|Year : 2014 | Volume
| Issue : 4 | Page : 615-620
Ferula gummosa Boiss flower and leaf extracts inhibit angiogenesis in vitro
S Mirzaaghaei1, H Akrami1, MH Asadi2, H Mahdiuni1
1 Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran
2 Department of Biotechnology, International Center for Science, High Technology and Environmental Sciences, Kerman, Iran
|Date of Web Publication||1-Feb-2016|
Department of Biology, Faculty of Science, Razi University, Kermanshah
Source of Support: International Center for Science, High Technology and Environmental Sciences through grant no.1/2454,, Conflict of Interest: None
Background: Angiogenesis is a vital process in development as well as in tumor metastasis. Therefore, inhibition of tumor angiogenesis may be an approach for cancer therapy. In this study, we evaluated the effect of Ferula gummosa Boiss flower and leaf extracts on angiogenesis. Materials And Methods: Cell growth and cytotoxic effects of different concentrations (0-70 μg/mL) of F. gummosa Boiss flower and leaf extracts were evaluated on the growth of human umbilical vein endothelial cells (HUVECs) using Neutral Red assay. Then, wound healing, in vitro angiogenesis assay and quantitative VEGF gene expression analysis were conducted with the noncytotoxic concentrations of the ethanol extract. Results: Our results indicated that observed HUVECs viability was higher than 60% for both extracts after 24 hours treatment at concentration of 30 μg/mL or lower, whereas cytotoxic effects were observed at higher concentrations or after 48 hours treatment. F. gummosa Boiss flower and leaf extracts inhibited migration and angiogenesis capacity in a concentration-dependent manner (10-30 μg/mL), and down regulated VEGF transcription (20 μg/mL for flower extract and 30 μg/mL for leaf extract). Conclusions: Our findings revealed that F. gummosa Boiss flower and leaf extracts may contain antiangiogenic compounds, which could be used in preparation of new therapeutic agents for inhibition of tumor angiogenesis. To the best of our knowledge, this is the first report of antiangiogenic effects of F. gummosa Boiss flower and leaf extracts and more studies are needed to identify the effective components of the extracts.
Keywords: Angiogenesis, Ferula gummosa Boiss, herbal medicines, metastasis
|How to cite this article:|
Mirzaaghaei S, Akrami H, Asadi M, Mahdiuni H. Ferula gummosa Boiss flower and leaf extracts inhibit angiogenesis in vitro. Indian J Cancer 2014;51:615-20
| » Introduction|| |
Angiogenesis, the process of creating new blood vessels from preexisting ones, plays a key role in many physiological and pathological processes such as tumor growth and metastasis. Without angiogenesis, tumor mass cannot grow more than 1 mm in diameter and this is the basis of Judah Folkman's hypothesis in antiangiogenic cancer therapy. Endothelial cells release both angiogenic and antiangiogenic factors, since there is a balance between pro- and antiangiogenic factors, but as the proangiogenics exceed the antiangiogenic factors, "angiogenic switch" takes place.,, Antiangiogenic therapeutic drugs target endothelial cells, which are responsible for making new blood vessels in response to tumor induction. These endothelial cells are genetically stable and might not reveal problems in targeting their own tumor cells.,, Therefore, a promising approach in cancer therapy is focusing on antiangiogenic therapy, and developing drugs that target different aspects of angiogenesis.
Most of antiangiogenic drugs target vascular endothelial growth factor (VEGF) and its downstream receptors, which are the most important molecules in both physiologic and pathologic angiogenesis., However, unacceptable side effects of chemical antiangiogenic drugs persuade researchers to look forward to new, more effective with fewer side effects drugs.
