Anticancer potential of sclareol against human KB oral carcinoma cell line invitro

Sclareol, a labdane diterpene phytochemical in the various genus Salvia of the herbaceous plant has been anti-carcinogenic properties on numerous human cancer cell lines. Till known the anticancer properties of sclareol on human oral KB cancer cells are not revealed. Therefore, we scrutinized the viability, nucleate damage associated with apoptosis in KB cells treated with sclareol.The cytotoxicity of KB cells displayed to sclareol was evaluated by hexosaminidase and trypan blue exclusion assay. Apoptosis was noticed by Hoechst staining and affirmed by nucleate damage. We discovered that sclareol treatment for KB cells with expressively lessened cell proliferation or viability and evoked cell death in a dose-dependent manner compared with untreated control. Sclareol stimulated inhibition of growing of oral KB cells came along to be induction nucleate damage. Our data proved that sclareol on stimulated apoptosis in human oral KB cancer cells through nucleate damage. These observations recommend the anti-cancer properties of sclareol.


INTRODUCTION
Squamous cell carcinoma is the majority of oral cancer which originates from the squamous mucosa of the oral surface. In spite of the similar histologic phenotype, the molecular structure of oral cancer is extremely complex, which exposes numerous genetical mutations (Ram et al., 2011) &(Rothenberg andEllisen, 2012) . Squamous cell carcinoma of oral cancer is a high mortal and morbid in countries such as India Srilanka, Pakistan (Bagan and Scully, 2008) increasing risk in young people below 40 years old. High rates of this incidence occur in the southern region of Asia, some parts of Europe and Latin America (Warnakulasuriya, 2009) . Now days, oral cancer incidence has been increasing especially in developed countries (Chaturvedi et al., 2013) . Some speci ic methods such as drugs, radiotherapy, chemotherapy, surgery have been utilized for the initial stage of oral cancer. Radiotherapy and Surgery have been stimulating undesirable side effects due to the un-identify targeting on both normal and cancer cells (Warnakulasuriya, 2009). Due to recent advancements in surgery, imaging, systemic therapies, and radiation, overall average survival has been promoted by 15 percent in the last 50 years, however, only 5 percent of survival had occurred in the last 20 years (Chinn and Myers, 2015) . Because the potency of standard treatment is til now moderated, the novel therapeutic strategies are eagerly anticipated. These systematic procedures for therapy are not so ef icacious and thus the necessity of natural chemotherapeutic compounds has been an essential demand. The welldevised diet consist of fruits and vegetables was directly associated with the diminishing risk rate of cancer (Wang et al., 2014) .
Apoptosis kind of regulated programmed cell death which occurred genetically that controls the proliferation of multicellular organisms (Taraphdar et al., 2001) . Apoptosis represents a crucial role in the step-down of damaged or abnormal cells without sickening normal cells (Elmore, 2007) . Chemotherapeutics wreck down tumor cells and constrain their proliferation initially by upgrading tumor cell apoptosis (Yamamoto et al., 1999) . The ef icaciousness of anticancer drugs is valued by their capability of cancer cell detection and apoptosis promotion. Phytochemicals have low toxicity and also very low harmfulness to normal cell hence, which is used as a substitute method for cancer therapy (Mehta et al., 2010) . Apoptosis is characterized by morphological alterations such as membrane blebbing, cell lessening, chromatin contraction and DNA fragmentation (Ziegler and Groscurth, 2004) .

Drug treatment
Sclareol was dissolved in dimethyl sulfoxide (DMSO) as 100 mM stock solutions. Then the stock solution freshly diluted with a DMEM medium for the further assay.

Cell culture
National Centre for Cell Science (NCCS), Pune provided us human oral epidermal carcinoma cell line (KB cells). DMEM medium (added on 10% fetal bovine serum 1% penicillin, 1% streptomycin) has been used for KB cells growing. A moisturized incubator containing CO 2 at 37ºC has hatched these KB cells.

