G1 phase and induces apoptosis in human oral carcinoma (KB) cells possibly via oxidative DNA damage

Biomedicine & Pharmacotherapy 92 (2017) 661–671

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Original article

Umbelliferone arrest cell cycle at G0/G1 phase and induces apoptosis in human oral carcinoma (KB) cells possibly via oxidative DNA damage Annamalai Vijayalakshmi, Ganapathy Sindhu* Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamilnadu 608022, India

A R T I C L E I N F O

Article history: Received 4 March 2017 Received in revised form 25 May 2017 Accepted 28 May 2017 Keywords: Oral cancer Cell proliferation Apoptosis Umbelliferone Mitochondrial membrane potential DNA damage

A B S T R A C T

Umbelliferone (UMB) has widespread pharmacological activity, comprising anti-inflammatory, antioxidant, anti-genotoxic and anti-immunomodulatory but the anticancer activity remains unknown in human oral carcinoma (HOC) KB cells. MTT assay determinations was revealed that treatment of KB cells with UMB, prevent and reduce the cell proliferation with the IC50
200 mM as well as induces loss of cell viability, morphology change and internucleosomal DNA fragmentation in a concentration dependent manner. Acridine orange and ethidium bromide dual staining assay established that UMB induced apoptosis in KB cells in a dose dependent manner. Alkaline comet assay determination revealed UMB has the potential to increase oxidative DNA damage in KB cells through DNA tail formation significantly (p < 0.05). Furthermore, UMB brought a dose-dependent elevation of reactive oxygen species (ROS), which is evidenced by the DCF fluorescence, altered the mitochondrial membrane potential in KB cells. Similarly, we observed increased DNA damage stimulated apoptotic morphological changes in UMB treated cells. Taken together, the present study suggests that UMB exhibits anticancer effect on KB cell line with the increased generation of intracellular ROS, triggered oxidative stress mediated depolarization of mitochondria, which contributes cell death via DNA damage as well as cell cycle arrest at G0/G1 phase. The results have also provided us insight in the pharmacological backgrounds for the potential use of UMB, to target divergent pathways of cell survival and cell death. To conclude UMB could develop as a novel candidate for cancer chemoprevention and therapy, which is our future focus and to develop a connectivity map between in vivo and in vitro activity. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction Cancer is a deadly disease worldwide, which is representing a tremendous burden on society. Oral cancer (OC), an aggressive progression and miserable diagnosis, accounting 3–5%, achieves remarkable burden on public health in many parts of the world, especially in South and South East Asia. The key risk factors of OC include tobacco smoking and chewing, alcohol consumption and betel quid chewing with or without tobacco. Progress of OC is accompanied with too much cell proliferation, deregulation of cellular differentiation, inadequate apoptosis and genomic instability [1]. Even by way of the use of chemotherapeutic agents, including platinum, 5-fluorouracil, taxane, ifosfamide and methotrexate, their therapeutic efficacy is often compromised by the improvement of drug resistance during the course of tumor

* Corresponding author at: Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, Tamil Nadu, 608 002, India. E-mail address: [email protected] (G. Sindhu). http://dx.doi.org/10.1016/j.biopha.2017.05.128 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

progression and toxicity leading to poor clinical outcomes [2]. Chemopreventive is accordingly, ahead extensive attention as a promising and alternative approach for cancer control [3]. Successful cure through chemotherapeutic agents is mostly dependent on their facility to inhibit cell growth and/or induce differentiation. Plant-derived natural products or phytochemicals and their man-made derivatives are manufacturing a significant impact in modern drug discovery programs via pointing numerous human diseases, including cancer. Most of these natural compounds are often multi targeted in nature, which is generally a very desirable property for cancer therapy. In squamous cell carcinoma (SCC) characteristically implicate deregulations of manifold genes and accompanying cell-signaling pathways at numerous phases of cancer progression should be targeted in the treatment strategy. Cancer cell line derived from human tissue not only provides a fundamental platform to understand molecular biology of neoplasia, also served as a basis for the investigation of specific therapeutic strategies towards cancer types [4]. In vitro schemes allow both more precise dosing of drug and length of exposure in a uniform, chemically and experimentally defined medium.

