Anticancer activity of a sub-fraction of dichloromethane extract of Strobilanthes crispus on human breast and prostate cancer cells in vitro
© Yaacob et al; licensee BioMed Central Ltd. 2010
Received: 10 April 2010
Accepted: 5 August 2010
Published: 5 August 2010
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.
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.
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.
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.
Cancer is a major public health problem worldwide with millions of new cancer patients diagnosed each year and many deaths resulting from this disease. Chemotherapy remains the principal mode of treatment for various cancers. Tamoxifen, a non-steroidal anti-estrogen drug, is used in the treatment of estrogen receptor (ER)-positive breast cancer patients and as chemoprevention in high risk women  but is not effective against ER-negative breast tumours . The anthracycline doxorubicin is frequently used as a chemotherapeutic agent against metastatic breast cancers . Plant alkaloids like docetaxel and paclitaxel are considered highly active chemotherapeutic agents in various cancers including those of the breast and prostate [4, 5]. However, development of resistance to chemotherapeutic drugs impedes effective killing of the cancer cells, resulting in tumour recurrence. In addition, patients usually suffer from serious side-effects such as cardiac and other toxicities [6–8].
S. crispus has high mineral content and contains polyphenols, catechins, alkaloids, caffeine, tannins and vitamins  and bioactive components such as stigmasterol and β-sitosterol . The water extract of this plant was reported to contain compounds with very high binding affinity to protein molecules, hence, inhibiting the proliferation of retroviruses . Pharmacological studies have further shown the ability of S. crispus in preventing chemically-induced hepatocarcinogenesis in rats [17–19]. Administration of S. crispus extract also reduced the severity of hepatic necrosis in rats with diethylnitrosamine- and acetylaminofluorene-induced hepatocellular carcinoma and this was suggested to be due to the inhibition of enzymes involved in metabolic activation of the carcinogens . In vitro studies demonstrated that crude methanol (MeOH) extract of S. crispus was cytotoxic against HepG2 (liver), Caco-2 (colon) and MDA-MB-231 (breast) cancer cell lines while the chloroform extract was found to be cytotoxic to HepG2 and Caco-2 cells only . These authors also reported that stigmasterol and β-sitosterol isolated from S. crispus leaves were cytotoxic to Caco-2, HepG-2, MCF-7 as well as MDA-MB-231 (stigmasterol only) cells. The essential oils of this plant, however, did not display any cytotoxic activity in these cell lines, despite their high antioxidant content .
In the current study, the cytotoxicity of various sub-fractions of the dichloromethane (DCM) extract of S. crispus was determined and the apoptotic activity of one of the sub-fractions with high cytotoxic potential was further analysed in breast and prostate cancer cell lines. Various sub-fractions of the DCM extract of S. crispus were able to selectively induce cell death of breast and prostate cancer cell lines. One of the bioactive sub-fractions, SC/D-F9, was found to be relatively more potent than doxorubicin, paclitaxel, docetaxel and tamoxifen (low dose) in vitro, and induced cancer cell death via apoptosis.
S. crispus (L.) Blume (Acanthaceae) plants were collected from Tasek Gelugur, Pulau Pinang, Malaysia. The plant was authenticated and a voucher specimen of the plant (no. 11046) was deposited at the herbarium of the School of Biological Sciences, Universiti Sains Malaysia.
Fractionation of the active plant extract
The DCM extract above was tested and found to be cytotoxic (data not shown) and thus warranted further investigation. The DCM extract (approximately 2 g) was chromatographed on a glass column (50 mm i.d.) packed with silica gel 60, 0.040-0.063 mm (Merck, Darmstadt, Germany) (200 g). Gradient step elution was carried out using a combination of hexane, DCM and MeOH with an initial ratio of hexane-DCM-MeOH, 9:1:0 (v/v/v), followed by 4:1:0, 3:2:0, 2:3:0, 1:4:0, 0:1:0, 0:95:5, 0:9:1, 0:4:1, 0:3:2 and 0:2:3 (v/v/v). The volume of the solvent combination used in each gradient step was 400 ml. Nitrogen gas pressure was applied onto the column at 1 bar to increase the flow of the mobile phase. Eluents were collected in portions of 40 ml. Finally the column was flushed with MeOH.
