https://orcid.org/0000-0002-6980-6761
Abstract
This research aimed to evaluate the antiangiogenic activity of isolated flavonoid 4a,5,8,8a-tetrahydro-5-hydroxy-3,7,8-trimethoxy-2-(3,4-dimethoxyphenyl) chromen-4-one (TMF) from Tabebuia chrysantha. STAT3-MMP9 signalling is a signal transduction mechanism that promotes angiogenesis in various cancers.
The tumour xenografting chicken embryo chorioallantoic membrane (CAM) model-based ex vivo assay was used to evaluate the activity of TMF. The Western blot, densitometric analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to evaluate the activity of the MMP9. Zebrafish embryos were used to evaluate embryotoxicity, and in vitro free radical scavenging activity of flavonoid was also elucidated.
This research assessed the high level of STAT3, p-ERK, VEGF-R and MMP9 in the tissue extract of the control group, and also, the suppression of angiogenesis in the treatment groups was due to scavenged ROS and RNS, dephosphorylation of STAT3 and ERK, and suppression of MMP9 gene expression.
The isolated flavonoid named TMF from T. chrysantha functions as specific regulators of target proteins of angiosarcoma. The STAT3-MMP9 signalling may be used as an effective prognostic marker of angiosarcoma.
Introduction
Angiosarcoma is a fast-growing cancer that looks like an inflamed brush area with purpleish or reddish skin and can occur anywhere in the body such as breast, liver and spleen. Angiosarcomas make up about 1–2% of all sarcomas in the United States. The patient may feel deep algesia in the growing tumour area.[1] The researcher hypothesised that the phosphorylation of STAT3 regulates MMP-9 production during inflammation, invasiveness, and metastasis of ovarian cancer, breast cancer and angiosarcoma on the scalp.[2–4] STAT3-MMP9 signalling is a signal transduction pathway that promotes angiogenesis and inflammation in various cancers such as angiosarcoma and breast cancer in response to STAT3 protein. Matrix metalloproteinase 9 (MMP9) is an endopeptidase which actively involved during angiogenesis and inflammatory reactions. The Kasabach–Merritt syndrome (KMS) in infants is a life-threatening disease in the presence of vascular tumours (VTs).[5,6] VT contains a rich network of capillary vessels, and their growth or thrombosis can cause more severe clinical manifestations. Angiogenesis is a key and complex biological process in VT growth that favours the formation of new blood vessels (neovascularisation) from the pre-existing vascular tissue. Promotion or inhibition of angiogenesis is facilitated by membrane-bound or cytoplasmic protein factors such as vascular endothelial, epidermal, fibroblast and placental growth factors.[7] VEGF buttresses a rapid and complete angiogenic response of almost all living tissues via binding to its receptor.[8]
There is a growing interest in the pharmacological evaluation of lead phytoconstituents from traditional plants used in the rural community of India. The total flavonoid content of methanol extract of Tabebuia chrysantha (Jaq.) Nicholson (Bignoniaceae) was 118.4 mg QE/g of the extract.[9] According to their chemical structure, flavonoids are divided into four categories: flavones, flavanones, flavonols and anthocyanin. The 16 species of plants belonging to the Bignoniaceae family are undergone phytochemical analysis and reported a large predominance of flavones over flavonols.[10] Scientists reported the presence of flavones and flavonols in Tabebuia caraiba. The above findings motivated us to separate flavonoids from the extract by applying different dilution techniques.[11]
Our previous research demonstrated the apoptosis of EAT induced cancer tissue was due to suppression of sEGFR mediated pSTAT3 proteins.[9] Agents that target sEGFR potentially exert tumour-suppressive effect by reducing the secretion of proangiogenic protein factor VEGF which stimulates tumour neoangiogenesis.[12,13] Some tumours may resistant to sEGFR targeted drugs and use alternative VEGF mediated STAT3-MMP9 signalling for proliferation and angiogenesis. So modern cancer therapy warrants novel approaches that can target both EGFR and STAT3-MMP9 signalling.[14]