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Umbelliferone (7-hydroxycoumarin) is a naturally arising edible coumarin derivative of benzopyrone and dietary antioxidant. It occurs in many familiar families such as the most ear hawkeed. Among the various phytochemicals, UMB have good nutritive value and active phytocontituents of fruits, vegetables and plants (e.g. Carrot, coriander, golden Angelica, golden apple (Ageles marmelos Correa) and bitter orange (Citrus aurantium)). UMB reveals an extensive field of therapeutic properties including antioxidant, anti-hyperglycemic, anti-inflammatory, anti-genotoxic and immunomodulatory [5–8] properties. In the recent years, UMB has attracted more attention in the field of functional foods, nutritional supplements and medicines because of its various pharmacological and biological benefits, such as antibacterial, anti-thrombotic, vasodilators, anti-mutagenic, lipoxygenase inhibition, scavenging ROS, anti-metastatic, antinociceptive effect, radioprotective ability and anti-lipidperoxidative. However, previous studies also showed that UMB protects alcoholic fatty liver, detoxify reactive aldehyde, radiation mediated cellular damage in human blood lymphocytes, immuno modulators and treat vascular lesions. Muthu and Vaiyapuri, [9] has revealed that UMB suppress free radical mediated colon cancer by their powerful antioxidant property. Kanimozhi et al. [10] investigated that UMB initiation of apoptosis, augmented DNA damage from radiation induced human blood lymphocytes. Jimenez
Orozco and their coworkers [11] reported that UMB inhibited the G1/S transition of cell cycle, an action consistent with the cytostatic effect. Furthermore, RT-PCR and Flowcytometric studies have demonstrated cyclin D1 expression was indicate action of UMB on early events of G1 phase in human lung A427 adenocarcinoma cell line. Lopez gonzalez and coworkers [12] hypothesized that many of the observed in vitro studies on UMB (100 mg/ml) could be able to inhibit cell proliferation rate in tumor cell lines for 24 h and arrest cell cycle at G1 phase in cancer cell lines (1.3.11, 1.3.15. 3A5A and SK-MES-1) and when exposure of 160 mg/ml concentration of UMB during 4– 6 h shows cytotoxic effects, morphological changes and induction of apoptosis was observed in adenocarcinoma cell lines. With this knowledge we try to explore its use in oral cancer therapy. In the current study, we obligate an attempt to explore the anticancer effect of UMB on KB cell lines in a dose and time dependent manner which has not been previously studied. The dose was fixed and applied in the KB cells by the cytotoxicity test. To understand the interaction and interference with a widespread of biotic signals and functions by investigate the existence of cytotoxicity, cell morphology, mitochondrial membrane potential (MMP), apoptotic morphological changes, DNA damage and cell cycle arrest in KB cells. 2. Materials and methods 2.1. Chemicals and reagents UMB (Sigma Aldrich chemicals Pvt Ltd, Bangalore, India), KB cell lines (National Centre for Cell Science (NCCS) India), Dulbecco’s Modified Eagle Medium (DMEM) media, fetal bovine serum (FBS), Penicillin, MTT
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide, Acridine orange (AO), Ethidium bromide (EB), 20 -7’dichlorofluorescein diacetate (DCFH-DA), Rhodamine123 (Rh-123), Propidium iodide (PI), RNase, Tween-20 and phosphate buffer saline (PBS) (Himedia, Mumbai). Acetone, Ethanol and Dimethyl sulfoxide (DMSO) (Fisher Scientific, India). 2.2. Cell culture and assessment of growth and viability The human carcinoma cell line, KB was obtained from NCCS (Pune). KB cell line was used by the National Cancer Institute (NCI) for most of the in vitro anticancer drug-screening work and has

been thought to be derived from an epidermoid carcinoma of the oral cavity [13]. These KB cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 U ml
1 of penicillin and 100 mg ml
1 of streptomycin at 37  C in a humidified 95% air and 5% CO2 incubator. Cells were seeded in 6 or 12 well plates prior to the addition of UMB. The KB cells incubated with UMB (0– 500 mg/ml of KB cells) for 24 h. 2.3. Preparation of umbelliferone A stock solution of UMB, 1 g/L (0.006165 M) was prepared in 0.5% DMSO (w/v) and stored at 4  C. Further dilution was made in culture media to obtain the desired concentrations. The working solution was diluted using sterile distilled water so that the final concentrations of DMSO in the culture medium were not more than 0.01% (v/v). 0.01% DMSO was used as a vehicle control. MTT assay was used for cytotoxicity testing in KB cells, IC50 values were calculated. 2.4. Anti-proliferative MTT assay KB cells were seeded in 96 well plates at a concentration of 6000 cells per plate. Afterwards 24 h of incubation, the medium was extracted and replaced by media dosed with UMB at various concentrations of (50, 100, 150, 200, 250, 300, 400 and 450 mM/ well). KB cells treated with blank vehicles (DMSO) were used as vehicle control. A primary stock was prepared by dissolving UMB in DMSO at a concentration of 0.006165 M (1 g/1L) from which a secondary concentration of 450 mM was prepared in the media. Extra dilutions were prepared using the secondary stock. The whole volume of 100 ml was added to each well. After 48 h, the drug-containing medium was replaced with 100 ml of medium containing MTT (tetrazolium bromide solution in PBS 5 mg/ml). The yellow thiazolyl groups of MTT are abridged to purple tetrazolium crystals by viable cells which were dissolved in 100 ml of DMSO after 4 h incubation in the dark [14]. The absorbance was recorded at 540 nm using a multi-well plate reader (Bio-Rad). The OD of the untreated group was equivalent to 100% viable cells; cell viability was calculated as a percentage of the vehicle control. The IC50 value (a concentration that yields 50% lessening in the viable cell number) was determined by analysis between the concentration and normalized response (Percentage of cell viability). 2.5. Tryphan blue and colony survival assay Cell feasibility was measured by the capacity of living cells to eliminate tryphan blue vital dye [15]. Cells were seeded in 96-well micro plates at a concentration of 104 cells/well were treated within the presence and absence of UMB for 24 h. After this, the KB cells were trypsinzed from the micro plates, combined with any moving cells prevailing in the media and pelleted by centrifugation at 1000g for 10 min at 4  C. Cells were washed twice with PBS and tryphan blue was added at a final concentration of 0.2%. Living cells will be counted in a haemocytometer and articulated as the% of the whole count in vehicle control. A dose dependent study was accepted for UMB to find out maximum inhibition. 2.6. Measurement of intracellular reactive oxygen species assay Reactive oxygen species (ROS) were distinguished by determining the fluorescence intensity of dichlorofluorescein (DCFH). When non-fluorescent DCFH-DA flows into the cell via the plasma membrane and hydrolyzed in the interior cell to DCFH. Intracellular oxidation converts DCFH into the fluorescent form, DCF. After

