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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure statement
  8. References

Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme that converts isocitrate to alpha-ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development are unclear. We used a skin cell transformation model and mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter TPA and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with dimethylbenz[α]anthracene/TPA also showed decreases in IDH1 expression and activity. In non-promotable JB6 P-cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed, TPA-induced cell transformation. Finally, manganese superoxide dismutase overexpression suppressed tumor promoter induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha-ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress might contribute to IDH1 inactivation, because manganese superoxide dismutase, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02317.x, 2012)

Cellular metabolism is known to be altered in cancer cells. Cancer cells adapt to glycolysis and lactate fermentation for ATP production (the “Warburg effect”), although energy production is less efficient compared with oxidative phosphorylation in normal cells. This metabolic switch provides a growth advantage for cancer cells using metabolic intermediates as building blocks. The Warburg effect is mediated by metabolic enzyme dysregulation. Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are NADP+-dependent enzymes that convert isocitrate to α-ketoglutarate. However, there are several questions regarding the role of IDH1/2. At what stage of cancer development is IDH1/2 altered? Does IDH1/2 affect tumor promotion? Are IDH1 and IDH2 regulated similarly during early stages of cancer development? These fundamental questions will help in targeting IDH1/2 in cancer prevention.

We used murine skin epidermal JB6 cells (a well-established model to study tumor promotion) immortalized murine keratinocyte C50 cells, neoplastic papilloma SP1 cells and mouse skin epidermal tissues in early tumorigenesis studies. The tumor promoter TPA and UVC irradiation were used to promote JB6 cell transformation. Our previous studies using JB6 cells demonstrate that TPA decreases mitochondrial complex activities, membrane potential[1] and respiration,[2] suggesting that a metabolic shift might occur in early tumorigenesis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure statement
  8. References

Cell lines, reagents and treatment

Murine skin epidermal JB6 Cl-41 P+ (promotion-sensitive) and Cl 30-7b P– (promotion-resistant) cells were used to study tumor promotion (both cell lines were purchased from American Type Culture Collection, Manassas, VA, USA). Cells were grown in EMEM medium containing 4% FBS, 2 mM L-glutamine and 2.5 μg/mL penicillin and 2.5 μg/mL streptomycin in a 37°C incubator under 5% CO2.

Murine skin keratinocytes C50 and skin papilloma SP1 (obtained from Dr Terry Oberley at the University of Wisconsin-Madison) cells were grown in S-MEM medium supplemented with 8% Chelexed FBS, 2 mM of L-glutamine, 0.1 mM non-essential amino acid, 50 μM Ca2+, 2.5 μg/mL penicillin and 2.5 μg/mL streptomycin in a 37°C incubator under 5% CO2. C50 cells are an immortalized murine keratinocyte cell line;[3] neoplastic SP1 papilloma cell lines are produced in SENCAR mice.[4]

For cell culture, mycoplasma levels were routinely (once every 3 months) assessed using a MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, ME, USA).The results were negative.

Dimethylbenz[α]anthracene (DMBA; Sigma, St. Louis, MO,USA) was dissolved in DMSO (Sigma); the tumor promoter TPA (Sigma) was also prepared in DMSO. The TPA final concentration for cell culture studies was 100 nM, except for the soft agar assay, which was 5 nM.

For UVC irradiation, JB6 cells were grown in p100 dishes. The day before irradiation, normal growth medium was replaced with fresh growth medium containing only 0.1% FBS. Twenty-four hours later, growth medium was replaced with 2 mL PBS and cells received 20 J/m2 UVC irradiation (UVP, LLC, Model XX-15S, Upland, CA, USA). (This dose of UVC has been widely used in tumor promotion studies.) PBS was then replaced with normal growth medium and cells were incubated for 24 h.

