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| RESEARCH ARTICLE |
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| Year : 2022 | Volume
: 54
| Issue : 5 | Page : 329-337 |
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Antitumor effects of polyphenol-rich extract of Euphoria longana seed by vascular endothelial growth factor and transforming growth factor-beta signaling inhibition in experimentally induced oral cancer in rats
Labhu Lagariya1, Kinal Soni2, Jigna Samir Shah2
1 Zydus Corporate Park, Sarkhej, Gandhinagar Highway, Ahmedabad, Gujarat, India
2 Department of Pharmacology, Institute of Pharmacy, Nirma University, Sarkhej- Gandhinagar Highway, Ahmedabad, Gujarat, India
| Date of Submission | 14-Oct-2021 |
| Date of Decision | 07-Oct-2022 |
| Date of Acceptance | 09-Nov-2022 |
| Date of Web Publication | 13-Dec-2022 |
Correspondence Address:
Jigna Samir Shah
Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad - 382 481, Gujarat
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijp.ijp_802_21
ETHNOPHARMACOLOGICAL RELEVANCE: Oral cancers are found to have high risk in South Central Asia due to exposure of various risk factors. Euphoria longana Lam. (EL) has been traditionally used to relieve insomnia, prevent amnesia, and treat palpitation. In addition, EL has been reported to have anti-inflammatory, anti-cancer, and antioxidant activities. The investigation was aimed to evaluate the mechanism of action and antitumor activity of polyphenol-rich EL seeds extract against oral cancer induced by 4-Nitroquinoline-1-oxide (4-NQO).
MATERIALS AND METHODS: Seven groups of Sprague–Dawley rats were formulated: normal animals, oral cancer induced with 4-NQO, EL-treated normal control, EL-treated disease control from 0-day, EL-treated disease control from 60 days, 5-fluorouracil (5-FU)-treated disease control from day 60, and combined EL- and 5-FU-treated disease control animals from day 60. The animal tongue was smeared with 0.5% 4-NQO at frequency of thrice a week for 12 weeks to induce oral cancer. At the end of treatment, excised tongues were used for biochemical and tumour-specific parameters along with histopathology assessment.
RESULTS: Treatment with EL, 5-FU, and combination of both in diseased animals exhibited significant improvement in interleukin-6, vascular endothelial growth factor (VEGF), and Transforming growth factor beta (TGF-β) levels, antioxidant status together with histoarchitecture of the tongue tissue. In addition, the combination of both was slightly more effective than EL and 5-FU alone.
CONCLUSION: Our data suggest antitumor activity of Euphoria longana Lam. Extract against 4-NQO induced oral cancer in rats, which could be attributed to alteration in the VEGF and TGF-β signaling axis.
Keywords: 4-Nitroquinoline-1-oxide, 5-fluorouracil, Euphoria longana Lam, oral cancer, transforming growth factor beta, vascular endothelial growth factor
How to cite this article:
Lagariya L, Soni K, Shah JS. Antitumor effects of polyphenol-rich extract of Euphoria longana seed by vascular endothelial growth factor and transforming growth factor-beta signaling inhibition in experimentally induced oral cancer in rats. Indian J Pharmacol 2022;54:329-37 |
How to cite this URL:
Lagariya L, Soni K, Shah JS. Antitumor effects of polyphenol-rich extract of Euphoria longana seed by vascular endothelial growth factor and transforming growth factor-beta signaling inhibition in experimentally induced oral cancer in rats. Indian J Pharmacol [serial online] 2022 [cited 2023 Mar 27];54:329-37. Available from: https://www.ijp-online.com/text.asp?2022/54/5/329/363398 |
| » Introduction | |  |
