Fucosterol, propidium iodide (PI), RNase A triton X-100 dimethyl and sulfoxide (DMSO), were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All primary and secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The fluorescent probes DCFH-DA, DiOC₆, 4′-6-diamidino-2-phenylindole (DAPI), Fetal bovine serum (FBS), RPMI-1640 medium, L-glutamine, antibiotics were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA).

Human cancer cell lines, human lung cancer cell line (A-549), pancreas (MiaPaca-2), prostate (PC-3, CVCL_0035), breast (MCF-7), gastric cancer cell line (SNU-5), cervical cancer cell line (HeLa) and human normal cell line (fR2) were procured from Cancer Research Institute of Beijing, China, and it was maintained in DMEM and was supplemented with 10% FBS and antibiotics (100 µg/ml streptomycin and 100 U/ml penicillin G) in a incubator at 37°C (5% CO₂ and 95% air).

The anti-proliferation effect of fucosterol was evaluated against a panel of human cancer cell linesby MTT assay. Cells were grown at 1×10⁶ cells per well in 96-well plates for a time period of 12 h and then exposed to different concentration of fucosterol (0–160 µM) for 48–72 h. To each well, MTT solution (20 µl) was added. Prior to the addition of 500 µl of DMSO, the medium was completely removed. To solubilize MTT formazan crystals, 500 µl DMSO was added. ELISA plate reader was used for the determination of optical density. Since an lowest IC50 was observed for HeLa cervical cancer cell line, they were subjected to further evaluation at the doses of 0, 20, 40 and 80 µM of fucosterol dose for 24, 48 and 72 h. We selected one dose lower and one dose higher than the IC50 of fucosterol.

For clonogenic assay, cervical cancer HeLa cells at the exponential growth phase were harvested and counted with a hemocytometer. Seeding of the cells was done at 200 cells per well, incubated for a time period of 48 h to allow the cells to attach and then to the cell culture different doses (0, 20 40 and 80 µM) of fucosterol was added. After treatment, the cells were again incubated for 6 days, washing was done with PBS and methanol was used to fix colonies and then stained with crystal violet for about 30 min before being counted under light microscope.

HeLa cells at a density of 2×10⁵ cells/well were seeded in 6-well plates were administrated with 0, 20, 40 and 80 µM fucosterol for 48 h. The cells were then subjected to DAPI staining. Afterwards, the cell sample was studied and photographs taken under fluorescence microscopy as previously described (7).

HeLa cells were seeded at a density of 2×10⁵ cells/wel in a 6-well plate and kept for 24 h and treated with (0–100 µM) fucosterol for 48 h at 37°C in 5% CO₂ and 95% air. Thereafter cells from all samples were collected, washed 2 times by PBS and re-suspended in 500 µl of DCFH-DA (10 µM) for ROS estimation and DiOC₆ (1 µmol/l) for ΔΨ_m at 37°C indark room for 30 min. The samples were then examined instantly using flow cytometer as described previously in literature (8).

The cells seeded in 6 well plates (2×10⁵ cells/well) and fucosterol was administrated to the cells at the doses of 0, 20,40 and 80 µM followed by 24 h of incubation. DMSO was used as a control. For estimation of DNA content, PBS was used to wash the cells and fixed in ethanol at −20°C. This was followed by re-suspension in PBS holding 40 µg/ml PI and, RNase A (0.1 mg/ml) and Triton X-100 (0.1%) for 30 min in a dark room at 37°C. Afterwards, analysis was carried out by flow cytometry as reported previously (9).

Cell migration assay was carried out by Boyden chamber assay with some modifications. Cells at the density of 5×10⁴ cells/well were suspended in 2% FBS medium and placed in the upper chamber of 8 µm pore size transwells. After wards, medium supplemented with 10% FBS was added to lower chamber. This was followed by an incubation of 48 h. On the upper surface of the membrane, unmigrated cells were removed while as on the lower surface of the membrane the migrated cells were fixed in methanol (100%) and Giemsa stained. The cell migration was estimated by counting the number of the migrated cells under a microscope.

The fucosterol administrated cells were harvested and lysed. The protein concentrations of the lysates were quantified by BCA assay using specific antibodies. β-actin was used as a control. From each sample equal amounts of protein were loaded and separated by electrophoresis on a 12% denaturing SDS gel. Afterwards, the proteins were then electroblotted on polyvinylidene difluoride membranes (0.45 m pore size).

All experiments were carried out in triplicates and expressed as mean ± standard deviation (SD). Statistical analysis was carried by Students t-test and one way ANOVA (in case of comparisons between more than two groups) using Tukey's HSD test. GraphPad prism 7 software (GraphPad Software, Inc, USA). The values were considered significant at *P<0.01, ** P<0.001, ***P<0.0001.

To identify the anti-proliferative role of fucosterol was evaluated against a panel of human cancer cell lines (Table I). However, fucosterol exhibited selective anticancer activity against cervical cancer HeLa cells in a dose dependent manner and exhibited an IC₅₀ 40 µM (Table I and Fig. 1B). In the colony formation assay, we observed that fucosterol administration reduced the number of colonies in a dose-dependent manner (Fig. 1C).

In order to confirm apoptotic cell death induced by fucosterol Annexin V/PI staining was performed. Flow cytometric results showed that the percentage of apoptotic cell population increased to 12.2, 37 and 62 % in HeLa cancer cells after 48 h at the concentrations of 20, 40 and 80 µM, respectively as compared to untreated control (Fig. 2). Thus the results indicate that the extract caused apoptotic cell death in a concentration dependent manner.

The pro-apoptotic potential of fucosterol observed through DAPI staining study suggested that fucosterol might induce generation of intracellular ROS. Therefore, we calculated the ROS level at varied concentrations of fucosterol for 48 h. The results showed that the intracellular ROS levels of treated cells increased 110 to 305% as compared to untreated cells (Fig. 3A). Our result suggested that fucosterol is a potent molecule for activating ROS in HeLa cells to trigger the apoptosis.

ROS generationis related to mitochondrial dysfunction. It disrupts the outer mitochondrial potential to release the death-promoting proteins (10). Therefore, we examined whether fucosterol reduces the ΔΨ_m in HeLa cells treated with fucosterol at varied concentrations. Fucosterol treated HeLa cells showed a significant reduction in ΔΨ_m in a dose-dependent manner (Fig. 3B).

It was observed that the percentage of cells was considerably increased in G2 at the concentrations of 0 to 80 µM concentrations of fucosterol causing G2 arrest (Fig. 4). Additionally the populations of HeLa cells G2 phase were marginally increased at a dose of 20 µM, reasonably increased at 20 µM, and dramatically increased at 40 µM. This fucosterol-induced G2 increase of HeLa cancer cells was observed to exhibit a dose-dependent pattern.

Further, we investigated fucosterol can inhibit the migration of cervical cancer cells at the IC₅₀ concentration (40 µM). The results of transwell assays showed that fucosterol reduced the migratory capability of cervical cancer HeLa cells (Fig. 5).

The fact that fucosterol could modulate the protein expressions of m-TOR/PI3K/Akt signalling pathway, we carried out the western blot analysis. The findings are shown in (Fig. 6) and indicate an interesting outcome. Compared to the untreated control cells, fucosterol treated cells showed a concentration-dependent downregulation of m-TOR and pm-TOR proteins. It also caused marked downregulation of PI3K/Akt protein expressions. Thus it may be concluded that fucosterol induced anticancer and apoptotic effects partly via m-TOR/PI3K/Akt signalling pathway.