Animal treatments

For the present study, 10 DBA/2 female mice (6–8 weeks old, purchased from the Jackson Laboratory, Bar Harbor, ME, USA), 10 manganese superoxide dismutase (MnSOD) transgenic (in C57BL/6 background)[5] and 10 non-transgenic littermates were housed in the LSUHSC-S Animal Resource Facility under standard regulations. The LSUHSC-S Animal Facility is AAALAC approved and maintains a consultation team of two veterinarians. The program is monitored by the National Institute of Health Office for Protection from Research Risk and the U.S. Department of Agriculture. Animals were provided food and water ad libitum. All studies were performed under an approved institutional animal protocol. Animals were killed at the termination of the study. This method is consistent with recommendations by the Panel on Euthanasia of the American Veterinary Medical Association.

Mice were separated into two groups (Control and DMBA/TPA, n = 5 per group) and hair on the backs of mice was shaved. Two days later, for the DMBA/TPA group, a single dose of 100 nmol DMBA was painted on the backs of mice. After 2 weeks, 4 μg of TPA was applied to the same area for 24 h. The control group received DMSO treatment each time. Mice were then killed, skin tissues were removed and skin epidermal cells were collected, as described previously.[6, 7]

Immunofluorescent staining of JB6 P+ cells

JB6 cells (5 × 104) were seeded in eight-well Lab-Tek chamber slides with covers (Nalge Nunc International, Naperville, IL, USA) in 400 μL medium per well and incubated overnight. Then, 24 h after plating, cells were treated with TPA (100 nM) or equivolume DMSO for 24 h. Cells were washed and fixed with 10% formalin for 15 min at room temperature. After rinsing with PBS (pH 7.4), cells were permeabilized with 400 μL of 1% Triton X-100 for 10 min at room temperature. Anti-IDH1 or anti-IDH2 (N-20; W-16, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were added (1:32 dilution in PBS containing 0.1% BSA) and incubated at 37°C for 1 h, followed by incubation with anti-goat IgG-FITC (1:32 dilution in PBS containing 0.1% BSA) for 1 h. Cells were washed with PBS and stained with DAPI, followed by mounting with VECTASHIELD mounting medium for fluorescence (H-1000; Vector Laboratories, Burlingame, CA, USA). Fluorescence was observed using a wide-field inverted microscope (Nikon Eclipse TE300, Melville, NY, USA).

Measurements of oxygen consumption of skin cells stripped from mouse skin tissues

Stripped skin cells resuspended in mitochondrial isolation buffer (0.225 M mannitol, 0.075 M sucrose, 1 mM EGTA, pH 7.4) were suspended in a thermostated closed vessel at 37°C. Oxygen consumption was measured polarographically using a Clark-type O2 electrode (Yellow Spring Instruments; Yellow Springs, OH, USA). Rates of mitochondrial O2 consumption were determined as cyanide-sensitive rates after addition of sodium cyanide to the final concentration of 300 μM.[8] Rates of oxygen consumption were collected from each experiment and data from at least three experiments were combined and plotted as nmol O2/min/mg (protein).

Isocitrate dehydrogenase 1 siRNA and vector transfections

Cells were seeded (2 × 105 cells/well) in six-well tissue culture plates, and incubated at 37°C in a 5% CO2 incubator until 70–80% confluent. For each transfection, 2 μL of IDH1 siRNA duplex (sc-60830; Santa Cruz Biotechnology) were diluted into 100 μL of siRNA transfection medium (sc-36868; Santa Cruz Biotechnology). In a separate tube, 2 μL of transfection reagent (sc-29528; Santa Cruz Biotechnology) were diluted into 100 μL of siRNA transfection medium. The dilutions were mixed gently together and incubated for 30 min at room temperature, as previously described.[2] Fluorescein conjugated control siRNA (sc-36869; Santa Cruz Biotechnology) was used to monitor transfection efficiency.

