A and B subunits of SDH. Immunoprecipitation of PRODH/POX resulted in co-immunoprecipitation of both SDHA and SDHB. In addition, PRODH/POX co-immunoprecipitated with Complex II when an anti-Complex II was used (Fig. 3d). These data indicate that PRODH/POX and Complex II co-localize on the surface of the mitochondrial matrix membrane within the length of the DSP spacer arm (12 angstroms).

Succinate inhibits PRODH/POX-dependent ROS generation
Our data suggested that PRODH/POX and proline control mitochondrial respiration through ROS-mediated downregulation of ETC proteins. In addition, our data showed that succinate inhibits catalytic activity of PRODH/POX. We were intrigued whether succinate would modulate the PRODH/POX effect on the ETC. DLD-POX cells were grown in 0.2 ng/ml of DOX to allow PRODH/POX expression, alone or in combination with 5, 10, or 20 mM of methyl-succinate. Cells were then treated with general ROS indicator 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA) to detect total cellular ROS. PRODH/POX expression resulted in a threefold increase in detectable ROS generation versus uninduced control; this ROS generation was inhibited by succinate in a dose-dependent manner (Fig. 4a).
To determine whether succinate inhibition of PRODH/ POX protected against the PRODH/POX-dependent downregulation of respiration, we grew DLD-VEC and DLD-POX cells in media containing proline alone or together with methyl-succinate. DLD-VEC control cells showed no change in basal and maximal respiration whether treated with proline, methyl-succinate, or a combination of both (Fig. 4b). In contrast, PRODH/POX-expressing cells showed a marked reduction of respiration in response to proline treatment that was inhibited by co-treatment with methyl-succinate (Fig. 4c). Taken together, this data indicates that succinate inhibits the effects of PRODH/POX and proline on respiration through the inhibition of PRODH/ POX-and proline-derived ROS.

Succinate inhibition of PRODH/POX restores levels of ETC component proteins
Because succinate inhibited PRODH/POX-dependent ROS generation, we investigated whether succinate treatment could modulate PRODH/POX-mediated down-regulation of ETC proteins. DLD-POX cells were grown in either 20 ng/ml DOX or 0.2 ng/ml DOX to allow PRODH/POX expression alone or in combination with increasing concentrations of succinate. To link effects on ETC proteins with effects on ROS (Fig. 4a), we used increasing concentrations of succinate. Expression of PRODH/POX resulted in down-regulation of subunits of Complexes I-IV of the ETC (Fig. 5, lane 1 vs lane 2). Addition of increasing PRODH/POX activity is regulated by succinate and SDH inhibitors and co-localizes with SDH on the mitochondrial inner membrane. a PRODH/POX and SDH share similar sensitivity to SDH inhibitors except AA5. Mouse mitochondria (440 µg protein) were incubated in reaction buffer containing either 10 mM of succinate or proline alone (Control) or in the presence of 1 mM TTFA, 1 mM Carboxin, or 5 µM AA5 at 37 °C for 10 min. Absorbance was measured at 600 nm and results calculated as a percent of unreacted control dye. Data shown represent mean ± SEM (n = 3) of comparisons against their respective controls. *p < 0.001; + p < 0.05. b Doublereciprocal analysis of TTFA-dependent inhibition of PRODH/POX activity. DLD-POX lysate containing 100 µg protein was incubated with 1, 2, 5, or 10 mM of proline and 0, 0.5, 1 and 2 mM of TTFA in assay buffer at 37 °C for 20 min. Absorbance at 440 nm was used to measure the amount of OAB-P5C complex formed and a P5C standard curve used to calculate the P5C concentration. c Double-recipro-cal analysis of succinate-dependent inhibition of PRODH/POX activity. DLD-POX lysate containing 100 µg protein was incubated with 1, 2, 5, or 10 mM of proline and 0, 0.5, 1, and 5 mM of succinate in assay buffer at 37 °C for 20 min. Absorbance at 440 nm was used to measure the amount of OAB-P5Ccomplex formed and a P5C standard curve used to calculate the P5C concentration. d Co-immunoprecipitation of PRODH/POX and Complex II. 4 mg of mouse mitochondria was cross-linked with DSP, then solubilized and incubated with either PRODH/POX (POX IP) or Complex II antibody (CII IP). Control samples were incubated with an equal concentration of a non-specific mouse IgG (CON). Samples were incubated with beads coated with anti-mouse IgG overnight. Cross-linker was cleaved and proteins solubilized with SDS-PAGE buffer. Control and IP lysates were immunoblotted versus a 5 % input control (IN) using PRODH/ POX, SDHA, and SDHB antibodies. Data is representative of three separate experiments concentrations of succinate resulted in restoring protein levels for Complexes I-III (Fig. 5, lanes 3-6). Levels of the COX IV subunit of Complex IV were not restored by succinate treatment, but it was the only exception of all of the ETC subunits examined.