Herbal medicines, which have been used for long time in different parts of the world, are one of the most reliable alternative candidates for antiangiogenic drugs. Plants extracts are composed of many different complex compounds, which may have effects on different aspects of a process concomitantly, and exhibit fewer side effects in comparison with nonnatural drugs. Nowadays, the search for new chemopreventive and chemotherapeutic agents has been increased to find new drugs, which are effective in malignant tumors therapy.,
Feula gummosa Boiss of apiaceae family is a native wild traditional medical plant of Asia, which has anticonvulsant, anticatarrhal, and antinociceptive effects and has been used in treating stomach pains and epilepsy.,
In this study antiangiogenic activity of ethanol extracts of F. gummosa Boiss flower and leaf were investigated on human umbilical vein endothelial cells (HUVECs).
| » Materials and Methods|| |
Ferula gummosa Boiss were collected from Damavand Mountains in northern Iran. After harvesting, flowers and leaves were dried in dark, at room temperature for 7 days. The dried flowers and leaves were ground in a blender and ca. 30 g of them were put in a Soxhelt apparatus separately and were mixed with petroleum ether (~3 times) until discoloration. Afterwards, the two almost resin-free suspensions were centrifuged (at 3000 g for 10 minutes); the obtained pellets were re-mixed with 300 mL ethanol and were stirred for 4 days. After centrifugation (at 3000 g for 15 minutes), the supernatants were evaporated under vacuum to dryness, using a Heidolph rotary evaporator, and the resultant powders (henceforth referred to as ethanol extract of flowers (EF) or leaves (EL)) dissolved in dimethylsulfoxide (DMSO) in different concentrations.
HUVECs were obtained from Medical Biology Research Center of Kermanshah University of Medical Sciences. The cells were maintained in Dulbecco Modified Eagle's Medium (DMEM) (Gibco, Belgium):Roswell Park Memorial Institute medium (RPMI) 1640 (Sigma-Aldrich, USA) at the ratio 1:1, containing 10% Fetal Bovine serum (FBS) (Biocrom AG), and supplemented with, 100 U/mL penicillin (Sigma-Aldrich, USA) and 100 μg/mL streptomycine (Sigma-Aldrich, USA) incubated at 37°C in a humidified atmosphere of 5% CO2. The culture media were changed twice a week.
Cell viability of HUVECs was assessed after each EF and EL treatment, by the Neutral Red (NR) (Sigma-Aldrich, USA), uptake assay. HUVECs were cultured initially at a concentration of 3 × 104 cells/well on 24-well plates in DMEM: RPMI 1640 at the ratio 1:1, containing 10% FBS and supplemented with antibiotics. HUVECs achieved approximately 50% confluent monolayer within 24 hours, the medium of each well was replaced by fresh DMEM: RPMI 1640 (1:1) containing 3% FBS, supplemented with appropriate antibiotics. Then, different concentrations of EF and EL (10, 20, 30, 40, 50, 60, and 70 μg/mL for 24-hour treatment and 5, 10, 15, 20, 25, 30, and 35 μg/mL for 48-hour treatment) were added to each well of HUVECs and incubated in a humidified atmosphere of 5% CO2 at 37°C. Each assay included three control wells containing highest concentration of applied DMSO (0.25% v/v) as control. The medium and dead cells were washed out with phosphate buffered saline (PBS) at the end of a specified time period (24 and 48 hours). Then, the cells were incubated for 2 hours with 500 μL of serum free DMEM: RPMI 1640 (1:1) culture medium supplemented with 33 μg/mL of NR dye at 37°C, 5% CO2 and 90% humidity. After the incubation, the medium containing NR dye was washed out using FBS. Afterwards, 500 μL of destaining solution including 15% v/v acetic acid and 45% ethanol in water was added to each well and incubated in a dark shaking incubator at 37°C for 15 minutes. The absorbance of colored solution of controls and treated cells were measured at 540 nm using blank (destaining solution) as the reference. Cell viability of treated HUVECs with different concentrations ofEF and EL was obtained by comparing optical density (OD) of treated HUVECs with untreated HUVECs as controls in 540 nm. Each sample was assessed in triplicate and the assay was repeated at least twice.
For performing three-dimensional angiogenesis assay, HUVECs were loaded on cytodex 3-microcarriers beads in 10% FBS containing DMEM: RPMI 1640 (1:1) medium supplemented with antibiotics at 37°C, 5% CO2 and 90% humidity incubator by flicking cell suspension and initially prepared microcarriers, every 20 minutes in a 4-hour period and then incubated in a 20-hour period in the same condition without flicking.