For IDH1 vector transfection experiments, JB6 P+ cells were seeded in p35 dishes in 2-mL growth medium. At 70–80% confluency, cells were transfected with either 2 μg GFP or IDH1 containing AAV9 shuttle vector, which was mixed with the FuGENE HD Transfection Reagent (Roche, Indianapolis, IN, USA) (FuGENE:DNA ratio = 7:2), following instructions provided by the manufacturer. The vector incorporated a hybrid cytomegalovirus/chicken beta actin (CBA) promoter, human IDH1 and a bovine growth hormone polyadenylation sequence, which was flanked on both sides with 142 base pair terminal repeats (TR) from adeno-associated virus serotype 2: TR-CBA-IDH1-pA-TR. The control plasmid expressed GFP instead of IDH1.[9] After 24 h, cells were collected and subjected to the soft agar assay.

Preparation of whole cell lysate

Collected skin cells were suspended in 250 μL of PBS containing proteinase inhibitor cocktail (Calbiochem, La Jolla, CA, USA). Cells were sonicated on ice for two strokes (10 s per stroke) using a Fisher Sonic Dismembrator (Model 100, Scale 2). After incubating on ice for 30 min, cell lysate was centrifuged at 18 000 ×g for 20 min, and the supernatant was collected and designated as whole cell lysate.

Western blot analysis

Whole cell lysate was used for the assay. Antibodies against IDH1 (sc-49996), IDH2 (sc-55668) and GAPDH (sc-32233) were purchased from Santa Cruz Biotechnology.

NADP+-dependent Isocitrate dehydrogenase activity assay

NADP+-dependent IDH activity (including both IDH1 and IDH2) was measured in a 400-μL assay solution containing 33 mM of potassium phosphate buffer (pH 7.0), 0.167 mM of NADP+, 3.3 mM of Mg2+, 0.167 mM of threo-DS-isocitrate and 10 μL (approximately 20 μg protein) of freshly isolated whole cell lysate. The activity of IDH was monitored through reduction of NADP+ to NADPH, recorded by spectrophotometry at 340 nm for 5 min.

Determination of alpha-ketoglutarate

Levels of α-ketoglutarate (α-KG) were determined using the Alpha-Ketoglutarate Assay Kit (BioVision Inc., Milpitas, CA, USA; K677-100) following instructions provided by the manufacturer. This assay kit can detect α-KG at nmol levels. whole cell lysate was diluted to 2 μg/μL in PBS and deproteinized by passing through a 10-kD cut-off membrane (VWR, 82031-348). For each sample, 50 μL of whole cell lysate filtrate was used.

Anchorage-independent growth assay in soft agar

Soft agar-based cell transformation assay was carried out in six-well plates. The bottom agar was made using 1.25% agar, 2× EMEM medium, 10% FBS, PBS, glutamine and penicillin, and was incubated in a 50°C water bath for 15 min. The mixture (0.5% agar) was then divided and treated with various treatments, as previously described.[2]

Statistical analysis

Student's t-test was used for two-group comparison, and one-way anova followed by Newman–Keuls post-test were used for multi-group comparisons. Data are mean ± SE; P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure statement
  8. References

Isocitrate dehydrogenase 1 expression and activity are decreased by TPA and UVC irradiation treatments

The skin epidermal JB6 Cl41 (P+) cell model is the only characterized skin cell model to study tumor promotion.[10] Tumor promoter TPA (100 nM) or UVC irradiation (20 J/m2) (the concentration of TPA and dose of UVC have been used extensively in our and others' studies) were used to treat mouse skin epidermal JB6 P+ cells for 24 h. Cells were lysed in PBS containing proteinase inhibitors and subjected to western blot analysis and NADP+-dependant IDH activity assays. In Figure 1A,B, IDH1 expression was suppressed by both TPA and UVC treatments, whereas expression levels of IDH2 were not altered by either TPA or UVC irradiation. Correspondingly, NADP+-dependent activities of IDH were reduced by approximately 28% in TPA-treated samples and 41% in UVC-treated samples. In Figure 1C, immunofluorescent signals of IDH1 (green) were reduced similarly in TPA-treated cells, whereas those of IDH2 (green) were not affected. The nucleus was stained blue (DAPI).