Tumour xenografting chorioallantoic membrane (CAM) model is valuable in developmental studies and cancer biology.[15,16] The inflammatory cells release large amounts of MMPs and VEGFs to stimulate angiogenesis. The MMP2, MMP9 and VEGF-A signalling are actively involved during angiogenesis in gastric carcinoma, angiosarcoma and vascular tumour.[8,17] Various studies indicated that site-specific promoter demethylation of DNA in tumour cells influences the regulation of MMP9 gene expression.[18,19] Cellular infiltration and osteoarthritis are due to the overexpression of MMP9 gene expression in cartilage tissues.[20]
Several promising angiosarcoma-targeting agents are currently in use: VEGF-R-targeted drug (e.g. bevacizumab), tyrosine kinase receptor inhibitors (e.g. sorafenib, sunitinib), platelet-derived growth factor receptor inhibitor (e.g. pazopanib).[21–23] Although single-agent therapy was successful for many patients but patients with advanced age and significant clinical comorbidities may not qualify for therapy.[24] Angiosarcoma response to VEGF-targeted agents is limited, and biomarkers are required to identify patients most likely to respond to treatment.[25] Researchers reported that flavonoids and iso-flavonoids possess multiple bioactivities and can either kill or resensitise conventional chemotherapeutics to resistant cancer cells.[26] So we are in search of a novel angiosarcoma-targeting agent that has minimal side effects and null tumour resistance nature and can be suitable for old patients with angiosarcoma.
The present study aimed to characterise fraction D (compound 4), which was isolated but not analysed in our previous research, and to evaluate its antiangiogenic effect in comparison with standard podophyllotoxin using EAT cells loaded in CAM model (ex vivo) and Zebrafish embryo. Before that, the cytotoxicity of the TMF was examined in vitro by MTT and trypan blue dilution assay against EAT cells.
Material and Methods
Authentication of plant
The plant T. chrysantha (Jaq.) Nicholson (Bignoniaceae) was authenticated by M. S. Mondal, Botanical Survey of India, Kolkata, India, and a voucher specimen (CNH/5-1(750) /2018/Tech-II/980) has been preserved in the College of Pharmacy, KLEF deemed to be University, Guntur, for future reference.
Materials and ethics statement
EAT cell suspension with the token number (140/CTS/EATS) and Zebrafish (05/CTS/ZFISH) were procured from CLINITECH solution, Hyderabad, India, and preserved perfectly at Biotechnology Research Laboratory of KL University. All of the experiments were conducted with the approval of the Institutional Animal Ethical Committee (IAEC) of the Southern Institute of Medical Sciences (SIIMS) college of pharmacy (Regd. No.1523/PO/a/11/CPCSEA), India, dated 5/9/2014.
Separation, quantification and quality control of phytoconstituents
Methanol extract (250 g) was subjected to column chromatography on silica gel (100–200 mesh–Merck) packed and eluted with mixture of n-hexane, dichloromethane and methanol of increasing polarity to obtain fractions. The elution started with 100% hexane and increased with solvent polarity ethyl acetate and methanol. The hexane–dichloromethane combinations gave four colourless compounds and the yields were 450 mg (A), 225 mg (B), 325 mg (C) and 250 mg (D), respectively. Fraction A (5.0 g) was purified by LH-20 column eluted with 80% CH2Cl2–MeOH (2 l) and further applied to a radial chromatography with 95% hexane–EtOAc (200 ml) to afford compound 1. Fraction B (7.5 g) was purified by LH-20 column eluted with 80% CH2Cl2–MeOH (2 l) and further applied to a chromatogram with 50% hexane–CH2Cl2 (200 ml) to obtain compound 2. Compound 3 was separated by LH-20 column eluted with 50% CH2Cl2–MeOH (2 l) from fraction C (5.0 g). Fraction D (7.2 g) was purified by LH-20 column eluted with 50% CH2Cl2–MeOH (2 l) to give compound 4.
The isolated compound was analysed with a UV spectrophotometer (LAB INDIA 1800), IR spectroscopy and the HPLC chromatography (Shimadzu Analytical, Chennai, India; SPD detector) with a gradient elution technique (water + acetonitrile). All the optimised conditions such as gradient time, flow rate and column temperature were maintained by following my previous research and Bala et al.[27] The isolated compound was stored at −80°C till further used, to maintain its stability and purity.