Head-and-neck cancer has been described as an uncontrolled and abnormal cell growth along with injuries in pharynx, nasal cavity, oral cavity, larynx, and paranasal sinuses. Among these, 90% oral cancer patients are of Oropharyngeal squamous cell carcinoma (OSCC).[1] According to the WHO, there are 657,000 new cases and more than 330,000 mortality was reported of oral cancer and pharynx every year. Oral cancers comprise the foremost subsites of the oral cavity, lip, pharynx as well as nasopharynx and found to have high risk in South Central Asia due to the exposure of risk factors. GLOBOCAN 2018 worldwide estimates 354,864 new cases of lip and oral cancer out of which 246,420 are male and 108,444 are female. Furthermore, 177,384 deaths are estimated from which 119,693 are male and 57,691 are female. The incidence rate is more than double in men as compared with women.[2] Among blacks, the incidence rate is found to be reduced by 2.3% every year whereas is augmented by 1.2% every year amongst whites from the year 2006 to 2015. The death rate of the pharynx and oral cavity cancers has augmented by 1% every year from the year 2012 to 2016.[3] Major risk factors of OSCC include smoking habit, alcohol and tobacco consumption, betel quid and areca chewing as well as human papillomavirus infection.[4],[5],[6]
Vascular endothelial growth factor (VEGF) is an angiogenic factor which plays a significant role in the p of angiogenesis in oral cancer development. Tissue inhibitors of metalloproteinases prevent VEGF binding to VEGF Receptor 2 and thereby inhibit VEGF-induced signaling and angiogenesis. The release of other angiogenic factors is facilitated by the binding of heparin-binding VEGF forms to cell surface and proteoglycans extracellular matrix (ECM). Rise in microvessel density is correlated with the growth in VEGF expression by oral squamous cell carcinoma.[7] Various inflammatory cytokines and pro-inflammatory mediators such as tumor necrosis factor-1α (TNF-1α), interleukin-1 β (IL-1 β), hypoxia-inducible factor-1α, IL-6, and cyclooxygenase-2 are released due to hypoxic cell conditions caused by genetic alteration which ultimately which lead to chronic inflammation in the tissues causing ECM degradation.[8]
Various diagnostic techniques as well as treatment options are available for treating head and neck cancer including chemotherapeutics agents as well as herbal compounds. Among which, phenolic compounds have been found to be effective as anti-cancer agents. Polyphenols are obtained primarily from plants. Various herbal chemotherapeutic drugs have been extracted from Chinese medicines such as polyphenols, vincristine, vinblastine, and camptothecin, and studied for various kinds of cancer. Longan flowers and seeds have also been found to be rich in polyphenols, epicatechin, gallic acid, proanthocyanidin, and ellagic acid.[9] Dimocarpus Longan (Euphoria longana Lam [EL]. [Lour.]) belongs to lychee family, Sapindaceae, and found in subtropical climate regions.[10] It has been used as traditional Chinese medicine for stomachic, vermifuge, febrifuge and as an antidote to poison as well as for insomnia, forgetfulness, and palpitation.[11] Reports also possess the beneficial effects in the treatment of cancer, diabetes, hernia, eczema, acariasis, insomnia, hemorrhages, scrofula, and fatigue.[12] Apart from this, EL fruit pulp is used in food and beverages industry for the production of canned syrup and juice. Seeds obtained from the pulp are also utilized for beneficial health product development.
The longana seeds are rich in polyphenols as compared with the fruits. Various phenolic composites such as corilagin, gallic acid, isomallotinic acid, ellagic acid, isomallotinic acid, 4-O-a-L-arabinofuranoside, geranin, methyl brevifolin, A-type proanthocyanidin trimer, (−)-epicatechin, and chebulagic acid are present in longana seeds. Ellagic acid, gallic acid, and corilagin [Figure 1] are the main phenolic excipients from the stated compounds. Due to the rich polyphenols, longana seeds exhibit antioxidant properties. | Figure 1: Chemical structures of Ellagic acid, Gallic acid and Corilagin
Click here to view |
These seeds have been reported to have 2,2-diphenyl-1-picrylhydrazyl scavenging and anti-matrix metalloproteinase activity.[13] Apart from antioxidant and anti-cancer properties, gallic acid and elagic acid also possess anti-plasmodial and antimicrobial activities by blocking nuclear factor kappa-B activation (nF-kB).