image

Figure 1. TPA (A) and UVC irradiation (B) downregulated isocitrate dehydrogenase 1 (IDH1). JB6 P+ cells were grown in p100 dishes. (A) Cells were treated with TPA (final concentration of 100 nM) or vehicle DMSO (0.01%, control) for 24 h. (B) Cells were irradiated by UVC (20 J/m2) followed by a 24-h incubation. Control: no UVC treatment. GAPDH served as the loading control. Experiments were repeated three times and a representative result is shown. *P < 0.05 compared with the control. (C) Immunofluorescent staining of JB6 P+ cells. JB6 cell growth in eight-well chambers was treated with TPA (100 nM) or DMSO (0.01%, control) for 24 h. Anti-IDH1 or anti-IDH2 antibodies were incubated with fixed and permealized cells followed by incubation with FITC-conjugated secondary antibodies. DAPI was used to stain the nucleus.

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Isocitrate dehydrogenase 1 downregulation occurs in tumor promotion-sensitive cells but not promotion-resistant cells

To determine the specificity of decreased IDH1 expression and activity to tumorigenesis, promotion-sensitive and promotion-resistant JB6 cells were treated with TPA. Expression levels of both IDH1 and IDH2 were determined in both cell lines. In Figure 2, TPA reduced expression of IDH1 in P+ cells, accompanied by a decrease in NADP+-dependent enzymatic activity. However, in P– cells, IDH1expression and enzymatic activity were increased. Similar to previous studies, expression of IDH2 was maintained at similar levels in P+ and P– cells, and DMSO and TPA-treated samples.

image

Figure 2. Comparison of isocitrate dehydrogenase (IDH) expression (A) and activity (B) levels between tumor promotable JB6 P+ and non-promotable P– cells. JB6 cells grown in p100 dishes were used. Cells were treated with TPA (final concentration of 100 nM) or vehicle DMSO (0.01%, Ctrl) for 24 h and whole cell lysate was prepared. GAPDH served as the loading control. Experiments were repeated three times and a representative result was shown. *P < 0.05 compared with control (DMSO treatment). #P < 0.05 compared with TPA-treated JB6 P+ cells.

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Skin papilloma SP1 cells display decreased isocitrate dehydrogenase 1 expression and activity compared with normal keratinocyte C50 cells

Skin papilloma SP1 cells and immortalized skin keratinocyte C50 cells were used to confirm that IDH1 downregulation exists in skin tumor cells. In Figure 3, IDH1 expression was greatly reduced in SP1 cells compared with that in C50 cells. Similarly, IDH enzymatic activity was reduced by 46% in SP1 cells compared with that in C50 cells. However, expression levels of IDH2 were similar between these two cell lines.

image

Figure 3. Comparison of isocitrate dehydrogenase (IDH) expression (A) and activity (B) levels between immortalized skin keratinocyte C50 and papilloma SP1 cells. Cells grown in p100 dishes were harvested and whole cell lysate was prepared. Succinate dehydrogenase subunit B (SDHB) served as the loading control. Experiments were repeated three times and a representative result is shown. *P < 0.05 compared with sample from C50 cells.

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DMBA/TPA treatment decreases isocitrate dehydrogenase 1 expression in vivo

To verify that the effects on IDH1 expression by tumor promoters occur in vivo, DMBA and TPA were applied to mouse skin, skin epidermal cells were stripped and whole cell lysate was prepared for western blot analysis and IDH activity assay. In Figure 4, it is evident that DMBA/TPA treatment suppressed IDH1 expression, accompanied by an approximate 32% reduction in its enzymatic activity. Similar to the in vitro results, expression of IDH2 was not altered in mouse skin tissues after DMBA/TPA treatment.

image

Figure 4. DMBA/TPA treatment caused decreases in isocitrate dehydrogenase (IDH) expression (A) and activity (B) in mouse skin tissues. DBA/2 mice were used. Shaved mouse skin received a single application of 100 nM DMBA. Two weeks later, the skin received a single application of 4 μg TPA. Twenty-four hours later, skin tissues were collected and used. GAPDH served as the loading control. Experiments were repeated three times and a representative result is shown. *P < 0.05 compared with the control (DMSO treatment).

