Journal of Life Sciences Volume 7 Number (4)
J LS
Journal of Life Sciences
Volume 7, Number 9, September 2013 (Serial Number 65)
Contents
Biochemistry and Molecular Biology
913 Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling in
Paracoccidioides brasiliensis Infection
Elis Araujo Morais, Estefânia Mara do Nascimento Martins, Viviane Cristina Fernandes, Íria Gabriela Dias dos Santos, Gerluza Aparecida Borges Silva, Dawidson Assis Gomes and Alfredo Miranda de Goes
Induction of Apoptosis in HeLa Cells by Methanolic Extract of Litsea cubeba Fruit Residue from Essential Oil Extraction
Trisonthi Piyapat, Miyagawa Kana and Tamura Hirotoshi 935
Pros and Cons in Therapeutic Evaluation of Paraoxonase 1 in Nerve Agent Toxicity
Manojkumar Valiyaveettil, Yonas Alamneh, Bhupendra P. Doctor, Alfred M. Sciuto and Madhusoodana P. Nambiar
Efficacy and Safety of Dif1stat® for the Treatment of Secondary Dyslipidemia in Chronic Kidney Disease
Fabio Mazza and Claudia Stefanutti
950 Mesenchymal Stem Cells Express Low Levels of Cardiomyogenic Genes and Show Limited
Plasticity towards Cardiomyogenic Phenotype
Juliana Lott de Carvalho, Danilo Roman Campos, Maira Souza Oliveira, Jader Santos Cruz, Nathalia Martins Breyner, Dawidson Assis Gomes and Alfredo Miranda de Goes
965 Morphological Evaluation of Adhesion and Proliferation of Osteoblast Like Cells Grown on
Gelatin/Genipin Scaffold
Gabriella Teti, Adriana Bigi, Monica Mattioli-Belmonte, Roberto Giardino, Milena Fini, Antonio Mazzotti and Mirella Falconi
971 Genetic Diversity among Some Sheep Breeds in Sulaimani Governorate Using RAPD-PCR Technique
Yousif Muhammad Salih Al-Barzinj and Muqdad Kamal Ali
Agricultural Science and Ecology
Contribution of Different Agronomic Practices and the Fungus Beauveria bassiana on the Coffee Berry Borer
Alberto Fernández Turro, Ernesto Castañeda Hidalgo, Francisco Simón Ricardo, Everton Kort Kamp Fernandes, Juana Iris Durán Cos and Odisa Baqué Fuentes
Effect of Water Stress in Grape Berries Cabernet Sauvignon (Mendoza, Argentina) during Four Years Consecutives
Leonor Deis and Juan Bruno Cavagnaro
1002 Long-Term H 2 O and CO 2 Trends in Conifer Disc Tree Rings and Meteorological Parameters
Ageev Boris Grigor’evich, Gruzdev Aleksandr Nikolaevich, Bondarenko Svetlana Leonidovna and Sapozhnikova Valeria Aleksandrovna
1009 Qualitative and Environmental Aspects Study of Water Distributed in West Algeria: The Case of
Sidi Bel Abbés City
Habib Meliani, Mohamed Benyahia and Mohamed Ali Bouzidi
September 2013, Vol. 7, No. 9, pp. 913-927
Journal of Life Sciences, ISSN 1934-7391, USA DAVID PUBLISHING
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling in Paracoccidioides brasiliensis Infection
1 2 Elis Araujo Morais 1 , Estefânia Mara do Nascimento Martins , Viviane Cristina Fernandes , Íria Gabriela Dias dos Santos 3 , Gerluza Aparecida Borges Silva 3 , Dawidson Assis Gomes 1 and Alfredo Miranda de Goes 1
1. Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901, Brazil 2. Albert Einstein College of Medicine, Yeshiva University, New York, NY 10033, USA 3. Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901,
Brazil
Received: March 14, 2013 / Accepted: June 14, 2013 / Published: September 30, 2013.
Abstract: VEGF (Vascular endothelial growth factor) signaling is critical for endothelial cell survival and maintenance of the vasculature. Deregulation of VEGF signaling contributes to the physiopathology of many diseases. However, the ways in which infection with Paracoccidioides brasiliensis affects VEGF signaling and the influence of immunization with rPb27 (recombinant protein Pb27) on VEGF signaling have not yet been studied. Animals were immunized with rPb27 and subsequently infected with a virulent strain of P. brasiliensis. The fungal load was evaluated by measuring CFU (colony-forming unit) and histology was performed to evaluate the inflammatory reaction. At the two time points analyzed, the PC (positive control) and TREAT (treated) animals had decreased levels of pulmonary VEGF compared to basal levels. However, in the immunized (Pb27) and treated mice (Pb27 + TREAT), VEGF expression remained unchanged after infection. In the case of VEGFR-2, the Pb27 and Pb27 + TREAT groups showed increased levels of expression. Regarding the levels of the eNOS enzyme, only the Pb27 group did not reduce the expression levels relative to baseline. The immunization with rPb27 kept VEGF signaling, NO production and increased VEGFR-2 expression, after infection with P. brasiliensis. Thus, the authors infer that immunization with rPb27 protects mice from the disruption of VEGF signaling in Paracoccidioides brasiliensis infection.
Key words: rPb27, VEGF, VEGFR, eNOS, Paracoccidioides brasiliensis, paracoccidioidomycosis, inflammatory infiltrate.
1. Introduction fungus [5, 6]. The resulting systemic disorder primarily involves the lungs and disseminates to other
PCM (Paracoccidioidomycosis) is a systemic organs and systems, but the lung is commonly the mycosis caused by the dimorphic fungus most affected organ [7, 8]. In the lung, P. brasiliensis Paracoccidioides brasiliensis. The disease is converts to its parasitic yeast form and may cause considered to be the most prevalent systemic fungal destruction of the alveoli and bronchial tree. The infection in Brazil and is present in many Latin infection may also lead to chronic pulmonary American countries [1-4]. P. brasiliensis infection is insufficiency resulting from the development of acquired upon the inhalation of airborne spores fibrosis. This framework changes over time and can derived from the saprophytic mycelium form of the
be consolidated into a number of chronic
Corresponding author: Alfredo Miranda de Goes, Ph.D., granulomatous processes, resulting in hypoxemia and associate professor, research fields: biochemistry, immunology,
ventilatory dysfunction [9, 10].
biomaterials and tissue engineering. E-mail: goes@icb.ufmg.br.