The following day, cell-coated beads were mixed with collagen solution including collagen type I, 10× DMEM: RPMI 1640 (1:1) medium, 23 mg/mL NaHCO3 and FBS with the ratio of 7.5:1:1:0.5, on ice, respectively. Then, 100 μL of the cell-coated beads were mixed with the collagen solution and loaded into each 96-well plate. After solidification of collagen solution, 3% FBS containing DMEM: RPMI 1640 (1:1) with different concentrations of EF and EL including 10, 15, 20, and 25 μg/mL of the flower extract and 10, 15, 20, 25, and 30 μg/mL of the leaf extract were added into each 96-well plate of HUVECs. Results were monitored under a microscope for 3 days.
Tube formation assay was performed by Extracellular Matrices (ECM) gel (Sigma-Aldrich, USA) according to the manufacturer's protocol. Briefly, 100 μl of gel was dispensed to the each well of 24-well plates and incubated at 37°C, in 5% CO2 incubator for 30 minutes to form a matrix. HUVECs were seeded onto the solidified gel at the concentration of 3 × 104 cells per well, allowing 10 and 20 μg/mL of the EF, 10, 20, and 30 μg/mL of the EL in antibiotics and 5% FBS containing DMEM: RPMI 1640 (1:1) for each well and incubated at 37°C, 5% CO2 and 90% humidity incubator within 24 hours. The tube like structure formation was evaluated under inverted microscope (CETI, UK). The average tube lengths were measured in four randomly chosen microscopic fields using Adobe Photoshop software (Adobe Photoshop 8, CS/ME).
The capacity of HUVECs to migrate was studied through wound healing assay. The cells were seeded at high density on a 24-well plate in DMEM: RPMI 1640 (1:1), containing antibiotics and 10% FBS. The following day, a wound was created in confluent monolayer of cells in 24-well plate by scratching a spatula across the cells. Detached cells were washed out gently using PBS and 500 μL of DMEM: RPMI 1640 (1:1) containing antibiotics and 3% FBS were added to each well of a 24-well plate. Cells were treated with 10 and 20 μg/mL of the flower extract and 20 and 30 μg/mL of the leaf extract,and incubated at 37°C, 5% CO2 and 90% humidity incubator. The migrated cells from the edge of the wound in treated wells by EF and EL were compared with the control wells in 24 hours.
Total ribonucleic acid (RNA) was extracted from HUVECs using RNeasy Plus Mini kit (QIAGEN, USA), according to the protocol of the manufacturer. The yield and purity of RNA were estimated by 1% agarose gel electrophoresis and its optical density at 260/280 nm ratio. complementary deoxyribonucleic acids (cDNAs) were synthesized from total RNA (1 μg) with QuantiTect ® Reverse Transcription Kit (QIAGEN, USA) according to the manufacturer's instruction. Primers for polymerase chain reaction (PCR) and real-time polymerase chain reaction (RT-PCR) were designed using Gene Runner and online primer design softwares such as Primer 3 and primer-BLAST. The VEGF and GAPDH amplicon sizes were 187 and 162 bp, respectively.
The following primers were designed for GAPDH and VEGF:
GAPDH forward primer, 5'-CCT GCA AAT GGG ACT TAC G- 3'; GAPDH reverse primer, 5'-AAA AAC CCT TAT CGC ATT CAA AC-3';
VEGF forward primer, 5'- CTACCTCCACCATGCCA AGT -3'; VEGF reverse primer, 5'- CACACAGGATGGC TTGAAGA -3'. It is notable that the VEGF primer detects all transcript variants of VEGF-A gene.