Structural elucidation of TMF
The structure of TMF was characterised using 1D and 2D NMR spectroscopy and compared with available data from the literature and respective research.[28]
In vitro free radical scavenging activity screening of TMF
1, 1-diphenyl-2-picryl-hydrazyl (DPPH) and nitric oxide (NO) radical scavenging activity
The above two activities for tumour extract were extrapolated by observing the modified procedure of Mondal et al.[29]
Hydroxyl radical (HO) scavenging activity
The activity was extrapolated with a standard curve of ascorbic acid using a UV-Vis spectrophotometer at 517 nm and by following the method of Elizabeth and Rao.[30]
EAT grafted Chick CAM antiangiogenesis model ex vivo
Thirty fertile chicken eggs were incubated at 37°C in a 60–70% relative humidity for 72 h. On the 5th day after tumour cell inoculation, the tumour xenografts became visible and were supplied with vessels of CAM origin, taken for study.
Preparation of CAM grafted with EAT cells and inoculation of TMF and podophyllotoxin
The CAMs were grouped into six groups. Groups I and II were marked as DMSO control/normal control and VEGF-A (10 ng/ml) control (inducer control), respectively. 2 × 107 cells/ml EAT cells have been implanted directly on the CAM of Groups III–VI using a cotton plug for localising the cells. The white background tumour cells were cleaned from CAM by normal saline and DMSO solution. Five filter paper discs were prepared after soaking in 5 µl of the treatment solutions: Group III membranes were treated with TMF (10 µg/ml), Group IV and Group V CAMs treated with TMF 17 and TMF 25 µg/ml, respectively, Group VI membranes treated with PDL 0.7 µg/ml (standard). On day 16, the developing embryos were sacrificed by cryopreservation and fixation in 4% paraformaldehyde. The biomaterials are harvested and examined for vascular infiltration using imaging techniques.
Microscopic examination of CAM membrane
The 5-µm section CAM membranes were prepared (fixed in formaldehyde solution, 3.7% and sealed in paraffin) with a Spencer micrometer, marked by haematoxylin–eosin dye and examined by microscope.
Measurement of protein concentration
25% CAM tissue homogenate was prepared using 0.05 m phosphate buffer (pH 7.6) and centrifugation at 100 000 g for 60 min.
Evaluation of STAT3, MMP9 and VEGF-R expression: Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR)
The protein content of the CAM tissue homogenate was estimated by the Bradford assay procedure. The protein samples were passed over 12% SDS–PAGE for separation. The gel was then transferred overnight to the polyvinyl difluoride (PVDF) membrane at 48°C. The following primary antibodies were used: rabbit anti-VEGF-R (GTX102643; Gene Tex, Irvine, CA, USA), anti-MMP-9, monoclonal antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotech, Dallas, TX, USA), anti-ERK and anti-STAT3 antibodies (Cell Signaling, Danvers, MA, USA). The bands of all proteomics were detected using the available software (LI-COR Biosciences, Lincoln, NE, USA).[9]
The qRT-PCR assay was performed and statistically analysed using 2−ΔΔCT methodology in SYBR Green master mix (Applied Biosystems 7300, Foster City, CA, USA) with cycling conditions as follows: 95°C for 15 s and 60°C for 1 min for 40 cycles.[31] Total RNA from cells exposed to different treatments was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. For STAT3 detection, total RNA (2 μg) was reverse transcribed into cDNA with AMV Reverse Transcriptase (Promega, Madison, WI, USA). GAPDH was used as the internal control. The primers used in our study are listed in Table 1.