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Isocitrate dehydrogenase 1 knockdown enhances, whereas isocitrate dehydrogenase 1 overexpression suppresses skin cell transformation

To detect whether IDH1 downregulation contributes to skin cell transformation, siRNA to IDH1 or control siRNA was transfected into JB6 P+ cells. After incubation for 24 h, cells were collected and soft agar assays were performed. TPA induced cell transformation and knockdown of IDH1 (confirmed by western blot analysis; Fig. 5A) slightly increased colony formation, and enhanced TPA-induced tumorigenicity of JB6 cells (Fig. 5A,B). Conversely, when IDH1 expression was elevated via gene transfection (Fig. 5C), TPA-induced cell transformation was greatly reduced (Fig. 5D), further suggesting that IDH1 can inhibit tumor promotion.

image

Figure 5. Knockdown of isocitrate dehydrogenase (IDH) enhanced whereas overexpression of IDH1 suppressed TPA-induced skin cell transformation. JB6 P+ cells were used. Expression levels of IDH1 were detected in siRNA-transfected (A) or IDH1-transfected (C) cells using western blot analysis. GAPDH served as the loading control. Soft agar colony formation assay was performed using siRNA-transfected (B) or IDH1-transfected (D) cells. The final concentration of TPA was 5 nM. Experiments were repeated three times and a representative result is shown. *P <0.05 compared with control/DMSO group; #P < 0.05 compared with control/TPA group.

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Manganese superoxide dismutase overexpression prevents decreases in isocitrate dehydrogenase 1 expression in dimethylbenz[α]anthracene/TPA-treated mouse skin epidermal tissues

To investigate whether carcinogen-induced oxidative stress contributes to IDH1 downregulation, MnSOD transgenic mice and their wild-type littermates were treated with DMBA and TPA. Our previous studies have demonstrated that MnSOD overexpression suppresses DMBA/TPA-induced skin tumor formation, which is mediated, at least in part, by reducing DMBA/TPA-induced oxidative stress.[6] DMBA/TPA treatment suppressed IDH1expression in non-transgenic mice, whereas expression levels of IDH1 in MnSOD overexpression mice were not reduced after 24 h TPA treatment (Fig. 6A). IDH2 expression remained at similar levels between non-transgenic and MnSOD transgenic, and between DMSO and DMBA/TPA treatment. However, decreased IDH1 expression did not lead to decreased levels of α-ketoglutarate, the product of IDH1 (Fig. 6B). Nevertheless, mitochondrial respiration was decreased by DMBA/TPA treatment in non-transgenic mice, not in MnSOD overexpression mice (Fig. 6C).

image

Figure 6. Effects of manganese superoxide dismutase (MnSOD) overexpression on isocitrate dehydrogenase (IDH) expression (A), α-ketoglutarate levels (B) and mitochondrial respiration (C) after DMBA/TPA treatment in mouse skin epidermal tissues. MnSOD transgenic mice (TgH, in C57BL/6 background) and their non-transgenic littermates (nTg) were used. Mice received a topical application of DMBA (100 nM) and, 2 weeks later, received a single application of 4 μg TPA. Twenty-four hours later, skin tissues and skin cells were stripped and used for these studies. GAPDH served as the loading control. *P < 0.05 compared with its own control (DMSO treatment).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure statement
  8. References

Wild-type IDH converts isocitrate to α-KG, which activates downstream dioxygenases. Therefore, further insight into the normal function of IDH1 and IDH2 and their effects on downstream metabolism-regulated signal transduction pathways in IDH1 and IDH2-dysregulated cancers has therapeutic potential. However, several important questions need to be answered:

  1. At what stage of cancer development does the functional change of IDH1/2 occur?
  2. What is the role of IDH1/2 in early carcinogenesis?
  3. Does it play an important role in chemoprevention?