Corilagin is an ellagitannins, stated to possess immunomodulatory, anti-inflammatory, antifungal, activities, and anticancer effects on lung, colon, cervical, and hepatic cancer cells in vitro.[14] Evidences indicated that corilagin exerts anticancer effects by targeting Transforming growth factor beta (TGF-β)/AKT/ERK/Smad pathway in ovarian cancer cells by reducing cell growth. Furthermore, other in vitro cell line studies on Hey, SKOv3ip, and HO-8910PM-Snail concluded the cell growth inhibition at G2/M phase.[15] It also inhibits NF-kB translocation by reducing pro-inflammatory cytokines and mediators at genetic and protein levels.
Further, the water-soluble longana seed polysaccharide fraction showed antiproliferative effects on the A549 lung cancer cell line by increasing the release of lactate dehydrogenase. The cell cycle analysis of the longana extract also demonstrated the increased cell cycle arrest in G1 phase. Increased S phase arrest was also observed through cleavage of Poly-ADP ribose polymerase (PARP) and activation of caspase 3 as well as caspase 9 in A549 cells.[16]
In the light of the above reports, the study was conducted to assess the antineoplastic activity of Euphoria longana Lam.(EL) seed extract in 4-Nitroquinoline-1-oxide (4-NQO)-induced oral cancer and investigate the downstream signaling pathways involved in the same.
| » Materials and Methods | | |
Materials
4-NQO (≥98% purity) was purchased from Sigma-Aldrich. Fruits of Euphoria longana Lam. Were bought from Millennium Farm Fresh, Mumbai. 5-fluorouracil (5-FU) was received as a gift sample from Sun Pharma, Vadodara. 0.5% of propylene glycol along with 4-NQO was smeared on the tongue with the use of paint brush (size-number 4) to induce oral cancer.
Extraction of the seed
Authentication of the fruits of Euphoria longana Lam. was carried out at the Department of Botany, Gujarat University, Ahmedabad, Gujarat, India. Fresh fruits of longana were used for pharmacognostic evaluation and some preliminary phytochemical tests. The seeds of longana fruits were dried at 75°C for 24 h using hot air oven. Dried seeds were crushed and blended to get a fine powder. Obtained fine powder was extracted using hot water at 70°C–75°C for 1 h. This procedure was repeated thrice at ratio of 1:4 w/v. The obtained extract was concentrated and put on a vacuum rotary evaporator to get polyphenols yield in dry form at 50 rpm, 50 mbar pressure, and 55°C.[17] For the estimation of polyphenols, thin-layered chromatography (TLC) was performed using the benzene: methanol: formic acid (8:3:2 v/v) as mobile phase.
Phytochemical analysis
For the estimation of phenol, carbohydrate, flavonoids, proteins, tannins, glycoside, steroids, alkaloids, and saponins preliminary identification test of Euphoria longana Lam. Extract was carried out. TLC was performed for qualitative analysis of longana extract. The tests were performed for flavonoids, carbohydrates, tannins, proteins, steroids, alkaloids, saponins, and phenolics compounds to confirm the presence of various phytochemical moieties using the yield of acetone fraction.
Experimental protocol
According to Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines, Ministry of Social Justice and Empowerment, Government of India, the study protocol was approved by Institutional Animal Ethics Committee. IP/PCOL/MPH/15-1/003.
Animals
Sprague–Dawley male albino rats aging 9 weeks and weighing 300–400 g (n = 42) were selected for the study. All controlled conditions such as temperature (23°C ± 2°C), humidity (55% ± 5%), and 12 h light and dark cycle were maintained and laboratory conventional diet (Pranav Agro Private Limited) ad libitum was provided along with ultraviolet-filtered water.
