The cells were then treated with either Tamoxifen (Sigma-Aldrich, St. expression of the genes. Results DCM-DS was cytotoxic to the MCF-7 cells in a time-and dose-dependent manner. The IC50 values of DCM-DS at 24, 48 and 72?hours were 20.3??2.8, 17.8??1.5 and 15.5??0.5?g/mL, respectively. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 cells at low concentration (12.5 and 25?g/mL) and high concentration (50?g/mL), respectively. Although Annexin-V/PI-flow cytometry analysis has confirmed that DCM-DS induced apoptosis in MCF-7 cells, the distinct characteristics of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies were not observed under microscope. DCM-DS induced formation of ROS in MCF-7 cells. Nevertheless, co-treatment with antioxidants did not attenuate the cell death at low concentration of DCM-DS. The pro-apoptotic gene was up-regulated whereby anti-apoptotic genes and were down-regulated in a dose-dependent manner. Western blot analysis has confirmed that DCM-DS significantly up-regulated the expression of pro-apoptotic JNK1, pJNK and down-regulated anti-apoptotic AKT1, ERK1 in MCF-7 cells. Conclusion DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via multiple signalling pathways. It shows the potential of DCM-DS to be developed to target the cancer cells with mutant caspase-3. (Griffith ex Hook. F. and Thomson) Martelli (Family: Dilleniaceae), commonly known as exhibited anti-cervical and colon cancer properties in rodents (Patent ID: 20120003490) [21]. In addition, root dichloromethane total extract of (DCM-DS) from sequential solvent extraction exhibited strong cytotoxicity towards human MCF-7 breast malignancy cells [22]. Therefore, DCM-DS has a great potential to be developed as evidence-based complementary and option medicine for the treatment of breast cancer. Nevertheless, the underlying mechanisms of DCM-DS-induced Cefditoren pivoxil cytotoxicity in caspase-3 deficient MCF-7 breast malignancy cells remain to be elucidated. This study investigated the cell cycle profile, mode of cell death and signalling pathways of DCM-DS-treated human caspase-3 deficient MCF-7 breast malignancy cells. Methods Herb material Fine powder of was supplied by Primer Herber Sdn. Bhd., Malaysia. The plants authentication was performed with the parts of the plants (flower, leaves, Cefditoren pivoxil stems and roots) at the Biodiversity Unit, Institute of Bioscience, Universiti Putra Malaysia, Malaysia (voucher specimen number SK1937/11). Preparation of plant extract DCM-DS from sequential solvent extraction exhibited strong cytotoxicity towards human MCF-7 breast malignancy cells [22]. Therefore, DCM-DS was employed for the current study with modification around the extraction method (Patent ID: 20120003490). Briefly, 100?g of the powdered root was macerated with 500?L of hexane (1:5, w/v) (Friedemann Schmidt, Francfort, Germany) for 2?days at room heat with occasional shaking at 200?rpm (IKA KS 260 basic, IKA, Staufen, Germany). The mixture was then centrifuged at 2000 for 5?min. The supernatant was filtered through Whatman filter paper No. 1. The residue was re-extracted until the colour disappeared, dried in the oven (40C for 24?hours) and further macerated with dichloromethane (DCM) (Friedemann Schmidt, Francfort, Germany). The combined DCM total extracts were pooled and DCM was removed under reduced pressure (Rotavapor R210, Buchi, Flawil, Switzerland). The percentage of yield for DCM-DS was calculated as: (weight of extract/weight of powdered root) 100%. Cell culture The human MCF-7 breast malignancy Ppia and non-tumourigenic MCF10A cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7 cells were produced in phenol-red-free RPMI 1640 with L-glutamine (Nacalai Tesque, Kyoto, Japan), supplemented with 10% foetal bovine serum (FBS) (PAA, Pasching, Austria) and 1% penicillinCstreptomycin (PAA, Pasching, Austria). MCF-10A cells Cefditoren pivoxil were cultured in DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (PAA, Pasching, Austria), 20?ng/mL epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA), 0.5?mg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 100?ng/mL cholera toxin (Sigma-Aldrich, St. Louis, Cefditoren pivoxil MO, USA), and 10?g/mL insulin (Sigma-Aldrich, St. Louis, MO, USA). The cells used for each experiment were of less than 20 passage number. Determination of cell viability The stock concentration (30?mg/mL) of DCM-DS total extract was prepared in dimethyl sulfoxide (DMSO) (Friedemann.