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Antifungal therapy is required to control the VEGFR2-mediated downstream signaling activate Paracoccidioidomycosis. Extended periods of therapy
eNOS, resulting in the release NO [30], which is are usually required for a good clinical response [11].
considered responsible for maintaining the The conventional treatment of PCM relies on homeostasis of the airways. The mechanism by which sulfonamides, amphotericin B and azole derivatives.
NO promotes cell survival has been directly linked to However, new and more effective alternatives for
increased neovascularization and cell migration [21, PCM treatment have been studied, and antigens used
for immunization showed protection against P. Deregulated VEGF signaling is found in the brasiliensis [12]. Among the antigens of P.
physiopathology of diseases such as asthma, brasiliensis, the recombinant protein rPb27, which has
emphysema, pulmonary hypertension and acute been studied, is especially remarkable because it has
respiratory distress syndrome [24, 33]. However, the shown significant potential for the induction of a
effect of P. brasiliensis on the expression of VEGF protective immune response against PCM in previous
and VEGFR-2 has not been studied. Therefore, in this reports [12-18].
study, authors evaluated the effect of P. brasiliensis The maintenance of the microvasculature in the
infection and the influence of rPb27 immunization lung is critical for gas exchange, integrity of the
with or without antifungal therapy on the expression alveolar structure and tissue repair [19]. VEGF
of VEGF, VEGFR-2 and eNOS after infection. (vascular endothelial growth factor) is an important
2. Materials and Methods
regulator of vascular development [20-22], and lung microvascular endothelial cells produce significant
2.1 Animals
amounts of VEGF, which contributes to endothelial Adult male BALB/c mice (6-8 weeks old) were maintenance and homeostasis within the lungs in an
purchased from the vivarium of ICB-UFMG (Belo autocrine manner [23, 24]. VEGF is a highly
Horizonte, MG, Brazil) and divided into six groups of vascular-specific signaling molecule and its biological
five animals each: NC (negative control): group activity is dependent on its interaction with specific
without any intervention; PC (positive control): receptors (VEGF-R1, 2, and 3), which are expressed
infected only; ADJ (adjuvant): injected with not only by endothelial cells but also by activated
Corynebacterium parvum (R.V Special Manipulations, macrophages and alveolar type II epithelial cells.
Rio de Janeiro, RJ, Brazil) and Al(OH) 3 Endothelial survival is mediated via VEGFR-2/kinase
(Pesamar-Sanosi-synthelado, RJ, Brazil) as adjuvant; insert KDR (domain-containing receptor) [25-27].
immunized (Pb27): immunized with rPb27; treated Several lines of investigation have revealed that the
and immunized (Pb27 + TREAT): immunized with inhibition of the VEGF-VEGFR-2 signaling pathway
rPb27 and treated with fluconazole; and TREAT results in decreased lung alveolarization and arterial
(treated): infected and treated with fluconazole. The density [20].
specifications of the groups and the interventions eNOS (endothelial nitric oxide synthase) plays a
made in the animals during the experiment are shown central role in maintaining vascular integrity [20] and
in Table 1. The experiments were performed three studies have demonstrated that eNOS/NO plays an
times to determine the repetition of the results. important role in many VEGF-induced processes [28].
2.2 P. brasiliensis Strain
NO (nitric oxide) is also known to be an essential mediator of endothelial cell migration and
Virulent P. brasiliensis, strain Pb18, was VEGF-induced angiogenesis [29]. VEGF and maintained in yeast form in YPD agar medium [0.5%
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Table 1 Experimental groups.
Groups Immunization Infection Treatment NC --- PC -
3 10 5 cells of Pb18
ADJ
C. parvum + Al(OH) 3 3 10 5 cells of Pb18
Pb27
C. parvum + Al(OH) 3 + Pb27
3 10 5 cells of Pb18
fluconazole TREAT -
Pb27 + TREAT
C. parvum + Al(OH) 3 + Pb27
3 10 5 cells of Pb18
3 10 5 cells of Pb18
fluconazole
yeast extract, 0.5% peptone, 1.5% D-glucose, 1.5%
2.5 Antifungal Therapy
agar, pH 7.0] at 36 °C. The viability of fungal After 30 days of infection, animals were treated suspensions was determined by staining with Janus every day for one month. The group of mice received Green B vital dye method (Merck. Darmstadt, doses of 10 mg/kg of fluconazole (Mantena Germany) and was always higher than 90%. The laboratories limited) every 24 h. All drug virulence of the human isolate was checked in each administration was intraperitoneally. experiment by infecting intratracheally BALB/c mice
and recovering the yeast cells from their organs [12].
2.6 Lung CFU Measurement from Intratracheally Infected BALB/c Mice Immunized with rPb27 and/or
2.3 Immunization
Treated with Fluconazole
Groups of male BALB/c mice were immunized by The analysis of organ CFUs was performed 30 and the subcutaneous injection of 50 µg of rPb27
90 days post infection (dpi). The organs were removed, (GenBanK-NCBI accession number AAC49615) in
weighed, homogenized in PBS (pH 7.2) and plated on the presence of 100 µg of C. parvum (R.V Special
solid brain heart infusion medium (BHI, Difco
Laboratories, Detroit, MI, USA) supplemented with animals were boosted three times at fifteen-day
Manipulations) and 1 mg Al(OH) 3 as adjuvant. The
4% fetal calf serum and 5% P. brasiliensis spent intervals with the same amount of antigen, seven days
culture medium (Pb18) as growth factors. Gentamicin after last immunization the challenge infection was
was added at a concentration of 40 mg/L. The plates performed [12, 13, 15].
were incubated at 36 °C, and the CFUs were counted
2.4 Intratracheal Infection after 20 days of incubation. The results are expressed as the log 10 of the number of CFUs of viable yeast
Male BALB/c mice were inoculated intratracheally cells of P. brasiliensis per gram of tissue per mouse in with 3 5 10 viable yeast cells of virulent P. each experimental group (log 10 /g).
brasiliensis, which was grown on YPD-agar and
2.7 Lung Fixation, Histology and IHC suspended in sterile PBS as described by Fernandes et
(Immunohistochemistry)
al. [12]. Briefly, mice were anesthetized intramuscularly with 40 µL of a solution containing
The lungs were excised, fixed in 4% 57% ketamine (Dopalen, Vetbrands, Brazil) and 43%
paraformaldehyde and embedded in paraffin. Sections xylazine (Dopaser, Laboratório Calier do Brazil
(5 µm) were obtained and used for histopathology and LTDA, Brazil). After approximately 10 min, their
IHC. In the histology, the sections were stained with necks were extended, the trachea was exposed at the
hematoxylin-eosin and examined microscopically. The level of the thyroid, and yeast cells were injected in 50
inflammatory infiltrate area was measured 90 days µL of PBS using a 30-gauge needle. The incisions
after the challenge infection using a MacBiophotonics were sutured with 4-0 silk [13].
