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RESEARCH ARTICLE
Year : 2020 | Volume
: 52
| Issue : 6 | Page : 488-494
Antiproliferative effect of Acacia nilotica (L.) leaf extract rich in ethyl gallate against human carcinoma cell line KB
Kalaivani Thiagarajan1, Shalini Mohan1, Tapas Kumar Roy2, Rajasekaran Chandrasekaran1
1 Department of Biotechnology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India
2 Division of Plant Physiology and Biochemistry, Indian Institute of Horticultural Research, Bengaluru, Karnataka, India
Correspondence Address:
Dr. Rajasekaran Chandrasekaran
Department of Biotechnology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore - 632 014, Tamil Nadu
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijp.IJP_223_17
OBJECTIVES: The objective of this study is to analyze the antiproliferative activity of Acacia nilotica (L.) leaf ethanolic extract against cancer KB cells and to determine the mode of cancer cytotoxicity.
MATERIALS AND METHODS: In this study, high-performance liquid chromatography and liquid chromatography-mass spectrometry analysis were done to confirm the presence of ethyl gallate as a major bioactive phenolic in the leaf ethanolic extract of A. nilotica, further dose-dependent (0–120 μg/mL) antiproliferative effect was investigated in human carcinoma cell line KB. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, reactive oxygen species, mitochondrial membrane potential loss, DNA damage, and apoptosis were evaluated.
RESULTS: A. nilotica leaf ethanolic extract (ANLEE) showed effective concentration (EC50) of 40 μg/mL. Interference of growth was significantly (P < 0.05) high in KB cells treated with ANLEE when compared to untreated control, but less when compared to the reference drug paclitaxel. In addition, the in vivo acute toxicity study demonstrated the safe limit of administration of 2000 mg/kg body weight ANLEE by the histological analysis in rats. The results from the present study indicate that mitochondria and DNA of KB cells are severely affected leading to apoptosis.
CONCLUSIONS: ANLEE is a prospective source for cancer therapy and therefore should be highlighted to explore on its wide range of safety in rats and efficacy against human carcinoma cell line KB.
Keywords: Acacia nilotica (L.), cancer, cytotoxicity, ethyl gallate, liquid chromatography-mass spectrometry
Oral cancer is a predominant carcinoma, prevalent across India as well as throughout the globe due to etiological factors such as smoking, drinking, tobacco, and betel chewing.[1],[2] About 350,000 new cases are diagnosed each year globally. Even after advanced treatments, survival of the oral cancer patients has never improved.[3] As a result, it is critical to find therapeutics that can act as monotherapy or in combination with other anticancer drugs for the treatment of cancer.
Currently, available anticancer drugs produce many serious adverse reactions. Hence, there is a desire to find newer anticancer compounds that are having a better safety profile. Complementary and alternative medicine uses traditional medicinal plants as an alternative for treating various diseases, including cancer with less side-effects, unlike synthetic drugs. Hence, plants that are considered safe are investigated for screening anticancer drugs and numerous anticancer compounds from plants are used as therapeutics such as taxanes, vinca alkaloids, and camptothecin.[4]
Acacia nilotica (L.) commonly known as babool tree belonging to the family Fabaceae and subfamily Mimosoideae are widely distributed in tropical and subtropical countries. Leaves of A. nilotica were used to treat mouth, skin, and bone cancers by traditional healers, across Chhattisgarh, India. The bark and gums are also used for treating cancers of ears, eyes, or testicles in different parts of Africa.[5]Acacia spp. is the source of numerous bioactive principles such as gallic acid, kaempferol, naringenin, and catechin to name a few.[6]
Plant extracts rich in phenolics act as antioxidants inside a normal cell and enhances free radical generation in cancer cells. This dual behavior has been well documented for plant extracts or plant-derived phenolics in different cancer cell lines.[7] These plant-derived phenolics damage DNA through oxidative damage leading to cell death in cancer cells. Our previous studies revealed that A. nilotica leaf ethanolic extract (ANLEE) has cytotoxic effect on HeLa cells.[5] The plant also reported to possess anticancer activity on the solid tumor model of Dalton's ascitic lymphoma.[8] Ethyl gallate is a major bioactive phenolic from ANLEE reported to act against human carcinoma cell line KB.[9],[10] Therefore, the present study was aimed to investigate the antiproliferative property of ethyl gallate obtained from the leaf ethanolic extracts of A. nilotica against KB cells with reference to paclitaxel.