Oral cancer induced by 4-nitroquinoline-1-oxide
4-NQO acts as carcinogen by binding to the guanine residue of DNA through causing intracellular oxidative stress. This leads to reactive oxygen species (ROS) production such as superoxide dismutase and hydrogen peroxide causing tumor progression. Oral cancer occurs by 4-NQO shows similarity to tobacco-induced cancer. Apart from the similarity in presence of carcinogen, histological and molecular changes are also similar between oral cancer induced by 4-NQO and human oral carcinogenesis.
After 5 days of acclimatization, standard deviation (SD) rats were grouped into seven different sets based on the treatment regime. These groups were as follows: Group I normal control animals (NC), Group II 280 mg/kg longana-treated animals neuronal ceroid lipofuscinoses, Group III diseased control animals in which oral cancer was induced using 4-NQO (DC), Group IV 280 mg/kg longana-treated diseased animals from day 0 (DCL 0), Group V 280 mg/kg longana-treated diseased animals from day 60 (DCL 60), Group VI standard-treated animals in which diseased rats were gavage with 24.5 mg/kg 5-FU (DS), Group VII diseased animals treated with a combination of 280 mg/kg longana and 24.5 mg/kg 5-FU, respectively, from day 60 to 30 days. All the treatments were administered in rats by oral gavage (P.o). Oral cancer was induced by spreading 0.5% 4-NQO in propylene glycol using soft and small paint brush on the tongue with frequency of thrice a week for 12 weeks as the latency period for induction of oral cancer by 4-NQO is 2 months. Furthermore, vehicle-controlled animals were treated with only propylene glycol thrice a week for 12 weeks. The treatment was started after that based on each treatment regime. Human doses of 5-FU and longana were converted to animal doses considering factors of body weight and surface area.
Oxidative stress parameters
On completion of treatment, rats were euthanized and tongues were removed to perform various parameters such as oxidative stress parameters, tumor-specific parameters, and histopathological study.
Malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) estimations were carried out from homogenate of excised tumor. Homogenate was prepared using 0.01 M Tris-HCL buffer having pH 7.4. MDA levels were estimated by mixing 0.2 ml homogenate with 8% sodium lauryl sulfate (SLS) with equal quantity of homogenate. The thiobarbituric acid (1.5 ml in 1% tris HCL), acetic acid (1.5 ml in 20% HCL) and distilled water were added to the mixture of homogenate and SLS. The whole mixture was heated at 95°C for 45 min and cooled down to room temperature. n-butanol and pyridine in ration of 15:1 were added to the entire mixture and absorbance was read against 532 nm. GSH levels were estimated using Moron et al. 1979 method.[18] According to the method, the homogenate was mixed with 10% trichloroacetic acid (1 ml) and kept at water bath for 30 min. After 30 min, mixture was centrifuged at 3000 rpm for 10 min at 4°C and supernatant was collected. 0.3 M disodium hydrogen phosphate (2 ml), 5, 5'-dithiobic-(2-nitrobenzoic acid) (0.04%), and freshly prepared DTNB in 1% sodium citrate were then mixed with supernatant and read against blank at 412 nm. Likewise, SOD was estimated by mixing supernatant with 0.001 M EDTA, carbonate buffer having pH 9.7, and 0.003 M epinephrine by reading absorbance against blank at 480 nm till 3 min at each 30 s intervals. Quantitative estimation of rat IL-6 was performed in vitro using rat IL-6 ELISA kit at 450 nm.
Tumor-specific parameters
Tumor expressions were evaluated by the expression of VEGF and TGF-β. Briefly, TGF-β levels were estimated using ELISA assay kit. The assay was performed using the quantitative sandwich enzyme immunoassay technique. Here, the substrate was added after conjugation of antibody with reporter enzyme, and intensity of the developed color was measured. VEGF rat ELISA kit was used to measure the amount of VEGF bound to the substrate. The color developed was measured at 450 nm.
Histological examination
Changes in the layers such as mucosa, sub-mucosa, papillae, and core of the muscular layer of the tongue were examined using histopathological study.