Hexosaminidase Assay
The cell viability was dictated by hexosaminidase assay according to [Landegren, et al., 1984]. The 30, 000 cells were sowed in a 96 well plate and treated with sclareol at 5 -50 µM for 24hrs. After that, the medium was removed and citrate containing pnitrophenol N-acetyl-beta-D-glucosaminidase, was added (60µL per well) in to well then incubated for 30min at room temperature. Subsequently, terminating the reaction by adding 90µL of 50mM glycine containing 5mM of EDTA (pH 10.4) and absorbance was measured with Read well touch, ELISA plate reader (Robonic, India) at 405nm. The % cell viability was calculated by using the following formula: Y-axis for % of proliferation and X-axis for the concentration of the sclareol, hereby graph was plot-ted. 5, 10, 20 µM/mL for sclareol, were the obtained values, and were farther utilized for assessment of effects on nucleus, morphology and apoptotic effects on DNA. Figure 2 shows the experiment was carried out by mean ±S.D using a one way ANOVA. The peak value is ≤ 0.05 was signi icantly different from the control sample.

Trypan blue assay
Trypan blue dye exclusion assay was used for the evaluation of cell membrane integrity of live and dead cells using KB cells. The 3000 cells were sowed 96-well plate and were treated with several concentrations of sclareol (5-50µM) for 24hrs. Afterward, these cells were rinsed off with PBS and then cells detached by trypsin/EDTA (200 µL) and sub-sequently 50µL of trypan blue was summed up to every well and plate was incubated for 5min. Hereafter, 20µL of trypan blue aliquot was laid on a Neubauer hemacytometer. The number of viable cells was counted with the help of microscopy. The following formula is used for calculation of the number of viable cells V iable cell count X dilution X 10 4 N Where N is the number of hemacytometer squares was counted. The percent of viability was calculated as: V iable cell count T otal cell count X 100

Hoechst staining
Nucleate disruption an indicator of apoptosis was analyzed by Hoechst staining . Concisely, cells sowed in 6 well plates were treated with sclareol 5, 10 and 20µM and incubated for 24hrs. Cells were stained by Hoechst for 30 min in a dark at 37 • C. After removing the medium, the cells were rinsed twice off with PBS. Stained cells were successively examined by using a luorescent microscope (Floid Cell Imaging Station, Life technologies, USA).

PI staining
Propidium iodide (PI) staining was done by the method of [Sirpu et al., 2018]. 5000 cells were placed in 6 well plates and were treated with sclareol 5, 10 and 20µM for 24hrs. Before stained with 10µL PI for 20min, these cells had gently been ixing in methanol: acetic acid (3:1 v/v) for 10min. Condensed and fragmented nuclei were observed by a luorescent microscope (Floid Cell Imaging Station, Life technologies, USA). Therefore we concluded that sclareol induced nucleate morphology of apoptotic cell death.

Statistical analysis
The biological data perception was held by one way ANOVA-Duncan's test. Data are presented as means ± S.D was described at p≤ 0.05.

Sclareol inhibited cell growth in KB cells
To further investigation, the cytotoxic effect of sclareol was determined by hexosaminidase assay and Trypan blue assay. KB cells had cautiously been treated with sclareol for 24hrs and the outcome exhibited in a dose-dependent manner for both assays. Hexosaminidase assays ≤ 20 µM and Trypan blue assay ≥18.5µM led to a drastic cell number decrement compared with untreated control Figure 1 and Figure 2 . Both assays in prescribed seemed to be preferred for triggering inhibition of cell growth. So 5, 10, 20µM sclareol had been used in the following experiments.

