Anticancer activity of a sub-fraction of dichloromethane extract of Strobilanthes crispus on human breast and prostate cancer cells in vitro

Abstract

Background: The leaves of Strobilanthes crispus (S. crispus) which is native to the regions of Madagascar to the Malay Archipelago, are used in folk medicine for their antidiabetic, diuretic, anticancer and blood pressure lowering properties. Crude extracts of this plant have been found to be cytotoxic to human cancer cell lines and protective against chemically-induced hepatocarcinogenesis in rats. In this study, the cytotoxicity of various sub-fractions of dichloromethane extract isolated from the leaves of S. crispus was determined and the anticancer activity of one of the bioactive sub-fractions, SC/D-F9, was further analysed in breast and prostate cancer cell lines.

Methods: The dichloromethane extract of S. crispus was chromatographed on silica gel by flash column chromatography. The ability of the various sub-fractions obtained to induce cell death of MCF-7, MDA-MB-231, PC-3 and DU-145 cell lines was determined using the LDH assay. The dose-response effect and the EC50 values of the active sub-fraction, SC/D-F9, were determined. Apoptosis was detected using Annexin V antibody and propidium iodide staining and analysed by fluorescence microscopy and flow cytometry, while caspase 3/7 activity was detected using FLICA caspase inhibitor and analysed by fluorescence microscopy.

Results: Selected sub-fractions of the dichloromethane extract induced death of MCF-7, MDA-MB-231, PC-3 and DU-145 cells. The sub-fraction SC/D-F9, consistently killed breast and prostate cancer cell lines with low EC50 values but is non-cytotoxic to the normal breast epithelial cell line, MCF-10A. SC/D-F9 displayed relatively higher cytotoxicity compared to tamoxifen, paclitaxel, docetaxel and doxorubicin. Cell death induced by SC/D-F9 occurred via apoptosis with the involvement of caspase 3 and/or 7.

Conclusions: A dichloromethane sub-fraction of S. crispus displayed potent anticancer activities in vitro that can be further exploited for the development of a potential therapeutic anticancer agent.

Figure 1

Strobilanthes crispus. Strobilanthes crispus (Acanthaceae) Blume, also known by its vernacular name of 'pecah beling' or 'pecah kaca', is a flowering shrub distributed throughout the regions of Madagascar to the Malay Archipelago [11]. The plant can either be found wild in scrublands and river banks or cultivated. A mature plant may grow up to a height of 2 m. The stems are grayish in colour, the branches are dark green, the top surface of the leaves are darker green compared to the surface below, oblong to lanceolate in shape with the sides slightly crenated. Both surfaces of the leaves are very scabrid. The corollas are pubescent and yellowish while the calyx segments are covered with patent long and short hairs [12,35].

Figure 2

Flow chart of extraction procedure for S. crispus. S. crispus leaves were macerated with hexane followed by DCM and fractionated by column chromatography using different combinations of hexane, DCM and MeOH into 15 sub-fractions of differing polarity.

Figure 3

Cytotoxicity of dichloromethane sub-fractions of S. crispus on breast and prostate cancer cell lines. MCF-7 (A), MDA-MB-231 (B), DU-145 (C) and PC-3 (D) cells were treated with various dichloromethane sub-fractions of S. crispus (100 μg/ml) for 24, 48 and 72 hr and cytotoxicity was measured using the LDH release assay. Each value represents the mean of triplicate determinations.

Figure 4

Dose-response effect and EC₅₀ values of SC/D-F9 on breast and prostate cancer cell lines. Dose- and time-dependent effect of a bioactive sub-fraction of dichloromethane extract of S. crispus, SC/D-F9 on MCF-7 (A), MDA-MB-231 (B), DU-145 (C) and PC-3 (D) cells using the LDH assay (prepared in triplicates). The EC₅₀ values were plotted against each time point for the determination of the respective constant EC₅₀ values (E to H).