Statistical analysis
The results were analyzed and evaluated through one-way ANOVA following Tukey's multiple comparison test and presented as mean ± standard error of mean Statistically significant value was considered as P < 0.05 for all the data.
| » Results | | |
Phytochemical analysis
Phytochemical analysis resulted in the absence of flavonoid, carbohydrate, tannins, saponins, and phenolic compounds in polyphenol rich fraction of E. longana.
Qualitative estimation of major chemical constituents
Merck silica gel 60 F254 and mobile phase as benzene: methanol: formic acid (8:3:2 v/v) was used to perform TLC of Polyphenol-rich fraction (2 mg/ml). After the development of spots, plate was removed and read at 283 nm. The sample showed the presence of corilagin, gallic acid, and ellagic acid at Rƒ value 0.73, 0.44, as well as 0.63 correspondingly as shown in [Figure 2]. | Figure 2: Qualitative estimation of major chemical constituent using TLC
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Effect of treatment on 4-nitroquinoline-1-oxide induced oral cancer on body weight, water intake, and food intake
Prominent reduction in body weight, water intake, and food intake was observed in diseased rats as compared to normal rats at the end of 12 weeks due to 4-NQO administration. Body weight, water, and food intake were significantly lowered in longana, 5-FU alone and combined 5-FU and longana-treated groups when compared with diseased animals. Minor increase water intake, food intake, and body intake were observed in the animals treated with longana (280 mg/kg) from 0 day. Reduction in water intake, food intake and body weight were observed in combined treatment of EL with 5-FU groups as compared to each individual treatment groups [Figure 3], [Figure 4], [Figure 5]. | Figure 3: Effect of Euphoria longana, 5-Fluorouracil and combination of Euphoria longana with 5-FU on body weight in oral cancer induced by 4-NQO in rats NC- Normal control group, NCL- Normal control treated with 280 mg/kg longana only, DC- Disease control group, DCL 0 - Disease control treated with longana 280 mg/kg from day 0, DCL 60 - Disease control treated with longana 280 mg/kg from day 60, DS- Disease control group treated with the dose of 24.5 mg/kg 5-fluorouracil, DSL- Disease control group treated with the dose of 24.5 mg/kg-fluorouracil and 280 mg/kg longana.
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 | Figure 4: Effect of Euphoria longana, 5-Fluorouracil and combination of Euphoria longana with 5-FU on food intake in oral cancer induced by 4-NQO in rats. NC- Normal control group, NCL- Normal control treated with 280 mg/kg longana only, DC- Disease control group, DCL 0 - Disease control treated with longana 280 mg/kg from day 0, DCL 60 - Disease control treated with longana 280 mg/kg from day 60, DS- Disease control group treated with the dose of 24.5 mg/kg 5-fluorouracil, DSL- Disease control group treated with the dose of 24.5 mg/kg-fluorouracil and 280 mg/kg longana.