The Rotor-Gene 3000 System (Corbett Research, Australia) was used to quantify the transcript gene expression level of VEGF in HUVECs using Quanti Tect™ SYBR® Green PCR kit (QIAGEN, USA) and the reactions were performed according to the protocol of the manufacturer. Amplification efficiency of reactions were determined by constructing a standard curve for each amplification experiment through an initial dilution of 1:1 of cDNA samples and a 2-fold serial dilution series. The analysis was performed according to the 2-ΔΔCt method based on the threshold cycle (Ct) values for VEGF as target gene and GAPDH as an endogenous control gene. Each sample was assessed in duplicate and the assay was repeated at least two times.
All data in the different experimental groups were obtained in at least two independent experiments, were expressed as the mean ± SD, with SPSS (Version 16:SPSS. Link. USA) and were analyzed using Student's t-test. One-way analysis of variance (ANOVA) was used for analyzing data between different groups. P values less than 0.05 were considered as statistically significant.
| » Results|| |
Results of NR uptake assay indicated that cell viability of HUVECs decreased after treating with the EF and EL of F. gummosa Boiss in comparison with the nontreated control. The viability percentage of HUVECs were exposed to EF and EL, was higher than 60% for both extracts after 24 hours treatment at concentration of 30 μg/mL or lower and are shown in [Figure 1] and [Figure 2], respectively. The EF and EL had cytotoxic effects on HUVECs in a dose and time dependent manner (P < 0.05) with the IC50 value of 39.21 and 14.66 μg/mL for EF and 38.05 and 23.93 μg/mL for EL in 24- and 48-hours treatment, respectively. P values of both EF and EL at 30 μg/mL were 0.001 in 24 hours. While, P values of EF at 10 μg/mL and EL at 25 μg/mL were 0.001 in 48 hours.
|Figure 1: The viability effect of F. gummosa Boiss flower extract on HUVECs. The cell viability of HUVECs treated with various concentrations of flower extract for 24 and 48 hours (10, 20, 30, 40, 50, 60, and 70 μg/mL for 24.hour treatment and 5, 10, 15, 20, 25, 30, and 35 μg/mL for 48-hour treatment) was evaluated relative to HUVECs untreated by Neutral Red assay. Assays were done in triplicate. Error bars represent mean ± SD of triple samples, (*, P < 0.01)|
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|Figure 2: The effect of F. gummosa Boiss leaf extract on HUVEC viability. The cell viability of HUVECs were evaluated by neutral red assay. HUVECs treated with various concentrations of leaf extract for 24 and 48 hours (10, 20, 30, 40, 50, 60, and 70 μg/mL for 24-hour treatment and 5, 10, 15, 20, 25, 30, and 35 μg/mL for 48-hour treatment) was evaluated relative to HUVECs untreated. Assays were done in triplicate with triple samples. Error bars, mean ± SD, (*, P < 0.01)|
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To evaluate the effect of the flower and leaf extracts on angiogenesis, an in vitro 3-D collagen cytodex model was accomplished with HUVECs treated with different concentrations of EF and EL for 72 hours. Both extracts inhibited in vitro angiogenesis in a concentration dependent manner. The EF inhibited 64.5% and 85.5% of sprout formation, using the cytodex-3 microcarriers, at the concentrations of 10 and 15 μg/mL, respectively, and a complete inhibition was observed at higher concentrations. The results of the EL showed 77%, 80%, and 96% inhibition of endothelial cells differentiation and sprout formation from the cytodex-3 microcarriers, at the concentrations of 15, 20, and 25 μg/mL, respectively, and a complete inhibition at higher concentrations [Figure 3].