Since both the decrease in PRODH/POX-generated ROS and the mitigation of the PRODH/POX-mediated decrease in ETC protein levels appeared to be dependent on the concentration of succinate, we plotted the individual ETC proteins versus succinate and showed a general concentration dependence (Fig. 5b). This suggested that succinate-dependent inhibition of PRODH/ POX was a common mechanism. With this hypothesis, we compared the values for ETC proteins in combination. Furthermore, we included the values for ETC proteins at 0.2 ng/ml DOX from Fig. 2e. These values represent mean ± STD of ETC proteins. With SUC = 0, 0.635 ± 0.091, N = 10, SUC = 10 mM, mean = 0.782 ± 0.122, N = 5, different from SUC = 0 with p = 0.0203; SUC = 20 mM, values were 0.768 ± 0.156, N = 5, different from SUC = 0, with p = 0.0546. These values and statistical differences support our working hypothesis of a common mechanism underlying the changes in ETC proteins.

Discussion
In this study, we have examined the interaction of PRODH/ POX with Coenzyme Q1 and with the ETC. While PRODH/POX supports respiration in the short term, PRODH/POX expression results in a decrease of total cellular respiration through down-regulation of proteins of the ETC. Most interestingly, the effects of PRODH/POX on respiration and the ETC can be inhibited by succinate, resulting in a potential regulatory loop. c Succinate inhibits the decrease in respiration induced by PRODH/ POX and proline. DLD-POX cells were grown for 48 h in 0.2 ng/ ml of DOX to allow PRODH/POX expression. Oxygen consumption rate (OCR) was measured and a respiratory profile was established as described for (b). Comparison of 10 mM succinate to 5 mM PRO, + p < 0.01. Comparison of 5 mM POX to 5 MM POX + 10 mM SUC, *p < 0.05. Data represents mean ± standard error of the mean (n = 3) for all panels Our analysis of the relationship between PRODH/POX and CoQ1 suggested that POX can transfer proline-derived electrons directly to CoQ1 without the formation of a tertiary complex. In addition, selective inhibitors indicated that PRODH/POX did not require the activity of Complex I or Complex II to support respiration. This is consistent with studies in other diverse model systems of PRODH/POX activity, including Z. meas (corn) mitochondria, purified S. cerevisiae PRODH/POX, and PutA from E. coli (Elthon and Stewart 1982;Wanduragala et al. 2010;Moxley et al. 2011;Abrahamson et al. 1983). In addition, PRODH/POX dependence on a functional ETC in our DLD-POX expression system was almost identical to that of isolated mouse mitochondria. Thus, we believe our DLD expression system accurately reflects the relationship between PRODH/ POX and the ETC.
PRODH/POX was necessary and sufficient to support proline-dependent oxidative respiration during acute nutrient stress, consistent with previous publications showing its pro-survival role during nutrient stress (Pandhare et al. 2009;Liu et al. 2012). In addition, extended expression of PRODH/POX (48-96 h) resulted in the down-regulation of basal oxidative respiration and maximal respiratory capacity. This effect was dependent on the duration and intensity of POX expression as well as the addition of exogenous proline, and was inhibited by the co-treatment with NAC. Reduction of respiratory capacity correlated with reduction in levels of protein subunits of Complexes I-IV of the ETC, which was reversed by co-treatment of cells with either DHP or NAC. These effects are most readily explained as resulting from proline oxidation and ROS generation by PRODH/POX.
Inhibition of PRODH/POX by TTFA was through competitive inhibition. Both TTFA and carboxin have previously been shown to compete for the ubiquinone-binding site of SDH that is formed when the 4 SDH subunits combine to form the holoenzyme (Sun et al. 2005;Ruprecht et al. 2009). Based on this, one possibility we considered was that PRODH/POX required the ubiquitin-binding site of SDH for its activity. However, AA5, which has been shown to bind deeper in the ubiquinone-binding pocket of SDH (Miyadera et al. 2003;Horsefield et al. 2006), showed very little inhibition of PRODH/POX. In addition, the interaction of PRODH/POX with CoQ1 indicated direct binding to PRODH/POX. We think it more likely that the SDH inhibitors compete for the ubiquitin-binding site on PRODH/POX. The observation that TTFA and carboxin can inhibit PRODH/POX and SDH to a similar degree at the concentrations used may warrant caution in their use as SDH-selective inhibitors.