Image J software analyzer. Three sections from each
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
mouse were analyzed using a microscope with the 2 anti-VEGF (SC152- Santa Cruz Biotechnology), magnification; sections were then scanned using JVC
rabbit anti-VEGFR-2 (Ab39638-Abcam, Cambridge, TK-1270/RGB micro camera. Using a digital pad, in
MA, USA) and rabbit anti-GAPDH (SC 25778-Santa each section, the total lung area and the area of intense
Cruz Biotechnology, Inc, CA, USA). After washing, inflammatory infiltrate were measured (areas with
the bound antibody was detected using an anti-rabbit increased cellularity but without obstruction of alveoli
antibody (1:10.000 dilutions) conjugated to were not measured), and the results were expressed in
peroxidase (Sigma-Aldrich, St Louis, MO, USA).
Blots were visualized by enhanced chemiluminescence percentage of area with inflammatory infiltrate was
square millimeters (mm 2 ). From these values, the
and quantitatively analyzed using Image software. calculated.
2.9 cDNA Synthesis and Quantitative Real Time PCR With regard to the IHC, briefly, the lung sections
(qPCR) Analysis
were deparaffinized in xylene and rehydrated in alcohol, and then the endogenous peroxidase was inhibited with
Total RNA was extracted using TRIzol (Life 10% hydrogen peroxide. The sections were stained with
Technologies, Grand Island, NY, USA). The RNA
a primary antibody overnight at 4 °C. Immunodetection was extracted from the samples according to the was performed using a goat anti-rabbit IgG conjugated
manufacturers. The samples were treated with to peroxidase (Sigma- Aldrich) and 3,3’- RNase-free DNase (Promega, Madison, WI, USA) diaminobenzidine (DAB) (K3468- DAKO, Glostrup,
and the synthesis of cDNA was performed with Denmark). The nuclei were counterstained with Harris
H Minus First Strand cDNA Synthesis hematoxylin solution. For negative control of the each
RevertAid TM
Kit (Fermentas Life Sciences Inc, Ontario, Canada) section, the primary antibody was omitted from one
using an oligo(dT) adapter primer. section of each of the samples.
The reactions were performed in triplicate using SYBR Green PCR Master Mix (Applied Biosystems,
2.8 Lung Tissue Homogenate and WB (Western Blot) Carlsbad, CA, USA) according to the manufacturer’s
Lung tissue (0.1 g) was homogenized in 1 mL of instructions. The reactions were carried out in a lysis buffer (100 mM NaCl, 50 mM Tris base, 5 mM
96-well plate using an ABIPrism 7900 (Applied EDTA, 50 mM sodium pyrophosphate decahydrate,
Biosystems, Carlsbad, CA, USA). Gene expression 1% NP-40, 0.3% TRITON, sodium deoxycholate
- was quantified using the comparative Ct (2 ΔΔCt ) 0.5%, 20 mM sodium fluoride and a complete
method and β-actin were used as endogenous control protease cocktail inhibitor (Sigma-Aldrich)). The
genes for data normalization, and the results represent homogenate was kept on ice for 45 min and then
the relative quantification of gene expression centrifuged at 16.500 ×g for 15 min at 4 °C and the
normalized to the constitutively expressed β-actin supernatant was stored at -20 °C until further use.
gene. The NC (negative control) was used to obtain The protein concentrations were determined using
the basal levels of expression [35]. the Bradford method [34]. The protein samples (30
The full sequences of genes used for the primer µg) were separated on a 6-15% gradient SDS-PAGE
confection were withdrawals of NCBI with gel by electrophoresis and transferred onto a PVDF
identification number (Gene ID): eNOS (18127), (polyvinylidene fluoride) membrane (Millipore, MA,
VEGF (22339), VEGFR-2 (16542) and β-ACTIN USA). The membranes were blocked with 5% nonfat
(11461). The qPCR amplification was carried out milk and incubated with the relevant primary
using the primers (Prodimol Biotecnologia S/A, Belo antibody. The primary antibodies used were rabbit
Horizonte, MG, Brazil) listed in Table 2.
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Table 2 Primer sequences used for quantitative qPCR.
Primer Sequence 5 ′-3′ (Gene ID) Forward
Forward β-ACTIN GGA TGC AGA AGG AGA TTA CTA CTG
2.10 Statistical Analysis TREAT groups showed a significant reduction in the CFU recovered from the lungs compared to the PC
The statistical analysis about determine the and TREAT groups. No fungal colonies were significance of the data was performed using Prism recovered from the ADJ and Pb27 groups, 90 days
5.0 software (GraphPad, CA, USA). The data were after infection, and the mice from Pb27 + TREAT normally distributed, and the values obtained from the group had a 90% reduction in lung CFUs in different groups of mice were compared using a comparison to the PC and TREAT groups (Fig. 1B). one-way ANOVA (analysis of variance) with
Bonferroni post- tests. Data were considered statistically significant at P < 0.05.
2.11 Ethics Statement This work was approved by the CETEA/UFMG
(Committee on the Ethics of Animal Experimentation of Federal University of Minas Gerais) under permit number: 203/2010 and was carried out in strict accordance with the regiment of ethics commission on the use of Animals. All surgery was performed with use of anesthesia, and all efforts were made to minimize suffering.