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development of the KB cells with various concentrations (50, 100, 150, 250, 350 and 450 mM) of UMB for 24 h, fluorescent dye DCFHDA was added to the cells and then kept in incubator 30 min only. Finally, the cells were washed with PBS to remove the excess dye. Fluorescent intensity was prepared with excitation and emission filters set at 488 and 530 nm, respectively. Fluorescence microscopic images were taken using a blue filter (450–490 nm). 2.7. Measurement of mitochondrial membrane potential The study of mitochondria and changes in the mitochondrial membrane potential (MMP) has developed an emphasis of apoptotic analysis. The possible effect of UMB in disturbing MMP was evaluated using the lipophilic cationic fluorescent probe Rh-123 for mitochondria. Rh-123 green fluorescent monomer at depolarized or a red fluorescent aggregate at hyper polarized membrane potential [16]. Rh-123 localizes in the mitochondria of living cells owing to the somewhat high negative electric potential through the inner membrane of the mitochondria. After UMB treatment, at various concentrations (50
450 mM) Rh-123 was supplementary to attain a final concentration of 10 mg/ml and the KB cells were incubated for 30 mins at 37  C. Then the cells were swept away with PBS and observed under fluorescence microscope using blue filter. 2.8. Acridine orange/Ethidium bromide dual staining assay DNA-binding dyes AO/EB were used for the morphological apoptotic and necrotic cells [17]. After treatment with various concentration of UMB (50, 100, 150, 250, 350 and 450) for 48 h, the KB cells was detached, washed by cold PBS and then stained with a mixture of AO (100 mg ml
1) and EB (100 mg ml
1) at room temperature for 5 min. The stained KB cells were observed by a fluorescence microscope at 40 x magnifications. The KB cells were separated into four classes as follows: living cells normal green nucleus appeared, early apoptotic condensed or fragmented form of bright green nucleus with chromatin, late apoptotic chromatin condensation or fragmentation orange-stained nuclei and necrotic cells (uniformly orange-stained cell nuclei). In each experiment, 300 cells/concentration were counted. 2.9. Alkaline single-cell gel electrophoresis comet assay DNA damage was estimated by alkaline single-cell gel electrophoresis (SCGE) comet assay according to the method of [18]. A layer of 1% NMPA was equipped on microscopic slides. Subsequently KB cells (50 ml) were mixed with 200 ml of 0.5% LMPA. The suspension was pipetted out and placed on the precoated slides. Slides was immersed in cold lysis solution at pH 10 (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris pH 10, 1% Triton X-100, 10% DMSO) and kept at 4  C for 60 mins. To allow denaturation of DNA, the slides were positioned in alkaline electrophoresis buffer at pH 13 and left for 25 min. Subsequently, slides were removed to an electrophoresis tank with fresh alkaline electrophoresis buffer and electrophoresis was performed at field strengths of 1.33 V/cm for 25 min at 4  C. Slides were neutralized in 0.4 M Tris (pH 7.5) for 5 mins and stained with 20 mg/ml of EB. For visualization of DNA damage, observations will be made using a 40 objective in an epifluorescent microscope equipped with an excitation filter of 510–560 nm and a barrier filter of 590 nm. Images were captured with a digital camera with networking capability and analyzed by image analysis software CASP. DNA damage was quantified by tail moment, tail length and olive tail moment. Olive tail moment is the product of the distance (in the x direction) between the midpoint of gravity of the head and the midpoint of gravity of the tail and the tail% DNA. The tail length indicates the distance of DNA crossing

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from the nuclear core and is used to assess the extent of DNA damage. Olive tail moment = (tail mean
head mean)  tail% DNA/ 100; tail moment is = tail length  tail% DNA (tail intensity)/100; tail intensity is = 100 head% DNA. 2.10. Cell cycle analysis For flow cytometric analysis of cell cycle, KB Cells at a density of 1.5  105 cells/ml were treated vehicle control and UMB at concentrations of 100, 200 and 300 mM for 24 h. All adhering and floating cells were harvested and washed with cold PBS; icecold 70% ethanol was fixed the cells and incubated at
20  C overnight. The cells incubated with 500 ml of hypotonic PI solution (10 ml sodium citrate, 0.25 mg of PI, and 0.4 mg of RNase and 0.3 ml of tween-20 for 10 ml) at 37  C for 30 min staining in the dark at 4  C. Flow cytometry analysis was performed by the central instrumentation scientific laboratory (CISL) of Annamalai University uses a flow Cytometer (BD FACS Aria III) equipped with a 633, 488 and 375 nm air-cooled Argon laser collecting at least 10,000 events. The DNA profile indicated the relative abundance of the cell population in sub-G1, G0/G1, S and G2/M phases. 2.11. Statistical analysis The data are expressed as mean  SD statistical comparisons were performed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test (DMRT) using SPSS version 17.0 for windows. The results were considered statistically significant values p < 0.05. 3. Results 3.1. Effect of UMB on cell viability, growth of KB cells in a dose dependent manner The cytotoxicity effect of UMB in KB cells has been assessed by MTT assay. Fig. 1B shows the% of viable cells was significantly decreased (p < 0.05) with respect to treatment of UMB with increasing concentrations after 24 h. The MTT assay indicated that cell viability was significantly decreased to 80, 72, 63 and 44% when cells were exposed to UMB at concentrations of 50, 100, 150 and 200 mM/ml respectively. 50% of viable cells were observed at 200 mM on KB cells at 24 h. From this observation the IC50 values of UMB were considered as 200 mM. The survival of KB cells decreased significantly in a concentration dependent manner with an IC50 (the concentration causing 50% growth inhibition) value at 200 mM/ml 0.48  0.052. There is complete destruction (p < 0.001) of cells above 450 mM/ml. A change in cell morphology with a decrease in the cell number was observed in cells treated with 450 mM/ml concentration of UMB (Fig. 1A). In contrast, no change was formed in the cell morphology was observed with the vehicle treated KB cells under the same conditions. It is likely that UMB 150 mM/ml concentration affected the KB cells, whereas the significantly (p < 0.05) induced anti-proliferative responses were observed in the KB cells. 3.2. Measurement intracellular reactive oxygen species level in UMB In this study, ROS produced in KB cells exposed to various concentrations of UMB has been accessed using DCFH-DA (Fig. 2). Level of ROS was observed by green fluorescence in vehicle control (Fig. 2A). UMB treated KB cells were depicted weak background fluorescence, while treatment with high dose UMB showed bright DCF fluorescence. The ROS observed in (Fig. 2B) KB cells exposed with 50, 100 and 150 mM/ml concentrations were moderately changed the ROS (p < 0.001) 40, 41 and 42% respectively, when