Sequences of primers used for qRT-PCR
| STAT3 | Forward Reverse | 5′-CTGGCCTTTGGTGTTGAAAT-3′ 5′-AAGGCACCCACAGAAACAAC-3′ |
| MMP9 | Forward Reverse | 5′-GAGTCAACGGATTTGGTCGT-3′ 5′-GACAAGCTTCCGTTCTCAG-3′ |
| GAPDH | Forward Reverse | 5′-GAGTCAACGGATTTGGTCGT-3′ 5′-GACAAGCTTCCGTTCTCAG-3′ |
| STAT3 | Forward Reverse | 5′-CTGGCCTTTGGTGTTGAAAT-3′ 5′-AAGGCACCCACAGAAACAAC-3′ |
| MMP9 | Forward Reverse | 5′-GAGTCAACGGATTTGGTCGT-3′ 5′-GACAAGCTTCCGTTCTCAG-3′ |
| GAPDH | Forward Reverse | 5′-GAGTCAACGGATTTGGTCGT-3′ 5′-GACAAGCTTCCGTTCTCAG-3′ |
Sequences of primers used for qRT-PCR
| STAT3 | Forward Reverse | 5′-CTGGCCTTTGGTGTTGAAAT-3′ 5′-AAGGCACCCACAGAAACAAC-3′ |
| MMP9 | Forward Reverse | 5′-GAGTCAACGGATTTGGTCGT-3′ 5′-GACAAGCTTCCGTTCTCAG-3′ |
| GAPDH | Forward Reverse | 5′-GAGTCAACGGATTTGGTCGT-3′ 5′-GACAAGCTTCCGTTCTCAG-3′ |
| STAT3 | Forward Reverse | 5′-CTGGCCTTTGGTGTTGAAAT-3′ 5′-AAGGCACCCACAGAAACAAC-3′ |
| MMP9 | Forward Reverse | 5′-GAGTCAACGGATTTGGTCGT-3′ 5′-GACAAGCTTCCGTTCTCAG-3′ |
| GAPDH | Forward Reverse | 5′-GAGTCAACGGATTTGGTCGT-3′ 5′-GACAAGCTTCCGTTCTCAG-3′ |
Quantitative densitometric analysis of STAT3, MMP9
The band intensity of GAPDH, STAT3, MMP9 were determined using Quantity One software (Bio-Rad) at the CLINITECH Solutions.[32]
Results
Phytochemical screening and structure elucidation
Our previous research demonstrated the total phenolic, flavonoid and quinone contents of METC were 120.72 mg GAE/g of extract, 118.4 mg QE/g of extract and 0.54 µg/g of extract, respectively, and also elucidated the structure of 2-hydroxynaphthalene-1,4-dione (compound 1); λmax = 452 nm, β-lapachone (2,2-dimethyl-3,4-dihydro-2H-benzo [h]chromene-5,6-dione) (compound 2); λmax = 279 nm and 2-((dimethylamino)methyl)-3-methoxynaphthalene-1,4-dione (compound 3); λmax = 213 nm (Table 2). The HPLC retention time for three compounds was 3.096, 2.784 and 3.691, respectively (Figure 1).[9] The structure of compound 4 named 4a,5,8,8a-tetrahydro-5-hydroxy-3,7,8-trimethoxy-2-(3,4-dimethoxyphenyl)chromen-4-one (TMF) was characterised by using UV, HPLC, IR, 1H NMR and 13C NMR spectrum (Figures S1 and 2). The HPLC retention time of TMF was 2.460 min (Figure 3), and IR data were observed as OH: 3374.31 cm−1, OMe: 2923.07 cm−1, C=O: 1711.80 cm−1 (Figure S2).
Structure of isolated compounds from Tabebuia chrysantha
 |  |  |
| 2-Hydroxynaphthalene-1,4-dione | β-lapachone | 2-((dimethylamino)methyl)-3-methoxynaphthalene-1,4-dione |
| | |
| 2-Hydroxynaphthalene-1,4-dione | β-lapachone | 2-((dimethylamino)methyl)-3-methoxynaphthalene-1,4-dione |
Structure of isolated compounds from Tabebuia chrysantha
| | |
| 2-Hydroxynaphthalene-1,4-dione | β-lapachone | 2-((dimethylamino)methyl)-3-methoxynaphthalene-1,4-dione |
| | |
| 2-Hydroxynaphthalene-1,4-dione | β-lapachone | 2-((dimethylamino)methyl)-3-methoxynaphthalene-1,4-dione |
HPLC chromatogram of three isolated compounds from Tabebuia chrysantha.