3. Results
3.1 Fungal Loads in Lungs of Mice after Challenge Infection
The CFU analysis performed 30 and 90 dpi showed
significantly fewer fungi which recovered from the
Fig. 1 Protection assay during experimental PCM.
lungs of animals (CFU) immunized with rPb27. In
Colony-forming units (expressed as Log/g) detected in the lungs of mice 30 (A) and 90 (B) dpi. Each bar represents the
these animals, there was a 75% reduction in the CFUs
mean and standard deviation of five animals per group and
30 days post infection compared to the PC and ADJ
repeated three times (n = 15). The * indicates a significant
groups (Fig. 1A).
difference (P < 0.05) in comparison with NC. The groups
After 90 days of infection, all groups showed represented in the figure are: NC (negative control) PC
(positive control), ADJ (adjuvant), immunized (Pb27),
reduction of the fungal load in comparison with the
TREAT (treated), and immunized and treated (Pb27 +
results of 30 dpi. However the ADJ, Pb27 and Pb27 +
TREAT).
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
3.2 Lung Histology significant reduction in VEGF expression after 30 days of infection with P. brasiliensis in PC and ADJ
The histology of the lungs was performed animals compared with NC. However, there was no qualitatively for the demonstration of inflammatory decrease in VEGF expression in the Pb27 group (Fig. 4). reactions. At the two time points investigated, the After 90 days of infection, animals in the PC, lungs of animals of PC and TREAT showed multiple TREAT and ADJ groups had reduced VEGF foci of inflammation, containing yeast cells of P. expression, and again, there was no decrease in VEGF brasiliensis. The mice of Pb27 + TREAT group expression in the immunized mice (Pb27 and Pb27 + presented compact infiltrates and showed a TREAT) (Fig. 4B). The IHC experiments confirmed considerable reduction in the size of lesions after 90 qualitatively the results obtained with the WB,
days of infection in comparison with PC (Figs. 2 and showing lower levels of VEGF expression in PC, ADJ
3). The Pb27 group, at the two time points investigated, showed limited areas with a compact cellular and TREAT animals compared to the NC and Pb27 infiltration that did not form into a considerable lesion
(Fig. 5A and 5B).
(Figs. 2 and 3).
3.4 VEGFR-2 Expression Levels in Mouse PCM To investigate the protective activity induced by
Model Lungs
vaccination with rPb27, the authors measured the extent of the inflammatory infiltrate, 90 days after
The authors also examined the effects of P. infection, using Image J software. Analysis of the PC
brasiliensis infection on VEGFR-2 expression in and TREAT groups showed that approximately 40%
mouse lungs IHC. After 30 days of infection, the NC of the area of the lungs had intense inflammatory
and the Pb27 groups showed more marked areas of the infiltrate. Among the groups the ADJ had greater area
VEGFR-2 expression than the PC group (Fig. 6A). of inflammatory infiltrate. The immunized groups
After 90 days of infection, Pb27 and Pb27 + TREAT (Pb27 and Pb27 + TREAT) had smaller areas of
groups showed higher levels of VEGFR-2 expression inflammatory infiltrate even the Pb27 group showed
in the lungs (Fig. 6B).
that only 10% of the field of lungs had intense Additionally, the authors also observed that the inflammatory infiltrate and with no significant relative mRNA expression of VEGFR-2 by qPCR differences with the NC. All other groups showed
after 30 and 90 dpi was significantly increased in the significant differences in the area of inflammatory
immunized groups (Pb27 and Pb27 + TREAT) infiltrate compared with the NC. Comparison with the
compared to the other groups and basal levels. The PC showed that the immunized groups (Pb27 and
Pb27 group had higher levels of VEGFR-2 mRNA Pb27 + TREAT) had significant lower areas of
expression compared to the other groups (Fig. 7A and inflammatory infiltrate. In contrast, the TREAT group
B).
did not show statistic differences with the PC group.
3.5 The eNOS Expression Levels in the Lungs of Mice These results confirm the efficiency of Pb27 in
Infected with P. brasiliensis
modulating the inflammatory response. VEGF-induced VEGFR-2 downstream signaling
3.3 VEGF Expression Levels in Mouse PCM Model leads to the activation of eNOS, a key enzyme linked
Lungs to endothelial survival and function. Therefore, the
To investigate the expression of VEGF in mouse effect of PCM on mRNA eNOS levels was assessed lungs infected with P. brasiliensis, WB and IHC were
by qPCR. After 30 days of infection, the level of performed. The results obtained by the WB showed a
eNOS in the lungs of the PC group was significantly
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Fig. 2 Histological analysis of the lungs of mice subjected to 30 days of P. brasiliensis infection. After 30 days of infection with P. brasiliensis, mouse lungs were stained with HE and analyzed for histology. The representative figure of each group is
shown with low magnification (4 ) in the left panel and higher magnification (10) in the right of the panel. The groups are as
follows: (NC) negative control (A), (PC) positive control (B), (ADJ) adjuvant (C), and (Pb27) immunized (D). The black
arrows indicate the location of fungal cells in the tissue.
reduced compared to basal levels and to those found levels of eNOS similar to baseline; in the other groups, in the Pb27 group (Fig. 8A). However, 90 days after
the mRNA expression of eNOS was significantly infection, only the immunized group had expression
lower than basal levels (Fig. 8B).
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Fig. 3 Histological analysis of the lungs of mice subjected to 90 days of P. brasiliensis infection. After 90 days of infection with P. brasiliensis, mouse lungs were stained with HE and analyzed by histology. The representative figure of each group is
shown with low magnification (4 ) in the left of the panel and high magnification (10) in the right of the panel. The groups
are as follows: (NC) negative control (A), (PC) positive control (B), (ADJ) adjuvant (C), (TREAT) treated (D), (TREAT + Pb27) immunized and treated (E), and (Pb27) immunized (F). The black arrows indicate the location of fungal cells in the tissue.
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Fig. 4 Western blot of VEGF expression. Representative WB of the lung tissue homogenate shows VEGF expression, (A) 30 dpi and (B) 90 dpi. The histograms represent the mean ± SE (pool of five animals per group and repeated three times (n = 15)
of VEGF expression. The * indicates that P < 0.05 compared to the positive control animals. The # indicates that P < 0.001
compared to the negative control and Pb27 animals.
Fig. 5 Immunohistochemical for VEGF expression in mouse lungs infected with P. brasiliensis. IHC to evaluate VEGF expression (A) 30 dpi and (B) 90 dpi. The microscopy was performed at 10x magnification. The groups are as follows: (NC) negative control, (PC) positive control, (ADJ) adjuvant and (Pb27) immunized. The representative figure of each group is shown and the microscopy was performed at 4x magnification. The groups are as follows: (NC) negative control; (PC)
positive control; (ADJ) adjuvant; (Pb27) immunized (TREAT) treated; and (Pb27+TREAT) immunized and treated.