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Fig. 1. Effect of UMB on morphological characteristics and cell viability of HOC KB cells was assessed by MTT assay. (A) Morphological changes in vehicle control and UMB treated HOC KB cells for 24 h. The images captured by light microscope (Nikon, Eclipse TS100, Japan) showing (a) intact morphology of vehicle control KB cells, (b–h) show UMB treated with KB cells at different concentration (50, 100, 200, 250, 300, 400, and 500 mM) respectively. (B) Results are expressed as HOC KB cells treated with either vehicle or UMB (50–450 mM) for 24 h. Values were presented as mean  SD of three independent experiments (one way analysis of variance [ANOVA]) followed by Duncan’s multiple range test (DMRT). Asterisks indicate statically different from vehicle control: *p < 0.05 and **p < 0.001.

related with untreated cells. Treatment with 250, 350 and 450 mM/ ml concentrations was showed significant increase in ROS production (p < 0.05) 43, 53 and 57% in a dose dependent manner. Amongst all the doses tested, 450 mM/ml of UMB showed the maximum generation of ROS (p < 0.05) 57% in KB cells. In contrast, treatment with UMB (24 h) resulted in a dose dependent enhance in ROS production as shown by increased DCF staining in the nucleus. 3.3. Effects of UMB on mitochondrial membrane potential in KB cells The Rh-123 is a lipophilic cationic dye, highly specific for mitochondria, which is widely used to monitor mitochondrial health in cell death studies. The MMP was investigated using Rh123 after 24 h exposure of KB cells to various concentrations of (50–450 mM/ml) UMB. The fluorescent dye ratio was observed, as evidenced by low in the intensity of the red and green fluorescence ratio (Fig. 3A). The decline in MMP was observed in concentrations (350 and 450 mM/ml) of UMB compared to the vehicle control and significant levels (p < 0.01) at 54 and 50%. Fluorescence microscopic images (Fig. 3B) represents the accumulation of Rh-123 fluorescence dye from orange red to green compared in vehicle

control and the accumulation found to be decreased in UMB treated KB cells at various concentrations (50, 100, 150 and 350 mM/ml) p < 0.1, 0.05, 0.01, 0.001 respectively. 3.4. Effects of UMB induce apoptosis in KB cells to inhibit cell proliferation Apoptosis is a functional configuration of cell death characterized by morphological structures and extensive DNA fragmentation [19]. Thus, to regulate whether the development inhibitory activities of UMB were resulted in the initiation of apoptosis and the morphological fluctuations of KB cells were examined using AO/EB staining assay to confirm cell apoptosis. AO is accumulated by both viable and non-viable cells and emits green fluorescence if intercalated into ds DNA and EB is accumulated only by non-viable cells and emits red fluorescence by intercalation into DNA. Thus, viable cells have a normal green nucleus, however early apoptotic cells are cheerful green nucleus with compared with shortened or fragmented orange chromatin. As can be seen in Fig. 4A early apoptotic cells with yellow dots and late apoptotic cells with orange dots in KB cell nuclei treated with UMB concentration 150– 450 mM/ml. The maximum increase (p < 0.05) in the quantity of

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Fig. 2. UMB induces intracellular ROS production in KB cells by DCFH-DA staining assay. (A) KB cells were treated with UMB at various concentrations (50–450 mM) for 24 h, stained with DCFH-DA dye. Then the digital images were captured by Fluorescence microscope (Nikon, Eclipse TS100, Japan). a) vehicle control (green florescence) b) UMB 50 mM; c) UMB 100 mM; d) UMB 150 mM; e) UMB 250 mM; f) UMB 350 mM; g) UMB 450 mM; (c– e) depicted weak background florescence arrow mark represent clearly visible DCF fluorescence; (e–g) depicted bright DCF florescence in HOC KB cells treated with UMB in a dose dependent manner. (B) Intracellular ROS measurement by spectrofluoremetry. Results in a dose dependent enhance in ROS production as shown by increased% DCF staining ratio. Values were presented as mean  SD of six experiments in each group ANOVA followed by DMRT. Asterisks indicate statically different from vehicle control: *p < 0.05 and **p < 0.001.

Fig. 3. (A) Effect of UMB on the MMP of KB cells. KB cells were treated with different concentration UMB for 24 h, stained with Rh-123 and the mitochondrial depolarization patterns of KB cells were observed. Results the gradual decrease of red/green fluorescence indicates a decrease MMP in a dose dependent manner were analyzed by fluorescent microscope (Nikon, Eclipse TS100, Japan) at 400 x magnification. In the fluorescent image shows a) vehicle control (Rh accumulation) b) UMB 50 mM; c) UMB 100 mM; d) UMB 150 mM; e) UMB 250 mM; f) UMB 350 mM; g) UMB 450 mM (No Rh accumulation). B) Quantification of MMP in the spectrofluorometry. Values are given as mean  SD of six experiments in each concentration ANOVA followed by DMRT. Asterisks indicate statically different from vehicle control: *p < 0.05; **p < 0.1; ***p < 0.01 and ## p < 0.01.