NMR spectra of TMF.
The HPLC chromatogram of TMF from Tabebuia chrysantha (water + acetonitrile).
In vitro free radical scavenging activity of TMF
The TMF showed the dose-dependent DPPH, NO and OH radical scavenging activity comparable to the standard ascorbic acid antioxidant curve. The IC50 were found to be 15.53 ± 2.8, 15.62 ± 3.2 and 20.08 ± 1.5 µg/ml, respectively, as shown in Figure 4.
DPPH, NO and OH free radical scavenging activity of TMF: Values are represented as mean ± SEM of five individual experiments (n = 5) where **P < 0.01 and *P < 0.001 were considered as highly significant.
Chick chorioallantoic membrane assay
The normal control/DMSO control (A) and positive control/VEGF control (B) showed a marked arteriovenous growth around the embryo. Representative micrographs of the three tested specimens are depicted in C–E. The micrograph C (TMF 10) did not affect growing blood vessels. The micrograph D (TMF 17) and micrograph E (TMF 25) showed arteriovenous malformation tissues as well as the lymphatic malformation surrounding embryo (Figure 5).
TMF suppressed EAC cells induced angiogenesis in the CAM assay (ex vivo model). (a) Control, DMSO alone showed evidence of angiogenesis and growth of embryo (b) VEGF-A (10 ng/ml), a network of new blood vessels was established (see arrow) at the point of application (c) TMF (10 µg/ml) started suppression of blood vessel formation (d) TMF (17 μg/ml) (e) TMF (20 µg/ml) (f) PDL (0.7 µg/ml) showed evidence of angiogenesis suppression. Representative photomicrographs are shown. Magnification ×50.
The Western blotting, densitometric and qRT-PCR analysis
We observed the high density of p-ERK, STAT3 and MMP-9 in the control group (Figure 6) which confirmed a positive linkage between the p-ERK, STAT3 and MMP-9. The quantified results from TMF-17 and TMF-25 µg/ml treatment groups suggested that p-ERK regulated STAT3 and similarly STAT3 controlled the activity of MMP-9 gene expression. The qRT-PCR report illustrated the dose-dependent decrease in relative mRNA levels of STAT3 and MMP9 (Figure 7). The R2 value for the data sets (3 ng–0.4 pg of protein target) was 0.896, indicated TMF targets on tissue proteins during angiogenesis suppressive action.
Weston blot analysis. This gel pattern is representative of three independent experiments. TMF inhibits ERK and STAT3-mediated MMP9 survival pathway. (a) CAM cell extract was preincubated with indicated concentrations of TMF for 12 h. Total extracts were analysed by Western blot analysis using the indicated antibodies for VEGF-R, p-ERK, STAT3 and MMP9. GAPDH level was used as a loading control. Data represented as mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different when compared with control. (b) and (c) Quantitative densitometry analysis of STAT3 and p-ERK. The experiments were repeated three times. ***P < 0.05; **P < 0.01; *P < 0.001. (d) Quantitative densitometry analysis of MMP9.
Quantitative expression of STAT3 and MMP-9 in EAC cells. Total RNAs were extracted, and qRT-PCR performed for the determination of relative mRNA levels of MMP-9 and STAT3. *P < 0.001.
Statistical analysis
The results (mean ± SEM) were analysed by one-way analysis of variance (ANOVA) followed by Dunnett's test using GraphPad Software, InStat version 3.05, San Diego, CA, USA.
Discussion
Previous research revealed that sEGFR targeted ERK and STAT3 activation promoted tumour cell proliferation. Targeting EGFR, VEGF-R and extracellular signal-regulated kinase (ERK1/2)-mediated MMP9-STAT3 signalling pathway for getting the antiangiogenesis effect is a novel mechanism for effective cancer treatment.