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Fig. 6 Immunohistochemical analysis of VEGFR-2 expression in mouse lungs. IHC to evaluate VEGF expression (A) 30 dpi and (B) 90 dpi. Microscopy was performed at 10x magnification. The representative figure of each group and the microscopy was performed at 4x magnification. The groups are as follows: (NC) negative control; (PC) positive control; (ADJ) adjuvant; (Pb27) immunized; (TREAT) treated; and (Pb27 + TREAT) immunized and treated.
4. Discussion
second time point (90 dpi), the immunization reduced the fungal load to undetectable levels. These results
The rPb27 was first described by McEwen, et al. are consistent with the results obtained by Reis et al.
[36] and has demonstrated great potential in the [15] and confirm the ability of the rPb27 protein to
immunodiagnostics of PCM [13, 16, 36-38]. Previous protect mice against PCM. The combined
immunization experiments in mice showed that Pb27 administration of rPb27 and fluconazole also resulted
was able to provide protection against a challenge in a significant reduction in the fungal load, which infection in both prophylactic and therapeutic was 90% compared to the PC. However, the fungal
protocols and it has therefore become a strong vaccine load was not reduced in the TREAT group, probably candidate for PCM [12-15, 36]. In this work, the
due to the short treatment time.
authors confirmed efficacy of the protective effect of The fungal load was also at undetectable levels for rPb27 immunization in prophylactic protocol against
the ADJ group 90 dpi. The adjuvant is an agent that PCM and reported the effect of the immunization on
stimulates the immune system and increases the the expression of VEGF, VEGFR and eNOS.
response to a vaccine without having any specific The fungal load was examined in the lungs of
antigenic effect itself. Some adjuvant are often used to infected mice and the quantification of CFUs in the
modify the effects of an immunization protocol by lungs showed a protective effect of rPb27
stimulating the immune response more vigorously and immunization against infection. It was observed that
thus providing increased immunity to a particular immunized mice had a significant decrease in lung
disease. The immune response has evolved to CFUs when compared with controls, 30 dpi, the
recognize antigens of P. brasiliensis and the presence immunization reduced the CFU number by 75% in
of an adjuvant in conjunction with this fungal comparison to control groups (PC and ADJ). At the
infection which can greatly increase the innate
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Fig. 7 Relative expression of VEGFR-2 mRNA in mouse Fig. 8 Relative expression of eNOS mRNA in mouse lungs lungs infected with P. brasiliensis as assessed by qPCR.
infected with P. brasiliensis as assessed by qPCR. eNOS VEGFR-2 mRNA expression (A) 30 dpi and (B) 90 dpi. The
mRNA expression (A) 30 dpi and (B) 90 dpi. The histograms represent the mean ± SE of the VEGFR-2
histograms represent the mean ± SE of the eNOS expression (of five animals per group and repeated three
expression (of five animals per group and repeated three times (n = 15). The * indicates that P < 0.05 compared to
times (n = 15). The * indicates that P < 0.05 compared to the negative control animals. The NC (negative control) was
the negative control animals. The NC (negative control) was used to obtain the basal levels of expression. The groups are
used to obtain the basal levels of expression. The groups are as follows: (NC) negative control; (PC) positive control;
as follows: (NC) negative control; (PC) positive control; (ADJ) adjuvant; (Pb27) immunized; (TREAT) treated; and
(ADJ) adjuvant; (Pb27) immunized; (TREAT) treated; and (Pb27 + TREAT) immunized and treated.
(Pb27 + TREAT) immunized and treated.
immune response to the antigen by augmenting the In this context the authors infer that in addition to activity of dendritic cells and macrophages, decreasing the CFUs in the lung, the immunization mimicking a natural infection.
reduce obstructions from airways after infection (with Thus, the adjuvant used in this work by itself may
lower intensity of inflammatory infiltrates and tissue have induced an inflammatory infiltrate, however, in
damage) and thus they confirm the protective capacity association with Pb27, it helps this protein to establish
of Pb27 showed by Reis et al. [15]. the protective effect and down modulation of the
The Pb27 + TREAT group had a reduced infiltrates inflammatory response showing lungs without fungal
in the lungs compared to the PC, however, the results cells and unobstructed airways with a preserved lung
obtained only with the protein were better. The parenchyma as well few inflammatory infiltrates.
combination of fluconazole with rPb27 protein may Based on that, we can infer that others adjuvant by
have interfered in the protective effects of the itself does not induce inflammatory reactions which
immunization. Furthermore, comparing the groups must be investigated.
that received some type of treatment (immunization
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
/fluconazole) the group with the largest lesions was was greater 30 dpi and similar 90 dpi. The TREAT the TREAT group.
group was similar to the PC at the two time points. The fluconazole antifungal therapy has significant
The biological effects of VEGF are mediated activity in human PCM and is used most commonly in
through its binding to specific receptors expressed on cases of central nervous system involvement by the
the cell surface [19]. The protein-tyrosine kinase fungus, nevertheless its use is not the most receptor FLk-1(VEGFR-2) is an important factor in recommended form of treatment against endothelial survival and the maintenance of the paracoccidioidomycosis, and the most recommended
vasculature [21, 22, 49]. In this work, the immunized therapy has been mainly itraconazole. Though, animals showed a significant increase in the expression recently fluconazole was associated with rPb27
levels of VEGFR-2 in comparison with PC animals. protein and had proven additive efficiency in
Studies have shown that VEGF-VEGFR-2 signaling therapeutic treatment of mice infected with P.
enhances the expression of eNOS, and eNOS brasiliensis [12], in this work the authors showed that
inhibitors disrupt NO production and significantly the association of this drug with the rPb27 on the
change the effects of VEGF. Thus, the improvement prophylactic protocol do not have the same additional
in endothelial function by VEGF expression is effect. Other antifungal agents should be tested in
associated with increased eNOS and NO release [28, association with the protein to obtain better results.