Ethyl gallate and paclitaxel are also reported to act in the same manner in cancer cells.[9],[10] On this basis, an effort has been made to investigate the antiproliferative activity of ANLEE against human carcinoma cell line KB using paclitaxel as a reference.[10],[11] As KB cells can rapidly grow to overcome the effect of some anticancer drugs, paclitaxel is an exception with effective growth inhibition.[10] This study will also provide information on anticancer activity of ethyl gallate when present along with other ingredients of ANLEE.
Materials and MethodsPlant extract and phytochemical analysis
Tender leaves of Acacia nilotica (L.) Wild. ex. Delile subsp. indica (Benth.) Brenan was gathered at the vicinity of Vellore Institute Technology, Vellore, Tamil Nadu, India. Dr. G.V.S. Murthy, BSI-SRC, Coimbatore, has identified and validated (Voucher number: 1035). The air-dried leaves were powdered and extracted with 100% ethylated spirit[11] in a Soxhlet apparatus has given 40% yield.
High-performance liquid chromatography analysis
High-performance liquid chromatography (HPLC) analysis was performed to identify ethyl gallate in leaf ethanolic extract.[12] The results were verified with standard ethyl gallate (retention time, 8.58) at 291 nm [Figure 1].
Figure 1: High-performance liquid chromatography chromatogram of ethyl gallate in Acacia nilotica leaf ethanolic extract at 291 nm (retention time 8.3)Liquid chromatography-mass spectrometry analysis
ACQUITY UPLC-BEH C18 column 1.7 μm (2.1 mm × 50 mm) was used at 25°C ± 1°C. The mobile phase contains 0.5% formic acid in methanol (A) and 0.25% formic acid in acetonitrile (B), respectively, in the ratio of A: B (5:95) with an isocratic flow rate of 0.1 mL/min. Standard and ANLEE were dissolved in A and B in the ratio of 1:1 of 10 mL and diluted to 1:100. 4 μL samples were injected after filtering in a 0.2 μm nylon filter.[13] Data were analyzed using MassLynx software (Waters Corporation, Milford, MA, USA).
Maintenance of cell cultures
KB cells were cultured in Dubelco's Modified Eagle's Medium complemented with 10% fetal bovine serum, 1% glutamine, penicillin (100 U/mL), and streptomycin (100 U/mL) and kept at 37°C in 5% CO2 atmosphere. After reaching 80% confluence, KB cells were planted at 5 × 104 cells/well in 24-well plates earlier to the exposure of test substances.[10] 1 mg/mL stock of ANLEE or 0.5% of paclitaxel was prepared in dimethyl sulphoxide (DMSO) (w/v) and kept at 4°C until use.
Cell viability assay
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is colorimetrically evaluated for cell viability.[14] KB cells were introduced in 24 well plates at 5 × 104 cells/well prior to exposure of ANLEE and paclitaxel (0–120 μg/mL). Cells with 0.01% DMSO served as untreated control. MTT was added to KB cells after 24 h of exposure to test substances and kept undisturbed for 4 h. Then, formazan crystals were dissolved in 200 μL of DMSO, and OD was measured at 570 nm. The percentage cell viability and percentage cytotoxicity were calculated using the following formulae: Cell Viability = (Abs @570nm [test/control] × 100) % Cytotoxicity = (1 – [test/control]) × 100. The optimal doses of ANLEE for further parameters were taken based on the IC50 values obtained.