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 | Figure 5: Effect of Euphoria longana, 5-Fluorouracil and combination of Euphoria longana with 5-FU on water intake in oral cancer induced by 4-NQO in rats. NC- Normal control group, NCL- Normal control treated with the dose of 280 mg/kg longana only, DC- Disease control group, DCL 0 - Disease control treated with longana 280 mg/kg from day 0, DCL 60 - Disease control treated with longana 280 mg/kg from day 60, DS- Disease control group treated with the dose of 24.5 mg/kg 5-fluorouracil, DSL- Disease control group treated with the dose of 24.5 mg/kg-fluorouracil and 280 mg/kg longana
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Oxidative stress parameters
Substantial (P < 0.05) rise in MDA levels was detected in the diseased animals as compared with the normal animals at the end of 12 weeks. However, MDA levels were prominently reduced (P < 0.05) in the 280 mg/kg longana-treated animals, 24.5 mg/kg treated animals and combined-treated animals when compared with DC animals. Further, in animals treated with longana together with 5-FU group, substantial reduction in levels of MDA was observed as compared with the animals treated with 5-FU and longana alone group [Table 1]. On the contrary, a significant (P < 0.05) rise of SOD and GSH was observed in the animals treated with 4-NQO and Euphoria longana Lam. (280 mg/kg) group, 24.5 mg/kg 5-FU along with combined longana and 5-FU treatment as compared to disease control animals. However, GSH and SOD levels were reduced considerably (P < 0.05) in diseased animals as compared with normal animals. While the treatment with combination of EL with 5-FU showed slightly high levels of GSH as compared with animals treated with 5-FU alone and longana alone with no significant increase in SOD levels [Table 1]. | Table 1: Effect of Euphoria longana Lam. 5-Fluorouracil and combination of both (Euphoria longana Lam. + 5-fluorouracil) on malondialdehyde, reduced glutathione, super oxide dismutase and interleukin-6 levels in oral cancer induced by 4-nitroquinoline-1-oxide in rats
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Inflammatory marker interleukin-6
Disease control animals showed a significant rise in IL-6 levels as compared to normal animas. Significant reduction in the IL-6 levels was observed in Euphoria longana Lam. (from 0 day of induction), Euphoria longana Lam. (from 60 days of induction), 5-FU, and combined therapy of EL as well as 5-FU-treated animals than disease control animals. Overall, Euphoria longana Lam.-treated animals exhibited significant declining phenomena from 0 to 60th day of oral cancer induction [Table 1].
Tumour-specific parameters
Vascular endothelial growth factor level
VEGF levels (P < 0.05) were prominently higher in disease-control animals than NC animals. Furthermore, a significant decrease was observed in individual therapy of 24.5 mg/kg 5-FU and 280 mg/kg longana-treated animals when compared with diseased animals. Likewise, animals treated with combined therapy of 5-FU and longana showed high reduction in VEGF levels when compared with animals treated using alone therapy of 5-FU and longana [Table 2]. | Table 2: Effect of Euphoria longana Lam. 5-fluorouracil and combination of both (Euphoria longana Lam. + 5-fluorouracil) on vascular endothelial growth factor levels and transforming growth factor β levels in 4-nitroquinoline-1-oxide induced oral cancer in rats
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Transforming growth factor beta
Diseased animals had significantly higher (P < 0.05) TGF-β levels than NC animals. Whereas, animals treated with alone therapy of 5-FU (24.5 mg/kg) and longana (280 mg/kg) had decreased TGF-β levels than diseased animals. Likewise, combined-treated animals showed high reduction in TGF-β levels as compared to animas treated with alone therapy of 5-FU and Longana [Table 2].
Histopathological study
The histopathological evaluation of excised tongue of SD rats was carried out and mucosa, submucosa, papillae, and core of muscular layer were observed at ×100. Inflammation and mucosal damage were not observed in normal animals [Figure 6]a, however, severe and moderate dysplasia was observed only in oral cancer-induced animals [Figure 6]b Significant reduction in mucosal damage was observed with longana-treated animals from day 0 as compared with animals treated with longana from day 60 [Figure 6]c and [Figure 6]d. In addition, 5-FU (24.5 mg/kg)-treated animals showed reduced dysplasia as compared with disease-control animals [Figure 6]e. Furthermore, animals treated with 5-FU and longana combination showed higher mucosal damage and dysplasia when compared with alone therapy of 5-FU- and longana-treated animals [Figure 6]f. | Figure 6: Histopathological examination of rat tongue (a) Normal control, (b) Disease control, (c) Disease control treated with EL from day 0, (d) DC treated with EL from day 60, (e) Disease control treated with 5-Fluorouracil from day 60, (f) Disease control treated with longana + 5-FU from day 60.