44]. It is also known that NO produced by eNOS is an VEGF is a signaling protein that stimulates
essential mediator of endothelial cell migration and angiogenesis and vascularization and is part of the
VEGF-induced angiogenesis [31, 50]. Because this system that restores oxygen to the tissues where blood
enzyme plays a central role in maintaining vascular circulation is inadequate [39-43]. Many lung diseases
integrity and is activated by VEGF [20, 44], authors are characterized by changes in the expression of this
determined the levels of lung eNOS after infection molecule [36, 42, 44], including damage caused by
with P. brasiliensis. The results allow us to suggest cigarette smoke [20-22, 45], pulmonary infection by
that P. brasiliensis infection alters or negatively Pseudomonas aeruginosa [46], asthma [47], modulates the expression of this enzyme and that
emphysema, pulmonary hypertension and acute immunization prevents changes in its expression after respiratory distress syndrome [24, 33]. Furthermore,
infection, whereas, among the groups studied only the recently VEGFR-2 was considered to be valuable
Pb27 not showed reduced levels of eNOS 30 dpi and predictive biomarkers in the development of ALI
90 dpi.
(acute lung injury) and ARDS (acute respiratory distress syndrome) [48]. In these diseases, the
5. Conclusion
expression of VEGF/VEGFR is reduced and lung In conclusion, the analysis of results supports the function is impaired.
hypothesis that P. brasiliensis infection alters In this study, the authors show that, as in the
pulmonary levels of VEGF-VEGFR-2 signaling, diseases mentioned above, PCM disrupted VEGF
consequently modifying the levels of eNOS, and thus expression in mouse lungs. This reduction was
most likely disrupts the endothelial cell functions. showed in all tests made in PC mouse 30 and 90 dpi.
Animals infected with P. brasiliensis have decreased The immunized (Pb27 and Pb27 + TREAT) group
expression of these molecules. However, showed that the immunization increases the VEGF
immunization with the recombinant protein Pb27, in expression if compared with the other groups. When
addition to reduces fungal load and inflammatory compared with the NC the levels of the expression
infiltrate in the lungs, maintain VEGF expression at
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
basal levels, increases VEGFR-2 expression, and
6 (2) (1993) 89-117. [8] M. Armas, C. Ruivo, R. Alves, M. Goncalves, L. Teixeira,
consequently, maintains eNOS expression at basal Pulmonary paracoccidioidomycosis: A case report with
levels 90 days after infection. Thus, we showed that high-resolution computed tomography findings, Rev. Port. immunization maintains key functions that are
Pneumol. 18 (4) (Jul-Aug 2012) 190-193. normally inhibited by infection with P. brasiliensis,
[9] S.H.M. Silva, A.L. Colombo, M.H.S.L. Blotta, F. Queiroz-Telles, A.B. Balthazar, J.D. Lopes, et al.,
and the expression of the VEGF, VEGFR-2 and eNOS Diagnosis of paracoccidioidomycosis by detection of
possibly can be related to the maintenance of the antigen and antibody in bronchoalveolar lavage fluids, structure of the lung, proliferation and migration of
American Society for Microbiology 13 (12) (2006) endothelial cells, pulmonary regeneration and
1363-1366.
F.C.D. Silva, T.I.E. Svidzinski, E.V. Patussi, C.P. homeostasis [20, 23, 24, 51]. Cardoso, M.M.D.O. Dalalio, L. Hernandes, Morphologic
Acknowledgments organization of pulmonary granulomas in mice infected
with Paracoccidioides brasiliensis, Am. J. Trop. Med. 80 This research was supported by Fundação de
(5) (2009) 798-804.
G. Visbal, G. San-Blas, J. Murgich, H. Franco, Amparo a Pesquisa do Estado de Minas Gerais
Paracoccidioides brasiliensis, paracoccidioidomycosis, (FAPEMIG), Conselho Nacional de Desenvolvimento
and antifungal antibiotics, Curr. Drug Targets Infect Científico e Tecnológico (CNPq), Coordenação de
Disord 5 (3) (2005) 211-226.
Aperfeiçoamento de Pessoal de Nível Superior [12] V.C. Fernandes, E.M. Martins, J.N. Boeloni, J.B. Coitinho, R. Serakides, A.M. Goes, Additive effect of
(CAPES) and Pró-Reitoria de Pesquisa da UFMG rPb27 immunization and antifungal therapy in (PRPQ). Thank Laboratory of Cellular and Molecular
experimental paracoccidioidomycosis, PLoS One 6 (3) Immunology (LICM).
V.C. Fernandes, J.B. Coitinho, J.M. Veloso, S.A. Araujo,
References
E.P. Pedroso, A.M. Goes, Combined use of Paracoccidioides brasiliensis recombinant rPb27 and
[1] R. Martinez, Paracoccidioidomycosis: The dimension of rPb40 antigens in an enzyme-linked immunosorbent the problem of a neglected disease, Rev. Soc. Bras. Med.
assay for immunodiagnosis of paracoccidioidomycosis, J. Trop. 43 (4) (2010) 480.
Immunol Methods 367 (1-2) (2011) 78-84. [2] M.R. Fortes, H.A. Miot, C.S. Kurokawa, M.E. Marques,
V.C. Fernandes, E.M. Martins, J.N. Boeloni, J.B. S.A. Marques, Immunology of paracoccidioidomycosis,
Coitinho, R. Serakides, A.M. Goes, The combined use of An. Bras. Dermatol. 86 (3) (2011) 516-524.
brasiliensis Pb40 and Pb27 [3] V.L. Calich, C.A. Vaz, E. Burger, Immunity to
Paracoccidioides
recombinant proteins enhances antifungal therapy effects Paracoccidioides brasiliensis infection, Res. Immunol.
in experimental paracoccidioidomycosis, Microbes Infect 149 (4-5) (1998) 407-417.
13 (12-13) (2011) 1062-1072.
[4] M.A. Shikanai-Yasuda, F.F. de Q. Telles, R.P. Mendes, [15] B.S. Reis, V.C. Fernandes, E.M. Martins, R. Serakides, A.L. Colombo, M.L. Moretti, Guidelines in
A.M. Goes, Protective immunity induced by rPb27 of paracoccidioidomycosis, Rev. Soc. Bras. Med. Trop. 39
Paracoccidioides brasiliensis, Vaccine 26 (43) (2008) (3) (2006) 297-310.
5461-5469.
[5] A. Pina, P.H. Saldiva, L.E. Restrepo, V.L. Calich, [16] L.S. Santos, V.C. Fernandes, S.G. Cruz, W.C. Siqueira, Neutrophil role in pulmonary paracoccidioidomycosis
A.M. Goes, E.R Pedroso, Profile of total IgG, IgG1, IgG2, depends on the resistance pattern of hosts, J. Leukoc Biol.
