Plant material and extraction

The roots of Dorstenia psilurus (D. psilurus) were collected at Komako in the West Region of Cameroon and identified by Mr Victor NANA, of the National Herbarium of Cameroon, in December 2010. A voucher specimen (1649/SRF/CAM) was deposited at the National Herbarium Yaounde, Cameroon. The roots of D. psilurus (DP) were air-dried and ground. The powdered plant material (150 g) was macerated in MeOH (1 l) for 24 h at room temperature and then repeated once. The diluted extract was concentrated under reduced pressure to afford 40 g of a dark residue.

Cell culture

Human promyelocytic leukemia (HL-60 cells) and prostate cancer (PC-3 cells) were obtained from European Collection of Cells Culture (ECCC), Sigma Aldrich, India. They were grown in RPMI-1640 medium containing 10% Foetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 μg/ml medium). The cells were culture in the incubator (Thermocom Electron Corporation, USA) at 37°C, 5% CO 2 ; 98% humidity. Cells were used for different assays during logarithmic growth phase while the untreated control cultures received only the vehicle (DMSO <0.1%).

Cells viability and treatments

The human promyelocytic leukemia (HL-60 cells) and prostate cancer (PC-3 cells) were seeded in different 96 well plates containing 15x103 and 6x103 cells/100 μl/well, respectively. The cultured cells were then treated the same (triplicate wells per condition) by the addition of 100 μl of serial dilutions of the DP extract dissolved in DMSO to give a final concentration of 30, 10 and 1 μg/ml. For PC-3, the extract was added after 24 h of incubation. In addition, the DMSO alone was added to another set of cells as the solvent control (DMSO <0.1%). The cells were then incubated for another 48 h prior to the addition of 20 μl of 2.5 mg/ml solution of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) into each well. The incubation was continued for another 3 h before the media was removed. A mixture of DMSO (150 μl) was added to each well and mixed to ensure dissolving of the crystal formazan before the absorbance at 570 nm was measured. Three replications of each experiment were performed and fifty percent of inhibitory concentration (IC 50 ) of each extract was calculated.

DNA content and cell cycle phase distribution

HL-60 cells (1x106 cells/2 ml/well) were treated with DP at 20, 50, 100 μg/ml for 24 h. They were harvested and washed with 1 ml of PBS, then centrifuged 400 g for 5 min at 4°C. The pellet was suspended in 100 μl of PBS and 900 μl of hypertonic buffer (PI-25 μg/ml, RNAase-40 μg/ml, sodium citrate-0.1% and Triton-100X-0.03%) and incubated at 37°C in dark for 20 min. Finally, cells were analyzed immediately on flow cytometer FACSCalibur (Becton Dickinson, USA). The data were collected in list mode on 10,000 events and illustrated in a histogram, where the number of cells (counts) is plotted against the relative fluorescence intensity of PI (FL-2; λem: 585 nm; red fluorescence). The resulting DNA distributions were analyzed by Modfit (Verity Software House Inc., Topsham, ME) for the proportions of cells in G 0 -G 1 , S- phase, and G 2 -M phases of the cell cycle [20].

Hoechst 33258 staining of cells for nuclear morphology

HL-60 cells (2x106 cells/3 ml/well) were treated with DP extract at different concentration of extract for 24 h. They were collected, centrifuged at 400 g and washed once with PBS. A solution of Hoechst (Hoechst, 10 μg/ml; citric 10 mM; Na 2 HPO 4 0.45 M; Tween-20 0.05%) was added in each tube and kept in the dark at room temperature for 30 min. The mixture was then washed once with PBS and the pellet resuspended in 100 μl of PBS/glycerol (1:1). The solution (10 μl) was poured into the slide and observed for nuclear morphology alterations under fluorescence microscope (Olympus X 70, magnification 20 X).

Mitochondrial membrane potential (MMP) assay

HL-60 cells (1x106 cells/2 ml/well) were treated with DP extract at different concentrations for 24 h. Thirty minutes before the end of the experiment, the cell culture was treated with Rhodamine-123 (200nM) and keep in the dark for 30 mn. Cells were then collected, centrifuged (400 g; 4°C; 5 min), the pellet was washed with 1 ml of PBS and centrifuged as mentioned earlier. The fluorescence intensity of 10,000 events was analyzed in FL-1 channel on BD FACSCalibur (Becton Dickinson, USA) flow cytometer. The decrease in fluorescence intensity because of mitochondrial membrane potential loss was analyzed in FL-1 channel and the change of in potential membrane (∆ψm) was assessed by comparing fluorescence.

Reactive oxygen species (ROS) assay

ROS production was monitored by flow cytometry using 2’, 7’-dichlorodihydrofluorescin diacetate (DCFH 2 -DA). This dye is a stable non polar compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield 2’,7’-dichloro dihydrofluorescin (DCFH), which is trapped within the cells. Hydrogen peroxide or low molecular weight peroxides produced by the cells oxidize DCFH to the highly fluorescent compound 2’,7’-dichlorofluorescein (DCF). Thus, the fluorescence intensity is proportional to the amount of hydrogen peroxide produced by the cells. Briefly, HL-60 cells (1x106 cells/2 ml/well) were treated with DP at different concentration for 24 h. Thirty minutes before the end of the experiment, the cell culture was treated with DCFH 2 -DA (50 μM) and keep in the dark. Cells were then collected, centrifuged (200 g; 4°C; 5 min) and the pellet was washed with 1 ml of PBS and centrifuged as mentioned earlier. The pellet was suspended in 500 μl of PBS and the fluorescence was assessed by comparing two fluorescence emission 480 nm/530 nm using a flow-cytometer (BD-LSR).

Statistical analysis

The viability experiments were done in triplicates and each data point represents the average of at least 3 independent experiments. The data was expressed as mean ± SD. In order to carry out statistical analysis, the data was analyzed using SPSS (Version 11.5; SPSS Inc.,) and M.S. Office, Excel software. One way analysis of variance technique was applied to observe the significance between the groups. The post hoc test Duncan’s multiple range test was performed to know the significant difference among the groups. Entire statistical analysis was carried out at p < 0.05.