Based on our results, both UV irradiation (in vitro) and TPA (in vitro and in vivo) cause downregulation of IDH1; and knockdown of IDH1 enhances skin cell transformation, suggesting that IDH1 may suppress tumor promotion during early stage skin tumorigenesis. Importantly, no mutation in IDH1 has been detected in non-melanoma skin cancer.[11] Interestingly, mitochondria localized IDH2 is not downregulated at this early stage of tumorigenesis; however, downregulation of IDH1 is associated with decreased mitochondrial respiration. Our speculation is that downregulation of IDH1 might be connected to the cytosolic metabolic shift (e.g. glycolysis), which eventually impacts mitochondrial respiration.

How does IDH1 downregulation contribute to tumorigenesis? α-KG, the enzymatic product of IDH, is a known inhibitor of HIF-1α because of its effect as a cofactor on the activity of prolyl hydroxlases that increase the turnover of HIF-1α. HIF-1α contributes to skin tumorigenesis,[12] and is markedly increased in skin epidermal hyperplasia.[13] Because IDH1 produces α-KG, a HIF-1α inhibitor, one possible mechanism of action might be that IDH1 downregulation leads to activation of HIF-1α signaling.

Our results did not reveal a decrease in intracellular levels of α-KG in TPA-treated mouse skin. Although wild-type IDH1 activity is likely compromised, α-KG can be alternatively produced via glutamine; the latter is known to take place at a higher rate in cancer cells.

This study highlights the importance of metabolic changes during early stage tumorigenesis. IDH1 downregulation is associated with our previous observations that mitochondrial membrane potential and complex activities are decreased upon tumor promoter treatment.[1] In addition, IDH1 downregulation is accompanied by pyruvate kinase M2 upregulation,[2] suggesting that complex and collaborative metabolic changes occur at the early stage of cancer development.

How does tumor promoter TPA and UVC irradiation suppress IDH1? IDH1 regulation is largely unknown; although enzymatically, the levels of substrates and products are able to modulate its activity. Because TPA and UV irradiation, as well as other oncogenic activation events, generate reactive oxygen species (ROS), a causative factor of tumorigenesis, we speculate that IDH1 is inactivated by ROS. It has been shown that ROS can be prevented by elevated MnSOD levels and MnSOD overexpression has been shown to suppress tumorigenesis.[6, 14] Our results demonstrate that overexpression of MnSOD not only prevents carcinogen-induced decreases in IDH1 expression and activity, but also upregulates IDH1 after carcinogen treatment. Similar results were also observed in promotion-resistant JB6 P– cells. Consistently, MnSOD expression and activity in P– cells are higher than in P+ cells.[15] Although localized in mitochondrial matrix, increased MnSOD activity can also reduce extra-mitochondrial ROS by maintaining mitochondrial respiration.[16] Therefore, oxidative stress might play an important role in inactivating IDH1 during early tumorigenesis. The exact mechanism of action needs to be determined in future studies.

In summary, our study provides new insight into the role of IDH1 in tumor promotion, which reveals that IDH1 may suppress cell transformation and tumor promotion in early skin tumorigenesis. Therefore, inducing IDH1activity may serve as a novel chemopreventive strategy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure statement
  8. References

The authors wish to thank Dr Terry Oberley at the University of Wisconsin for providing us with the cell lines, Dr Lynn Harrison in the Department of Molecular and Cellular Physiology at LSUHSC-Shreveport, the National Institutes of Health [NS057656 to S. N. W.], and the Michael J. Fox Foundation (R. L. K.).

Disclosure statement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure statement
  8. References

The authors have no conflict of interest to declare.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure statement
  8. References