IgG3 and IgG4 levels in sera of patients with 79 (6) (2006) 1202-1213.
paracoccidioidomycosis: Treatment follow-up using [6]
A.H. Tavares, L.S. Derengowski, K.S. Ferreira, S.S. Silva, Mexo and rPb27 as antigens in an ELISA, Mem Inst C. Macedo, A.L. Bocca, et al., Murine dendritic cells
Oswaldo Cruz 107 (1) (2012) 1-10. transcriptional modulation upon Paracoccidioides
A.F. Marques, M.B. Silva da, M.A.P. Juliano, L.R. brasiliensis infection, PLoS Negl. Trop. Dis. 6 (1) (2012)
Travassos, C.P. Taborda, Peptide immunization as an 1459.
adjuvant to chemotherapy in mice challenged [7]
E. Brummer, E. Castaneda, A. Restrepo, intratracheally with virulent yeast cells of Paracoccidioidomycosis: An update, Clin Microbiol. Rev.
Paracoccidioides brasiliensis, Antimicrob Agents
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
in Paracoccidioides brasiliensis Infection
Chemother 50 (8) (2006) 2814-2819. [29] S.E. Michaud, S. Dussault, J. Groleau, P. Haddad, A. [18]
A.F. Marques, M.B. da Silva, M.A. Juliano, J.E. Munhõz, Rivard, Cigarette smoke exposure impairs VEGF-induced L.R. Travassos, C.P. Taborda, Additive effect of P10
endothelial cell migration: Role of NO and reactive immunization and chemotherapy in anergic mice
oxygen species, J Mol Cell Cardiol 41 (2) (2006) challenged intratracheally with virulent yeast of
275-284.
Paracoccidioides brasiliensis, Microbes Infect. 10 [30] T. Tanimoto, Z.G. Jin, B.C. Berk, Transactivation of (12-13) (2010) 1251-1258.
vascular endothelial growth factor (VEGF) receptor [19] N. Ferrara, T. Davis-Smyth, The biology of vascular
Flk-1/KDR is involved in sphingosine endothelial growth factor, Endocr. Rev. 18 (1) (1997)
1-phosphate-stimulated phosphorylation of Akt and 4-25.
endothelial nitric-oxide synthase (eNOS), J Biol Chem [20]
I. Edirisinghe, S.R. Yang, H. Yao, S. Rajendrasozhan, S.
277 (45) (2002) 42997-43001.
Caito, D. Adenuga, et al., VEGFR-2 inhibition augments [31] R.F. Filho, B. Zilberstain, Óxido nítrico: o simples cigarette smoke-induced oxidative stress and
mensageiro percorrendo a complexidade, Metabolismo, inflammatory responses leading to endothelial
síntese e funções, Revista da Associação Medica dysfunction, Faseb J 22 (7) (2008) 2297-2310.
Brasileira 46 (3) (2000) 265-271.
[21] J.A. Marwick, I. Edirisinghe, G. Arunachalam, C.S. [32] N. Kang-Decker, S. Cao, S. Chatterjee, J. Yao, L.J. Egan, Stevenson, W. Macnee, P.A Kirkham, et al., Cigarette
D. Semela, et al., Nitric oxide promotes endothelial cell smoke regulates VEGFR2-mediated survival signaling in
survival signaling through S-nitrosylation and activation rat lungs, J Inflamm (Lond) 7 (1) (2010) 11.
of dynamin-2, J Cell Sci 120 (Pt 3) (2007) 492-501. [22] J.A. Marwick, C.S. Stevenson, J. Giddings, W. MacNee,
G. Korpanty, E. Smyth, L.A. Sullivan, R.A. Brekken, K. Butler, I. Rahman, et al., Cigarette smoke disrupts
D.N. Carney, Antiangiogenic therapy in lung cancer: VEGF165-VEGFR-2 receptor signaling complex in rat
Focus on vascular endothelial growth factor pathway, lungs and patients with COPD: Morphological impact of
Exp Biol Med (Maywood) 235 (1) (2010) 3-9. VEGFR-2 inhibition, Am J Physiol Lung Cell Mol
[34] D.A.M. Zaia, C.T.B.V. Zaia, J. Lichtig, Determination of Physiol 290 (5) (2006) 897-908.
total protein by spectrophotometry: Advantages and [23] N.F. Voelkel, R.W. Vandivier, R.M. Tuder, Vascular
disadvantages of proposed methods, Quím Nova 21 (6) endothelial growth factor in the lung, Am J Physiol Lung
(1998) 787-793.
A. Giulietti, L. Overbergh, D. Valckx, B. Decallonne, R. [24] N.F. Voelkel, C. Cool, L. Taraceviene-Stewart, M.W.
Cell Mol Physiol 290 (2) (2006) 209-221.
Bouillon, C. Mathieu, An overview of real-time Geraci, M. Yeager, T. Bull, et al., Janus face of vascular
quantitative PCR: Applications to quantify cytokine gene endothelial growth factor: The obligatory survival factor
expression, Methods 25 (4) (2001) 386-401. for lung vascular endothelium controls precapillary artery
[36] J. McEwen, B.L Ortiz, A. García, A. Florez, S. Botero, A. remodeling in severe pulmonary hypertension, Crit Care
Restrepo, Molecular cloning, nucleotide sequencing, and Med 30 (5 Suppl) (2002) 251-256.
characterization of a 27-kDa antigenic protein from [25] Y. Abadie, F. Bregeon, L. Papazian, F. Lange, B.
Paracoccidioides brasiliensis, Fungal Genet Biol 20 (2) Chailley-Heu, P. Thomas, et al., Decreased VEGF
(1996) 125-131.
concentration in lung tissue and vascular injury during [37] M.M. Correa, A.M. Bedoya, M.P. Guerrero, J. Mendez, ARDS, Eur Respir J 25 (1) (2005) 139-146.
A. Restrepo, J.G. McKeen, Diagnosis of [26] M.J. Cross, Claesson-Welsh. FGF and VEGF function in
paracoccidioidomycosis by a dot blot assay using a angiogenesis: Signalling pathways, biological responses
recombinant Paracoccidioides brasiliensis p27 protein, and therapeutic inhibition, Pharmacological Sciences 22
Mycoses 50 (1) (2006) 41-47.