Intracellular reactive oxygen species analysis
The amount of reactive-oxygen species (ROS) generated by KB cells was measured using dye DCFH-DA)[10]. KB cells (5 × 104 cells/well) were exposed to ANLEE (0–50 μg/mL) and paclitaxel (20 μg/mL) selected from the MTT assay. After 24 h of treatment, KB cells were suspended in phosphate buffer saline (PBS) and nurtured with 13 mM DCFH-DA for 30 min at 37°C. Excess dye was removed using PBS, and the fluorescence was measured and quantified with spectrofluorimeter, and the images were documented under the fluorescence microscope (450–490 nm).
DNA damage analysis
DNA damage was determined by comet assay.[15],[16] KB cells (50 μL) treated with ANLEE and paclitaxel for 24 h were mixed with 1% low melting point agarose (200 μL) and overlaid on glass slides coated with agarose (1%). It is kept in cold Tris-NaCl lysis buffer of pH 10 under 4°C for 1 h. Slides were treated with an alkaline buffer of pH 13 for 25 min, followed by electrophoresis in the fresh buffer for 20 min. Neutralized using tris buffer (0.4 M, pH 7.5) 5 min and stained with EtBr, further visualized by an epifluorescence microscope. Images were captured and analyzed with the comet assay software project (CASP).[17] For the visual classification and quantification of DNA damage, 100 cells were chosen to measure the tail length, tail moment, and olive tail moment.[17]
Mitochondrial membrane potential loss
A lipophilic cationic dye, rhodamine-123, is extremely precise to mitochondria and generally used to measure the membrane potential (MMP) loss.[18] The KB cells (5 × 104 cells/well) were exposed to 20, 30, 40, and 50 μg/mL leaf extract of A. nilotica and 20 μg/mL of paclitaxel for 24 h. Then, rhodamine-123 (10 μg/mL) added, nurtured for 30 min, these cells were washed with PBS buffer and visualized under the fluorescence microscope.
Apoptotic morphological analysis
Apoptosis involves the condensation and fragmentation of DNA. Cells undergoing apoptosis are visualized by acridine orange (AO) and EtBr dual-dye staining.[11] After 24 h of treatment of KB cells with ANLEE (0–50 μg/mL), paclitaxel (20 μg/mL), fix the cells with methanol and glacial acetic acid in the ratio of 3:1 at 37°C for 1 h. Then, stain with AO/EtBr (100 μg/mL) in the ratio of 1:1 in PBS for 5 min, wash with PBS and visualized under ultraviolet illumination in ×40 objective using a Nikon fluorescence microscope.
Acute toxicity study
Experimental animals
Female albino Wister age group of 6–8 weeks were obtained from Vellore Institute of Technology, Vellore, India. Animal handlings were done according to the Institutional Regulations, OECD and NIH guidelines (VIT/IAEC/V/017/2012).[12] Thirty rats were segregated into five groups according to their body weight, 15 h before drug administration, only water was provided. 1 mL of ANLEE was administered orally by gavage.[12] Group 1 animals served as vehicle control (0.1% EtOH); Groups 2–5 administered with 250, 500, 1000, and 2000 mg/kg body weight of ANLEE, respectively. Animals were observed for mortality till 14 days. Then, animals were sacrificed by cervical decapitation; liver and kidney tissues were collected, washed in saline, stored in 4% formalin. The hematoxylin and eosin stain was used in the histopathological analysis.[12]
Statistical analysis
The results were represented as mean with standard deviation and standard error. Data were analyzed using the one way-ANOVA and DMRT in SPSS version 16.0 (SPSS, Chicago, IL, USA). Values were corresponded as the means of six determinations and significance were considered at P < 0.05. Effective concentration (EC50) values were calculated using graph pad prism software.