A small sample of each eluent was evaluated using thin layer chromatography and those eluents which showed similar chemical composition were combined and concentrated under vacuum to yield a total of 15 DCM sub-fractions designated as SC/D-F1, SC/D-F2, SC/D-F3, SC/D-F4, ..., SC/D-F15 (Figure 2).
A concentrated stock solution of S. crispus extracts and fractions was prepared in dimethyl sulphoxide (DMSO) and stored at -20°C until required. Prior to analysis, the samples were diluted in an appropriate growth culture medium with the final concentration of DMSO in culture of ≤ 0.1%.
Human breast (MCF-7 and MDA-MB-231) and prostate (PC-3 and DU-145) cancer cell lines and normal breast epithelial cell line (MCF-10A) were obtained from the American Type Culture Collection (ATCC) (Rockville, USA). MCF-7 and DU-145 cells were cultured in RPMI-1640 medium, MDA-MB-231 cells in Dulbecco's modified Eagle's medium (DMEM), PC-3 cells in Ham F12K medium and MCF-10A in DMEM:Ham F12K (1:1) medium, all supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco BRL, USA) and 10% fetal bovine serum (Hyclone, USA) and maintained at 37°C in a humidified atmosphere of 5% CO2 in air.
The cytotoxicity of S. crispus DCM sub-fractions was determined using the LDH Cytotoxicity Detection Kit (Roche Diagnostics, Germany) which quantifies the release of lactate dehydrogenase (LDH) from the cells into the culture medium. The cells were seeded in 24-well plates at a density of 1 × 105 (PC-3, DU-145 and MCF-10A), 2 × 105 (MCF-7) or 1 × 106 cells/ml (MDA-MB-231) and grown until about 70% confluence. The medium was then replaced with fresh medium containing 2% fetal bovine serum prior to treatment with the fractions of the extracts for up to 72 hr. The control cells received not more than 0.1% DMSO which was used as a solvent for the extract. Maximum LDH release (high control) was determined by solubilising cells with 1% (w/v) Triton X-100 and spontaneous LDH release (low control) was determined by incubating cells with the medium alone. Cell-free supernatants from the cultures were collected and transferred to 96-well plates for measurement of LDH activity. A reduction reaction of tetrazolium salts, INT, to a red formazan salt was used as an indicator of LDH activity in the supernatant. Absorbance was read at 490 nm and 620 nm reference absorbance by using a microplate reader (Versamax, USA). Results were expressed as % cytotoxicity [(experimental value-low control/high control-low control) × 100]. Effective concentration (EC50) values were expressed as μg/ml concentration of the extract that causes 50% cell growth inhibition.
Annexin V-FLUOS Assay
Apoptosis was determined using the Annexin-V FLUOS Staining Kit (Roche, Germany) in combination with propidium iodide, according to the manufacturer's instructions. Briefly, cells were cultured in chamber slides or T25 flasks at a density of 5 × 105 cells/ml and allowed to attach overnight, followed by treatment with the SC/D-F9 or anticancer drugs for 24 or 48 hr. Cells were then washed with phosphate-buffered saline (PBS) and incubated with Annexin-V FLUOS labeling solution (containing 2 μl Annexin-V-FLUOS labeling reagent and 2 μl propidium iodide solution in 100 μl incubation buffer) for 10-15 min at room temperature. Analysis was carried out by fluorescence microscopy (Nikon, USA) and flow cytometry (FACS Calibur, Becton-Dickinson, USA). A minimum of 10,000 events were collected for analysis.
Determination of Caspase 3/7 Activity
Cells were cultured in chamber slides as above for detection of active caspase 3/7 by fluorescence microscopy using the Carboxyfluorescein FLICA Apoptosis Detection Kit (Immunochemistry Technologies, LLC), according to the manufacturer's instructions. Cells were labeled with green fluorescent FAM-VAD-FMK and caspase-3/7 activity was analysed by fluorescence microscopy.
Determination of Total Phenolic Content
Total phenol estimation was determined according to a previously described method . A 0.2 N Folin-Ciocalteu reagent (500 μl) was added to 1 ml aliquots of the extract (1 mg/ml) and vigorously mixed by vortexing. The mixture was then incubated at room temperature for 3 min. Subsequently, 4 ml sodium carbonate solution (7.5% w/v) was added and the mixture was incubated at room temperature for 60 min. Finally, the absorbance was measured at 760 nm using a spectrophotometer (Shimadzu, Japan) and the measurement was compared to a standard curve of gallic acid. The mean value (± SD) of triplicate analyses is expressed as mg gallic acid equivalents per g plant material (GAE/g).