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Fig. 4. Effect of UMB induces apoptotic incidence. (A) KB cells treated within vehicle control and UMB at various concentrations (50–450 mM) at 24 h, stained with dual dye EB/AO and then analyzed by fluorescence microscopy. White arrow indicates green florescence; Orange arrow indicates apoptotic bodies; Blue arrow indicates apoptotic cells; Yellow arrow indicates necrotic cells. UMB induces apoptosis by generating ROS and disruption of MMP. (B) Percentage of apoptotic cells were calculated by scoring apoptotic and viable cells. The values are given as mean  SD of six experiments in each group ANOVA followed by DMRT. Asterisks indicate statically different from vehicle control: * p < 0.05.

apoptotic cells was observed as 95, 98 and 100 percent in 250, 350 and 450 mM/ml concentration of UMB treated cells respectively related to vehicle control (Fig. 4B). 3.5. Effects of UMB on DNA damage (Comet assay) In this context, the amount of DNA damage was assessed in vehicle control and UMB (50, 100, 150, 250, 350 and 450 mM/ml) treated KB cell lines. A substantial enhance in the level of DNA damage was observed in KB cells after UMB treatment. Fig. 5A represents the photomicrograph of comet in KB cells. The vehicle control exhibited large non-fragmented intact nucleoid. The tail DNA was observed in UMB exposed cells, which looked as a comet during SCGEs. Treatment with UMB at various concentrations (50– 450 mM/ml) resulted in DNA damage dose dependently. Digital images were further examined via CASP software that permits quantitative measurements of several comet assays terminalpoints, in specific, the mean average of tail length, % of DNA in the tail, olive tail moment and tail moment (Fig. 5B). UMB (50, 100, 150, 250, 350 and 450 mM/ml) treatment significantly increased the tail length, percentage of DNA in tail, olive tail moment and tail moment of KB cells in a concentration manner. Among all doses tested, 450 mM/ml of UMB showed maximum tail length (23%) p < 0.05; tail DNA (44%) p < 0.01, olive tail moment (14%), tail moment (19%) p < 0.01. 3.6. Effects of UMB on cell cycle distribution of KB cells The vehicle control showed a typical distribution of cell cycle phases. UMB had a significant impact on the cell cycle. Effects of UMB on cell cycle distribution in KB cells were studied, after exposure to UMB at various concentrations 100, 200 and 300 mM/ ml for 24 h and vehicle control. To identify whether the growth inhibitory effect of UMB was caused by specific perturbation of cell cycle related events, the DNA contents of KB cells were measured

by means of flowcytometric analysis (Fig. 6A). UMB treatment inhibited proliferation of KB cells in a concentration dependent manner (IC50–200 mM). We observed a reduction in the percentage of UMB treated KB cells in the S phase (from 0.5 to 0.2%) and in G2/M phase (0.4–0.2%) when compared to the vehicle control and the DMSO treated KB cells along the cell cycle. Although there was a consistent trend toward accumulation (p < 0.02 and p < 0.1) of UMB-treated KB cells in the G1 phase (from 80.0 to 54.3%) of the cell cycle. This effect was statistically significant p  0.02 in UMB treated with 100 and 300 mM and p  0.01 in UMB treated 200 mM concentrations. The additive effect of G1 phase accumulation likely accounts for the observed decrease in S and G2 phase accumulation, followed increases in the population of cells in sub G1 phase in a dose dependent manner. This increase in sub-G1 was G1/S transition. These results indicate that UMB regulates the G1/S phases of the cell cycle in KB cells and thus affects their proliferation pattern. 4. Discussion Several studies over the last decades have demonstrated the anti-carcinogenic effects of UMB in various cellular and animal models. We report for the first time, the anti-cancer activity via induction of cell cycle at the G0/G1 phase arrest and apoptosis in KB cells. Natural compounds have always been preferred choice of all as it plays an immense role in the healthcare and in traditional medicine system of the world. Results from various in vitro readings also point to that coumarin derivatives exhibited antiproliferative properties in incorporation human and animal derived cell lines. Werber and their coworkers [23] found the anti tumor activity of UMB in several tumor cell lines such as gastric carcinoma, colon carcinoma (Caco-2) a hepatoma-derived cell line (Hep2) and lymphoblastic cell lines (CCRF CEM). Kielbus et al., [24] has been reported that UMB reduced cell viability and

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Fig. 5. Effect of UMB on comet parameters (DNA damage) was assessed by alkaline single cell gel electrophoresis (A) KB cells was treated with UMB different concentration (50–450 mM) for 24 h, stained with PI. Then the digital images were further examined by fluorescence microscope (Nikon, Eclipse TS100, Japan). a) vehicle control (largely non fragmented intact DNA), b) UMB 50 mM; c) UMB 100 mM; d) UMB 150 mM; e) UMB 250 mM; f) UMB 350 mM; g) UMB 450 mM; (b–g) shows released or broken DNA. B) Digital images were further examined via CASP software that permits quantitative measurements. The four comet parameters: (I) tail length; (II) tail intensity. (III) olive tail moment; (IV) tail moment in HOC KB cells. Values are given as mean  SD of six experiments in each concentration ANOVA followed by DMRT. Asterisks indicate statically different from vehicle control: *p < 0.05; **p < 0.1; ***p < 0.01; # p < 0.02 and ## p < 0.01.

migration of RK3 laryngeal cancer cells in a dose dependant manner. UMB induces apoptosis which could be the major cause for its anti proliferative activity that has been observed in many cancer cell lines. Yu and his coworkers [25] reported that UMB induced cell cycle arrest at S phase in HepG2 cells. UMB attributed to the potential role and act as an anticancer agent via induction of apoptosis, cell cycle arrest and DNA fragmentation in HepG2 cancer cells. Jimenez
orozco and their coworkers [11] reported that UMB can affect the cell cycle progression of human adenocarcinoma cell line A427. Moreover, the UMB showed greater cytostatic activity and cell cycle arrest at transition G1/S in human adenocarcinoma cell line A427. Lopez gonzalez and coworkers [12] observed that UMB could able to inhibit cell proliferation rate at 100 mg/ml concentration for 24 h and cell cycle arrest in G1 phase in cell lines (1.3.11, 1.3.15. 3A5A and SK-MES-1) and when exposure of 160 mg/ml concentration of UMB during 4– 6 h of exposure shows cytotoxic effects, morphological changes