ResultsThe confirmation of ethyl gallate as a major bioactive compound in ANLEE by the HPLC analysis [Figure 1]. Following this, an liquid chromatography-mass spectrometry (LC-MS) analysis was carried out due to its sensitivity in identifying the major components in the extract. The mass spectra of ethyl gallate and its mass fragmented product ions in ANLEE is illustrated in [Figure 2]. The representative multiple reactions monitoring (MRM) chromatogram of standard and ANLEE containing ethyl gallate (~100%) is depicted in [Figure 3].
Figure 2: Liquid chromatography-mass spectrometry analyses of Acacia nilotica leaf ethanolic extract showing mass spectra of full scan product ion of ethyl gallate (molecular weight-198) and its chemical structure
Figure 3: Multiple reactions monitoring chromatograms. (a) Standard ethyl gallate (b) Ethyl gallate in Acacia nilotica leaf ethanolic extractANLEE significantly decreased the KB cell viability in a dose-dependent manner. 98% cytotoxicity was obtained at 80 μg/mL of ANLEE with an IC50 value of 40 μg/mL and 100% cytotoxicity at 40 μg/mL of paclitaxel with an IC50 of 20 μg/mL concentration, respectively. Inhibition of KB cell growth was significantly less in ANLEE when compared to paclitaxel. Based on the results obtained, ANLEE (0–50 μg/mL) and paclitaxel (20 μg/mL) were preferred for further experiments. The results from cell viability assay indicate that the concentration of phytochemicals plays an important role on cytotoxicity and is directly proportional to KB cell death.
In this study, KB cells treated with ANLEE showed dose-dependent ROS generation when compared to the untreated control. Among different concentrations of A. nilotica leaf ethanolic extract (20, 30 40 and 50 μg/mL) tested, 50 μg/mL showed the maximum ROS generation (95%) with no significant difference in the activity with 20 μg/mL of paclitaxel. Relative fluorescence intensity of ROS by spectrofluorimetric quantification and fluorescent microscopic images of treated and untreated KB cells are demonstrated in [Figure 4] (a & b). The effective concentrations (EC50) for all the parameters are represented in [Table 1].
Figure 4: Effect of Acacia nilotica leaf ethanolic extract on intracellular reactive oxygen species generation in KB cells. (a) Relative fluorescence intensity of ANLEE and paclitaxel are expressed in % of control. Values are represented as mean ± standard devitation of six experiments. Bars not sharing a common superscript differ significantly from untreated control at P < 0.05. (b) Fluorescent microscopic images of reactive oxygen species generation in KB cells. G1, untreated control (0 μg/mL); G2-G5, ANLEE treatment (20, 30, 40 and 50 μg/mL); G6, paclitaxel (20 μg/mL)
Table 1: Effective concentration 50 (EC50) values exhibited by Acacia nilotica leaf ethanolic extract against KB cell proliferationIn comet assay, dissimilar grades of DNA damage were observed in 20, 30, 40, and 50 μg/mL of ANLEE treated KB cells when compared to the untreated control. Control cells presented mostly nonfragmented DNA, whereas 50 μg/mL of ANLEE treatment expressed extremely fragmented DNA. Quantitative measurements of comet assay endpoints such as tail length, tail moment, and olive tail moment were calculated, and their digital images were analyzed using CASP software. ANLEE 50 μg/mL significantly increased the tail length and modified the tail moment and olive tail moment in KB cells when compared to the untreated control as depicted in [Figure 5] (a & b). Furthermore, no significant difference in the percentage tail length was noticed between 50 μg/mL of ANLEE and 20 μg/mL of paclitaxel.
