MATERIALS AND METHODS

Media: Dulbecco's modified eagle medium (DMEM) and Dulbecco's Phosphate Buffered Saline (PBS) were purchased from MP Biomedicals Inc, USA. Fetal Calf Serum (FCS) was obtained from Gibco, Canada. Coomassie blue, pencillin-streptomycin, Trypan blue and Trypsin-EDTA were purchased from Sigma, USA.

Preparation of tested agents: The powder form of PM 701 and its fractions (PMF and PMFK) were prepared according to Khorshid et al. (2009) each fraction was dissolved in DMEM, 2% FBS at a concentration of 2 mg mL^-1 before using. The solution was then filter sterilized using a 0.2 μ syringe filter to prepare the working solution.

Cell culture: Breast carcinoma cells (MCF-7) were purchased from National Cancer Institute, Cairo University, Egypt. These cells were preserved as cell line in tissue Culture Unit at King Fahad Medical Research Center (KFMRC) in King Abdulaziz University (KAU).

MCF-7 cells were cultured in DMEM supplemented with 10 % FCS, 100 U mL^-1 penicillin and 100 μg mL^-1 streptomycin. Cells were grown in 75 cm^-2 tissue culture flasks in a 37°C incubator with a humidified mixture of 5% CO₂ and 95% air (Ptak et al., 2009).

Cytotoxicity assay: Cells were detached with 0.025% trypsin-EDTA. The cells were resuspended in 10 mL of medium to make single cell suspension and viable cells were counted by trypan blue exclusion in haemocytometer and diluted with medium to give a final concentration of 1x10⁵ cells/well. The 1 mL well^-1 of these cell suspensions were seeded in 24-well microtiter plates and incubated to allow for cell attachment.

Cell count using hemocytometer: A cell suspension fixed in a volume of cells (e.g.. 1 mL). 200 μL of this suspension was mix with an equal volume of trypan blue. Mixed solution was transferred using a pipette to a hemocytometer and live cells were counted. The numbers of cells were calculated per mL, using the following formula (Pollard and Walker, 1989; Khorshid et al., 2005).

Treatment with lyophilized PM 701 or it's fractions: Cytotoxicity assays were performed using short incubation for only 24 h in serials dilutions of examined drug, that were used to estimate the IC₅₀ concentration. The long incubation for more than 72 h, with a concentration lower or higher than IC₅₀ concentration, was also used with only crude substrate (PM 701) to confirm the cytotoxicity effect.

Short incubation for IC₅₀ estimation: Lyophilized PM 701 or PMF, PMFK, were diluted in medium. Serial dilutions were prepared 0, 0.5 and 7.5 μg mL^-1 of media each concentration was added to each well of the plate in 3 replicates of cancer cells (treated), after the selected time of treatment, the medium was aspirated. Then cells washed with PBS, trypsinized and counted by two methods counter coulter and Hemocytometer using trypan blue dye exclusion test (Khorshid et al., 2005; Moshref et al., 2006; Pollard and Walker, 1989; Khorshid and Moshref, 2006).

Fixing and staining cells: Each group of cells were plated onto Petri dishes in DMEM media for 24 h, then the media changed with examined media (with different concentrations) and control media and incubated at 37°C for 24 h.

Each group of cells were fixed in 4% formaldehyde for 5 min at room temperature after double washing with 1 X PBS each for 5 min. Then cells stained with Coomassie blue for 5-10 min followed by repeated washing with tap water (Khorshid et al., 2005).

Statistical analysis: The data were expressed as the Mean±SD of the optical density obtained from three independent experiments (each experiment was performed in three replicate wells). Statistical analysis was performed with SPSS and graph pad statistical programs.

RESULTS

The MCF-7 human breast cancer cell line was treated with PM 701 and its fractions at different concentrations. Cells were plated with 1x10⁵ cells/well in 24-well tissue culture plates and incubated in tested agent for 24 h at 37°C.

IC₅₀ estimation (short incubation): This experiment was preformed to estimate the IC₅₀ concentration of all tested agents.

The cytotoxic effect of lyophilized PM 701 was studied by incubated the human breast cancer cells, MCF-7 for 24 h in DMEM media with serial concentration of the drug. PM 701 and its fractions inhibited the proliferation of MCF-7 cells in a dose-dependent manner. The IC₅₀ of PM 701 is 0.35 μg mL^-1 of media, which was significantly low as compared to IC₅₀ of PMF and PMFK (3 and 3.5 μg mL^-1 of media, respectively).

The fixed and stained MCF-7 cells showed that they decreased in number when incubated in media containing the examined substrate for 24 h.

Effect of lyophilized PM 701 on MCF-7 cells: First results indicated that lyophilized PM 701 inhibited the proliferation of cancer cells and the IC₅₀ was about 0.35 μg of PM 701 mL of media, (Table 1, Fig. 1-4).

Effect of PMF on MCF-7 cells: Treatment of MCF-7 with serial concentrations of PMF fraction in incubated media showed inhibition of the cell proliferation with IC₅₀ near the concentration 3 μg of PMF mL^-1 of media (Table 2, Fig. 2-4).

Table 1:	The effect of lyophilized PM 701 on the growth of MCF-7 cancer cells after 24 h of incubation comparing with non treated cancer cells (control)

Table 2:	The effect of PMF on the growth of MCF-7 cancer cells after 24 h of incubation comparing with non treated cancer cells (control)

Fig. 1:

The effect of lyophilized PM 701 on MCF-7 cell line after 24 h of incubation, PM 701 appeared cytotoxic to the cancer cells in a dose-dependent manner

Fig. 2:

The effect of PMF on MCF-7 cell line after 24 h of incubation, PMF appeared cytotoxic to the cancer cells in a dose-dependent manner

Fig. 3:

The effect of PMFK on MCF-7 cell line after 24 h of incubation, PMFK appeared cytotoxic to the cancer cells in a dose-dependent manner

Effect of PMFK on MCF-7 cells: This part indicated that the serial concentrations of PMFK in incubated media inhibited the proliferation of MCF-7 cells and the IC₅₀ was determined near the concentration 3.5 μg of PMFK mL^-1 of media (Table 3, Fig. 3-4).

Long incubation: This experiment was carried on for only crude concentration of PM 701 to confirm the cytotoxic effect of this agent against MCF-7 cells after long incubation for more than 96 h, while the same result could apply to PMF and PMFK that separated from PM 701.

The incubation of MCF-7 cells in higher and lower concentrations than IC₅₀ concentration of PM 701 confirmed the results of short incubation, long incubation of MCF-7 cells in 2 and 20 μg of PM 701 mL^-1 of media inhibited the proliferation of cancer cells in both concentration compared with control non treated cancer cells, whereas Fig. 5 and 6 showed that MCF-7 cells decreased in numbers when PM 701 was added to the incubated media. It also showed that the cells incubated in 2 μg died mostly after incubation for 48 h.

Table 3:	The effect of PMFK on the growth of MCF-7 cancer cells after 24 h of incubation comparing with non treated cancer cells (control)