and induction of apoptosis in adenocarcinoma cell lines. Yang and his coworkers [26] were clearly highlighted the potential of UMB as a effective leading compound against human bladder carcinoma (cell line E-J) due to the effect on the growth inhibition. UMB also reported to exert both antitmour and antimetastasis properties during treatment to human lung cancer cell lines (A427, Calu-1) [8]. Finn and their coworkers [27] were demonstrated that UMB regulated the mitogen-activated protein kinase (MAPK) pathway and melanogenesis in human malignant melanoma cells. Further they attempted to elucidate the exact nature of the relationship between drug exposure and signal transduction. Jayakumar and their coworkers [28] investigated the several effect of UMB treatment such us radiation induced anti-genotoxic effects, reduced radiation induced apoptosis and protect against radiation induced loss of colony forming units at different doses (6.5–50 mM) on spleenic lymphocytes. In the current study, we reported UMB mediated cytotoxicity, DNA fragmentation, altered MMP and

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Fig. 6. (A) Effect of UMB on cell cycle distribution in KB cells was detected by flowcytometry. Cell cycle distributions of vehicle control and UMB treated KB cells with various concentrations (100, 200 and 300 mM) for 24 h were determined by PI staining in flow cytometry (a) Vehicle control cells. Sub-G1
19%, G0/G1
26.3%, S
2.8% and G2/M
4.2%; b) KB cells were treated with DMSO for 24 h. Sub-G1
18%, G0/G1
22.1%, S
5.0% and G2/M
4.9%; c) KB cells were treated by 100 mM UMB for 24 h. Sub-G1
21.4%, G0/G1
60%, S
0.5% and G2/M
0.4%; d) KB cells were treated by 200 mM UMB for 24 h. Sub-G1
28.5%, G0/G1
48.5%, S
0.1% and G2/M
0.3%; e) KB cells were treated by 300 mM UMB for 24 h. Sub-G1
31.5%, G0/G1
54.3%, S
0.2% and G2/M
0.2% and quantification in (B) Histograms showing the number of the channel (horizontal axis) against DNA content (vertical axis). UMB caused a dose dependent G1 phase accumulation and a corresponding decrease in the percentage of cells entering S and G2/M phase. The values are presented as mean standard deviation of three determinations and where indicated, by *, **, # showed a significant difference p < 0.01, 0.02, 0.1 respectively relative to the relevant vehicle control.

apoptosis in KB cells in a time and dose dependent manner. Based on our findings, we propose that UMB directly inhibit cell viability and growth by inducing apoptosis of KB cells through a mitochondria-dependent apoptotic pathway. MTT assay is commonly employed to assess the cytotoxic prospective of the test compound established on the viability of the test (cancer) cells owing to damage in mitochondrial and lysosome membranes that eventually trigger cell death. These assays serve as sensitive and integrated measures of cell integrity and of the inhibition of cell proliferation. Metabolically energetic viable cells can cleave MTT into a purple product which can be measured colorimetrically [30]. In the contemporary study, UMB significantly inhibited growth of KB cells in a dose dependent manner from 50 to 450 mM. The morphological variations were obtained in KB cells as increased number of rounded colonies and growth inhibition were observed when compared with vehicle control. Finally, this assay revealed the cytotoxic prospective of UMB an IC50 of 200 mM concentrations in KB cells. This result suggests that KB cells more vulnerable to UMB exposure. Yu et al., [25] tested the anticancer effect of UMB on HepG2 cell line at various dose and time (12, 24 & 48 h) dependent manner. They reported that UMB was found to exhibits significant anti cancer effect at 24 h treatment via MTT assay. Werber et al., [23] demonstrated that UMB effectively prevent the development of human gastric carcinoma cell line, Caco-2, HepG2 and CCRF CEM in a concentration dependent manner. UMB did not induce any cytotoxicity up to 124 mM in human blood lymphocytes during acute toxicity study [10]. In addition, UMB was found to exhibits low toxicity in normal human fibroblastic skin cell line (HS613.SK cells) with IC50 only at 494 mM [27]. ROS are generally perceived as toxicants that induce various deleterious effects, like cell dysfunction, death or malignant transformation [31]. Evidence survives that ROS are constructively involved in many signaling pathways that vehicle control development and maintain cellular homeostasis. ROS generation was effectively enhanced by UMB in both cell types and led to apoptosis. Intracellular ROS shows a major role in apoptotic

induction by affecting DNA fragmentation and nuclear damage [32]. The generation of intracellular ROS by UMB was measured using DCFH-DA based assays in KB cells. DCFH-DA fluorescent assay could help to examine the ROS generating potential of the cytotoxic compounds, an indicator of peroxides and superoxide accumulation. Under increased ROS, activated chromatin condensation, DNA fragmentation takes place which gives late apoptotic signals were appeared by AO/EB and comet assay technique in this study. Even though UMB is a well-known antioxidant, the compound was displayed to impose both redox and replication stress in cells, resulting in growth arrest. UMB has been suggested as a pro-oxidant in various cancer cell lines [33]. In the present finding, UMB at various concentrations of 50–450 mM significantly enhanced the ROS generation as compared to untreated KB cells. In the present study, intracellular ROS levels were examined by DCFH-DA in vehicle control and UMB treated KB cell lines by staining with DCA, thus it is important whether UMB stimulates ROS generation in KB cells. DCFH-DA acts as an indicator of peroxides and superoxide accumulation. At microscopic observation UMB exposed KB cells displayed green fluorescence, which evidences the excess making of ROS. These over production of ROS in the UMB treated KB cells suggest that UMB arbitrated apoptosis through the generation of ROS. UMB interact with free radicals to convert them to more stable products, which is terminated the radical chain reaction [34]. Though it’s having only one hydroxyl group, it has more intensity as well as activity against ROS during peroxidation, which is bringing the apoptosis in cancer cells. Thus UMB as an effective anticancer drug that not only brought out the significant normal cells producing peroxide products and also overcome the adverse effects produced from cancer cells. This proved that UMB is the anticancer drug against oral cancer and protect cells, increasing their oxidative capacity, which may also due to the anti-proliferative activity of UMB. High level of ROS is produced especially when cells undertake chemical or environmental stress, might be one of the causal factors most important to the cell cycle arrest or apoptosis.