Morphological changes using an inverted phasecontrast microscope
The apoptotic features of the sclareol had smartly been investigated by perceiving the morphological modi ication in KB cells for 24hrs. The morphological modi ication typically exhibited following phenomena, chromatin cleavage, nucleate condensation, cell shrinkage, membrane blebbing, (Rasola and Geuna, 2001) & (Moongkarndi et al., 2004) . These characteristics are distinctive stylemark modi ication changes and appreciated in cell deaths which are broadly utilized to quanti ication and identi ication of apoptosis. The morphological modi ication had been realized lucidly with help Inverted phase-contrast microscope Figure 3. By visualizing the untreated control cells we understood that cells' original adhesive behavior kept on stable by attached to their loor culture dishes with no loating. Sclareol treatment (5, 10, 20µM) revealed that remarkable morphological modi ication such as forming round shape, shrinkage, abnormal shape of the plasma membrane and lost nearby cell contact. Figure 3 shows the sclareol treated cells showed condensed nuclei, cell shrinkage and membrane blebbing, apoptotic bodies. The percentage of a conluence of the cells notably decreased to 75% at 24 h for sclareol treatment.

Effect of sclareol stimulated nucleate damage
Hoechst staining could be used to detect condensation and DNA fragmentation. Figure 3 showed that has untreated control cells have been no morphological changes and sclareol treatment (5, 10, 20µM) has been shrunken, fragmented and brightly luorescent with condensed chromatin were compared to untreated control cells by using luorescence microscopy (20×) imaging station, life technologies.
Oral cancer is a kind of utmost common cancers, which paralyzes the lifestyle of so many people, thence contributes to the burden economically and psychosocially on so many countries. So the necessity of chemotherapeutic drugs for curing oral cancer is extremely essential. Existing drug for oral cancer such as cisplatin and methotrexate, which leads to adverse outcomes. Hence the seeking for an innate chemotherapeutic agent without any adverse outcomes is always in extremely needing  . Our results showed cytotoxicity assay using hexosaminidase and trypan blue has been clearly revealed that sclareol signi icantly inhibited oral squamous cell carcinoma after an incubation period of 24hrs by hexosaminidase assays. The dye exclusion assay showed that sclareol can effectively inhibit cell proliferation in the sense different concentrations which decrease % cell viability. Similarly sclareol own inhibition of cell growth and induction apoptosis in MG63 human cancer cells (Wang et al., 2015b) .
Chemotherapeutics used its primary mechanism to ruin tumor cells by using stimulated apoptosis. Apoptosis is powerfully consociated with chemotherapeutic sensibility (Yamamoto et al., 1999) . Therefore, the resolution of the morphological modi ication to described apoptosis was clearly visualized by using an inverted phase-contrast microscope. On belated periods of exposure of KB cells which undergo the necrosis was assessed depend on medication that impacts the plasma membrane (Elmore, 2007) . According to our further demonstration, that, PI and Hoechst staining has been utilized for cell apoptosis detection. DNA fragmentation of apoptotic cell nuclei, condensa-tion and cellular damage Figure 4 and Figure 5 has been showed by sclareol treated KB cells. Emphatically, the DNA of apoptotic cells displays oligonucleosomal fragments are a characterized laddering pattern. Hence force, sclareol had found a morphological modi ication of apoptosis in these cells by using a cell luorescence microscope and sclareol stimulates the arrest of the cell cycle in those cells. (Wang et al., 2015a). As per the detection in control cells, KB cells seemed to be inviolate to have the nuclei as less or no luorescent. Apoptotic cells were puffed-up and had sporadic membranes and chromatin indicated luorescent bright blue. Because of feeble membrane unity, PI seeps into shrunken cells and their DNA fragmentation is commonly conceived to be the hallmark of apoptosis. Various studies have proved a certain correlation between the fragmentation of DNA and apoptosis (Kapoor and Kakkar, 2012) & (Kumari and Kakkar, 2012) . The results backing our discovered data that crucial changes occurred in cell death could take place DNA fragmentation which is the reason for considering cell death of apoptotic (Cohn et al., 1992) .

CONCLUSIONS
The present study could be for the irst time, which showed cytotoxicity sclareol on oral squamous cell carcinoma in which apoptosis or programmed cell death performs a crucial role. Sclareol should also be considered as a reliable chemotherapeutic agent in oral cancer treatment. Even so, additional investigations are inevitable to validate its therapeutic claims to analyze its impact on the invivo model.