ANTI-CANCER ACTIVITY OF ASTER TATARICUS ON SCC-9 HUMAN ORALSQUAMOUS CARCINOMA
Abstract
Background:
Oral squamous carcinoma is a head and neck cancer, which is one of the types ofmalignant cancers. Present study evaluates the anticancer activity of Astertataricus (AT) on SCC-9 human oral squamous carcinoma.
Materials and Methods:
Ethanol extract of AT was prepared by a standard procedure of maceration. AT extractwas used in different concentrations like 10, 20, 40, 80, 160, 320 and 640 μg/mlfor the evaluation of its anticancer activity. Effect of AT extract on SCC9 cells wereobserved by microscope and cytotoxicity by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Moreover,clonogenic assay was used for the estimation of effect of AT extract on colony formingability of SCC9 cells.
Result:
Result of the study suggested that treatment with AT extract causes cytotoxicity toSCC9 cancerous cells. In addition, AT extract treatment reduces clonogenic potential ofSCC9 cell and it also inhibits the proliferation of cell significantly (p<0.001)in G2/M phase.
Conclusion:
Thus, given study concludes that AT extract effectively attenuates the growth of SCC-9cancerous cells by the virtue of its cytotoxic and anti clonogenic activity.
Introduction
Oral squamous carcinoma is the 6th most malignant cancer across the globe(Krishna et al., 2013). Every year around 600000 cases reported and around 350000 deathsoccur due to it. Squamous cell carcinoma is the commonest type of oral cancer (Forman etal., 2014). Head and neck cancer may originate from the different anatomical regions such aslarynx, nasopharynx, lip, oropharynx and neck cavity. In 70 - 75 % cases of head andneck cancer the commonest cause is tobacco and alcohol (Anantharaman et al., 2013).Moreover, other risk factor that causes head and neck cancer is human papilloma virus (HPV)infection, poor oral hygiene, deficiency of vitamins and malnutrition (de Camargo Cancela etal., 2010).
However, there are various treatment options available for head and neck cancer such asradiotherapy, chemotherapy and surgery (Qi et al., 2010). Although these therapies possessbeneficial effects but some time they cause severe toxicity and thereby affect the posttreatment life of patient. Moreover, resistance of cancer cell from the therapy emerges newproblem, which is required to develop an alternate therapy for the management of it(Saraswathy and Gong, 2013).
Aster tataricus (AT) traditionally used in China last from 2000 years formedicinal purpose. Roots of AS contain the chemical constituents like triterpenes andsaponins (Dongliang and Yu, 1993). Other reported chemicals that were isolated from rootssuch as epifriedelinol, caffeoylquinic acids, astersaponins, shionone and aster peptides (Yuet al., 2015). Traditionally it has been used as antibacterial, antifungal, anticancer andmanagement of chronic bronchitis and tuberculosis (Bown, 1995). Literature suggested thatroot extract of AS possess anti-inflammatory, expectorant and antitussive activity (Yu etal., 2015). Caffeoylquinic acids and epifriedelinol possess strong antioxidant andanti-inflammatory (Duke and Ayensu, 1985; Ma et al, 2011; Peluso et al, 1995). Thus, thegiven study evaluates the anti cancer activity of AT on human oral squamous carcinoma.
Material and Methods
Extraction of plant
Root of AS was procured from local supplier and authenticated from Institute of MedicinalPlant Development, Beijing (Specimen No: IMPD/10/2016). Extraction of AS roots was done bymaceration. Dried root was powdered and kept it with ethanol for 72 hrs. Thereafterethanol was evaporated from the extract at low temperature by rota vapor apparatus.Percentage yield of extract was found to be 8.2 % w/w.
Cell and culture media
SCC-9 and NIH/3T3 cell lines were procured from Shanghai Institutes for BiologicalSciences, Shanghai, China and all the protocols were approved from Ethics Committee of the306th Hospital of PLA, China. In a 5% CO2 humidified incubator at 37°C cells lines were incubated. DMEM medium was used with (10% v/v) fetalbovine serum as a culture medium for the present study. Medium used in this study wasreplaced at a specific interval of time and cell were trypsinized routenly till it growsup to 80-90% of confluency.
Aster tataricus extract treatment
Extract of AT used for the treatment after diluted in the culture medium to achieveserial concentrations. Culture media with 1% DMSO was used to treat controlcells.
Evaluation of morphology of SCC-9 cell lines
Light microscope was used to observe the changes of SCC-9 cells morphology after thetreatment with AT extract. Cell lines (5 × 104 cells/well) were kept in 96well plate and for one day treated with AT extract with different concentrations of 0 to640 μg/ml. Moreover, as an additional control fibroblast cells (NIH/3T3) of mouseembryo was used in this study. Morphological changes in the cell line were monitored bythe help of phase contrast microscope.
Estimation of cell viability by MTT assay
MTT assay was used to assess cellular viability. In which cell (5×103cells/well) were placed at 96 well plate and treated with AT extract at a differentconcentration for the period of the duration of 3 days. MTT assay was performed asdescribed by Xiao et al. MTT (50μΓ) was added to the well plate and keep itfor incubation for 4 hr. at 37°C. Thereafter from the reaction mixture supernatantwas washed out and subsequently dimethylsulfoxide (200μl) was added to all the wellplate at room temperature. The absorbance was estimated at 570 nm wavelength (Xiao et al,2015).