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Mitochondria play a fundamental role in incorporating and directing death signal towards the caspase cascade by this activator in authority for a variety of crucial events in apoptotic route, such as fluctuations in electron transport, loss of MMP [35] and release of caspase activators. MMP alteration is an indicator during the early stage of apoptosis [36]. Cancer cell displays comparatively reduce resting membrane potentials paralleled with normal multiplying cells [37]. MMP changes were measured by the uptake of the cationic fluorescent dye Rh-123 fluorescence in KB cells at different concentration. Treatment with UMB resulted in the increased depolarization of the MMP as revealed by the elevated fluorescence intensity (Fig. 3B) for Rh-123 absorption compared to vehicle control. We observed accumulation of Rh-123 in the mitochondria of vehicle control cells (Fig. 3A). Whereas the UMB treated cells showed no uptake of Rh-123. This indicates that MMP has been altered during UMB treatment. In this report, we showed that UMB induced attenuation of MMP possibly through ROS production, which promote mitochondria membrane permeability and subsequent induction of apoptosis. Disturbances of apoptosis in cancer cells have been studied in detail and induction of apoptosis in these cells is one of the strategies for anticancer drug development [38]. In this study, we present evidence demonstrating that treatment of KB cells with a variety of concentrations of UMB resulted in dose and time dependent sequences of events marked by apoptosis, such as loss of cell viability; inter nucleosomal DNA fragmentation and G1/G0 phase accumulation. This study also defines those events, most of which are used as biomarkers of apoptosis, that were associated with UMB induced apoptotic cell death in KB cells. Apoptosis is controlled by two major pathways, a mitochondrial pathway [39] and a membrane death receptor (DR) pathway [40]. Mitochondrial dysfunctions, including the loss of MMP and release of cytochrome c (cyt c) from the mitochondria into the cytosol are associated with apoptosis [39]. Apoptosis is a physiologically formed pattern of cell death categorized by morphological structures and widespread DNA fragmentation [19]. Thus to determine whether the growing inhibitory activities of UMB were linked to the induction of apoptosis, the morphological changes of KB cells were investigated using AO/EB staining assay to confirm cell apoptosis. AO is occupied by both viable and non-viable cells discharges green fluorescence if interpolated into double stranded nucleic acid (ds DNA) and EB is occupied only by non-viable cells and releases red fluorescence by intercalation into DNA. Thus, live cells have a green, while the early apoptotic cells are the bright green nucleus with condensed or fragmented chromatin and the late apoptotic cells shows condensed and fragmented orange chromatin [41]. We observed KB cells exposed with UMB for 24 h exhibited early apoptotic cells (50 and 100 mM; bright green nuclei), late apoptotic cells (150, 250 and 350 mM; orange nuclei) and necrotic cells (450 mM; red nuclei). Therefore, it might be concluded that UMB treatment could induce apoptosis significantly in (100– 450 mM) KB cells than the vehicle control KB cells. UMB treated KB cells demonstrated significant apoptosis related morphological alterations, such as apoptotic body formation and chromatin condensation. In concurrence with the findings of the present study, UMB induced DNA damage has also been reported in HepG2 cells [25]. This could be due to the higher internalization of the drug UMB inside the cells, causes an increase in ROS levels and subsequent loss of MMP resulted in apoptotic morphological changes. Our results are in good agreement with the results of Yu et al., [25] reported that PI staining shows induction of apoptosis by UMB on HepG2 cells. Our data indicate that UMB induces apoptosis of KB cells through a mitochondria- dependent pathway. Cell-free systems have been used to demonstrate that mitochondrial release of cytC into the cytosol is rate limiting in terms of the activation of

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caspases and endonucleases [39]. Cytosolic cytC activates caspases (including caspase-3), triggering apoptosis [42]. In this study, we provide evidence demonstrating that UMB-induced apoptosis of KB cells are mediated by loss of mitochondrial membrane potential, increased cytosolic translocation of cytC, activation of caspase-3 and degradation of PARP. Moreover, ROS can also play an important role in apoptosis by regulating the activity of certain enzymes involved in the celldeath pathway [43–45]. All of these factors point to a significant role for intracellular oxidative metabolites in the regulation of apoptosis. It was also reported that many stimuli, such as anticancer drugs, could induce cells to produce ROS and which mediate mitochondrion-initiated apoptosis by inducing the loss of MMP [46,47]. Growth inhibition and ROS generation induced by UMB in KB cells indicate that ROS production probably causes apoptotic cell-death via the mitochondrial pathway. Damage to DNA by reactive oxygen metabolites (superoxide radical, hydrogen peroxide radical and precursors of number of oxygen derived radical including hydroxyl radical) is a significance of oxidative stress that dismiss nonstop yield single or double stranded DNA breaks, purine, pyrimidine or deoxyribose modifications and DNA cross-links also induce a number of oxidative DNA adducts, including 8-Oxo-20 -deoxyguanosine, have been implicated in the tumorigenic process [48]. The comet assay remains used to analyze the stimulus of DNA damage. In our study UMB treated KB cells 24 h in a dose dependent manner. Damaged DNA was travelling during electrophoresis from the nucleus towards the anode, creating a shape of a “comet” with a head (cell nucleus with intact DNA) and a tail (released or broken DNA). This assay is capable to detect DNA cross links, imperfect excision repair events, alkali-labile sites, such as single-strand breakage events [49,50]. We observed or depicted, UMB treatment caused extensive DNA damage which is evident by the formation of comet, according with% tail DNA and olive tail moment. The comet assay indicated the reason for improved DNA damage in UMB might be owing to increased generation of ROS. Our results found that UMB (250–450 mM) showed an enhanced cleavage of DNA into lay to rest nucleosomal fragments indicates more apoptosis when compared with a lower concentration of UMB (50 and 100 mM) treated group. Our results are in lines with the previous findings, which supported that UMB induces DNA fragmentation in HepG2 cells. Reports shows that UMB has been considered to act as antimutagen by modifying DNA replication and/or DNA repair after DNA damage by mutagen. It has been suggested that UMB exhibits antimutagenic and/or anticarcinogenic effects at various in vitro and in animal systems which may be linked to its antioxidant nature as well as alkylation of UMB improves its antimutagenic activity [20]. Further, UMB treatment to human blood lymphocytes 30 min before radiation significantly reduced the DNA damage [21]. Pillai et al., [22] have been demonstrated that UMB prevented mutations induced by benzo(a)pyrene and hydrogen peroxide and also exhibited good superoxide scavenging ability in the PMSNADH assay. One such phytophenolic compound, UMB has anti-cancer, which had been reported earlier by Revathi and Manju [29], from their in vivo studies in hepatic carcinoma rats. Moreover, they carry various biological, biochemical and pharmacological activities, suggesting that they significantly affect basic cell functions such as growth, differentiation and/or programmed cell death (PCD). Results from various in vitro readings also point to that coumarin derivatives exhibited anti-proliferative properties in cooperation human and animal derived cell lines. In the current study, we reported UMB mediated cytotoxicity, DNA fragmentation, altered MMP, apoptosis and cell cycle arrest in KB cells in a time and dose dependent manner. In the current work, UMB markedly