|Figure 3: Inhibitory effect of F. gummosa Boiss flower and leaf extracts on in vitro 3-dimensional angiogenesis. HUVECs which loaded on cytode × 3-microcarriers beads were embedded on solidified collagen gel in 96-well plate. Culture medium treated with various concentrations of flower and leaf extracts as well as untreated control were added into each 96-well plate. (a) 72 hours later, sprouting was assessed by macroscopic comparison of (original magnification × 100) A- control (0.5% v/v DMSO), B- 10 μg/mL, C- 15 μg/mL, D- 20 μg/mL, E- 25 μg/mL, A’- control, B’- 15 μg/mL, C’- 20 μg/mL, D’- 25 μg/mL, E’- 30 μg/mL (letters with prime indicate the leaf extract treatment). (b) The number of sprouts per total 30 beads was counted in 10 different fields by microscopy. Assay organized in three independent triplicated experiments. Error bars represent mean ± SD, (*, P < 0.05 vs. control group)|
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Treating HUVECs seeded on a 2-D matrigel matrix with EF at the concentrations of 10 and 20 μg/mL for 24 hours, revealed 67.7% and 92.6% inhibition, respectively in formation of tube like structures, and treating with theELat the concentrations of 20 and 30 μg/mL for 24 hours displayed 54.5% and 92.4% inhibition of tube-like structure formation, respectively [Figure 4].
|Figure 4: Effect of F. gummosa Boiss flower and leaf extracts on in vitro tube formation by HUVECs. HUVECs were cultured on solidified ECM gel. Culture medium was treated with 10 and 20 μg/mL of flower and 20 and 30 μg/mL of leaf extracts and untreated control were added into each 24-well plate. (a) After 24 hours, the tube formation was measured using mean tube lengths of treated cells in comparison with the control (original magnification × 100) A- control, B- 10 μg/mL, C- 20 μg/mL, A’- control, B’- 20 μg/mL, C’- 30 μg/mL (letters with prime sign indicate the leaf extract treatments). (b) Total tube lengths were determined and triplicate assays organized in three independent experiments. Error bars represent mean ± SD, (*, P < 0.05 vs control group)|
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The effect of flower and leaf extracts on endothelial cells migration in nontoxic concentrations were evaluated using wound healing assay. EF and EL remarkably inhibited cell migration of HUVECs in comparison with untreated HUVECs as control within 24 hours. The flower extract suppressed cell migration at concentrations of 10 and 20 μg/mL, and leaf extracts inhibited cell migration at concentrations of 20 and 30 μg/mL [Figure 5].
|Figure 5: Effect of F. gummosa Boiss flower and leaf extracts on HUVECs migration. A “wound” was created in a confluent HUVECs and fresh medium containing 10 and 20 μg/mL of flower and 20 and 30 μg/mL of leaf extracts as well as untreated control were replaced. (a) 24 hours later HUVECs migration was detected (original magnification × 72) (letters with prime sign indicate the leaf extract treatments). a- Control, b- 10 μg/mL, c- 20 μg/mL, a’- control, b’- 20 μg/mL, c’- 30 μg/mL. (b) After 24 hours HUVECs migration were counted in five separate fields in each sample. Error bars represent mean ± SD. Assays were performed in three independent experiments in triplicate, (*, P < 0.01 vs. control group)|
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The effect of F. gummosa Boiss flower and leaf extracts on VEGF transcripts in comparison with GAPDH were examined by relative quantification in RT-PCR. Data analysis of the RT-PCR results showed that the transcripts level of VEGF decreased 78% and 83% in HUVECs treated with 20 and 30 μg/mL concentrations of F. gummosa Boiss flower and leaf extracts compared with nontreated control HUVECs in 24 hours, respectively [Figure 6].
|Figure 6: Inhibitory Effect of F. gummosa Boiss flower and leaf extracts on VEGF gene expression in HUVECs. After 24 hours of treating HUVECs by flower and leaf extracts, VEGF gene expression measured using real-time RT-PCR relative to GAPDH gene as endogenous control. Error bars represent mean ± SD. Assays were organized in three independent experiments in duplicate|
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| » Discussion|| |
Angiogenesis is comprised of several sequential steps, including stimulation of endothelial cells, their activation, proliferation, migration, tube formation, and sprouting, which requires ECM degradation, anastomosis, basement membrane reformation, and stabilization utilizing pericytes and smooth muscle cells.,, Since introducing antiangiogenic therapy as an aspect of treating cancer in 1971 by Folkman, different antiangiogenic compounds have been reported. Botanicals contain various phytochemical complexes that may target different pathways involved in tumor development such as different aspects of tumor angiogenesis.