Figure 5

Comparison of the cytotoxic effect of SC/D-F9 with tamoxifen, paclitaxel and doxorubicin in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with 8.5 and 10.0 μg/ml SC/D-F9, respectively, 5 and 15 μM tamoxifen (T5, T15), 10 and 100 nM paclitaxel (P10, P100) and 50 and 250 nM doxorubicin (Dx50, Dx250), for 24 and 48 hr. The control cultures contained the solvent (0.1% DMSO). The percentages of cell death induced were determined using the LDH release assay and each data represents the mean ± SD from three independent experiments. Statistical analysis was determined using the Student T test with * p < 0.05; ** p < 0.01; *** p < 0.001, compared to control.

Figure 6

Comparison of the cytotoxic effect of SC/D-F9 with docetaxel, doxorubicin and paclitaxel on prostate cancer cells. DU-145 and PC-3 cells were treated with 7.2 and 7.4 μg/ml SC/D-F9, respectively, 5 and 20 nM docetaxel (Dc5, Dc20), 10 and 100 nM doxorubicin (Dx10, Dx100) and 5 and 50 nM paclitaxel (P5, P50) and, for 24 and 48 hr. The control cultures contained 0.1% DMSO. The percentages of cell death induced were determined using the LDH release assay and each data represents the mean ± SD from three independent experiments. Statistical analysis was determined using the Student T test with * p < 0.05; ** p < 0.01; *** p < 0.001, compared to control.

Figure 7

The effect of SC/D-F9, tamoxifen, paclitaxel, doxorubicin and docetaxel on MCF-10A cells. MCF-10A cells were treated with SC/D-F9 (8.5 and 17.0 μg/ml), 5 and 15 μM tamoxifen (T5, T15), 10 and 100 nM paclitaxel (P10, P100), 50 and 250 nM doxorubicin (Dx5, Dx250) as well as 10 and 100 nM docetaxel (Dc10, Dc100) for 24, 48 and 72 hr. The control cultures contained 0.1% DMSO. Each value represent the mean ± SD from three independent experiments. Statistical analysis was determined using the Student T test with * p < 0.05; ** p < 0.01; *** p < 0.001, compared to control.

Figure 8

Apoptosis of breast and prostate cancer cells by SC/D-F9, tamoxifen and paclitaxel. MCF-7 (A-C) and MDA-MB-231 (D-F) cells were treated with DMSO (0.1%), SC/D-F9 (8.5 or 10.0 μg/ml, repectively), and tamoxifen (15 μM) for 24 hr while the PC-3 (G-I) and DU-145 (J-L) cells were treated with DMSO (0.1%), SC/D-F9 (7.4 and 7.2 μg/ml, respectively) and paclitaxel (50 nM) for 48 hr. The cells were stained with annexin V-FITC antibody (green staining) and propidium iodide (red staining) and observed using a fluorescence microscope (20× magnification).

Figure 9

Percentage distribution of SC/D-F9-induced apoptotic and necrotic breast and prostate cancer cells. MCF-7 and MDA-MB-231 cells were treated with DMSO (0.1%), SC/D-F9 (8.5 or 10.0 μg/ml, repectively), and tamoxifen (15 μM) for 24 hr while the PC-3 and DU-145 cells were treated with DMSO (0.1%), SC/D-F9 (7.4 and 7.2 μg/ml, respectively) and paclitaxel (50 nM) for 48 hr. The cells were stained with annexin V-FITC antibody and propidium iodide and analysed by flow cytometry.

Figure 10

Activation of caspase 3/7 in breast and prostate cancer cells by SC/D-F9. MCF-7 and MDA-MB-231 cells were treated with DMSO (0.1%) or SC/D-F9 (8.5 or 10.0 μg/ml, repectively) for 24 hr while the PC-3 and DU-145 cells were treated with DMSO (0.1%) or SC/D-F9 (7.4 and 7.2 μg/ml, respectively) for 48 hr. The cells were reacted with the green fluorescent-labeled caspase inhibitor, FAM-VAD-FMK, and observed under a fluorescent microscope.