(4) (2001) 201-207. [38] B.L. Ortiz, S. Diez, M.E. Uran, J.M. Rivas, M. Romero, [27] N.C. Rivron, E.J. Vrij, J. Rouwkema, S.L. Gac, A. Van
V. Caicedo, et al., Use of the 27-kilodalton recombinant den Berg, R.K. Truckenmüller, et al., Tissue deformation
protein from Paracoccidioides brasiliensis in spatially modulates VEGF signaling and angiogenesis,
serodiagnosis of paracoccidioidomycosis, Clin Diagn Lab Proc Natl Acad Sci USA 109(18) (2012) 6886-6891.
Immunol 5 (6) (1998) 826-830.
[28] S. Ben-Quan, D.Y. Lee, H.F. Zioncheck, Vascular
H.F. Dvorak, L.F. Brown, M. Detmar, A.M. Dvorak, endothelial growth factor governs endothelial
Vascular permeability factor/vascular endothelial growth nitric-oxide synthase expression via a KDR/Flk-1
factor and the significance of microvascular receptor and a protein kinase C signaling pathway, The
hyperpermeability in angiogenesis, American Journal of Journal of Biological Chemistry 274 (46) (1999)
Pathology 237 (1999) 97-132.
33057-33063. [40] H.J.H. Marti, Vascular endothelial growth factor, in:
Immunization with rPb27 Protects Mice from the Disruption of VEGF Signaling
927
in Paracoccidioides brasiliensis Infection
Bioscience; MCBDIL Database [Internet], Landes mouse lung, J Cell Physiol. (2012), doi: Bioscience, Madame Curie, Bioscience Austin (TX),
10.1002/jcp.24140.
2000. [47] M. Balantic, M. Rijavec, M. Skerbinjek Kavalar, S. [41] H.J.H. Marti, M. Bernaudin, A. Bellail, H. Schoch, M.
Suskovic, M. Silar, M. Kosnik, et al., Asthma treatment Euler, E. Petit, et al., Hypoxia-induced vascular
outcome in children is associated with vascular endothelial growth factor expression precedes
endothelial growth factor A (VEGFA) polymorphisms, neovascularization after cerebral ischemia, American
Mol Diagn Ther 16 (3) (Jun 1, 2012) 173-180. Journal of Pathology 156 (3) (2000) 965-976.
[48] T. Wada, S. Jesmin, S. Gando, Y. Yanagida, A. Mizugaki, [42]
G. Neufeld, T. Cohen, S. Gengrinovitch, Z. Poltorak, S.N Sultana, et al., The role of angiogenic factors and Vascular endothelial growth factor (VEGF) and its
their soluble receptors in acute lung injury (ALI)/acute receptors, The FASEB Journal 13 (1999) 9-22.
respiratory distress syndrome (ARDS) associated with [43] S.A. Marques, Paracoccidioidomycosis: Epidemiological,
critical illness, Journal of Inflammation (2013) doi: clinical and treatment up-date, Anais Brasileiros de
10.1186/1476-9255-10-6.
Dermatologia 78 (2) (2003) 135-150. [49] Y. Kasahara, R.M. Tuder, L. Taraseviciene-Stewart, T.D. [44] N.F. Cerqueira, W.B. Yoshida, Óxido Nítrico, Revisão,
Le Cras, S. Abman, P.K Hirth, et al., Inhibition of VEGF Acta Cirúrgica Brasileira 17 (6) (2002) 417-423.
receptors causes lung cell apoptosis and emphysema, J [45] X.J. Guan, L. Song, F.F. Han, Z.L. Cui, X. Chen, X.J.
Clin Invest 106 (11) (2000) 1311-1319. Guo, et al., Mesenchymal stem cells protect cigarette
[50] I.M. Bird, Endothelial nitric oxide synthase activation smoke-damaged lung and pulmonary funtion partly via
and nitric oxide function: New light through old windows, VEGF-VEGF receptors, J Cell Biochem 114 (2) (2013)
Journal of Endocrinology 210 (3) (2011) 239-241. 323-335.
[51] M. Jakkula, T.D. Le Cras, S. Gebb, K.P. Hirth, R.M. [46]
E.C. Breen, J.L. Malloy, K. Tang, F. Xia, Z. Fu, R.E. Tuder, N.F. Voelkel, et al., Inhibition of angiogenesis Hancock, et al., Impaired pulmonary defense against
decreases alveolarization in the developing rat lung, Am J Pseudomonas aeruginosa in VEGF gene inactivated
Physiol Lung Cell Mol Physiol 279 (3) (2000) L600-607.
September 2013, Vol. 7, No. 9, pp. 928-934
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Induction of Apoptosis in HeLa Cells by Methanolic Extract of Litsea cubeba Fruit Residue from Essential Oil Extraction
1 2 Trisonthi Piyapat 2 , Miyagawa Kana and Tamura Hirotoshi 1. United Graduate School of Agricultural Sciences, Ehime University, Ehime 790-8566, Japan
2. Department of Applied Biological Science Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan
Received: May 31, 2013 / Accepted: August 01, 2013 / Published: September 30, 2013.
Abstract: Anticancer activity in vitro of Litsea cubeba fruit extracts was investigated, focusing on the fruit residue from essential oil extraction. The methanol extract was fractionated by an Amberlite® XAD-7 column. Cell viability, cell proliferation and cell death were determined using conversion of WST-8, BrdU incorporation and measurement of released LDH, respectively. Activation of caspase-3/-7 was detected using Z-DEVD-R substrate and morphological characteristics of apoptotic cells were revealed by DAPI staining. It was found that 80-100% methanol fractions (RME-4B, -5A, -5B and -5C) were effective against HeLa cell viability and
also promoted cell death. RME-5A and -5B were highly effective in suppressing DNA replication (IC 50 4.89 and 3.26 g/mL at 48 h) and also in activation of caspase-3/-7 (9 and 17 times of untreated population at 12 h). The presence of apoptotic bodies was clearly observed. The results of this study suggested that L. cubeba fruit residue has remarkable apoptosis induction potential for further use in cancer drug research and for waste management in the essential oil industry.
Key words: Litsea cubeba, lauraceae, apoptosis, caspase-3/-7, HeLa cell.