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| » Discussion | | |
The polyphenolic fraction of longana mainly comprises of polyphenols such as corilagin, gallic acid, and ellagic acid. Seeds of Longana have been used from a long time for the treatment of acariasis, eczema, hernia, and wound hemorrhages. Along with the seed, fruits have also shown beneficial effects on spleen, heart, and the nervous system. Dried fruits have also been considered as tonic for the treatment of neurasthenic neurosis as well as insomnia when used in the form of decoction.[11] A toxicological study of longan seed extract was also carried out which resulted in showing no toxicity and considered to be safe.[18]
Euphoria longana Lam. Seed extract is reported to possess memory-enhancing activity which may be attributed to increase in pCREB, BrdU, BDNF, and DCX expression within the hippocampus. Signaling mediators such as phospholipase C gamma, ERK, and phosphatidyl-inositol 3 kinase are stimulated by binding of BNDF and its receptors. Immunomodulatory effect of longana has been reported due to polysaccharide-protein complex (LP3) formation.[19] It has also been reported that longana down-regulates the pancreatic lipase activity as well as fatty acid synthase (FAS) (sterol regulatory element that binds to FAS) and SREBP-1c (Protein-1c) gene expression showing hypolipidemic effect.[20] Seed extract of longana has also been used for the treatment of yeast infection as an antifungal agent.[21]
Antiproliferative effect was produced by the water-soluble extract of longana on A549 lung cancer cell line. An early mitochondria apoptosis was also triggered by longana in A549 cells causing G1 phase arrest, PARP cleavage and caspase 3 and caspase 9 activation.[16] Apart from these, apoptosis was also observed in various cell lines such as C33A, MDA-MB-231, and Hep G2 cell line of cervix, breast, and hepatic-cellular carcinoma, respectively. S-phase amplification was also observed in C33A cells treated with LSE and MDA-MB-231 cells.[22] Phenolic compounds present in longana seeds such as ellagic acid and gallic acid have acted as significant angiogenic inhibitors and shown inhibition on VEGF activity by interrupting VEGF secretion as well as expression of SW480 cells along with abrogate the gelatinase activity of cell proliferation and HUVECs tube formation.[23] Khan et al. synthesized gold nanoparticles (AuNPs) using fruit juice of longan as a capping, stabilizing, and reducing agent and found that these AuNPs exhibited dose-dependent cytotoxicity when tested in MCF-7 cell line and depicted significant fluorescence emission and antioxidant activity. Further, the capping of AuNPs also assisted Bax (pro-apoptotic protein) and Bcl-2 (anti-apoptotic protein) regulation.[24]
Considering the above facts, our study was designed to estimate the anticancer action in seed extract of longana by investigating the mechanism of action using experimental model of chemical-induced oral cancer in rats.
Initially, the presence of active chemical constituents gallic acid, ellagic acid, and corilagin were confirmed by performing preliminary confirmational tests as well as by performing TLC. The dark spots for these polyphenols were obtained on TLC at Rf 0.63, 0.73, and 0.44, respectively. 5-FU has been standard drug of choice for the treating oral cancer. Hence, animal dose of 5-FU was selected as 24.5 mg/kg on which tumor growth was inhabited in rats. Ellagic acid and Gallic acid are stated to have VEGF inhibitory activity[25],[26] while corilagin possesses TGF-β inhibitory activity.[15] In our study, we found that growth factors such as VEGF and TGF-β are effectively down-regulated by longana alone and when used in combination with chemotherapeutic agent. Longana also contains minor amount of geranin, a type of polyphenol, which is reported to exhibit inhibitory effect on NF-kB. Geraniin in combination with corilagin showed NF-kB blockade in skin cancer.[15] This showed that polyphenols reduce degree of resistance when given as adjuvant with other chemotherapeutic agent.