Aerial parts of F. gummosa Boiss have been reported tocontain organic substances of terpene class including a- and b-pinenes. Terpenes are one of the most frequent secondary metabolites in higher plants. Some papers have reported medicinal plants with anticancer activity to compose of the terpenes as their main compounds., Antiangiogenic activity of terpens has also been indicated in some reports. Ginkgo biloba extract consists of terpens as one of the main compounds and shows anticancer and antiangiogenic activity through down regulating VEGF, and also triterpens extracted from Agaricales Fungi indicate antitumor activity via antiangiogenic properties.,, Mastic oil from Pistacia lentiscus variation chia has inhibitory activity against proliferation, survival, angiogenesis, and inflammatory responses of tumor. Artemisia annua (Chinese wormwood) contains 95% artemisinin, and other related terpenes and flavonoids, and inhibits angiogenesis by decreasing VEGF expression.
In this study, in order to assess the effect of EF and EL on angiogenesis, we used two in vitro angiogenesis models: in vitro 3-D angiogenic sprouting and 2-D capillary tube formation models. Our experimental results indicate that bothEF and EL suppress sprouting and tube formation of endothelial cells in a dose dependent manner. The results of the antiangiogenic activity of EF and EL with therapeutic plant extracts and their composition indicated a relationship between antiangiogenic activity a- and b-pinenes and in F. gummosa Boiss flower and leaf extract compounds.
There is, however, a difference between the concentration of flower and leaf extracts that used to have maximum inhibition efficiency on angiogenesis in both models of angiogenesis assays. That is, EF has more suppression efficiency than EL on angiogenesis in both models, and this data suggested that the EF may contain more angiogenesis inhibiting compounds than the EL of F. gummosa Boiss.
The results of EF and EL Cell viability assays indicated that the viability effects of both were significantly higher in 24 hours than in 48 hours, which demonstrated both extracts have dose and time dependent viability effects on HUVECs. In contrast, treatment of HUVECs by F. gummosa Boiss leaf extract had a noticeably higher rate of viability loss than the flower extract in 48 hours.
Comparison of the results of the flower and leaf extracts antiangiogenic and cytotoxic activities, in two time intervals, suggested two distinct mechanisms of cytotoxic and antiangiogenic activities.In vitro angiogenesis assay results were indicated no discernible difference between antiangiogenic activity of the EF and EL in 24 hours for 2-D tube formation model and in 72 hours for 3-D sprout formation assay. However, the cytotoxic effect of both flower and leaf extracts increased for a longer time interval. These results suggested that there may be no direct relationship between the antiangiogenic and cytotoxic activities of EF and EL on HUVECs.
Wound healing assay of EF and EL on HUVECs demonstrated that both extracts inhibited HUVECs migration, a process that plays a key role in angiogenesis capacity of endothelial cells, which supported antiangiogenesis activity of EF and EL.
VEGF and its receptors play a fundamental role in endothelial cells survival, proliferation, migration, vessel permeability, and angiogenesis. Evaluation of VEGF transcription level in HUVECs treated with EF and EL indicated that both extracts have the capacity to down regulate VEGF gene expression and with regard to the role for VEGF in tumor angiogenesis, we suggest that EF and ELmay suppress angiogenesis through VEGF signaling. To our knowledge, this is the first report that showed significant antiangiogenesis capacity of EF and ELand more studies need to identify the effective components of the extracts.
| » Conclusion|| |
Our study indicated that EF and ELhave antiangiogenesis effects in noncytotoxic concentrations on HUVECs. Consequently, with regard to the medical properties and also nutritional usage of F. gummosa Boiss in Iran, our findings may present F. gummosa Boiss as a new anticancer therapeutic plant that can be utilized in anticipating new drug compositions with fewer side effects.
| » Acknowledgment|| |
The financial supports from the Research Council of International Center for Science, High Technology and Environmental Sciences through grant no. 1/2454 are gratefully acknowledged. The authors are profoundly grateful to Roghaye Gharaei, Shafagh Heidari and Kamran Mansouri for their contribution in completing this work.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]