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suppressed the proliferation of KB cells. These results suggest that UMB may be useful for the prevention and/or treatment of patients with oral squamous cell carcinogenesis. As it is known that chemotherapeutic agents induce apoptosis and that DNA strand breaks may be an indication of on-going PCD, we searched for morphological signs of apoptosis and for its molecular hallmark, the DNA damage by comet assay. UMB treated groups showed long DNA tail, indicating DNA damage was not observed vehicle cotrol KB cells. This could be due to the generation of ROS by UMB. The mechanistic role of ROS generating anticancer agents is only beginning to be understood, the mechanism of most anticancer agents was believed to be mainly due to the direct the interaction with DNA and interfering with DNA regulatory machinery and to the initiation of DNA damage via production of ROS. Under prooxidant conditions, highly reactive radicals can damage DNA along with protein and lipid components, which may lead to PCD. Apparently, the mechanism of action of UMB affecting the cells is not the same in all cancer cell types. In KB cells, the UMB-induced inhibition of proliferation may be through G0/G1 arrest. One feature of malignant cells is the lack of vehicle control on their cell cycle, mainly at the transition G1/S [51,52]. Cyclin D1 is expressed early in phase G1 and is degraded prior to entrance into S-phase in normal cells [53]. In recent findings were shown that UMB was inhibiting the G1/S transition in A-427 cell line at 160 mg/ml (1 mM) concentration due to decrease of cyclin D1 expression. This has led to action on its early events in G1 phase [11]. Our results were corroborated with these findings. Profound studies were shown that UMB inhibited in vitro DNA synthesis and topoisomerase II functions, the machinery associated with cell division and its regulatory proteins. This important observation as they allowed to possible identification of key components of cell cycle progression machinery may be modulated by drug treatment. UMB arrests the G0/G1 phase in the cell cycle. The nature of the cytotoxicity effects observed, along with their effects on key cellular events such a regulation of cell cycle progression. Treatment of KB cells with UMB (100, 200 and 300 mM) resulted in an increase in the proportion of cells in G1/G0 phase, along with fewer cells entering S and G2/M phase consequently, a reduction in the percentage of cells re-entering the Sub-G1 phase. The effects of this latter compound were more apparent at lower drug concentrations. This result is in agreement with the cytotoxicity data shown in Fig. 1B. As seen with the cell proliferation assay, UMB produced biological effects. A visual inspection of flow cytometric histograms showed in my study no evidence of cell death, using the study conditions specified here (Fig. 6). However, there is an apparent early G1 phase accumulation, characterized by a widening of the Sub-G1 phase and the heightening of the G1 phase signal, which may account for the G1 and S phase overlaps (Fig. 6B). These observations are important as they allowed the possible identification of key components of cell cycle progression machinery that may be modulated by drug treatment. In our present studies elucidate the UMB produced dose dependent cytotoxicity by increasing intracellular ROS, executed apoptotic program by fragmentation of chromatin DNA alterations in MMP, exhibited antitumor activities in KB cells via induction of cancer cell apoptosis (PCD) and cell cycle arrest at G0/G1 phase. In conclusion, our collective data suggested that UMB prevents cell growth of HOC KB cell lines through induction of cell cycle arrest G0/G1 phase arrest and apoptosis by dose and time dependent manner. UMB induces apoptosis by ROS generation, loss of MMP and cytC release through intra nucleosomes DNA fragmentation and also cell cycle arrest due to G1 phase accumulation in KB cells dose dependently. Our data confirm the potential of UMB as an agent of chemotherapeutic and cytostatic activity in human oral carcinogenesis. KB cells are in pro-

oxidant conditions, UMB may direct interaction with DNA and interfering regulatory machinery to initiate DNA damage due ROS production along with protein and lipid contents lead to PCD. These data suggest that UMB may possess anticancer properties and, therefore, may be potentially valuable for application in food and drug products. Conflict of interest The authors declare that there are no conflicts of interest related to this work. Acknowledgments The author(s) sincerely thank Indian Council of Medical Research, India for providing financial support for this research project, in the form of Senior Research Fellowship (ICMR SRF
IRIS ID: 2015
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