Cancer development leads to the increase in ROS-free radical formation. Due to higher ROS, more oxidative stress occurs to the cells and MDA levels increased. 4-NQO is a potent carcinogen which causes intracellular oxidative stress as well as metabolic product of 4-NQO binds to guanine residues of DNA. Molecular and cellular damage caused by carcinogen caused by tobacco and 4-NQO are similar.[27]
Various defensive mechanisms are involved in the generation of free radicals such as GSH, SOD, and MDA. In cancerous condition, levels of GSH and SOD decrease whereas MDA levels increases. Reports have also concluded that a significant reduction in MDA levels as well as increase in SOD, GSH, GSH-Px, TNF, MPO, and IL-1 levels were observed in the brain tissue of focal cerebral ischemia/reperfusion injury treated by longana seed extract. These effects were mainly observed due to the availability of polyphenols ellagic acid and gallic acid in seed extract. The treatment of longana also leads to substantial blockage of NO generation caused by inhibition of histamine release and generation of pro-inflammatory cytokines.[28] Results from our study also depicts decreased SOD and GSH activity as well as increased MDA levels in the oral cancer induced by 4-NQO in rats. MDA levels were reduced significantly in the animals treated with longana alone as well as combined therapy with 5-FU at the end of the 12th week. Furthermore, higher oxidative stress due to low SOD and GSH levels as well as high MDA level was observed in the disease-control animals. On the contrary, GSH levels and SOD levels were increased significantly and levels of MDA were significantly lower in the animals treated with alone longana and combined with 5-FU. These results suggest that longana plays a protective role against the generation of free radical.
As IL-6 is inflammatory marker, serum level of IL-6 increases due to severe inflammation and cell necrosis. In addition to this, tumor IL-6 expression to both mRNA and protein levels are also directly correlated with a higher tumor stage. Hence, when treated with an extract of Euphoria longana Lam., the level of IL-6 was decreased significantly from 0 to day 60. No bleeding was observed in 4-NQO-induced oral cancer animals for the first 2 weeks however, mild bleeding was observed at the end of 3 weeks. Further, diseased animals treated with longana extract (280 mg/kg) from day 0 showed no bleeding even after 30 days. This indicated protective effect of longana suggesting the use as a preventive therapy for oral cancer.
NC group was compared with DC group and results from out showed increased VEGF levels while VEGF levels were found to be decreased in 5-FU (24.5 mg/kg), longana (280 mg/kg) as well as combined therapy-treated animals. VEGF levels were further reduced in 5-FU and longana combined-treated animals. This result is indicative of protective role of longana in the oral cancer which might be due to the inhibition of VEGF signaling axis. Furthermore, it is suggestive that resistance of the chemotherapeutic drug can be reduced or delayed using longana if given in the combination with another chemotherapeutic agent. TGF-β signaling plays an essential role for the development and progression of oral cancer. Based on cell condition, role of TGF-β changes. In normal cell it plays the role of tumor suppressor while in the cancerous process it plays tumor promoter role. Hence, targeting TGF-β inhibition might act as a protective effect in the treatment of oral cancer. In the current study, DC animals showed increased levels of TGF-β as compared to the NC animals. On the other side, TGF-β levels were decreased in animals treated with alone as well as combination therapy of 5-FU (24.5 mg/kg) and longana (280 mg/kg). These results evidenced the reduction in tumor progression by inhibition of TGF-β. However, reduction in the chemotherapy-induced drug resistance might be observed if longana was combined with another chemotherapeutic agent.
From the NC group, the histopathological analysis of the rat tongue showed no inflammation and damage in papillae, submucosa, mucosa and muscular core layer, and hyperplastic lesions. However, moderate and severe dysplasia was observed in tongue of oral cancer-induced animals. Significant mucosal damage reduction was observed combined as well as alone therapy of 5-FU and longana-treated animals. Whereas, combined therapy of 5-FU and longana treated animals exhibited further reduction in dysplasia and mucosal damage when compared with alone therapy of 5-FU and longana.
| » Conclusion | | |
It has been concluded from the data that longana and 5-FU combination showed a prominent effect in controlling oral cancer by reducing the oxidative stress, inflammatory, and tumor markers levels. TGF-β and VEGF levels inhibition depicts anti-cancer effect. Thus, the polyphenol-rich extract of Euphoria longana Lam. seeds can be used as an adjuvant treatment regime with 5-FU or in combination with other chemotherapeutic agents for the treatment of oral cancer and thereby improving the quality of life for cancer patients.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]
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