Figure 5: Effect of Acacia nilotica leaf ethanolic extract on DNA damage in KB cells. (A) Relative fluorescence intensity of ANLEE and paclitaxel are expressed in % of control. Values are represented as mean ± standard devitation of six experiments. Bars not sharing a common superscript differ significantly from untreated control at P < 0.05. (B) Fluorescent microscopic images of DNA damage (% tail length, % tail moment and % olive tail moment) in KB cells. G1, untreated control (0 μg/mL); G2-G5, ANLEE treatment (20, 30, 40 and 50 μg/mL); G6, paclitaxel (20 μg/mL)As per comet assay, concentration-specific DNA damage was observed in ANLEE (0–50 μg/mL) treated KB cells when compared to the untreated control. Control cells presented mostly nonfragmented DNA, whereas 50 μg/mL of ANLEE treatment expressed extremely fragmented DNA. The quantity of DNA present at the tail of the comet is linearly related to DNA break frequency. ANLEE at 50 μg/mL enhanced the tail length and modified the fragments configuration as compared with control [Figure 5]a & [Figure 5]b. Furthermore, there is no observed difference in the percentage of tail length noticed.
ROS generation is also an important factor for MMP loss. In this study, ANLEE had some restrictions in allowing rhodamine-123 to pass through the mitochondrial membrane. ANLEE increased the MMP loss in KB cells [Figure 6]a & [Figure 6]b. ANLEE (0–50 μg/mL) treatments significantly increased the mitochondrial depolarization in KB cells in a dose-dependent manner when compared to the untreated control. Among the treatments, 50 μg/mL of ANLEE exhibited the highest mitochondrial dysregulation but was significantly less when compared to paclitaxel (20 μg/mL).
Figure 6: Effect of Acacia nilotica leaf ethanolic extract on mitochondrial membrane potential loss in KB cells. (a) Relative fluorescence intensity of ANLEE and paclitaxel are expressed in % of control. Values are represented as mean ± standard deviation of six experiments. Bars not sharing a common superscript differ significantly from untreated control at P < 0.05. (b) Fluorescent microscopic images of membrane potential loss in KB cells. G1, untreated control (0 μg/mL); G2-G5, ANLEE treatment (20, 30, 40 and 50 μg/mL); G6, paclitaxel (20 μg/mL)Our results also demonstrated that ANLEE has dose-dependent apoptosis effect on KB cells and due to increased ROS generation. Relative fluorescence intensity of apoptosis in ANLEE and paclitaxel expressed in the percentage of control [Figure 7]a. Quantitative analysis revealed 81% of apoptosis at 50 μg/mL of ANLEE. Fluorescence microphotographs of KB cells indicating the apoptotic morphological changes in different treatments [Figure 7]b.
Figure 7: Effect of Acacia nilotica leaf ethanolic extract on apoptosis in KB cells. (a) Relative fluorescence intensity of ANLEE and paclitaxel are expressed in % of control. Values are represented as mean ± standard deviation of six experiments. Bars not sharing a common superscript differ significantly from untreated control at P < 0.05. (b) Fluorescent microscopic images of apoptosis in KB cells. G1, untreated control (0 μg/mL); G2-G5, ANLEE treatment (20, 30, 40 and 50 μg/mL); G6, paclitaxel (20 μg/mL)Histopathology analysis
In this study, control and A. nilotica leaf ethanolic extract treated liver and kidney tissues revealed no evidence of toxicity even at 2000 mg/kg body weight indicating its safety upon oral administration [Figure 8]. The overall summary of ANLEE-induced KB cell death in vitro is depicted on [Figure 9].
