Fucoxanthin induced apoptotic cell death in oral squamous carcinoma (KB) cells

Abstract

Fucoxanthin (Fx) is an active compound commonly found in the many types of seaweed with numerous biological activities. The main goal of this investigation is to explore the effect of Fx against the cell proliferation, apoptotic induction and oxidative stress in the oral squamous (KB) cell line. Cytotoxicity of Fx was determined by MTT assay. The intracellular ROS production, mitochondrial membrane potential (MMP) and apoptosis induction in KB cells were examined through DCFH-DA, Rhodamine-123 and DAPI, and dual staining techniques. Effect of Fx on the antioxidant enzymes and lipid peroxidation in the KB cells was studied through the standard procedures. Fx treated KB cells showed morphological changes and reduced cell survival, which is exhibited by the cytotoxic activity of 50 µM/ml (IC50) Fx against the KB cells. The Fx treatment considerably induced the apoptotosis cells (EB/AO) and decreased the MMP (Rh-123) in KB cells. Further, it was pointed out that there was an increased lipid peroxidation (LPO) with decreased antioxidants (CAT, SOD and GSH). These results concluded that Fx has the cytotoxic effect against KB cells and has the potential to induce the apoptosis via increased oxidative stress. Hence, the Fx can be a promising agent for the treatment of oral cancer and it may lead to the development of cancer therapeutics.

Keywords: Antioxidant; Cytotoxicity; Lipid peroxidation; Oral cancer; Reactive oxygen species.

Figure 1

Chemical structure of Fx.

Figure 2

Effect of Fx on cell viability and morphological characteristics of OC cells by MTT assay. (A) Depicts Fx treated with cancer cells at various concentrations (Control, 25 to 200 µM/ml) respectively. B) Morphological changes in control and Fx treated KB cells for 24 hr. Results are expressed as cancer cells treatment with either control or Fx for 24 h. Values were presented as mean ± SD of asterisks independent experiments ANOVA followed by DMRT. Asterisks indicate statistically different from control *p < 0.05.

Figure 3

Effect of Fx on the intracellular ROS levels in the KB cell. (A) Fluorescence microscopic showing the production of intracellular ROS using DCFH-DA staining in OC cells (KB). White arrow mark represents clearly visible DCF fluorescence in cancer cells treated with Fx in various concentration manners. (B) Intracellular ROS examined by spectrofluorometer. The values were presented as mean ± SD of asterisks independent experiments ANOVA followed by DMRT. Asterisks indicate statistically different from control *p < 0.05.

Figure 4

Effects of Fx on the level of MMP in the KB cells. (A) Effect of Fx on the MMP of HOC cells (KB). OC cells were treated with various concentrations Fx for 24 h, stained with Rh-123 and the mitochondrial depolarization patterns of cancer cells were observed. Results the gradual decrease of red/green fluorescence indicates a decrease MMP in a various concentration manner were investigated by fluorescent microscope. In the fluorescent image shows control (Rh accumulation); Fx (25 and 50 µM/ml) (No Rh-123 accumulation). B) Quantification of MMP in the spectrofluorometry. Values are given as mean ± SD of three experiments in each concentration ANOVA followed by DMRT. Asterisks indicate statically different from control * p < 0.05.

Figure 5

Effects of Fx on the induction of apoptosis in the KB cells. (A) OC cells (KB) treatment within control and Fx at different doses at 24 h, stained with AO/EB and then evaluated by fluorescence microscopy. White arrow indicates green florescence; Orange arrow indicates apoptotic bodies; Blue arrow indicates apoptotic cells; Yellow arrow indicates necrotic cells. Fx induced apoptosis by generating ROS and interruption of MMP. (B) % of apoptotic cells were measured by scoring apoptotic and viable cells (KB). The values are given as mean ± SD of three experiments in each group ANOVA followed by DMRT. Asterisks indicate statistically different from control * p < 0.05.

Figure 6

Effect of Fx on LPO and antioxidants levels in KB cells. Fx induced LPO and modulates cellular antioxidant levels in HOC cells (KB). The values are given as mean ± SD of three experiments in each group ANOVA followed by DMRT. Asterisks indicate statistically different from control * p < 0.05.