Although succinate inhibition of PRODH/POX has been investigated (Kowaloff et al. 1977;Norden and Matanganyidze 1974), to our knowledge this is the first study to examine the effects of this inhibition on PRODH/POXmediated ROS generation and mitochondrial metabolism. Our analysis showed that succinate inhibits POX through  (Fig. 4a). a Whole cell lysates were harvested, and protein expression of PRODH/POX and of subunits of Complex I (NDUFA10), Complex II (SDHA, SDHB), Complex III (CIII-R, Reiske Fe-S subunit), and Complex IV (COX IV) was analyzed by Western blotting. The band intensities shown below each panel were quantified using Image Studio, normalized to β-actin control, and expressed as the level relative to untreated control (lane 1 20 ng/ml DOX). b Since succinate decreased PRODH/POX-mediated ROS in a dose-dependent manner (Fig. 4a) and the levels of proteins in a suggested a succinate concentration effect, we constructed a combination plot of protein levels as the fraction of control against succinate concentrations (see text). For the value with treatment by POX (0.2 ng/ml DOX) without succinate, we combined the values from a with those obtained in Fig. 2e. We then compared the values statistically. The value with 10 mM succinate was different from that without succinate, *p = 0.020. At 20 mM succinate, the difference was borderline significant, + p = 0.055 uncompetitive inhibition. This mechanism makes it very unlikely that the observed effects are due to non-selective inhibition of PRODH/POX by succinate. Treatment with succinate inhibited PRODH/POX-dependent ROS production and prevented PRODH/POX-dependent ROS effects on oxidative respiration. Succinate also protected ETC component proteins from PRODH/POX ROS-mediated down-regulation with almost the same efficacy as DHP and NAC.
As an uncompetitive inhibitor, the affinity of succinate is for the enzyme-substrate complex of PRODH/POX and proline rather than for the enzyme binding site for proline. Additionally, our data show that PRODH/POX and SDH co-localize on the mitochondrial inner membrane, where local substrate concentrations would have the potential to affect the activities of both enzymes. Thus, in the presence of low levels of proline, higher levels of succinate could act to inhibit PRODH/POX activity and ROS generation. This may provide an additional level of regulation of PRODH/POX stress signaling until cellular levels of TCA cycle intermediates fall below a critical point. The specific role that SDH plays in the transmission of the PRODH/ POX-generated ROS signal remains to be elucidated, but a coordinated role with PRODH/POX would be consistent with the observation that SDH-mediated ROS generation was essential for a hypoxia-dependent stress response in mouse lung sections (Paddenberg et al. 2003(Paddenberg et al. , 2012 and that Complex II has been found to be a site of ROS generation resulting from proline oxidation (Goncalves et al. 2014).
The data presented here introduce a novel relationship between PRODH/POX, proline, and succinate and the regulation of respiration. Depending on cellular context, this relationship could provide an additional point of regulation between the identified inducers of PRODH/POX and regulation of cellular energy levels and routing of metabolites. More intriguing is the possibility that the proline found in extracellular collagen may serve both as a source of energy for tumors and tissues through proline catabolism, and also as a signaling and metabolic link between the extracellular environment during development and tumor formation. It is becoming increasingly apparent that dysregulation of metabolism and the resulting change in metabolite levels affect not only cell transformation (Ward et al. 2010;Lu et al. 2013), but influence tumor cell aggressiveness and metastatic potential, as has been shown recently in ovarian cancer and ovarian cancer stem cells (Vermeersch et al. 2014(Vermeersch et al. , 2015. The role of PRODH/POX as a direct mediator of ROS-dependent signaling and central mediator of metabolic intermediates will, no doubt, continue to play an interesting role in cellular development and transformation.

Summary
This work shows for the first time the regulation of respiratory fitness by PRODH/POX through downregulation of ETC proteins, and the inhibition of these effects by succinate. The existence of a regulatory loop between PRODH/ POX, the ETC, and succinate would link the various stress pathways that induce PRODH/POX with ETC regulation and TCA cycle metabolite flux. Changes in proline and succinate homeostasis may be a mechanism of epigenetic gene regulation during differentiation and tumorigenesis, and this possibility should be investigated further.