Figure 8: Histopathology sections of liver and kidney tissues (a and c) control rat liver and kidney tissues (b and d) Acacia nilotica leaf ethanolic extract at 2000 mg/kg body weight treated rat liver and kidney tissues. Oral administration of ANLEE did not show any signs of toxicity or damage in liver and kidney tissues indicating its safety
Figure 9: Overall summary of antiproliferative effect ofAcacia nilotica leaf ethanolic extract on KB cells. ANLEE inhibited KB cell proliferation by eliciting oxidative stress mediated by reactive oxygen species generation, membrane potential loss, DNA damage and apoptosis Discussion The plant A. nilotica was reported to possess anticancer activity against Dalton's ascitic lymphoma induced solid tumor model.[8] Ethyl gallate is a major bioactive phenolic in ANLEE has reported to act against human carcinoma and other cell lines KB.[5],[9] The present study HPLC analysis, LC-MS analysis, and the representative MRM chromatogram [Figure 1], [Figure 2], [Figure 3] were congruent with the previous report on standard ethyl gallate by Gao et al., 2009.[13]
The difference in cytotoxicity of ANLEE and paclitaxel against KB cell may be due to the presence of a complex mixture of substances in A. nilotica leaf extract.[10] Similarly, plant-derived triterpene has been reported to inhibit the neuroectodermal cancer cell growth.[5] Results from cell viability assay indicate that the concentration of phytochemicals plays an important role on cytotoxicity.
ROS are the derivatives of molecular oxygen metabolism involved in the normal physiology of living organisms.[19] Earlier reports suggest that anticancer drugs lift the oxidative stress in cancer cells by triggering ROS and RNS.[20] Certain polyphenols have been well characterized as prooxidants based on their dose-dependent ROS generation in cancer cells. Paclitaxel generates oxidants followed the trend as in AS2, A549, H157, and H460 cells.[9] ANLEE showed dose-dependent ROS generation, and several studies support these findings that plant phenolics such as ethyl gallate and caffeic acid exhibit prooxidant activity at higher concentrations.[8]
It has been reported that polyphenols boost DNA damage in cancer cells.[21] Based on this report, the DNA damage by ANLEE in KB cells were analyzed by comet assay. DNA is one of the crucial targets for killing cancer cells. Our results manifest the observation in percentage comet formed in ANLEE treated KB cells and expressed extremely fragmented DNA. Quantitative measurements of comet assay were calculated, and their digital images were analyzed using CASP software, which is the key parameters for measuring DNA damage[15],[16] The quantity of DNA present at the tail of the comet is linearly related to DNA break frequency.
ROS generation is also an important factor for MMP loss. It is evident from the earlier reports that dietary phenolics like isothiocyanate sulforaphane influenced the alteration in mitochondrial collapse leading to apoptosis in cancer cells.[22] Mitochondria are involved in regulating cell death, leading to coordinated cell degradation ensuring the organization of apoptotic bodies and hence considered as a marker for apoptosis.
One of the importance of cancer chemotherapy is to discover leads that cause apoptosis in cancer cells.[21] Many anticancer drugs act as pro-oxidants targeting mitochondria by eliciting apoptosis.[21] Our results have also demonstrated that ANLEE has dose-dependent apoptosis effect on KB cells and it is due to increased ROS generation. Relative fluorescence intensity of apoptosis in ANLEE shows fluorescence microphotographs of KB cells indicating the apoptotic morphological changes in different treatments. The histopathology analysis of the liver and kidney tissues revealed no evidence of toxicity even at 2000 mg/kg body weight indicating its safety upon oral administration. Earlier studies also have shown the safety limit of ANLEE based on the biochemical analysis in liver and serum.[12]
ConclusionsIn vitro studies are important to screen any therapeutic compounds before stepping into the in vivo studies. To conclude, this is the first report of A. nilotica leaf ethanolic extract induced antiproliferative activity against human carcinoma cell line KB by the generation of ROS, loss of MMP, damage of DNA, and apoptosis. ANLEE also possesses a wide margin of safety on the acute oral administration to rats. From the present results and the earlier report on ethyl gallate against KB cell growth, we infer that the activity of ethyl gallate is slightly hindered when present along with other components in ANLEE. In future, comparative studies need to be carried out determining their signalling mechanism in cancer cell death.
Acknowledgements
The authors are grateful to Vellore Institute of Technology management and the Dean SBST for providing facilities, their constant support and encouragements.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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