Determination of Total Flavonoid Content
Total flavonoid content was determined according to the method of Sakanaka et al.. The extract (250 μl, 1 mg/ml) was mixed with distilled water (1.25 ml) and sodium nitrite [75 μl, 5% (w/v)] and incubated at room temperature for 6 min. Subsequently, aluminium chloride solution [150 μl, 10% (w/v)] was added and the mixture was further incubated for 5 min before the addition of sodium hydroxide (0.5 ml, 1 M). Thereafter, distilled water (275 μl) was added and vortexed. Finally, the absorbance was measured at 510 nm using a spectrophotometer and the measurement was compared to a standard curve of catechin. The value (mean of triplicate analyses) is expressed as mg catechin equivalents per g plant material (CE/g).
Data are presented as mean ± standard deviation (SD) of three independent experiments and statistical significance was determined using Independent Student T test and the SPSS software (SPSS Science Inc.)
Results and Discussion
MCF-7 cell line is estrogen receptor (ER)-dependent and carries the wild type tumour suppressor p53 gene, while the highly aggressive MDA-MB-231 is an ER-independent breast cancer cell line, and is a p53 mutant [25, 26]. PC-3 and DU-145 are androgen-insensitive prostate cancer cells . PC-3 is of an aggressive phenotype while DU-145 cells have a more moderate metastatic potential [28, 29]. In addition, both prostate cancer cells do not express normal p53 gene. The selective cytotoxic effects of the different DCM sub-fractions observed against the various cancer cell lines tested may be hormone-dependent or -independent, p53-related or influenced by other properties of the cancer cell lines, although these characteristics are yet to be determined.
Androgens regulate prostate cancer cell growth and differentiation. Current medical therapy for prostate cancer patients includes anti-androgens which inhibit the binding of androgens to the androgen receptor, as well as gonadotrophin-releasing hormone (GnRH) analogues which downregulate GnRH receptors leading to the inhibition of androgen production . This would therefore lead to apoptosis of prostate cancer cells. However, treatment for hormone-resistant prostate cancer is limited and addition of anti-androgens may produce only a transient biochemical response . DU-145 and PC-3 cells are both androgen receptor-positive but androgen non-responsive  and hence SC/D-F9 may provide a potential complementary therapeutic agent for hormone resistant prostate cancer.
Analysis of SC/D-F9 showed that it has a total phenolic content of 29.0 ± 0.867 mg/g of gallic acid and a total flavonoid content of 59.0 ± 0.333 mg/g of catechin. These amounts are higher than those reported on some other plant extracts [33, 34] and these may contribute to the anticancer effects of SC/D-F9 observed in this study, although this has yet to be confirmed. In addition, it has earlier been reported that S. crispus extract has high antioxidant activity that may be attributed to the presence of catechin as well other flavonoids . Since SC/D-F9 is shown to effectively induce apoptosis in androgen non-responsive prostate cancer cells as well as ER-positive and ER-negative breast cancer cells in the current study, the potential of the S. crispus plant to be developed further as a cancer therapeutic agent should be explored. Apoptosis is a tightly regulated process controlled by various intracellular signaling molecules involved in different pathways . Work is currently underway to identify the bioactive component(s) in SC/D-F9 to further understand the mechanism of action of S. crispus.
A large number of novel anticancer drugs have been discovered from natural products in the past and new ones are continually being developed. These cytotoxic natural products may be able to play a significant role in treating selected cancers by working in concert with conventional chemotherapeutic drugs thereby improving their efficacy or reducing their toxicity. We have shown that S. crispus has potent anticancer activities in vitro and could therefore, potentially be a source for a pharmacologically active product suitable for development as a chemotherapeutic agent.
This study was supported by the Research University Grant of Universiti Sains Malaysia (1001.PPSP.813002) and MOSTI/IPharm Research Initiative Grant. NNNMK and SNAZA are sponsored under the Universiti Sains Malaysia Fellowship Scheme. The authors thank Baharuddin Sulaiman of the School of Biological Sciences, Universiti Sains Malaysia, for authenticating the plant material
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