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Fig. 2 | BMC Cancer

Fig. 2

From: XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis

Fig. 2

(a) Embelin inhibits cell viability in BC cells. (a) Breast cancer cells; CAL-120, EVSAT, MCF-7 and MDA-MB-231cells were treated with increasing doses of embelin ranging between 0 and 50 μM concentration. Cell viability assays were performed using MTT as described in Materials and methods. The graph displays the mean +/− SD (standard deviation) of three independent experiments with replicates of six wells for all the doses and vehicle control for each experiment * p < 0.01 and ** p < 0.001, statistically significant (Students t-test). (b) Embelin treatment induces apoptosis in BC cells. BC cells were treated with increasing doses of embelin for 24 h and cells were analysed for apoptosis after staining with annexin V/PI dual staining by flow cytometry. (c) Embelin inhibits expression of XIAP and induces cleavage of caspases-9, −3 and PARP in BC cells. EVSAT and MDA-MB-231 cells were treated with 25 and 50 μM embelin for 24 h. After cell lysis, equal amounts of proteins were separated on SDS-PAGE, and immunoblotted with antibodies against XIAP, caspase-9, caspase-3 and GAPDH as indicated. (d) XIAP siRNA transfection down-regulates XIAP expression and activates caspases in BC cells. EVSAT and MDA-MB-231 cells were transfected with either scrambled siRNA or specific siRNA against XIAP for 48 h. Following transfection, proteins were isolated and probed with antibodies against XIAP, caspase-9, caspase-3, PARP and GAPDH. (e) Embelin down-regulates XIAP transcript in BC cells. EVSAT cells were treated with 25 and 50 μM Embelin for 24 h and RNA were isolated, reverse transcribed into cDNA. Serial dilutions of untreated EVSAT cells cDNA were used to generate a standard curve for GAPDH and XIAP expression. Following treatment, quantitative RT-PCR was performed on cDNA of PTC cells treated with and without 25 and 50 μM Embelin for the expression of XIAP and GAPDH. Absolute qRT-PCR analysis was performed using ABI-7900HT Fast Real-Time PCR system. The results were plotted on a bar graph and standard deviation calculated. Three replicates for each sample were used. (f) Embelin-induced apoptosis in BC cells is caspase dependent. MDA-MB-231 cells were either pre-treated with universal caspase inhibitor, zVAD-fmk for 3 h followed by treatment with embelin for 24 h. Proteins were isolated and probed with antibodies against caspase-9, caspase-3, PARP and GAPDH

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