Estimation of AT extract effect on stages of SCC-9 cell cycle
SCC-9 cells (1×106 cells/well) were seeded in a 6 well plate andincubated for 32 hr. After a specific period of incubation DMEM media was replaced byserum (1%) and incubated it for synchronization of cell cycle. These cells weretreated with 10, 20, 40, 80, 160, 320 and 640 μg/ml of AT extract for 24 hr andthereafter PBS was used to wash all the cells, later 70% ethanol at 4° C wasused to fix it for 24 hr. Later cells were washed and incubated with DNase free-RNase-A(100 μg/ ml) for one hour at 37°C and propidium iodide was used for thestaining of it. Cell cycle was estimated by using flowcytometer (Becton Dickinson, USA)(Chen et al., 2012).
Clonogenic assay
cells were (5×103 cells/ well) poured in to a 6 well plate andincubated for one day at 37 °C. These cells were treated with AT extract at 50,100, 200 and 400 μg/ml for one day. Thereafter culture media was exchanged by DMEMcontaining FBS (10%) and this process of replacement of media with fresh onerepeated after 5 day. On 21st day cells were washed with PBS and crystal violet(0.25%) was used for staining for 30 min. Number of colonies were counted bystereomicroscope and its plating efficiency was determined (Franken et al., 2006).
Statistical analysis
Data of given manuscript represented as mean ± SD (n=10). All the results wereanalyzed statistically by one way ANOVA and post hoc study by Dunnett. In this studyvalues p<0.05 was considered as significant.
Result
Effect of AT extract on cell morphology
The effect of AT extract on morphological characteristics of SCC9 cell line was shown inFig.1. AT extract treated SCC9 cells were lost thecharacteristics as contact between adjacent cell lost and cell shrunken in size. Theeffect was found in a dose dependent manner.
Evaluation of effect of AT extract on cell by MTT assay
Effect of AT extract on SCC9 cells was estimated by MTT assay as shown in Fig.2. It was observed that treatment with AT extractinhibit the proliferation of SCC9 cells in a dose dependent manner as at a lowconcentration (10-40 μg/ml) AT extract inhibit the proliferation by below20%. Whereas at 80 and 160 μg/ml concentration it shows inhibition ofproliferation between 20-30% and at higher concentration such as 320 and 640μg/ml AT extract inhibits above 50% proliferation of SCC9 cell.
Effect of AT extract on SCC9 cell by clonogenic assay
Surviving fractions was found to be significantly decreases when SCC9 cells were treatedwith AT extract for the period of one day. This decrease in survival fraction of SCC9 cellwith AT extract was found to be in a dose dependent manner as shown in Fig 3.A.
Control group of SCC9 cells has shown 100% cloning ability. Moreover, treatmentwith AT extract significantly (p<0.001) declines the cloning ability of SCC9 cellsas compare to control group in a dose dependent manner as shown in Fig. 3B.
Effect of AT extract on SCC9 cell cycle
Effect of AT extract on SCC9 cells at various stages of cell cycle was shown in Fig.4. It was observed that treatment with AT extractsignificantly (p<0.001) increases the cells at G2/M phase compared to controlgroup. Whereas, the cell in G0/G1 phase decreases in AT extract treated group compared tocontrol group.
Discussion
Management of cancer becomes complex as due to resistance of cancer cell towardsconventional treatment options. Literature reveals that phytomedicine possess stronganticancer activity and promises alternative therapy for the management of cancer (Xavier etal., 2009). This study evaluates the anticancer activity of Aster tataricusextract on SCC-9 cancer cells. Effect of AT extract on SCC9 cancer cells was evaluated byobserving the morphology, MTT assay, clonogenic assay and cells in which phase of cellcycle. Previous report suggested that phytomedicine do the morphological changes in oralcancer cell line and the result of the give study shows that treatment with AT extractcauses similar changes in the morphology of SCC9 cell line (King et al., 2007). Reportsuggested that AT contain several chemically active substances that possess several medicalproperties. Caffeoylquinic acid is the chemically active compound that reported to havestrong antioxidant and anti-inflammatory property (Duke and Ayensu, 1985; Ma et al, 2011;Peluso et al, 1995). Moreover, it has effect on cell viability and effective against lungcancer (In et al, 2016). Thus, cytotoxic potential of AT extract was estimated by MTT assay.In which mitochondrial dehydrogenase that is present only in viable cell reduces tetrazoliumsalt and converted it in to formazan (Mosmann, 1983). Our study shows the reduction oftetrazolim salt in a dose dependent manner in AT extract treated SCC9 cell line. Moreover,clonogenic ability of cancer cell is the important characteristics and estimation of it wasdose by clonogenic assay (Katz et al., 2008). Cytotoxic drugs show anticlonogenic activityand thereby inhibit the growth of tumor cell. Result of this study suggests that treatmentwith AT extract significantly (p<0.01) decreases cloning ability of SCC9 cells in adose dependent manner. In addition, effect of AT extract was observed over the cell cycleand observed that treatment with AT extract significantly (p<0.01) increases the nocells in G2/M phase compared to control group.
Conclusion
This study concludes that Aster tataricus extract possess anticanceractivity on human oral squamous carcinoma cell line.
Acknowledgement
This study was supported by the State key laboratory of aerospace medicine foundation andapplication of open issue (Differentiation gene research of periodontal ligamentosteoblastic differentiation under simulated weightlessness based on microarray datascreening of rabbits, SMFA14K03).and the Capital health development research project ofBeijing, China (Grant No.2014-4-5051).
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