The isolated trimethoxyflavone (TMF) belongs to polymethoxyflavones (PMFs) that bear three methoxy groups on a basic benzo-γ-pyrone skeleton. A wide range of pharmacological activity is associated with PMFs, including antiproliferative,[33] anti-inflammatory and antioxidant,[27,34] anticarcinogenic,[35] selective cytotoxicity, antiangiogenesis and reduction in lymphocyte infiltration.[36] Researchers also demonstrated that PMFs have chemoprotective efficacy against colon carcinogenesis. A decrease in cytosolic and nuclear accumulation of β-catenin, phosphorylation of AKT, phosphorylation of STAT3 in a dose-dependent manner was hypothesised to be an underlying molecular mechanism of chemoprevention.[37,38] Further, upon long-term PMFs feeding reduced the expression of inflammatory enzymes (iNOS and COX2) and protein expression of MMP9, VEGF and cyclin D1 was observed.[34]
The deoxypodophyllotoxin from Pulsatilla koreana has antiangiogenic activity in vitro or in vivo.[39] The time required for the exponential growth of CAM with the vasculature (6 cm2 up to 65 cm2) in developing egg cells is 10 days.[40,41] The histological CAM images showed there was devascularisation and reduced inflammatory elements for CAMs submitted to TMF.
VEGF-A-mediated signal transduction pathway involved in angiogenesis/vasculogenesis and mitogenesis during embryonic growth and development.[42,43] VEGF-A has two distinct tyrosine kinase-linked receptors: VEGFR1 and VEGFR2 on endothelial cells. At the cellular level, VEGF-A binds with the VEGFR2, results in MAPK/ERK activation, nitric oxide (NO) production and phosphatidylinositol-3-kinase (PI3K)/Akt activity.[44,45] The oxidative stress by ROS and RNS in mitochondria of tumour cells increases the production of ERK and STAT3 proteins causing inflammation and proliferation.[46,47] The natural polyphenols inhibit ERK protein, causing a reduction in p-STAT3.[48]
The nuclear regulation of ERK signalling with other phosphorylated proteins in the cell is very important to regulate the normal function of cells such as the cell cycle, cell proliferation and cell development.[49,50] Also, researchers expressed there was a possibility that generated ROS and RNS/NO in tumour cells regulate MMP9 expression and/or activation.[51–53] Flavonoids/flavones are direct scavengers of ROS and RNS.[54] Henceforth, it was believed that TMF as a flavone scavenged ROS and RNS free radicals and thereby suppress activation of both STAT3 and MMP9.
Using EAT cells as an ex vivo model, this research observed high-density p-STAT3 and MMP9 in normal control and VEGF-A control CAM. These results suggested that the pSTAT3 regulated MMP-9 production in the carcinoma disease condition, which might be responsible for arteriovenous growth and invasiveness. The results of the immunoblot (Figure 5) of the different groups were observed on the digital images. The positive control group (EAT induced) analysed the upregulation of STAT3, ERK and MMP9 expression and the drug treatment group observed the downregulation of STAT3 leading to suppression of the MMP9 gene. Marked low levels of arteriovenous growth and disorganised endothelial cells surrounding embryo were observed in the TMF- and podophyllotoxin-treated groups, compared to the positive control group (Figure 2).
So the proposed angiogenesis suppressive action TMF is suppression of VEGF-A mediated STAT3-MMP9 signalling pathway.
Conclusion
This research suggested a low dose extract of Tabebuia chrsantha and/or its lead compounds can be used as a novel product to suppress angiogenesis and cell proliferation-associated vascular tumours (angiosarcoma). A flavonoid 4a,5,8,8a-tetrahydro-5-hydroxy-3,7,8-trimethoxy-2-(3,4-dimethoxyphenyl)chromen-4-one (TMF) was isolated from METC and concluded that TMF functions as specific regulators of target protein-associated angiosarcoma. The research data hypothesised about TMF regulation of STAT3-MMP9 signalling may serve a therapeutic marker in controlling angiosarcoma.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest to disclose
Author's contributions
SPP, UPP and DP carried out the design, conduct of the study, ex vivo analysis, chromatographic analysis and wrote the manuscript. All authors read and approved the final manuscript. BRJ, USG collected plant and helped in isolation and animal experiment. CG and DPP co-ordinated the research work.
Funding
This research has no research grant.
Acknowledgements
This research would not be fruitful without the help of KL University and Jadavpur University, Kolkata, India.
