br Statistical analysis br The results were elicited with
2.8. Statistical analysis
The results were elicited with triplicate value and expressed as a mean ± SD. All the data were analyzed statistically by One-way ANOVA and student t-test using SPSS software student’s version-16. A p value < 0.05 was considered statistically significant.
3.1. Effect of fucoidan on cell viability assay
Cell viability by MTT assay was evaluated in HepG2 cancer cells constituted with fucoidan/quercetin at different concentrations (0–200 μg/ml) for 48 h (Fig. 1). The pragmatic results revealed up to 98% cell viability in HepG2 cells cultured without fucoidan/quercetin whereas fucoidan/quercetin at 50, 100 and 200 μg/ml constituted cell viability was about 71, 60 & 40/80, 65 & 45%, respectively. Outcome of the results proved that the fucoidan/quercetin treatment was reduced the cell viability significantly (p ≤ 0.05) as below as 50% and the de-cline rate was significant in a concentration dependent manner. How-ever, the effect was more pronounced by fucoidan when compared to the quercetin standard (Fig. 1).
3.2. Effect of fucoidan on colony formation assay and wound healing assay
The clonogenic effect of fucoidan in HepG2 cancer cells was ex-amined using colony formation assay. The HepG2 cancer cells were Toxicology Reports 6 (2019) 556–563
Fig. 1. Inhibition of cell viability in HepG2 cancer cells by fucoidan constituted with nuance of concentration for 48 h. The values are presented as mean ± SD in triplicate and significance (p ≤ 0.05) determined by student t-test between fucoidan treated vs untreated control. Dark bar indicate quercetin and light bar fucoidan.
constituted fucoidan with nuance of concentrations (0–200 μg/ml) and maximum concentration of quercetin standard (200 μg/ml). As the re-sponse of fucoidan towards colony forming potential of HepG2 cancer cells was found to be 78, 55 & 37% at 200, 100 & 50 μg/ml compared with untreated control (100%), respectively (Fig. 2). Besides, the in-hibition effect of fucoidan on colony formation was observed to be more or less equal (37%) to the standard of quercetin (40%) at their maximum concentration (200 μg/ml). Thus, the result clearly revealed that fucoidan has significant (p ≤ 0.05) anti-proliferative effect against HepG2 cancer cells.
Cell migration is one of the essential processes during the cancer cell development and metastatic spread. The suppressive effect of fucoidan on HepG2 cancer cell migration was determined by wound healing assay. The HepG2 cell migration was estimated with or without fu-coidan at different concentrations (50, 100 & 200 μg/ml). Consequently, there was a gradual suppression of HepG2 cell migration observed about 74, 62, and 47% when constituted fucoidan with nuance of concentration 50, 100 & 200 μg/ml compared with untreated control (90%), respectively. However, the effect of fucoidan on HepG2 cell migration suppression was recorded to be concentration-dependent and significantly (p ≤ 0.05) higher (47%) than that of quercetin stan-dard (57%).
The N-octanoyl-L-Homoserine lactone phase distribution of exponentially proliferative HepG2 cancer cells was examined with PI staining by flowcytometer. Histogram of the cell cycle phase illustrated that the HepG2 cancer cells were categorized into G0/G1, S, and G2/M phases (Fig. 4). Compared with untreated control, fucoidan constituted cells had significant (p ≤ 0.05) accumulation of proliferative cells in the G0/G1 phase of the cell cycle in a concentration dependent manner and also significantly (p ≤ 0.05) decreased cells in the S phase of the cell cycle (Table 2). The accumulated proliferative cells in the G0/G1 phase of cell cycle were regarded about 15–35% (50–200 μg/ml) higher than that of untreated control cells (50%) and comparable to the quercetin (87%) at their maximum concentrations (200 μg/ml). Thus, fucoidan also reduced cell population in the G2/M and S phases of cell cycle were about ∼3-4 times lesser than that of untreated control cells (29.4-20.6%). These results substantiated that the fucoidan was appropriately induced HepG2 cancer cell arrest in the G0/G1 phase of the cell cycle.
Fig. 2. Fucoidan on clonogenic effect of HepG2 cancer cells was analyzed by colony formation assay. (A) Colony formation image was cap-tured under inverted light microscope. (B) Colony cells calculated and expressed as the percentages of means ± standard deviation of three independent experiments. *P ≤0.05, vs. the untreated control (0 μg/ml).
3.4. Fucoidan induces genetic damage and apoptosis in HepG2 cells
The fucoidan induced DNA damage in HepG2 cells was studied with comet assay kit by single gel electrophoresis (Fig. 5). The occurrence of genetic damage was scoured in terms of various parameters like head, tail and tail moment by comet assay software (Table 1). There was no significant effect on DNA damage in untreated HepG2 cancer cells whereas fucoidan constituted with nuance of concentration (50–200 μg/ml) was observed to be significantly promoted the DNA damage. Hence, the amount of head damage was regarded to be de-creased in the range of 0.93-0.59 besides tail amount damage increased in the range of 2.8–11.25 while increasing the concentrations of fu-coidan when compared to the untreated control cells. Thus, fucoidan significantly enhanced the tail moment damage was directly reflected in terms of DNA damage occurred in HepG2 cells. The enhanced tail moment value was about 3.8, 7.1 & 12.8 folds higher than that of un-treated control cells. The effect of fucoidan on genetic damage in HepG2 cancer cells was recorded as like as quercetin standard. On the other hand, the effect of fucoidan on apoptotic characteristics of the HepG2 cancer cells was examined as fucoidan induced apoptosis in fucoidan constituted HepG2 cancer cells through annexin V-FITC assay kit analyzed in four groups of cells by flowcytometer (Fig. 6A).The total apoptotic cells including the addition of early and late apoptosis were observed after 48 h of the fucoidan treatment. Total apoptotic cells recorded were about 20–40% at 50–200 μg/ml of fucoidan concentra-tion. Further, fucoidan was significantly (p ≤ 0.05) enhanced the total apoptotic cells (40%) better than the standard of quercetin (44%) at their maximum concentration (200 μg/ml). These results indicated that the inhibition and arrest of HepG2 cancer cell proliferation by fucoidan